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UBC Theses and Dissertations

Characterization of Bacteroides melaninogenicus and its role in mixed anaerobic infections Mayrand, Denis 1978

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CHARACTERIZATION OF BACTEROIDES MELANINOGENICUS AND ITS ROLE IN MIXED ANAEROBIC INFECTIONS by DENIS MAYRAND B . S c , U n i v e r s i t e de M o n t r e a l , 1969 M . S c , U n i v e r s i t e de M o n t r e a l , 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA DOCTOR OF PHILOSOPHY IN May, 1978 D e n i s Mayrand, 1978 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g ree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f M i c r o b i o l o g y The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 Date 30/5/78 ABSTRACT The pigmented o r a l B a c t e r O i d e s were c h a r a c t e r i z e d w i t h r e g a r d t o t h e i r p a t h o g e n i c , c o l l a g e n o l y t i c , h e m a g g l u t i n a t i n g and m e t a b o l i c p r o p e r t i e s . I s o l a t e s were o b t a i n e d f r o m h e a l t h y and d i s e a s e d g i n g i v a l s u l c i o f 396 c h i l d r e n , a d u l t s and mongrel dogs. F i f t y - t h r e e o f the i s o l a t e s p r o v e d t o be members of the s u b s p e c i e s I i . m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s . The re m a i n d e r were e i t h e r 15. m e l a n i n o g e n i c u s s s . i n t e r m e d i u s o r m e l a n i n o g e n i c u s . A l l i s o l a t e s d e s i g n a t e d a s a c c h a r o l y t i c u s were p a t h o g e n i c i n t h e g u i n e a p i g i n f e c t i v i t y a s s a y . The s u b s p e c i e s i n t e r m e d i u s and m e l a n i n o g e n i c u s were n o t p a t h o g e n i c . The s u b s p e c i e s a s a c c h a r o l y t i c u s p o s s e s s e d a c e l l bound oxygen s e n s i t i v e c o l l a g e n a s e , a c e l l bound and s o l u b l e oxygen s e n s i t i v e h e m a g g l u t i n i n , and produced b u t y r i c and p h e n y l a c e t i c a c i d s . These p r o p e r t i e s were u n i q u e t o a s a c c h a r o l y t i c u s and were n o t d e t e c t e d w i t h o t h e r o r g a n i s m s . A l l i s o l a t e s r e q u i r e d hemin. The p a t h o g e n i c o r g a n i s m s p o s s e s s e d a h e a t s e n s i t i v e t o x i n w h i c h i n d u c e d f l u i d a c c u m u l a t i o n i n l i g a t e d mouse i l e a l l o o p s . I n f e c t i v i t y o f 13. m e l a n i n o g e n i c u s a s a c c h a r o l y t i c u s was dependent on t h e p r e s e n c e o f a second o r g a n i s m . An i n f e c t i v e c o n s o r t i u m c o n s i s t i n g o f 13. m e l a n i n o g e n i c u s a s a c c h a r o l y t i c u s and K l e b s i e l l a pneumoniae was d e f i n e d . N e i t h e r o r g a n i s m was i n f e c t i v e a l o n e b u t t h e K l e b s i e l l a c o u l d be r e p l a c e d by organisms o f a number o f d i f f e r e n t g e n e r a . The number and p r o p o r t i o n s o f J3. m e l a n i n o g e n i c u s and K. pneumoniae r e q u i r e d to e s t a b l i s h a l e s i o n was d e t e r m i n e d . The n a t u r e o f t h e i n f e c t i o n appeared t o be d e t e r m i n e d by t h e l e n g t h o f the l a g p e r i o d p r e c e d i n g t h e i n i t i a t i o n o f growth o f 15. m e l a n i n o g e n i c u s . A r a p i d o n s e t o f growth l e d t o t h e s e v e r e s p r e a d i n g f o r m o f t h e d i s e a s e whereas a s l o w i n i t i a t i o n o f growth r e s u l t e d i n t h e f o r m a t i o n o f a l o c a l i z e d s e l f l i m i t i n g a b s c e s s . J3. m e l a n i n o g e n i c u s depends on t h e second o r " h e l p e r " o r g a n i s m t o pro d u c e a r e q u i r e d growth f a c t o r w h i c h i s n o t p r e s e n t a t t h e i n o c u l a t i o n s i t e . The growth f a c t o r was shown t o be s u c c i n a t e w h i c h was a b l e t o r e p l a c e t h e hemin r e q u i r e m e n t . The dependency oh s u c c i n a t e p r o duced by K. pneumoniae was d e m o n s t r a t e d i n a g a r medium, i n l i q u i d c u l t u r e and i n t h e i n f e c t i v i t y a s s a y . Any o r g a n i s m w h i c h produced s u c c i n a t e was a b l e t o s t i m u l a t e growth o f JJ. t n e l a h i n o g e n i c u s on a g a r medium and c o u l d r e p l a c e K.. pneumoniae as a member of the i n f e c t i o u s c o n s o r t i u m . The need f o r t h e second o r g a n i s m c o u l d be e l i m i n a t e d by i n o c u l a t i n g 13. m e l a n i n o g e n i c u s t o g e t h e r w i t h a g a r - i m m o b i l i z e d s u c c i n a t e o r hemin. Growth o f I i . m e l a n i n o g e n i c u s i s dependent on t h e p r e s e n c e o f l a r g e q u a n t i t i e s of s u c c i n i c a c i d s u g g e s t i n g t h a t t h e compound i s used i n energy m e t a b o l i s m and i s n o t i n c o r p o r a t e d i n t o c e l l u l a r c a r b o n . T h i s a s s u m p t i o n i s s u p p o r t e d by t h e o b s e r v a t i o n t h a t o n l y 0.5% o f the s u c c i n a t e c a r b o n can be f o u n d i n t h e c e l l , t h e r e m a i n d e r o f t h e m e t a b o l i z e d s u c c i n a t e i s e x c r e t e d as b u t y r a t e . Hemin b l o c k s t h e m e t a b o l i s m o f s u c c i n a t e . The f a t t y a c i d metabo-l i t e s a r e q u a l i t a t i v e l y s i m i l a r b u t q u a n t i t a t i v e l y d i f f e r e n t i n c e l l s grown i n hemin f r e e s u c c i n a t e medium. i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i i L IST OF FIGURES x ACKNOWLEDGEMENTS x i i I . . INTRODUCTION A. B a c t e r o i d e s m e l a r i i n b g e r i i c u s 1 1. B i o c h e m i c a l h e t e r o g e n e i t y 2 2. A n t i g e n i c s t r u c t u r e and s e r o l o g i c a l h e t e r o g e n e i t y 6 3. G e n e t i c v a r i a t i o n 7 4. P a t h o g e n i c p r o p e r t i e s o f I i . m e l a n i n o g e n i c u s 8 a. Adherence 8 b. I n v a s i v e n e s s 8 c. T o x i n p r o d u c t i o n 9 d. E n d o t o x i c a c t i v i t y 10 B. Mi x e d a n a e r o b i c i n f e c t i o n s 11 I I . MATERIALS AND METHODS A. Organisms 17 1. _B. m e l a n i n o g e n i c u s 17 2. Ot h e r organisms 17 B. A n a e r o b i c growth 18 C. Growth media 19 1. B. m e l a n i n o g e n i c u s 19 a. S t a n d a r d medium 19 b. B a s a l medium 19 c. V i t a m i n K medium 19 V TABLE OF.CONTENTS Ccontinued) Page I I . MATERIALS AND METHODS d. Supplemented media 19 2. Other organisms 20 D. I n f e c t i v i t y 20 E. E n u m e r a t i o n o f b a c t e r i a 22 1. T o t a l count 22 2. V i a b l e count 22 a. C o l o n y f o r m i n g u n i t s 22 b. Most p r o b a b l e number 22 F. C o l l a g e n a s e and p r o t e a s e a c t i v i t y 23 1. C o l l a g e n a s e 23 2. P r o t e a s e 25 G. H y d r o x y p r o l i n e a s s a y 25 H. F a t t y a c i d d e t e r m i n a t i o n 26 1. P r e p a r a t i o n o f samples 26 a. G a s - l i q u i d chromatography (GLC) 26 i . P r e p a r a t i o n o f column 26 i i . O p e r a t i n g c o n d i t i o n s 26 b. T h i n - l a y e r chromatography (TLC) 29 I . P u r i f i c a t i o n o f growth f a c t o r 29 1. Growth a s s a y 29 2. C o n c e n t r a t i o n 30 3. Chromatography 30 a. P r e p a r a t i o n and c a l i b r a t i o n , 30 b. Chromatography of growth f a c t o r .... 31 v i TABLE OF CONTENTS ( c o n t i n u e d ) Page I I . MATERIALS AND METHODS J . G l u c o s e d e t e r m i n a t i o n 31 K. C e l l f r a c t i o n a t i o n . . 31 L. H e m a g g l u t i n a t i o n . 37 M. I l e a l l o o p a s s a y . 37 N. C h e m i c a l s ' 38 0. P o r p h y r i n and,hemin e x t r a c t i o n 38 I I I . RESULTS P a r t I . C h a r a c t e r i z a t i o n o f jB. m e l a n i n o g e n i c u s 39 A. I s o l a t i o n o f organisms 39 B. C h a r a c t e r i z a t i o n o f i s o l a t e s 41 1. F a t t y a c i d p r o d u c t i o n 41 2. H e m a g g l u t i n a t i o n 48 3. C o l l a g e n a s e 55 4. Requirement f o r v i t a m i n K 58 5. P r o t e o l y t i c a c t i v i t y 62 6. P a t h o g e n i c i t y 62 C. F l u i d a c c u m u l a t i o n 64 P a r t I I . S t u d i e s on Mi x e d I n f e c t i o n s 68 A. I s o l a t i o n o f an i n f e c t i o u s c o n s o r t i u m 68 B. E f f e c t o f 1-20 on the p a t h o g e n i c i t y o f o t h e r I i . m e l a n i n o g e n i c u s i s o l a t e s 71 C. V i a b i l i t y and i n f e c t i o n 74 D. Number of c e l l s r e q u i r e d t o i n i t i a t e an i n f e c t i o n 74 v i i TABLE OF CONTENTS ( c o n t i n u e d ) Page I I I . RESULTS P a r t I I . E. Age of c e l l s g rowth 77 F. Numbers o f K. pneumoniae 81 P a r t I I I . N a t u r e of t h e M i c r o b i a l I n t e r d e p e n d e n c e 83 A. E v i d e n c e f o r n u t r i t i o n a l dependence 83 1. Growth s t i m u l a t i o n on agar 83 2. Growth s t i m u l a t i o n i n l i q u i d media 86 B. C o - c u l t i v a t i o n o f B. m e l a n i n o g e n i c u s and K. pneumoniae 88 C. Growth of s t r a i n K110 i n K. pneumoniae c u l t u r e f i l t r a t e .... 94 D. I d e n t i f i c a t i o n o f the growth f a c t o r 94 1. P r o p e r t i e s 94 2. M e t a b o l i t e a n a l y s i s 94 3. P r o d u c t i o n of s u c c i n a t e 96 4. Growth s t i m u l a t i o n by s u c c i n i c a c i d 99 5. I s o l a t i o n o f the growth f a c t o r 106 E. Growth s t i m u l a t i o n by o t h e r b a c t e r i a 106 F. In v i v o s t u d i e s 109 G. S u c c i n i c a c i d 114 1. S u c c i n i c a c i d u t i l i z a t i o n by 33. m e l a n i n o g e n i c u s 114 2. S u c c i n i c a c i d 116 a. I n c o r p o r a t i o n i n t o c e l l u l a r m a t e r i a l 116 B. A n a l y s i s o f the c u l t u r e s u p e r n a t a n t s 118 IV. DISCUSSION ' 123 V. LITERATURE CITED 130 v i i i L IST OF TABLES T a b l e T i t l e Page I . D e s c r i p t i o n o f d i s e a s e 21 I I . C o l l a g e n a s e a s s a y 24 I I I . S o u r c e o f 13. m e l a n i n o g e n i c u s i s o l a t e s 40 IV. Summary of f a t t y a c i d p r o d u c t i o n o f 47 B. m e l a r i i n b g e r i i c u s  V. E f f e c t o f the s o u r c e of r e d b l o o d 51 c e l l s on HA a c t i v i t y V I . E f f e c t o f a e r a t i o n on HA a c t i v i t y 53 of s t r a i n K110 V I I . HA o f o r a l B. m e l a n i n o g e n i c u s i s o l a t e s 54 V I I I . V i t a m i n K r e q u i r e m e n t s o f 61 13. m e l a n i n o g e n i c u s i s o l a t e s I X . Summary o f B. m e l a n i n o g e n i c u s 65 c h a r a c t e r i z a t i o n s t u d i e s X. I l e a l l o o p a s s a y 67 X I . I n f e c t i v i t y o f v a r i o u s b a c t e r i a l m i x t u r e s 72 X I I . E f f e c t o f i s o l a t e 20 on o r a l 73 13. m e l a n i n o g e n i c u s i s o l a t e s X I I I . B i o c h e m i c a l c h a r a c t e r i s t i c s o f 1-20 75 XIV. C e l l v i a b i l i t y and i n f e c t i o n 76 XV. Number o f c e l l s r e q u i r e d t o i n d u c e an i n f e c t i o n 78 XVI. E f f e c t o f v a r y i n g t h e r e l a t i v e p r o p o r t i o n o f s t r a i n K110 and K l e b s i e l l a on t h e c o u r s e o f i n f e c t i o n ^2 X V I I . E f f e c t o f g l u c o s e and hemin c o n c e n t r a t i o n on th e growth o f pur e c u l t u r e s ' o f _B. m e l a n i n o g e n i c u s s t r a i n K110 and K. pneumoniae .... 87 i x L IST OP TABLES Ccontimied) T a b l e T i t l e Page X V I I I . Growth of s t r a i n K110 i n K. pneumoniae f i l t r a t e . 98 XIX. Growth s t i m u l a t i o n o f s t r a i n K110 by o t h e r b a c t e r i a 110 XX. I n f e c t i v i t y o f s t r a i n K110 when combined w i t h o r a l b a c t e r i a I l l XXI. E f f e c t o f hemin and s u c c i n a t e on i n f e c t i v i t y o f s t r a i n K110 112 X X I I ' E f f e c t o f hemin and s u c c i n i c a c i d on v o l a t i l e f a t t y a c i d p r o d u c t i o n by B. m e l a n i n o g e n i c u s ......... 115 14 X X I I I . D i s t r i b u t i o n o f C l a b e l l e d s u c c i n a t e 117 XXIV. M e t a b o l i s m o f s u c c i n a t e 122 X LIST OF FIGURES F i g u r e T i t l e Page 1. Gas c h r o m a t o g r a p h i c a n a l y s i s o f f a t t y a c i d s 28 14 2. F r a c t i o n a t i o n o f C - l a b e l l e d I i , m e l a n i n o g e n i c u s ... 33 3. Diagram of gas chromatograph 36 4. Gas chromatography o f v o l a t i l e f a t t y a c i d s p roduced by B. m e l a n i r i o g e n i c u s 43 5. Gas chromatography of n o n - v o l a t i l e f a t t y a c i d s p r o d uced by 15. m e l a r i i r i o g e h i c u s 46 6. I n f l u e n c e o f c u l t u r e age on HA a c t i v i t y 50 7. J5. m e l a n i n o g e n i c u s a s a c c h a r o l y t i c u s c o l l a g e n a s e a c t i v i t y 57 8. C o l l a g e n a s e a c t i v i t y as a f u n c t i o n o f c u l t u r e age 60 9. Growth of J3. m e l a n i n o g e n i c u s K110 i n t h e p r e s e n c e and absence of hemin and v i t a m i n K 70 10. E f f e c t o f i n o c u l u m age on growth 80 11. Growth s t i m u l a t i o n on agar . ... 85 12. E f f e c t o f i n o c u l u m o r i g i n on the growth of s t r a i n K110 90 13. Dependence of s t r a i n K110 on K. pneumoniae i n mixed c u l t u r e s 92 14. Growth and s u c c i n i c a c i d p r o d u c t i o n i n c u l t u r e s of K. pneumoniae 98 15. Growth r e s p o n s e of s t r a i n K110 i n s u c c i n a t e supplemented b a s a l medium 101 16. Growth c h a r a c t e r i s t i c s of s t r a i n K110 i n s u c c i n a t e or hemin supplemented b a s a l medium .... 103 17. E f f e c t o f pH on t h e growth of s t r a i n K110 105 x i L I ST OF FIGURES ( c o n t i n u e d ) F i g u r e T i t l e Page I 8 - F r a c t i o n a t i o n o f the K. pneumoniae c u l t u r e f i l t r a t e on Sephadex G-15 108 19. F a t t y a c i d a n a l y s i s o f s t r a i n K110 c u l t u r e s u p e r n a t a n t 120 ACKNOWLEDGEMENTS To Dr. J.J.R. C a m p b e l l f o r a c c e p t i n g .me i n h i s d e p a r t m e n t , h i s u n d e r s t a n d i n g and h i s g u i d a n c e . To Dr. B a r r y C. M c B r i d e f o r h i s encouragement, c r i t i c i s m and g u i d a n c e t h r o u g h o u t t h i s work. H i s c r e a t i v i t y and open-mindedness i n r e s e a r c h was g r e a t l y a p p r e c i a t e d . I a l s o w i s h t o than k Thomas Edwards, Mary G i s s l o w and Susan J e n s e n f o r h e l p f u l comments, s u g g e s t i o n s and s c i e n t i f i c d i s c u s s i o n . To t h e M e d i c a l R e s e a r c h C o u n c i l and the U n i v e r s i t y o f B r i t i s h C o l u m b i a f o r f i n a n c i a l s u p p o r t . To my w i f e A n n i e , f o r a l l h e r u n d e r s t a n d i n g and m o r a l s u p p o r t I d e d i c a t e t h i s t h e s i s . 1 I . INTRODUCTION PART A. BACTEROIDES MELANINOGENICUS B a c t e r o i d e s m e l a n i n o g e n i c u s i s a g r a m - n e g a t i v e , n o n - m o t i l e , non-s p o r u l a t i n g a n a e r o b i c r o d - The s p e c i e s i s u s u a l l y d e f i n e d as s h o r t r o d s b u t o f t e n appears as ro d s w i t h rounded o r p o i n t e d ends w h i c h v a r y i n s i z e and shape f r o m s m a l l c o c c o i d forms t o l o n g f i l a m e n t o u s o r g a n i s m s . The or g a n i s m ' s p r i n c i p a l h a b i t a t i s t h e o r a l c a v i t y where i t i s found i n t h e g i n g i v a l c r e v i c e (21,94) and i n s u p r a g i n g i v a l human d e n t a l p l a q u e (21,59,98). I t i s a l s o p r e s e n t i n t h e mouths of dogs. I n the human g i n g i v a l c r e v i c e I5_. m e l a n i n o g e n - i c u s r e p r e s e n t s 5 t o 8% of the t o t a l c u l t i v a b l e f l o r a ( 4 , 2 7 , 5 8 ) . T h i s f i g u r e may n o t r e p r e s e n t t h e e x a c t p e r c e n t a g e o f t h e b a c t e r i a i f t h e v a r i o u s problems i n h e r e n t i n such s t u d i e s ; e.g., samplings t r a n s p o r t v i a b i l i t y , d i s p e r s i o n methods and c u l t u r a l c o n d i t i o n s , a r e t a k e n i n t o a c c o u n t . The r o l e o f 13. m e l a n i n o g e n i c u s i n i n f e c t i o n and i t s a b i l i t y t o degrade c o l l a g e n have made i t a pr i m e s u s p e c t as an i m p o r t a n t e t i o l o g i c agent i n p e r i o d o n t a l d i s e a s e . U n f o r t u n a t e l y , t h e r e i s as y e t no e v i d e n c e t o s u b s t a n t i a t e t h i s h y p o t h e s i s . Throughout t h e y e a r s , j J . m e l a n i n o g e n i c u s has a t t r a c t e d the i n t e r e s t o f a number of i n v e s t i g a t o r s b u t d e t a i l e d a n a l y s e s c o n c e r n i n g the b i o c h e m i c a l p r o p e r t i e s o f t h e o r g a n i s m have been hampered by d i f f i c u l t i e s i n g r o w i n g t h e o r g a n i s m i n pur e c u l t u r e . F u r t h e r m o r e , i s o l a t e s c o n s i d e r e d t o be 15. m e l a n i - n o g e n i c u s r e p r e s e n t a h e t e r o g e n e o u s group. A l t h o u g h the c h a r a c t e r i s t i c f e r m e n t a t i o n p a t t e r n w i t h i n t h e s p e c i e s was e s t a b l i s h e d i n e a r l y s t u d i e s ( 9 0 ) , _B. m e l a n i n o g e n i c u s was s t i l l r e c o g n i z e d as a s i n g l e s p e c i e s w i t h f e r m e n t a t i v e and n o n - f e r m e n t a t i v e v a r i a n t s . A n a l y s i s o f f e r m e n t a t i o n p a t t e r n s l e d Moore and Holdeman (72) t o r e c o g n i z e t h r e e s u b s p e c i e s : J3. m e l a n i n o g e n i c u s s s . m e l a n i n o g e n i c u s , B. m e l a n i n o g e n i c u s s s . i n t e r m e d i n s and 15. m e l a n i n o g e n i c u s  s s . a s a c c h a r o l y t i c u s . V a r i o u s methods and p r o c e d u r e s (37,58,93,102,103,116) 2 a r e now a v a i l a b l e t o answer t h e i d e n t i f i c a t i o n p r o b l e m , and s t u d i e s i n t h i s d i r e c t i o n (36,92) i n d i c a t e t h a t t h e r e a r e s u b s t a n t i a l d i f f e r e n c e s between s u b s p e c i e s o f IS. m e l a n i n o g e n i c u s . The s u b s p e c i e s d i f f e r f r o m each o t h e r i n c e l l w a l l c o m p o s i t i o n , g u a n i n e p l u s c y t o s i n e c o n t e n t , m a l a t e dehydrogenase -m o b i l i t y and some b i o c h e m i c a l t e s t s s u c h as e s c u l i n and s t a r c h h y d r o l y s i s (92,116). T e s t s have been d e v e l o p e d f o r r o u t i n e s e p a r a t i o n o f t h e t h r e e s u b s p e c i e s of _B. m e l a n i n o g e n i c u s and i t has r e c e n t l y been s u g g e s t e d t h a t t h e r e m i g h t be an e c o l o g i c a l s u b d i v i s i o n between t h e s u b s p e c i e s ( 9 2 ) . A f t e r a c a r e f u l a s s e ssment, t h e subcommittee on Gram-negative a n a e r o b i c r o d s of t h e I n t e r n a t i o n a l Committee on S y s t e m a t i c B a c t e r i o l o g y p r o p o s e d t h a t a s a c c h a r o -l y t i c s t r a i n s of _B. m e l a n i n o g e n i c u s s h o u l d be r e c l a s s i f i e d as a s e p a r a t e s p e c i e s , B a c t e r o i d e s a s a c c h a r o l y t i c u s , whereas t h e s a c c h a r o l y t i c s t r a i n s s h o u l d r e t a i n t h e i r c u r r e n t d e s i g n a t i o n ( 3 8 ) . F o r t h e p u r p o s e s of t h i s t h e s i s , however, t h e f o r m e r n o m e n c l a t u r e w i l l be u s e d , i . e . , 15. a s a c c h a r o - l y t i c u s w i l l be r e f e r r e d t o as 15. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s . A l t h o u g h r e c e n t s t u d i e s seem t o be more r e a l i s t i c i n terms o f r e c o g n i z i n g s i g n i f i c a n t d i f f e r e n c e s between the t h r e e s u b s p e c i e s t h e r e a r e s t i l l s t r a i n s w h i c h do n o t f i t t h e e x i s t i n g p a t t e r n s of c l a s s i f i c a t i o n and t h e r e f o r e t h e s p e c i e s c a n s t i l l be r e g a r d e d as h e t e r o g e n e o u s . My i n t e n t i o n h e r e i s t o g i v e a b r i e f summary o f t h i s h e t e r o g e n e i t y under f o u r g e n e r a l h e a d i n g s . 1. B i o c h e m i c a l h e t e r o g e n e i t y The most s t r i k i n g c h a r a c t e r i s t i c of j i . m e l a n i n o g e n i c u s i s i t s a b i l i t y t o p r o d u c e a c e l l a s s o c i a t e d b l a c k pigment when grown i n t h e p r e s e n c e o f e x c e s s heme. The u s u a l p r o c e d u r e i s t o grow t h e o r g a n i s m on b l o o d agar medium c o n t a i n i n g l a k e d b l o o d . Two t o s e v e n days a f t e r t h e c o l o n i e s f i r s t appear t h e y w i l l change f r o m w h i t e t o brown o r d a r k b l a c k , o f t e n t h i s i s t h e s o l e c r i t e r i o n f o r i d e n t i f y i n g an i s o l a t e as IS. m e l a n i n o g e n i c u s . I t was f i r s t t h o u g h t t h a t t h e pigment was m e l a n i n 3 (74) but T r a c y (109) r e p o r t e d t h a t under c e r t a i n c o n d i t i o n s , 13. m e l a n i - n o g e n i c u s p r oduces a b l a c k compound c o n s i s t i n g of c o l l o i d a l f e r r o u s s u l p h i d e . Schwabacher j i t . a l . (91) and more r e c e n t l y Duerden (11) p r e s e n t e d d a t a showing t h a t t h e pigment was h e m a t i n . Most s t r a i n s of j3. m e l a n i n o g e n i c u s r e q u i r e hemin o r a r e s t i m u l a t e d by i t and many r e q u i r e v i t a m i n K f o r growth (19,20,50,51,52,64,85). A number o f b i o s y n t h e t i c p r e c u r s o r s of v i t a m i n K can a c t as growth promot-e r s f o r j3. m e l a n i n o g e n i c u s . B i o c h e m i c a l s t u d i e s (7,90) have shown t h a t a l l s t r a i n s can grow w e l l i n s u g a r - f r e e p e p t i d e c o n t a i n i n g b r o t h , s u g g e s t i n g t h a t t h e o rganisms have the p o t e n t i a l t o f e r m e n t amino a c i d s . E x p e r i m e n t s u s i n g l a b e l l e d p r o t e i n i n d i c a t e t h a t s t r a i n s of 13. m e l a n i - n o g e n i c u s r e a d i l y (112) f erment amino a c i d s when they a r e p r e s e n t as p e p t i d e s . T h i s s u g g e s t s t h a t p e p t i d e s a r e more e a s i l y t r a n s p o r t e d i n t o t h e c e l l t h a n a r e f r e e amino a c i d s . More r e c e n t l y ( 7 0 ) , i t was found t h a t t h e a d d i t i o n of i n d i v i d u a l amino a c i d s t o a t r y p t i c a s e - y e a s t e x t r a c t - h e m i n medium a f f e c t e d growth r a t e s and the f i n a l y i e l d of s a c c h a r o l y t i c and a s a c c h a r o l y t i c s t r a i n s o f _B. m e l a n i n o g e n i c u s . These r e s u l t s a r e c o n s i s t e n t w i t h t h o s e of p r e v i o u s s t u d i e s ( 7 ) , showing t h a t t h e s e m i c r o o r g a n i s m s r e p r e s e n t a b i o c h e m i c a l l y h e t e r o g e n e o u s group. Sawyer e t . a l . (90) s t u d i e d the b i o c h e m i c a l c h a r a c t e r i s t i c s of 31 s t r a i n s of j3. m e l a n i n o g e n i c u s . A l l s t r a i n s were a c t i v e l y p r o t e o l y t i c , a t t a c k i n g r e c o n s t i t u t e d n e u t r a l s a l t - e x t r a c t e d c o l l a g e n and g e l a t i n . A l l s t r a i n s p r o d u c e d B^S and r e q u i r e d or were s t i m u l a t e d by hemin. Three d i s t i n c t l y d i f f e r e n t f e r m e n t a t i v e p a t t e r n s were o b s e r v e d . A s i m i l a r s t u d y done by C o u r a n t and Gibbons (7) showed the same r e s u l t s . Sawyer et_. a l . (90) a n a l y z e d t h e a c i d end p r o d u c t s of t h e 13. m e l a n i n o g e n i c u s i s o l a t e s and f ound t h a t t h e o r g a n i s m s produced l a c t i c , a c e t i c , p r o p i o n i c and b u t y r i c a c i d s and s m a l l amounts of f o r m i c and 4 v a l e r i c a c i d . S a c c h a r o l y t i c s t r a i n s produced m o s t l y . l a c t i c , s u c c i n i c and a c e t i c a c i d s whereas n o n - s a c c h a r o l y t i c s t r a i n s produced l a r g e amounts o f p r o p i o n i c and b u t y r i c a c i d . Wahren and G i b bons (112) r e p o r t e d t h a t they were u n a b l e t o d e t e c t the p r o d u c t i o n o f l a c t i c , f o r m i c and s u c c i n i c a c i d s . I t was f o u n d t h a t a d d i t i o n of g l u c o s e t o t r y p t i c a s e - y e a s t e x t r a c t medium d i d n o t i n c r e a s e growth n o r was the g l u c o s e m e t a b o l i z e d t o v o l a t i l e a c i d end p r o d u c t s . Recent s t u d i e s by F i n e g o l d and B arnes (16) showed b a s i c d i f f e r e n c e s i n the a c i d end p r o d u c t s p roduced by the two f e r m e n t -a t i v e groups of JS. m e l a n i n o g e n i c u s . They found t h a t i s o b u t y r i c a c i d was p r o d uced by b o t h g r o u p s , whereas n - b u t y r i c a c i d was p r o d u c e d o n l y by a s a c c h a r o l y t i c s t r a i n s and s u c c i n i c a c i d was p r o duced o n l y by s a c c h a r o -l y t i c s t r a i n s . The f a c t t h a t b o t h heme.and v i t a m i n - K a r e r e q u i r e d f o r g r o w t h of J3. m e l a n i n o g e n i c u s s u g g e s t s t h a t an e l e c t r o n t r a n s p o r t s y s t e m may be i n -v o l v e d i n energy m e t a b o l i s m . R i z z a e t . a l . (80) l o c a t e d 99% of t h e c ytochromes i n a p a r t i a l l y p u r i f i e d membrane f r a c t i o n . The cytochromes c o u l d be o x i d i z e d by a i r o r f u m a r a t e or r e d u c e d i n t h e p r e s e n c e of 5mM NADH. Whether o r not t h e e l e c t r o n t r a n s p o r t system f u n c t i o n s i n t h e m e t a b o l i s m of JB. m e l a n i n o g e n i c u s remains u n d e f i n e d . The l i p i d s and r e l a t e d compounds i n the c y t o p l a s m i c membrane were s t u d i e d by two groups of i n v e s t i g a t o r s . LaBach and W h i t e (46) and R i z z a e t . a l . (81) i d e n t i f i e d p h o s p h a t i d y l e t h a n o l a m i n e and two p h o s p h o s p h i n g o -l i p i d s , c e r a m i d e p h o s p h o r y l g l y c e r o l (CPG) and c e r a m i d e p h o s p h o r y l e t h a n o -l a m i n e (CPE) w h i c h a c c o u n t e d f o r about h a l f of the p h o s p h o l i p i d s . I t was a l s o f ound t h a t t h e l i p i d c o m p o s i t i o n of JS_. m e l a n i n o g e n i c u s was s i m i l a r t o t h a t o f o t h e r g r a m - n e g a t i v e b a c t e r i a i n t h a t t h e m a j o r p a r t o f t h e e x t r a c t a b l e f a t t y a c i d s were p r e s e n t as p h o s p h o l i p i d s . The d i a c y l p h o s p h o l i p i d s were r e p r e s e n t e d i n JS. m e l a n i n o g e n i c u s by p h o s p h a t i d y l 5 e t h a n o l a m i n e , p h o s p h a t i d y l g l y c e r o l , p h o s p h a t i d y l s e r i n e , p h o s p h a t i d i c a c i d and, f o r one s t r a i n , c a r d i o l i p i n . L ev e t . a l . ( 5 4 ) , i n v e s t i g a t i n g some a s p e c t s o f n u t r i t i o n o f a rumen s t r a i n of 33. m e l a n i n o g e n i c u s , found t h a t s u c c i n a t e was a growth f a c t o r f o r the b a c t e r i u m . T h i s f i n d i n g was s u p p o r t e d by the f o l l o w i n g o b s e r v -a t i o n s : (1) s u c c i n a t e s i g n i f i c a n t l y i n c r e a s e d the g r o w t h r a t e when added t o a c u l t u r e supplemented w i t h v i t a m i n K and b l o o d , (2) s u c c i n a t e r e p l a c e d t h e heme r e q u i r e m e n t when added to a v i t a m i n K supplemented c u l t u r e medium and (3) s u c c i n a t e p a r t i a l l y r e p l a c e d t h e v i t a m i n K r e q u i r e m e n t when added t o a b l o o d supplemented b r o t h . The l a s t two o b s e r v a t i o n s s u g g e s t e d t h a t t h e r e were two pathways of s u c c i n a t e meta-b o l i s m , m e d i a t e d by heme and v i t a m i n K r e s p e c t i v e l y . The a u t h o r s n o t e d t h a t t h e rumen s t r a i n formed cytochrome-b f r o m the heme. Cya n i d e i n h i b i t e d c e l l s grown i n t h e p r e s e n c e of b l o o d b u t d i d not i n h i b i t c e l l s grown i n v i t a m i n K p l u s s u c c i n a t e . T h i s was i n t e r p r e t e d t o mean t h a t cytochrome was n o t produced i n t h e l a t t e r c a s e . L e v and M i l f o r d (53,55) r e p o r t e d t h a t v i t a m i n K had a s p e c i f i c e f f e c t on s p h i n g o l i p i d b i o -s y n t h e s i s . U s i n g r a d i o a c t i v e l y l a b e l l e d p r e c u r s o r s t h e y d e m o n s t r a t e d t h a t v i t a m i n K i n d u c e d the f o r m a t i o n of 3-keto d i h y d r o s p h i n g o s i n e s y n t h e t a s e (56) and thus s t i m u l a t e d t h e s y n t h e s i s of t h i s n o v e l m i c r o b i a l l i p i d . The r e l a t i o n s h i p between heme, v i t a m i n K and s u c c i n a t e i n B. m e l a n i n o g e n i c u s i s n o t u n d e r s t o o d . I t i s n o t known whether s t r a i n s o f B_. m e l a n i n o g e n i c u s w h i c h do not r e q u i r e v i t a m i n K s y n t h e s i z e t h e m o l e c u l e de novo o r whether they have e v o l v e d a l t e r n a t e m e t a b o l i c systems w h i c h do n o t r e q u i r e t h e m o l e c u l e . A l s o , t h e r e i s no e v i d e n c e t h a t v i t a m i n K o r s u c c i n a t e p l a y s any r o l e i n t h e energy p r o c e s s e s o f the c e l l s . F i n a l l y , none of t h e s e s t u d i e s have i n d i c a t e d whether t h e compounds behave d i f f e r e n t l y i n each of the s u b s p e c i e s . 6 A s t u d y (115) on a n t i b i o t i c s u s c e p t i b i l i t y showed t h a t I3_. m e l a n i n o - g e n i c u s s s . a s a c c h a r o l y t i c u s was s e n s i t i v e t o p e n i c i l l i n s , c e p h a l o -s p o r i n s , b a c i t r a c i n , c h l o r t e t r a c y c l i n , c h l o r a m p h e n i c o l , e r y t h r o m y c i n and r i f a m p i c i n and r e s i s t a n t t o c o l i s t i n , p o l y m y x i n B, neomycin and s t r e p t o m y c i n . However, no i n d i c a t i o n of t h e a n t i b i o t i c s u s c e p t i b i l i t y of the two s a c c h a r o l y t i c s u b s p e c i e s was g i v e n . A more r e c e n t s t u d y (92) showed t h a t m e a s u r i n g s e n s i t i v i t y t o v a ncomycin and c o l i s t i n w i l l d i f f e r e n t i a t e between s a c c h a r o l y t i c and a s a c c h a r o l y t i c s t r a i n s . I t was f o u n d t h a t most a s a c c h a r o l y t i c s t r a i n s were s e n s i t i v e t o v a n comycin and r e s i s t a n t t o c o l i s t i n whereas b o t h s a c c h a r o l y t i c groups were r e s i s t a n t t o v a ncomycin and s e n s i t i v e t o c o l i s t i n . Shah e t . a l . d e m o n s t r a t e d t h a t the a s a c c h a r o l y t i c s t r a i n s of J3. m e l a n i n o g e n i c u s have a g u a n i n e - p l u s - c y t o s i n e (G+C) c o n t e n t of 49 t o 51.7 mol % whereas t h e i n t e r m e d i u s s t r a i n s have a G+C c o n t e n t of 39 t o 43.9 mol % and t h e m e l a n i n o g e n i c u s s u b s p e c i e s have a G+C c o n t e n t o f 38 t o 39.8 mol % ( 9 2 ) . 2. A n t i g e n i c s t r u c t u r e and s e r o l o g i c a l h e t e r o g e n e i t y The a n t i g e n i c c o m p o s i t i o n of B a c t e r o i d e s c e l l s has been shown t o be s p e c i e s s p e c i f i c and . p o t e n t i a l l y u s e f u l i n s p e c i a t i o n ( 9 ) . The a n t i -g e n i c i t y of 13 s t r a i n s of J i . m e l a n i n o g e n i c u s i s o l a t e d f r o m v a r i o u s s o u r c e s was s t u d i e d by C o urant and Gibbons (7) who c o n c l u d e d t h a t t h e t h i r t e e n a n t i s e r a p r o duced seemed t o be s p e c i e s - s p e c i f i c . However, t h e f o l l o w i n g e v i d e n c e i n d i c a t e d t h a t J i . m e l a n i n o g e n i c u s s t r a i n s were s e r o l o g i c a l l y h e t e r o g e n e o u s : (a) t h e r e was c r o s s r e a c t i v i t y between a l l t h i r t e e n s t r a i n s , (b) a n t i b o d i e s t o J i . m e l a n i n o g e n i c u s were d e t e c t e d i n a l l of the s i x t e e n human serum samples examined u s i n g sheep e r y t h r o -c y t e s p o l y s e n s i t i z e d w i t h l i p o p o l y s a c c h a r i d e (LPS) f r a c t i o n s of e i g h t _B. m e l a n i n o g e n i c u s s t r a i n s , (c) c r o s s - r e a c t i o n c o m p a r a b l e t o t h a t o b s e r v e d w i t h whole c e l l s was a l s o f ound i n h e m a g g l u t i n a t i o n i n h i b i t i o n s t u d i e s u s i n g whole c e l l s o f f o r t y - e i g h t s t r a i n s o f 13. m e l a n i n o g e n i c u s to s p e c i f i c a l l y i n h i b i t t he r e a c t i o n between e i g h t d i f f e r e n t a n t i s e r a and e r y t h r o c y t e s s e n s i t i z e d w i t h LPS d e r i v e d f r o m t h e homologous s t r a i n . More r e c e n t l y , a f l u o r e s c e n t a n t i b o d y p r o c e d u r e was d e v e l o p e d (47) w h i c h showed t h a t human 13. m e l a n i n o g e n i c u s s t r a i n s c o u l d be d i v i d e d i n t o t h r e e s p e c i f i c s e r o g r o u p s c o r r e s p o n d i n g t o t h e b i o c h e m i c a l s u b s p e c i e s a l r e a d y known. The f l u o r e s c e n t a n t i b o d y c o n j u g a t e s were s p e c i f i c and no c r o s s r e a c t i o n was o b s e r v e d w i t h o t h e r anaerobes or aer o b e s t e s t e d . 3. G e n e t i c v a r i a t i o n G e n e t i c s t u d i e s on 13. m e l a n i n o g e n i c u s a r e n o n - e x i s t e n t and no s p e c i f i c d e t a i l s have been p u b l i s h e d c o n c e r n i n g the g e n e t i c p r o p e r t i e s and v a r i a -b i l i t y o f the or g a n i s m . Known examples of p h e n o t y p i c v a r i a b i l i t y w h i c h m i g h t r e f l e c t g e n e t i c v a r i a t i o n a r e f a m i l i a r t o a l l w o r k e r s i n t h e f i e l d . C o l o n y f o r m i s u s u a l l y smooth b u t rou g h v a r i a n t s a r e fo u n d . P i g m e n t -a t i o n i s a l s o n o t i c e a b l y v a r i a b l e , b e i n g g e n e r a l l y b l a c k , b u t d i f f e r e n t shades of brown can be o b s e r v e d . Other p r o p e r t i e s , such as the r e q u i r e -ments f o r heme and v i t a m i n K and c o l l a g e n a s e p r o d u c t i o n might a l s o r e f l e c t some g e n e t i c v a r i a t i o n , a l t h o u g h t h i s has n o t been p r o v e n . B a c t e r i o p h a g e c a p a b l e o f i n f e c t i n g 13. m e l a n i n o g e n i c u s have n o t been i s o l a t e d ( 4 1 ) . The organisms have not been found t o p o s s e s s p l a s m i d s n o r have they been shown t o a c q u i r e p l a s m i d s f r o m d i f f e r e n t gram n e g a t i v e s p e c i e s ( 6 , 1 0 ) . A n t i b i o t i c r e s i s t a n c e i s not r e l a t e d t o t h e e x i s t e n c e o f p l a s m i d s w i t h i n 13. m e l a n i n o g e n i c u s . 8 4. P a t h o g e n i c p r o p e r t i e s of B, m e l a n i n o g e n i c u s a. Adherence Adherence has r e c e n t l y been found t o be an i m p o r t a n t e c o l o g i c a l f a c t o r i n s p e c i f i c d i s e a s e p r o c e s s e s (12,17,22,66,117). One method of s t u d y i n g adherence i s t o e v a l u a t e t h e h e m a g g l u t i n a t i n g a c t i v i t y o f an o r g a n i s m . Okuda and Takazoe (73) found t h a t t w e n t y - n i n e of f i f t y - n i n e s t r a i n s o f B. m e l a n i n o g e n i c u s s t u d i e d had h e m a g g l u t i n a t i n g a c t i v i t y . They a l s o showed t h a t a l l h e m a g g l u t i n a t i n g s t r a i n s p o s s e s s e d f i m b r i a - l i k e s t r u c t u r e s ( p i l i ) whereas n o n - h e m a g g l u t i n a t i n g s t r a i n s d i d not have p i l i . U n f o r t u n a t e l y , t h e s t r a i n s were n o t s p e c i a t e d and i t i s not known i f t h e r e was any c o r r e l a t i o n of h e m a g g l u t i n a t i n g a c t i v i t y and t h e p r e s e n c e of p i l i w i t h any p a r t i c u l a r s u b s p e c i e s of B. m e l a n i n o g e n i c u s . b. I n v a s i v e n e s s B a c t e r i a l s p e c i e s t h a t a r e e a s i l y p h a g o c y t i z e d and do not p o s s e s s mechanisms f o r i n t r a c e l l u l a r s u r v i v a l a r e g e n e r a l l y n o n - i n v a s i v e . W i t h one e x c e p t i o n ( 1 0 6 ) , I i . m e l a n i n o g e n i c u s does n o t p o s s e s s any known c a p s u l a r m a t e r i a l o r a n t i p h a g o c y t i c s u r f a c e components. The f a c t t h a t 13. m e l a n i n o g e n i c u s s t r a i n s do n o t i n f e c t by t h e m s e l v e s i m p l i e s t h a t t h e c e l l s do n o t behave as i n t r a c e l l u l a r p a r a s i t e s . However, some s t r a i n s can e l a b o r a t e c e l l a s s o c i a t e d e x t r a c e l l u l a r enzymes w h i c h may enhance t h e i r i n v a s i v e p r o p e r t i e s . These enzymes i n c l u d e c o l l a g e n a s e ( 2 1 ) , p r o t e a s e s and h y a l u r o n i d a s e ( 1 ) . A l t h o u g h J3_. m e l a n i n o g e n i c u s i s t h e o n l y o r g a n i s m i n d i g e n o u s t o t h e o r a l c a v i t y known t o p r o d u c e a c o l l a g e n a s e (20,28,29), the r e l a t i o n s h i p between c o l l a g e n a s e p r o d u c t i o n and t h e p a t h o g e n i c i t y of the o r g a n i s m e i t h e r i n o r a l l e s i o n s or i n mixed i n f e c t -i o n s remains u n c l e a r . However, Kestenbaum e t . a l . (43) d e m o n s t r a t e d a p o s i t i v e c o r r e l a t i o n between c o l l a g e n a s e a c t i v i t y and i n f e c t i v i t y f o r 9 f o u r 13. m e l a n i n o g e n i c u s s t r a i n s i n a g u i n e a p i g system. I t has been shown by Kaufman ^ t . a l . (40) t h a t a p a r t i c u l a t e f r a c t i o n p r e p a r e d f r o m a u t o l y s e d c e l l s of J3. m e l a n i n o g e n i c u s s s . a s a c - c h a r o l y t i c u s p o s s e s s i n g c o l l a g e n o l y t i c a c t i v i t y c o u l d enhance a f u s o -b a c t e r i a l i n f e c t i o n . H e a t i n g e l i m i n a t e d b o t h enzyme a c t i v i t y and t h e e f f e c t of the e x t r a c t on i n f e c t i o n , s u g g e s t i n g t h a t c o l l a g e n a s e may p l a y an i m p o r t a n t r o l e i n the d i s e a s e p r o c e s s . T h i s does n o t e x c l u d e t h e p o s s i b i l i t y t h a t o t h e r h e a t s e n s i t i v e f a c t o r s may c o n t r i b u t e t o t h e p a t h o g e n i c i t y of t h e c e l l s . I t i s u n l i k e l y t h a t e n d o t o x i n was i n v o l v e d as i t s h o u l d n o t be i n a c t i v a t e d by h e a t , c. T o x i n p r o d u c t i o n There a r e no p u b l i s h e d r e p o r t s of e x o t o x i n p r o d u c t i o n by B. m e l a n i n o g e n i c u s . However, the f a c t t h a t some m i x t u r e s of b a c t e r i a c o n t a i n i n g _B. m e l a n i n o g e n i c u s i n j e c t e d s u b c u t a n e o u s l y i n t o the g r o i n of a g u i n e a p i g cause a r a p i d and s p r e a d i n g i n f e c t i o n , r e s u l t i n g i n e x t e n s i v e f l u i d a c c u m u l a t i o n (up t o 25% of the body w e i g h t ) t e n d s t o s u p p o r t the h y p o t h e s i s t h a t t h e s t r a i n s can produce a f l u i d a c c u m u l a t i n g t o x i n . V i b r i o c h o l e r a t o x i n i n d u c e s a s i m i l a r r e s p o n s e i n the g u t . P u r i f i e d p r e p a r a t i o n s of t h i s t o x i n have: (1) d i a r r h e a g e n i c a c t i v i t y i n e x p e r -i m e n t a l a n i m a l s (2) t h e a b i l i t y t o c a u s e i n c r e a s e d s k i n c a p i l l a r y a c t i v i t y and (3) the a b i l i t y t o i n d u c e f l u i d a c c u m u l a t i o n where i n j e c t e d i n t o l i g a t e d i l e a l l o o p s of r a b b i t s ( 7 9 ) . P r e l i m i n a r y e x p e r i m e n t s by B.C. M c B r i d e ( p e r s o n a l communication) have i n d i c a t e d t h a t c u l t u r e s u p e r n a t a n t s o b t a i n e d from 13. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s p o s s e s s a c h o l e r a t o x i n - l i k e a c t i v i t y as measured by the v a s c u l a r p e r m e a b i l i t y a s s a y d e s c r i b e d by C r a i g ( 8 ) . 10 d. E n d o t o x i c a c t i v i t y The e n d o t o x i c a c t i v i t y o f .B. m e l a n i n o g e n i c u s has been s t u d i e d i n g r e a t e r d e t a i l . The u n i q u e c o m p o s i t i o n of t h i s c e l l u l a r m a t e r i a l has been n o t e d by H o f s t a d (33,34,35). P h e n o l e x t r a c t e d l i p o p o l y s a c c h a r i d e (LPS) was f o u n d t o be composed of a number of n e u t r a l s u g a r s i n c l u d i n g g l u c o s e and rhamnose; n e i t h e r 2 - k e t o - 3 - d e o x y o c t o n i c a c i d (KDO) nor h e p t o s e was p r e s e n t . The s t r u c t u r e o f t h e e n d o t o x i n i s t h e r e f o r e d i f f e r -e n t f r o m t h e " c l a s s i c a l " LPS d e s c r i b e d f o r t h e g r a m - n e g a t i v e e n t e r i c b a c t e r i a . H o f s t a d a l s o f o u n d (34) t h a t LPS p r e p a r a t i o n s f r o m a number o f o r a l _B. m e l a n i n o g e n i c u s s t r a i n s were s e r o l o g i c a l l y a c t i v e i n complem-ent b i n d i n g and a n t i b o d y n e u t r a l i z i n g c a p a c i t y as measured by i n h i b i t i o n o f h a e m a g g l u t i n a t i o n . A l l p r e p a r a t i o n s c r o s s - r e a c t e d i n such t e s t s as r i n g p r e c i p i t a t i o n , complement f i x a t i o n , i n d i r e c t h a e m a g g l u t i n a t i o n and d o u b l e d i f f u s i o n i n a g a r . However, t h e y a l s o c o n t a i n e d t y p e s p e c i f i c d e t e r m i n a n t s i d e n t i f i e d by n o n - i d e n t i c a l agar p r e c i p i t a t i o n bands. The e n d o t o x i c a c t i v i t y of the LPS p r e p a r a t i o n s was c o n f i r m e d by s e v e r a l c r i t e r i a i n c l u d i n g p o s i t i v e Schwartzman r e a c t i o n i n r a b b i t s , mouse l e t h a l i t y , s u s c e p t i b i l i t y t o b a c t e r i a l i n f e c t i o n o f LPS t r e a t e d m i c e and l e u c o c y t o m i g r a t i o n . A r e c e n t s t u d y (65) of t h r e e s t r a i n s o f m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s showed s i m i l a r r e s u l t s : (1) t h e absence of KDO and h e p t o s e i n LPS (2) u n u s u a l f a t t y a c i d c o m p o s i t i o n and (3) l o w b i o l o g i c p o t e n c y . C h a r a c t e r i z a t i o n of a c o l l e c t i o n o f a l l t h r e e s u b s p e c i e s o b t a i n e d f r o m c l i n i c a l o r a l samples s h o u l d h e l p t o e s t a b l i s h w h i c h of t h e s e sub-s p e c i e s a r e p r e v a l e n t i n the v a r i o u s s i t e s o f t h e mouth. W h i l e d i f f e r -e nces have been found between s t r a i n s w i t h r e g a r d t o enzyme a c t i v i t y , i n d o l e , e s c u l i n and s t a r c h h y d r o l y s i s , NH^, ^ S , f e r m e n t a t i o n p a t t e r n s and end p r o d u c t a n a l y s i s , no c o r r e l a t i o n between any of t h e above and 11 p a t h o g e n i c i t y has been e s t a b l i s h e d . The o b v i o u s b i o c h e m i c a l and s e r o l o g i c a l h e t e r o g e n e i t y o f t h e pigmented B a c t e r o i d e s c r e a t e s t h r e e problems w h i c h can be r e a d i l y i d e n t i f i e d : (1) The i m p o r t a n c e o f e s t a b l i s h i n g the r e l a t i o n s h i p between th e t h r e e s u b s p e c i e s o f J i . m e l a n i n o g e n i c u s and mixed a n a e r o b i c i n f e c t i o n s (more s p e c i f i c a l l y , p e r i o d o n t a l d i s e a s e ) , (2) The need f o r d e v e l o p i n g . a r o u t i n e p r o c e d u r e f o r s p e c i a t i n g t h e organisms and (3) The c o r r e l a t i n g of p a t h o g e n i c i t y w i t h a b i o c h e m i c a l p r o p e r t y t h a t can be r e a d i l y d e m o n s t r a t e d . PART B. MIXED ANAEROBIC INFECTIONS The i n d i g e n o u s m i c r o b i a l f l o r a w h i c h i s found i n c l o s e a s s o c i a t i o n w i t h t h e human g i n g i v a i s m o r p h o l o g i c a l l y , p h y s i o l o g i c a l l y and a n t i g e n i c a l l y d i v e r s e . A n a e r o b i c b a c t e r i a c o m p r i s e a l a r g e p e r c e n t a g e of the g i n g i v a l m i c r o f l o r a and i t i s n o t u n r e a s o n a b l e t o assume t h a t t h e y may be i n v o l v e d i n the i n i t i a t i o n o f p a t h o g e n i c p r o c e s s e s . O f t e n , t h e i n f e c t i o n s i n v o l v e - s e v -e r a l s p e c i e s o r g e n e r a of b a c t e r i a t h a t behave i n a c o o p e r a t i v e f a s h i o n t o pro d u c e s e p s i s . Examples of mixed i n f e c t i o n i n c l u d e b r a i n and l u n g a b s c e s s e s , a p p e n d i c i t i s , a n i m a l and human b i t e wounds and p e r i o d o n t a l d i s e a s e s . M i x e d i n f e c t i o n s have r e c e n t l y been r e v i e w e d i n a c r i t i c a l f a s h i o n (14,15,25,26,99). C o o p e r a t i v e o r s y n e r g i s t i c i n f e c t i o n s have been s t u d i e d i n a number of a n i m a l model systems and i n a n a t u r a l i n f e c t i o n , sheep f o o t r o t (82,83,84). The l a t t e r i s known as o v i n e i n t e r d i g i t a l d e r m a t i t i s and i s i n r e a l i t y a number of d i f f e r e n t d i s e a s e s a s s o c i a t e d w i t h an i n v a s i v e i n f e c t i o n by a mixed p o p u l a t i o n o f a n a e r o b i c and f a c u l t a t i v e o r g a n i s m s . A r e l a t i v e l y m i l d form of the d i s e a s e o c c u r s when F u s i f o r m i s n e c r o p h o r u s , v a r i o u s d i p t h e r o i d s and C o r y n e b a c t e r i u m pyogenes i n v a d e the s u r f a c e t i s s u e s and cause s u p e r f i c i a l n e c r o t i z i n g i n f e c t i o n . A more s e r i o u s c o n d i t i o n can d e v e l o p from t h i s 12 i n f e c t i o n i f F_. n e c r o p h o r u s and C^ . pyogenes p e n e t r a t e deeper i n t o t h e t i s s u e s c r e a t i n g a c o n d i t i o n known as i n f e c t i v e b u l b a r n e c r o s i s . Two o t h e r d i s e a s e s , b e n i g n f o o t - r o t and v i r u l e n t f o o t - r o t may be r e g a r d e d as o v i n e i n t e r d i g i t a l d e r m a t i t i s c o m p l i c a t e d by t h e p r e s e n c e of low o r h i g h v i r u l e n c e s t r a i n s of F u s o b a c t e r i u m nodosus. I n e x p e r i m e n t a l f o o t - r o t , the o r g a n i s m s were i n f e c t i v e o n l y when i n o c u l a t e d t o g e t h e r . The e v i d e n c e s u g g e s t s t h a t each member con-t r i b u t e d t o t h e p a t h o g e n i c i t y and t h a t t h e sum of t h e i r p a t h o g e n i c p r o p e r t i e s was s u f f i c i e n t t o overcome h o s t d e f e n c e s . M i c r o b i a l s y n e r g y has been d e m o n s t r a t e d i n i n t r a - a b d o m i n a l i n f e c t i o n i n t h e r a t . I n t h i s c a s e , i t was shown t h a t a b s c e s s f o r m a t i o n o c c u r r e d when the a n a e r o b e s , B a c t e r o i d e s f r a g i l i s o r F u s o b a c t e r i u m v a r i u m were i n o c u l a t e d w i t h E s c h e r i c h i a c o l i (75,76,113). L e s i o n s d i d n o t f o r m when the o rganisms were i n o c u l a t e d i n d i v i d u a l l y . The r e a s o n s f o r m i c r o b i a l i n t e r d e p e n d e n c y have not been e l u c i d a t e d . The mouse has been used t o s t u d y mixed i n f e c t i o n s of the l i v e r ( 3 1 ) . I t was f ound t h a t p a r t i c u l a r p a i r s o f b a c t e r i a p r o duced an i n f e c t i o n more f r e q u e n t l y t h a n p u r e c u l t u r e s . V e s z p r e m i r e p o r t e d i n 1905 (111) t h a t m i c r o b i a l m a t t e r o b t a i n e d from the human mouth w o u l d cause mixed i n f e c t i o n s i n r a b b i t s . A l t h o u g h i t i s s t i l l g e n e r a l l y a g r e e d t h a t no s i n g l e s p e c i e s i n s u c h a m i x t u r e can c a u s e a l e s i o n , many r e p o r t s have shown t h a t mixed c u l t u r e s o f t h e s e o r g a n i s m s can p r o d u c e l e s i o n s . P a u l (78) found t h a t f o u l gangrenous a b s c e s s e s c o u l d be p r o duced i n r a b b i t s and g u i n e a p i g s w i t h mixed c u l t u r e s c o n t a i n i n g s p i r o c h e t e s , f u s i -f o r m b a c i l l i and c o c c i . K i r t c h e w s k i and S e g u i n ( 4 5 ) , r e p o r t e d t h a t t h e y c o u l d i n f e c t g u i n e a p i g s w i t h a m i x t u r e of s p i r o c h a e t e s , f u s i f o r m b a c i l l i and e i t h e r an a l p h a h e m o l y t i c s t r e p t o c o c c u s or a S t a p h y l o c o c c u s a l b u s . Smith (95) i n i t i a t e d l e s i o n s w i t h m i c r o b i a l d e b r i s o b t a i n e d from i n d i v i d u a l s w i t h V i n c e n t ' s a n g i n a . The i n o c u l u m was shown t o c o n t a i n a number of s t a p h y l o c o c c i , a h e m o l y t i c s t r e p t o c o c c u s , a v i b r i o , two f u s i f o r m s and a treponeme. He found t h a t a m i x t u r e o f s p i r o c h e t e s and f u s i f o r m s d i d n o t produce an i n f e c t i o n i n g u i n e a p i g s . However, the s u c c e s s i v e a d d i t i o n o f o t h e r b a c t e r i a i n d u c e d i n f e c t i o n s w h i c h were p r o g r e s s i v e l y more s e v e r e . S m ith n o t e d t h a t t h i s t y p e of i n f e c t i o n was n o t s p e c i e s s p e c i f i c s i n c e a w i d e v a r i e t y o f b a c t e r i a c o u l d p r o d u c e t h e same r e s u l t s . A c h a r a c t e r i s t i c mixed b a c t e r i a l p o p u l a t i o n , the s o - c a l l e d " f u s o -s p i r o c h a e t a l complex" was o f t e n o b s e r v e d i n t h e s e i n f e c t i o n s . E x p e r i m e n t a l mixed i n f e c t i o n s c o n t a i n i n g t h e same complex c o u l d be p r o d u c e d by subcutaneous i n o c u l a t i o n o f a n i m a l s w i t h human g i n g i v a l d e b r i s o r m a t e r i a l f r o m a v a r i e t y of a b s c e s s e s ( 4 4 , 8 6 ) . Rosebury and co-workers (87,88,89), i n a s t u d y o f a f u s o s p i r o c h a e t a l i n f e c t i o n , found t h a t a m i x t u r e o f f i f t e e n o r a l i s o l a t e s r e t a i n e d i t s i n f e c t i v i t y t h r o u g h s e v e r a l a n i m a l passages o r t h r o u g h s e v e r a l t r a n s f e r s as a mixed p o p u l a t i o n i n v i t r o b u t t h a t r e c o m b i n a t i o n o f pure c u l t u r e s of t h e i s o l a t e s f a i l e d t o pr o d u c e the " t y p i c a l " i n f e c t i o n . The r e s u l t s can be p a r t i a l l y e x p l a i n e d by t h e f a c t t h a t the a n a e r o b i c t e c h n i q u e employed was n o t adequate. T h i s m i ght have a f f e c t e d the r e c o v e r y of the v a r i o u s s p e c i e s w i t h i n t h e o r i g i n a l m i x t u r e and c o u l d e x p l a i n the i n a b i l i t y o f t he a u t h o r s t o pro d u c e " t y p i c a l " i n f e c t i o n s w i t h pure c u l t u r e s . Mergenhagen j i t . ajL. (68) found t h a t p u r e c u l t u r e s o f e i t h e r a S t a p h y l o c o c c u s  a u r e u s or an o r a l s t r e p t o c o c c u s s t r a i n p r o d uced o n l y l o c a l i z e d a b s c e s s e s . However, t h e y found a s y n g e r g i s t i c e f f e c t when b o t h i s o l a t e s were i n o c u l a t e d i n t o t h e g r o i n o f a g u i n e a p i g . The mechanism of i n v a s i o n was e x p l a i n e d by the f a c t t h a t the h y a l u r o n i d a s e f r o m the jS. aureus h e l p e d the s t r e p t o c o c c u s t o i n v a d e the t i s s u e s . S u c c e s s f u l t r a n s m i s s i b l e i n f e c t i o n s i n a n i m a l s i n o c u l a t e d w i t h d e f i n e d m i x t u r e s of m i c r o o r g a n i s m s were r e p o r t e d by s e v e r a l i n v e s t i g a t o r s (3,32,42, 43,62,63,64,67,96). The t y p i c a l mixed a n a e r o b i c i n f e c t i o n u s i n g g i n g i v a l 14 c r e v i c e m a t e r i a l was f i r s t d e s c r i b e d by Macdonald et.. a l . ( 6 0 , 6 1 ) . The i n f e c t i o n c o u l d be o f two t y p e s : (a) a l o c a l i z e d a b s c e s s c o n t a i n i n g e x u d a t e m a t e r i a l w h i c h c o u l d be used t o t r a n s m i t the i n f e c t i o n t o a second a n i m a l , and (b) a r a p i d l y s p r e a d i n g n e c r o t i c i n f e c t i o n w h i c h p e r f o r a t e d e i t h e r t h e s k i n o r t h e a b d o m i n a l w a l l . I n the l a t t e r case t h e a n i m a l d i e d a l m o s t i m m e d i a t e -l y . E xudate a s p i r a t e d f r o m t h e l e s i o n was h i g h l y i n f e c t i o u s when i n o c u l a t e d i n t o a second a n i m a l . T r a n s m i s s i b l e mixed i n f e c t i o n s c o u l d be produced i n g u i n e a p i g s when s e v e n t e e n p u r e c u l t u r e s i s o l a t e d f r o m a g i n g i v a l i n f e c t i o n were recombined and i n j e c t e d s u b c u t a n e o u s l y i n t o t h e g r o i n o f a g u i n e a p i g . S u c c e s s i v e e l i m i n a t i o n o f organisms f r o m t h e o r i g i n a l c o m b i n a t i o n produced a m i n i m a l c o m b i n a t i o n o f f o u r o r g a n i s m s w h i c h was c a p a b l e o f c a u s i n g a t r a n s -m i s s i b l e i n f e c t i o n ( 6 1 ) . The m i n i m a l i n f e c t i o u s m i x t u r e c o n s i s t e d o f two s p e c i e s of B a c t e r o i d e s , a m o t i l e Gram n e g a t i v e r o d and a f a c u l t a t i v e d i p h t h e r o i d . One o f the B a c t e r o i d e s s p e c i e s was i d e n t i f i e d as I i . m e l a n i n o - g e n i c u s . Subsequent s t u d i e s (64,96) i n d i c a t e d t h a t t h i s m i c r o o r g a n i s m p l a y e d an e s s e n t i a l r o l e i n the i n f e c t i o n . M i x t u r e s o f human i n d i g e n o u s b a c t e r i a w i t h o u t J 3 . m e l a n i n o g e n i c u s were n o n - i n f e c t i v e . I n the p r e s e n c e o f 13. m e l a n i n o g e n i c u s t h e s e m i x t u r e s were u n i f o r m l y i n f e c t i v e . The r o l e o f the " h e l p e r " o r g a n i s m s has o n l y been p a r t i a l l y d e f i n e d (19,96,97,100). I t can be assumed t h a t h e l p e r organisms a s s i s t anaerobes by u s i n g up the oxygen, d e c r e a s i n g t h e Eh and/or p r o d u c i n g c a t a l a s e . O t h e r f a c t o r s t h a t might be i n v o l v e d a r e the a b i l i t y t o f o r c e e n t r y i n t o t i s s u e s , r e s i s t a n c e t o h o s t d e f e n s e s , t h e p r o d u c t i o n o f s u b s t a n c e s t h a t b l o c k humoral a n t i m i c r o b i a l a c t i o n o r n u t r i t i o n a l dependency. E v i d e n c e f o r t h e l a t t e r was p r o v i d e d i n an e x p e r i m e n t i n w h i c h a v i t a m i n K i n d e p e n d e n t s t r a i n o f 13. m e l a n i n o g e n i c u s was s u b s t i t u t e d f o r the o r i g i n a l n aphthoquinone dependent s t r a i n . The i n f e c t i o u s m i x t u r e c o u l d t h e n be r e d u c e d t o t h r e e o r g a n i s m s : J 3 . m e l a n i n o g e n i c u s , a B a c t e r o i d e s and the Gram-negative r o d . The d i p h t h e r o i d was no l o n g e r r e q u i r e d i m p l y i n g t h a t i t s r o l e i n the i n f e c t i o n was t o s u p p l y v i t a m i n K and thus s u s t a i n t h e growth of the pathogen. The r o l e of the o t h e r o r g a n i s m s has n o t been d e f i n e d . S i m i l a r r e s u l t s were o b t a i n e d by Takazoe and Nakamura (105) . A d d i t i o n a l e v i d ence f o r t h e p a t h o g e n i c i t y of B. m e l a n i n o g e n i c u s i s p r o v i d e d by t h e f a c t t h a t t h e o r g a n i s m has been i s o l a t e d f r o m a number of c l i n i c a l i n f e c t i o n s (2,5,15,25,26,49,71,74,97,107,108,114). O r a l mixed a n a e r o b i c i n f e c t i o n s a r e g e n e r a l l y found t o have s e v e r a l c h a r a c t e r i s t i c s . (1) The e t i o l o g i c a g e n t s a r e members of the n ormal i n d i -genous b a c t e r i a l f l o r a o f the mouth. Hence the same s p e c i e s a r e f o u n d i n h e a l t h y and i n d i s e a s e d i n d i v i d u a l s . The c o m p l e x i t y of t h e m i c r o b i a l p o p u l a t i o n and t h e p roblems i n v o l v e d i n c u l t i v a t i n g some o f i t s members makes i t d i f f i c u l t t o e s t a b l i s h t h e e t i o l o g y o f d i s e a s e . (2) The p a t h o g e n i c members of s u c h i n f e c t i o n s a r e o n l y m i n i m a l l y v i r u l e n t under most c o n d i t i o n s . There i s a l s o v e r y l i t t l e i n f o r m a t i o n about t h e mechanisms by w h i c h i n d i v i d u a l o r g a n i s m s a s s i s t each o t h e r i n the g e n e r a l s e q u e n t i a l e v e n t s t h a t make up the i n f e c t i o u s p r o c e s s . (3) P r e d i s p o s i n g f a c t o r s may p l a y an i m p o r t a n t i f n o t e s s e n t i a l r o l e i n the i n i t i a t i o n of such i n f e c t i o n s , s i n c e a change i n t h e e n v i r o n m e n t a l c o n d i t i o n s e n a b l e s some members of the e c o s y s t e m t o become e i t h e r more p r o m i n e n t and/or t o m a n i f e s t some of t h e i r p a t h o g e n i c p r o p e r t i e s . Our o b j e c t i v e i s t o g a i n an u n d e r s t a n d i n g o f the e c o l o g i c a l r e l a t i o n s h i p s w h i c h e x i s t among the b a c t e r i a r e q u i r e d t o i n i t i a t e and s u s t a i n a d e f i n e d mixed a n a e r o b i c i n f e c t i o n and t o i d e n t i f y t h o s e p r o p e r t i e s w h i c h would make J3. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s a pathogen. 16 S p e c i f i c t o p i c s t o be c o v e r e d i n t h i s t h e s i s i n c l u d e : 1. C h a r a c t e r i s t i c s o f 13. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s ; 2. D e f i n i t i o n o f t h e s i m p l e s t mixed i n f e c t i o n u s i n g an i n v i v o a s s a y ; 3. N u t r i t i o n a l c h a r a c t e r i z a t i o n o f the o rganisms r e s p o n s i b l e f o r the i n f e c t i o n and a s s a y f o r t h e i r enzymes and m e t a b o l i c p r o d u c t s ; 4. S t u d i e s on t h e e f f e c t o f growth o f d i f f e r e n t m i c r o b i a l s p e c i e s on J each o t h e r i n v i t r o and . i n v i v o 5. R o l e of s u c c i n a t e as growth f a c t o r f o r _B. m e l a n i n o g e n i c u s i n v i v o and i n v i t r o . I I . MATERIALS AND METHODS Organisms 1. 15. m e l a n i n o g e n i c u s 13. m e l a n i n o g e n i c u s s u b s p e c i e s a s a c c h a r o l y t i c u s s t r a i n s K110 and CR2A were o b t a i n e d f r o m Dr. P. A. Mashimo. These a r e c o l l a g e n o l y t i c s t r a i n s o r i g i n a l l y i s o l a t e d by Macdonald and co-workers (60) f r o m p a t i e n t s w i t h d i a g n o s e d g i n g i v i t i s . O ther s t r a i n s o f _B. m e l a n i n o g e n i c u s were i s o l a t e d i n t he l a b o r a t o r y f r o m g i n g i v a l s c r a p i n g s t a k e n f r o m p e r s o n s w i t h v a r y i n g s t a t e s o f o r a l h e a l t h o r f r o m dog's g i n g i v a . Samples were s t r e a k e d on f r e s h l y poured b l o o d agar p l a t e s ( D i f c o b l o o d agar base w i t h 5% l a k e d human b l o o d ) . A f t e r f o u r t o seven days of a n a e r o b i c i n c u b a t i o n (see below) a t 37°C, b l a c k c o l o n i e s were p i c k e d f r o m the p l a t e s and sub-c u l t u r e d on t h e same medium u n t i l p u r e c u l t u r e s were o b t a i n e d . I s o l a t i o n of v i t a m i n K r e q u i r i n g s t r a i n s o f 15. m e l a n i n o g e n i c u s was pe r f o r m e d as f o l l o w s : t h e 15. m e l a n i n o g e n i c u s i s o l a t e s were s t r e a k e d on b l o o d a g a r p l a t e s and the p l a t e s were t h e n i n o c u l a t e d w i t h a c e n t r a l s t r e a k o f S t a p h y l o c o c c u s a u r e u s . Growth of v i t a m i n K r e q u i r i n g s t r a i n s was e v i d e n t n ear t h e S_. a u r e u s s t r e a k . I t i s w o r t h n o t i n g t h a t v i t a m i n K r e q u i r i n g s t r a i n s o f 13. m e l a n i n o g e n i c u s tended t o grow as mixed c o l o n i e s , i . e . , i n a s s o c i a t i o n w i t h o t h e r o rganisms on the o r i g i n a l i s o l a t i o n p l a t e s . 2. O t h e r organisms Samples f r o m d e n t a l a b s c e s s e s were p l a t e d and i n c u b a t e d a n a e r o b i c a l l y on b l o o d a g a r p l a t e s . I s o l a t e d c o l o n i e s were s u b c u l t u r e d a e r o b i c a l l y and a n a e r o b i c a l l y and c h a r a c t e r i z e d m i c r o s c o p i c a l l y as f o l l o w s : 1-20 g r a m - n e g a t i v e f a c u l t a t i v e r o d 1-21 g r a m - n e g a t i v e a n a e r o b i c r o d 1-22 g r a m - n e g a t i v e a n a e r o b i c r o d 1-23 g r a m - n e g a t i v e a n a e r o b i c f i l a m e n t o u s r o d 1-25 g r a m - p o s i t i v e a n a e r o b i c r o d 1-35 g r a m - n e g a t i v e a n a e r o b i c r o d 1-45 gr a m - n e g a t i v e a n a e r o b i c r o d I s o l a t e 20 was c h a r a c t e r i z e d by s t a n d a r d p r o c e d u r e s (77) and p r o v e d t o be K l e b s i e l l a pneumoniae. F u s o b a c t e r i u m n u c l e a t u r n and V e i l l o n e l l a  a l c a l e s c e n s were i s o l a t e d f r o m human d e n t a l p l a q u e and c h a r a c t e r i z e d a c c o r d i n g t o s t a n d a r d m o r p h o l o g i c a l , n u t r i t i o n a l and m e t a b o l i c end p r o d u c t a n a l y s i s . A c t i n o m y c e s v i s c o s u s 15987 was o b t a i n e d f r o m the A m e r i c a n Type C u l t u r e C o l l e c t i o n . E s c h e r i c h i a c o l i was o b t a i n e d f r o m t h e d e p a r t m e n t a l c o l l e c t i o n . S t a p h y l o c o c c u s a l b u s was i s o l a t e d f r o m m a n n i t o l s a l t p l a t e s i n o c u l a t e d w i t h s k i n swabs. B. A n a e r o b i c Growth Tubes o r p l a t e s were u s u a l l y i n c u b a t e d i n a n a e r o b i c j a r s ( T o r b a l l , T o r s i o n B a l a n c e , C l i f t o n , New J e r s e y ) e v a c u a t e d and f l u s h e d w i t h m -.CO^ (95:5) ( C a n a d i a n L i q u i d A i r , M o n t r e a l ) o r i n an a n a e r o b i c g l o v e box (Coy Mfg., Ann A r b o r , M i c h i g a n ) c o n t a i n i n g an atmosphere of N2:H2:C02 (85:10:5) a t a t e m p e r a t u r e o f 37°C. H u m i d i t y i n the g l o v e box was m a i n t a i n e d between 45 and 55% by a d d i n g t r a y s o f s i l i c a g e l . I t was found t h a t c o n t a m i n a t i o n problems were m i n i m i z e d i f t h e h u m i d i t y was k e p t a t 45%. Oxygen l e v e l s i n t h e chamber were m o n i t o r e d e v e r y two days w i t h a t r a c e - o x y g e n a n a l y z e r (Lockwood and M c L o r i e I n c . , Horsham, P a ) . An oxygen l e v e l o f two t o f i v e ppm was c o n s i d e r e d t o be a c c e p t a b l e . I n some i n s t a n c e s o r g a n i s m s were c u l t u r e d i n p r e - r e d u c e d media i n s t o p p e r e d tubes u s i n g c o n v e n t i o n a l a n a e r o b i c t e c h n i q u e s as d e s c r i b e d by Holdeman and Moore ( 3 7 ) . Growth M e d i a 1. 13. m e l a n i n o g e n i c u s The f o l l o w i n g media were used t o c u l t i v a t e 13. m e l a n i n o g e n i c u s : a. S t a n d a r d medium L i q u i d c u l t u r e s of 13. m e l a n i n o g e n i c u s were m a i n t a i n e d i n t h e t r y p t i c a s e - y e a s t e x t r a c t medium d e s c r i b e d by Gibbons and Macdonald ( 2 0 ) . G l u c o s e , menadione and NaHCO^ were r o u t i n e l y o m i t t e d f r o m t h e medium. b. B a s a l medium The b a s a l medium c o n s i s t e d o f t r y p t i c a s e (17 g / 1 ) , y e a s t e x t r a c t (3 g / 1 ) , K^HPO^ (2.5 g/1) and N a C l (5 g / 1 ) . I3_. m e l a n i n o g e n i c u s r e q u i r e s hemin f o r growth b u t a p p a r e n t l y m a i n t a i n s enough of the compound t o s u s t a i n growth t h r o u g h one t r a n s f e r i n l i q u i d media. I n o r d e r t o s t u d y c e l l s i n a hemin d e f i c i e n t s t a t e i t was n e c e s s a r y t o grow them once i n b a s a l medium. C e l l s o b t a i n e d f r o m t h e b a s a l medium w i l l n o t grow when s u b c u l t u r e d a second time i n hemin f r e e media. These or g a n i s m s w i l l be r e f e r r e d t o as hemin d e p l e t e d ]3. m e l a n i n o g e n i c u s . c. V i t a m i n K i medium L i q u i d medium f o r v i t a m i n K r e q u i r i n g s t r a i n s was p r e p a r e d as f o l l o w s : f i l t e r s t e r i l i z e d v i t a m i n ( 0 . 5 % i n 95% e t h a n o l ) was added to s t e r i l i z e d 2% a g a r w h i c h had been c o o l e d t o 50°C. The m i x t u r e was d i s p e n s e d i n t o t u b e s i n 1 m l q u a n t i t i e s and a l l o w e d t o s o l i d i f y . Ten ml o f t h e s t a n d a r d medium was the n added a s e p t i c a l l y . d. Supplemented media When needed, the f o l l o w i n g supplements were added s e p a r a t e l y o r t o g e t h e r t o e i t h e r t h e s t a n d a r d medium or the b a s a l medium: sodium t h i o -_3 g l y c o l a t e ( F i s h e r ) 0.05%; v i t a m i n K-^  (Sigma) 10 g/1; g l u c o s e ( F i s h e r ) -3 0.1%; hemin (Sigma) 5 x 10 g/1; s u c c i n i c a c i d (Eastman), (see t e x t f o r c o n c e n t r a t i o n s ) . I n a l l c a s e s , t h e compounds were added t o t h e medium b e f o r e s t e r i l i z a t i o n and the pH was a d j u s t e d t o 7.0 w i t h e i t h e r HC1 o r NaOH. 2. Other organisms K l e b s i e l l a pneumoniae was grown i n t r y p t i c a s e soy b r o t h o r i n g l u c o s e supplemented b a s a l medium. A l l t h e o t h e r organisms were m a i n t a i n e d on g l u c o s e o r b r a i n - h e a r t i n f u s i o n supplemented b a s a l medium. A l l o r g a n i s m s were s t o r e d i n the f o l l o w i n g manner. B a c t e r i a were grown i n s u i t a b l e l i q u i d medium t o t h e m i d - l o g a r i t h m i c growth phase. S t e r i l e g l y c e r o l was added t o the b r o t h (10% v./v) and a l i q u o t s were f r o z e n a t -70°C i n s m a l l p l a s t i c v i a l s . D. I n f e c t i v i t y C e l l s t o be used f o r i n o c u l a t i o n o f a n i m a l s were h a r v e s t e d f r o m b l o o d a g a r p l a t e s o r f r o m b r o t h c u l t u r e s and resuspended i n 1 ml of s t e r i l e p h o s p h a t e -b u f f e r e d s a l i n e c o n t a i n i n g 0.05% sodium t h i o g l y c o l a t e (PBS + t h i o ) , pH 7.0. G u i n e a p i g s w e i g h i n g 150 t o 200 g were i n j e c t e d s u b c u t a n e o u s l y i n t h e g r o i n a r e a w i t h 0.5 ml of e i t h e r i n v i t r o c u l t u r e d c e l l s o r e x u d a t e a s p i r a t e d from an i n f e c t e d g u i n e a p i g . The a n i m a l s were o b s e r v e d f o r up t o f o u r weeks. The c r i t e r i a f o r e v a l u a t i n g a p o s i t i v e i n f e c t i o n were: (a) t h e p r e s e n c e of an a b s c e s s ( p u s t u l a r o r n e c r o t i c ) ; (b) the t r a n s m i s s i b i l i t y o f t h e d i s e a s e . The l a t t e r was d e m o n s t r a t e d by i n j e c t i n g m a t e r i a l a s p i r a t e d f r o m a l e s i o n i n t o a second a n i m a l t o pr o d u c e a s i m i l a r p a t h o l o g y . T h i s p r o c e s s was r e p e a t e d a t l e a s t t w i c e i n o r d e r t o e s t a b l i s h t h a t t h e c e l l s were g r o w i n g . Exudate was a s p i r a t e d f r o m i n f e c t e d g u i n e a p i g s u s i n g a s t e r i l e d i s p o s a b l e s y r i n g e w h i l e the a n i m a l was under l i g h t e t h e r a n a e s t h e s i a . The exud a t e was examined f o r m i c r o b i a l c o n t a m i n a t i o n by p l a t i n g on b l o o d a g a r and i n c u b a t i n g i t a e r o b i c a l l y and a n a e r o b i c a l l y . The c r i t e r i a u sed t o c h a r a c t e r i z e an i n f e c t i o n a r e d e s -c r i b e d i n T a b l e I . TABLE I DESCRIPTION OF DISEASE Symbol Used D e s c r i p t i o n (-) No e v i d e n c e o f i n f e c t i o n a n i m a l n o r m a l (+) L o c a l i z e d , i n f l a m e d a r e a w h i c h i s f l a t and e v e n t u a l l y becomes h a r d (±) S m a l l n o d u l a r r e g i o n ; e x u d a t e d i f f i c u l t t o o b t a i n (+) L o c a l i z e d a b s c e s s l e s s t h a n 2 cm i n d i a m e t e r ; e x u d a t e (++) L o c a l i z e d a b s c e s s 2 cm o r more i n d i a m e t e r (+++) L o c a l i z e d a b s c e s s w i t h e x t e n s i o n , n e c r o t i c , not f a t a l (I I I I ) S p r e a d i n g i n f e c t i o n ; f a t a l i n 18 ho u r s t o 3 days (P) Used f o r passage I n o r d e r t o s u p p l y b a c t e r i a w i t h a s l o w l y d i s s e m i n a t i n g s o u r c e of hemin o r s u c c i n a t e a 2% agar s o l u t i o n was mixed w i t h sodium s u c c i n a t e ( f i n a l c o n -c e n t r a t i o n 0.5 M) o r hemin (5 u g / m l ) . The a g a r was a l l o w e d t o g e l and was t h e n pushed f i r s t t h r o u g h a f i n e n y l o n mesh and f i n a l l y t h r o u g h an 18 gauge n e e d l e . The s u c c i n a t e - a g a r was mixed w i t h b a c t e r i a and i n j e c t e d s u b c u t a n e o u s -l y i n t o the g r o i n o f a g u i n e a p i g . E. E n u m e r a t i o n o f B a c t e r i a 1. T o t a l count:. T o t a l c o u n t s were p e r f o r m e d m i c r o s c o p i c a l l y i n a P e t r o f f - H a u s s e r c o u n t i n g chamber. 2. V i a b l e count V i a b l e c o u n t s were d e t e r m i n e d by m e a s u r i n g c o l o n y f o r m i n g u n i t s or by t h e most p r o b a b l e number (MPN) method. a. C o l o n y f o r m i n g u n i t s Samples of pu r e o r mixed c u l t u r e s were s e r i a l l y d i l u t e d and p l a t e d a n a e r o b i c a l l y on a n a e r o b i c a l l y s t o r e d t r y p t i c a s e - s o y - h e m i n a g a r or b l o o d a g a r u s i n g the s p r e a d p l a t e t e c h n i q u e . I t was f o u n d t h a t t h i s t e c h n i q u e gave i n c o n s i s t e n t r e s u l t s a t h i g h d i l u t i o n s o f p u r e c u l t u r e s o f J3. m e l a n i n o g e n i c u s . I ndeed, i t was found t h a t s t r a i n K110 —6 o f t e n d i d n o t grow a t d i l u t i o n s g r e a t e r t h a n 10 b. Most p r o b a b l e number S e r i a l l y d i l u t e d _B. m e l a n i n o g e n i c u s was i n o c u l a t e d i n t o a n a e r o b i c l i q u i d o r a n a e r o b i c s e m i - s o l i d medium and i n c u b a t e d a n a e r o b i c a l l y . F i v e t u b e s of medium were i n o c u l a t e d p e r d i l u t i o n . A f t e r 72 h o u r s , t h e number of t u r b i d tubes o b s e r v e d f o r each of t h e d i l u t i o n s s t u d i e d were c o u n t e d and compared w i t h p u b l i s h e d t a b l e s (69) t o f i n d t h e M.P.N. p e r i n o c u l u m o f the f i r s t d i l u t i o n . H i g h e r r e c o v e r i e s were o b t a i n e d w i t h t h i s method but t h e r e s u l t s were v e r y much l e s s t h a n t h o s e o b t a i n e d f o r t o t a l c o u n t s . C o l l a g e n a s e and P r o t e a s e A c t i v i t y 1. C o l l a g e n a s e C o l l a g e n a s e was measured as d e s c r i b e d by G i s s l o w and M c B r i d e ( 2 3 ) . A c i d - s o l u b l e c o l l a g e n was e x t r a c t e d from f r e s h f e t a l c a l f - s k i n as d e s -c r i b e d by G a l l o p and S e i f t e r ( 1 8 ) , e x c e p t t h a t p a r t i c u l a t e m a t t e r was removed by f i l t r a t i o n . L y o p h i l i z e d c o l l a g e n was s t o r e d i n s t o p p e r e d f l a s k s a t -20°C. T h i s m a t e r i a l was s o l u b i l i z e d i n 0.01% c o l d a c e t i c a c i d a t a c o n c e n t r a t i o n o f 2 mg/ml, and a c e t y l a t e d w i t h a c e t i c -1 ^ C - a n h y d r i d e i n benzene a t pH 8.5. The l a b e l l e d m i x t u r e was t h e n a c i d i f i e d w i t h g l a c i a l a c e t i c a c i d and the a c e t y l a t e d c o l l a g e n d i a l y z e d a g a i n s t c o l d 14 14 d i s t i l l e d w a t e r t o remove C - a c e t i c a c i d . The C - c o l l a g e n was l y o p h i -l i z e d and s t o r e d a t -20° u n t i l needed. A t y p i c a l r e a c t i o n m i x t u r e i s shown i n T a b l e I I . The a s s a y components were i n c u b a t e d f o r f i f t e e n m i n u t e s b e f o r e a d d i t i o n of s u b s t r a t e and i n c u b a t i o n was c o n t i n u e d a t room t e m p e r a t u r e . The r e a c t i o n was s t o p p e d and n o n ^ h y d r o l y z e d c o l l a g e n was p r e c i p i t a t e d by t h e a d d i t i o n of 0.04 N p h o s p h o t u n g s t i c a c i d (PTA) and 2N HC1 t o a f i n a l c o n c e n t r a t i o n o f 0.01N and 0.5N r e s p e c t i v e l y . A 0.1 ml a l i q u o t of the r e a c t i o n m i x t u r e s was added t o a " m i c r o f u g e " tube (Beckman I n s t r u m e n t s , I n c . , I r v i n e , C a l i f o r n i a ) c o n t a i n i n g 50 y l each of PTA and HC1. The samples were l e f t a t room t e m p e r a t u r e f o r t e n m i n u t e s and t h e n c e n t r i f u g e d f o r f i v e m i n u t e s i n a Beckman-Spinco m i c r o f u g e (model 152, Beckman I n s t r u m e n t s , I n c . ) . 100 y l of t h e s u p e r n a t a n t was c o u n t e d . The c o n t r o l sample f o r each a s s a y c o n t a i n e d b u f f e r o r un-i n o c u l a t e d medium i n p l a c e of c e l l s . TABLE I I COLLAGENASE ASSAY Components 14 C - c o l l a g e n ( 0 . 1 % I n 0.01% a c e t i c a c i d ) T r i s - H C l b u f f e r (0.05M, pH 7.2) w i t h C a C l 2 (0.005M) C y s t e i n e (.005M) C e l l s u s p e n s i o n ( l O x ) Volume (ml)  Sample C o n t r o l 0.2 0.1 0.1 0.1 0 r2 0.2 0.1 25 2. P r o t e a s e P r o t e o l y t i c a c i t i v i t y was d e t e r m i n e d by m e a s u r i n g a c t i v i t y a g a i n s t A z o c o l l ( C a l b i o c h e m ) or c a s e i n (Hammarsten). The r e a c t i o n m i x t u r e f o r the A z o c o l l a s s a y c o n t a i n e d : T r i s - H C l b u f f e r (0.05M, pH 7.2), 4.8 m l ; n e u t r a l i z e d c y s t e i n e h y d r o c h l o r i d e i n t h e same b u f f e r (0.05M), 0.2 m l ; and s u p e r n a t a n t f r o m s e d i m e n t e d c u l t u r e , 0.5 m l . The r e a c t i o n components were p r e - i n c u b a t e d a t 37°C f o r 15 m i n u t e s b e f o r e a d d i t i o n o f 20 mg of A z o c o l l . I n c u b a t i o n was t h e n c o n t i n u e d a t t h e same t e m p e r a t u r e i n a s h a k i n g w a t e r b a t h . Two m i l l i l i t e r samples were removed a t v a r i o u s t i m e i n t e r v a l s , c h i l l e d i n i c e , and f i l t e r e d t o remove i n s o l u b l e s u b s t r a t e . The amount of dye r e l e a s e d was d e t e r m i n e d by mea-s u r i n g t h e a b s o r b a n c e of the f i l t r a t e a t 520 nm. The s u b s t r a t e f o r the c a s e i n a s s a y was p r e p a r e d by d i s s o l v i n g 1 g of c a s e i n i n 100 ml of phosphate b u f f e r (pH 7.4), and t r e a t i n g 15 m i n u t e s i n a b o i l i n g w a t e r b a t h . The r e a c t i o n m i x t u r e c o n t a i n e d 2.5 ml c a s e i n , 1.0 ml o f n e u t r a l i z e d c y s t e i n e h y d r o c h l o r i d e (0.05 M), 0.5 ml o f s u p e r n a t a n t f r o m a sedimented c u l t u r e o r 100 y l of a 10 t i m e s c o n c e n t r a t e d c e l l s u s -p e n s i o n and b u f f e r t o 5. 0 ml. The m i x t u r e was i n c u b a t e d a t 37°C and t h e r e a c t i o n t e r m i n a t e d by t h e a d d i t i o n o f 5.0 ml of 10% t r i c h l o r o a c e t i c a c i d . A f t e r 30 m i n u t e s a t room t e m p e r a t u r e t h e c o n t e n t s o f t h e tubes were f i l t e r e d and t h e a b s o r b a n c e (280 nm) o f t h e f i l t r a t e s d e t e r m i n e d . G. H y d r o x y p r o l i n e A s s a y H y d r o x y p r o l i n e was a s s a y e d by t h e method of Le a c h ( 4 8 ) . C i t r a t e - e x t r a c t e d c o l l a g e n was h y d r o l y z e d i n vacuo i n 6N HC1 by h e a t i n g 108°C f o r 18 h o u r s . The h y d r o l y z e d samples were n e u t r a l i z e d w i t h NaOH, d i l u t e d t o 100 y g p r o t e i n / m l and f i l t e r e d p r i o r t o a s s a y i n g f o r h y d r o x y p r o l i n e . F a t t y A c i d D e t e r m i n a t i o n 1. P r e p a r a t i o n o f samples V o l a t i l e and n o n - v o l a t i l e a c i d s were a n a l y z e d as d e s c r i b e d by H o l d e -man and Moore ( 3 7 ) . C u l t u r e s u p e r n a t a n t s were a c i d i f i e d w i t h aqueous H^SO^ (50% v / v ) and t h e v o l a t i l e f a t t y a c i d s e x t r a c t e d i n t o e t h e r . The e t h e r was t h e n d r i e d w i t h anhydrous MgSO^. N o n - v o l a t i l e a c i d s were f i r s t m e t h y l a t e d by e i t h e r one of t h e f o l l o w i n g methods: 2 m l o f m e t h a n o l o r 1 ml o f b o r o n - t r i f l u o r i d e - m e t h a n o l , 14% w/v ( A p p l i e d S c i e n c e L a b o r a t o r i e s ) was added t o a 1.5 ml a l i q u o t of the a c i d i f i e d c u l t u r e . The tubes were s t o p p e r e d and l e f t o v e r n i g h t a t room? t e m p e r a t u r e . The m e t h y l a t e d a c i d s were th e n e x t r a c t e d i n t o c h l o r o f o r m . Known s t a n d a r d s of v o l a t i l e and m e t h y l a t e d f a t t y a c i d s were p r e p a r e d w i t h each s e t of samples. a. G a s - l i q u i d chromatography (GLC) i . P r e p a r a t i o n o f column Column p a c k i n g m a t e r i a l was p r e p a r e d by m i x i n g 10 gm; of a c i d washed Chromosorb W (60-80 mesh) ( J . M a n v i l l e Co.) w i t h 1.1 g of R e s o f l e x LAC-1-R296 d i s s o l v e d i n 20 ml of c h l o r o f o r m . The m i x t u r e was mixed g e n t l y u n t i l t h e C H C l ^ had e v a p o r a t e d and was t h e n packed i n t o a 6' x 1/4" U-shaped tub e . A two f o o t column was p r e p a r e d f o r a n a l y s i s of t h e m e t h y l e s t e r of p h e n y l a c e t i c a c i d , i i . O p e r a t i n g c o n d i t i o n s Samples were a n a l y z e d i n a B e n d i x model 2500 gas chromatograph (Ca n a d i a n Dynamics, Vancouver, B.C.) e q u i p p e d w i t h a hydrogen f l a m e i o n i z a t i o n d e t e c t o r . The c a r r i e r gas f l o w was s e t a t 90 c c / min.and t h e oven t e m p e r a t u r e a t 120°C f o r v o l a t i l e a c i d s and 125°C f o r n o n - v o l a t i l e a c i d s . F i g u r e s l a and l b show t h e r e s u l t s o b t a i n e d when a m i x t u r e of t h e s t a n d a r d v o l a t i l e f a t t y a c i d s and 27 F i g u r e 1. A. V o l a t i l e f a t t y a c i d s . E q u i m o l a r amounts o f s t a n d a r d f a t t y a c i d s were mixed and e x t r a c t e d as d e s c r i b e d i n the t e x t . The r e t e n t i o n t i m e s , i n m i n u t e s , f o r t h e compounds t e s t e d a r e as f o l l o w s : a c e t i c a c i d , 5; p r o p i o n i c a c i d , 7.5; i s o b u t y r i c a c i d , 8.6; b u t y r i c a c i d , 12; i s o v a l e r i c a c i d , 14.4. B. M e t h y l a t e d f a t t y a c i d s . E q u i m o l a r amounts of s t a n d a r d f a t t y a c i d s were mi x e d , m e t h y l a t e d and e x t r a c t e d as d e s c r i b e d i n t h e t e x t . R e t e n t i o n t i m e s i n m i n u t e s a r e : l a c t i c a c i d , 3.3; f u m a r i c a c i d , 8.7; s u c c i n i c a c i d , 12.6. m e t h y l a t e d f a t t y a c i d s were chromatographed by t h i s method, b. T h i n l a y e r chromatography (TLC) Ten u l of e i t h e r c h l o r o f o r m o r e t h e r e x t r a c t e d f a t t y a c i d s were s p o t t e d on c e l l u l o s e t h i n l a y e r chromatogram s h e e t s (Eastman Kodak, R o c h e s t e r , New Y o r k ) and chromatographed i n a s o l v e n t system c o n t a i n -i n g , n - b u t a n o l : d i e t h y l a m i n e : w a t e r (85:1:14). When th e s o l v e n t f r o n t had m i g r a t e d 10 cm f r o m t h e o r i g i n t h e chromatogram was removed and d r i e d c a r e f u l l y t o remove d i e t h y l a m i n e . D i e t h y l a m i n e s a l t s of the a c i d s were d e t e c t e d by s p r a y i n g w i t h n i n h y d r i n (0.2%) i n w a t e r s a t u r a t e d n - b u t a n o l . The a c i d s a l t s appeared as p a l e brown s p o t s a g a i n s t a f a i n t b l u e b a c kground. The R^ v a l u e s o b t a i n e d by t h i s method a r e as f o l l o w s : a c e t i c a c i d , 0.42; p r o p i o n i c a c i d , 0.54; b u t y r i c a c i d , 0.70; v a l e r i c a c i d , 0.74; s u c c i n i c a c i d , 0.53; f u m a r i c a c i d , 0.60; l a c t i c a c i d , 0.70. T h i s method was used t o t e s t t h e 14 p u r i t y o f C l a b e l l e d s u c c i n i c and b u t y r i c a c i d . Samples of t h e s e compounds were s p o t t e d t o g e t h e r w i t h n o n - r a d i o a c t i v e s t a n d a r d s and chromatographed as d e s c r i b e d above. The r a d i o a c t i v e chromatograms 14 were t h e n c u t i n t o 1 cm bands and a n a l y z e d f o r C; t h e non r a d i o -a c t i v e s t a n d a r d s were i d e n t i f i e d as d e s c r i b e d e a r l i e r . P u r i f i c a t i o n o f Growth F a c t o r 1. Growth a s s a y C u l t u r e s s u s p e c t e d t o have g r o w t h - p r o m o t i n g a c t i v i t y were t e s t e d i n the f o l l o w i n g growth a s s a y . A 10 ml a l i q u o t of the c u l t u r e was sedimented (10 min a t 10,000 x g) and the s u p e r n a t a n t was f i l t e r - s t e r i l i z e d u s i n g a 0.3 m i c r o n f i l t e r ( M i l l i p o r e ) . The f i l t r a t e was t h e n i n o c u l a t e d w i t h a hemin d e p l e t e d c u l t u r e of J3. m e l a n i n o g e n i c u s . 30 2. C o n c e n t r a t i o n M a t e r i a l w h i c h was known t o have g r o w t h - p r o m o t i n g a c t i v i t y was c o n -c e n t r a t e d a c c o r d i n g t o t h e f o l l o w i n g scheme: c u l t u r e grown a t 37°C sedimented 10 min a t 10,000 x g 1 s u p e r n a t a n t f r o z e n and f r e e z e - d r i e d r e h y d r a t e d I f i l t e r s t e r i l i z e d I growth a s s a y U s u a l l y , t h e c o n c e n t r a t e was r e h y d r a t e d t o a f i n a l c o n c e n t r a t i o n 20 t i m e s t h a t o f t h e o r i g i n a l s u p e r n a t a n t . The r e h y d r a t e d m a t e r i a l was f i l t e r -s t e r i l i z e d and a s s a y e d f o r growth a c t i v i t y as d e s c r i b e d e a r l i e r , e x c e p t t h a t 0.5 ml of c o n c e n t r a t e was added t o a d e p l e t e d medium b e f o r e i n o c u l -a t i o n . I n a l l c a s e s , o p t i c a l d e n s i t y (OD-,^) r e a d i n g s of b o t h g r o w t h boU a s s a y s were s i m i l a r . 3. Chromatography a. P r e p a r a t i o n and c a l i b r a t i o n A 2.6 x 40 cm column (Vt=192 ml) ( P h a r m a c i a Canada L t d . , D o r v a l , Quebec) was packed w i t h Sephadex G-15 (Pharmacia Canada L t d . ) washed w i t h d i s t i l l e d w a t e r and e q u i l i b r a t e d w i t h phosphate b u f f e r , 50 mM pH 6.6. The column was c a l i b r a t e d w i t h N a C l , g l y c i n e , g l u c o s e and r a f f i n o s e , t h e i r m o l e c u l a r w e i g h t s b e i n g r e s p e c t i v e l y 58, 75, 180 and 504. S e v e n t y - f i v e f r a c t i o n s of 3 ml were c o l l e c t e d . The c h l o r i d e i o n was d e t e c t e d by p r e c i p i t a t i o n w i t h 0.1N s i l v e r n i t r a t e . G l y c i n e was d e t e c t e d w i t h n i n h y d r i n and the s u g a r s were d e t e c t e d by t h e a n t h r o n e a s s a y . 31 b. Chromatography o f growth f a c t o r The column was l o a d e d w i t h 5 m l of 2 0 t i m e s c o n c e n t r a t e d g r o w t h -p r o m o t i n g m a t e r i a l and e l u t e d w i t h phosphate b u f f e r (50 mM, pH 6.6). S e v e n t y - f i v e f r a c t i o n s of 2 m l each were c o l l e c t e d . The f r a c t i o n s were put on i c e u n t i l the end of the r u n and t h e n t r a n s f e r r e d i n t o the a n a e r o b i c chamber and f i l t e r - s t e r i l i z e d . A l l f r a c t i o n s were l e f t o v e r n i g h t t o e q u i l i b r a t e w i t h t h e atmosphere i n t h e chamber and checked v i s u a l l y f o r c o n t a m i n a t i o n b e f o r e a s s a y i n g . The f r a c t i o n s were a s s a y e d f o r (a) g r o w t h - p r o m o t i n g a c t i v i t y (0.8 ml added t o 4 ml o f b a s a l medium i n o c u l a t e d w i t h h e m i n - d e p l e t e d K110) and (b) v o l a t i l e and n o n - v o l a t i l e f a t t y a c i d s by gas chromatography. J . G l u c o s e D e t e r m i n a t i o n G l u c o s e measured by e i t h e r t h e a n t h r o n e a s s a y (110) o r by t h e p e r o x i d a s e -g l u c o s e o x i d a s e (PGO) a s s a y (Sigma C h e m i c a l Co., S t . L o u i s , M i s s o u r i ) as des-c r i b e d i n Sigma t e c h n i c a l b u l l e t i n No. 510. K. C e l l F r a c t i o n a t i o n 14 14 The d i s t r i b u t i o n o f C i n c e l l s grown i n C-succinate medium was determined by a m o d i f i c a t i o n of t h e c e l l f r a c t i o n a t i o n p r o c e d u r e d e s c r i b e d by S u t h e r l a n d and W i l k i n s o n ( 1 0 1 ) . The scheme i s o u t l i n e d i n F i g u r e 2. S i x t e e n o r f o r t y -e i g h t hour c u l t u r e s were removed f r o m the chamber and t h e a b s o r b a n c e a t 660 nm was d e t e r m i n e d . R a d i o a c t i v e C O 2 was measured by a d d i n g 1.0 ml of the c u l t u r e t o a Thunberg tube. ^ S O ^ (0.5 ml o f 50% v/v) was added t o t h e c u l t u r e and the tube q u i c k l y s t o p p e r e d , v o r t e x e d and the e v o l v e d gas t r a p p e d i n 20% KOH. 14 B o t h t h e t r a p and the a c i d i f i e d c u l t u r e were a s s a y e d f o r C i n a s c i n t i l l a t i o n c o u n t e r . 32 14 F i g u r e 2. F r a c t i o n a t i o n o f C - l a b e l l e d B. m e l a n i n o g e n i c u s . C e l l s were grown i n b a s a l m e d i a supplemented w i t h 1,4 o r 2,3 14 C - s u c c i n a t e and h a r v e s t e d 16 or 48 h r s a f t e r i n o c u l a t i o n . A t each s t e p i n t h e e x t r a c t i o n a sample of the e x t r a c t e d p e l l e t and 14 t h e s o l u b l e s u p e r n a t a n t were a s s a y e d f o r C. or 48 hour c u l t u r e s Wash c e l l s (2x) C o l d TCA (10%) St A c i d i f i e d EtOH CHCL -MeOH V Hot TCA (5%) 1 0.1 N NaOH s u p e r n a t a n t s r e t a i n e d f o r f a t t y a c i d a n a l y s i s i n t e r m e d i a t e p o o l i n o r g a n i c i o n s l ow m o l e c u l a r w e i g h t p e p t i d e s j j a l c o h o l s o l u b l e p r o t e i n s j ~ l i p i d s - 0 RNA and DNA c e l l w a l l m a t e r i a l g l y c o p r o t e i n s , p r o t e i n s The r e m a i n i n g c u l t u r e was c e n t r i f u g e d t o s e p a r a t e t h e growth medium fr o m the c e l l s . The c u l t u r e s u p e r n a t a n t was s t o r e d a t -20°C f o r subsequent a n a l y s i s and t h e c e l l s were washed t w i c e w i t h c o l d (4°C) PBS and res u s p e n d e d i n 1 m l of d i s t i l l e d H^O. The c e l l s u s p e n s i o n was mixed w i t h 1 ml of c o l d 10% TCA and i n c u b a t e d f o r 20 min b e f o r e c e n t r i f u g a t i o n . The s u p e r n a t a n t was saved and the p e l l e t mixed w i t h 2 m l of 75% a c i d - e t h a n o l (pH 2.5) and i n c u b a t e d a t 45°C f o r 15 m i n and t h e n c o o l e d and c e n t r i f u g e d . The s u p e r n a t a n t was saved and the p e l l e t mixed w i t h 3 ml o f c h l o r o f o r m - m e t h a n o l (2:1) f o r 20 min a t room t e m p e r a t u r e . A f t e r c e n t r i f u g a t i o n t h e p e l l e t was mixed w i t h 2 ml o f 5% TCA and i n c u b a t e d a t 90°C f o r 20 min. The p e l l e t f r o m t h e e x t r a c t i o n was r e -suspended i n 0.5 ml of 0.1N NaOH and i n c u b a t e d a t 45°C f o r 15 min. A l l f r a c t i o n s were p l a c e d i n m e t h a n o l - t o l u e n e - l i q u i f l u o r (400:600:42, v/v/v) 14 s c i n t i l l a t i o n f l u i d and C was measured i n a I s o c a p 300 s c i n t i l l a t i o n c o u n t e r ( N u c l e a r C h i c a g o , I n c . ) 14 C u l t u r e s u p e r n a t a n t s were a s s a y e d f o r C l a b e l l e d v o l a t i l e and non-v o l a t i l e f a t t y a c i d s on a gas chromatograph equipped w i t h b o t h f l a m e i o n -14 i z a t i o n and C d e t e c t o r s . V o l a t i l e f a t t y a c i d s were e x t r a c t e d i n t o e t h e r and m e t h y l a t e d n o n - v o l a t i l e a c i d s were e x t r a c t e d i n t o c h l o r o f o r m as d e s c r i b e d e a r l i e r . The GC s e t - u p i s shown i n F i g u r e 3. I n e s s e n c e , t h e r a d i o a c t i v e sample p a s s e d t h r o u g h the GC column and was e i t h e r d e t e c t e d by t h e fl a m e i o n i z a t i o n d e t e c t o r o r a l t e r n a t i v e l y b y - p a s s e d t o a c o m b u s t i o n chamber where th e t e m p e r a t u r e was m a i n t a i n e d a t 700°C. The sample was t h u s combusted t o C O 2 and w a t e r . Water was removed i n a magnesium p e r c h l o r a t e t r a p and the 14 C O 2 p a s s e d t h r o u g h a c o u n t i n g chamber. I t was found t h a t c h l o r o f o r m formed a d e r i v a t i v e w h i c h b l o c k e d the e n t r a n c e o r t h e e x i t o f the com b u s t i o n chamber. The p r o b l e m was overcome by p a s s i n g the c h l o r o f o r m peak i n t o t h e f l a m e d e t e c t -o r and t h e n s w i t c h i n g t h e two-way v a l v e so t h a t t h e r e m a i n i n g e l u a n t passed 35 F i g u r e 3. Diagram of Gas Chromatography 1. GC column 2. Two-way v a l v e 3. Flame i o n i z a t i o n d e t e c t o r 4. D i g i t a l i n t e g r a t o r 5. R e c o r d e r 6. Combustion chamber 7. Water t r a p 8. R a d i o a c t i v e m o n i t o r 9. Carbon d i o x i d e t r a p i n t o t h e c o m b u s t i o n tube. T h i s p r o b l e m d i d not o c c u r w i t h e t h e r - e x t r a c t e d m a t e r i a l s . The e f f i c i e n c y o f t h e gas chromatograph r a d i o a c t i v e m o n i t o r ( N u c l e a r C h i c a g o ) was measured by comparing the c o u n t s o b t a i n e d when known r a d i o a c t i v e s t a n d a r d s such as s u c c i n i c and b u t y r i c a c i d were i n j e c t e d i n t o t h e gas chromatograph w i t h r e s u l t s o b t a i n e d f r o m the same samples when t h e y were counted by t h e s c i n t i l l a t i o n m o n i t o r . The e f f i c i e n c y was 57% f o r b u t y r i c a c i d and 28% f o r s u c c i n i c a c i d . L. H e m a g g l u t i n a t i o n C e l l s f r o m a 48 hour c u l t u r e were h a r v e s t e d by c e n t r i f u g a t i o n , washed t w i c e and r e s u s p e n d e d i n PBS + $ - m e r c a p t o e t h a n o l (50 mM). The o p t i c a l d e n s i t y (660 nm) of t h e c e l l s u s p e n s i o n was a d j u s t e d t o 3.0. H e m a g g l u t i n a t i o n was measured i n m i c r o t i t e r p l a t e s u s i n g a 2.5% s u s p e n s i o n o f f o r m a l i n i z e d human r e d b l o o d c e l l s (RBC). R e s u l t s were r e c o r d e d a f t e r one hour i n c u b a t i o n a t room t e m p e r a t u r e . I n some i n s t a n c e s h e m a g g l u t i n a t i o n was measured by m i x i n g 0.2 ml o f b a c t e r i a l c e l l s (O.D. 660 = 3.0) w i t h 0.2 ml o f 2.5% RBC. C e l l s p o s s e s s i n g h e m a g g l u t i n a t i o n a c t i v i t y clumped the RBC w i t h i n 10 m i n u t e s . The e x t e n t o f c l u m p i n g was a s s e s s e d v i s u a l l y and s c o r e d on a 0 t o +4 b a s i s . M. I l e a l Loop A s s a y The e f f e c t of c u l t u r e f i l t r a t e and v i a b l e c e l l s on f l u i d a c c u m u l a t i o n i n l i g a t e d i l e a l l o o p s o f a d u l t mice was measured as d e s c r i b e d by K a s a i and Burrows ( 3 9 ) . A n i m a l s were a n a e s t h e t i z e d w i t h sodium p e n t o b a r b i t a l and a l a p a r o t o m y p e r f o r m e d i n o r d e r t o expose the i l e u m . The i l e u m was s e c t i o n e d by l i g a t i o n w i t h s u r g i c a l t h r e a d a t measured i n t e r v a l s . I n m i c e i t was p o s s i b l e t o f o r m two l i g a t e d l o o p s and i n r a b b i t s as many as s i x . The t e s t 38 m a t e r i a l was i n j e c t e d i n t o one l o o p and an a p p r o p r i a t e c o n t r o l s o l u t i o n i n t o a n o t h e r l o o p i n t h e same a n i m a l . . A f t e r 18-24 h o u r s i n c u b a t i o n , the a n i m a l was s a c r i f i c e d and t h e f l u i d a c c u m u l a t e d i n t h e l o o p was measured. The measurement was made by c o r r e l a t i n g t h e w e i g h t of t h e i n t a c t l o o p w i t h i t s l e n g t h . The w e i g h t - l e n g t h r a t i o o f e x p e r i m e n t a l l o o p s was compared w i t h t h e c o n t r o l . A l i g a t e d s e c t i o n o f i l e a l l o o p was c o n s i d e r e d t o show f l u i d a c c u m u l a t i o n i f i t s w e i g h t / l e n g t h r a t i o was two t i m e s t h a t of t h e c o n t r o l s e c t i o n . To i n s u r e t h a t f a l s e p o s i t i v e and f a l s e n e g a t i v e r e s u l t s d i d n o t i n v a l i d a t e t h e d a t a , each sample was t e s t e d i n e i g h t d i f f e r e n t a n i m a l s ; o n l y samples g i v i n g p o s i t i v e r e s u l t s i n a t l e a s t t h r e e a n i m a l s were c o n s i d e r e d a c t i v e . As a r e f e r e n c e each e x p e r i m e n t i n c l u d e d an a n i m a l i n o c u l a t e d w i t h p u r i f i e d c h o l e r a t o x i n . The t o x i n was g r a c i o u s l y donated by the N a t i o n a l I n s t i t u t e of H e a l t h ( B e t h e s d a , M a r y l a n d ) . N. C h e m i c a l s The siderochomes r h o d o t o r u l i c a c i d , D e s f e r a l and e n t e r o b a c t i n were o b t a i n -ed f r o m Dr. J.B. N e i l a n d s . 0. P o r p h y r i n and Hemin E x t r a c t i o n P o r p h y r i n and hemin were e x t r a c t e d w i t h e t h y l a c e t a t e and h y d r o c h l o r i c a c i d by the o n e - s t e p p r o c e d u r e d e s c r i b e d by F a l k ( 3 ) . 39 I I I . RESULTS PART I . CHARACTERIZATION OF B. MELANINOGENICUS The t h r e e s u b s p e c i e s o f the b l a c k pigmented o r a l B a c t e r o i d e s have n o t been c h a r a c t e r i z e d w i t h r e s p e c t t o t h e i r p a t h o g e n i c i t y i n t h e g u i n e a p i g model i n f e c t i o n , n o r i s t h e r e much known about t h e m e t a b o l i c , c o l l a g e n o l y t i c , p r o t e o -l y t i c and h e m a g g l u t i n a t i n g a c t i v i t y of t h e o r g a n i s m s . The p u r p o s e of t h i s s e c t i o n o f the t h e s i s i s t o d e s c r i b e e x p e r i m e n t s d e s i g n e d t o c h a r a c t e r i z e a l a r g e number of Ti. m e l a n i n o g e n i c u s i s o l a t e s w i t h r e g a r d t o t h e i r p a t h o g e n i c i t y and a number of r e l a t e d p r o p e r t i e s . A. I s o l a t i o n o f o r g a n i s m s I n o r d e r t o e v a l u a t e the p a t h o g e n i c i t y of t h e pigmented B a c t e r o i d e s i t was n e c e s s a r y t o i s o l a t e a l a r g e number of s t r a i n s f r o m a v a r i e t y of d i f f e r e n t o r a l s o u r c e s . Organisms were i s o l a t e d f r o m a number o f d i f f e r -ent s o u r c e s i n o r d e r t o g u a r d a g a i n s t t h e p o s s i b i l i t y t h a t t h e r e m i g h t be v a r i a t i o n w i t h i n a s u b s p e c i e s w h i c h would be r e f l e c t e d by i t s p r e s e n c e w i t h i n a p a r t i c u l a r e cosystem. I t was d e c i d e d t o sample the c r e v i c u l a r f l o r a f r o m d i s e a s e d and h e a l t h y s i t e s i n c h i l d r e n and a d u l t s and t h e s u p r a g i n g i v a l p l a q u e a d j a c e n t t o t h e g i n g i v a i n a d u l t m o n g r e l dogs. The o b j e c t i v e was t o b u i l d a c o l l e c t i o n o f o r g a n i s m s ; t h e r e was no a t t e m p t t o d e t e r m i n e t o t a l numbers. The number of i s o l a t e s and t h e i r p l a c e of o r i g i n i s shown i n T a b l e I I I . A t o t a l o f 396 o rganisms were i s o l a t e d f r o m f i v e major e c o s y s t e m s . A f t e r i n o c u l a t i o n t h e p l a t e was p l a c e d i n an a n a e r o b i c j a r and r e t u r n e d t o t h e l a b o r a t o r y where i t was p l a c e d i n t h e a n a e r o b i c chamber and the o r g a n i s m s s t r e a k e d t o o b t a i n i s o l a t e d c o l o n i e s . B l a c k pigmented c o l o n i e s were f i r s t o b s e r v e d a f t e r f o u r days i n c u b a t i o n but c o l o n i e s were n o t p i c k e d u n t i l t h e s e v e n t h day 40 TABLE I I I SOURCE OF B. MELANINOGENICUS ISOLATES Source G i n g i v a l H e a l t h ' Number of I s o l a t e s C h i l d r e n 7-12 y e a r s o l d Good Poor 42 4 C h i l d r e n 12-18 y e a r s o l d Good Poor 110 54 A d u l t s Good Poor 100 40 Dogs T o t a l ND 46 396 G i n g i v a l h e a l t h was a s s e s s e d a c c o r d i n g t o t h e c r i t e r i a o f Loe and S i l n e s s ( 5 7 ) . A r a t i n g o f good means a g i n g i v a l h e a l t h s c o r e of 1 o r l e s s and po o r means 2 or more. Not d e t e r m i n e d . 41 when a l l t h e pigmented c o l o n i e s had appeared. A p p r o x i m a t e l y 90% o f t h e samples c o n t a i n e d b l a c k c o l o n i e s w h i c h were s u b s e q u e n t l y i d e n t i f i e d as B a c t e r o i d e s m e l a n i n o g e n i c u s . Grey or l i g h t brown c o l o n i e s were n o t sub-c u l t u r e d . B e s t r e s u l t s were o b t a i n e d when g i n g i v a l m a t e r i a l was s t r e a k e d onto b l o o d agar p l a t e s w h i c h had been i n c u b a t e d i n an o x y g e n - f r e e e n v i r o n m e n t f o r 24 h o u r s . Many samples c o n t a i n e d two t y p e s of b l a c k c o l o n i e s , one was 3-4 mm i n d i a m e t e r and grew as a d i s t i n c t i s o l a t e d c o l o n y , t h e second was s m a l l e r and grew i n a s s o c i a t i o n w i t h non-pigmented o r g a n i s m s . I t was assumed t h a t t h e s e were v i t a m i n K i n d e p e n d e n t and v i t a m i n K dependent s t r a i n s as i t i s known t h a t many organisms s e c r e t e enough of t h e v i t a m i n t o s u s t a i n t h e growth of J i . m e l a n i n o g e n i c u s . P u r i f i c a t i o n o f t h e v i t a m i n K r e q u i r i n g s t r a i n s r e q u i r e d two t o f o u r t r a n s f e r s t o s e p a r a t e them from t h e con-t a m i n a n t s . The i s o l a t i o n of v i t a m i n K r e q u i r i n g s t r a i n s was f a c i l i t a t e d by a p p l y i n g a s i n g l e s t r e a k of S_. a u r e u s o v e r an a g a r p l a t e s t r e a k e d w i t h the B a c t e r o i d e s . S a t e l l i t e c o l o n i e s formed i n t h e a r e a a d j a c e n t t o t h e S t a p h y l o c o c c u s s t r e a k l i n e , p r e s u m a b l y b e c a u s e enough v i t a m i n K d i f f u s e d i n t o t h i s a r e a t o s u p p o r t growth. B. C h a r a c t e r i z a t i o n o f i s o l a t e s 1. F a t t y a c i d p r o d u c t i o n The f i r s t s t e p i n c h a r a c t e r i z i n g the organisms was t o s u b d i v i d e them on t h e b a s i s o f the a c i d i c end p r o d u c t s produced d u r i n g growth. Organisms were c u l t u r e d f o r 48 h o u r s i n the s t a n d a r d medium o r v i t a m i n K-supplemented l i q u i d medium and the s u p e r n a t a n t s a n a l y z e d f o r v o l a t i l e and n o n - v o l a t i l e f a t t y a c i d s by gas chromatography. The e l u t i o n p r o f i l e o b s e r v e d when v o l a t i l e a c i d s were chromatographed i s shown i n F i g u r e 4. Two b a s i c p a t t e r n s were e v i d e n t : i n one c a s e f i v e a c i d s 42 F i g u r e 4. Gas chromatography of v o l a t i l e f a t t y a c i d s p r o duced by J3. m e l a n i n o g e n i c u s i s o l a t e s . V o l a t i l e f a t t y a c i d s were e x t r a c t e d f r o m a c i d i f i e d c u l t u r e s u p e r n a t a n t . The peaks were i d e n t i f i e d by comparing t h e i r r e t e n t i o n t i m e s w i t h t h o s e of s t a n d a r d s ( F i g u r e 1 A ) : a, a c e t i c a c i d ; p, p r o p i o n i c a c i d ; i s o B, i s o b u t y r i c a c i d ; B, b u t y r i c a c i d ; i s o V, i s o v a l e r i c a c i d . 8 12 MINUTES 16 20 c o u l d be d e t e c t e d and i n t h e o t h e r c a s e t h e r e were n e v e r more t h a n t h r e e . I n t h e f o r m e r t h e r e was a l a r g e b u t y r i c a c i d peak w h i c h was m i s s i n g e n t i r e l y i n the l a t t e r p r o f i l e . The r e l a t i v e c o n c e n t r a t i o n s of t h e a c i d s were u s u a l l y i d e n t i c a l i n t h e b u t y r a t e p r o d u c i n g o r g a n -i s m s . Two e x c e p t i o n s were n o t e d i n w h i c h a much h i g h e r l e v e l o f i s o v a l e r i c a c i d was p roduced. T y p i c a l e l u t i o n p r o f i l e s f o r t h e non-v o l a t i l e f a t t y a c i d s a r e shown i n F i g u r e 5. Once a g a i n two b a s i c p a t t e r n s were e v i d e n t : one group of organisms produced s u c c i n i c a c i d and the o t h e r a compound w h i c h was r e t a i n e d f o r 24.5 m i n u t e s on t h e column. The compound w h i c h e l u t e d l a s t was shown t o be p h e n y l a c e t i c a c i d by mass s p e c t r o s c o p y and gas chromatography (Susan J e n s e n , p e r s o n a l c o m m u n i c a t i o n ) . The f a t t y a c i d p r o f i l e s were q u a l i t a t i v e l y s i m i l a r r e g a r d l e s s of t h e medium i n w h i c h t h e o rganisms were grown. The i s o l a t e s were thus s u b d i v i d e d i n t o two groups on t h e b a s i s of a c i d p r o d u c t i o n . T a b l e I V summarizes t h i s d a t a . The o rganisms p r o -d u c i n g b u t y r i c a c i d and p h e n y l a c e t i c a c i d c o r r e s p o n d t o t h e s u b s p e c i e s known as 15. m e l a n i n o g e n i c u s a s a c c h a r o l y t i c u s and the s u c c i n i c a c i d p r o d u c i n g o rganisms a r e e i t h e r i n t h e s u b s p e c i e s i n t e r m e d i u s o r m e l a n i n o g e n i c u s . I t was i m p o s s i b l e t o d i f f e r e n t i a t e between t h e l a t t e r two o r g a n i s m s on the b a s i s of f a t t y a c i d p r o d u c t i o n . F a t t y a c i d p r o -d u c t i o n was not c o n s i s t e n t w i t h i n a s u b s p e c i e s , i . e . , some i s o l a t e s p r o d uced i s o b u t y r i c a c i d and/or l a c t i c a c i d whereas o t h e r i s o l a t e s d i d n o t . The p r e s e n c e of p h e n y l a c e t i c a c i d i n c u l t u r e s of 15. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s has n o t been n o t e d p r e v i o u s l y and s h o u l d p r o v i d e a u s e f u l t o o l i n i d e n t i f y i n g t h i s o r g a n i s m i n mixed i n f e c t i o n s . 45 F i g u r e 5. Gas chromatography of n o n - v o l a t i l e f a t t y a c i d s produced by ]3_. m e l a r i i r i b g e r i i c u s . M e t h y l a t e d f a t t y a c i d s were e x t r a c t e d f r o m a c i d i f i e d c u l t u r e s u p e r n a t a n t s . The peaks were i d e n t i f i e d by comparing t h e i r r e t e n t i o n t i m e s w i t h t h o s e of s t a n d a r d s ( F i g u r e I B ) : 1, l a c t i c a c i d ; pa, p h e n y l a c e t i c a c i d ; s, s u c c i n i c a c i d . 10 15 20 MINUTES 25 B 8 12 16 MINUTES 20 TABLE IV SUMMARY OF FATTY ACID PRODUCTION BY B. MELANINOGENICUS Organism Acids"'" Number of I s o l a t e s A B S PA J i . m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s + + + 53 J i . m e l a n i n o g e n i c u s s s . i n t e r m e d i u s o r s s . m e l a n i n o g e n i c u s + + 343 A, a c e t i c a c i d ; B, b u t y r i c a c i d ; S, s u c c i n i c a c i d ; PA, p h e n y l a c e t i c a c i d . 48 2. H e m a g g l u t i n a t i o n (HA) When human r e d b l o o d c e l l s were mixed w i t h I i . m e l a n i n o g e n i c u s s t r a i n K110 t h e y r a p i d l y a g g r e g a t e d i n d i c a t i n g t h a t the o r g a n i s m p o s s e s s e d p o w e r f u l h e m a g g l u t i n a t i n g a c t i v i t y . I t was o f i n t e r e s t t o d e t e r m i n e i f t h i s p r o p e r t y was a s s o c i a t e d w i t h a p a r t i c u l a r s u b s -s p e c i e s of J 3 . m e l a n i n o g e n i c u s . The optimum c o n d i t i o n s f o r a s s e s s i n g HA a c t i v i t y were e s t a b l i s h e d w i t h s t r a i n K110 p r i o r t o e v a l u a t i n g t h e f r e s h i s o l a t e s . The i n -f l u e n c e of c u l t u r e age on HA a c t i v i t y was d e t e r m i n e d . F i v e hundred ml of s t a n d a r d medium was i n o c u l a t e d w i t h a r a p i d l y g r o w i n g c u l t u r e o f 13. m e l a n i n o g e n i c u s and samples were removed a t v a r i o u s times.- B o t h c e l l s and c u l t u r e s u p e r n a t a n t s were a s s a y e d f o r HA a c t i v i t y . The r e s u l t s a r e shown i n F i g u r e 6. The p r o d u c t i o n of c e l l bound HA i n c r e a s e s w i t h c u l t u r e age, r e a c h i n g a maximum a t 24 h o u r s . C e l l f r e e HA does n o t appear u n t i l t h e c u l t u r e i s i n t h e l a t e e x p o n e n t i a l growth phase and c o n t i n u e s t o i n c r e a s e a f t e r g r o w t h has c e a s e d . P r e s u m a b l y t h i s i s due t o l i b e r a t i o n o f c e l l u l a r HA due t o c e l l l y s i s . Red b l o o d c e l l s f r o m a number of a n i m a l s and f r o m d i f f e r e n t human b l o o d t y p e s were t e s t e d i n t h e HA a s s a y . The r e s u l t s a r e shown i n T a b l e V. I n t h i s c a s e HA a c t i v i t y was measured by t h e tube t e s t . F r o g RBC showed t h e s t r o n g e s t a c t i v i t y and g u i n e a p i g the l e a s t , b u t a l l c e l l t y p e s were c a p a b l e o f h e m a g g l u t i n a t i n g w i t h J 3 . m e l a n i n o g e n i c u s . Subsequent s t u d i e s were done w i t h human f o r m a l i n i z e d RBC because t h e y were easy t o p r e p a r e i n l a r g e q u a n t i t i e s . C e l l s o b t a i n e d f r o m c o l o n i e s grown on b l o o d agar or s t a n d a r d medium agar p o s s e s s e d HA a c t i v i t y . 49 F i g u r e 6. I n f l u e n c e o f c u l t u r e age on HA a c t i v i t y . B. m e l a n i n o g e n i c u s s t r a i n K110 was grown i n s t a n d a r d medium. • #, a b s o r b a n c e ; A c e l l bound HA; • • , HA i n s u p e r n a t a n t . 50 TABLE V EFFECT OF THE SOURCE OF RED BLOOD CELLS ON HA ACTIVITY Source of RBC HA A c t i v i t y G u i n e a p i g +2 R a b b i t +3 C h i c k e n +3 Fr o g +4 Hors e +3 Sheep +3 Human A +3 Human 0 +3 Human B +3 F o r m a l i n i z e d Human A +3 F o r m a l i n i z e d Human 0 +3 F o r m a l i n i z e d Sheep +3 F o r m a l i n i z e d F r o g +3 The e f f e c t o f oxygen on HA was d e m o n s t r a t e d i n an e x p e r i m e n t i n w h i c h washed c e l l s and s u p e r n a t a n t f r o m a 48 hour c u l t u r e o f s t r a i n K110 were s u b j e c t e d t o v i g o r o u s a e r a t i o n p r i o r t o t h e a s s a y ( T a b l e V I ) . A l l HA a c t i v i t y was l o s t upon a e r a t i o n , b u t the a c t i v i t y c o u l d be r e s t o r e d by t h e a d d i t i o n of t h e r e d u c i n g agent 3 _ m e r c a p t o e t h a n o l ($ME) (25 mM). HA a c t i v i t y was i n c r e a s e d f o u r f o l d by a d d i t i o n of 3ME t o c o n t r o l c e l l s w h i c h had not been d e l i b e r a t e l y a e r a t e d . C y s t e i n e and t h i o e r y t h r i t o l were e q u a l l y e f f e c t i v e i n r e s t o r i n g HA a c t i v i t y t o a e r a t e d c e l l s . R e d u c i n g a g e n t s had no e f f e c t on t h e HA a c t i v i t y of c e l l s o r s u p e r n a t a n t w h i c h were h a r v e s t e d and a s s a y e d i n t h e a n a e r o b i c chamber. The o p t i m a l pH f o r HA a c t i v i t y was 7.0 but good a c t i v i t y c o u l d be d e m o n s t r a t e d i n t h e range of pH 5.0 t o 8.0. Above and below t h e s e pH v a l u e s t h e b a c t e r i a l c e l l s began t o clump t h u s i n v a l i d a t i n g t h e a s s a y . HA o c c u r r e d a t 4 , 22 and 37 C. On t h e b a s i s of t h e s e r e s u l t s , a l l HA a s s a y s were p e r f o r m e d i n 25 mM 3ME a t pH 7.0 and a t room t e m p e r a t -u r e w i t h c e l l s o b t a i n e d from a 48 hour b r o t h c u l t u r e . The r e s u l t s o b t a i n e d f o r t h e 396 i s o l a t e s a r e shown i n T a b l e V I I . A l l s t r a i n s d e s i g n a t e d a s a c c h a r o l y t i c u s e x h i b i t e d s t r o n g HA a c t i v i t y whereas the o t h e r i s o l a t e s c o m p l e t e l y l a c k e d t h i s p r o p e r t y . The p o s s i b i l i t y t h a t th e n o n ^ h e m a g g l u t i n a t i n g i s o l a t e s r e q u i r e d d i f f e r e n t c o n d i t i o n s was c o n s i d e r e d . A number of t h e s e organisms were t e s t e d w i t h g u i n e a p i g , r a b b i t , f r o g and f r e s h and f o r m a l i n i z e d human RBC b u t no HA was d e t e c t e d . The p r e s e n c e or absence of r e d u c i n g a g e n t s had no e f f e c t on a c t i v i t y , n e i t h e r d i d c h a n g i n g the pH or t e m p e r a t u r e . Thus i t appears t h a t the s u b s p e c i e s a s a c c h a r o l y t i c u s has a u n i q u e s t r u c t u r e on i t s c e l l w a l l w h i c h promotes a t t a c h m e n t t o e r y t h r o c y t e s . TABLE V I EFFECT OF AERATION ON HA ACTIVITY OF STRAIN K110 A e r a t i o n 0ME HA T i t e r C e l l s + - 0 C e l l s + + 10 C e l l s - - 9 C e l l s - + 11 S u p e r n a t a n t + - 1 S u p e r n a t a n t + + 8 S u p e r n a t a n t - - 7 S u p e r n a t a n t - + 10" T i t e r = t h e number o f w e l l s on a m i c r o t i t e r p l a t e w h i c h were HA p o s i t i v e . TABLE V I I HA OF ORAL B. MELANINOGENICUS ISOLATES S t r a i n Number HA P o s i t i v e I s o l a t e s / T o t a l Number C e l l s S u p e r n a t a n t m e l a n i n o g e n i c u s  a s a c c h a r o l y t i c u s 53/53 53/53 m e l a n i n o g e n i c u s  i n t e r m e d i u s o r m e l a n i n o g e n i c u s 0/343 0/343 The m i c r o t i t e r a s s a y system was not a r e l i a b l e method f o r a s s e s s i n g the amount of HA p r e s e n t on t h e c e l l s because of the i n -a b i l i t y t o r e p r o d u c e a c o n s i s t e n t end p o i n t . T h i s may be due t o b a c t e r i a l c e l l c l u m p i n g w h i c h would have t h e e f f e c t of r e d u c i n g the number of organisms c a p a b l e of b i n d i n g t o the RBC. I n d i v i d u a l i s o -l a t e s d i f f e r e d i n t h e i r tendency t o a u t o a g g r e g a t e and t h u s t h e s i m p l e t u b e t e s t was used f o r a s s e s s i n g HA a c t i v i t y . However, t h e HA a c t i v i t y i n t h e c u l t u r e s u p e r n a t a n t s c o u l d be a c c u r a t e l y and r e p r o -d u c i b l y a s s e s s e d by t h e m i c r o t i t e r a s s a y . L a r g e d i f f e r e n c e s were n o t e d i n t h e amount o f HA a c t i v i t y p r e s e n t i n t h e s u p e r n a t a n t s f r o m d i f f e r e n t i s o l a t e s . P o s s i b l y t h e s e d i f f e r e n c e s a r e r e l a t e d t o i n d i v i d u a l v a r -i a t i o n i . i n t h e r a t e o f r e l e a s e o f c e l l w a l l o r o u t e r membrane c o n s t i t -u e n t s . 3. C o l l a g e n a s e B a c t e r o i d e s m e l a n i n o g e n i c u s has been r e p o r t e d t o possess a c e l l bound c o l l a g e n a s e w h i c h i s b e l i e v e d t o be a s s o c i a t e d w i t h p a t h o g e n -i c i t y . I n o r d e r t o d e t e r m i n e i f the c o l l a g e n a s e was a s s o c i a t e d w i t h any p a r t i c u l a r s u b s p e c i e s of JB. m e l a n i n o g e n i c u s , 48-hour c u l t u r e s . w e r e 14 h a r v e s t e d and i n c u b a t e d w i t h C c o l l a g e n . The r e s u l t s of a t y p i c a l a s s a y a r e shown i n F i g u r e 7. J i . m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s p o s s e s s e s a c e l l bound c o l l a g e n a s e . The enzyme i s oxygen s e n s i t i v e and f u l l a c t i v i t y i s e x p r e s s e d o n l y when a r e d u c i n g agent i s i n c o r p -o r a t e d i n t o t h e b u f f e r . The o t h e r J i . m e l a n i n o g e n i c u s s u b s p e c i e s d i d n o t d e m o n s t r a t e any c o l l a g e n o l y t i c a c t i v i t y i n t h e p r e s e n c e o r absence of a r e d u c i n g agent. The p o s s i b i l i t y t h a t the a c e t y l a t e d c o l l a g e n was not a s u i t a b l e s u b s t r a t e was examined by a s s a y i n g the r e l e a s e of h y d r o x y p r o l i n e f r o m u n m o d i f i e d a c i d s o l u b l e f e t a l c a l f s k i n c o l l a g e n 56 F i g u r e 7. _B. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s c o l l a g e n a s e a c t i v i t y . The a s s a y i s d e s c r i b e d i n M a t e r i a l s and Methods and the c o m p l e t e r e a c t i o n m i x t u r e i s shown i n T a b l e I I . ^ , complete r e a c t i o n - m i x t u r e ; 0 0, c o m p l e t e r e a c t i o n m i x t u r e a e r a t e d p r i o r t o a d d i t i o n of r e d u c i n g a g e n t ; # #, no a e r a t i o n and no r e d u c i n g a g e n t ; • a e r a t i o n and no r e d u c i n g a g e n t . 57 f 1200 TIME(hours) and i n s o l u b l e r a t tendon c o l l a g e n . I n n e i t h e r c a s e was t h e r e any e v i d e n c e t h a t the o r g a n i s m s c o u l d a t t a c k t h e s e s u b s t r a t e s . The p r o d u c t i o n o f c o l l a g e n a s e p a r a l l e l e d g r o w t h u n t i l t h e . c u l t u r e was i n t h e s t a t i o n a r y phase ( F i g u r e 8 ) . A t t h i s t i m e t h e r e was a sudden and r a p i d l o s s of c e l l a s s o c i a t e d c o l l a g e n a s e a c t i v i t y ^ t h e r e f o r e c o l l a g e n a s e was a l w a y s a s s a y e d i n c e l l s o b t a i n e d f r o m e x p o n e n t i a l l y g r o w i n g c u l t u r e s . The s p e c i f i c a c t i v i t y of t h e c e l l bound c o l l a g e n a s e v a r i e d w i d e l y among the d i f f e r e n t i s o l a t e s . S p e c i f i c a c t i v i t y i s e x p r e s s e d as the yg of c o l l a g e n s o l u b i l i z e d p e r hour by one ml of c e l l s (AbsorbanceggQ= 1.0). The s p e c i f i c a c t i v i t y of c o l l a g e n a s e p o s i t i v e c u l t u r e s ranged f r o m 85 t o 150 yg/hr/O.D. w i t h the m a j o r i t y of i s o l a t e s c l u s t e r e d a r o u n d 100 yg/hr/O.D. .B. m e l a n i n o g e n i c u s s t r a i n K110 had a s p e c i f i c a c t i v i t y of 112 yg/hr/O.D. 4. Requirement f o r v i t a m i n K The r e q u i r e m e n t f o r v i t a m i n K as a growth f a c t o r was a s s e s s e d i n a l l 396 i s o l a t e s . C e l l s grown i n v i t a m i n K media p o s s e s s adequate amounts of the compound t o s u s t a i n growth t h r o u g h one s u b c u l t u r e i n v i t a m i n K f r e e medium. T h e r e f o r e i t was n e c e s s a r y to f i r s t c u l t u r e t h e i s o l a t e i n l i q u i d medium c o n t a i n i n g no v i t a m i n K and t h e n sub-c u l t u r e t o s i m i l a r medium and medium supplemented w i t h v i t a m i n K. The r e s u l t s d e s c r i b e d i n T a b l e V I I I i n d i c a t e t h a t v i t a m i n K depend-ency was n o t r e l a t e d t o a p a r t i c u l a r s u b s p e c i e s ; t h e r e were as many v i t a m i n K r e q u i r i n g o r g a n i s m s among t h e 15. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s as t h e r e were among th e s a c c h a r o l y t i c s t r a i n s . There was no c o r r e l a t i o n between v i t a m i n K dependency and t h e s o u r c e o f t h e human i s o l a t e s . A v e r y h i g h p r o p o r t i o n of the 15. m e l a n i n o g e n i c u s s s . 5 9 F i g u r e 8. C o l l a g e n a s e a c t i v i t y as a f u n c t i o n of c u l t u r e age. F i v e hundred m l of s t a n d a r d medium was i n o c u l a t e d w i t h a 48 hour c u l t u r e o f 13. m e l a n i n o g e n i c u s s s ; a s a c c h a r o l y t i c u s (A, = 1.0). A t t h e t i m e s i n d i c a t e d , samples were w i t h d r a w n and 660 t h e A,rr. and c o l l a g e n a s e a c t i v i t y measured. ooO 0 #, a b s o r b a n c e (660 nm); 0 0, c o l l a g e n a s e a c t i v i t y . 60 TABLE V I I I VITAMIN K REQUIREMENTS OF B. MELANINOGENICUS ISOLATES G i n g i v a l H e a l t h I s o l a t e s R e q u i r i n g V i t a m i n K (%) Good Poor 13. m e l a n i n o g e n i c u s S a c c h a r o l y t i c s t r a i n s s s . a s a c c h a r o l y t i c u s o f B. m e l a n i n o g e n i c u s C h i l d r e n Age 7-12 + 0 10 Age 7-12 + N.O. 1 15 Age 12-18 + N.O. 13 Age 12-18 + 3 3 2 8 A d u l t s + 0 13 A d u l t s + 10 7 Dogs N.D. 3 N.D. 90 68 None o b t a i n e d 'Represents 1 out o f 3 s t r a i n s 'Not d e t e r m i n e d a s a c c h a r o l y t i c u s s t r a i n s i s o l a t e d f r o m dog g i n g i v a r e q u i r e d v i t a m i n K, i n d i c a t i n g t h a t t h i s e c o s y s t e m i s s e l e c t i v e f o r t h i s p a r t i c u l a r v a r i a n t . 5. P r o t e o l y t i c a c t i v i t y Whole c e l l s and c u l t u r e s u p e r n a t a n t s were a s s a y e d f o r p r o t e a s e a c t i v i t y u s i n g the A z o c o l l and c a s e i n a s s a y s . A l l i s o l a t e s c h a r a c t e r -i z e d as _B. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s produced a c e l l a s s o -c i a t e d p r o t e a s e c a p a b l e o f h y d r o l y z i n g b o t h s u b s t r a t e s . S a c c h a r o -l y t i c i s o l a t e s o f jB. m e l a n i n o g e n i c u s b r o k e down i n t o a group p o s s e s s -i n g a c e l l a s s o c i a t e d p r o t e a s e a c t i v e a g a i n s t b o t h A z o c o l l and c a s e i n and a second group w h i c h was n o t a c t i v e a g a i n s t e i t h e r s u b s t r a t e . There was no c o r r e l a t i o n between p r o t e a s e a c t i v i t y and t h e s o u r c e o f the i s o l a t e s . The p r o t e a s e i s s e n s i t i v e t o oxygen and s h o u l d be a s s a y e d i n t h e p r e s e n c e of a r e d u c i n g agent t o i n s u r e f u l l a c t i v i t y . 6. P a t h o g e n i c i t y The p a t h o g e n i c i t y of t h e _B. m e l a n i n o g e n i c u s i s o l a t e s was a s s e s s e d i n the g u i n e a p i g model system. The o r g a n i s m to be t e s t e d was i n o c u l -a t e d onto b l o o d a g a r , i n c u b a t e d f o r f o u r days and t h e n s c r a p e d f r o m t h e p l a t e and r e s u s p e n d e d i n PBS + t h i o g l y c o l a t e . The o p t i c a l d e n s i t y (660 nm) of t h e m i x t u r e was a d j u s t e d t o 1.0 and a sample p l a t e d on b l o o d a g a r t o d e t e r m i n e i f t h e ; p r e p a r a t i o n was a pure c u l t u r e . As t h e pigmented B a c t e r o i d e s a r e o f t e n dependent on " h e l p e r " o r g a n i s m s t o i n i t i a t e an i n f e c t i o n , a n o n - p a t h o g e n i c m i x t u r e of h e l p e r o r g a n i s m s was d e f i n e d . T h i s m i x t u r e w i l l be d i s c u s s e d l a t e r i n t h e s e c t i o n d e a l i n g w i t h s t u d i e s on t h e c h a r a c t e r i z a t i o n o f mixed i n f e c t i o n s . The s u s p e n s i o n o f B a c t e r o i d e s was mixed w i t h the c o n s o r t i u m o f h e l p e r o r g a n i s m s and i n j e c t e d i n t o t h e g u i n e a p i g . An i s o l a t e was c o n s i d e r e d t o be n o n - p a t h o g e n i c i f i t d i d n o t i n d u c e the f o r m a t i o n o f a l e s i o n w i t h i n 4 weeks. The i s o l a t e was c o n s i d e r e d p a t h o g e n i c i f i t formed a l e s i o n c o n t a i n i n g m a t e r i a l w h i c h was c a p a b l e of i n f e c t i n g a second a n i m a l . The p u r i t y of the i n f e c t i o u s m i x t u r e s was a s s e s s e d by s t a n d -a r d m i c r o b i o l o g i c a l p r o c e d u r e s . The r e s u l t s i n d i c a t e d that" a l l i s o l a t e s c h a r a c t e r i z e d as J i . m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s were i n f e c t i v e i n t h i s a s s a y whereas none o f the o t h e r i s o l a t e s i n -duced f o r m a t i o n o f a l e s i o n . The i n f e c t i v i t y o f p u r e c u l t u r e s o f a s a c c h a r o l y t i c s t r a i n s o f J i . m e l a n i n o g e n i c u s was found t o v a r y i n a n o n - p r e d i c t a b l e f a s h i o n and c o u l d n o t be c o r r e l a t e d w i t h t h e s o u r c e o f t h e i s o l a t e s . The n a t u r e of t h e l e s i o n s was v a r i a b l e r a n g i n g from s m a l l l o c a l i z e d a b s c e s s e s t o r a p i d l y s p r e a d i n g n e c r o t i c i n f e c t i o n s w h i c h k i l l e d t h e a n i m a l w i t h i n 18 t o 24 h o u r s . The l e s i o n s w h i c h formed r a p i d l y e v e n t u a l l y c o v e r e d t h e e n t i r e a b d o m i n a l and t h o r a c i c r e g i o n and i n some c a s e s s p r e a d i n t o t h e neck. The s k i n o v e r l y i n g t h e s e a r e a s was s e p a r a t e d from the u n d e r l y i n g t i s s u e and hung l o o s e l y when t h e a n i m a l s t o o d u p r i g h t ; o f t e n t h e s k i n became d i s c o l o r e d and the h a i r f e l l o u t . G e n e r a l l y s p e a k i n g t h e i s o l a t e s w h i c h e s t a b l i s h e d a v i s i b l e i n f e c t i o n w i t h i n the f i r s t 8 h o u r s produced t h e r a p i d l y s p r e a d i n g f a t a l d i s e a s e . L o c a l i z e d a b s c e s s e s o c c u r r e d i n a n i m a l s i n w h i c h t h e r e was no a p p a r e n t l e s i o n 24 h o u r s a f t e r i n o c u l a t i o n . M a t e r i a l a s p i r a t e d f r o m t h e r a p i d l y s p r e a d i n g l e s i o n s was a t h i n s e r o u s f l u i d w h i c h c o n t a i n e d l a r g e numbers o f b a c t e r i a b u t v e r y few w h i t e b l o o d c e l l s . I n some c a s e s t h e r e were l a r g e numbers of r e d b l o o d c e l l s i n d i c a t i n g t h a t the organisms had i n v a d e d the c i r c u l a t o r y system. L o c a l i z e d a b s c e s s e s were f i l l e d w i t h t h i c k p u s t u l a r m a t e r i a l c o n t a i n i n g l a r g e numbers o f w h i t e b l o o d c e l l s . The m a t e r i a l a s p i r a t e d f r o m a l e s i o n u s u a l l y p r o d u c e d a more r a p i d l y p r o g r e s s i n g d i s e a s e t h a n d i d the o r i g i n a l i n o c u l u m ; i f the m i x t u r e was passaged t h r o u g h a number of a n i m a l s i t i n v a r i a b l y p r o duced a r a p i d l y s p r e a d i n g i n f e c t i o n . The enhancement o f p a t h o g e n i c i t y by s e r i a l passage s u g g e s t s t h a t a t t e n -u a t i o n o f t h e o r g a n i s m (B. m e l a n i n o g e n i c u s ) has o c c u r r e d i n t h e o r a l c a v i t y o r d u r i n g l a b o r a t o r y c u l t i v a t i o n and t h a t under a p p r o p r i a t e c o n d i t i o n s more v i r u l e n t v a r i a n t s can a r i s e . A summary of the c h a r a c t e r i z a t i o n s t u d i e s i s shown i n T a b l e I X . The b l a c k pigmented c o l o n i e s w h i c h a r e p a t h o g e n i c b e l o n g t o t h e sub-s p e c i e s .B. m e l a n i n o g e n i c u s a s a c c h a r o l y t i c u s . P a t h o g e n i c i t y c a n be c o r r e l a t e d w i t h t h e p r e s e n c e of c o l l a g e n a s e and a p o w e r f u l HA a c t i v i t y . The p a t h o g e n i c o r g a n i s m s a r e a l s o m e t a b o l i c a l l y d i s t i n c t p r o d u c i n g b u t y r i c a c i d and an u n u s u a l a r o m a t i c compound, p h e n y l a c e t i c a c i d . There i s no c o r r e l a t i o n between t h e human s o u r c e of the i s o l a t e and i t s p a t h o g e n i c i t y . Dogs appear t o p o s s e s s a h i g h p r o p o r t i o n o f v i r u l e n t o r g a n i s m s . Dependence on v i t a m i n K cannot be c o r r e l a t e d w i t h any p a r t i c u l a r s u b s p e c i e s . Not a l l s a c c h a r o l y t i c s t r a i n s o f J3. m e l a n i n o g e n i c u s p o s s e s s p r o t e o l y t i c a c t i v i t y . C. F l u i d a c c u m u l a t i o n R a p i d l y s p r e a d i n g l e s i o n s a r e c h a r a c t e r i z e d by t h e a c c u m u l a t i o n o f a l a r g e volume of l i q u i d exuded i n t o the p o c k e t s formed by the i n f e c t i n g o r g a n i s m s . The p o s s i b i l i t y t h a t the e x u d a t e r e s u l t e d f r o m the a c t i o n o f a t o x i n was examined i n t h e i l e a l l o o p a s s a y . Whole c e l l s and c u l t u r e s u p e r n a t a n t s were examined. M a c r o m o l e c u l a r c o n s t i t u e n t s i n t h e c u l t u r e s u p e r n a t a n t were c o n c e n t r a t e d 20 t i m e s by u l t r a f i l t r a t i o n (Amicon) on a PM-10 membrane. The p r o c e d u r e c o n c e n t r a t e s m o l e c u l e s l a r g e r t h a n TABLE IX SUMMARY OF B. MELANINOGENICUS CHARACTERIZATION STUDIES C o l l a g e n a s e HA B u t y r i c S u c c i n i c P h e n y l a c e t i c P r o t e a s e V i t a m i n P a t h o g e n i c i t y A c i d A c i d A c i d K J3. m e l a n i n o g e n i c u s + + + + + v l s s . a s a c c h a r o l y t i c u s 13. m e l a n i n o g e n i c u s s s . i n t e r m e d i n s and - - - + - v v s s . m e l a n i n o g e n i c u s "'"variable + 10,000 MW b u t does not a f f e c t the s a l t c o n c e n t r a t i o n . C o n t r o l s c o n s i s t e d o f e i t h e r h e a t k i l l e d c e l l s o r u n i n o c u l a t e d growth medium. The o r i g i n a l e x p e r i m e n t s were done ±a r a b b i t i l e a l l o o p s b u t t h e s e a n i m a l s were d i f -f i c u l t t o work w i t h and subsequent e x p e r i m e n t s were done w i t h m i c e . The r e s u l t s o f e x p e r i m e n t s i n m i c e a r e d e s c r i b e d i n T a b l e X. B o t h whole c e l l s and c u l t u r e s u p e r n a t a n t s c o n t a i n a f a c t o r w h i c h s t i m u l a t e s i n t e s t i n a l c e l l s t o r e l e a s e f l u i d i n t o t h e lumen o f t h e i l e u m . The c h o l e r a - l i k e t o x i n a c t i v i t y was d i f f i c u l t t o s t u d y b e c a u s e o f a n i m a l v a r i a b i l i t y and l a r g e d i f f e r e n c e s i n t o x i c a c t i v i t i e s between c u l t u r e s o f t h e same or g a n i s m . The r e a s o n s f o r t h e v a r i a b i l i t y were n o t e s t a b l i s h e d b u t may be t h e r e s u l t o f a t o x i n w i t h a low l e v e l o f a c t i v i t y . H e a t i n g t h e c e l l s o r c u l t u r e s u p e r n a t a n t a t 90°C f o r 10 m i n u t e s d e s -t r o y e d a c t i v i t y i n d i c a t i n g t h a t t h e t o x i c f a c t o r i s p r o b a b l y p r o t e i n and n o t l i p o p o l y s a c c h a r i d e . The t o x i n may be s e n s i t i v e t o o x i d a t i o n and thus i n a c t i v a t e d i n t h e i l e u m , however, a d d i t i o n o f r e d u c i n g a g e n t s t o t h e i n o c u l u m d i d n o t have any e f f e c t on a c t i v i t y . The f a t e o f r e d u c i n g a g e n t s i n t h e i l e u m was n o t i n v e s t i g a t e d and i t i s p o s s i b l e t h a t t h e y were r a p i d l y d e s t r o y e d and thus u n a v a i l a b l e t o t h e t o x i n . A t t e m p t s t o c u l t u r e _B. m e l a n i n o g e n i c u s f r o m i l e a l l o o p s a t the c o n c l u s i o n o f an e x p e r i m e n t were u n s u c c e s s f u l i n d i c a t i n g t h a t the oxygen t e n s i o n was h i g h enough t o k i l l t h e o r g a n i s m s . I l e a l l o o p s were examined 1, 4, 8, 16, 18, 24 and 36 hours a f t e r i n o c u l a t i o n . B e s t r e s u l t s were o b t a i n e d between 18 and 24 h o u r s . O f t e n a c u l t u r e o r s u p e r n a t a n t would be p o s i t i v e i n 2 o r 3 a n i m a l s and not i n a n o t h e r , t h e r e f o r e a minimum of e i g h t a n i m a l s was t e s t e d w i t h each p r e p a r a t i o n . TABLE X ILEAL LOOP ASSAY Inocu l u m Whole c e l l s J i . m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s P-2 CR2A 2D D-3 V i b r i o c h o l e r a h e a t - k i l l e d P-2 o r 2D Weight of l o o p s (mg)  E x p e r i m e n t a l C o n t r o l 2330 2080 1360 1280 1340 360 340 280 320 620 370 390 S u p e r n a t a n t s P-2 CR2A 2D 520 690 840 560 240 260 h e a t e d s u p e r n a t a n t 280 275 L e n g t h o f l o o p s (cm)  E x p e r i m e n t a l C o n t r o l E x p e r i m e n t a l / C o n t r o l 8.5 9.0 5.0 7.5 6.0 6.0 5.5 5.5 7.0 8.5 5.0 5.5 4.44 4.54 5.95 2.34 2.96 0.846 5.5 5.0 4.5 5.5 3.5 5.0 0.929 2.01 3.59 5.5 5.0 0.926 PART I I . STUDIES ON MIXED INJECTIONS The o b j e c t i v e of t h i s s e c t i o n i s t o d e t a i l t h e s t u d i e s w h i c h shed l i g h t on t h e n a t u r e of t h e mixed i n f e c t i o n . _B. m e l a n i n o g e n i c u s s s . a s a c c h a r o - l y t i c u s s t r a i n K110 was chosen as t h e t e s t o r g a n i s m f o r t h e s e s t u d i e s S t r a i n K110 was f o u n d t o r e q u i r e hemin b u t n o t v i t a m i n K as seen i n F i g u r e 9. The pH o f the medium a f t e r growth was 6.8 w i t h o r w i t h o u t added g l u c o s e . A e s c u l i n and s t a r c h were n o t h y d r o l y z e d . A. I s o l a t i o n o f an i n f e c t i o u s c o n s o r t i u m The s u l c u l a r f l o r a f r o m a p e r i o d o n t a l l e s i o n i n a p a t i e n t w i t h advanced p e r i o d o n t i t i s was i n o c u l a t e d i n t o t h e g r o i n o f a g u i n e a p i g . The f o l l o w i n g day t h e a n i m a l had d e v e l o p e d a l a r g e l e s i o n c o n t a i n i n g a t h i n , s e r o u s , d a r k - c o l o r e d , f o u l s m e l l i n g e x u d a t e . I n o c u l a t i o n o f a second a n i m a l w i t h as l i t t l e as 0.1 ml o f t h i s e x u d a t e produced an i d e n t i c a l r a p i d l y s p r e a d i n g i n f e c t i o n . A u t o p s y of t h e a n i m a l s showed marked t i s s u e n e c r o s i s i n t h e a b d o m i n a l r e g i o n w i t h p e r f o r a t i o n o f the p e r i t o n e a l w a l l i n one of the a n i m a l s . M i c r o s c o p i c a n a l y s i s o f t h e e x u d a t e r e v e a l e d a v a r i e t y o f gram (-) and gram (+) c o c c i and r o d s . The exudate f r o m t h e second a n i m a l was s e r i a l l y d i l u t e d under a n a e r o b i c c o n d i t i o n s , p l a t e d on b l o o d a g a r and i n c u b a t e d a n a e r o b i c a l l y . The p l a t e s -7 -9 i n o c u l a t e d w i t h h i g h d i l u t i o n s (10 t o 10 ) of exud a t e c o n t a i n e d a number of c o l o n y t y p e s i n c l u d i n g some w h i c h were b l a c k pigmented. The b l a c k c o l o n i e s were shown t o be Ji_. m e l a n i n o g e n i c u s ss. a s a c c h a r o l y t i c u s and t h e s t r a i n was d e s i g n a t e d GP 14. A l l c o l o n i e s on the h i g h d i l u t i o n p l a t e s were i s o l a t e d i n pure c u l t u r e . A n a l y s i s of t h e i s o l a t e s showed t h a t t h e r e were f i v e d i f f e r e n t o rganisms w h i c h c o u l d be d i s t i n g u i s h e d on t h e b a s i s of c e l l u l a r morphology and gram s t a i n (see M a t e r i a l s and M e t h o d s ) . A m i x t u r e o f the f i v e o r ganisms was n o t i n f e c t i v e u n l e s s i t was combined 69 F i g u r e 9. Growth of J5. roelaninogenicu s s t r a i n K110 i n the p r e s e n c e and absence of hemin and v i t a m i n K« The B a s a l medium supplemented w i t h e i t h e r hemin (5 x 10 g/1) —6 or v i t a m i n K (1 x 10 g/1) or w i t h b o t h compounds was i n o c u l a t e d w i t h c e l l s o b t a i n e d from growth i n unsupplemented B a s a l medium. In o c u l u m was 1% o f c u l t u r e volume (A,,_ = 1.0). ooU 0 0, hemin supplemented b a s a l medium; • • , hemin p l u s : v i t a m i n K supplemented b a s a l medium; #• #, b a s a l medium; | 1 , v i t a m i n K supplemented b a s a l medium. 10 20 30 40 TIME (hours) 71 w i t h I i . m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s . A l t h o u g h f i v e d i f f e r e n t o r g a n i s m s had been f o u n d i n l a r g e numbers i n t h e i n f e c t e d a n i m a l s i t was n o t known how many were r e q u i r e d t o i n i t i a t e i n f e c t i o n . An e x p e r i m e n t was d e s i g n e d t o e s t a b l i s h the minimum number of s p e c i e s c a p a b l e of i n i t i a t i n g an i n f e c t i o n . The c e l l s were grown on l a k e d b l o o d a g a r p l a t e s u s i n g t h e wheel p l a t e t e c h n i q u e . P l a t e s were i n c u b a t e d f o u r days a t 37°C i n t h e a n a e r o b i c chamber. C e l l s were s c r a p e d f r o m the agar s u r f a c e , r e s u s p e n d e d i n PBS + t h i o g l y c o l a t e and i n j e c t e d i n t o a n i m a l s t o g e t h e r w i t h ]3. m e l a n i n o g e n i c u s s t r a i n K110. The r e s u l t s a r e shown i n T a b l e X I . The e x p e r i m e n t s p r o v e d t h a t i s o l a t e 1-20 was the o n l y o r g a n i s m r e q u i r e d by s t r a i n K110 t o i n i t i a t e an i n f e c t i o n . T h i s h e l p e r o r g a n i s m c o u l d not be r e p l a c e d by any c o m b i n a t i o n of two, t h r e e o r f o u r o f the o t h e r i s o l a t e s . S t r a i n K110 w i l l n o t i n f e c t by i t s e l f when grown under t h e s e c o n d i t i o n s . Organisms n o t r e q u i r e d f o r i n f e c t i v i t y do n o t i n h i b i t t he i n f e c t i o u s p o t e n t i a l o f t h e m i n i m a l i n f e c t i o u s m i x t u r e n o r do t h e y appear t o s t i m u l a t e i n f e c t i v i t y . I n o t h e r words t h e y have no i n f l u e n c e on t h e c o u r s e o f the i n f e c t i o n and c a n be c o n s i d e r e d as s a p r o -p h y t i c c o n t a m i n a n t s . B. E f f e c t o f 1-20 on t h e p a t h o g e n i c i t y o f other B. m e l a n i n o g e n i c u s i s o l a t e s A number o f 13. m e l a n i n o g e n i c u s i s o l a t e s were examined t o d e t e r m i n e i f t h e y were i n f e c t i v e when i n j e c t e d t o g e t h e r w i t h 1-20. The same i n v i v o p r o c e d u r e was used f o r g r o w i n g t h e c e l l s . T a b l e X I I shows the r e s u l t s when s u c h c o m b i n a t i o n s were i n j e c t e d i n t o t h e a n i m a l s . A g a i n , one can o b s e r v e t h a t J3_. m e l a n i n o g e n i c u s i s o l a t e s were n o t i n f e c t i v e u n l e s s t h e y were combined w i t h 1-20. The o r g a n i s m 1-20 was i d e n t i f i e d by a s e r i e s o f b i o c h e m i c a l t e s t s as K l e b s i e l l a pneumoniae ( T a b l e X I I I ) . The r e s u l t s i n d i c a t e d t h a t t h e TABLE X I INFECTIVITY OF VARIOUS BACTERIAL MIXTURES M i x t u r e I n j e c t e d R e s u l t s 20 21 22 23 25 (-) K110 20 21 22 23 25 (+++) o r (++-H-) K110 20 21 22 23 (++) P K110 20 21 22 25 (++) P K110 20 21 23 25 (++) P K110 20 22 23 25 (++) P K l l O 21 22 23 25 (-) K l l O 20 22 23 (+++) P K l l O 20 22 25 (++) P K l l O 20 21 25 (++) P K l l O 20 23 25 (+++) P K l l O 20 21 23 (++) P K l l O 20 21 22 (+) K l l O 23 25 (+) K l l O 20 22 (++) o r (++-»-) K l l O 20 23 (++) o r (++• I-) K l l O 21 25 (+) K l l O 20 21 (++) o r (++-I-) K l l O 21 23 (-) K l l O 20 25 (++) o r (++-»•) K l l O 22 23 (+) K l l O 20 (+++) o r (++• H-) K l l O 21 (+) K l l O 22 (-) K l l O 23 .<+> K l l O 25 (+) K l l O C~) 20 , (") TABLE X I I EFFECT OF ISOLATE 20 ON ORAL B. MELANINOGENICUS ISOLATES C e l l s I n j e c t e d R e s u l t s m o (-) K110 + 20 (+++) P2 (-) P2 + 20 (+++)' DI (-) DI + 20 (++++) D2 (-) D2 + 20 (+++) D3 (-) D3 + 20 (-) GP 14 (-) GP 14 + 20 (+++) 20 (-) i s o l a t e c l o s e l y r e s e m b l e d K. pneumoniae t y p e 3 as d e s c r i b e d by H e n r i k s e n ( 2 4 ) . The g r a m - n e g a t i v e , n o n - s p o r i n g , n o n - m o t i l e r o d produced o n l y s u c c i n i c , l a c t i c and a c e t i c a c i d s when grown a n a e r o b i c a l l y on g l u c o s e . No c o l l a g e n a s e or p r o t e a s e was d e t e c t e d , n o r d i d the o r g a n i s m p o s s e s s a c h o l e r a t o x i n - l i k e a c t i v i t y when a s s a y e d i n t h e i l e a l l o o p a s s a y . C. V i a b i l i t y and i n f e c t i o n The p o s s i b i l i t y t h a t t h e n a t u r e of t h e s y n e r g i c e f f e c t m i ght be the r e s u l t o f i n t e r a c t i o n s n o t r e q u i r i n g b a c t e r i a l growth was i n v e s t i g a t e d w i t h l i v i n g and dead c e l l s . Organisms were h a r v e s t e d f r o m t h e i r r e s p e c t -i v e g r o w t h media by c e n t r i f u g a t i o n and t h e n suspended i n PBS. 15. m e l a n i n o g e n i c u s s t r a i n K110 was k i l l e d by v i g o r o u s l y a e r a t i n g the c e l l s and K. pneumoniae by e x p o s u r e t o CHCl^. I n t h e l a t t e r c a s e CHCl^ was removed by p r o l o n g e d s p a r g i n g w i t h a i r a t 37°C. The e f f e c t i v e n e s s o f t h e k i l l i n g p r o c e d u r e s was a s s e s s e d by p l a t i n g t h e o r g a n i s m s on b l o o d a g a r . T a b l e XIV shows t h e r e s u l t s o b t a i n e d . I t i s c l e a r t h a t b o t h m i c r o o r g a n i s m s had t o be a l i v e t o s u c c e s s f u l l y i n f e c t an a n i m a l . I t was a l s o f ound t h a t a s t e r i l e e xudate p r e p a r e d f r o m an i n f e c t e d g u i n e a p i g d i d n o t cause a b s c e s s f o r m a t i o n . D. Number of c e l l s r e q u i r e d t o i n i t i a t e an i n f e c t i o n H a v i n g d e f i n e d a s i m p l e mixed i n f e c t i o n model, i t was t h e n of i n t e r e s t (a) t o d e t e r m i n e i f t h i s b a s i c s y s t e m c o u l d i n d u c e an i n f e c t i o n u s i n g c e l l s grown i n l i q u i d medium, and (b) t o q u a n t i t a t e , i f p o s s i b l e , the number of organisms r e q u i r e d t o i n i t i a t e t he d i s e a s e . C e l l s o f s t r a i n K110 were grown i n s t a n d a r d medium as d e s c r i b e d e a r l i e r and c u l t u r e s o f K. pneumoniae were grown i n t r y p t i c a s e - s o y b r o t h . Organisms were grown f o r 48 hours and t o t a l numbers d e t e r m i n e d u s i n g a P e t r o f f - H a u s s e r chamber. An a l i q u o t o f each o r g a n i s m was c e n t r i f u g e d and the c e l l s were r e s u s p e n d e d TABLE XIII BIOCHEMICAL CHARACTERISTICS OF 1-20 Gas from glucose at 37°C <+ Nitrate reduced + Carbohydrates (acid from) Adonitol + Arabinpse + Glucose + Glycerol + Inositol + Inulin + Lactose + Raffinose + Rhamnose + Sorbitol + Sucrose + Trehalose + Xylose + Citrate MR • + VP Indole Urea hydrolyzed Hemolysis - (gamma) TABLE X I V CELL VIABILITY AND INFECTION C e l l s I n j e c t e d I n f e c t i o n l i v e K110 + l i v e 20 (+++) l i v e 20 (-) l i v e K110 (-) l i v e K110 + dead 20 (-) dead K110 + l i v e 20 (-) dead K110 + dead 20 (-) Each c e l l m i x t u r e was t e s t e d i n 2 a n i m a l s . 7 7 i n PBS + t h i o g l y c o l a t e t o an A ^ ^ Q of 1 . 0 . They were mixed i n t h e p r o -p o r t i o n s i n d i c a t e d and i n j e c t e d . T a b l e XV shows the r e s u l t s o b t a i n e d . The e x p e r i m e n t i n d i c a t e d t h a t t h e minimum number of b a c t e r i a r e q u i r e d t o p r o d u c e an i n f e c t i o n was a p p r o x i m a t e l y 1 0 ^ . T h i s f i g u r e was o b t a i n e d by c o u n t i n g c e l l s I n a P e t r o f f - H a u s s e r c o u n t i n g chamber and thus r e f l e c t s t o t a l c o u n t s . T o t a l v i a b l e c o u n t s measured by p l a t i n g o r by MPN gave v a l u e s r a n g i n g f r o m 0 . 1 t o 2 0 % o f t h e m i c r o s c o p i c c o u n t . Because o f t h i s i n c o n s i s t e n c y i t was n o t p o s s i b l e t o a c c u r a t e l y a s s e s s t h e number of .B. m e l a n i n o g e n i c u s r e q u i r e d . The most i m p o r t a n t i n f o r m a t i o n o b t a i n e d f r o m t h i s e x p e r i m e n t r e l a t e s t o the n a t u r e of the i n f e c t i o n i n d u c e d by d i f f e r e n t numbers of c e l l s . When s m a l l e r numbers were i n j e c t e d t h e l e s i o n was s l o w t o d e v e l o p and i n v a r i a b l y became l o c a l i z e d , whereas i n j e c t i o n o f l a r g e numbers of c e l l s c aused t h e more a c u t e , r a p i d l y s p r e a d i n g f o r m of t h e d i s e a s e . E. Age of c e l l s and g r o w t h The age of IS_. m e l a n i n o g e n i c u s c u l t u r e s has been shown t o a f f e c t a number of i t s p r o p e r t i e s . For example, c e l l f r e e HA a c t i v i t y i n c r e a s e s d u r i n g the s t a t i o n a r y growth phase and c o l l a g e n a s e a c t i v i t y d e c r e a s e s . I t was of i n t e r e s t t o d e t e r m i n e i f the age of the c e l l s i n an i n o c u l u m i n f l u e n c e d subsequent growth ..in v i t r o . F i g u r e 1 0 shows the i n f l u e n c e of the age o f t h e i n o c u l u m on the g r o w t h of s t r a i n K l l O and K. pneumoniae. S t a n d a r d b r o t h was i n o c u l a t e d w i t h a 4 8 hour c u l t u r e o f J3_. m e l a n i n o g e n i c u s and i n c u b a t e d a t 3 7 ° . Samples were removed 1 2 , 2 4 and 4 8 hours a f t e r i n o c u l a t i o n and s u b c u l t u r e d i n t o f r e s h medium. The p r o c e d u r e f o r K l e b s i e l l a was the same w i t h t h e e x c e p t i o n t h a t o rganisms were grown i n t r y p t i c a s e soy b r o t h . The 1 2 , 2 4 and 4 8 hour i n o c u l a were s t a n d a r d i z e d w i t h r e g a r d t o o p t i c a l d e n s i t y 6 6 0 and volume. The r e s u l t s i n d i c a t e t h a t TABLE XV NUMBER OF CELLS REQUIRED TO INDUCE AN INFECTION Number of C e l l s I n j e c t e d x 1 0 ~ 6 . 1 I n f e c t i o n K110 + 20 5 5 (-) 10 10 (±) 600 600 (±) 750 750 (+) 1250 1250 (+) 2000 2000 (++) or (+++) , C e l l s were i n o c u l a t e d i n a volume of 0.5 m l . The d a t a r e p r e s e n t s the r e s u l t s f r o m f o u r s e p a r a t e a n i m a l s . 79 F i g u r e 10. E f f e c t o f i n o c u l u m age on growth. B. -melaninogenicus s t r a i n K110 was grown i n s t a n d a r d medium. K l e b s i e l l a pneumoniae was grown i n t r y p t i c a s e soy b r o t h . Ten m l of b r o t h was i n o c u l a t e d w i t h 0.2 m l of c e l l s ( k r r r . = 0.1). 660 A, !B. m e l a n i n o g e n i c u s : 1, 48 h r ; 2, 24 h r ; 3, 12 h r . B, K. pneumoniae: 1, 12 h r ; 2, 24 h r ; 3, 48 h r . K. pneumoniae behaves i n a manner t y p i c a l o f most or g a n i s m s i n t h a t the l o n g e r the i n o c u l u m has been i n s t a t i o n a r y phase t h e l o n g e r t h e l a g p e r i o d p r e c e d i n g r a p i d g r o wth. The o p p o s i t e s i t u a t i o n was o b s e r v e d w i t h s t r a i n K110 w h i c h had t h e s h o r t e s t l a g p e r i o d when t h e i n o c u l u m had been t a k e n f r o m an o l d c u l t u r e . P o s s i b l y t h e c e l l s a c c u m u l a t e a r e q u i r e d n u t r i e n t d u r i n g s t a t i o n a r y phase w h i c h s t i m u l a t e s growth i n a new e n v i r o n m e n t . A number of " i n v i v o " e x p e r i m e n t s d e m o n s t r a t e d t h a t c e l l s f r o m a 48 h o u r c u l t u r e o f s t r a i n K110 a r e more i n f e c t i o u s t h a n younger c e l l s . F. Numbers o f K. pneumoniae The e f f e c t o f v a r y i n g t h e p r o p o r t i o n s o f c e l l s i n t h e i n f e c t i o u s m i x t u r e was i n v e s t i g a t e d t o d e t e r m i n e optimum c o n c e n t r a t i o n s and t h r e s h -o l d v a l u e s . C e l l numbers were d e t e r m i n e d by m i c r o s c o p i c c o u n t and a r e not i n t e n d e d t o r e p r e s e n t t h e number of v i a b l e c e l l s . The e x p e r i m e n t was p e r f o r m e d w i t h two d i f f e r e n t c u l t u r e s o f IS. m e l a n i n o g e n i c u s , one o f w h i c h appeared t o be more i n f e c t i v e t h a n t h e o t h e r . The r e s u l t s o u t l i n e d i n T a b l e XVI i n d i c a t e t h a t an i n f e c t i o u s m i x t u r e must c o n t a i n a c e r t a i n minimum number of B a c t e r o i d e s and t h a t below t h i s number i n c r e a s i n g t h e number of K l e b s i e l l a has no e f f e c t , on i n f e c t i o n . I n t h e e x p e r i m e n t s d e s c r i b e d h e r e t h e r e was no i n f e c t i o n when 8 x 10^ B a c t e r o i d e s were i n j e c t e d r e g a r d l e s s o f the number of K l e b s i e l l a p r e s e n t i n t h e m i x t u r e . The minimum number of B a c t e r o i d e s n e c e s s a r y t o i n i t i a t e a d i s e a s e p r o c e s s 8 8 was 10 or g a n i s m s b u t o n l y when combined w i t h 10 K l e b s i e l l a . Any r e d u c t -i o n i n the number of K l e b s i e l l a y i e l d e d a n o n - i n f e c t i o u s m i x t u r e . The i n f e c t i o n became more s e v e r e as t h e number of K l e b s i e l l a was i n c r e a s e d 8 8 f r o m 1 x 10 t o 9 x 10 . I n c r e a s i n g the number of K l e b s i e l l a beyond 9 1 x 10 d i d n o t i n c r e a s e t h e p a t h o g e n i c i t y of the m i x t u r e . There was TABLE XVI EFFECT OF VARYING THE RELATIVE PROPORTION OF STRAIN K l l O  AND KLEBSIELLA ON THE COURSE OF INFECTION C e l l s I n j e c t e d x 10 I n f e c t i o n K l l O + 20 9 1 (++++) E x p e r i m e n t 1 7. 5 5 2. 5 5 (++++) (++++) 2. 5 7. 5 (+++) 1 9 (+) 0 10 (-) E x p e r i m e n t 2 1 10 (++) 1 9 (++) 1 7. 5 (+) 1 5. 0 (+) 1 1 (±) 1 0. 5 (-) 0. 8 10 (-) 0. 8 5 (-) 0. 8 1 (-) 0. 8 0. 5 (-) C e l l s were i n j e c t e d i n a volume of 0.5 m l . Each c e l l c o n c e n t r a t i o n was t e s t e d i n a minimum of f o u r a n i m a l s . 83 o f t e n a c o n s i d e r a b l e d i f f e r e n c e i n t h e p a t h o g e n i c i t y of d i f f e r e n t c u l t u r e s and t h u s i t i s d i f f i c u l t t o compare the r e s u l t s o f one e x p e r i m e n t w i t h a n o t h e r . L a r g e d i f f e r e n c e s i n t h e r e l a t i v e p r o p o r t i o n s o f t h e two s p e c i e s do n o t i n f l u e n c e i n f e c t i v i t y p r o v i d e d t h e r e i s a c e r t a i n minimum number of B a c t e r o i d e s p r e s e n t . PART . I l l . NATURE OF THE MICROBIAL INTERDEPENDENCE The s t u d i e s d e s c r i b e d i n t h e p r e v i o u s s e c t i o n d e f i n e d an i n f e c t i o u s m i x -t u r e r e q u i r i n g J i . m e l a n i n o g e n i c u s and one a d d i t i o n a l o r g a n i s m . The c h a r a c t e r of the d i s e a s e and the numbers o f o r g a n i s m s r e q u i r e d t o i n i t i a t e an i n f e c t i o n have been d e t e r m i n e d but t h e r e i s no i n d i c a t i o n o f why a second o r g a n i s m i s r e q u i r e d . Two p o s s i b i l i t i e s a r e : (a) 13. m e l a n i n o g e n i c u s r e q u i r e s a n u t r i e n t s y n t h e s i z e d by t h e second o r g a n i s m w h i c h i s n o t a v a i l a b l e i n t h e e c osystem o r (b) t h e second o r g a n i s m p r o v i d e s a p a t h o g e n i c component needed t o complement t h e p a t h o g e n i c armament of t h e B a c t e r o i d e s . E v i d e n c e f o r a n u t r i t i o n a l dependency i s p r o v i d e d i n t h e e x p e r i m e n t s d e s c r i b e d i n t h e f o l l o w i n g s e c t i o n s . A. E v i d e n c e f o r n u t r i t i o n a l dependence 1. Growth s t i m u l a t i o n on a g a r B a s a l a g a r medium c o n t a i n i n g g l u c o s e (0.25%) and i n some ca s e s - 3 hemin (5.0 x 10 g/1) was i n o c u l a t e d w i t h a 48 hour c u l t u r e of hemin d e p l e t e d jB. m e l a n i n o g e n i c u s s t r a i n K110 and f i v e o t h e r o r g a n i s m s . The B a c t e r o i d e s were s p r e a d e v e n l y o v e r t h e a g a r s u r f a c e i n o r d e r t o form a lawn. The o t h e r b a c t e r i a l s p e c i e s were i n o c u l a t e d as d i s c r e t e -s p o t s i n d e s i g n a t e d a r e a s of the p l a t e . The agar was f r e s h l y p r e p a r e d or had been s t o r e d under a n a e r o b i c c o n d i t i o n s . The r e s u l t s of the e x p e r i m e n t can be seen i n F i g u r e 11. B a c t e r o i d e s formed s a t e l l i t e c o l o n i e s on h e m i n - f r e e media around A. v i s c o s u s , K. pneumoniae and 84 F i g u r e 11. Growth s t i m u l a t i o n on a g a r . B a s a l medium supplemented w i t h g l u c o s e (0,25%) and b a s a l medium supplemented w i t h hemin (5 x 10 g/1) and g l u c o s e (0.25%) was i n o c u l a t e d w i t h Ti_. m e l a n i n o g e n i c u s s t r a i n K l l O and f i v e o t h e r o r g a n i s m s . S t r a i n K l l O was s p r e a d e v e n l y o v e r t h e s u r f a c e o f t h e p l a t e . The^ o t h e r b a c t e r i a were i n o c u l a t e d as d i s c r e t e s p o t s . The p l a t e s were i n c u b a t e d a n a e r o b i c a l l y f o r seven days a t 37°. 1, A. v i s c o s u s ; 2, K. pneumoniae; 3, S^ . a l b u s ; 4, F u s o b a c t e r i u m ; 5, _E. c o l l . 0 0 Uv-86 E . c o l i b u t n o t i n t h e a r e a o f t h e jp_. a l b u s o r F u s o b a c t e r i u m . Media c o n t a i n i n g hemin s u p p o r t e d the growth of B a c t e r o i d e s and no dependency on t h e o t h e r organisms was e v i d e n t . The h i s t o r y o f the i n o c u l u m was i m p o r t a n t ; b e s t r e s u l t s were o b t a i n e d when 13. m e l a n i n o g e n i c u s had been c u l t u r e d i n h e m i n - f r e e medium p r i o r t o p l a t i n g . I t was a l s o i m p o r t a n t n o t t o use a heavy i n o c u l u m because t h e r e was a t e n d e n c y t o d e v e l o p a background of " i n d e p e n d e n t " c o l o n i e s due e i t h e r t o c r o s s f e e d i n g o r c r y p t i c g r o w th. The 13. m e l a n i n o g e n i c u s c o l o n i e s were s l o w t o appear presumably because t h e r e was a l a g p e r i o d c r e a t e d by t h e s l o w d i f f u s i o n of t h e growth f a c t o r f r o m t h e c o l o n y i n t o t h e a g a r . A l t h o u g h t h i s e x p e r i m e n t d i d n o t show t h a t the f a c t o r produced by K. pneumoniae was hemin, i t p o i n t e d o u t t h e n u t r i t i o n a l dependency of 13. m e l a n i n o g e n i c u s on K. pneumoniae. 2. Growth s t i m u l a t i o n i n l i q u i d media. I n o r d e r t o i d e n t i f y the growth f a c t o r i t was n e c e s s a r y t o f o r m -u l a t e a l i q u i d medium w h i c h c o u l d : (a) s u p p o r t the growth of b o t h organisms when s p e c i f i c components were added; (b) d u p l i c a t e the p l a t e phenomenon; and (c) s e r v e f o r subsequent a n a l y s i s of the growth f a c t o r ( s ) . What was r e q u i r e d was a medium i n w h i c h s t r a i n K110 d i d n o t grow i n the absence of hemin b u t w h i c h a l l o w e d growth of the K l e b s i e l l a . S e v e r a l p o s s i b i l i t i e s were t e s t e d . T a b l e X V I I d i s p l a y s t h e r e s u l t s o b t a i n e d when s t r a i n K110 and K. pneumoniae were grown a n a e r o b i c a l l y i n b a s a l medium i n the p r e s e n c e or absence of hemin and g l u c o s e . A number of p o i n t s a r e a p p a r e n t f r o m t h i s T a b l e . K l e b s i e l l a r e q u i r e s an exogenous s u p p l y of g l u c o s e t o s u p p o r t a n a e r o b i c growth; B a c t e r o i d e s w i l l grow o n l y when hemin i s p r e s e n t . A hemin c o n c e n t r a t i o n TABLE X V I I EFFECT OF GLUCOSE AND HEMIN CONCENTRATION ON THE GROWTH  OF PURE CULTURES OF B. MELANINOGENICUS STRAIN K l l O AND K. PNEUMONIAE Medium^ Supplement Absorbance (660 nm) S t r a i n K l l O K. Pneumoniae Hemin (g/1) G l u c o s e (g/1) 24 h r s . 48 h r s . 24 h r s . 48 h r s . 1 5.0 x 1 0 ~ 4 5.0 .08 .23 .73 .80 2 2.5 x 1 0 " 3 5.0 .12 .44 .75 .85 3 5.0 x 1 0 ~ 3 5.0 .14 1.22 .74 .82 4 5.0 x 1 0 " 4 0 .16 .49 .15 .13 5 2.5 x 1 0 ~ 3 0 .24 .86 .16 .15 6 5.0 x 1 0 ~ 3 0 .96 1.16 .16 .15 7 0 2.5 .08 .30 .78 .77 8 5.0 x 1 0 ~ 4 2.5 .14 .45 .81 .78 9 2.5 x 1 0 ~ 3 2.5 .19 .72 .84 .79 10 5.0 x 1 0 ~ 3 2.5 .44 1.55 .85 .78 11 0 1 .15 .47 .52 .38 12 5.0 x 1 0 ~ 3 1 .92 .97 .52 .39 B a s a l medium was supplemented w i t h g l u c o s e and hemin as i n d i c a t e d and s t o r e d a n a e r o b i c a l l y f o r 24 h o u r s p r i o r t o i n o c u l a t i o n . I n o c u l u m 0.1 ml p e r 10 m l of c u l t u r e medium, ab s o r b a n c e (660) 1.0. S t r a i n K l l O i n o c u l u m was o b t a i n e d f r o m c e l l s grown i n s t a n d a r d b r o t h - a n d K:; ..pneumoniae from c e l l s grown i n t r y p t i c a s e soy b r o t h . 88 -3 o f 5 x 10 g/1 i s t h e minimum c o n c e n t r a t i o n r e q u i r e d t o i n i t i a t e r a p i d g r o w th. The background growth of B a c t e r o i d e s i n c u l t u r e s con-t a i n i n g no hemin r e s u l t e d f r o m u s i n g an i n o c u l u m w h i c h had n o t been c u l t u r e d i n h e m i n - f r e e medium^ a p p a r e n t l y endogenous r e s e r v e s a r e s u f f i c i e n t t o s u p p o r t a low r a t e o f growth. Growth o f K l e b s i e l l a i s c o m p l e t e w i t h i n 24 h o u r s . F i n a l l y , i t i s i n t e r e s t i n g t o n o t e t h a t growth o f s t r a i n K l l O was r e t a r d e d by g l u c o s e . The e f f e c t o f t h e s o u r c e o f t h e i n o c u l u m on t h e growth of s t r a i n K l l O i n h e m i n - f r e e medium i s shown i n F i g u r e 12. C e l l s o b t a i n e d f r o m growth i n hemin-f r e e media were u n a b l e t o grow when s u b c u l t u r e d i n t o t h e same medium, whereas c e l l s o b t a i n e d f r o m t h e s t a n d a r d (hemin c o n t a i n i n g ) medium were a b l e t o grow b ut a t a much reduced r a t e . The i m p o r t a n t f a c t o b t a i n e d f r o m t h i s e x p e r i m e n t i s t h a t a s i n g l e passage t h r o u g h hemin f r e e medium i s s u f f i c i e n t t o e x h a u s t endogenous s u p p l i e s of the growth f a c t o r thus y i e l d i n g an i n o c u l u m w h i c h i s t o t a l l y dependent on an exogenous s u p p l y o f the f a c t o r . T h i s conf i r m e d the a s s u m p t i o n t h a t s t r a i n K l l O grew i n the p r e v i o u s e x p e r i m e n t s because o f i t s endogenous s u p p l i e s and n o t bec a u s e o f t h e p r e s e n c e o f g l u c o s e . B. C o - c u l t i v a t i o n o f B. m e l a n i n o g e n i c u s and K. pneumoniae The i n f l u e n c e of K l e b s i e l l a on the growth of the B a c t e r o i d e s was i n v e s t i g a t e d i n t h e g l u c o s e supplemented b a s a l medium. Both o r g a n i s m s were i n o c u l a t e d i n t o a n a e r o b i c medium a t the same t i m e and growth was measured. The B a c t e r o i d e s i n o c u l u m was grown i n h e m i n - f r e e medium f o r 48 h o u r s and t h e K l e b s i e l l a i n o c u l u m was t a k e n f r o m a 12 hour c u l t u r e grown i n t r y p t i c a s e soy b r o t h ( b o t h c u l t u r e s were a d j u s t e d t o an of 1.0). The r e s u l t s a r e shown i n F i g u r e 13. There was no growth i n t h e medium i n o c u l a t e d w i t h B a c t e r o i d e s . The k-f.,_:n of t h e medium i n o c u l a t e d 89 Figure 12. Effect of inoculum origin on the growth of strain KllO. Basal medium supplemented with glucose (0.1%) (A&B) and basal medium supplemented with glucose (0.1%) and hemin (5 x 10 g/1) (c) were inoculated with strain KllO. # ft inoculum grown in standard medium or basal medium; A. A, inoculum grown in standard medium; | 1 , inoculum grown in basal medium. 90 10 20 30 40 50 TIME(hours) 91 F i g u r e 13. E f f e c t of K. pneumoniae on growth of s t r a i n K110 i n l i q u i d media. K. pneumoniae and hemin d e p l e t e d s t r a i n K110 were i n o c u l a t e d i n t o b a s a l medium supplemented w i t h g l u c o s e ( 0 . 1 % ) . A, s t r a i n K110; B, K. pneumoniae; C, s t r a i n K110 and K. pneumoniae. 92 93 w i t h K l e b s i e l l a i n c r e a s e d t o a p p r o x i m a t e l y 0.48 w i t h i n 12 h o u r s and t h e n remained s t a t i o n a r y . I n t h e l a t t e r c a s e , growth was l i m i t e d by the amount of g l u c o s e a v a i l a b l e s i n c e g l u c o s e c o u l d n o t be d e t e c t e d i n t h e medium when t h e c e l l s r e a c h e d s t a t i o n a r y phase. A d i p h a s i c growth c u r v e was n o t e d i n medium i n o c u l a t e d w i t h b o t h o r g a n i s m s , t h e i n i t i a l i n c r e a s e i n a b s o r b a n c e c o r r e s p o n d e d t o t h e growth of K. pneumoniae. The second growth phase was shown t o r e s u l t f r o m the growth of B_. m e l a n i n o g e n i c u s s i n c e two enzyme a c t i v i t i e s a s s o c i a t e d w i t h s t r a i n K110 ( c o l l a g e n a s e and p r o t e a s e ) appeared i n t h e second h a l f o f t h e d i p h a s i c c u r v e . I n a d d i t i o n and co n c o -m i t a n t l y , t h e f a t t y a c i d s and p l a t e c o u n t s i n d i c a t e d t h a t 13. m e l a n i n o g e n i c u s was g r o w i n g . P r i o r t o t h e second growth phase i t was n o t p o s s i b l e t o d e t e c t b u t y r i c o r p h e n y l a c e t i c a c i d , b o t h of w h i c h a r e produced d u r i n g g r o w t h of ]3. m e l a n i n o g e n i c u s . A g l u c o s e c o n c e n t r a t i o n o f 0.1% was chosen because h i g h e r c o n c e n t r a t i o n s d i d n o t c r e a t e a medium w h i c h s u p p o r t e d the growth of s t r a i n K110. O t h e r e x p e r i m e n t s have d e t e r m i n e d t h a t t h e f a i l u r e t o s t i m u l a t e growth was due a t l e a s t i n p a r t t o t h e p r o d u c t i o n o f enough a c i d t o r e d u c e t h e pH t o a l e v e l w h i c h i n h i b i t e d t h e growth of s t r a i n K110. The c o - c u l t i v a t i o n e x p e r i m e n t i s i m p o r t a n t because i t most c l o s e l y p a r a l l e l s t h e s i t u a t i o n w h i c h o c c u r s in. v i v o d u r i n g a mixed i n f e c t i o n . The e x p e r i m e n t p r o v e s t h a t K l e b s i e l l a s u p p o r t s the growth of B a c t e r o i d e s i n a n u t r i e n t d e f i c i e n t e n v i r o n m e n t . The p o s s i b i l i t y t h a t i t c r e a t e s a s u i t a b l e r e d o x p o t e n t i a l i s e l i m i n a t e d because growth t o o k p l a c e i n an a n a e r o b i c environment i n r e d u c e d media. 94 C. Growth o f s t r a i n K l l O i n K. priemoniae c u l t u r e f i l t r a t e The n u t r i t i o n a l dependency of the B_. m e l a n i n o g e n i c u s on t h e K l e b s i e l l a c o u l d a l s o be d e m o n s t r a t e d when a h e m i n - d e p l e t e d s t r a i n K l l O was added t o a s t e r i l e c u l t u r e f i l t r a t e f r o m K. pneumoniae. I n t h i s e x p e r i m e n t , K l e b s i e l l a was grown i n g l u c o s e (0.10%) supplemented b a s a l medium and a t v a r i o u s t i m e s a f t e r i n o c u l a t i o n a 10 ml a l i q u o t of the c u l t u r e was removed and f i l t e r s t e r i l i z e d . The f i l t r a t e was t h e n i n o c u l a t e d w i t h s t r a i n K l l O and g rowth was measured a f t e r 48 h o u r s ( T a b l e X V I I I ) . A l l t h e s t e p s i n t h i s t y p e of e x p e r i m e n t were done a n a e r o b i c a l l y . The r e s u l t s c o n f i r m e d t h e e v i d e n c e t h a t K l e b s i e l l a produced a growth s t i m u l a t o r y f a c t o r f o r s t r a i n K l l O and t h a t the f a c t o r was produced as e a r l y as mid-l o g a r i t h m i c growth phase. A s i m i l a r e x p e r i m e n t u s i n g K. pneumoniae as i n o c u l u m and s t r a i n K l l O c u l t u r e f i l t r a t e showed t h a t g r o w t h of t h e f a c u l t a t i v e o r g a n i s m was n o t enhanced by s t r a i n K l l O . D. I d e n t i f i c a t i o n of t h e growth f a c t o r 1. P r o p e r t i e s V a r i o u s t e s t s were p e r f o r m e d t o c l a r i f y t he i d e n t i t y o f the unknown n u t r i t i o n a l f a c t o r . The compound was s t a b l e i n d e f i n i t e l y a t 37°C, s t a b l e t o a u t o c l a v i n g , n o t o x y g e n - s e n s i t i v e and d i a l y s a b l e . I t p a s s e d t h r o u g h a TJM-05 membrane i n d i c a t i n g a m o l e c u l a r w e i g h t of l e s s t h a n 500. B a t c h t r e a t m e n t o f the c o n c e n t r a t e d f i l t r a t e w i t h D o w e x - l - C l i n d i c a t e d t h a t t h e compound was n e g a t i v e l y c h a r g e d . 2. M e t a b o l i t e a n a l y s i s A t t e m p t s t o e x t r a c t hemin o r p o r p h y r i n - l i k e m o l e c u l e s from s t e r i l e c u l t u r e f i l t r a t e s o f K. pneumoniae were u n s u c c e s s f u l . S i m i l a r l y , i t was n o t p o s s i b l e t o r e p l a c e the growth f a c t o r by s u p p l e m e n t i n g the b a s a l medium w i t h i r o n c h e l a t i n g m o l e c u l e s s u c h as D e s f e r a l , TABLE X V I I I GROWTH OF STRAIN K110 IN K. PNEUMONIAE FILTRATE P r e g r o w t h of K. pneumoniae g l u c o s e supplemented (0.1%) b a s a l medium Time ( h r s ) Abs o r b a n c e ( 6 6 0 nm) pH 0 0.02 7.0 7 .22 6.8 9.5 .465 6.4 24 .56 6.2 50 .49 6.0 Growth of B. m e l a n i n o g e n i c u s 24 h r s 48 h r s . 0.02 0.02 .15 0.28 .86 1.31 .42 1.01 .43 0.92 r h o d o t o r u l i c a c i d , e n t e r o b a c t i n and f e r r i c h o m e i n v a r i o u s c o n c e n t -r a t i o n s (100, 10 and 1 y g / m l ) . These r e s u l t s a r e i n agreement w i t h t h e f a c t t h a t t h e s e t y p e s of compounds a r e n o t s y n t h e s i z e d under a n a e r o b i c c o n d i t i o n s . S i n c e t h e f a c t o r s u p p l i e d by K. pneumoniae was n o t hemin o r a p o r p h y r i n d e r i v a t i v e and p r e sumably n o t a p r o t e i n ( b o i l i n g and a u t o -c l a v i n g r e s i s t a n t ) i t was d e c i d e d t o a n a l y z e the c u l t u r e s u p e r n a t a n t f o r m e t a b o l i c end p r o d u c t s . 3. P r o d u c t i o n o f s u c c i n a t e K l e b s i e l l a was grown i n g l u c o s e supplemented (0.1%) b a s a l medium and the c u l t u r e s u p e r n a t a n t was a n a l y z e d f o r the p r e s e n c e o f v o l a t i l e and n o n - v o l a t i l e f a t t y a c i d s . K. pneumoniae was found t o produce s m a l l amounts of a c e t i c and l a c t i c a c i d and l a r g e amounts of s u c c i n i c a c i d . T h i s was of p a r t i c u l a r i n t e r e s t because of the known s t i m u l a t o r y e f f e c t of s u c c i n a t e on t h e growth of the pigmented rumen B a c t e r o i d e s . The b i o s y n t h e s i s of s u c c i n a t e by K. pneumoniae d u r i n g a n a e r o b i c growth i s d e s c r i b e d i n F i g u r e 14. A 500 ml g l u c o s e supplemented (0.1%) b a s a l medium was made a n a e r o b i c by i n c u b a t i n g o v e r n i g h t i n t h e a n a e r o b i c chamber and was t h e n i n o c u l a t e d w i t h K l e b s i e l l a . Samples were w i t h d r a w n a t the t i m e s i n d i c a t e d and a s s a y e d f o r growth and s u c c i n i c a c i d . A t t h e o n s e t o f t h e s t a t i o n a r y g r o w t h phase t h e s u c c i n i c a c i d c o n c e n t r a t i o n was a p p r o x i m a t e l y 600 yg/ml; m e t a b o l i s m c o n t i n u e d i n the absence of an i n c r e a s e i n c e l l numbers and by 48 h o u r s the s u c c i n i c a c i d c o n c e n t r a t i o n had r e a c h e d 919 yg/ml. These r e s u l t s s u p p o r t e d t h e i d e a t h a t s u c c i n i c a c i d was a p o t e n t i a l growth f a c t o r . 97 F i g u r e 14. Growth and s u c c i n i c a c i d p r o d u c t i o n i n c u l t u r e s of K. pneumoniae. 5 0 0 m l of b a s a l medium supplemented w i t h g l u c o s e (0.1%) was i n o c u l a t e d w i t h 5 m l of a 48 hour c u l t u r e o f K. pneumoniae (A,^_ = 1.0) and i n c u b a t e d a n a e r o b i c a l l y a t 37°. • O A,,„; 660 660 0 0, s u c c i n i c a c i d . 98 l O O O L 8 16 24 32 40 48 TIME (hours) 99 4. Growth s t i m u l a t i o n by s u c c i n i c a c i d The p o s s i b i l i t y t h a t s u c c i n i c a c i d c o u l d r e p l a c e hemin as a growth f a c t o r was examined by i n o c u l a t i n g a s u c c i n a t e s u p p lemented, hemin-f r e e b a s a l medium w i t h a hemin d e p l e t e d i n o c u l u m of J3. m e l a n i n o g e n i c u s s t r a i n K l l O . The r e s u l t s o f . t h e e x p e r i m e n t a r e shown i n F i g u r e 15. I t i s c l e a r t h a t s u c c i n i c a c i d can r e p l a c e hemin a l t h o u g h the amount of s u c c i n i c a c i d r e q u i r e d f o r s t r a i n K l l O t o r e a c h maximum o p t i c a l d e n s i t y i s h i g h . I t i s i n t e r e s t i n g t o n o t e t h a t when hemin was p r e s e n t i n t h e s u c c i n a t e supplemented medium, t h e growth r e s p o n s e was t h e same r e g a r d l e s s of the s u c c i n a t e c o n c e n t r a t i o n . Thus, s u c c i n a t e d i d n o t have an a d d i t i v e e f f e c t when combined w i t h hemin. The co n -c e n t r a t i o n o f s u c c i n a t e r e q u i r e d f o r maximal growth r e l a t i v e t o t h e c o n c e n t r a t i o n o f hemin i s v e r y h i g h s u g g e s t i n g t h a t s u c c i n a t e i s used as an energy s o u r c e r a t h e r t h a n as a p r e c u r s o r f o r heme b i o s y n t h e s i s . The growth r e s p o n s e t o s u c c i n a t e i s not l i n e a r , t h e r e i s a t h r e s h o l d v a l u e below w h i c h v e r y l i t t l e g r o w t h o c c u r s . F i g u r e 16 d i s p l a y s t h e growth c h a r a c t e r i s t i c s o f s t r a i n s K l l O i n t h e same media s e e n i n t h e p r e v i o u s f i g u r e . The growth r a t e i n t h e p r e s e n c e o f 750, 850 and 1000 yg/ml of s u c c i n a t e o r hemin i s i d e n t i c a l . However, i n a l l t h e s u c c i n a t e c o n c e n t r a t i o n s a s s a y e d , growth was p r e c e d e d by a l o n g e r l a g p e r i o d t h a n was o b s e r v e d i n t h e p r e s e n c e o f hemin. The l a g p e r i o d of 10 t o 15 h o u r s was a l s o found when mixed c u l t u r e s o f t h e two o r g a n i s m s were grown i n g l u c o s e - s u p p l e m e n t e d medium (see F i g u r e 1 3 ) . The e f f e c t o f pH on growth of s t r a i n K l l O i n s u c c i n a t e supplemented b a s a l medium was d e t e r m i n e d . F i g u r e 17 shows t h e r e s u l t s o b t a i n e d when the pH o f t h e medium was v a r i e d f r o m 5.5 t o 7.5. The maximum growth u n d e r t h e s e c o n d i t i o n s (pH 6.5) c o r r e s p o n d e d t o t h e pH o f t h e 100 F i g u r e 15. Growth r e s p o n s e o f s t r a i n K110 i n s u c c i n a t e supplemented b a s a l medium. -3 B a s a l medium supplemented w i t h hemin (5 x 10 g/1) and b a s a l medium supplemented w i t h s u c c i n a t e was i n o c u l a t e d w i t h a 48 hour c u l t u r e o f hemin d e p l e t e d s t r a i n K110 and i n c u b a t e d f o r 48 hours a t 37°. 0 J0, s u c c i n a t e supplemented b a s a l medium; • • , hemin p l u s s u c c i n a t e supplemented b a s a l medium. 25 50 100 250 500 1000 2500 SUCCINATE, CONCENTRATION (ug/ml) 102 F i g u r e 16. Growth c h a r a c t e r i s t i c s o f s t r a i n K110 i n s u c c i n a t e or hemin supplemented b a s a l medium. C o n d i t i o n s were i d e n t i c a l t o t h o s e d e s c r i b e d i n F i g u r e 15. 103 10 20 30 40 50 TIME (hours) 104 F i g u r e 17. E f f e c t o f pH on the g r o w t h o f s t r a i n K l l O . One hundred m l of s u c c i n a t e supplemented (1 mg/ml) b a s a l medium pH 5.5, 6.0, 6.5, 7.0 and 7.5 was i n o c u l a t e d w i t h hemin d e p l e t e d s t r a i n K l l O (1 m l , A f i f t n = 1.0). 10 20 30 40 50 TIME (hours) 106 c u l t u r e s u p e r n a t a n t a f t e r g r o w t h o f K. pneumoniae i n g l u c o s e -supplemented (0.1%) basal-medium (see T a b l e X V I I I ) . 5. I s o l a t i o n o f the g r o w t h f a c t o r The e x p e r i m e n t s d i s c u s s e d i n t h e p r e c e d i n g pages s t r o n g l y s u g g e s t e d t h a t s u c c i n a t e was the growth f a c t o r p r o d u c e d by K. pneumoniae. An i s o l a t i o n p r o c e d u r e e m p l o y i n g g e l f i l t r a t i o n was used t o i s o l a t e the growth f a c t o r . The growth f a c t o r was o b t a i n e d f r o m 200 m l o f a 10 hour c u l t u r e of a n a e r o b i c a l l y grown K l e b s i e l l a . The c u l t u r e s u p e r n a t a n t was c o n c e n t r a t e d 20 t i m e s by f r e e z e - d r y i n g and 5 m l o f c o n c e n t r a t e was a p p l i e d t o a column c o n t a i n i n g Sephadex G-15. The e l u a n t was a s s a y e d f o r f a t t y a c i d s and g rowth p r o m o t i n g a c t i v i t y ( F i g u r e 1 8 ) . Growth s t i m u l a t i o n was c o n f i n e d t o a s i n g l e s y m m e t r i c a l peak w h i c h c o r r e s p o n d e d t o t h e p o s i t i o n where s u c c i n i c a c i d e l u t e d f r o m the column. There was e x c e l l e n t c o r r e l a t i o n between t h e amount of s u c c i n i c a c i d p r e s e n t i n the f r a c t i o n and t h e amount o f growth s t i m u l a t i o n . A c e t i c and l a c t i c a c i d s were d e t e c t e d i n t h e e l u a n t b u t t h e y were w e l l s e p a r a t e d f r o m the growth f a c t o r . A s t a n d a r d c o n t a i n i n g s u c c i n i c a c i d e l u t e d a t t h e same p o i n t as the . . s u c c i n i c a c i d f r o m the c u l t u r e s u p e r n a t a n t . The r e s u l t s o b t a i n e d showed t h a t t h e compound p r o d u c e d by K. pneumoniae was s u c c i n i c a c i d and t h a t i t s e r v e d as the growth p r o m o t i n g f a c t o r f o r _B. m e l a n i n o - g e n i c u s s t r a i n K110. E. Growth s t i m u l a t i o n by o t h e r b a c t e r i a The r e l a t i o n s h i p between endogenously produced s u c c i n a t e and the growth of the B a c t e r o i d e s was e v a l u a t e d f u r t h e r by a s s e s s i n g t h e a b i l i t y of a number of organisms t o s t i m u l a t e growth. P r o d u c t i o n o f t h e growth 107 F i g u r e 18. F r a c t i o n a t i o n o f t h e K..pneumoniae c u l t u r e f i l t r a t e on Sep had ex G-?15. F i v e ml o f c o n c e n t r a t e d c u l t u r e f i l t r a t e was a p p l i e d t o a Sephadex G-15 column (2.6 ic 40 cm) and e l u t e d w i t h phosphate b u f f e r (50 TSM, pH 6.6). F r a c t i o n s were a s s a y e d f o r v o l a t i l e and n o n - v o l a t i l e f a t t y a c i d s and f o r growth p r o m o t i n g a c t i v i t y . • #, AggQ; O O, s u c c i n i c a c i d ; Q Q , l a c t i c a c i d ; ^ Q , a c e t i c a c i d . ABSORBANCE (660nm) 80T 109 f a c t o r was measured i n t h e c o - c u l t i v a t i o n a s s a y and i n t h e c u l t u r e f i l t r a t e a s s a y . The r e s u l t s d e p i c t e d i n T a b l e X I X show t h a t E_. c o l i and A. v i s c o s u s , o r g a n i s m s w h i c h produce s u c c i n i c a c i d , s t i m u l a t e the growth of s t r a i n K l l O . Organisms w h i c h do n o t p r o d u c e s u c c i n i c a c i d do not s t i m u l a t e growth. The growth f a c t o r p r o duced by E_. c o l i B23 was c h a r a c t e r i z e d by t h e p r o c e d u r e s d e s c r i b e d f o r K l e b s i e l l a . The compound had s i m i l a r p r o p e r t i e s and e l u t e d a t the same p o s i t i o n f r o m a Sephadex G-15 column. These e x p e r i m e n t s a r e a d d i t i o n a l e v i d e n c e t h a t s u c c i n a t e i s t h e growth f a c t o r p r o d uced by K. pneumoniae. These e x p e r i m e n t s were c o n s i s t e n t w i t h t h e r e s u l t s o b t a i n e d when s u c c i n a t e p r o d u c i n g organisms were grown i n t h e p r e s e n c e of 13. m e l a n i n o g e n i c u s on agar p l a t e s ( F i g u r e 1 1 ) . Other e x p e r i m e n t s have shown t h a t s u c c i n i c a c i d was n o t produced when K l e b s i e l l a was grown a e r o b i c a l l y ; f i l t r a t e s f r o m a e r o b i c a l l y grown c u l t u r e s d i d n o t s u p p o r t the growth of J3_. m e l a n i n o g e n i c u s . F. I n v i v o s t u d i e s Once t h e i n v i t r o growth f a c t o r had been i d e n t i f i e d , i t was of i n t e r e s t t o d e t e r m i n e i f i t c o u l d r e p l a c e K. pneumoniae i n the i n f e c t -i v i t y a s s a y . A number of e x p e r i m e n t s d e s i g n e d t o examine t h i s p o s s i b i l -i t y a r e summarized i n T a b l e s X X and X X I . One e x p e r i m e n t a l a p p r o a c h was t o i n o c u l a t e g u i n e a p i g s w i t h s t r a i n K l l O and a v a r i e t y o f organisms w h i c h d i d or d i d n o t produce s u c c i n a t e . E_. c o l i and A. v i s c o s u s s y n t h e -s i z e s u c c i n i c a c i d and a r e c a p a b l e of f o r m i n g an i n f e c t i v e c o n s o r t i u m w i t h s t r a i n K l l O . N e i t h e r o r g a n i s m was i n f e c t i v e when i n o c u l a t e d by i t s e l f . B o t h organisms s u p p o r t e d t h e development of t h e r a p i d l y s p r e a d i n g f a t a l f o r m of t h e d i s e a s e . S t r a i n OBM-3, a s a c c h a r o l y t i c TABLE XIX GROWTH STIMULATION OF STRAIN K110 BY OTHER BACTERIA B a c t e r i a P r e s e n c e o f Growth S t i m u l a t i o n S u c c i n a t e R e s u l t  I s o l a t e 35 ( g r a m - n e g a t i v e r o d ) A. v i s c o s u s + + F_. n u c l e a t u m - -E. c o l i B23 + + S_. aureus - -5. s a n g u i s -L a c t o b a c i l l u s sp. - -_S_. a l b u s - -V e i l l o n e l l a sp. Pseudomonas a e r u g i n o s a - -I l l TABLE XX . INFECTIVITY OF STRAIN K l l O WHEN COMBINED WITH ORAL BACTERIA C e l l s Injected"*" C o n d i t i o n o f A n i m a l Day 1 2 3 4 K l l O + K l e b s i e l l a +++ dead K l e b s i e l l a _ _ -K l l O + E. c o l i +++ dead E. c o l i -K l l O + A. v i s c o s u s ++ +++ dead A. v i s c o s u s - - ~ K l l O + _S. s a n g u i s -K l l O + V e i l l o n e l l a -V e i l l o n e l l a -K l l O + F u s o b a c t e r i u m -F u s o b a c t e r i u m _ _ - -K l l O + L a c t o b a c i l l u s -L a c t o b a c i l l u s _ - -K l l O + 1-35 -1-35 ~ K l l O + A- m e l a n i n o g e n i c u s OBM-3 ± ++ ++ ++ B. m e l a n i n o g e n i c u s OBM-3 - -"Number of c e l l s i n j e c t e d : 1 x 10 ... I n each c a s e c e l l s were h a r v e s t e d f r o m c u l t u r e s i n t h e e a r l y s t a t i o n a r y growth phase. TABLE XXI EFFECT OF HEMIN AND SUCCINATE ON INFECTIVITY OF STRAIN K l l O 1 C e l l s I n j e c t e d C o n d i t i o n o f A n i m a l Day 1 1 1 A K l l O -K l l O + hemin -K l l O + s u c c i n a t e _ _ - -K l l O + hemin a g a r ++ +++ dead K l l O + s u c c i n a t e agar ++ ++ ++ K l l O + a g a r - -K l l O + K l e b s i e l l a +++ dead Each a n i m a l was i n j e c t e d w i t h 1 x 10 c e l l s i n a volume o f 1 m l . S u c c i n a t e -3 c o n c e n t r a t i o n was 0.5M and hemin was 5x10 g/1. 113 B. m e l a n i n o g e n i c u s , p r o d u c e s s u c c i n a t e and forms an i n f e c t i v e c o n s o r t i u m . I n t h i s c a s e t h e d i s e a s e d e v e l o p s s l o w l y and i s r e s t r i c t e d to f o r m a t i o n o f a l o c a l i z e d p u s t u l a r a b s c e s s . S. s a n g u i s , V e i l l o r i e l l a , F u s o b a c t e r i u m and L a c t o b a c i l l u s do n o t pr o d u c e s u c c i n i c a c i d and were n o t i n f e c t i v e . The d a t a do n o t p r o v e t h a t s u c c i n i c a c i d i s t h e f a c t o r r e q u i r e d f o r i n f e c t i v i t y b u t p r o v i d e s t r o n g p r e s u m p t i v e e v i d e n c e . P r o o f t h a t s u c c i n i c a c i d i s the f a c t o r r e q u i r e d t o i n d u c e f o r m a t i o n of a p a t h o g e n i c l e s i o n was o b t a i n e d f r o m s t u d i e s i n w h i c h p u r e c u l t u r e s o f J i . m e l a n i n o g e n i c u s s t r a i n K l l O were i n o c u l a t e d w i t h s u c c i n a t e ( T a b l e XXI) . I n t h e i n i t i a l e x p e r i m e n t s c e l l s were mixed w i t h hemiri-s u c c i n a t e and i n j e c t e d i n t o t h e a n i m a l b u t u n f o r t u n a t e l y l e s i o n s d i d n o t form. A p o s s i b l e e x p l a n a t i o n f o r t h i s l a c k o f s u c c e s s was t h a t the growth f a c t o r d i f f u s e d away f r o m t h e i n j e c t i o n s i t e b e f o r e j i . m e l a n i n o - g e n i c u s had begun g r o w i n g . An a t t e m p t was made t o s l o w d i f f u s i o n by i n c o r p o r a t i n g t h e growth f a c t o r i n t o an agar m a t r i x . H o p e f u l l y , d i f f u s i o n would be s l o w enough t h a t J i . m e l a n i n o g e n i c u s would have a c c e s s to-.the m o l e c u l e when i t was r e a d y t o i n i t i a t e growth. When t h i s was done pu r e c u l t u r e s o f the o r g a n i s m were found t o be i n f e c t i v e . Hemin s u p p o r t e d development o f t h e r a p i d l y s p r e a d i n g f a t a l d i s e a s e and s u c c i n a t e the l e s s s e v e r e l o c a l i z e d d i s e a s e . T h i s i s i n k e e p i n g w i t h the i n v i t r o s t u d i e s w h i c h showed t h a t t h e r e was a l o n g e r l a g p e r i o d when s t r a i n K l l O was grown w i t h s u c c i n a t e . These e x p e r i m e n t s d i s c o u n t t h e p o s s i b i l i t y t h a t t h e " h e l p e r " o r g a n i s m i s r e q u i r e d t o r e d u c e the 0„ t e n s i o n . S u c c i n i c a c i d 1. S u c c i n i c a c i d u t i l i z a t i o n by B, m e l a n i n o g e n i c u s Gas c h r o m a t o g r a p h i c a n a l y s i s of \B. m e l a n i n o g e n i c u s grown i n hemin f r e e s u c c i n a t e - s u p p l e m e n t e d medium has shown t h a t the s u c c i n i c a c i d was o n l y p a r t i a l l y m e t a b o l i z e d . F o r example, when t h e i n i t i a l c o n c e n t r a t i o n of s u c c i n a t e was 500 and 1000 y g / m l , 225 and 474 ug/ml r e s p e c t i v e l y r emained a f t e r an i n c u b a t i o n p e r i o d of 48 h o u r s . The a d d i t i o n o f hemin t o the same med i a c o m p l e t e l y b l o c k e d the m e t a b o l i s m of s u c c i n a t e . Whether the s u c c i n a t e s e r v e d as an energy s o u r c e o r f o r t h e s t r u c t u r a l c a r b o n needs t o be d e m o n s t r a t e d . However, t h e amount of s u c c i n i c a c i d m e t a b o l i z e d i n t h e hemin d e p l e t e d medium s u g g e s t s t h a t s u c c i n a t e may be u t i l i z e d f o r e n e r g e t i c p u r p o s e s . The e f f e c t of hemin and s u c c i n i c a c i d on the p r o d u c t i o n o f v o l a t i l e f a t t y a c i d s by 13. m e l a n i n o g e n i c u s i s shown i n T a b l e X X I I . The r e s u l t s show t h a t i n the p r e s e n c e of hemin (and r e g a r d l e s s of t h e amount of s u c c i n i c a c i d p r e s e n t ) , the p a t t e r n and t h e q u a n t i t y of t h e end p r o d u c t s d i d n o t change w i t h t h e e x c e p t i o n o f a c e t i c a c i d w h i c h i n c r e a s e d by 40% i n t h e p r e s e n c e of 2500 ug of s u c c i n i c a c i d . S e v e r a l s i g n i f i c a n t changes can be o b s e r v e d when s t r a i n K l l O i s grown on b a s a l medium supplemented w i t h s u c c i n i c a c i d : (a) T h e r e i s a f o u r f o l d i n c r e a s e i n the amount of a c e t i c a c i d produced, (b) I n c r e a s i n g c o n c e n t r a t i o n s of s u c c i n a t e r e d u c e the q u a n t i t y of a c e t a t e s y n t h e s i z e d , (c) S y n t h e s i z e d b u t y r a t e p r o d u c t i o n i s i n c r e a s e d and i s o b u t y r a t e p r o d u c t i o n d e c r e a s e d , (d) There a r e s m a l l i n c r e a s e s i n t h e amount of p r o p i o n a t e and i s o v a l e r a t e . There were no changes i n t h e c o n c e n t r a t i o n o f n o n - v o l a t i l e f a t t y a c i d s . TABLE X X I I EFFECT OF HEMIN AND SUCCINIC ACID ON VOLATILE FATTY ACID PRODUCTION BY B. MELANINOGENICUS yM/mg C e l l Dry Weight S u c c i n i c a c i d 1 250 500 1000 2500 Hemin +H -H +H -H +H -H +H -H A c e t i c 12.74 49.12 14.05 44.72 15.61 45.13 17.83 43.13 P r o p i o n i c 1.73 2.46 1.77 2.46 1.94 2.40 2.20 2.16 I s o b u t y r i c 8.84 1.03 8.33 3.05 8.70 4.25 8.27 4.22 B u t y r i c 31.02 35.95 30.42 44.90 31.44 46.77 31.59 46.01 I s o v a l e r i c 16.80 15.31 15.97 19.48 16.39 21.39 16.11 20.46 C o n c e n t r a t i o n i n yg/ml a t z e r o t i m e . -3 C o n c e n t r a t i o n (5.0 x 10 g / l i t e r ) 116 2. S u c c i n i c a c i d The f a t e o f s u c c i n i c a c i d d u r i n g growth o f J i , m e l a n i n o g e n i c u s was s t u d i e d by g r o w i n g t h e o r g a n i s m i n t h e p r e s e n c e of e i t h e r 1,4 14 14 C - s u c c i n a t e o r 2,3 C - s u c c i n a t e . The o b j e c t i v e was t o d e t e r m i n e i f s u c c i n a t e was m e t a b o l i z e d t o CO^ o r some o t h e r e x c r e t e d end p r o d u c t o r was i n c o r p o r a t e d i n t o c e l l u l a r m a t e r i a l . S t r a i n K l l O was grown i n b a s a l media supplemented w i t h s u c c i n a t e (1000 yg/ml) and 14 e i t h e r 1,4 o r 2,3 C - s u c c i n a t e and i n b a s a l medium supplemented -3 w i t h s u c c i n a t e (1000 yg/ml) and hemin (5 x 10 g/ml) and e i t h e r 14 1,4 or 2,3 C - s u c c i n a t e . The m e d i a were i n o c u l a t e d w i t h hemin d e p l e t e d s t r a i n K l l O and i n c u b a t e d a t 37°C. A t v a r i o u s t i m e s a f t e r i n o c u l a t i o n samples were removed f r o m t h e a n a e r o b i c chamber f o r d r y w e i g h t d e t e r m i n a t i o n and c e l l f r a c t i o n a t i o n . a.. I n c o r p o r a t i o n i n t o c e l l u l a r m a t e r i a l 14 The d i s t r i b u t i o n o f C w i t h i n t h e c e l l c o u l d be i m p o r t a n t i n s u g g e s t i n g whether s u c c i n a t e was a p r e c u r s o r f o r a s p e c i f i c c e l l u l a r component. C e l l s were sedimented by c e n t r i f u g a t i o n and washed t w i c e i n c o l d PBS t o remove e x t r a c e l l u l a r l a b e l . The p e l l e t r e m a i n i n g a f t e r the second wash was resuspended i n one ml of d i s t i l l e d w a t e r and e x t r a c t e d as d e s c r i b e d . The r e s u l t s o f th e a n a l y s i s a r e p r e s e n t e d i n T a b l e X X I I I . An a n a l y s i s o f t h e 14 r e s u l t s shows t h r e e i m p o r t a n t f a c t s . (1) The f a t e of C i n 14 1,4 o r 2,3 C - s u c c i n a t e was the same. The s m a l l d i f f e r e n c e s 14 o b t a i n e d , s l i g h t l y h i g h e r CO^ c o u n t s i n 1,4 C - s u c c i n a t e and 14 s l i g h t l y h i g h e r v a l u e s i n t h e v a r i o u s f r a c t i o n s i n 2,3 C s u c c i n a t e , c a n be e x p l a i n e d by the p o s i t i o n o f the l a b e l i t s e l f . TABLE X X I I I DISTRIBUTION OF C LABELLED SUCCINATE  F r a c t i o n 1 4 C ( c p m ) 5 S u c c i n a t e S u c c i n a t e + Hemin 1,4 2 ,3 1 ,4 2 ,3 Medium 1 . 4 x 1 0 6 1 . 52 X 1 0 6 1 . 38 X 1 0 6 1 . 54 X 1 0 6 4 Whole c u l t u r e 1 . 35 x 1 0 6 1 . 47 X 1 0 6 1 . 34 X 1 0 6 1 . 53 X 1 0 6 S u p e r n a t a n t 1 . 2 1 x 1 0 6 1 . 38 X 1 0 6 1 . 24 X 1 0 6 1 . 37 X 1 0 6 P e l l e t 3 1 . 8 x 1 0 3 3. 2 X 1 0 3 1 . 7 X 1 0 3 3. 8 X 1 0 3 C O 2 i n t r a p 4. 6 x 1 0 3 6 X 1 0 2 1 . 5 X 1 0 4 1 . 8 X 1 0 3 C o l d TCA 0 4 X 1 0 2 0 2 . 8 X 1 0 2 A c i d EtOH 9 . 6 x 1 0 2 1 . 4 X 3 1 0 2 . 4 X 1 0 2 1 . 0 X 1 0 3 CHCl 3-MeOH 3 X 1 0 2 6 . 6 X 1 0 2 4. 2 X 1 0 2 1 . 1 X 1 0 3 Hot TCA 0 4. 0 X 1 0 2 1 . 6 X 1 0 2 5. 6 X 1 0 2 NaOH 4. 5 x 1 0 2 5. 1 X 1 0 2 5. 2 X 1 0 2 7. 9 X 1 0 2 S u c c i n a t e c o n c e n t r a t i o n : 1 mg/ml + x l tC s u c c i n a t e , 0.05 u C i . -3 14 Hemin c o n c e n t r a t i o n : 5 x 10 g/1, C - s u c c i n a t e 0.05 u C i . P e l l e t r e s uspended a f t e r second wash. C e l l s were h a r v e s t e d when t h e c u l t u r e was i n s t a t i o n a r y phase. T o t a l c o u n t s / f r a c t i o n . 118 (2) L e s s t h a n 0.5% of the ^ ^C was a s s o c i a t e d w i t h t h e c e l l s . A l m o s t a l l t h e l a b e l was p r e s e n t i n t h e c u l t u r e s u p e r n a t a n t . 14 (3) Hemin d i d n o t i n f l u e n c e the d i s t r i b u t i o n o f C. T h e . r e s u l t s shown i n T a b l e X X I I I were o b t a i n e d f r o m a 48 hour c u l t u r e . S i m i l a r r e s u l t s were o b t a i n e d w i t h c e l l s h a r v e s t e d f r o m younger c u l t u r e s . b. A n a l y s i s of the c u l t u r e s u p e r n a t a n t The i n c l u s i o n o f s u c c i n a t e i n the growth medium made s i g n -i f i c a n t changes i n t h e q u a n t i t y o f a c i d s formed d u r i n g growth ( T a b l e X X I I ) . I n l i g h t of the f i n d i n g s t h a t o n l y 0.3% o r l e s s o f t h e s u c c i n a t e was i n c o r p o r a t e d i n t o c e l l s ( T a b l e ' X X I I I ) and t h a t , a s i g n i f i c a n t p e r c e n t a g e of s u c c i n a t e d i s a p p e a r e d from the growth medium i n s u c c i n a t e supplemented b a s a l medium, i t was r e a s o n a b l e t o assume t h a t t h e m a j o r i t y of t h e s u c c i n a t e m e t a b o l i z e d by t h e c e l l s had been e x c r e t e d as a m e t a b o l i c end p r o d u c t . T h i s p o s s i b i l i t y was i n v e s t i g a t e d by a s s a y i n g c u l t u r e s u p e r n a t a n t s f o r 14 C l a b e l l e d p r o d u c t s . The s u p e r n a t a n t s were o b t a i n e d f r o m the 14 c u l t u r e s of or g a n i s m s w h i c h were a n a l y z e d f o r c e l l a s s o c i a t e d C. The r a d i o a c t i v e s u p e r n a t a n t s were e x t r a c t e d f o r v o l a t i l e and m e t h y l a t e d f a t t y a c i d s as d e s c r i b e d e a r l i e r and a n a l y z e d by gas chromatography ( F i g u r e 1 9 ) . A n a l y s i s of t h e s u p e r n a t a n t s o b t a i n e d f r o m c u l t u r e s grown w i t h o u t hemin i n d i c a t e t h a t the org a n i s m s m e t a b o l i z e s u c c i n a t e t o a s i n g l e v o l a t i l e f a t t y a c i d under t h e s e growth c o n d i t i o n s . 14 There were no C l a b e l l e d compounds,, o t h e r t h a n s u c c i n a t e p r e s e n t i n n o n - v o l a t i l e f a t t y a c i d e x t r a c t s o f t h e s u p e r n a t a n t . When 14 hemin was added t o t h e medium t h e r e were no C — l a b e l l e d 119 F i g u r e 19. F a t t y a c i d a n a l y s i s o f s t r a i n K l l O c u l t u r e s u p e r n a t a n t . A. C h r o m a t o g r a p h i c a n a l y s i s of the - v o l a t i l e f a t t y a c i d s . V o l a t i l e f a t t y a c i d s were e x t r a c t e d f r o m t h e c u l t u r e s u p e r n a t a n t o b t a i n e d f r o m t h e growth of s t r a i n K l l O i n b a s a l 14 medium supplemented w i t h 2,3 C - s u c c i n a t e . The s o l i d l i n e r e f e r s t o a n a l y s i s of t h e gas chromatograph e l u a n t i n t h e f l a m e i o n i z a t i o n .'detector and the b r o k e n l i n e t o r e f e r s t o 14 a n a l y s i s o f the same e l u a n t i n the C d e t e c t o r . B. C h r o m a t o g r a p h i c a n a l y s i s of the n o n - v o l a t i l e f a t t y a c i d s . 121 compounds p r e s e n t i n t h e v o l a t i l e f a t t y a c i d e x t r a c t s . 14 The r e t e n t i o n time of the C v o l a t i l e f a t t y a c i d was i d e n t i c a l t o t h a t o f b u t y r i c a c i d and the compound c o - m i g r a t e d 14 w i t h a known sample o f C - b u t y r i c a c i d . The c o n c l u s i o n t h a t t h e compound was b u t y r a t e w a s v e r i f i e d by t h i n l a y e r chroma-t o g r a p h y . There was no r a d i o a c t i v i t y i n a c e t i c o r l a c t i c a c i d . The a c i d p r o d u c t s were s i m i l a r r e g a r d l e s s of whether t h e c e l l s 14 were grown w i t h 1,4 o r 2,3 C - s u c c i n a t e . The r e l a t i o n s h i p between the amount o f s u c c i n a t e m e t a b o l i z e d and the b u t y r a t e s y n t h e s i z e d i s o u t l i n e d i n T a b l e XXIV. As n o t e d p r e v i o u s l y , hemin b l o c k s the u t i l i z a t i o n o f s u c c i n a t e ; t h i s i s r e f l e c t e d by the amount of s u c c i n a t e r e m a i n i n g i n t h e 14 medium and t h e i n s i g n i f i c a n t q u a n t i t y of C - b u t y r a t e s y n t h e s i z e d . 14 The amount o f C p r e s e n t i n b u t y r i c a c i d c o r r e l a t e s c l o s e l y w i t h t h e amount of s u c c i n a t e m e t a b o l i z e d i n h e m i n - f r e e media. The s p e c i f i c a c t i v i t y o f t h e b u t y r a t e i s l o w e r t h a n t h a t o f s u c c i n a t e p r e s umably because i t i s s y n t h e s i z e d f r o m a v a r i e t y of p r e c u r s o r s . TABLE XXIV METABOLISM OF SUCCINATE Me d i a supplement I s o t o p e S u c c i n a t e m e t a b o l i z e d (yci) B u t y r a t e produced (UC1) S u c c i n a t e 14 1,4 C - s u c c i n a t e 0.35 14 2,3 C - s u c c i n a t e 0.38 0.33 0.35 S u c c i n a t e p l u s hemin^ 14 1,4 C - s u c c i n a t e 0.05 14 2,3 C - s u c c i n a t e 0 0 0 1 14 1,4 o r 2,3 C - s u c c i n a t e 0.05 y C i / m l . " S u c c i n a t e 1 mg/ml. 3 -3 S u c c i n a t e 1 mg/ml; hemin 5 x 10 g/1. 123 IV. DISCUSSION P a s t work i n s e v e r a l l a b o r a t o r i e s (36,92,103,116) has l e d t o t h e i d e n t i f i c a t i o n of t h r e e s u b s p e c i e s w i t h i n t h e s p e c i e s 13. m e l a n i n o g e n i c u s . These have been d e f i n e d on t h e b a s i s of p h y s i o l o g i c a l , b i o c h e m i c a l and s e r o -l o g i c a l p r o p e r t i e s . No c o r r e l a t i o n between t h e s e p r o p e r t i e s and p a t h o g e n i c i t y has y e t been f o u n d . More r e c e n t l y , d i f f e r e n c e s between t h e a s a c c h a r o l y t i c and s a c c h a r o l y t i c s p e c i e s have l e d t o t h e p r o p o s a l t h a t t h e f o r m e r s h o u l d be r e -c l a s s i f i e d as B. a s a c c h a r o l y t i c u s whereas the s a c c h a r o l y t i c s t r a i n s s h o u l d r e m a i n as j3. m e l a n i n o g e n i c u s ( 3 8 ) . S t u d i e s i n t h i s l a b o r a t o r y tend t o v a l i d a t e the r e c e n t p r o p o s a l i n t h a t e x i s t i n g s u b s p e c i e s o f 13. m e l a n i n o g e n i c u s c a n be c l a s s i f i e d i n two groups o n l y . However, our c l a s s i f i c a t i o n i s based on a number of new taxonomic c r i t e r i a w h i c h t a k e n t o g e t h e r w i t h the s t a n d a r d c r i t e r i a can f u r t h e r d i f f e r e n t i a t e s t r a i n s known as _B. m e l a n i n o g e n i c u s . These c r i t e r i a a r e p r o d u c t i o n o f p h e n y l a c e t i c a c i d , HA a c t i v i t y , c o l l a g e n a s e a c t i v i t y and p a t h o g e n i c i t y . The r e s u l t s have shown t h a t t h e p r o d u c t i o n o f p h e n y l a c e t i c a c i d was common to a l l a s a c c h a r o l y t i c s t r a i n s . S a c c h a r o l y t i c s t r a i n s d i d n o t p r o d u c e the compound. T h i s u n u s u a l end p r o d u c t has n o t been found b e f o r e because many w o r k e r s d i d n o t a n a l y s e the n o n - v o l a t i l e a c i d end p r o d u c t s of a n a e r o b i c gram-n e g a t i v e r o d s . The compound can s e r v e as a marker a l o n g w i t h b u t y r i c and s u c c i n i c a c i d s f o r r o u t i n e s e p a r a t i o n o f J3. a s a c c h a r o l y t i c u s f r o m J3. m e l a n i - n o g e n i c u s . F u r t h e r m o r e , the compound m i g h t have some i m p o r t a n c e i n c l i n i c a l d i a g n o s i s s i n c e i t has been found i n exu d a t e f r o m a n i m a l s (S. J e n s e n , p e r s o n a l c o m m u n i c a t i o n ) . HA a c t i v i t y was p r e s e n t i n a l l J3. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s s t r a i n s . Most r e a c t i o n s were s t r o n g , r a p i d and c o m p l e t e w i t h i n f i v e m i n u t e s . The a s s a y c o u l d be used as a n o t h e r c l i n i c a l t o o l t o i d e n t i f y a s a c c h a r o l y t i c 124 s t r a i n s of 13. m e l a n i n o g e n i c u s . I t would a l s o be p o s s i b l e t o do t h e a s s a y d i r e c t l y f r o m p l a t e s u s i n g r e s u s p e n d e d c o l o n i e s . S i n c e the HA a c t i v i t y i s s t a b l e , t h e r e a c t i o n c o u l d be r u n e i t h e r a t 22° o r 37°C. I t i s i m p o r t a n t t o n o t e t h a t r e d u c i n g c o n d i t i o n s r e q u i r e d i n b o t h HA and c o l l a g e n a s e a s s a y s a r e c o n s i s t e n t w i t h the f a c t t h a t t h e or g a n i s m s l i v e i n a n a e r o b i c c o n d i t i o n s . A l t h o u g h some w o r k e r s (92,103,116) have r a i s e d t h e p o s s i b i l i t y of an e c o l o g i c a l s u b d i v i s i o n between s a c c h a r o l y t i c and a s a c c h a r o l y t i c s t r a i n s of Ti. m e l a n i n o g e n i c u s , our s t u d i e s of human s t r a i n s showed t h a t t h e r e was no r e l a t i o n s h i p between t h e s o u r c e o f i n o c u l u m and t h e Ji_. m e l a n i n o g e n i c u s sub-s p e c i e s . A p o s s i b l e e x p l a n a t i o n f o r t h e poor r e c o v e r y of B_. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s i n p r e v i o u s s t u d i e s m i ght be t h a t t h i s s p e c i e s (HA p o s i t i v e ) b i n d s t o t h e g i n g i v a l e p i t h e l i u m and i s t h e r e f o r e not i n t h e b u l k of s u p r a g i n g i v a l p l a q u e , b u t r a t h e r c o n f i n e d t o a t h i n l a y e r a d j a c e n t t o t h e g i n g i v a . The f l o r a o f dogs was found t o be u n u s u a l s i n c e most of the s t r a i n s i s o l a t e d f r o m t h i s s o u r c e were a s a c c h a r o l y t i c and gave p o s i t i v e r e s u l t s i n t h e i n f e c t -i v i t y a s s a y . T h i s may e x p l a i n why dog b i t e s f r e q u e n t l y become i n f e c t e d . From t h e n u t r i t i o n a l p o i n t o f v i e w , our s t u d i e s showed t h a t a l l s t r a i n s i s o l a t e d r e q u i r e d hemin f o r growth. V i t a m i n K r e q u i r e m e n t was n o t found t o be s p e c i e s - s p e c i f i c . No c o r r e l a t i o n was found between v i t a m i n K dependency and t h e s o u r c e o f the human i s o l a t e s b u t a v e r y h i g h p r o p o r t i o n o f the a s a c c h a r o -l y t i c s t r a i n s i s o l a t e d f r o m dogs r e q u i r e d the f a c t o r i n d i c a t i n g a p o s s i b l e s e l e c t i o n i n t h i s e n v i r o n m e n t . No c o r r e l a t i o n was found between p r o t e o l y t i c a c t i v i t y and the s o u r c e of t h e i s o l a t e s . However, a l l s t r a i n s of Ti. m e l a n i - n o g e n i c u s ss. a s a c c h a r o l y t i c u s produced a c e l l a s s o c i a t e d p r o t e a s e c a p a b l e o f h y d r o l y z i n g b o t h c a s e i n and A z o c o l l . The p r o t e a s e found i n t h e s e organisms i s n ot l i k e l y t o be c o l l a g e n a s e s i n c e c a s e i n was a t t a c k e d . 125 The r e s u l t s f r o m t h e i n f e c t i v i t y a s s a y s i n d i c a t e d t h a t a l l s t r a i n s of J 3 . m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s were i n f e c t i v e whereas none of t h e s a c c h a r o l y t i c s t r a i n s i n d u c e d a r e s p o n s e i n the a n i m a l . Two t y p e s of i n f e c t -i o n were seen w i t h i n t h e p o s i t i v e a s s a y s . One t y p e was l o c a l i z e d whereas t h e o t h e r was a s p r e a d i n g i n f e c t i o n . I n t h e l a t t e r c a s e , d e a t h was p r o b a b l y due t o b a c t e r e m i a and p e r i t o n i t i s . The d i f f e r e n c e between t h e two t y p e s c o u l d be r e l a t e d t o the growth r a t e of the s t r a i n s t e s t e d . Organisms m a n i f e s t i n g a r a p i d growth r a t e c o u l d q u i c k l y overcome the body d e f e n c e s whereas the body c o u l d m o b i l i z e i t s d e f e n c e s i n t i m e t o " l o c a l i z e " s l o w e r g r o w i n g c e l l s . F u r t h e r s t u d i e s on mixed i n f e c t i o n s l e d t o t h e c o n c l u s i o n t h a t c e l l s must be a l i v e t o i n i t i a t e and s u s t a i n t h e i n f e c t i o n . I n d e ed, dead c e l l s or even s t e r i l e e x u d a t e c o u l d n o t p r o d u c e an i n f e c t i o n . I t i s t h e n r e a s o n a b l e t o assume t h a t t h e d i s e a s e produced w i t h v i a b l e c e l l s i s n o t s i m p l y a h y p e r -s e n s i t i v e r e s p o n s e t o t h e b a c t e r i a l a n t i g e n s . P a t h o g e n i c i t y of B. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s can be r e l a t e d t o a number of f a c t o r s . The r e s u l t s r e p o r t e d h e r e have shown t h a t t h e s e s t r a i n s p o s s e s s a c h o l e r a - l i k e t o x i n . The i n c o n s i s t e n t a c t i v i t y of t h e t o x i n may be due t o a low l e v e l of v i r u l e n c e o r t o i n s t a b i l i t y o f the m o l e c u l e i n t h e t e s t e n v i r o n m e n t . I t seems h i g h l y u n l i k e l y t h a t the t o x i c m a t e r i a l i s a l i p o p o l y s a c c h a r i d e - l i k e e n d o t o x i n s i n c e h e a t i n g d e s t r o y e d i t s a c t i v i t y . The two t y p e s of i n f e c t i o n s i n d i c a t e t h a t t h i s c h o l e r a - l i k e t o x i n may not a l w a y s be e x p r e s s e d . The r a p i d l y s p r e a d i n g i n f e c t i o n m i ght a l s o be due t o c o l l a g e n a s e a c t i v i t y b u t h e r e a g a i n , t h e r e s u l t s s u g g e s t t h a t the a c t i v i t y may n o t a l w a y s be e x p r e s s e d . H y a l u r o n i d a s e a c t i v i t y c o u l d n o t be d e t e c t e d i n t h e a s a c c h a r o -l y t i c s t r a i n s o f J 3 . m e l a n i n o g e n i c u s (M. G i s s l o w , p e r s o n a l c o m m u n i c a t i o n ) . The i n c r e a s i n g p a t h o g e n i c i t y a s s o c i a t e d w i t h a n i m a l passage of t h e e x u d a t e c o n t a i n i n g .B. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s s u g g e s t s t h a t under c e r t a i n c o n d i t i o n s t h e o r g a n i s m can p r o d u c e v a r i a n t s w h i c h a r e more v i r u l e n t . I t a l s o s u g g e s t s t h a t a t t e n u a t i o n can o c c u r i n t h e n a t u r a l e n vironment ( o r a l c a v i t y ) and/or i n c o n t i n u o u s i n v i t r o c u l t i v a t i o n . S i n c e t h e m i c r o o r g a n i s m s r e q u i r e a r e d u c e d e n v i r o n m e n t , the a n i m a l model used i n our e x p e r i m e n t s m i g h t n o t be t h e b e s t model. Indeed t h e model i s i n c o n t r a s t w i t h l e s i o n s e n c o u n t e r e d i n t h e o r a l c a v i t y w h i c h may be n e c r o t i c and t h e r e f o r e l e s s a e r o b i c . Two r e a s o n s c o u l d e x p l a i n t h e f a c t t h a t l a r g e numbers o f c e l l s a r e r e q u i r e d t o cause the i n f e c t i o n i n t h e a n i m a l system: (1) low l e v e l p a t h o g e n i c i t y of t h e s t r a i n s as d i s c u s s e d e a r l i e r and (2) s e n s i t i v i t y t o oxygen p r e s e n t i n t h e i n f e c t e d s i t e . I n t h i s c a s e , one c a n i m a g i n e t h a t l a r g e numbers of c e l l s would p r o t e c t each o t h e r . Clumps of a n a e r o b i c c e l l s c r e a t e m i c r o e n v i r o n m e n t s w h i c h p r o t e c t the c e l l p o p u l a t i o n f r o m o x i d a t i o n . I n a d d i t i o n f a c u l t a t i v e h e l p e r organisms c o u l d r e d u c e oxygen t e n s i o n and c r e a t e an a n a e r o b i c e n v i r o n m e n t . E i t h e r mechanism wou l d g u a r a n t e e s u r v i v a l o f 13. m e l a n i n o g e n i c u s , promote i t s growth and e n s u r e t h a t O ^ - s e n s i t i v e systems such as h e m a g g l u t i n a t i o n and c o l l a g e n a s e would f u n c t i o n . To u n d e r s t a n d any mixed i n f e c t i o n p r e s u p p o s e s t h a t s u c h a b a c t e r i a l m i x -t u r e i s the minimum number of s p e c i e s r e q u i r e d t o produce t h e d i s e a s e . The f i r s t c o n d i t i o n i s t h e n t o d e l i n e a t e t h e s p e c i e s i n v o l v e d i n t h e d i s e a s e f r o m t h e " a c c i d e n t a l " ones. The a d v a n t a g e s of h a v i n g a s i m p l e s y s t e m ( i n terms of s i m p l i c i t y , two s p e c i e s would be i d e a l ) a r e t h a t i t i s e a s i e r t o s t u d y t h e i n t e r a c t i o n s between t h e o r g a n i s m s i n v o l v e d . No o v e r l y complex i n v i t r o a p p a r a t u s i s needed and t h e e f f e c t s o f v a r i o u s a d d i t i o n s (such as a n t i b i o t i c s ) w o u l d be more r e v e a l i n g . I n c o n t r a s t t o the f i n d i n g s of o t h e r w o r k e r s ( 6 0 , 1 0 4 ) , s t u d i e s i n t h i s l a b o r a t o r y have shown t h a t i t i s p o s s i b l e t o o b t a i n a s i m p l e two o r g a n i s m system. J3. m e l a n i n o g e n i c u s p l a y e d an e s s e n t i a l r o l e i n t h e d i s e a s e . T h i s s u g g e s t e d t h a t the " h e l p e r " o r g a n i s m s d i d n o t p o s s e s s i m p o r t a n t p a t h o g e n i c 127 p r o p e r t i e s . The r e s u l t s showed t h a t t h e s u c c e s s f u l m i x t u r e c o n s i s t e d o f J i . m e l a n i n o g e n i c u s and K. pneumoniae. The c h a r a c t e r i s t i c s o f t h e t r a n s m i s s i b l e mixed i n f e c t i o n p r oduced by t h e i n j e c t i o n o f g u i n e a p i g s w i t h o r g a n i s m s c u l t u r e d o r i g i n a l l y f r o m g i n g i v a l m a t e r i a l were s i m i l a r t o t h o s e d e s c r i b e d by Macdonald and co-workers ( 6 3 ) . The r e l a t i o n between t h e two b a c t e r i a was n o t r e s t r i c t e d t o t h i s p a i r s i n c e a number of o t h e r c o m b i n a t i o n s i n v o l v i n g d i f f e r e n t s t r a i n s o f 13. m e l a n i n o - g e n i c u s s s . a s a c c h a r o l y t i c u s were i n f e c t i v e . The r e v e r s e s i t u a t i o n where the h e l p e r o r g a n i s m was i n t e r c h a n g e d was a l s o found t o be s u c c e s s f u l . Q u a n t i t a -9 t i v e s t u d i e s showed t h a t h i g h numbers (1 x 10 ) of j3. m e l a n i n o g e n i c u s were r e q u i r e d a l o n g w i t h K. pneumoniae t o prod u c e an i n f e c t i o n . When t h i s r e q u i r e -ment was met, t h e r e s u l t s i n d i c a t e d t h a t i n c r e a s i n g numbers o f K. pneumoniae i n c r e a s e d t h e s e v e r i t y o f the i n f e c t i o n . E x p e r i m e n t s w i t h h e m i n - f r e e a g a r showed t h a t K. pneumoniae was p r o v i d i n g an e s s e n t i a l n u t r i t i o n a l f a c t o r t o t h e 33. m e l a n i n o g e n i c u s . A common l i q u i d medium was d e v i s e d t o f a c i l i t a t e the i s o l a t i o n and i d e n t i f i c a t i o n o f t h e growth f a c t o r . I t was found t h a t t h e b a s a l medium supplemented w i t h g l u c o s e (0.1%) r e p r o d u c e d the r e s p o n s e found on s o l i d medium. P r e l i m i n a r y e x p e r -i m e n t s had a l s o shown t h a t i f s t r a i n K l l O was d e p l e t e d b e f o r e h a n d o f i t s endogenous s u p p l i e s of hemin i t c o u l d n o t grow i n such a medium. Growth of s t r a i n K l l O i n hemin-supplemented b a s a l medium r e s u l t e d i n a p a t t e r n o f v o l a t i l e f a t t y a c i d p r o d u c t i o n s i m i l a r t o t h a t d e s c r i b e d by Holdeman and Moore (37) but d i f f e r e n t f r o m t h a t r e p o r t e d by Wahren and Gibbons (112) f o r t h i s s t r a i n . I t i s i n t e r e s t i n g t o n o t e t h a t t h e f a t t y a c i d s produced d u r i n g growth of s t r a i n K l l O i n mixed c u l t u r e were s i m i l a r t o t h o s e found when s t r a i n K l l O was grown i n pure c u l t u r e . A s t e r i l e f i l t r a t e o b t a i n e d f r o m a c u l t u r e of K l e b s i e l l a a l s o . s h o w e d 128 g r o w t h - p r o m o t i n g a c t i v i t y f o r s t r a i n K l l O . The compound was i d e n t i f i e d as s u c c i n i c a c i d and was a b l e t o r e p l a c e hemin i n t h e g r o w t h medium. The s u c c i n a t e was needed i n r e l a t i v e l y h i g h c o n c e n t r a t i o n t o promote t h e growth o f the s t r i c t anaerobe. The e x p e r i m e n t s w i t h s u c c i n a t e supplemented b a s a l medium i n d i c a t e d t h a t a t h r e s h o l d v a l u e of a p p r o x i m a t e l y 250 yg/ml had t o be r e a c h e d b e f o r e s u c c i n a t e c o u l d s t i m u l a t e the growth of .B. m e l a n i n o g e n i c u s . C e l l f r a c t i o n a t i o n e x p e r i m e n t s showed t h a t most o f the l a b e l l e d s u c c i n a t e was n o t i n c o r p o r a t e d i n t o c e l l c a r b o n . I n o t h e r words, s u c c i n a t e was not used t o s y n t h e s i z e a s p e c i f i c c e l l u l a r component. The r e s u l t s i n d i c a t e d t h a t the compound c o u l d be used as an energy s o u r c e or an e l e c t r o n s i n k . R e g a r d l e s s 14 of whether the o r g a n i s m was grown on 1,4- o r 2,3- C - s u c c i n a t e o n l y one l a b e l l e d p r o d u c t other., t h a n s u c c i n a t e was e v e r d e t e c t e d . The f e r m e n t a t i o n 14 p r o d u c t was i d e n t i f i e d as b u t y r i c a c i d . The i n a b i l i t y t o d e t e c t C - a c e t a t e 14 and t h e s m a l l amount of CC^ produced s u g g e s t s t h a t s u c c i n a t e i s s i m p l y r e d u c e d and n o t m e t a b o l i z e d f u r t h e r . No r a d i o a c t i v i t y was f ound i n e i t h e r a c e t a t e or l a c t a t e . I t was a l s o found t h a t n o t a l l o f the s u c c i n a t e was u t i l i z e d by the c e l l s . The r e s u l t s c o u l d be e x p l a i n e d i f s u c c i n a t e was a c t i n g as an e l e c t r o n s i n k and the o r g a n i s m r a n out o f a s u p p l y of o x i d i z a b l e s u b s t r a t e . E x p e r i m e n t s w i t h mixed c u l t u r e s and f u r t h e r a n a l y s i s of the p r o d u c t s show t h a t a f o o d c h a i n e x i s t s i n t h e s y s t e m s t u d i e d . G l u c o s e i s m e t a b o l i z e d by K. pneumoniae under a n a e r o b i c c o n d i t i o n s t o produce l a r g e amounts of s u c c i n i c a c i d . The a c i d i s t h e n consumed by J3_. m e l a n i n o g e n i c u s s s . a s a c c h a r o l y t i c u s t o s u p p o r t i t s growth. The n u t r i t i o n a l dependency of s t r a i n K l l O was d e f i n i t i v e l y shown when v a r i o u s o rganisms p r o d u c i n g s u c c i n a t e were found t o enhance the growth of s t r a i n K l l O i n t h e two i n v i t r o growth a s s a y s and i n t h e i n f e c t i v i t y a s s a y 129 whereas organisms n o t p r o d u c i n g s u c c i n i c a c i d gave n e g a t i v e r e s u l t s i n a l l a s s a y s . The p o s i t i v e r e s u l t s o b t a i n e d a r e a l s o n o t l i k e l y t o be due t o e n d o t o x i c a c t i v i t y f r o m t h e h e l p e r o rganisms s i n c e p o s i t i v e r e s u l t s were o b t a i n e d when G r a m - p o s i t i v e , s u c c i n a t e p r o d u c i n g organisms were c o i n j e c t e d w i t h _B. m e l a n i n o g e n i c u s s t r a i n K l l O . The most i m p o r t a n t and d e f i n i t i v e e v i d e n c e i n d i c a t i n g t h e n u t r i t i o n a l dependency was o b t a i n e d by i n f e c t i n g a n i m a l s w i t h p u r e c u l t u r e s o f s t r a i n K l l O t o g e t h e r w i t h a s l o w l y d i f f u s i n g s o u r c e of s u c c i n a t e o r hemin. The e x p e r i m e n t e l i m i n a t e s t h e p o s s i b i l i t y t h a t . i n the model s y s t e m the c r i t i c a l f u n c t i o n o f t h e " h e l p e r " o r g a n i s m i s t o r e d u c e oxygen t e n s i o n . I n t h i s a s s a y l a r g e numbers of c e l l s , many of them p r e s e n t as clumps, p r o b a b l y p r o v i d e s u f f i c i e n t p r o t e c t i o n a g a i n s t oxygen. I n a n a t u r a l i n f e c t i o n where fewer organisms a r e i n v o l v e d t h e second o r g a n i s m may f a c i l i t a t e t h e e x p r e s s i o n o f p a t h o g e n i c i t y by c r e a t i n g an a n a e r o b i c environment i n a d d i t i o n t o s u p p l y i n g s u c c i n a t e . The i m p o r t a n c e of s u c c i n a t e as the r e q u i r e d f a c t o r was f u r t h e r s u b s t a n t i a t e d by t h e o b s e r v a t i o n t h a t i n f e c t i o n o c c u r r e d when s t r a i n K l l O was combined w i t h s u c c i n a t e p r o d u c i n g o r g a n i s m s . I n c o n c l u s i o n , i t c a n be s t a t e d t h a t J 3 . m e l a n i n o g e n i c u s s s . a s a c c h a r o - l y t i c u s c a n e x p r e s s i t s p a t h o g e n i c i t y when i t i s p l a c e d i n a s u i t a b l e e n v i r o n m e n t where i t s u n i q u e growth r e q u i r e m e n t s a r e s a t i s f i e d . F u t u r e s t u d i e s s h o u l d be d i r e c t e d towards c h a r a c t e r i z i n g i t s p a t h o g e n i c p r o p e r t i e s and d e f i n i n g ; . t h e m e t a b o l i s m o f s u c c i n a t e . 130 V. LITERATURE CITED 1. A i s e n b e r g , M.S. and A.D. A i s e n b e r g . 1951. H y a l u r o n i d a s e i n p e r i o d o n t a l d i s e a s e . O r a l S u r g . O r a l Med. O r a l P a t h o l . _4:317-320. 2. A l l i s o n , F. J r . 1975. A n a e r o b i c b a c t e r i a l i n f e c t i o n i n man. South Med. J . j>8:1088-1092. 3. A l t e m e i e r , W.A. 1942. The p a t h o g e n i c i t y of a p p e n d i c i t i s p e r i t o n i t i s . S u r g e r y 11:374-384. 4. Bowden, G.H. and J.M. H a r d i e . 1971. A n a e r o b i c organisms f r o m the human mouth, p. 177-205. I n D.A. Shapton and R.G. Board ( e d . ) . I s o l a t i o n o f Anaerobes. Academic P r e s s , I n c . , New Y o r k . 5. Burdon, K.L. 1928. B a c t e r i u m m e l a n i n o g e n i c u m from n o r m a l and p a t h o -l o g i c a l t i s s u e s . J . I n f e c t . D i s . 42:161-171. 6. B u r t , S.J. and D.R. Woods. 1976. R f a c t o r t r a n s f e r t o o b l i g a t e anaerobe f r o m E s c h e r i c h i a c o l i . J . Gen. M i c r o b i o l . 93:405-409. 7. C o u r a n t , P.R. and R . J . Gibbons. 1967. B i o c h e m i c a l and i m m u n o l o g i c a l h e t e r o g e n e i t y of B a c t e r o i d e s m e l a n i n o g e n i c u s . A r c h . O r a l B i o l . 12:1605-1613. 8. C r a i g , J.P. 1965. A p e r m e a b i l i t y f a c t o r ( t o x i n ) f ound i n c h o l e r a s t o o l s and c u l t u r e f i l t r a t e s and i t s n e u t r a l i z a t i o n by c o n v a l e s c e n t c h o l e r a s e r a . N a t u r e 207:614-616. . 9. De l a C r u z , B. and C. Cuadra. 1969. A n t i g e n i c c h a r a c t e r i z a t i o n o f f i v e s p e c i e s of human B a c t e r o i d e s . J . B a c t e r i o l . 100:1116-1117. 10- D e l Bene, V.E., A. Rogers and W.E. F a r r a r J r . 1976. A t t e m p t e d t r a n s f e r of a n t i b i o t i c r e s i s t a n c e between B a c t e r o i d e s and E s c h e r i c h i a c o l i . J . Gen. M i c r o b i o l . 92:384-390. 11. Duerden, B . I . 1975. Pigment p r o d u c t i o n by B a c t e r o i d e s s p e c i e s w i t h v r e f e r e n c e t o s u b - c l a s s i f i c a t i o n . J . Med. M i c r o b i o l . 8^:113-125. 12. E l l e n , R.P. and R.J. G i b b o n s . 1974. P a r a m e t e r s a f f e c t i n g t h e a d herence and t i s s u e t r o p i s m s of S t r e p t o c o c c u s pyogenes. I n f e c t . Immunity 2:85-91. 13. F a l k , J.E. 1964. P o r p h y r i n s and M e t a l l o p o r p h y r i n s . BBA L i b r a r y E l s e v i e r Pub. Co. 14. F e l n e r , J.M. and V.R. D o w e l l J r . 1971. B a c t e r o i d e s b a c t e r e m i a . t--" Am. J . Med. 50:787-796. 15. F i n e g o l d , S.M. 1974. I n f e c t i o n s due t o a n a e r o b i c o r g a n i s m s o t h e r t h a n C l o s t r i d i a . I n _ P r a c t i c e o f m e d i c i n e , V o l . I l l , C h a p t e r 27. ^ H a r p e r and Row. 131 L i t e r a t u r e C i t e d ( c o n t i n u e d ) 16. F i n e g o l d , S.M. and E.M. B a r n e s . 1977. R e p o r t o f the ICSB t a x o n o m i c subcommittee on Gram-negative a n a e r o b i c r o d s . I n t . J . S y s t . B a c t e r i o l . 27:388-391. 17. F r o s t , A . J . 1975. S e l e c t i v e a d h e s i o n o f m i c r o o r g a n i s m s t o the d u c t u l a r e p i t h e l i u m o f the b o v i n e mammary g l a n d . I n f e c t . Immunity 12:1154-1156. 18. G a l l o p , P.M. and S. S e i f e r t . 1963. S o l u b l e c o l l a g e n s , p. 635-641. I n S.P. C o l o w i c k and N.O. K a p l a n ( e d . ) , Methods i n enzymology, V o l . 6. Academic P r e s s , I n c . , New Y o r k . 19. G i b b o n s , R.J. and J.B. Macdonald. 1960. Hemin and v i t a m i n K compounds as r e q u i r e d f a c t o r s f o r the c u l t i v a t i o n o f c e r t a i n s t r a i n s of B a c t e r o i d e s m e l a r i i r i o g e n i c u s . J . B a c t e r i o l . 80:164-170. 20. G i b b o n s , R.J. and J.B. Macdonald. 1961. D e g r a d a t i o n o f c o l l a g e n o u s s u b s t r a t e s by B a c t e r o i d e s m e l a n i n o g e n i c u s . J . B a c t e r i o l . 81: 614-621. 21. G i b b o n s , R . J . , B. K a p s i m a l i s and S.S. S o c r a n s k y . 1964. The s o u r c e o f s a l i v a r y b a c t e r i a . A r c h . O r a l B i o l . _9:101-103. 22. G i b b o n s , R.J. and J . v a n Houte. 1971. S e l e c t i v e b a c t e r i a l adherence t o o r a l e p i t h e l i a l s u r f a c e s and i t s r o l e as an e c o l o g i c a l d e t e r m i n a n t . I n f e c t . Immunity _3:567-573. 23. G i s s l o w , M.T. and B.C. M c B r i d e . 1975. A r a p i d s e n s i t i v e c o l l a g e n a s e a s s a y . A n a l . Biochem. 68:70-78. 24. Gorbach, S.L., K.B. Menda, H. T h a d e p a l l i and L. K e i t h . 1973. A n a e r o b i c m i c r o f l o r a o f t h e c e r v i x i n h e a l t h y women. Am. J . O b s t e t . G y n e c o l . 117:1053-1055. 25. Gorbach, S.L. and J.G. B a r t l e t t . 1974. A n a e r o b i c i n f e c t i o n s : O l d myths and new r e a l i t i e s . J . I n f e c . D i s . 130:307-309. 26. Gorbach, S.L. and J.G. B a r t l e t t . 1974. A n a e r o b i c i n f e c t i o n s . New En g l a n d J . Med. 290:1177-1184 p a r t I , 1237-1245 p a r t I I , 1289-1294 p a r t I I I . 27. H a r d i e , J.M. and G.H. Bowden. 1975. B a c t e r i a l f l o r a o f d e n t a l p l a q u e . B r . Med. B u l l . 31:131-136. 28. Hansmann, E., P. Co u r a n t and D.S. A r n o l d . 1967. C o n d i t i o n s f o r t h e d e m o n s t r a t i o n o f c o l l a g e n o l y t i c a c t i v i t y i n B a c t e r o i d e s m e l a n i n o - g e n i c u s . A r c h . O r a l B i o l . 12:317-319. 132 L i t e r a t u r e C i t e d ( c o n t i n u e d ) 29. Hausmann, E. and E. Kaufman, 1969. C o l l a g e n a s e a c t i v i t y i n a p a r t -i c u l a t e f r a c t i o n ^ f r o m . ' I t e ^ t ^ ^ Biochem. B i o p h y s . A c t a . 194:612-615. 30. H e n r i k s e n , S.D. 1954. S t u d i e s on th e K l e b s i e l l a group (Kaufmann). I I . B i o c h e m i c a l r e a c t i o n s . A c t a P a t h o l . M i c r o b i o l . Scand. 34: 259-265. 31. H i l l , G.B., S. O s t e r h o u t and P.C. P r a t t . 1974. L i v e r a b s c e s s p r o d u c t i o n by n o n - s p o r e - f o r m i n g a n a e r o b i c b a c t e r i a i n a mouse model. I n f e c t . Immunity. j ) : 599-603. 32. H i t e , K.E., M. Loc k e and H.C, H e s s e l t i n e . 1949. S y n e r g i s m i n e x p e r i m e n t a l i n f e c t i o n s w i t h n o n s p o r u l a t i n g a n a e r o b i c b a c t e r i a . J . I n f e c t . D i s . 84:1-9. 33. H o f s t a d , T. 1968. C h e m i c a l c h a r a c t e r i s t i c s o f B a c t e r o i d e s m e l a n i n o g e n i c u s e n d o t o x i n . A r c h . O r a l B i o l . 13:1149-1155. 34. H o f s t a d , T. 1969. S e r o l o g i c a l p r o p e r t i e s o f l i p o p o l y s a c c h a r i d e from o r a l s t r a i n s of B a c t e r o i d e s m e l a n i n o g e n i c u s . J . B a c t e r i o l . 97:1078-1082. 35. H o f s t a d , T. 1970. B i o l o g i c a l a c t i v i t i e s o f e n d o t o x i n f r o m B a c t e r o i d e s m e l a n i n o g e n i c u s . A r c h . O r a l B i o l . 15:343-348. 36. H o l b r o o k , W.P., B . I . Duerden and A.G. Deacon. 1977. The c l a s s i f i c a t i o n of B a c t e r o i d e s m e l a n i n o g e n i c u s and r e l a t e d s p e c i e s . J . A p p l . B a c t e r i o l . 42:259-273. 37. Holdeman, L.V. and W.E.C. Moore. 1972. B a c t e r o i d e s . I n Anaerobe L a b o r a t o r y Manual. Holdeman, L.V. and W.E.C. Moore ( e d . ) . V P I Anaerobe L a b o r a t o r y , B l a c k s b u r g , V i r g i n i a . 38. I n t e r n a t i o n a l Committee on S y s t e m a t i c B a c t e r i o l o g y . 1977. Subcommittee on Gram-negative a n a e r o b i c r o d s . M i n u t e s o f the M e e t i n g s , 29 and 30 May 1976. I n t . J . S y s t . B a c t e r i o l . 27:61. 39. K a s a i , G.J. and W. Burrows. 1966. The t i t r a t i o n o f c h o l e r a t o x i n and a n t i t o x i n i n th e r a b b i t i l e a l l o o p . J . I n f e c t . D i s . 116:606-614. 40. Kaufman, E . J . , P.A. Mashimo, E. Hausmann, C.T. Hanks and S.A. E l l i s o n . 1972. F u s o b a c t e r i a l i n f e c t i o n : enhancement by c e l l - f r e e e x t r a c t s o f B a c t e r o i d e s m e l a n i n o g e n i c u s p o s s e s s i n g c o l l a g e n o l y t i c a c t i v i t y . A r c h . O r a l B i o l . 17:577-580. 41. K e l l e r , R. and N. Traub. 1974. The c h a r a c t e r i z a t i o n .of I i . f r a g i l i s b a c t e r i o p h a g e r e c o v e r e d f r o m a n i m a l s e r a : o b s e r v a t i o n s on t h e n a t u r e o f B a c t e r o i d e s phage c a r r i e r c u l t u r e s . J . Gen. V i r o l . 24:179-189. L i t e r a t u r e C i t e d ( c o n t i n u e d ) Kestenbaum, R.C. and S. W e i s s . 1962. The p r o d u c t i o n o f e x p e r i m e n t a l i n f e c t i o n s w i t h c o m b i n a t i o n s o f m i c r o o r g a n i s m s o f human o r i g i n , ( a b s t r . ) , IADR 4 0 t h m e e t i n g . Kestenbaum, R . C , J . M a s s i n g and S. Weis s . 1964. The r o l e o f c o l l a g e n a s e i n m ixed i n f e c t i o n s c o n t a i n i n g B a c t e r o i d e s m e l a n i n o g e n i c u s . ( a b s t r . ) , IADR 42nd m e e t i n g . K l i n e , B.S. 1923. E x p e r i m e n t a l gangrene. J . I n f e c . D i s . 32:481-483. K r i t c h e w s k i , B. and P. S e g u i n . 1920. S p i r o c h e t o s e s b u c c a l e s -r e p r o d u c t i o n e x p e r i m e n t a l e e t t r a i t e m e n t . Rev. Stomat. ( P a r i s ) 2_2: 613-647. L a b a c h , J.P. and D.C. W h i t e . 1969. I d e n t i f i c a t i o n o f c e r a m i d e p h o s p h o r y l e t h a n o l a m i n e and c e r a m i d e p h o s p h o r y l g l y c e r o l i n t h e l i p i d s o f an a n e r o b i c b a c t e r i u m . J . L i p i d Res. 10:528-534. Lambe, D.W. J r . 1974. D e t e r m i n a t i o n o f B a c t e r o i d e s m e l a n i n o g e n i c u s s e r o g r o u p s by f l u o r e s c e n t a n t i b o d y s t a i n i n g . App. M i c r o b i o l . 28: 561-567. L e a c h , A.A. 1960. Notes on a m o d i f i c a t i o n o f the Neuman and Logan Method f o r t h e d e t e r m i n a t i o n o f h y d r o x y p r o l i n e . Biochem. J . 74:70-71. L e d g e r , W.J. 1975. A n a e r o b i c i n f e c t i o n s . Am. J . O b s t e t . G y n e c o l . 123:111-118. Lev, M. 1958. Ap p a r e n t r e q u i r e m e n t f o r v i t a m i n K of rumen s t r a i n s o f F u s i f o r m i s n i g r e s c e n s . N a t u r e 181:203-204. L e v , M. 1959. The g r o w t h - p r o m o t i n g a c t i v i t y o f v i t a m i n K and s e v e r a l a n a l o g u e s f o r a rumen s t r a i n of F u s i f o r m i s n i g r e s c e n s . J . Gen. M i c r o b i o l . 20:697-703. L e v , M. 1968. V i t a m i n K d e f i c i e n c y i n F u s i f o r m i s n i g r e s c e n s . I . I n f l u e n c e on whole c e l l s and c e l l e n v e l o p e c h a r a c t e r i s t i c s . J . B a c t e r i o l . 95:2317-2324. L e v , M. and A.F. M i l f o r d . 1971. V i t a m i n K s t i m u l a t i o n o f s p h i n g o l i p i d s y n t h e s i s . Biochem. B i o p h y s . Res. Comm. 45:358-362. L e v , M., K.C. K e n d e l l and A.F. M i l f o r d . 1971. S u c c i n a t e as a growth f a c t o r f o r B a c t e r o i d e s m e l a n i n o g e n i c u s . J . B a c t e r i o l . 108: 175-178. Lev, M. and A.F. M i l f o r d . 1972. E f f e c t of v i t a m i n K d e p l e t i o n and r e s t o r a t i o n on s p h i g o l i p i d m e t a b o l i s m i n B a c t e r o i d e s m e l a n i n o g e n i c u s . J . L i p i d Res. 13:364-370. 134 L i t e r a t u r e C i t e d ( c o n t i n u e d ) 56. L e v , M. and A.F. M i l f o r d . 1973. The 3 ^ k e t o d i h y d r o s p h i n g o s i n e s y n t h e t a s e of B a c t e r o i d e s m e l a n i n o g e n i c u s : I n d u c t i o n by v i t a m i n K. A r c h . Biochem. B i o p h y s . 157:500-508. 57. Loe, H. and J . S i l n e s s . 1963. P e r i o d o n t a l d i s e a s e i n p regnancy. I . P r e v a l e n c e and s e v e r i t y . A c t a Odont. Scand. 21:533-551. 58. L o e s c h e , W.J. and R . J . Gibbons. 1965. A p r a c t i c a l scheme f o r i d e n t i f i c -a t i o n of t h e most numerous o r a l Gram n e g a t i v e a n a e r o b i c r o d s . A r c h . O r a l B i o l . 3.0:723-725. 59. L o e s c h e , W.J., R.N. H o c k e t t and S.A. Syed. 1972. The predominant c u l t i v a b l e f l o r a o f t o o t h s u r f a c e p l a q u e removed f r o m i n s t i t u t i o n a l -i z e d s u b j e c t s . A r c h . O r a l B i o l . 17:1311-1326. 60. Macdonald, J.B., R.M. S u t t o n and M.L. K n o l l . 1954. The p r o d u c t i o n o f f u s o s p i r o c h a e t a l i n f e c t i o n s i n g u i n e a p i g s w i t h recombined p u r e c u l t u r e s . J . I n f e c . D i s . 95^:275-284. 61. M a c d o n a l d , J.B., R.M. S u t t o n , M.L. K n o l l , E.M. Madlener and R.M. G r a i n g e r . 1956. The p a t h o g e n i c components of an e x p e r i m e n t a l f u s o s p i r o c h a e t a l i n f e c t i o n . J . I n f e c . D i s . 98:15-20. 62. Macdonald, J.B., R.J. Gibbons and S.S. S o c r a n s k y . 1960. B a c t e r i a l mechanisms i n p e r i o d o n t a l d i s e a s e . Ann. N.Y. Acad. S c i . 85: 467-478. 63. Macdonald, J.B. and R.J. G i b b o n s . 1962. The r e l a t i o n s h i p of i n d i g e n o u s b a c t e r i a t o p e r i o d o n t a l d i s e a s e . J . of Dent. Res. 41:320-326. 64. Macdonald, J.B., S.S. S o c r a n s k y and R.J. Gibbons. 1963. A s p e c t s o f the p a t h o g e n e s i s of mixed a n a e r o b i c i n f e c t i o n s of mucous membranes. J . o f Dent. Res. 42:529-544. 65. Mansheim, B . J . , A.B. Onderdonk and D.L. K a s p e r . 1978. Immunochemical and b i o l o g i c s t u d i e s of l i p o p o l y s a c c h a r i d e of B a c t e r o i d e s m e l a n i n o - g e n i c u s s u b s p e c i e s a s a c c h a r o l y t i c u s . J . Immunol. 120:72-78. 66. Mardh, P.-A. and L. Westrom. 1976. Adherence of b a c t e r i a t o v a g i n a l e p i t h e l i a l c e l l s . I n f e c t . Immunity 13:661-666. 67. M eleney, F.L. 1931. B a c t e r i a l s y n e r g i s m i n d i s e a s e p r o c e s s w i t h a c o n f i r m a t i o n o f t h e s y n e r g i s t i c b a c t e r i a l e t i o l o g y o f a c e r t a i n t y p e of p r o g r e s s i v e gangrene of the stomach w a l l . Ann. S u r g . 19:961-981. 68. Mergenhagen, S.E., J.C. Thonard and H.W. S c h a r p . 1958. S t u d i e s on s y n e r g i s t i c i n f e c t i o n s I . E x p e r i m e n t a l i n f e c t i o n s w i t h a n a e r o b i c s t r e p t o c o c c i . J . I n f e c . D i s . 103:33-34. 135 L i t e r a t u r e C i t e d ( c o n t i n u e d ) 69. M e y n e l l , G.G. and E. M e y n e l l . 1965. Theory and p r a c t i c e i n e x p e r i m e n t a l b a c t e r i o l o g y . Cambridge U n i v . P r e s s . 70. M i l e s , D.O., J.K. Dyer and J.C, Wong. 1976, I n f l u e n c e of amino a c i d s on t h e growth of B a c t e r o i d e s m e l a n i n o g e n i c u s . J . B a c t e r i o l . 127: 899-903. 71. M i r a g l i a , G.J. 1974. P a t h o g e n i c a n a e r o b i c b a c t e r i a . CRC C r i t i c a l Reviews i n M i c r o b i o l . : 161-181. 72. Moore, W.E.C. and L.V. Holdeman. 1973. New names and c o m b i n a t i o n s i n t h e g e n e r a B a c t e r o i d e s C a s t e l l a n i and C h a l m e r s , F u s o b a c t e r i u m K n o r r , E u b a c t e r i u m P r e v o t , P r o p i o n i b a c t e r i u m D e l w i c h and L a c t o b a c i l l u s O r l a - J e n s e n . I n t . J . S y s t . B a c t e r i o l . 23:69-74. 73. Okuda, K. and I . Takazoe, 1974. H a e m a g g l u t i n a t i n g a c t i v i t y o f B a c t e r o i d e s m e l a n i n o g e n i c u s . A r c h . O r a l B i o l . 19:415-416. 74. O l i v e r , W.W. and W.B. Wherry. 1921. Notes on some b a c t e r i a l p a r a s i t e s of t h e human mucous membranes. J . I n f e c . D i s . 28:341-344. 75. Onderdonk, A.B., W.M. W e i h s t e i n , N.M. S u l l i v a n / J . G . B a r t l e t t and S.L. Gorbach. 1974. E x p e r i m e n t a l i n t r a - a b d o m i n a l a b s c e s s e s i n r a t s : Q u a n t i t a t i v e b a c t e r i o l o g y o f i n f e c t e d a n i m a l s . I n f e c t . Immunity 10:1256-1259. 76. Onderdonk, A.B., J.G. B a r t l e t t , T. L o u i s , N. S u l l i v a n - S e i g l e r and S.L. Gorbach. 1976. M i c r o b i a l s y n e r g y i n e x p e r i m e n t a l i n t r a -a b d o m i n a l a b s c e s s . I n f e c t . Immunity, 13:22-26. 77. Orskov, I . and S.T. Cowan. 1974. I n B e r g e y ' s Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y , R.E. Buchanan and N.E. Gibbons ( e d . ) , p. 290-322. W i l l i a m s and W i l k i n s Co., B a l t i m o r e , Md. 78. P a u l , E. 1910. Deutsche M o n a t s s c h r . f . Zahnh. 28:22. 79. P i e r c e , N.F., W.B. Greenough I I I and C.C.J. C a r p e n t e r J r . 1971. V i b r i o c h o l e r a e n t e r o t o x i n and i t s mode of a c t i o n . B a c t . Reviews 35:1-13. 80. R i z z a , V., P.R. S i n c l a i r , D.C. W h i t e and P.R. C o u r a n t . 1968. E l e c t r o n t r a n s p o r t system of t h e p r o t o h e m e - r e q u i r i n g anaerobe B a c t e r o i d e s  m e l a n i n o g e n i c u s . J . B a c t e r i o l . 96:665-671. 81. R i z z a , V., A.N. T u c k e r and D.C. W h i t e . 1970. L i p i d s of B a c t e r o i d e s m e l a n i n o g e n i c u s . J . B a c t e r i o l . 101:84-91. 82. R o b e r t s , D.S. 1967. The p a t h o g e n i c s y n e r g y of F u s i f o r m i s n e c r o p h o r u s and C O r y r i e b a c t e r i u m pyogenes. I . I n f l u e n c e of t h e l e u c o c i d a l e x o t o x i n of F_. n e c r o p h o r u s . B r . J . Exp. P a t h . 48:665-673. 136 L i t e r a t u r e C i t e d ( c o n t i n u e d ) 83. R o b e r t s , D.S. 1967. The p a t h o g e n i c s y n e r g y o f F u s i f o r m i s n e c r o p h o r u s and C o r y n e b a c t e r i u m pyogenes. I I . The r e s p o n s e o f F. n e c r o p h o r u s to a f i l t e r a b l e p r o d u c t o f C. pyogenes. B r . J . Exp. P a t h . 48:674-679. 84. R o b e r t s , D.S. 1969. S y n e r g i c mechanisms i n c e r t a i n m i x e d i n f e c t i o n s . J . I n f e c . D i s . 120:720-724. 85. R o b i n s , D.J., R.B. Yee and R. B e n t l e y . 1973. B i o s y n t h e t i c p r e c u r s o r s o f v i t a m i n K as growth p r o m o t e r s f o r B a c t e r o i d e s m e l a n i n o g e n i c u s . J . B a c t e r i o l . 116:965-971. 86. R o s e b u r y , T., J.B. Macdonald and A.R. C l a r k e . 1950. A b a c t e r i o l o g i c s u r v e y o f g i n g i v a l s c r a p i n g s f r o m p e r i o d o n t a l i n f e c t i o n s by d i r e c t e x a m i n a t i o n , g u i n e a p i g - i n o c u l a t i o n and a n a e r o b i c c u l t i v a t i o n . J . Dent. Res. 29:718-731. 87. Rosebury, L., A.R. C l a r k , S.G. E n g e l and F. T e r g i s . 1950. S t u d i e s o f f u s o s p i r o c h e t a l i n f e c t i o n s I . P a t h o g e n i c i t y f o r g u i n e a p i g s of i n d i v i d u a l and combined c u l t u r e s o f s p i r o c h e t e s and o t h e r a n a e r o b i c b a c t e r i a d e r i v e d f r o m human mouth. J . I n f e c . D i s . 87:217-225. 88. Rosebury, T., A.R. C l a r k , F. T e r g i s and S.G. E n g e l . 1950. S t u d i e s of f u s o s p i r o c h e t a l i n f e c t i o n I I . A n a l y s i s and a t t e m p t e d q u a n t i t a t i v e r e c o m b i n a t i o n o f the f l o r a o f f u s o s p i r o c h e t a l i n f e c t i o n a f t e r r e p e a t e d g u i n e a p i g passage. J . I n f e c . D i s . 87:226-233. 89. R o s e b u r y , T., A.R.1 C l a r k , J.B. Macdonald and D.C. O ' C o n n e l l . 1950. S t u d i e s o f f u s o s p i r o c h e t a l i n f e c t i o n I I I . J . I n f e c . D i s . 87:234-248. 90. Sawyer, S . J . , J.B. Macdonald and R.J. Gib b o n s . 1962. B i o c h e m i c a l c h a r a c t e r i s t i c s of B a c t e r o i d e s m e l a n i n o g e n i c u s . A r c h . O r a l B i o l . 2:685-691. 91. Schwabacher, H., D.R. L u c a s and C. R i m i n g t o n . 1947. B a c t e r i u m m e l a n i n o -genicum - a misnomer. J . Gen. M i c r o b i o l . 1_:109-120. 92. Shah, H.N., R.A.D. W i l l i a m s , G.H. Bowden and J.M. H a r d i e . 1976. Comparison of the b i o c h e m i c a l p r o p e r t i e s o f B a c t e r o i d e s m e l a n i n o - g e n i c u s from human d e n t a l p l a q u e and o t h e r s i t e s . J . App. B a c t e r i o l . 41:473-492. 93. S l i f k i n , M. and H.J. H e r c h e r . 1974. Paper chromatography as an a d j u n c t i n t he i d e n t i f i c a t i o n . o f a n a e r o b i c b a c t e r i a . App. M i c r o b i o l . 27: 500-505. 94. S l o t s , J . 1976. The predominant c u l t i v a b l e o r g a n i s m s i n j u v e n i l e p e r i o d -o n t i t i s . Scand. J . Dent. Res. 84:1-10. 137 • L i t e r a t u r e C i t e d ( c o n t i n u e d ) 95. S m i t h , D.T. 1930. F u s o s p i r o c h e t a l d i s e a s e of the l u n g s p r o duced w i t h c u l t u r e s f r o m V i n c e n t ' s a n g i n a . J , I n f e c . Dis.; 46:303-310. 1.96. S o c r a n s k y , S.S. and R.J. Gibbons. 1965. R e q u i r e d r o l e o f B a c t e r o i d e s  m e l a n i n o g e n i c u s i n m i x e d a n a e r o b i c i n f e c t i o n s . J . . " I n f e c . D i s . 115:247-253. 97. S o c r a n s k y , S.S. 1970. R e l a t i o n s h i p o f b a c t e r i a t o t h e e t i o l o g y o f p e r i o d o n t a l d i s e a s e . J . Dent. Res. 49:203-222. 98. S o c r a n s k y , S.S. and S.D. M a n g a n i e l l o . 1970. The o r a l m i c r o b i o t a o f man f r o m b i r t h t o s e n i l i t y . J . P e r i o d o n t . 42:485-496. 99. S o c r a n s k y , S.S. 1977. M i c r o b i o l o g y o f p e r i o d o n t a l d i s e a s e . P r e s e n t s t a t u s and f u t u r e c o n s i d e r a t i o n s . J . P e r i o d o n t . ' 48_:497-504. 100. S t o n e , J . J . and J.D. M a r t i n , J r . 1972. S y n e r g i s t i c n e c r o t i s i n g c e l l u l i t i s . Ann. Surg. 175:702-711. 101. S u t h e r l a n d , I.W. and J.F. W i l k i n s o n . 1971. I n Methods i n M i c r o b i o l o g y . J.R. N o r r i s and D.V. Ribbons (ed.) V o l . 5B p. 345-383. Academic P r e s s , London. 102. S u t t e r , V.L., V.L. Vargo and S.M. F i n e g o l d . Wadsworth A n a e r o b i c B a c t e r i o l o g y Manual 2nd e d i t i o n 1975. Department o f C o n t i n u i n g E d u c a t i o n i n H e a l t h S c i e n c e s U n i v e r s i t y E x t e n s i o n , and The S c h o o l of M e d i c i n e , UCLA. 103. S w i n d l e h u r s t , C A . , H.A. Shah, CW. P a r r and R.A.D. W i l l i a m s . 1977. Sodium d o d e c y l s u l p h a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f p e p t i d e s f r o m B a c t e r o i d e s m e l a n i n o g e n i c u s . J . App. B a c t e r i o l . 43: 319-324. 104. Takazoe, I . and T. Nakamura. 1971. E x p e r i m e n t a l mixed i n f e c t i o n by human g i n g i v a l c r e v i c e m a t e r i a l . B u l l . Tokyo Dent. C o l l . 12:85-93. 105. Takazoe, I . , T. Nakamura, T. Homma, A. Yamamoto, Y. Hashimoto, K. Okuda and M. Tanaka. 1971. Growth-promoting e f f e c t of C o r y n e b a c t e r i u m upon B a c t e r o i d e s . B u l l . Tokyo Dent. C o l l . 12:95-97. 106. Takazoe, I . , M. Tanaka and T. Homma. 1971. A p a t h o g e n i c s t r a i n o f B a c t e r o i d e s m e l a n i n o g e n i c u s . A r c h . O r a l B i o l . 16:817-822. 107. T a k e u c h i , H., M. S u m i t a n i , K. Tsubakimoto and M. T s u t s u i . 1974. O r a l m i c r o o r g a n i s m s i n the g i n g i v a of i n d i v i d u a l s w i t h p e r i o d o n t a l d i s e a s e . J . Dent. Res. 53:136. 138 L i t e r a t u r e C i t e d ( c o n t i n u e d ) 108. T h a d e p a l l i , H., S.L. Gorbach and L, K e i t h . 1973. A n a e r o b i c i n f e c t i o n s o f the f e m a l e g e n i t a l t r a c t ; B a c t e r i o l o g i c and t h e r a p e u t i c a s p e c t s . Am. J . O b s t e t . G y n e c o l . 117:1034-1040. 109. T r a c y , 0. 1969. Pigment p r o d u c t i o n i n B a c t e r o i d e s . J . Med. M i c r o b i o l . .2:309-315. 110. T r e v e l y a n , W.E. and J.S. H a r r i s o n . 1952. S t u d i e s on y e a s t m e t a b o l i s m I . F r a c t i o n a t i o n and m i c r o d e t e r m i n a t i o n o f c e l l c a r b o h y d r a t e s . Biochem. J . 50:298-303. 111. V e s s p r e m i , D. 1905. C e n t r a l b l . f . B a k t . 38:136. 112. ' Wahren, A. and R.J. G i b b o n s . 1970. Amino a c i d f e r m e n t a t i o n by B a c t e r o i d e s m e l a n i n o g e n i c u s . A n t o n i e v a n Leeuwenhoek. 36:149-159. 113. W e i n s t e i n , W.M., A.B. Onderdonk, J.G. B a r t l e t t and S.L. Gorbach. 1974. E x p e r i m e n t a l i n t r a - a b d o m i n a l a b s c e s s e s i n r a t s : Development o f an e x p e r i m e n t a l model. I n f e c t . Immunity. 10:1250-1255. 114. W e i s s , C. 1943. The p a t h o g e n i c i t y of B a c t e r o i d e s m e l a n i n o g e n i c u s and i t s i m p o r t a n c e i n s u r g i c a l i n f e c t i o n s . S u r g e r y . 13: 683-691. 115. Werner, J . , G. P u l v e r e r and C. R e i c h e r t z . 1971. The b i o c h e m i c a l p r o p e r t i e s and a n t i b i o t i c s u s c e p t i b i l i t y of B a c t e r o i d e s m e l a n i n o - g e n i c u s . Med. M i c r o b i o l . Immunol. 157:3-9. 116. W i l l i a m s , R.A.D., G.H. Bowden, J.M. H a r d i e and H. Shah. 1975. B i o c h e m i c a l p r o p e r t i e s of B a c t e r o i d e s m e l a n i n o g e n i c u s s u b s p e c i e s . I n t . J . S y s t . B a c t e r i o l . 25:298-300. 117. W i l s o n , M.R. and A.W. Hohmann. 1974. Immunity t o E s c h e r i c h i a c o l i i n p i g s : A d h e s i o n o f e n t e r o p a t h o g e n i c E s c h e r i c h i a c o l i t o i s o l a t e d i n t e s t i n a l e p i t h e l i a l c e l l s . I n f e c t . Immunity 10:776-782. 

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