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Role of the outer membrane of Pseudomonas aeruginosa in antibiotic resistance Nicas, Thalia Ioanna 1982

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ROLE OF THE OUTER MEMBRANE OF PSEUDOMONAS A E R U G I N O S A I N A N T I B I O T I C RESISTANCE by Thalia B.Sc. M.Sc.  A  loanna  University University  Nicas  of Calgary, of Alberta,  1975 1977  THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE R E Q U I R E M E N T S FOR T H E DEGREE OF DOCTOR OF P H I L O S O P H Y in THE  FACULTY  (Department  We  accept  this  thesis  THE  OF GRADUATE  of Microbiology)  as c o n f o r m i n g  UNIVERSITY  OF  Thalia  to the required  BRITISH  September  (c)  STUDIES  loanna  COLUMBIA  1982  Nicas,  1982  standard  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I further  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 copying of t h i s t h e s i s f o r s c h o l a r l y purposes may department or by h i s or her  be granted by  the head of  representatives.  my  It is  understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  T h a l i a I. N i c a s  Department o f  Microbiology  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6  (3/81)  23. September  1982.  written  ABSTRACT  I t was demonstrated  t h a t i n d u c t i o n o f a major outer  p r o t e i n , HI, was a s s o c i a t e d with i n c r e a s e d r e s i s t a n c e t o c h e l a t o r s o f d i v a l e n t c a t i o n s such as EDTA and t o the c a t i o n i c a n t i b i o t i c s polymyxins  and aminoglycosides.  p r o t e i n HI was the major c e l l u l a r Mg  - d e f i c i e n t medium  with decrease i n c e l l  p r o t e i n i n c e l l s grown i n  (0.02 mM Mg  r e s i s t a n c e t o polymyxin.  Outer membrane  ) and i n mutants s e l e c t e d f o r  Increase i n p r o t e i n HI was a s s o c i a t e d 2+  I n d u c t i o n o f p r o t e i n HI 2+ was prevented by supplementation o f Mg - d e f i c i e n t medium with 0.5 mM M g , C a , M n 2+  Sn  2 +  .  2 +  envelope Mg  2 +  or S r  2 +  C e l l s grown i n C a , M n 2 +  .  , b u t not by Z n , B a , or 2 +  2+  or Z n  2 +  2 +  showed enhanced  levels  of these c a t i o n s as main major c e l l envelope a s s o c i a t e d  cation.  Only c e l l s grown i n the presence o f those c a t i o n s which  failed  t o prevent HI i n d u c t i o n were r e s i s t a n t t o c h e l a t o r s , polymyxin B and gentamicin. demonstrated  P r o t e i n HI overproducing c e l l s  also  a l t e r e d s t r e p t o m y c i n uptake.  I t was f u r t h e r demonstrated  t h a t aminoglycosides could  i n t e r a c t w i t h the outer membrane so as t o make i t more permeable 2+ . to other substances. Mg i n h i b i t e d aminoglycoside-mediated permeabilization. Both aminoglycosides and polymyxin B c o u l d be 2+ shown t o d i s p l a c e a s m a l l amount o f Mg envelope.  from the c e l l  A mutant severely d e f i c i e n t , i n outer membrane p r o t e i n F was i s o l a t e d .  Permeability  of t h i s s t r a i n was studied by  measuring h y d r o l y s i s of a chromogenic beta-lactam by p e r i p l a s mic  beta-lactamase.  I t was found that outer membrane permea-  b i l i t y of P. aeruginosa was low compared to E. c o l i and that l o s s of p r o t e i n F caused a f u r t h e r decrease. gest  that only a small p r o p o r t i o n  The r e s u l t s sug-  of p r o t e i n F molecules form  f u n c t i o n a l channels i n wild type c e l l s so that the h y d r o p h i l i c pathway of uptake across inefficient.  the outer membrane i s r e l a t i v e l y  C a t i o n i c a n t i b i o t i c s such as aminoglycosides and  polymyxins may use an a l t e r n a t e pathway of " s e l f promoted" permeation. cosides  I t i s proposed that EDTA, polymyxin and aminogly-  a c t by a t t a c k i n g  a c r i t i c a l divalent cation  s i t e on the l i p o p o l y s a c c h a r i d e .  binding  P r o t e i n HI i s proposed to act  by r e p l a c i n g d i v a l e n t c a t i o n s at t h i s s i t e , preventing the a c t i o n of these agents.  iv TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS  iv  L i s t of Tables  viii  L i s t of Figures  ix  L i s t of A b b r e v i a t i o n s  x  ACKNOWLEDGEMENTS  xi  INTRODUCTION  1  1.  Medical importance  2.  A n t i b i o t i c r e s i s t a n c e of Pseudomonas aeruginosa  2  3.  Mechanisms of a n t i b i o t i c r e s i s t a n c e  4  4.  The c e l l envelope  6  5.  The gram-negative outer membrane  8  a.  Lipopolysaccharide  9  b.  Protein  6.  of Pseudomonas aeruginosa  and p e r m e a b i l i t y  1  10  The outer membrane of P. aeruginosa and i t s role in a n t i b i o t i c permeability  12  1.  Media and growth c o n d i t i o n s  17  2.  Bacterial strains  17  3.  I s o l a t i o n of mutants d e f i c i e n t membrane p r o t e i n  4.  Bacteriophage  METHODS  i n outer  and b a c t e r i o c i n s t u d i e s  21 23  a.  Bacteriophages and p a r t i a l c h a r a c t e r i z a t i o n of t h e i r r e c e p t o r s  23  b.  Aeruginocin studies  25  c.  I s o l a t i o n of p r o t e i n s p e c i f i c phages from nature  26  d.  Bacteriophage  28  sensitivity  testing  CONTENTS  5.  (cont'd)  e.  I s o l a t i o n of p h a g e - r e s i s t a n t mutants  f.  Phage c h a r a c t e r i z a t i o n by a d s o r p t i o n to whole c e l l s  A n t i b i o t i c and c h e l a t o r s u s c e p t i b i l i t y  testing  a.  Antibiotics  b.  C h e l a t o r and a n t i b i o t i c b a c t e r i o l y s i s and killing  c.  Minimal i n h i b i t o r y c o n c e n t r a t i o n s  6.  S h i f t experiments  7.  Membrane i s o l a t i o n and c h a r a c t e r i z a t i o n of outer membrane, c e l l envelope, and whole c e l l proteins  8.  Determination of c e l l envelope c a t i o n  9.  Displacement of Mg2+ from the c e l l envelope by polymyxin B and aminoglycosides  levels  10.  Streptomycin uptake assays.  11.  Enhancement of n i t r o c e f i n p e r m e a b i l i t y by aminoglycosides and c h e l a t o r s  12.  Measurement of outer membrane p e r m e a b i l i t y by n i t r o c e f i n h y d r o l y s i s  13.  Other assays  RESULTS CHAPTER ONE S u s c e p t i b i l i t y to EDTA-Tris, Polymyxins, and Aminoglycosides 1.  S u s c e p t i b i l i t y to polymyxin B and EDTA-Tris i n M g ^ - s u f f i c i e n t and - d e f i c i e n t media +  2.  S u s c e p t i b i l i t y of polymyxin B r e s i s t a n t mutants  vi CONTENTS  (cont'd) Page  3.  Aminoglycoside r e s i s t a n c e  4.  S u b s t i t u t i o n of other c a t i o n s f o r M g  5.  Minimal i n h i b i t o r y c o n c e n t r a t i o n s polymyxin a t v a r i o u s Mg2+ l e v e l s  6.  Streptomycin uptake and b i n d i n g i n s u s c e p t i b l e and r e s i s t a n t s t r a i n s  48  7.  P e r m e a b i l i z a t i o n of the outer membrane by aminoglycosides  51  8.  Other p r o p e r t i e s of p o l y m y x i n - r e s i s t a n t mutants  52  a.  Resistance to chloramphenicol other a n t i b i o t i c s  52  b.  Ability  c.  Loss of v i a b i l i t y  9.  to accept  42 2 +  of  43 45  and  RPI  53  i n c o l d storage  53  Summary  54  CHAPTER TWO Outer Membrane C h a r a c t e r i z a t i o n  55  1.  Outer membrane p r o t e i n p a t t e r n s  55  2.  E f f e c t s of s h i f t  3.  D i v a l e n t c a t i o n c o n c e n t r a t i o n of c e l l envelopes and displacement of c a t i o n s by aminoglycosides and polymyxin B  62  4.  Comparison of EGTA and EDTA s u s c e p t i b l i t y of Ca + and Mg2+ grown c e l l s  63  5.  P r o t e i n H i i n d u c t i o n i n other Pseudomonas strains  67  6.  Summary  67  from low to high Mg + 2  60  2  vii CONTENTS  (cont'd) Page  CHAPTER THREE I s o l a t i o n and C h a r a c t e r i z a t i o n of a P o r i n D e f i c i e n t Mutant and Bacteriophage Studies  70  1.  I s o l a t i o n of outer membrane p r o t e i n d e f i c i e n t s t r a i n s by random heavy mutagenesis and i s o l a t i o n of phage s p e c i f i c f o r protein receptors  70  2.  Characterization isolate  76  3.  Bacteriophage s e n s i t i v i t y of outer membrane p r o t e i n mutants, mucoid i s o l a t e s and serotype s t r a i n s  78  4.  Summary  72  of a p o r i n - d e f i c i e n t  CHAPTER FOUR Measurement of Outer Membrane P e r m e a b i l i t y  84  Summary  87 90  DISCUSSION LITERATURE  CITED  109  vii i L I S T OF TABLES Page Table I  Bacterial  strains  18  II  B a c t e r i o p h a g e s o u r c e s and r e c e p t o r s  24  III  R e s i s t a n c e t o k i l l i n g by E D T A - T r i s and p o l y m y x i n B o f H103 and i t s p o l y m y x i n B r e s i s t a n t m u t a n t H181. Effect of Mg c o n c e n t r a t i o n i n the g r o w t h medium  41  IV  R e s i s t a n c e o f H103, i t s p o l y m y x i n d e r i v a t i v e s H181 a n d H185 and a r e v e r t a n t H207 t o v a r i o u s antibiotics  44  V  E f f e c t o f g r o w t h i n v a r i o u s d i v a l e n t c a t i o n s on i n d u c t i o n o f o u t e r membrane p r o t e i n H I , l y s i s and k i l l i n g by E D T A - T r i s , and k i l l i n g by p o l y m y x i n B and g e n t a m i c i n  46  VI  L e v e l s o f o u t e r membrane p r o t e i n H I , c e l l envelope M g c o n c e n t r a t i o n , and r e s i s t a n c e t o p o l y m y x i n s o f H103 and i t s p o l y m y x i n B r e s i s t a n t d e r i v a t i v e s H181 a n d H185 a n d a r e v e r t a n t , H207: e f f e c t s of varying Mg c o n c e n t r a t i o n s i n the medium  47  VII  D i v a l e n t c a t i o n s o f c e l l envelopes a f t e r growth in the presence of d i f f e r e n t c a t i o n s  64  VIII  E f f e c t s of EGTA-Tris in Mg and C a  66  2 +  2 +  2 +  2 +  and E D T A - T r i s on c e l l s grown  2 +  IX  R e s u l t s o f enrichment procedure f o r i s o l a t i o n of o u t e r membrane p r o t e i n r e c e p t o r - s p e c i f i c phage  75  X  B a c t e r i o p h a g e s e n s i t i v i t y o f o u t e r membrane p r o t e i n m u t a n t s , s e r o t y p e s t r a i n s and m u c o i d isolates  81  XI  Bacteriophage s u s c e p t i b i l i t y of serotype s t r a i n s o f JP. a e r u g i n o s a p e r f o r m e d u s i n g p h a g e s p r o p a g a t e d and c h a r a c t e r i z e d on P_. a e r u g i n o s a PA01 s t r a i n s  82  XII  R a t e o f n i t r o c e f i n h y d r o l y s i s b y i n t a c t c e l l s and o u t e r membrane p e r m e a b i l i t y c o e f f i c i e n t C o f P. a e r u g i n o s a H103 ( R P l ) , i t s d e r i v a t i v e c a r r y i n g p l a s m i a R P l , Z61 ( R P l ) and E. c o l i UB1636 ( R P l )  88  ix L I S T OF FIGURES Page Figure 1.  E f f e c t o f a d a p t a t i o n on M g - d e f i c i e n t medium, and o f m u t a t i o n t o p o l y m y x i n B r e s i s t a n c e , on s u s c e p t i b i l i t y t o l y s i s by p o l y m y x i n B and EDTA-Tris  40  2.  U p t a k e o f [3JH] s t r e p t o m y c i n a t two c o n c e n t r a t i o n s by t h e w i l d - t y p e s t r a i n H103 and t h e o u t e r membrane p r o d u c i n g s t r a i n H181  49  3.  Time r e q u i r e d f o r i n i t i a t i o n o f r a p i d u p t a k e o f s t r e p t o m y c i n (EDP-II) i n t h e w i l d - t y p e strain H103 a n d t h e o u t e r membrane p r o t e i n HI overproducing s t r a i n  50  4.  E f f e c t o f a d a p t a t i o n on M g - d e f i c i e n t medium and o f m u t a t i o n t o p o l y m y x i n B r e s i s t a n c e on levels of protein Hi  56  5.  E f f e c t o f growth i n d i f f e r e n t d i v a l e n t c a t i o n s on i n d u c t i o n o f p r o t e i n HI  59  6.  E f f e c t o f s h i f t from l o w t o h i g h o f p r o t e i n HI and s u s c e p t i b i l i t y and p o l y m y x i n B  61  7.  Induction of protein grown i n low M g  2 +  2 +  Mg on l e v e l s t o EDTA-Tris 2 +  HI i n Pseudomonas s t r a i n s  68  2 +  8.  Membranes o f h e a v i l y m u t a g e n i z e d s t r a i n s w i t h apparent p r o t e i n a l t e r n a t i o n s  72  9.  C e l l e n v e l o p e s o f w i l d - t y p e s t r a i n H103, i t s p o r i n d e f i c i e n t m u t a n t H283 and a r e v e r t a n t , H284  77  Model i l l u s t r a t i n g t h e proposed mechanism o f r e s i s t a n c e t o a m i n o g l y c o s i d e s , p o l y m y x i n B, and E D T A - T r i s i n P. a e r u g i n o s a w i t h h i g h l e v e l s o f p r o t e i n Hi  94  10.  X  L I S T OF  600 BM2 EDTA  ABBREVIATIONS  A b s o r b a n c e a t 600 nm. B a s a l medium number 2, a p h o s p h a t e b u f f e r e d medium ( G i l l e l a n d e t a l , 1 9 7 4 ) .  minimal  Ethylenediaminetetraacetetate.  EGTA  Ethyleneglycol-bis(beta-ethylether)N,N'tetraacetate.  LPS  Lipopolysaccharide.  MIC  Minimal  NTG  inhibitory  concentration.  N-methyl-N'-nitro-nitrosoguanidine.  PP2  Proteose  p e p t o n e number 2 medium.  SDS  Sodium d o d e c y l  sulphate.  xi  ACKNOWLEDGEMENTS  I wish to thank R.E.W. Hancock f o r h i s warm f r i e n d s h i p and c o n s i s t e n t encouragement.  I am g r a t e f u l f o r the support of a Frank Wesbrook s c h o l a r s h i p during most of my s t u d i e s .  1  INTRODUCTION  Pseudomonas aeruginosa i s a gram-negative found w i d e l y d i s t r i b u t e d  i n the environment.  bacterium  During the l a s t  2 0 - 2 5 years P. aeruginosa has a c q u i r e d n o t o r i e t y as a major o p p o r t u n i s t i c pathogen. emergence i n t h i s r o l e  One  of the primary reasons f o r i t s  i s i t s r e s i s t a n c e to commonly used  antibiotics. 1.  Med i c a l  importance'of Pseudomonas aerug inosa .  The  intro-  d u c t i o n of e f f i c i e n t a n t i - s t a p h y l o c o c c a l agents has played a major r o l e tions.  i n a l t e r i n g p a t t e r n s of h o s p i t a l - a c q u i r e d  Consequently, gram-negative  b a c t e r i a have r e p l a c e d  s t a p h y l o c o c c i as the major source of nosocomial aeruginosa i n p a r t i c u l a r a n t i s e p t i c s , and  infec-  infections.  P_.  i s w e l l known f o r i t s r e s i s t a n c e to  i s often d i f f i c u l t  to e r a d i c a t e .  Extensive  use of broad spectrum a n t i b i o t i c s has a l s o favoured the s e l e c t i o n of P. a e r u g i n o s a , so that i t i s now hazard i n the h o s p i t a l  a major p o t e n t i a l  environment.  P_. aeruginosa does not cause d i s e a s e i n h e a l t h y , uninjured i n d i v i d u a l s .  However, i n i n d i v i d u a l s with impaired  defense mechanisms, i t i s one of the most f r e q u e n t l y pathogens.  As modern medicine improves  the s u r v i v a l r a t e f o r  p a t i e n t s with severe burns, n e o p l a s t i c d i s e a s e and f i b r o s i s , an i n c r e a s i n g been noted  isolated  cystic  i n c i d e n c e of Pseudomonas i n f e c t i o n s  has  (Levine e_t a l . , 1 9 7 4 ; Pennington e_t a l . 1 9 7 5 ;  Reynolds e t a l . , 1 9 7 5 ) .  Treatment with immunosuppressive  and  2  c y t o t o x i c drugs and c o r t i c o s t e r o i d s , as w e l l as many h o s p i t a l techniques  (e.g., c a t h e t e r i z a t i o n ) which can r e s u l t i n  i n t r o d u c t i o n of organisms contributed  i n t o s u s c e p t i b l e t i s s u e s have  to the growing  prevalance of i n f e c t i o n s by gram-  negative b a c t e r i a , e s p e c i a l l y J?. a e r u g i n o s a . a predominant cause of burn-wound i n f e c t i o n s . some s t u d i e s have found by 5 days a f t e r  injury  P.  For example,  that i t c o l o n i z e s up t o 6 0 % of p a t i e n t s ( P r u i t t e_t a l . ,  1979).  The  P. aeruginosa i s a l s o extremely high among cancer especially resulting  aeruginosa i s  frequency of patients,  i n p a t i e n t s with acute leukemia or other d i s e a s e s i n neutropenia  r a t e s of such  (Rodriguez and Bodey, 1 9 7 9 ) .  Fatality  i n f e c t i o n s are r e p o r t e d to be as high as 5 0 - 8 0 %  (Rodriguez and Bodey, 1 9 7 9 ) .  P. aeruginosa i s a major pathogen  i n p a t i e n t s with c y s t i c f i b r o s i s , where i t i s the predominant bacterium a s s o c i a t e d with t e r m i n a l pulmonary i l l n e s s .  It i s  a l s o an important cause of h o s p i t a l - a c q u i r e d pneumonias, h o s p i tal-acquired urinary tract  i n f e c t i o n s , and one of the major  causes of i n f e c t i o n of p a t i e n t s undergoing  2.  invasive  procedures.  A n t i b i o t i c r e s i s t a n c e of Pseudomonas a e r u g i n o s a .  P. aeruginosa has been c h a r a c t e r i s t i c a l l y regarded as r e s i s t a n t to a n t i m i c r o b i a l agents, although i n t r o d u c t i o n of new c r o b i a l drugs  s p e c i f i c a l l y developed  antimi-  f o r anti-pseudomonal  t i v i t y has somewhat changed t h i s s i t u a t i o n  (Bryan, 1 9 7 9 ) .  small number of a n t i b i o t i c s which show high i_n v i t r o a g a i n s t P. aeruginosa i n c l u d e some aminoglycoside  acThe  activity  antibiotics  3  such as gentamicin, tobramycin, amikacin and t h e i r  recently  developed analogues such as n e t i l m i c i n and sisomycin, polymyxin a n t i b i o t i c s such as polymyxin B and c o l i s t i n , semi-synthetic beta-lactam a n t i b i o t i c s , lin,  i t s t h i e n y l analogue t i c a r c i l l i n ,  and a v a r i e t y o f  including  carbenicil-  and some new broad  spectrum c e p h a l o s p o r i n s such as cefotaxime and moxalactam. For  a l l anti-pseudomonal a n t i b i o t i c s c u r r e n t l y i n  use, e f f e c t i v e n e s s i s l i m i t e d by the narrow margin between safe and e f f e c t i v e doses (Young, 1979;  Bryan, 1979).  This i s par-  t i c u l a r l y t r u e f o r aminoglycosides, which are o t o t o x i c and can cause r e n a l damage, ( W e r s a l l ^t_ a_l. , 1969;  F a l c o ejt a_l. ,  1969)  and f o r polymyxins, which are g e n e r a l l y c o n s i d e r e d to be too t o x i c f o r p a r e n t e r a l use (Bryan, 1979).  Furthermore, there has  g e n e r a l l y been a poor c o r r e l a t i o n between the i n v i t r o s u s c e p t i b i l i t y t o a n t i m i c r o b i a l agents and i n v i v o t h e r a p e u t i c efficacy  (Davis, 1974;  F l i c k and C u f f , 1976).  One  possible  cause f o r t h i s i s the o c c u r r e n c e o f a d a p t i v e (non-mutational) r e s i s t a n c e i n v i v o , as has been demonstrated f o r a v a r i e t y of antibiotics in vitro al.,  1974;  (Brown and M e l l i n g , 1969;  Pechey and James, 1974).  P. a e r u g i n o s a which i s becoming t h a t the c e l l  G i l l e l a n d et  One o f the p r o p e r t i e s o f  c l e a r from r e c e n t s t u d i e s i s  envelope o f P_. aeruginosa i s h i g h l y  variable  depending on the growth c o n d i t i o n s t o which the organism i s subjected  (Brown and M e l l i n g , 1969; C o s t e r t o n _et jal. ,  Hancock and Carey, 1981; Hancock et a l . , with t h i s ,  i t now  1982).  In  1979;  agreement  appears t h a t the outer membranes o f c e l l s  grown i n v i v o are somewhat d i f f e r e n t from those grown i n v i t r o  4  (P.A. Sokol, D.E. script  Woods, C D .  Cox,  and B.H.  I g l e w s k i , manu-  i n p r e p a r a t i o n ; K. Poole and R.E.W. Hancock, unpublished  data). efficacy  Some of these changes may (Costerton et a l . ,  well affect  antibiotic  1979).  An a d d i t i o n a l cause of the poor c o r r e l a t i o n between i n v i v o and  iri v i t r o e f f e c t i v e n e s s of a n t i b i o t i c s 2+  onism of a n t i b i o t i c a c t i v i t y by Ca with polymyxin  2+ and Mg  , which occurs  and aminoglycoside a n t i b i o t i c s  Z i m e l i s and Jackson, 1973). d i v a l e n t c a t i o n antagonism greater significance negative b a c t e r i a  i s the antag-  (Newton,  1954;  In the case of a m i n o g l y c l o s i d e s , of a n t i b i o t i c a c t i o n  i s of much  i n P_. aeruginosa than i n other gram-  (Madeiros e_t a_l., 1971).  to 3 2 - f o l d have been observed  D i f f e r e n c e s of up  i n measurements of minimal  inhib-  i t o r y c o n c e n t r a t i o n s of gentamicin i n M u e l l e r Hinton medium with v a r y i n g l e v e l s of C a  2 +  and M g  2+  (Reller et a l . ,  1974).  It  2+  has been suggested by s e v e r a l workers that the s i t e of Mg Ca^  antagonism  Jackson, 1973;  i s the c e l l envelope Brown, 1975).  (Newton, 1954;  and  Z i m e l i s and  The study r e p o r t e d here  offers  evidence that the s i t e of competition i s the outer membrane. 3.  Mechanisms of a n t i b i o t i c r e s i s t a n c e .  Resistance of P_.  aeruginosa to a n t i m i c r o b i a l agents takes two g e n e r a l forms (Bryan, 1979).  One  of these i s r e s i s t a n c e to agents  a g a i n s t most other gram-negative "intrinsic"  resistance.  bacteria, generally  The second  form  i s emerging  to the more r e c e n t l y introduced anti-pseudomonal gentamicin and c a r b e n i c i l l i n .  effective termed resistance  agents such as  In many cases the l a t t e r form of  5  r e s i s t a n c e may  be a t t r i b u t e d to i n a c t i v a t i n g enzymes u s u a l l y  s p e c i f i e d by R f a c t o r s  (Bryan, 1979).  I n t r i n s i c r e s i s t a n c e does not, presence of R f a c t o r s , and P. a e r u g i n o s a . only  the  i s present i n n e a r l y a l l s t r a i n s of  A n t i b i o t i c i n a c t i v a t i n g enzymes appear to have  a minor r o l e in i n t r i n s i c r e s i s t a n c e .  somally-specified,  For example chroma-  i n d u c i b l e c e p h a l o s p o r i n a s e i s reported  present i n a l l s t r a i n s of P_. aeruginosa al.,  however, r e q u i r e  1965), which may  (Bryan, 1979;  i n p a r t account f o r r e s i s t a n c e  to  be  Sabath e_t to some  beta-lactam a n t i b i o t i c s . Other chromosomally-specified i n a c t i v a t i n g enzymes have not been found R e s i s t a n c e at the a l s o does not  l e v e l of the  (Bryan e_t a l . , 1975).  t a r g e t s of a n t i b i o t i c  appear to account f o r i n t r i n s i c  resistance.  example, Bryan et a l . (1975) have shown that p r o t e i n by c e l l - f r e e systems derived  activity  synthesis  from P. aeruginosa i s f u l l y  t i v e to i n h i b i t i o n by streptomycin, whereas whole c e l l s quite r e s i s t a n t .  S i m i l a r l y , Mirleman and  For  sensiare  Nuchamowitz (1979)  have shown that the enzymes which s y n t h e s i z e  peptidoglycan  in  P. aeruginosa are at l e a s t as s e n s i t i v e to b e n z y l p e n i c i l l i n as those of E_. c o l i , whereas whole c e l l s are much more r e s i s t a n t . As n e i t h e r a n t i b i o t i c  i n a c t i v a t i o n nor  altered targets  a n t i b i o t i c a c t i o n would seem to account f o r the high  of  antibiotic  r e s i s t a n c e of P. aeruginosa, a p o s s i b i l i t y to be considered that a n t i b i o t i c s are unable to reach t h e i r s i t e of Bryan and glycosides  colleagues  is  activity.  have shown that d e f e c t i v e uptake of amino-  (Bryan, 1979)  and  t e t r a c y c l i n e (Tseng and  Bryan,  6  1974)  c o r r e l a t e s with r e s i s t a n c e to these drugs.  In a d d i t i o n ,  enhanced p e r m e a b i l i t y of the outer membrane has been shown to c o r r e l a t e with  increased s u s c e p t i b l i t y  t o 27 d i f f e r e n t  i n the a n t i b i o t i c s u p e r s u s c e p t i b l e P_. aeruginosa (Angus et. a l . ,  1982).  impermeable to a n t i b i o t i c s  4.  explanation  is relatively  (Brown, 1975; Bryan, 1979).  T h e c e l l envelope and p e r m e a b i l i t y .  P. aeruginosa,  mutant Z61  Thus perhaps the most l i k e l y  of i n t r i n s i c r e s i s t a n c e i s that P. aeruginosa  agents  The c e l l envelope of  l i k e that of other gram-negative b a c t e r i a has  been shown by e l e c t r o n microscopy to c o n s i s t of three l a y e r s . These are the i n n e r , or cytoplasmic can, and the outer membrane.  membrane, the p e p t i d o g l y -  In some i n s t a n c e s , e s p e c i a l l y i n -  f e c t i o n s of c h i l d r e n with c y s t i c  f i b r o s i s , a capsule  of mucoid  m a t e r i a l may a l s o be p r e s e n t .  The space between the inner and  outer membrane, the p e r i p l a s m ,  i s the l o c a t i o n of a v a r i e t y of  degradative ics  enzymes, i n c l u d i n g those which i n a c t i v a t e a n t i b i o t -  (Benveniste  and Davies,  1973; Bryan, 1979).  The inner  membrane i s the s i t e of s p e c i f i c , e n e r g y - r e q u i r i n g , systems, as w e l l as components i n v o l v e d i n energy  transport  generation  and most o f the enzyme systems i n v o l v e d i n s y n t h e s i s of the external wall l a y e r s , peptidoglycan The  and the outer membrane.  inner membrane c o n s t i t u t e s a h i g h l y s e l e c t i v e  barrier  (Costerton e t a l . , 1974).  face of the cytoplasmic (Machtiger  permeability  Both the inner and outer  membrane are thought to be hydrophobic  and Fox, 1973), and i t has been demonstrated that  7  t h i s membrane does not c o n s t i t u t e an e f f e c t i v e b a r r i e r hydrophobic substances  (Teuber and M i l l e r , 1 9 7 7 ) .  the inner membrane i s r e l a t i v e l y substances.  against  In c o n t r a s t ,  impermeable to h y d r o p h i l i c  Such substances cross the inner membrane by means  of s u b s t r a t e s p e c i f i c t r a n s p o r t systems.  Thus h y d r o p h i l i c  a n t i b i o t i c s are unable to enter the cytoplasm  unless  they are  able to mimic a n a t u r a l s u b s t r a t e and use i t s t r a n s p o r t system, or d i s r u p t the membrane s u f f i c i e n t l y The  peptidoglycan  d i f f e r chemically 1975), although  to allow permeation.  of P. aeruginosa  does not appear to  from that of e n t e r i c organisms (Meadow,  i t does not appear to be c o v a l e n t l y l i n k e d to  an outer membrane p r o t e i n analogous to the major l i p o p r o t e i n of E_. c o l i  (Hancock e t a l . , 1 9 8 1 ) .  that the p e p t i d o g l y c a n  makes a major c o n t r i b u t i o n to the low  a n t i b i o t i c p e r m e a b i l i t y of P. As  However, i t seems u n l i k e l y  aeruginosa.  the t a r g e t s of a n t i b i o t i c  w i t h i n the cytoplasm envelope i t s e l f  a c t i v i t y are e i t h e r  (e.g., the ribosomes) or w i t h i n the c e l l  (e.g., the p e n i c i l l i n  binding p r o t e i n s exposed  on the outer s u r f a c e of the inner membrane), the common b a r r i e r which must be t r a v e r s e d i s the outer membrane. growing body of evidence of J?. aeruginosa  There i s a  that much of the i n t r i n s i c r e s i s t a n c e  may be accounted f o r on the b a s i s of the per-  m e a b i l i t y p r o p e r t i e s of i t s outer membrane.  The outer membrane  of gram-negative b a c t e r i a has a major r o l e as a p e r m e a b i l i t y barrier.  Nikaido  (Nikaido and Nakai, 1979) has d e s c r i b e d two  general pathways f o r d i f f u s i o n of small molecules across the  8  outer membrane, one f o r hydrophobic h y d r o p h i l i c compounds. unimportant  compounds and one f o r  The hydrophobic  pathway i s a p p a r e n t l y  i n the outer membrane o f organisms such as E. c o l i ,  Salmonella and P_. aeruginosa, which s y n t h e s i z e complete l i p o p o l y s a c c h a r i d e s although i t s e x i s t e n c e was demonstrated i n deep rough organisms.  The h y d r o p h i l i c pathway i s mediated by  s p e c i f i c i n t e g r a l membrane p r o t e i n s which form t r a n s membrane channels  o r pores.  Such molecules  " p o r i n s " (Nikaido and Nakai,  5.  are g e n e r a l l y termed  1979).  P r o p e r t i e s o f the outer membrane o f gram-negative b a c t e r i a .  The o u t e r membrane o f e n t e r i c b a c t e r i a has been e x t e n s i v e l y studied al.,  ( f o r reviews  see N i k a i d o and Nakae, 1979; DiRienzo et  1980; Osborn and Wu, 1980) and c o n s i d e r a b l e i n s i g h t has  been achieved with regard t o both outer membrane s t r u c t u r e and the r e l a t i o n s h i p o f s t r u c t u r e t o the f u n c t i o n o f the outer membrane as a p e r m e a b i l i t y b a r r i e r .  The major components o f the  o u t e r membrane a r e p r o t e i n , p h o s p h o l i p i d and l i p o p o l y s a c c h a r i d e (LPS).  L i k e other membranes, the outer membrane appears as a  b i l a y e r i n the e l e c t r o n microscope. brane o f E. c o l i 1979)  and Salmonella  Studies o f the o u t e r mem-  (reviewed by Nikaido and Nakae,  have shown t h a t the o u t e r membrane i s unusual, however,  i n the extreme assymetry o f d i s t r i b u t i o n o f membrane components:  v i r t u a l l y a l l the p h o s p h o l i p i d s a r e l o c a t e d on the i n n e r  face (except, p o s s i b l y i n c e r t a i n mutants) w h i l e v i r t u a l l y a l l the LPS i s on the outer s u r f a c e .  In c o n t r a s t , the p r o t e i n s o f  9  outer  membrane a r e  cases,  membrane  a. similar  0rskov  present  spanning  (Enderman  by  using  The  1977.  an a m p h i p a t h i c  a^. ,  LPS i s  portion,  l i p i d A,  out  from the the  repeating  cell  This  antisera,  allows  portion,  species,  and o f t e n  olamine phosphate fluorescent  divalent 1978) .  fine  contains  aeruginosa  This  result  account  hydrophobic rifampicin. ance of  in  P_.  to  in  such  for  antibiotics It  the  of  should  aeruginosa  the  contrast compounds  is the  of  noted  that  to hydrophobic  screened strains.  sugar  and and  sites  ethanstudies  for  bacteria  highly  with  carbon  (Schindler  et  impermeable of  and Nakae,  these D,  the high  a l . ,  a n d P_.  permeability  (Nikaido  resistance  within  variability  enteric  to  even  of  region,  LPS  the  oligosac-  Phosphate  this  of  simply  eight  such as a c t i n o m y c i n be  of  typing  less  the  LPS o f  which  portion  indicated binding  portion  of  in  a membrane w h i c h  compounds,  bilayers  would  this  properties  hydrophobic pholipid  in  have  the  c a n be  and a h e p t o s e .  by  embedded i n  portion,  a unique  is  reviewed  variability  shows  LPS  a  be  serological  some  molecule with  consists  which  R-core,  in  of  been  distal  wide  also present  probes  cations The  are  The  often  property,  the  to  polysaccharide  and shows  species.  structure  and has  believed  0 antigen,  units,  general  surface.  (2-keto-3-deoxyoctonate),  with  1978).  a l . ,  bacteria  proximal  given  et  gram-negative  all  polysaccharide,  single  and a r e ,  in  membrane and a h y d r o p h i l i c  a  layers,  The  hydrophobic  charide  both  Lipopolysaccharide.  ^t  extends  in  phos-  1979) .  organisms  to  erythromycin level  antibiotics  of  to  and  resist-  (Bryan,  1979)  10  strongly pathway  suggests exists  b.  in  Protein.  that  no s i g n i f i c a n t h y d r o p h o b i c  this  organism  The  responsible  for  hydrophilic  compounds.  appear  to  across  the  the  function outer  uptake of  (Hanke, trans are  1976),  low  diffusion apparent  outer  channels.  pore-forming  pholipid  or  in  studies  which  examined.  proteins  porin  is  lipid bilayers  N i k a i d o and c o w o r k e r s  thus  (Nikaido  channels  limiting  (Benz  the  appear size  et  Such  proteins  present The  major  comes  have  present  an in  They a r e p r e s e n t  et  generally  a l . ,  to have  The  into  in  phos-  1976a and 1976b)  1978).  Experiments  1979) have  a fairly  molecules  in  established  incorporated  a l . ,  all  closely  1978).  has been  (Nakae,  at  transmembrane  generally  and Nakae,  of  maltodex-  however,  are  for  nucleosides  and  1979).  and a r e  (DiRienzo  these  where  by  diameter,  far  required  substrates  form general  membrane s p a n n i n g ,  of  proteins  1976),  1976).  and N a k a e ,  to  compounds  1973),  32,000-42,000,  peptidoglycan  porin  a l . ,  uptake of  phospholipid-LPS vesicles  black  specific  et  largely  membrane  membrane  et_ a _ l . ,  which  of  so  into  that  the  (Nikaido  or  lished  of  Such p r o t e i n s ,  function  reconstitution  outer  membrane p e r m e a b i l i t y ,  bacteria  associated with  outer  (Szmeleman and Hufnung,  important  number,  the  (Hancock  B12 ( D i m a s i  molecular weight  copy  of  membranes a r e  These i n c l u d e p r o t e i n s  called porins,  gram-negative  outer  transport  chelates  vitamin  to  from p r o t e i n s  high  the  concentrations  contribution  of  A number o f  membrane.  and m a l t o s e  either.  permeability  in  iron  especially  very  the  proteins  uptake  able  estab-  constant to  cross  the  11  outer membrane, which consequently acts as a molecular E x c l u s i o n l i m i t s of E_. c o l i and measured as 550-650 d a l t o n s that of P. aeruginosa Nikaido,  1978).  permeability  has  The  1977;  (Decad and  p o r i n s have been  Nikaido,  f u n c t i o n of p o r i n s  Nikaido,  1977;  a l t e r n a t e p o r i n s which can be  induced  conditions  Nikaido,  1982) It  or prophage i n f e c t i o n  and  Lutkenhaus,  p o r i n species  been found i n s e v e r a l b a c t e r i a (Osborn and Wu,  Hancock and  (Hancock  in controlling  More than one  (von Meyenburg and  while  i n s t u d i e s of mutants d e f i c i e n t  (von Meyenburg and  B a v o i l et a l . , 1977).  Lugtenberg, 1970;  1976)  i s l a r g e r , 3000-9000 d a l t o n s  been confirmed  i n these p r o t e i n s  Salmonella  sieve.  1980),  has  and  by s p e c i f i c growth 1977;  Carey, 1980;  Tommassen  and  Hancock e_t a l . ,  (Schnaitman, 1974)  are a l s o known.  i s , however, unclear whether such p o r i n s are t r u l y capable  of mediating g e n e r a l i z e d p e r m e a b i l i t y "major" p o r i n s do. p o r i n s and  The  i n the same way  that  c o n t r i b u t i o n of p r o t e i n s other  than  s p e c i f i c t r a n s p o r t p r o t e i n s i s not known, but i t  would appear l i k e l y i s f o r the most p a r t  that t h e i r r o l e i n determining  permeability  indirect.  P o r i n a l t e r a t i o n s i n E_. c o l i  have been shown to  a f f e c t the uptake of some beta-lactams, chloramphenicol, tetracycline Chopra and  the  (Nikaido et a_l. , 1977;  E c c l e s , 1978;  Foulds,  Van  1976)  these p r o t e i n s are l a r g e l y r e s p o n s i b l e  Alphen et. _ a l . , and  and 1978;  i t would appear t h a t  f o r the p e r m e a b i l i t y  h y d r o p h i l i c a n t i b i o t i c s to the outer membrane.  of  Only a s i n g l e  i s o l a t e d example of an a n t i b i o t i c which uses a s p e c i f i c  outer  12  membrane t r a n s p o r t  protein  found:  which  ing  albomycin,  proteins  note  that  6.  is  a ferrichrome  outer  in  antibiotic  appear  its  differences  for  t o be  susceptibility  examined by  and C a r e y , have been  protein  of  enteric  unstable  sodium dodecyl  have  protein (Hancock pore  and C a r e y ,  with  during 1981).  1978; Hancock  a relatively  to  eight  1980). in  of  E. c o l i  to  be  the  and  signifi-  different  P_.  aeruginosa  (Stinnett  major  and  outer  has been  This  that  Eagon,  it  membrane demonstrated  molecule is  differs  unusually  sulphate,  and has  two  intra-chain  and Carey,  1979)  Two  inducible  These  and p r o t e i n  low e x c l u s i o n  g r o w t h on p h o s p h a t e - d e f i c i e n t A lipoprotein,  P.  and N i k a i d o , 1 9 7 8 ;  g r o w t h on g l u c o s e  1980),  membrane o f  membrane o f  a l s o been demonstrated.  induced during  in  to  laboratories  bacteria  (Hancock  protein  to  aeruginosa.  outer  a l . ,  important  role  appear  Porin activity  of  porins  there  found. et  is  been  transport-  and i t s  that  Six  from p o r i n s  bridges  the  it  outer  1979).  (Hancock  disulfide  the  contribute  P_.  several  F  to  aeruginosa  design,  of  membrane h a s  analogue.  similar to  w h i c h may w e l l  Mizuno and Kageyama,  proteins  P.  Although  overall  The p r o t e i n s  Hancock  1976) — a l t h o u g h  membrane o f  permeability.  outer  Ton A f e r r i c h r o m e  albomycin  Salmonella  1973;  the  a l . ,  The  been  uses  the  et  aeruginosa would  have  cross  (Braun  antibiotic  cant  to  I,  P,  are  as an  limit,  protein  sole  carbon  a  source  anion-selective which  appears  medium (Hancock  analogous  Dl,  to  the  et  Braun  a l . ,  13  l i p o p r o t e i n o f E. c o l i  ( B r a u n , 1975), h a s been d e m o n s t r a t e d i n  P. a e r u g i n o s a , a n d , a l t h o u g h t h i s p r o t e i n associated, of  i t may  t h e E_. c o l i  glycan  protein  I i s disputed  1981).  attached  the the  a s s o c i a t i o n o f t h e P.  substantially.  30%  peptido-  aeruginosa  1979; Hancock ejt  A second l i p o p r o t e i n  ( M i z u n o , 1979).  gel electrophoresis,  In sodium d o d e c y l  this protein  (H2) i s s e p a r a t e d  from a p r o t e i n o f s i m i l a r m o l e c u l a r weight described  by Hancock and C a r e y  (HI) o n l y (1979).  o f H2, and o f t h e o t h e r m a j o r o u t e r p r o t e i n s still  i n that  I n any c a s e , t h e amino a c i d c o m p o s i t i o n s o f t h e s e  has a l s o been d e m o n s t r a t e d  conditions  peptidoglycan  protein  ( M i z u n o and Kageyama,  two l i p o p r o t e i n s d i f f e r  sulphate  f r o m E. c o l i  i s covalently  whereas the c o v a l e n t  protein al.,  well differ  is  D2,  under  The  function  E, and G, i s  unknown. Although  i t h a s been e s t a b l i s h e d  that  the s i z e of  i n d i v i d u a l p o r i n c h a n n e l s i s l a r g e r i n P. a e r u g i n o s a t h a n i n enteric bacteria 1978) , t h e r e  (Benz and H a n c o c k , 1 9 8 1 ; Hancock and  i s evidence accumulating  that  t h e number o f  f u n c t i o n a l p o r e s and c o n s e q u e n t l y t h e t o t a l available via  membrane p e r m e a b i l i t y t y p e P.  aeruginosa  t h a t an a n t i b i o t i c Z61,  area of pore  f o r d i f f u s i o n i s much l o w e r , s o t h a t  the h y d r o p h i l i c pathway i s r e l a t i v e l y  selected  has been shown i n in (Angus e_t a l . , 1 9 8 2 ) .  low. vivo  total Low  susceptible  permeability outer  studies  of w i l d  These w o r k e r s  s u p e r s u s c e p t i b l e m u t a n t o f P.  as a n t i b i o t i c  Nikaido,  (Zimmerman  showed  aeruginosa, and  Rosselet,  1979) showed g r e a t l y e n h a n c e d o u t e r membrane p e r m e a b i l i t y ,  thus  14  p r o v i d i n g evidence that low outer membrane p e r m e a b i l i t y i s indeed a major determinant i n i n t r i n s i c r e s i s t a n c e to a n t i b i o t ics.  In Z61, increased  permeability  c e p t i b i l i t y were a s s o c i a t e d et a l . , 1982).  and a n t i b i o t i c  supersus-  with an LPS a l t e r a t i o n ( K r o p i n s k i  Low jLn v i v o p o r i n a c t i v i t y of w i l d type  cells  c o r r e l a t e s w e l l with the low pore forming a c t i v i t y of p r o t e i n F which has been found i n i n v i t r o s t u d i e s 1981).  These o b s e r v a t i o n s ,  (Benz and Hancock,  however, d i d r a i s e the question as  to whether the a c t u a l p o r i n was p r o t e i n F, or some minor contaminant c o p u r i f i e d with p r o t e i n F.  T h i s study r e p o r t s the  i s o l a t i o n of a mutant s e v e r e l y d e f i c i e n t i n p r o t e i n F. of p e r m e a b i l i t y  s t u d i e s with t h i s mutant confirm  Results  the pore-  forming f u n c t i o n of p r o t e i n F and i n d i c a t e that l e s s than 1% o f p r o t e i n F molecules form f u n c t i o n a l channels across  the outer  membrane. Another unusual property i s i t s unusually  of the LPS of P_. aeruginosa  high phosphate content  T h i s phosphate i s a s s o c i a t e d p a r t , as t r i p h o s p h a t e  (Drewry et, a l . , 1971).  with the core  (Wilkinson,  P_. aeruginosa a l s o has very high  1981).  r e g i o n , and i s , i n The c e l l envelope of  l e v e l s of d i v a l e n t  cations  (Brown and Wood, 1972), which may w e l l be a s s o c i a t e d with these phosphate groups since phosphate c a r r i e s a net negative at n e u t r a l pH. antibiotics,  While P. aeruginosa i s unusually  charge  r e s i s t a n t to  i t i s h i g h l y s u s c e p t i b l e to c h e l a t o r s of d i v a l e n t  c a t i o n s such as EDTA (Cox and Eagon, 1968), and to polymyxin antibiotics.  For example, although treatment of E. c o l i  with  15  EDTA i s i n s u f f i c i e n t to allow osmotic  l y s i s unless lysozyme i s  present  of P. aeruginosa  ( L e i v e , 1965), EDTA treatment  in osmotically f r a g i l e c e l l s .  Tris(hydroxymethylJaminomethane  ( T r i s ) maximizes t h i s EDTA e f f e c t Treatment with EDTA r e s u l t s  results  (Eagon and A s b e l l ,  1966).  i n the r e l e a s e of LPS-protein  complexes with low ( l e s s than 10%) p h o s p h o l i p i d content et a l . ,  1969; S t i n n e t t and Eagon, 1975).  (Rogers  These complexes can  be v i s u a l i z e d by e l e c t r o n microscopy  of f r e e z e f r a c t u r e d c e l l s  and appear to be d i s t i n c t aggregates  i n the plane of the  membrane  ( S t i n n e t t and Eagon, 1975).  I t has been  concluded  from these and other s t u d i e s (Roberts ejt a l . , 1970; Kenward e_t al.,  1979; Boggis  critical  e t a l . , 1979) that d i v a l e n t c a t i o n s play a  r o l e i n m a i n t a i n i n g the s t a b i l i t y of the outer  membrane. Polymyxins are amphipathic  molecules  h i g h l y c a t i o n i c peptide head and a hydrophobic al.,  1977).  c o n s i s t i n g of a tail  (Storm e_t  L i k e EDTA, polymyxins appear to a c t d i r e c t l y on  the outer membrane, and are known to bind with high a f f i n i t y to LPS  (Cooperstock,  1974; S c h i n d l e r and Osborn, 1979).  a c t i o n of polymyxins i s i n h i b i t e d by the presence cations  (Newton, 1954).  The  of d i v a l e n t  These o b s e r v a t i o n s have l e d to the  suggestion t h a t polymyxins and EDTA a c t a t a common s i t e on the o u t e r membrane, a d i v a l e n t c a t i o n b i n d i n g s i t e on the LPS which i s r e q u i r e d f o r outer membrane s t a b i l i t y The  (Brown, 1975).  study reported here provides experimental  dence supporting t h i s s u g g e s t i o n .  evi-  Furthermore, evidence i s  16  presented t h a t aminoglycoside a n t i b i o t i c s may a l s o be a c t i v e at t h i s same s i t e  i n P. aeruginosa, and that d i s r u p t i o n of t h i s  s i t e by aminoglycosides and s i m i l a r c a t i o n i c substances may provide  an a l t e r n a t e pathway across  study a l s o p r o v i d e s observation  an e x p l a n a t i o n  the outer membrane. f o r the  This  long-standing  that the s u s c e p t i b i l i t y of P. aeruginosa to EDTA 2+  and  polymyxin may be reversed  (Brown and M e l l i n g , 1969). crease  by growth i n Mg  I t i s demonstrated that t h i s i n -  in resistance i s associated  outer membrane p r o t e i n HI.  - l i m i t e d medium  with i n d u c t i o n of a major  I t i s suggested that H i acts to  r e p l a c e d i v a l e n t c a t i o n s at a c r i t i c a l  divalent cation  binding  s i t e on the LPS, p r o t e c t i n g t h i s s i t e from a t t a c k by EDTA and cationic  antibiotics.  17  METHODS  1.  M e d i a and g r o w t h c o n d i t i o n s .  ( D i f c o , PP2) was u s e d was  used as a r i c h medium.  B a s a l Medium No.  ( 1 9 7 4 ) , c o n t a i n i n g 10 mM succinate cose).  (BM2  The  2 (BM2) FeS04,  amounts s p e c i f i e d  was  d e f i c i e n t media  O t h e r c a t i o n s were added i n the t e x t .  potassium  0.5  (Mg  c o n t a i n e d 0.02  as c h l o r i d e Liquid  mM  salts  mM  i n the  c u l t u r e s were grown w i t h  v i g o r o u s a e r a t i o n a t 37°C e x c e p t where s t a t e d o t h e r w i s e . g l a s s w a r e u s e d w i t h d e f i n e d m e d i a was with d i s t i l l e d  e_t a l .  ( w t / v o l ) g l u c o s e (BM2 g l u -  u s u a l l e v e l o f MgS04 added Mg"  2  by G i l l e l a n d  e i t h e r 20 mM  a n d  no.  The m i n i m a l medium  described  s u c c i n a t e ) o r 0.4%  s u f f i c i e n t media). MgS04.  P r o t e o s e peptone  c l e a n e d by  All  autoclaving  water.  Nutrient broth  ( D i f c o ) was  used  t o grow c e l l s f o r  s t r e p t o m y c i n uptake a s s a y s .  2.  Bacterial  bacterial  strains.  strains  used  S o u r c e s and p r o p e r t i e s o f t h e in this  study are l i s t e d  Pseudomonas a e r u g i n o s a PA01 the w i l d  t y p e and r e f e r e n c e s t r a i n  brane of t h i s s t r a i n  H103  by d i e t h y l  H185  Hancock e t a l . ,  and H181  i n Table I .  H103  throughout.  has p r e v i o u s l y been w e l l  ( H a n c o c k and C a r e y , 1979; Strains  strain  principal  was  used  as  The o u t e r memcharacterized  1981).  were i n d e p e n d e n t l y i s o l a t e d  s u l f a t e mutagenesis  f o l l o w e d by s e l e c t i o n  on  from  18  TABLE 1.  Bacterial strains.  Strain  Properties  Source  HI 03  P. a e r u g i n o s a PAO 1 w i l d type  A. K r o p i n s k i (Queen's U n i v e r s i t y K i n g s t o n , Ont.)  H181 H185  Polymyxin B r e s i s t a n t mutants o f HI03  This study  H207  R e v e r t a n t o f HI81  This study  Z61  A n t i b i o t i c supers u s c e p t i b l e P. a e r u g i n o s a  W. Zimmerman (Ciba-Geigy, Basel Switzerland  H251  R e v e r t a n t o f Z61  B.L. Angus, U.B.C. (Angus e t a l 1982)  H283  Protein F-deficient mutant o f H103  This study  H284 H321 H324  R e v e r t a n t s o f H283  This study  AK43 AK1160 AK1012  LPS-deficient of H103  A. K r o p i n s k i  AK1213  Non-piliated of H103  AK1114  N o n - p i l i t e d , nonf l a g e l l a t e d mutant of H103  UB1636(RP1)  E. c o l i K12 t r p h i s s t r A l a c / amp t e t neo kan  mutants  mutant  r  r  r  A. K r o p i n s k i A. K r o p i n s k i  r  P.M. Bennet (University of Bristol, Bristol U.K.)  19  BM2 s u c c i n a t e Mutagenesis cells  in  agar  was  containing  carried  5 ml saturated  potassium phosphate were  then  diluted  and p l a t e d were  The  at  ance. agar  4°C The  were  0.8,  1979),  to  et  H283  membrane p r o t e i n  three  revertants  of is  Strains obtained  Kingston,  this  source.  30 m i n . to  media,  to  in  Cells  overnight, B.  Clones  retested  on  revertants  of  cultures of  0.06 M  25°C.  grow  then  similar  loss  at  sulphate.  overnight  polymyxin  which had  polymyxin  polymyxin  7 5 , and 0 . 8 ug/ml  H181  been  B  resist-  B o n BM2 for  poly-  succinate  H103, H181,  of  PAO w h i c h h a s  range  is  a mutant  F  (porin).  H283 w i t h  AK43, from A.  of  been  shown  to  antibiotics  revertant  of  Z61, have  been  (1982).  described  Ontario).  a non-piliated,  for  a wide  a l .  outer  strains  sulphate  50 u g / m l  and H251, a f u l l  Angus  and H324),  for  screening  weeks  75,  ethyl  five  Z61, a mutant  Strain  H321  with  resistance  susceptible  by  of  B  respectively.  Strain  described  0 . 1 ml of  PP2, allowed  H207 a n d  several of  (Zimmerman,  suspending  on n o n - s e l e c t i v e  levels  H185 a n d H 2 0 7 ,  polymyxin  pH 6 . 0  50 i n  i s o l a t e d by  for  plates  be h i g h l y  1 in  of  solution  buffer  revertant  a n d H185 w e r e held  by  o n BM2 s u c c i n a t e  subcultured  myxin.  out  50 u g / m l  wild  H103 s e v e r e l y  Isolation type  of  levels  deficient  this of  strain  porin  in and  (H284,  below.  AK1160  and AK1012 a r e  Kropinski  AK1213,  of  (Queens  a non-piliated  non-flagellated  strain,  were  LPS-altered  University, strain, also  and  AK1114,  obtained  from  20  RPl was introduced i n t o P. aeruginosa by c o n j u g a t i o n with E s c h e r i c h i a c o l i UB1636 ( R P l ) , k i n d l y provided by Bennett, U n i v e r s i t y of B r i s t o l , B r i s t o l , U.K. s i s t a n c e to a m p i c i l l i n cin,  RPl c a r r i e s r e -  (and c a r b e n i c i l l i n ) , neomycin, kanamy-  and t e t r a c y c l i n e .  Resistance to beta-lactam  i s mediated by a TEM-2 type beta-lactamase 1976).  P.M.  antibiotics  (Sykes and Mathews,  In the case of Z61, s e l e c t i o n was f o r r e s i s t a n c e to 100  ug/ml neomycin.  For a l l other s t r a i n s s e l e c t i o n was on 500  ug/ml c a r b e n i c i l l i n .  Z61 (RPl) was maintained on 200 ug/ml  neomycin while a l l other P. aeruginosa maintained on 200 ug/ml  (RPl) s t r a i n s were  tetracycline.  A s e t of 17 s e r o t y p e - s p e c i f i c s t r a i n s were a kind gift  from Dr. P. L i u , U n i v e r s i t y of L o u i s v i l l e ,  Kentucky.  Louisville,  These s t r a i n s were r e p r e s e n t a t i v e s of the I n t e r n a -  t i o n a l A n t i g e n i c Typing Scheme (IATS) (commercially marketed by D i f c o L t d . , D e t r o i t , Michigan) which c o n t a i n s as subsets the type s t r a i n s from a l l other commonly-used t y p i n g systems.  P. aeruginosa s e r o -  They were named as f o l l o w s :  type 1 (ATCC  33348), type 2 (ATCC 33349), type 3 (ATCC 33350), type 4 (ATCC 33351), type 5 (ATCC 33352), type 6 (ATCC 33354), type 7 (ATCC 33353), type 8 (ATCC 33355), type 9 (ATCC 33356), type 10 (ATCC 33357), type 11 (ATCC 33358), type 12 (ATCC 33359), type 13 (ATCC 33360), type 14 (ATCC 33361), type 15 (ATCC 33362), type 16 (ATCC 33363), type 17 (ATCC 33364). strain  Pseudomonas p u t i d a type  (ATCC 12633) was obtained from the American Type C u l t u r e  Collection  (ATCC), B r o c k v i l l e ,  Maryland.  21  Strains mucoid These  derivatives strains  Isolation  of  were  remained mucoid  3.  H325 a n d H329 w e r e  not  after  of  This  (1978),  who u s e d  mutants  for  repeated  of  mutants  with  no  ejt a l .  H103 w a s  collected  (1965 ).  sodium phosphate  was  Outer  b a s e d on  protocol  to is  6.0),  and  Washed  cells  (0.5 ml)  phase  1 mg/ml N - m e t h y l - N - n i t r o - n i t r o s o g u a n i d i n e  at  37°C f o r  tion,  resuspended  Plates were  were  also  survival  in  buffer,  examined  carried rate  after  out  after  diluted  collected  the  at  untreated  mutagenesis  in  by  cell  of of  50 mM twenty-fold to  1 ml  and  held  centrifuga-  on PP2  30°C.  of  that  added  and p l a t e d  72 h g r o w t h  with  this  then  Plate  agar. counts  suspension.  procedure  a l .  available.  (NTG),  1  et.  culture  once  were  heavy  classes  concentrated  of  were  Suzuki  b a s e d on  the  Cells  Proteins.  random  readily  washed  (pH  of  obtain  in  30 m i n .  7.  have  using  that  u s e d was  centrifugation,  and  phage  Membrane  attempted  procedure  to  on P P 2 .  A mid-logarithmic  buffer  same b u f f e r .  phage  subculture  was  selection  by  this  in  derived  resistance  Esherchia c o l i  mutagenesis  Adelberg  for  to  Deficient  procedure  it  which  The  resistant  Mutants  Isolation mutagenesis.  H103 s e l e c t e d  independently  was  The  about  0 .004%. Five These were single  were  transferred  colony  growth at  hundred  isolates  30°C, p l a t e s  supplemented with  colonies  were  to  PP2 p l a t e s ,  to  PP2 p l a t e s  were  stored  dimethyl  at  picked then  for  screening.  subcultured  and PP2 b r o t h . 4°C a n d b r o t h  sulfoxide  to  8% a n d  from  After cultures stored  at  22  -70°C.  Initial  plates,  and  frozen  The tein  and c o l l e c t e d  using  were  used  500 s t r a i n s  were  screened  using  Cultures  auxotrophs  was done  stocks  deficiencies  preparation. for  screening  which by  for  inocula  subsequent for  outer  a s i m p l i f i e d method were  were  grown  grown  in  for  studies. membrane  cell  Cell  at  30°C f o r  envelopes  pro-  envelope  30 mL BM2 g l u c o s e  i n PP2)  centrifugation.  f r o m PP2  (except  18 t o  were  42  h,  prepared  2+ in  3 0 mM T r i s  HC1  (pH  7.4)  with  method y i e l d e d  envelopes  which  membrane.  envelopes  were  mately  The  1 0 mg p r o t e i n / m l  polyacrylamide  gels  membrane p r o t e i n were  easily  (as  using  the  to  have  by  membranes were  e x a m i n e d by  sis  the  to  confirm  to  be  this  below)  enriched  in  to  This outer  approxi-  to  determine in major  the  (SDS) outer  proteins  procedure.  prepared in major  referred  from s t r a i n s outer  to  which  membrane  below,  SDS p o l y a c r y l a m i d e  alteration.  below.  in water  Deficiencies  deficiencies procedure  described  on sodium d o d e c y l s u l p h a t e  described  composition.  one-step  tended  and r u n  distinguished  as  resuspended  O u t e r membranes were appeared  Mg  and  gel  protein  these  outer  electrophore-  23  4.  Bacteriophage (a)  and b a c t e r i o c i n s t u d i e s .  Bacteriophages"and  receptors.  p a r t i a l c h a r a c t e r i z a t i o n of  A l l methods used  i n the handling of  their  bacteriophages  were d e s c r i b e d p r e v i o u s l y by Hancock and Reeves (1976).  Phages  were c h a r a c t e r i z e d using a p i l u s - d e f i c i e n t d e r i v a t i v e of  P.  aeruginosa PA01, altered  AK1144, and  two  l i p o p o l y s a c c h a r i d e (LPS)-  (rough) s t r a i n s , AK43 and AK1160, obtained from  Kropinski  (Queen's U n i v e r s i t y , Kingston, O n t a r i o ) .  sources and p u t a t i v e r e c e p t o r s are summarized Phages were obtained from 7, 21, 44, 68, 73, 109, M6  from T.L.  G101,  F116,  Pitt D3c  Fermentation, PB1  and B39  + 1 +  352,  1214,  A.  Phage  i n Table I I .  the f o l l o w i n g sources: C21,  F7, F8, F10,  2,  119X,  and  ( P u b l i c Health Laboratory, London, U.K.); , and D 3 c ~  1 +  from T. I i j i m a  Osaka, Japan); PLS27 and E79  from D.E.  Bradley (Memorial  (Institute for  from A. K r o p i n s k i ;  U n i v e r s i t y , S t . John's,  Newfoundland); SI from R. Warren ( U n i v e r s i t y of B r i t i s h Columbia);  and 176p  from J.D.  Geneva, S w i t z e r l a n d ) . plaques  using H103  AK1160 was  Piguet  ( I n s t i t u t e of Hygiene,  These phages were p u r i f i e d  as a host s t r a i n except  used as h o s t .  from  f o r PLS27, f o r which  A l l o t h e r phages were i s o l a t e d  l a b o r a t o r y of R.E.W. Hancock, U n i v e r s i t y of B r i t i s h  ability  The  pilus-  phages which were s e l e c t e d f o r  to form plaques on AK1144 ( p i l u s - d e f i c i e n t ) were  B6C,  and B6D  M6),  B5A  (independent  (from 119x), C7B  i n the  Columbia,  as host range mutants of phages which p l a t e d p o o r l y on or L P S - d e f i c i e n t s t r a i n s .  single  B6B,  i s o l a t e s d e r i v e d from 352), B9F  (from  (from 176p), B1A  (from  (from F7), C3A  24  TABLE I I . Bacteriophage sources and r e c e p t o r s  Phage  Lab name  73 119x M6 B39  A9A B5 B9 C9  Pitt Pitt Pitt Bradley  44 109 F8 1214 352 PBl SI C3A E79  A7 B4 B2 B7 B6 C8 D2 C3A Dl  Pitt Pitt Pitt Pitt Pitt Bradley Warrend Hancock ( C 2 7 ) Kropinskif  PLS27  D8  Kropinski  rough LPS  7 21 68 F10 C21 F116 G101 D3c+1+ A8A BIB B6B B6C  A2 A4 A8 B3 C3 D3 D4 D6 A8A BIB B6B B6C  Pitt Pitt Pitt Pitt Pitt I i jimag Iijima Iijima Hancock Hancock Hancock Hancock  protein protein protein protein protein protein protein protein protein protein protein protein  B9F B9E 2 D3c~l B5A C7B A9B B7A  B9F B9E Al D5 B5A C7B A9B B7A  Hancock Hancock Pitt Iijima Hancock Hancock Hancock Hancock  V1-V28  This  +  V1-V28 a  Source  Putative  pilus pilus pilus pilus  1 3  0  e  (68) (F7) (352) (352) (M6) (M6)  (119x) (B39) (73) (1214)  study  smooth smooth smooth smooth smooth smooth smooth smooth smooth  protein protein protein protein protein protein protein protein protein  See Nicas and Hancock, 1981.  b T.L. P i t t , P u b l i c H e a l t h L a b o r a t o r y , London, U.K. D.E. B r a d l e y , Memorial U n i v e r s i t y , St. John's, N f l d . R.A.J. Warren, U. o f B r i t i s h Columbia. c  d  R.E.W. Hancock, u. o f B r i t i s h Columbia (parent s t r a i n i n brackets) f A. K r o p i n s k i , Queen's U n i v e r s i t y , K i n g s t o n , Ont. 9 T. I i j i m a , I n s t i t u t e f o r Fermentation, Osaka, Japan.  e  Receptor  LPS LPS LPS LPS LPS LPS LPS LPS LPS  o r LPS o r LPS o r LPS o r LPS o r LPS o r LPS o r LPS o r LPS  3  25  C21), and A8A (from 68).  B7A was d e r i v e d  on AK43 (an L P S - a l t e r e d rough s t r a i n ) .  from 1214, s e l e c t e d  A9A was a contaminant  or d e r i v a t i v e o f 73 unable to form plaques on AK1144 or AK43. Phage s t o c k s were maintained at 4°C. r e g u l a r l y f o r t i t r e and phenotype,  Stocks were checked  and were r e p u r i f i e d every 18  months . Phages M6, B39, 73, and 119x were c h a r a c t e r i z e d as p i l u s - s p e c i f i c by t h e i r i n a b i l i t y ficient  strains.  to form plaques on p i l u s - d e -  Phages 44, 109, F8, E79, 1214, PB1, SI, 352,  and C3A were c h a r a c t e r i z e d as PAO smooth L P S - s p e c i f i c they f a i l e d  since  to form plaques on AK43 and other L P S - a l t e r e d  s t r a i n s and could be shown to adsorb to p u r i f i e d LPS. PLS27 has been c h a r a c t e r i z e d by J a r r e l l cific  and K r o p i n s k i as spe-  f o r PAO rough c o r e , and does not form plaques on smooth  strains C21,  Phage  ( J a r r e l l and K r o p i n s k i , 1981).  Phages 7, 21, 68, F10,  F116, G101, B6B, B6C, D 3 c ~ , B1A, and A8A 1+  formed  plaques w e l l on LPS a l t e r e d and p i l u s - d e f i c i e n t s t r a i n s and failed  to adsorb to LPS and thus appear to have p r o t e i n  receptors.  Phages 2, D 3 C 1 , +  +  B5A B7A, B9E, B9F, and C7B  formed plaques w e l l on p i l u s - d e f i c i e n t and w i l d - t y p e s t r a i n s , but  p o o r l y on L P S - a l t e r e d s t r a i n s but f a i l e d  to adsorb to LPS,  and thus may have LPS or L P S - a s s o c i a t e d p r o t e i n  (b)  Aeruginocin"studies.  receptors.  The a e r u g i n o c i n s used were a P.  aeruginosa typing s e t obtained from A. K r o p i n s k i , i n a d d i t i o n to  the a e r u g i n o c i n s from s t r a i n s H41, r e c e i v e d  from J . Govan,  U n i v e r s i t y of Edinburgh, S c o t l a n d , and PAF41 and PAH108 from B.  26  Holloway, Monash U n i v e r s i t y , C l a y t o n , A u s t r a l i a . The  r e c e p t o r s of these a e r u g i n o c i n s are as yet  u n c h a r a c t e r i z e d , but they p l a t e e q u a l l y w e l l on H103 although  some p l a t e only on AK43 (but not H103).  were prepared producing  Aeruginocins  using the method of Kageyama (1964).  s t r a i n s were grown at 30°C to an A600  o  t r e a t e d with 1 ug/ml mitomycin C (Sigma Chemical induce a e r u g i n o c i n p r o d u c t i o n .  remove the PEG.  (PEG)  t i n g t h i s p r e p a r a t i o n on b a c t e r i a l lawns.  when s t o r e d a t  0.6-0.8, then  f  Co.,  tended  MO.)  to  were pre-  then d i a l i s e d  A e r u g i n o c i n s u s c e p t i b i l i t y was  t i o n s were very unstable and  Aeruginocin  C u l t u r e supernatants  c i p i t a t e d with 10% p o l y e t h y l e n e g l y c o l  (c)  and AK1144  to  t e s t e d by  Aeruginocin  to l o s e a c t i v i t y  spot-  prepara-  rapidly  4°C.  I s o l a t i o n of p r o t e i n - s p e c i f i c phages"from  I s o l a t i o n of phages s p e c i f i c  nature.  f o r outer membrane p r o t e i n s  attempted using the enrichment method of Verhoef  was  e_t a l . (1977 ).  The b a s i s of t h i s method i s the use of a s t r a i n l a c k i n g a s p e c i f i c r e c e p t o r to adsorb  out the m a j o r i t y of phages, l e a v i n g  those phage which have the missing p r o t e i n as t h e i r r e c e p t o r . The  source of phages was  Iona I s l a n d sewage treatment  i n f l u e n t sewage from the  p l a n t , Vancouver, B.C.  Eight c o l -  l e c t i o n s were made, each on days f o l l o w i n g three r a i n - f r e e days i n the s p r i n g of 1980. d e b r i s and  Samples were c e n t r i f u g e d to remove  t r e a t e d with chloroform to reduce the p o s s i b i l i t y  encountering  pathogens.  U n d i l u t e d c h l o r o f o r m - t r e a t e d samples  (3 ml) were mixed i n equal volumes with 3.2%  agar and  2%  PP2  of  27  and  0.1 ml m i d - l o g a r i t h m i c c u l t u r e of H103  and  spread onto  PP2  p l a t e s to o b t a i n estimates of the number of phages p r e s e n t . Most samples contained 10-100 plaque s t r a i n s with apparent  forming  outer membrane p r o t e i n  units/ml.  Seven  deficiencies  ( i s o l a t e d by heavy mutagenesis as d e s c r i b e d below) were used i n these s t u d i e s as the adsorbing  strains.  AK1012, a L P S - d e f i c i e n t s t r a i n , and AK1213, a p i l u s def i c i e n t  s t r a i n , were used  order to reduce  as hosts f o r phage propagation i n  the p o s s i b i l i t y of i s o l a t i n g phages with LPS  or  pilus receptors. For each sewage sample, 20 ml of sample was with 20 ml double AK1012.  s t r e n g t h PP2  and 1 ml o v e r n i g h t c u l t u r e of  A f t e r o v e r n i g h t growth, the c e l l s were spun out  the supernatant  t r e a t e d with 0.5  ml c h l o r o f o r m .  t i t r e s of these p r e p a r a t i o n s on H103 plaque  forming  pooled, and aliquots.  u n i t s per ml.  i n seven  8-ml  s t r a i n with outer membrane p r o t e i n A f t e r 10 min  i n c u b a t i o n a t 37°, the t h i s a d s o r p t i o n pro-  then repeated using the same s t r a i n . to one  The  supernatant  i n PP2,  i n o c u l a t e d with AK1213, and  grown o v e r n i g h t a t 30° f o r a second  c y c l e of phage p r o p a g a t i o n .  Two  then d i l u t e d one  5  from 15 ml of a m i d - l o g a r i t h -  c e l l s were removed by c e n t r i f u g a t i o n , and  was  10  The phage p r e p a r a t i o n s were then  To each a l i q u o t , c e l l s  d e f i c i e n c i e s were added.  and  The phage  were 5 x 10^ to 4 x  the pooled p r e p a r a t i o n s d i s t r i b u t e d  mic phase c u l t u r e of one  cedure  mixed  f u r t h e r c y c l e s of a d s o r p t i o n and propagation were then  c a r r i e d out f o r each of the 7 p r e p a r a t i o n s .  Phage p r e p a r a t i o n s  28  were d i l u t e d  to about 10^ t o 10^ plaque-forming  ml b e f o r e a d s o r p t i o n .  u n i t s per  AK1012 was used f o r the t h i r d  propaga-  t i o n and AK1213 f o r the f o u r t h . The  f i n a l phage p r e p a r a t i o n s were then d i l u t e d and  p l a t e d with H103 on 1.6% agar o v e r l a y s to o b t a i n s i n g l e plaques.  Plaques were picked out of the agar using a Pasteur  p i p e t t e and suspended  i n 1 ml PP2 b r o t h .  These phage prepara-  t i o n s were then t e s t e d a g a i n s t H103, AK1012, AK1213, and the s t r a i n which had been used to 150 s i n g l e plaque  i n the a d s o r p t i o n procedure.  i s o l a t e s were t e s t e d  Phage which p l a t e d on w i l d  f o r each  Sixty  strain.  type P. aeruginosa but not on outer  membrane p r o t e i n d e f i c i e n t s t r a i n s were kept f o r f u r t h e r ing.  Each df these phage was r e - i s o l a t e d  S i n g l e plaques were obtained by s t r e a k i n g  from a s i n g l e  testplaque.  the phage p r e p a r a t i o n  onto PP2 with a l o o p , then slowly pouring 3 ml of PP2 with 1.6% agar onto the p l a t e from a p o i n t near the centre of the s t r e a k . This method r e s u l t e d  i n s i n g l e plaques without c a r r y i n g out  d i l u t i o n s of the s t o c k .  Five to 10 i s o l a t e s from each phage  were r e t e s t e d , and those with the d e s i r e d host range were kept.  (d)  Bacteriophage s e n s i t i v i t y t e s t i n g .  The method of  bacteriophage s e n s i t i v i t y t e s t i n g used was based on that of Hancock and Reeves (1976).  B a c t e r i a l lawns were prepared  o v e r n i g h t or m i d - l o g a r i t h m i c phase c u l t u r e s , d i l u t e d  from  1 i n 10  and spread on PP2 agar, e i t h e r i n PP2 agar o v e r l a y s (0.1 ml cells  i n 3 ml 1.6% agar) or by swabbing.  P l a t e s were allowed  29  to dry f o r 5 to 15 minutes at 25°C, and  phage suspensions con-  t a i n i n g 107-10*10 plaque forming units/ml the p l a t e , e i t h e r with a m u l t i p l e c r i b e d by Hancock and  (e)  syringe  onto  i n o c u l a t o r as des-  Reeves (1976), or with a  set to d e l i v e r 7.5-10 u l / s p o t . t i o n f o r 18-24  were spotted  micropipettor  P l a t e s were read  after  incuba-  h at 37°C or 40-48 h at 30°C.  I s o l a t i o n " o f ' p h a g e - r e s i s t a n t ~ mutants.  mutants were i s o l a t e d by c o - p l a t i n g 0.1  ml  Phage-resistant  mid-logarithmic  phase c u l t u r e with l O ^ - i o ^ plaque forming u n i t s of phage on p l a t e s i n PP2 picked, colony  agar o v e r l a y s .  A f t e r 24 h growth, c o l o n i e s were  then taken through three isolation.  The  third  PP2  s e r i e s of s t r e a k i n g and  i s o l a t e s were r e t e s t e d  single  f o r phage  sensitivity.  (f)  Phage c h a r a c t e r i z a t i o n by a d s o r p t i o n  Approximately 10^ 0.2  plaque-forming u n i t s of phage were mixed with  ml of o v e r n i g h t  f o r 10 min.  to whole c e l l s .  b a c t e r i a l c u l t u r e or PP2  These p r e p a r a t i o n s  and  held at  were then c e n t r i f u g e d  c e l l s , and  the supernatants were d i l u t e d and  mine phage  titres.  p l a t e d to  37°  to remove deter-  30  5.  A n t i b i o t i c and"chelator (a)  Antibiotics.  Gentamicin  g i f t s from Schering Co. Co.  s u s c e p t i b i l i t y testing. sulphate and  tobramycin  (Pte. C l a i r e , Quebec) and  (Indianapolis, Indiana).  C a r b e n i c i l l i n was  A y e r s t L a b o r a t o r i e s (Montreal, Quebec).  were  Eli Lilly  purchased  Streptomycin  from  sulphate,  neomycin s u l p h a t e , kanamycin, t e t r a c y c l i n e h y d r o c h l o r i d e , chloramphenicol, sulphate  benzyl p e n i c i l l i n ,  (8000 U/mg)  E, 12,470 U/mg)  and  r i f a m p i c i n , polymyxin B  c o l i s t i n methane s u l f o n a t e  were purchased  from Sigma Chemical  C e f s u l o d i n was  k i n d l y provided by Ciba Geigy A.G.  Switzerland).  N i t r o c e f i n was  O'Callaghan  () D  a generous g i f t  (polymyxin Co.  (Basel,  from Dr.  (Glaxo Group Research L t d . , Middlesex,  C.  U.K.).  C h e l a t o r and a n t i b i o t i c b a c t e r i o l y s i s " a n d k i l l i n g .  T e s t i n g of l y s i s by EDTA-Tris,  e t h y l e n e g l y c o l - b i s (2-amino-  e t h y l e t h e r ) N , N ' - t e t r a acetate  (EGTA)-Tris and polymyxin B  c a r r i e d out on c e l l s ance at 600  nm  i n m i d - l o g a r i t h m i c phase growth  resuspended i n 10 mM  T r i s - H C l b u f f e r (pH 8.5)  myxin B i n 30 mM The  A g g o was  EDTA or EGTA  at 25°C or 75 ug/mL of p o l y -  sodium phosphate b u f f e r (pH 7.4)  read at timed  To t e s t k i l l i n g i n , mid-log  (absorb-  (Agog) °f 0.30-0.60), which were c o l l e c t e d  c e n t r i f u g a t i o n a t 25°C and and 10 mM  was  at 37°C.  intervals. by EDTA-Tris,  c e l l s were c e n t r i f u g e d and  EGTA-Tris,  and  polymyx-  resuspended at 100-fold  31  dilution  i n e i t h e r 30 mM  75 ug/ml polymyxin mM  sodium phosphate b u f f e r pH 7.0,  B or e i t h e r 10 mM  T r i s - h y d r o c h l o r i d e , pH 8.5.  EDTA or 10 mM  A f t e r 5 min  with  EGTA i n 10  i n c u b a t i o n at 25°C,  c e l l s were d i l u t e d and p l a t e d f o r v i a b l e counts on PP2 agar i n PP2 agar  (0.6% agar) o v e r l a y s .  the procedure was  To t e s t k i l l i n g  by gentamicin,  modified s l i g h t l y because gentamicin i s  a c t i v e o n l y on r e s p i r i n g c e l l s  (Hancock, 1981).  c e l l s prepared as above were resuspended c o n t a i n i n g 5 ug/ml gentamicin  Centrifuged  i n BM2  growth medium  i n a d d i t i o n to s u c c i n a t e and  at normal l e v e l s but with no other c a t i o n s added, and incubated a t 37°C with vigorous a e r a t i o n f o r 5 min  the  iron cells  at which  time v i a b l e counts were c a r r i e d out as above.  (c) (MIC).  Determination of minimal  i n h i b i t o r y concentrations  For determinations i n d e f i n e d l i q u i d media, a n t i b i o t i c  r e s i s t a n c e was the s t a t e d Mg  measured i n 1 ml volumes of BM2 levels.  The  inoculum  used was  s u c c i n a t e with approximately  10^ c e l l s of an o v e r n i g h t c u l t u r e grown i n medium to the t e s t medium.  The  l e v e l of r e s i s t a n c e was  h i g h e s t a n t i b i o t i c c o n c e n t r a t i o n showing v i s i b l e  identical taken as the turbidity  a f t e r 24 h a t 37°C. Other measurements of MIC  were done on PP2 agar using  the method d e s c r i b e d by Angus e t a l (1982).  A multisyringe  32  a p p l i c a t o r was used to d e l i v e r to each p l a t e 24 drops of approximately 2 u l which contained d i l u t e d 18 h c u l t u r e s . incubation  6.  an estimated 1000 c e l l s  P l a t e s were read  from  a f t e r 18 and 48 h  at 37°C.  Shift"experiments.  Overnight c u l t u r e s  (1 ml) grown i n BM2-  2+ succinate  with 0.02 mM  Mg  were t r a n s f e r r e d to 200 ml of  the same medium and grown to an A g o o o f 0.15 t o 0.20, at 2+ which p o i n t Mg mM. min  was added to a f i n a l  concentration  Twenty ml of c u l t u r e were removed at t h i s p o i n t , and a t 15 i n t e r v a l s , and these samples used to t e s t polymyxin B and  EDTA-Tris s e n s i t i v i t y , and f o r p r e p a r a t i o n described 7.  of 0.5  of c e l l envelopes as  above.  Membrane~Isolation and C h a r a c t e r i z a t i o n of Outer~Membrane,  C e l l Envelope ~, and "Whole C e l l P r o t e i n s . a t i o n s , overnight  For whole c e l l  prepar-  or l o g a r i t h m i c phase c u l t u r e s were c e n t r i f u g -  ed and the c e l l s resuspended i n 2% SDS, 20 mM T r i s - H C l pH 8.0. A f t e r treatment at 100°C f o r 10 min, r e s i d u a l c e l l s were removed by c e n t r i f u g a t i o n a t 27,000 x g f o r 20 min. supernatant was sonicated  The r e s u l t i n g  (1 min, s e t t i n g 5, Biosonik  sonicator  ( B r o n w i l l S c i e n t i f i c , N.Y.)) to shear DNA and reduce v i s c o s i t y , and  the sample a p p l i e d d i r e c t l y to the g e l .  33  To prepare c e l l l o g a r i t h m i c phase resuspended  envelopes, c e l l s from o v e r n i g h t or  c u l t u r e s were c o l l e c t e d  i n 10 mM  sodium phosphate  by c e n t r i f u g a t i o n ,  buffer  (pH 7.4)  or 30  mM  2+  T r i s - H C l , pH 7.4  c o n t a i n i n g 2 mM  Mg  and 10 ug/ml p a n c r e a t i c  deoxyribonuclease I (Sigma Chemical Co.) and broken i n a French Press at 14,000 p s i . Whole c e l l s were removed by c e n t r i f u g a tion in  (1000 x g, 10 min)  and the r e s u l t i n g  the same b u f f e r and c e n t r i f u g e d  The c e l l  envelope p e l l e t was  supernatant d i l u t e d  at 160,000 x g f o r 2 h r .  resuspended  in deionized  water.  Outer membranes were prepared using the two methods d e s c r i b e d by Hancock and Carey  (1979).  One,  originally  des-  c r i b e d by Hancock and N i k a i d o (1978), employs a s i n g l e step and a f o u r - s t e p sucrose g r a d i e n t and y i e l d s two outer membrane f r a c t i o n s with i n d i s t i n g u i s h a b l e p r o t e i n composition.  The  second i s a more r a p i d method using only one sucrose g r a d i e n t which y i e l d s a s i n g l e outer membrane band  (Hancock  and Carey,  1979) . Sodium dodecyl sulphate (SDS) 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 was c o n t a i n i n g 0.07  performed  using the 14% acrylamide system  M NaCl i n the running g e l p r e v i o u s l y d e s c r i b e d  by Hancock and Carey  (1979).  Only i n the presence of 0.07  M  NaCl or with high acrylamide monomer c o n c e n t r a t i o n s are outer membrane p r o t e i n s Hi and H2 separated (Hancock 1979).  and  Carey,  S o l u b i l i z a t i o n c o n d i t i o n s were 88°C f o r 10 min i n  r e d u c t i o n mix c o n t a i n i n g  2-mercaptoethanol.  Ratios of p r o t e i n Hi to H2 were c a l c u l a t e d  from dens-  34  itometer (Helena blue  tracings  made o n a H e l e n a Q u i c k  Laboratories,  Beaumont,  R50-stained gels.  reference  since  it  and i t s  level  SDS g e l  electrophoresis  8. opes of  with  Determination of prepared  Kenward  et  absorption Liptak  S.  a l .  (1978)  Ma a n d D r . of  incubated with  of  37° w i t h  m y x i n was  then  envelopes  on  proteins  were  of  extracted  by  (to  aeration.  added and t h e then  lyophilized  cell  judged  Cell  by  the  by  (Department  envelmethod  determined by  Susan Jaspar  Engineering,  from the Cells  divalent  as  levels.  levels  out  the  a  and  University  of  Vancouver,  of  atomic Susan  British B.C.,  or  Physiology,  Columbia).  Mg  were  of  used as  proteins.  cation  and c a t i o n  Copp  1 mM K C N  15 m i n a t  above,  above  Civil  British  Displacement  analysis  cell  brilliant  H2 w a s  growth conditions  whole  carried  D.H.  and a m i n o g l y c o s i d e s .  Cell  major  Densitometer  Coomasie  Canadian M i c r o a n a l y t i c a l Corp.,  University  9.  the  envelope  described  spectroscopy  Columbia), Dr.  of  cell  of  of  little  as  (Department  of  O u t e r membrane p r o t e i n  was one  varied  Texas)  Scan J r .  grown t o prevent  envelope  cells  was  envelopes.  by  a n AgQO ° f  described  carried  uptake)  a further  out,  above, as  B  were  gentamicin  incubated as  polymyxin  0*60  aminoglycoside  Streptomycin,  prepared  cations  cell  or  for poly-  10 m i n . and  described  35  10. Streptomycin uptake'assays.  [ H]-dihydrostreptomycin  (1.2 Ci/mmol) (Amersham S e a r l e , O a k v i l l e , Ontario) was  diluted  by the a d d i t i o n of n o n r a d i o a c t i v e streptomycin to a s p e c i f i c activity ing  of 50 uCi/mg s t r e p t o m y c i n .  at 37°C to an A g o o  b r o t h , and  the uptake  o  C e l l s were grown by shak-  0.5-0.6 i n unmodified  f  nutrient  assay s t a r t e d by the a d d i t i o n , to the  growing  c e l l s , of streptomycin to the d e s i r e d f i n a l concentra-  tions.  At d e f i n e d times, 1.0 ml samples were removed and  cells um,  c o l l e c t e d by f i l t r a t i o n onto n i t r o c e l l u l o s e f i l t e r s  M i l l i p o r e , Bedford, Mass.) which had been presoaked  M LiCl  p r i o r to use.  with 3 ml of 0.1  The  method was leagues  filtered  then  (1978) and Bryan and  (1976) f o r streptomycin uptake  assays.  Trial  This col-  experi-  that the f i l t e r p r e p a r a t i o n and washing  techniques were c r i t i c a l , Bryan, 1976)  i n 0.1  i n a toluene-based s c i n t i l l a n t .  based on those of H o l t j e  ments demonstrated  (0.45  c e l l s were then washed twice  M L i C l , d r i e d a t 60°C f o r one hour,  assayed f o r r a d i o a c t i v i t y  1978;  the  as p r e v i o u s l y suggested  (Holtje,  but that the methods of e i t h e r of the above  authors were s a t i s f a c t o r y . N u t r i e n t broth was assays because  chosen as the medium f o r uptake  i t has r e l a t i v e l y low Mg  levels  (Nicas and  Bryan, 1978), and has p r e v i o u s l y been shown s u i t a b l e a l . , 1976). elsewhere was  BM2,  the phosphate  i n t h i s study was  found to i n h i b i t  (Bryan et:  b u f f e r e d minimal medium used  judged u n s u i t a b l e as high  phosphate  aminoglycoside uptake, n e c e s s i t a t i n g  use of l a r g e amounts of  label.  the  36  11. Enhancement o f n i t r o c e f i n p e r m e a b i l i t y by aminoglycosides and  chelators.  The assay used was modified  O'Callaghan e t a l . (1972). mg/ml benzyl  from that of  H103 growing i n the presence o f 0.2  p e n i c i l l i n or H103 (RPl) growing i n the presence  of 20 ug/ml t e t r a c y c l i n e was grown to an Agon o f 0.50 t o 0.60, harvested  by c e n t r i f u g a t i o n a t 25°C and resuspended i n 20 mM  sodium phosphate b u f f e r pH 7.0 a t an AgQO For s t u d i e s with  o  f  1.50 t o 2.0.  c h e l a t o r s , the c e l l s were then  d i l u t e d 1 i n 10 i n EDTA-Tris or EGTA-Tris at a f i n a l t i o n o f 10 mM EDTA or EGTA and 10 mM T r i s - H C l pH 8.5. min 0.65  concentraAfter 2  a t 25°C, 0.1 ml of c e l l suspension was q u i c k l y mixed ml of n i t r o c e f i n  with  (12.5 ug/ml, i n phosphate b u f f e r ) and the  h y d r o l y s i s of n i t r o c e f i n monitored s p e c t r o p h o t o m e t r i c a l l y by measurement of the i n c r e a s e h y d r o l y s i s f o r untreated  i n absorbance a t 540 nm.  Rates o f  c e l l s were a l s o measured, and h y d r o l -  y s i s r a t e s were expressed as the r a t i o of h y d r o l y s i s r a t e s i n treated c e l l s gentamicin,  to r a t e s of untreated  cells.  In s t u d i e s  with  1 ml of gentamicin was added t o 0.1 ml of c e l l  suspension to give a f i n a l concentation 2 min a t 25°C, 0.6 ml of n i t r o c e f i n  of 10-100 ug/ml.  After  (250 ug/ml) was added and  h y d r o l y s i s of the n i t r o c e f i n monitored as above.  12.  Measurement o f outer'membrane p e r m e a b i l i t y by n i t r o c e f i n  hydrolysis. Rosselet ed,  A method based on the technique of Zimmermann and  (1980) as modified  by Angus e t a^L. (1982) was develop-  s i n c e n e i t h e r the o r i g i n a l technique nor the m o d i f i c a t i o n  37  enabled a measurement of outer membrane p e r m e a b i l i t y f o r s t r a i n H283.  In p a r t i c u l a r , a r e l a t i v e l y  plasmic beta-lactamase  was  found d u r i n g the resuspension of  cells after centrifugation.  T h e r e f o r e , the technique  f u r t h e r m o d i f i e d as f o l l o w s : containing  s u b s t a n t i a l r e l e a s e of p e r i -  was  aeruginosa or E_. c o l i  strains  the RPl plasmid were grown o v e r n i g h t i n PP2 broth at  37°C i n the presence of 200 ug/ml t e t r a c y c l i n e or i n the case of Z61  (RPl) with 20 ug/ml t e t r a c y c l i n e to ensure  the p l a s m i d . was  r e t e n t i o n of  (Retention of the plasmid under these c o n d i t i o n s  confirmed by comparing p l a t e counts on PP2  ug/ml c a r b e n i c i l l i n or 200 ug/ml neomycin.)  and PP2 with  200  The o v e r n i g h t c u l -  tures were d i l u t e d 1 i n 20 i n t o f r e s h PP2 b r o t h and grown to an A  600 of 0.6  t o 0.8.  A 0.1 ml sample of c e l l s was  sample c u v e t t e of a Perkin-Elmer dual beam spectrophotometer.  (Oak Brook, 111.)  Another  1.5  m i c r o c e n t r i f u g e model 5412  Westbury, N.Y.).  The  cell-free  Lambda 3  ml sample was  at the same time and c e n t r i f u g e d f o r 1 min Eppendorf  placed i n the  taken  at 9000 x g i n an  (Brinkman  Instruments,  supernatant was  decanted  and  0.1 ml added to the r e f e r e n c e cuvette of a Perkin-Elmer Lambda 3 spectrophotometer. 0.8  To both r e f e r e n c e and  sample c u v e t t e s ,  ml of a 0.1 mg/ml s o l u t i o n of the chromogenic beta-lactam  nitrocefin  (O'Callaghan et. a l . ,  1972)  was  e n t i a l r a t e of c o n v e r s i o n of n i t r o c e f i n followed over time at an absorbance  added and  the  to n i t r o c e f o i c  of 540 nm  Perkin-Elmer model 581 s t r i p c h a r t r e c o r d e r .  differacid  using a coupled Since both  and r e f e r e n c e c u v e t t e s contained supernatants, the  sample  differential  38  r a t e of h y d r o l y s i s was a measure of whole c e l l nitrocefin.  Control  h y d r o l y s i s of  experiments showed that the rate of  h y d r o l y s i s d i d not i n c r e a s e  over time, i n d i c a t i n g that  cell  breakage was not o c c u r r i n g . In a l l experiments with the outer membrane p r o t e i n mutants H283 (RPl) and H181 ( R P l ) , the c u l t u r e used was checked f o r p r o t e i n F d e f i c i e n c y o r HI o v e r p r o d u c t i o n by examining whole c e l l p r o t e i n p r o f i l e s on SDS p o l y a c r y l a m i d e g e l s  after  each experiment.  13.  Other a s s a y s .  Schacterle  The p r o t e i n assay used was that of  and P o l l a c k  (1973).  Levels  of 2-keto-3-deoxyocton-  ate were estimated by the method of Osborn et. a l . (1963).  39  CHAPTER"ONE SUSCEPTIBILITY TO EDTA-TRIS, POLYMYXINS AND AMINOGLYCOSIDES  7+  1.  S u s c e p t i b i l i t y to-polymyxin B~and EDTA-Tris i n Mg^  s u f f i c i e n t and d e f i c i e n t media. Wild type J?. aeruginosa PA01 7+ 7+ s t r a i n H103, grown on Mg - s u f f i c i e n t medium (0.5 mM Mg was s e n s i t i v e to polymyxin B and EDTA k i l l i n g (Table I I I ) and 7+  lysis  (Fig. 1).  In c o n t r a s t , H103 grown under Mg  -deficient  7+  (0.02  mM  Mg  ) c o n d i t i o n s was 70 t o 700 f o l d more r e s i s t a n t to  these agents ( F i g . 1; Tables I I I and V ) , i n agreement with v i o u s l y published et a l . , 1974).  pre-  r e s u l t s (Brown and M e l l i n g , 1969; G i l l e l a n d  This r e s i s t a n c e could be reversed  by c u l t u r i n g  7+  H103 on Mg^ - s u f f i c i e n t medium All  l y s i s and k i l l i n g  f o r a few  generations.  experiments were done on mid-  l o g a r i t h m i c phase c e l l s a f t e r c o n t r o l experiments showed that s u s c e p t i b i l i t y t o l y s i s by EDTA-Tris and by polymyxin B v a r i e d with the growth phase of the c u l t u r e .  When 18 h ( l a t e s t a t i o n -  2+  ary phase) c u l t u r e s grown i n Mg  s u f f i c i e n t medium were  t r a n s f e r r e d to f r e s h medium, s u s c e p t i b i l i t y to l y s i s  increased  throughout e a r l y l o g a r i t h m i c phase, and reached a maximum i n middle and l a t e l o g a r i t h m i c phase.  As c e l l s entered  stationary  phase, s u s c e p t i b i l i t y decreased, so that 18 h c u l t u r e s were h i g h l y r e s i s t a n t to l y s i s , e x h i b i t i n g o n l y 5 t o 20% of the 2+  l e v e l of l y s i s seen i n mid-log c e l l s . d e f i c i e n t medium  followed  C e l l s grown i n Mg  a s i m i l a r p a t t e r n , except that de-  40  A.  EDTA-Tris  B.  Polymyxin  B  A  Time  (min)  Time  (min)  Figure"1. E f f e c t o f a d a p t a t i o n on Mg^ - d e f i c i e n t m e d i u m , and o f m u t a t i o n t o p o l y m y x i n B r e s i s t a n c e , on s u s c e p t i b i l i t y o f c e l l s t o l y s i s by p o l y m y x i n B and E D T A - T r i s . A , l y s i s by E D T A - T r i s ; B, l y s i s b y p o l y m y x i n B. Symbols: W i l d t y p e s t r a i n H103 g r o w n i n Mg -deficient (0.02) mM m e d i u m ( A ) ; s t r a i n H103, (A), p o l y m y x i n B r e s i s t a n t m u t a n t s H181, (•) a n d H185, (fP) a n d r e v e r t a n t H207, (O) g r o w n i n M g 2 + - s u f f i c i e n t (0.5 mM) m e d i u m .  TABLE I I I . Resistance to k i l l i n g by EDTA-Tris and polymyxin B of H103 and i t s polymyxin B r e s i s t a n t mutant H181. Effect of Mg2+ concentration i n the growth medium.  Strain  H103  04.  0.02  65  15  0.9  O.02  0.02  77  42  0.5  76  22  0.5  H181  a  Survivors (%) EDTA-Tris Polymyxin B a  Mg concentration during growth (mM)  C e l l s were treated for 5 minutes i n lOmM EDTA i n lOmM- Tris-HCl (pH8.5) or 75 ug/ml polymyxin B i n phosphate buffer (pH 7.4).  42  c r e a s e i n s u s c e p t i b i l i t y began somewhat e a r l i e r , i t h m i c phase growth,  and  in late  logar-  t h e s e c e l l s were more r e s i s t a n t  than  9 +  cells The  i n Mg  growth  s u f f i c i e n t medium d u r i n g a l l p h a s e s o f r a t e s of c e l l s growing  d e f i c i e n t medium w e r e i d e n t i c a l  i n Mg  2+  growth.  - sufficient  and  i n l o g phase, although  Mg  2+  lower  2+  l e v e l s o f Mg  (below  0.01  mM)  resulted  i n decreased  growth  2+  rate.  F o r c u l t u r e s grown i n 0.015-0.05 mM  mg  , the  growth  2+  yield  2.  was  proportional  t o t h e amount' o f Mg  S u s c e p t i b i l i t y of polymyxin  r e s i s t a n t mutants.  m y x i n B r e s i s t a n t m u t a n t s o f H103, resistant  t o E D T A - T r i s and  added.  strains  polymyxin  H181  B killing  and  Two  poly-  H185,  were  ( T a b l e I I I ) and  2+  lysis The  ( F i g . 1 ) , i r r e s p e c t i v e o f t h e medium Mg  m u t a n t p h e n o t y p e was  concentration.  s t a b l e f o r up t o 12 c o n s e c u t i v e  single  2+  colony  i s o l a t i o n s on Mg  r e v e r t a n t s o f H181 all  of the w i l d  below).  and  - s u f f i c i e n t medium. H185  (e.g., s t r a i n  type p r o p e r t i e s of s t r a i n  These d a t a s u g g e s t  single mutation  resulting  t h a t H181  and  S i x spontaneous  H207) had  regained  H103  (see F i g . 1  H185  each  i n phenotypic a l t e r a t i o n s  and  have a mimicking  9+  t h o s e o f t h e Mg 3.  -limited,  adaptively resistant strain  Aminoglycoside"resistance.  S i n c e Mg"  has  been  H103.  previously  shown t o a n t a g o n i z e t h e a c t i o n o f a m i n o g l y c o s i d e s , as w e l l polymyxins  ( Z i m e l i s and J a c k s o n , 1 9 7 3 ) ,  t h e m u t a n t s were  as  tested  f o r enhanced r e s i s t a n c e t o t h r e e r e p r e s e n t a t i v e a m i n o g l y c o s i d e a n t i b i o t i c s , g e n t a m i c i n , s t r e p t o m y c i n and  tobramycin.  Strains  43  H181 and H185 were c o n s i s t e n t l y 4 - f o l d more r e s i s t a n t to gentam i c i n and streptomycin and 2 - f o l d more r e s i s t a n t to tobramycin (Table I V ) . In c o n t r a s t they were e q u a l l y s u s c e p t i b l e t o c a r b e n i c i l l i n and t e t r a c y c l i n e when compared to s t r a i n H103. S t r a i n H103 grown i n Mg  - d e f i c i e n t medium was a l s o  shown t o be much more r e s i s t a n t to gentamicin than H103 grown 2+  i n Mg  - s u f f i c i e n t medium  (Table V) when r e s i s t a n c e was  measured i n a common assay medium.  S t u d i e s r e p o r t e d elsewhere  (Hancock, R a f f l e and N i c a s , 1981) have shown that H103 grown i n 2+  Mg  - d e f i c i e n t medium shows a marked decrease i n s e n s i t i v i t y to  killing  by gentamicin over a broad range of a n t i b i o t i c  trations.  concen-  The l e v e l of r e s i s t a n c e achieved was shown to be  s i m i l a r t o that of the polymyxin  r e s i s t a n t mutants H181 and  H185 . 4.  S u b s t i t u t i o n " o f o t h e r ' c a t i o n s for'Mg^  .  R e s u l t s of sup-  2+  plementation o f Mg shown i n Table V.  d e f i c i e n t medium with other c a t i o n s are These r e s u l t s confirmed and extended the  f i n d i n g s of Boggis et a l . (1979) with r e s p e c t to the e f f e c t s of d i f f e r e n t metal c a t i o n s on s u s c e p t i b i l i t y EDTA-Tris.  to polymyxin and  C e l l s grown with M g , C a , M n 2+  2 +  l e a s t 1000 times more s e n s i t i v e to polymyxin  2+  or S r  2 +  were a t  B, 100 times more  s e n s i t i v e to EDTA-Tris and 10 times more s e n s i t i v e to gentami2+  c i n than were c e l l s grown i n low Mg ; s u s c e p t i b i l i t y to l y s i s i n EDTA-Tris was a l s o much enhanced i n such c e l l s . In c o n t r a s t , 2+  c e l l s grown with the s e v e r a l other c a t i o n s  (Ba  2+  , Zn  2+  , Sn  ,  44  TABLE IV.  Resistance of H103, i t s polymyxin B resistant derivatives H181 and H185 and a revertant H207 to various a n t i b i o t i c s .  Resistance  Strain Gentamicin  Streptomycin  H103  1  8  H181  4  H185 H207  a  3.  (ug/ml) '  Tobramycin  b  Carbenicillin  Tetracycline  0.5  16  8  32  1  16  8  4  32  1  16  8  1  8  0.5  16  8  Resistance levels were determined as described i n Methods, using l i q u i d BM2 medium with 0.5 mM Mg2+.  k The differences observed, while not large, were reproducibly obtained i n 6-10 i n d i v i d u a l experiments.  45  3+ Al in  + ,  Na  ),  their  Tris  were  indistinguishable  relatively  low  and g e n t a m i c i n ,  from c e l l s  susceptibility  providing  that  to  grown i n  polymyxin  l o w Mg  B,  EDTA-  a common a s s a y m e d i u m w a s  used. 5.  Minimal inhibitory  Mg^" " l e v e l s .  Although  1  2+ Mg" increases  low is  tested  in  concentrations  of  polymyxin  growth  of  P_. a e r u g i n o s a  resistance  to  polymyxin  a common a s s a y m e d i u m , t h e  at  various  i n media  when  with  susceptibility  presence  of  added  ex-  2+ ternal  Mg  from the 1979).  can p r o t e c t polymyxin  The  MIC  both  action  to  resistant  (Newton,  and s u s c e p t i b l e  1964; Klemperer  et  cells a l . ,  polymyxin  B a n d c o l i s t i n ( p o l y m y x i n E) shown 2+ m T a b l e V I r e f l e c t t h e s e Mg effects. Thus, although c e l l s g r o w n i n 5 mM M g a r e c l e a r l y h i g h l y s u s c e p t i b l e t o k i l l i n g by 2+ p o l y m y x i n B i n t h e a b s e n c e o f Mg (Table III), they appear to 2 +  be  relatively  ence  of  Mg  2+  resistant  in  (Table VI).  MIC m e a s u r e m e n t s Since  at  this  Mg  done  2+  in  the  pres-  concentration  2+ there  was  60-fold in  B),  a large  at the  molar  excess  of  Mg  100 ug/ml p o l y m y x i n  B,  1000-fold  results  on the  LPS n o r m a l l y  sion).  Competition  explain  the  may b e  e x p l a i n e d by 2+  o c c u p i e d by between  Mg  Mg  in  0 . 5 mM Mg  (see  the  polymyxin at  4 ug/ml  competition  and t h e  2.5-fold difference in 2+  grown and t e s t e d  over  Fig.  10 a n d  polymyxins  resistances  relative  to  for  B  (e.g., polymyxa  Discus-  could of  site  also  H181 a n d H185  H103 grown i n  0.02  2+ mM Mg  ,  grown a t  (Table VI) whereas the r e s i s t a n c e s of the s t r a i n s 2+ t h e s e Mg c o n c e n t r a t i o n s were s i m i l a r when t e s t e d  in  TABLE V.  E f f e c t of growth in various divalent cations on induction of outer membrane protein HI, l y s i s and k i l l i n g by EDTA-Tris, and k i l l i n g by polymyxin B and gentamicin.  Mg2 during growth (mM) +  Other cations during growth (mM)  -  0.02  Induction o f protein H l a  +  % Lysis by EDTA-Tris"  11/5  Polymyxin B  % Survivors EDTA-Tris  0  Gentamicin  15.0  65.0  11.0  20.3  14.6  21.5  0. 02  Ba2+ (0.5)  +  0. 02  Sn2+ (0.5)  +  16.0  20.5  11.7  10.5  0.02  Zn2+ (0.5)  +  14.0  20. 2  13.0  19. 5  0.02  Ca2+ (0.5)  -  51. 5  O.01  O.l  1.56  0.02  Mn  (0.5)  -  53.5  <0.01  O.l  1.65  0.02  Sr2+ (0.5)  -  51.7  *0.01  O.l  0.68  -  52.3  SO. 01  <0.1  .2.01  0.5  2+  -  6. 0  Induction of Hi as judged from sodium dodecyl sulphate polyacrylamide gels o f c e l l envelope proteins, see Fig. 3. Lysis was measured as decrease i n A ^ Q Q a f t e r 1 5 min i n 1 0 mM EDTA,10 mM Tris-HCl pH 8 . 5 . C e l l s were treated f o r 5 min with 7 5 ug/ml polymyxin B i n phosphate buffer, 1 0 mM EDTA i n 1 0 mM Tris-hydrochloride, or 5 ug/ml gentamicin i n growth medium without added divalent cations.  TABLE V I .  L e v e l s o f outer membrane p r o t e i n H i , c e l l envelope Mg2+ c o n c e n t r a t i o n and r e s i s t a n c e t o polymyxins o f H103 and i t s polymyxin B r e s i s t a n t d e r i v a t i v e s H181 and H185 and a r e v e r t a n t H207: e f f e c t o f v a r y i n g Mg2+ c o n c e n t r a t i o n s i n the growth medium.  Strain  Mg  concentration d u r i n g growth (mM)  2 +  Outer Membrane Proteins:Ratio Hl:H2 a  Cations i n c e l l envelope (ug/mg p r o t e i n ) Mg Ca 2+  0.02 0. 5 5.0  4.7 0.7 0. 2  4.8 17.1 20. 5  1. 2 0. 9 0.8  H181  0.02 0. 5 5.0  6.3 4. 7 3.4  3.8 8.9 11. 6  1.4  0.02 0.5 5.0  6.4 -c 2.9  2.9 8.8 12.6  1.3  0. 5  0.8  H207  (ug/ml) Colistin d  b  2 +  H103  HI 8 5  Resistance Polymyxin B  1.1  1.0  10 1 4  50 4 25  100 25 100  200 100 200  100 25 100  200 100 200  C e l l envelopes were i s o l a t e d and s u b j e c t e d to SDS 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 . The r a t i o s o f p r o t e i n s H i t o H2 were c a l c u l a t e d from densitometer t r a c i n g o f s t a i n e d g e l s loaded with a standard amount o f p r o t e i n . Outer membrane p r o t e i n H2 was used as a r e f e r e n c e s i n c e i t was one o f t h e major p r o t e i n s o f t h e c e l l and i t s l e v e l s v a r i e d very l i t t l e with growth c o n d i t i o n s . b L e v e l s o b t a i n e d by atomic a d s o r p t i o n spectroscopy. "-" means not done. a  c  d Resistance  was determined i n l i q u i d  BM2 s u c c i n a t e u s i n g the given Mg2+ c o n c e n t r a t i o n s .  48  the absence of Mg^  (Table I I I ) .  The  r e l a t i o n s h i p of the  r e s i s t a n c e shown i n Table VI and p r o p e r t i e s of the outer membrane i s d e s c r i b e d below (Chapter  6.  Streptomycin  uptake and b i n d i n g i n ' s u s c e p t i b l e and  resistant strains. s t r a i n s H103  and  Two).  The  H181  p a t t e r n of streptomycin  uptake i n both  ( i l l u s t r a t e d by a t y p i c a l experiment F i g .  2) f o l l o w e d three phase k i n e t i c s as d e s c r i b e d by Bryan and c o l l e a g u e s f o r other s t r a i n s instantaneous and of  a l a t e r r a p i d uptake phase (EDP-II). experiments was  cyanide-treated Den  An e x t e n s i v e  (EDP-I) series  performed i n an attempt to demonstrate  [i.e.,  amount of streptomycin b i n d i n g to  non-streptomycin  t r a n s p o r t i n g (Bryan  E l z e n , 1977)] or -untreated w i l d type s t r a i n H103  mutant H181  cells.  statistical  (P >0.5)  strains  that there was  i n aminoglycoside  Thus, any apparent  b i n d i n g to the two  or  (data not shown),  a n a l y s i s of the data suggested  significant different strains.  and  For f i v e separate experiments done at e i g h t  d i f f e r e n t c o n c e n t r a t i o n s of streptomycin  two  E l z e n , 1976): an  b i n d i n g phase, an e a r l y slow uptake phase  d i f f e r e n c e s i n the apparent  Van  (Bryan and Van  no  b i n d i n g to the  d i f f e r e n c e s i n streptomycin  (e.g., as seen a t 10 ug/ml i n F i g .  2) were shown by more c a r e f u l a n a l y s i s to be not Scatchard a n a l y s i s of the data from one  significant.  experiment suggested  in  7 the order of 2 - 5 x 10 mycin per c e l l . on the c e l l  The  p o t e n t i a l binding s i t e s for s t r e p t o -  l a r g e number of n o n - s p e c i f i c binding  ( s i n c e there are only about 2 - 4 x 10°  sites  molecules  49  - 8  30 TIME (min) Figure 2. Uptake of [3H] streptomycin at two c o n c e n t r a t i o n s by the w i l d type s t r a i n H103 and the outer embrane p r o t e i n HI overproducing s t r a i n H181. Symbols: O H103, 2 ug/ml streptomycin; • H103, 10 ug/ml streptomycin; ^ H181, 2 ug/ml streptomycin; 4L H181, 10 ug/ml s t r e p t o m y c i n .  50  3  io  ~io  ioi  Streptomycin added (pg/ml)  Figure'3» Time r e q u i r e d f o r i n i t i a t i o n o f r a p i d u p t a k e o f streptomycin ( E D P - I I ) i n t h e w i l d t y p e s t r a i n s H103 ( © ) and t h e o u t e r membrane p r o t e i n H i o v e r p r o d u c i n g s t r a i n H181 ( £ ) . t h e p o i n t s r e p r e s e n t t h e means o f t h r e e e x p e r i m e n t s ; t h e g i v e n l i n e s were drawn by l i n e a r r e g r e s s i o n a n a l y s i s o f t h e p o i n t s w i t h c o r r e l a t i o n c o e f f i c i e n t ( r ) o f 0.98 f o r H103 and 0.97 f o r H181. 2  51  of  LPS per P. aeruginosa c e l l ) , and the high background  filter  a d s o r p t i o n of streptomycin even under the s t r i n g e n t washing procedures  used may w e l l have acted together to obscure  ed d i f f e r e n c e s  i n aminoglycoside  The major a l t e r a t i o n uptake seen  expect-  binding.  i n the k i n e t i c s of streptomycin  i n the r e s i s t a n t s t r a i n , H181, was that at a l l con-  c e n t r a t i o n s of a n t i b i o t i c used,  t r a n s i t i o n from  the e a r l y slow  phase of uptake EDP-I to the l a t e r r a p i d phase EDP-II was delayed  i n the r e s i s t a n t s t r a i n  c o n s i s t e n t l y observed  i n seven  ( F i g . 3 ) . This d i f f e r e n c e was separate experiments,  each using  s e v e r a l l e v e l s of a n t i b i o t i c .  7.  P e r m e a b i l i z a t i o n o f ' t h e outer membrane by aminoglycosides. The  a b i l i t y of EDTA and polymyxin  to i n t e r a c t with  and d i s r u p t the outer membrane of gram-negative b a c t e r i a i s w e l l known ( L e i v e , 1965; Cooperstock,  1974; G i l l e l a n d and  Murray, 1976; Michael and Eagon, 1966; Rosenthal 1977).  The a b i l i t y of aminoglycosides  and Strom,  to i n t e r a c t with the  outer membrane was i n v e s t i g a t e d by examining  t h e i r a b i l i t y to  enhance outer membrane p e r m e a b i l i t y to other agents. been reported elsewhere  I t has  (Hancock, R a f f l e and N i c a s , 1981) that  treatment with gentamicin made P. aeruginosa s u s c e p t i b l e to l y s i s by lysozyme, an.enzyme normally unable  to penetrate the  outer membrane to reach i t s s i t e of a c t i v i t y , p e p t i d o g l y c a n . C o n d i t i o n s known to block aminoglycoside did  not a f f e c t gentamicin-lysozyme  lysis.  t r a n s p o r t and k i l l i n g These c o n d i t i o n s  52  included  treatment  sodium a z i d e ,  Mg  lysis  of  and t h e  Nicas  the  completely  and R a f f l e ,  strain  H103 was  also  beta-lactam,  hydrolysis  intact  permeation of periplasmic increased  the  pretreatment  with  ug/ml g e n t a m i c i n present  during  hydrolysis lysozyme totally  8.  inhibited  H103, (MIC  As  Holloway,  of  strain  fact  of  15 u g / m l  with  gentamicin and 1 . 5 the  ug/ml, rate  to  nitrocefin  of  personal  could  of  be  .  polymyxin  resistant  chloramphenicol  reference  ug/ml  2 +  by  10  permeabilization 1 mM M g  the  be  H103 c e l l s  5 0 mM E D T A i n c r e a s e d  the  increased  could  and 1 . 7 - f o l d  of  of  membrane t o  hydrolysis  concentrations  rate  indicate  outer  untreated  in  hydrolysis  gentamicin-mediated  by  in  would the  rate  the  gentamicin  of  Resistance to  200-400  membrane b y  An i n c r e a s e d  gentamicin  with  1 mM  case  was p r e v i o u s l y of  of  actual  Treatment  10-fold.  The  that  a s s a y were  Other p r o p e r t i e s (a)  B.  [the  activity,  outer  cells  The  100 ug/ml  the  respectively].  the  non-growing  over  by  1981).  beta-lactam through  3.5 fold  Gentamicin-promoted  inhibited  nitrocefin.  beta-lactamase.  and  (chloramphenicol  examined by m e a s u r i n g  a chromogenic in  dinitrophenol  synthesis  A mutation.  Permeabilization of in  uncouplers  protein  str  was h o w e v e r  (Hancock,  2 +  KCN,  inhibitors  and t e t r a c y c l i n e ) lysozyme  with  strain  mutated to  used, high  compared t o  and o t h e r P.  antibiotics.  aeruginosa  PA01  chloramphenicol  10-20 for  communication).  strains  The  most  strain  resistance  isolates;  polymyxin  resistant  53  mutants H181 and H185 were found chloramphenicol  to have l o s t t h i s high l e v e l o f  r e s i s t a n c e , and had MIC of 20 ug/ml.  The  r e v e r t a n t s of H181 and H185 regained the same high l e v e l s of chloramphenicol  r e s i s t a n c e as the p a r e n t .  S t r a i n s H181 and  H185  d i d not d i f f e r from H103 with regard to t h e i r s u s c e p t i b i l i t y to rifampicin, cefsulodin, t i c a r c i l l i n ,  Cu  2 +  and A g , as 2 +  w e l l as c a r b e n i c i l l i n and t e t r a c y c l i n e as shown i n Table IV.  (b)  A b i l i t y to accept R P l .  The plasmid RPl could be con-  j u g a l l y t r a n s f e r r e d e a s i l y i n t o H103  from e i t h e r E. c o l i or  other P. aeruginosa s t r a i n s with a t r a n s f e r frequency of 1.02.4 x 1 0 ^ transconjugants per r e c i p i e n t .  In c o n t r a s t , the  -  polymyxin  r e s i s t a n t mutant H181 accepted  4  the plasmid at 1 0  lower  frequency,  2.0 x 10  to  —7  — f t  10 - f o l d  3  to 6.7 x 10  .  Frequen-  c i e s of c o n j u g a t i o n d i d not d i f f e r according to the a n t i b i o t i c used  for selection.  (c)  Loss of v i a b i l i t y  in cold storage.  I t was found  that  c u l t u r e s of H181 and H185 which had been stored at 4°C f o r seve r a l weeks gave r i s e to a high p r o p o r t i o n of r e v e r t a n t clones when s u b c u l t u r e d and t e s t e d f o r polymyxin attempt  resistance.  In an  to e x p l a i n t h i s phenomenon, the l o s s of v i a b i l i t y of  w i l d type and polymyxin  r e s i s t a n t mutants a f t e r storage  compared.  that a f t e r one week i n l i q u i d growth  I t was found  medium a t 4°C, v i a b l e counts of H103 showed c o n t r a s t to 1.2-4.6% s u r v i v a l of H181.  was  15-45% s u r v i v a l , i n  Of the s u r v i v i n g  H181  54  c l o n e s , only stored  0.3-1% remained polymyxin r e s i s t a n t .  Cultures  f o r 1-2 weeks at -70°C i n 10% dimethyl s u l f o x i d e showed  a s i m i l a r d i f f e r e n c e i n v i a b i l i t y between H103 and H181.  2+ 9.  Summary.  C e l l s grown i n Mg  - d e f i c i e n t medium  (0.02 mM  2+ Mg  ) were more r e s i s t a n t to the a c t i o n of EDTA, polymyxin 2+ B, and aminoglycosides than were c e l l s grown i n Mg -sufficient 2+ 2+ 2+ 2+ medium. Ca , Sr , or Mn could s u b s t i t u t e f o r Mg in r e v e r s i n g r e s i s t a n c e while s e v e r a l other c a t i o n s could n o t . Mutants s e l e c t e d  f o r polymyxin r e s i s t a n c e  resembled c e l l s grown  2+ i n low Mg EDTA. sides.  i n their resistance  to c a t i o n i c a n t i b i o t i c s and  These mutants a l s o showed a l t e r e d uptake of aminoglycoI t was shown that aminoglycosides could  the outer membrane  i n wild  type c e l l s so as to make i t more  permeable to other substances.  Aminoglycoside-mediated pj.  permeabilization  could  i n t e r a c t with  be i n h i b i t e d with Mg  .  55  CHAPTER  TWO  OUTER MEMBRANE C H A R A C T E R I Z A T I O N  1.  O u t e r membrane p r o t e i n  was  shown t o  cells  (Fig.  tein  G level  was b y  far  high  4,  i n c r e a s e d up t o compare g e l  was  gel  of  F;  3-fold.  electrophoresis  of  Mg  Table  as  whole  while  these  by  HI  (Fig.  4,  gels  SDS  proteins.  m u t a n t s H181 and H185 h a d  protein  the  pro-  conditions,  judged cell  HI  - l i m i t e d H103  VI),  Under  cellular protein  B resistant  levels  O u t e r membrane p r o t e i n  24-fold in  E with  depressed  the major  acrylamide polymyxin  be  patterns.  HI  polyThe  constitutively  B a n d C)  which  varied  2+ only  2-fold with  (Table VI). H181 els tion HI,  The  and H185. of  c h a n g i n g Mg  protein  level  G  (e.g., B or  was c o n c l u d e d  unrelated  to  strains.  Similarly,  pyruvate els of  of  as  protein  porin with  the  (protein higher  the  F)  levels  observed  source,  of  greatly  effect  appeared t o be u n a l t e r e d  in  in  be  Hi.  these  the  medium  in  the  +  slightly  strains  lev-  altera-  in  protein  levels  of  protein  adapted or  could HI  lower.  porin (see  is mutant  (e.g.,  depress  levels.  somewhat r e d u c e d However,  both  depressed  conditions  NH4 )  in  1979) h a d no  this  on p r o t e i n  appeared t o be protein  or  growth  limiting  appeared to  of  with  decrease  a variety  G without  D2 a l s o  strains  EDTA r e s i s t a n c e ,  that  in  G was a l s o d e p r e s s e d  Hancock and Carey,  resistance  a carbon  protein  protein  Since other  in polymyxin it  of  concentrations  Levels  The in  lev-  level  of  membranes  function  Chapter  Four).  56  A  B  C  D  E  F  Figure 4. E f f e c t of a d a p t a t i o n on Mg2+-deficient medium and mutation t o polymyxin B r e s i s t a n c e on l e v e l s of p r o t e i n H i . Gels A and B - whole c e l l p r e p a r a t i o n s of s t r a i n s H103 and H185, r e s p e c t i v e l y , grown with 5 mM Mg ; Gels C and D - outer membrane p r e p a r a t i o n s o f s t r a i n s H181 and H103, r e s p e c t i v e l y , grown with 0.5 mM Mg +; Gels E and F - c e l l envelopes of H103 grown with 0.02 mM Mg + and 5 mM M g , r e s p e c t i v e l y . In the p r o t e i n p a t t e r n s o f whole c e l l s , c e l l envelopes or outer membranes o f H103 grown on low (0.02 mM) M g o r the polymyxin B r e s i s t a n t mutants H181 and H185 grown on high (5 mM) Mg^+, a l a r g e i n c r e a s e i n outer membrane p r o t e i n H i was observed, while p r o t e i n G was somewhat decreased compared to H103 grown on high Mg +. The high molecular weight p r o t e i n seen i n g e l E but not i n g e l F c e l l envelopes i s an inner membrane p r o t e i n of unknown f u n c t i o n induced i n e i t h e r H103 o r H181 grown i n low M g . 2+  2  2  2+  2 +  2  2+  57  An i n n e r in  all  membrane p r o t e i n  strains  (e.g.,  of  Fig.  75,000  4,  d a l t o n s was a l s o  G e l E)  g r o w n o n Mg  observed  -deficient  2+ medium b u t Thus, the  it  never  was  strains  concluded that  phenomena r e p o r t e d  wild  type  H207,  weight  to  proteins  Table  protein  It weight)  also  All  cell  sufficient  of  per  1979),  the  type  they  are  molecular  distinct provided  by  Alberta). protein  p e r mg  and the  amount  of  (87+7  ug/mg)  did  cells  m e d i u m (P  regained  a similar  amount o f  polymyxin  deficient  had  to  concentrations  Edmonton,  protein  medium.  unrelated  sample k i n d l y  ( 5 4 + 16%)  mg o f  medium and e i t h e r 2+  a l l Mg  p i l i n has  Alberta,  envelope  is  revertants 2+  at  a pilin  between w i l d  g r o w n i n Mg  six  patterns  was d e t e r m i n e d t h a t of  protein  Although  (University  significantly  cells  this  (Parynchych,  2-keto-3-deoxyoctonate vary  -sufficient  as determined u s i n g  Parynchych  (dry  VI). HI  g r o w n o n Mg  here.  membrane p r o t e i n  (e.g.,  W.  in  grown i n  Mg  not  -  resistant  mutants  or  >0.5 b y  Student's  t  test). The membrane w e r e  change  in  levels  with  in part  by  of  protein  HI  in  the  outer  degree of r e s i s t a n c e to 2+ t w o p o l y m y x i n a n t i b i o t i c s a n d b y t h e Mg concentration of the c e l l e n v e l o p e s o f s t r a i n s H103, H181, H185 a n d H207 g r o w n a t v a r i o u s m e d i u m Mg concentrations (Table VI). Thus, a 7 - f o l d i n c r e a s e i n H I l e v e l s b e t w e e n s t r a i n s H 1 0 3 a n d H 1 8 1 , g r o w n i n BM2  minimal  reflected  the  the  s u c c i n a t e m e d i u m c o n t a i n i n g 0 . 5 mM Mg  a 25-fold  Similarly,  increase  i n polymyxin  s t r a i n H 1 0 3 g r o w n o n Mg  2+  2+  was  B and c o l i s t i n -deficient-  associated resistance.  medium had 7  58  f o l d higher  l e v e l s of p r o t e i n HI than the same s t r a i n grown on  2+  Mg  - s u f f i c i e n t medium, and was 1 0 - f o l d more r e s i s t a n t to poly 2+  myxins.  I t i s w e l l e s t a b l i s h e d that high Mg  i n the medium i n h i b i t  concentrations  the a c t i o n of polymyxins  In the experiments d e s c r i b e d  (Newton, 1954).  i n Table VI, an increase  i n poly2+  myxin r e s i s t a n c e was observed i n the presence of 5 mM Mg d e s p i t e l e v e l s of p r o t e i n HI s i m i l a r to that of c e l l s grown i n 0.5  mM M g . 2+  As d e s c r i b e d  above (Chapter One, S e c t i o n 5 ) , t h i 2+  e f f e c t could  be accounted f o r by competition  between Mg  and 2+  polymyxin B f o r binding  sites.  The l a r g e molar excess of Mg  over polymyxin a t 5 mM polymyxin could account f o r the higher MIC  i n high M g . 2+  T h i s could  a l s o e x p l a i n why the MIC of H181  2+  and  H185 i n 0.5 mM Mg  i n d i c a t e higher  r e s i s t a n c e than H103  2+  grown i n 0.02 mM Mg d e s p i t e the high degree of s i m i l a r i t y between these c e l l s i n t h e i r l e v e l of HI (Table VI) and r e s i s t 2+  ance to polymyxin k i l l i n g  i n the absence of Mg  (Table I I I ) .  2+  with other  R e s u l t s of supplementation of Mg - d e f i c i e n t medium d i v a l e n t c a t i o n s are shown i n F i g . 5. Induction of 2+  p r o t e i n Hi was prevented with 0.5 mM C a  2 +  i n Mg  , M n , or S r 2+  - d e f i c i e n t media supplemented  2 +  ,  as w e l l as 0.5 mM  M g , but not with 0.5 mM Z n , S n , B a 2+  mM N a  2  +  (data not shown).  2 +  2 +  ( F i g . 5), A l  3 +  ,  or 1  As shown i n Table V, c e l l s grow i n  c a t i o n s which prevent HI i n d u c t i o n are h i g h l y s u s c e p t i b l e to killing  by polymyxin, EDTA-Tris and gentamicin, whereas c e l l s  grown i n c a t i o n s which f a i l e d  to prevent i n d u c t i o n of HI were 2+ •  i n d i s t i n g u i s h a b l e from c e l l s grown i n low Mg^  -  r e l a t i v e l y low s u s c e p t i b i l i t y to these agents.  in their  59  A  B  C  D  E  F  G  H  Figure 5. E f f e c t o f growth i n d i f f e r e n t d i v a l e n t c a t i o n s on i n d u c t i o n of p r o t e i n HI. Sodium dodecyl sulphate p o l y a c r y l a m i d e g e l of c e l l envelopes from c e l l s grown i n the presence of d i f ferent divalent cations. Lane A, 0.02 mM M g ; Lane B, 0.5 mM Mg2+ Lanes C-H, 0.0 2 mM M g plus 0.5 mM C a ( C ) , 0.5 mM Mn (D), 0.5 mM S r (E) , 0.5 mM B a ( F ) , 0.5 mM S n (G) or 0.5 mM Z n (H). In order to ensure that a l l the p r o t e i n Hi r a n at the head modified p o s i t i o n , s o l u b i l i z a t i o n was c a r r i e d out a t 100°C f o r 10 min (as d e s c r i b e d by Hancock and Carey, 1979). This r e s u l t s i n p a r t i a l heat m o d i f i c a t i o n of p r o t e i n F to run with a lower r e l a t i v e m o b i l i t y a t the p o s i t i o n F* (Hancock & Carey, 1979). 2+  2+  2 +  ;  2+  2 +  2 +  2 +  2 +  60  2.  E f f e c t of s h i f t ' f r o m low to high Mg"  confirm  .  To f u r t h e r  the r e l a t i o n s h i p between the presence of outer membrane  p r o t e i n Hi and r e s i s t a n c e , s h i f t experiments were c a r r i e d o u t . 2+  C e l l s growing i n 0.02 mM  Mg  i n e a r l y l o g a r i t h m i c phase growth 2+  ( 600 ° A  f  0.15-0.2) were supplemented with Mg  t o 0.5 mM,  and  l e v e l s of Hi i n the c e l l envelope and s u s c e p t i b i l i t y to p o l y myxin B and EDTA-Tris were followed  over time.  As  described  2+  above, 0.02 mM  Mg  allows  the same growth r a t e as 0.5 mM  up to 2+  an A g o o o f 0.6, so these c e l l s could not be considered Mg l i m i t e d f o r growth. C o n t r o l experiments demonstrated that 2+  c e l l s grown i n 0.02 mM  Mg  showed i n c r e a s i n g l e v e l s of p r o t e i n 2+  Hi as the medium became depleted  i n Mg  .  However, the s h i f t  2+  to high Mg was performed on e a r l y l o g a r i t h m i c phase c e l l s , which showed only moderately higher l e v e l s of p r o t e i n Hi (2- to 2+  3-fold)  i n order  starvation  to avoid  any n o n - s p e c i f i c e f f e c t s of Mg  [e.g., s t r i n g e n t response  ( S t . John and Goldberg,  1980), ribosome e f f e c t s (e.g., Gestland, a l . , 1967)].  A d d i t i o n of M g  (about 42 min g e n e r a t i o n  2+  1966; S c h l e s s i n g e r e t  d i d not a l t e r the growth r a t e  time), and the c e l l s remained i n  l o g a r i t h m i c growth phase throughout the sampling p e r i o d .  The  r a t i o of outer membrane p r o t e i n HI to p r o t e i n H2, c a l c u l a t e d from densitometer t r a c i n g s of Coomassie blue  s t a i n e d g e l s was  used to estimate r e l a t i v e l e v e l s of p r o t e i n Hi i n c e l l lopes.  The time r e q u i r e d  enve-  f o r the r e l a t i v e l e v e l of p r o t e i n HI  61  Min. after addition of Mg Figure 6. E f f e c t of s h i f t from low to high Mg2 + on l e v e l s of p r o t e i n HI and s u s c e p t i b i l i t y to EDTA-Tris and polymyxin B. C e l l s growing in 0.02 mM Mg + r e c e i v e d M g to a f i n a l c o n c e n t r a t i o n of 0.5 mM at t i n e 0. Samples of c u l t u r e s were subsequently removed at i n t e r v a l s and assayed f o r p r o t e i n HI l e v e l s and s e n s i t i v i t y . A. Decrease i n r a t i o of p r o t e i n H 1 : H 2 measured from s t a i n e d g e l s of c e l l envelopes ( l e v e l s of p r o t e i n H2 are constant under the c o n d i t i o n s used). B. Increase i n s e n s i t i v i t y to b a c t e r i c i d a l a c t i o n of EDTA-Tris (0) or polymyxin B (f). C e l l s sampled at the given times were treated f o r 5 min with e i t h e r 10 mM EDTA i n 10 mM T r i s - H C l pH 8.5 or 75 ug/ml polymyxin B i n phosphate b u f f e r , then p l a t e d f o r v i a b l e counts. C. Increase i n s e n s i t i v i t y to b a c t e r i o l y t i c a c t i o n of EDTA-Tris. L y s i s was measured as the decrease i n A g o o a f t e r 15 min treatment with 10 mM EDTA-Tris i n 10 mM T r i s - H C l pH 8.5 2  2+  62  to decrease by one-half was estimated as about 38 min, c l o s e to the  time f o r one c e l l d i v i s i o n .  t e i n HI l e v e l s t a b i l i z e d  A f t e r 45 t o 60 min,  a t the l e v e l s p r e v i o u s l y  the pro-  seen i n c e l l s  2+ grown i n Mg killing  s u f f i c i e n t medium.  Increase i n s u s c e p t i b i l i t y to  by polymyxin B and EDTA-Tris and l y s i s by EDTA-Tris  followed  a very s i m i l a r time course ( F i g . 6 ) .  s h o r t l a g before the c e l l s  increased  b a c t e r i c i d a l a c t i o n of EDTA.  There was a  i n s e n s i t i v i t y to the  However, s e n s i t i v i t y to l y s i s by  EDTA more c l o s e l y p a r a l l e l e d decrease i n p r o t e i n HI. 3.  Divalent  cation concentration  o f c e l l envelopes and d i s -  placement o f c a t i o n s by aminoglycosides and polymyxin B. As i n d i c a t e d  i n Table V I , decrease i n l e v e l s of p r o -  t e i n HI was accompanied by an i n c r e a s e content. of Mg  2+  In experiments with w i l d  i n c e l l envelope Mg  type c e l l s and three l e v e l s  , there was a r e c i p r o c a l r e l a t i o n s h i p between Mg  2+  l e v e l s i n the c e l l envelope and p r o t e i n HI l e v e l s ( c o r r e l a t i o n c o e f f i c i e n t of 0.99 by l i n e a r r e g r e s s i o n ) .  For the p r o t e i n Hi  2+  overproducing mutants grown i n Mg HI l e v e l s were about 7 - f o l d g r e a t e r  - s u f f i c i e n t medium, p r o t e i n than w i l d  type l e v e l s ,  2+  while envelope Mg  was reduced about  2-fold.  The r e l a t i o n s h i p between decrease i n p r o t e i n HI l e v els  and increase  i n c e l l envelope M g  a c t by r e p l a c i n g M g antibiotics.  2+  2+  suggested that HI may  at a s i t e susceptible  to c h e l a t o r and  This was t e s t e d by examining whether polymyxin or  63  aminoglycoside  could a c t to d i s p l a c e Mg  7+  i n whole  cells.  C e l l s were p r e - t r e a t e d with KCN to prevent inner membrane up7+  take of aminoglycosides.  When c e l l s grown i n 0.5 mM Mg 2+  t r e a t e d with 50 ug/ml of polymyxin  B, c e l l  envelope Mg  content was reduced by about 10%. Treatment gentamicin or 50 ug/ml streptomycin reduced about  3.5 - 5%.  T h i s suggests  were  with 25 ug/ml 2+ Mg l e v e l s by  that these agents can a c t to  2+ d i s p l a c e Mg  , but that a r e l a t i v e l y  small number of s i t e s  are i n v o l v e d . 2+ Table V I I shows that when c e l l s were grown with Ca  ,  2+ 2+ Mn , or Zn as the major d i v a l e n t c a t i o n , high l e v e l s of the major d i v a l e n t c a t i o n were i n c o r p o r a t e d i n t o the c e l l envelope. 2+ The l e v e l s of Zn were s i g n i f i c a n t l y lower than the l e v e l s of Ca or M g (P <0.05 by Student's unpaired t - t e s t ) i n c e l l s 2 +  2+  7+  grown with 0.5 mM Ca 4.  7+  and Mg  , respectively.  Comparison of -EGTA ~and'EDTA s u s c e p t i b i l i t y ' o f ~ C a  2 +  and  2+ Mg  grown c e l l s .  Since c e l l s were always provided with  2+ some Mg  as a growth f a c t o r , and c e l l envelopes  a l l contained  7+  s i g n i f i c a n t l e v e l s of Mg"  (Table V) , i t was attempted to 2+ determine whether EDTA was e x e r t i n g i t s e f f e c t on Ca grown 2+ c e l l s by removal of the small amounts of Mg or by removal of 2+ 2+ the major c a t i o n Ca (Table I I I ) . C e l l s grown i n Ca were e q u a l l y s u s c e p t i b l e to l y s i s by EGTA (which can be regarded as 6,  2+ 2+ a Ca - s p e c i f i c c h e l a t o r ) and EDTA (which c h e l a t e s both Ca  TABLE VII.  Divalent cations of c e l l envelopes after growth i n the presence of d i f f e r e n t cations.  2+ Mg present during growth (mM)  Divalent cations i n c e l l envelope (nmol/mg dry wt) 2+ 2+ 2+ „Ca Mn Zn Mg  Other cations present during growth (mM)  2 +  -  0. 02  54.7  <18  0. 02  „ Ba +  (0.5)  48. 5  <18  0. 02  (0.5)  53. 0  <18  0.02  r, + Sn „ Zn  (0.5)  19.5  <L8  0.02  o Ca +  (0.5)  26.3  147  (0.5)  25.0  <18  (0.5)  26. 0  <L8  122.5  <18  0. 02 0. 02  2  2  2  0.5  2+ Mn Sr  2+ Ca  <1  54.7  95  114. 5  +  2  0. 5  <0.4  Total  (0.5)  76. 5  173.3 111  <0.4  136  <1  115.5  Determined by atomic absorption spectroscopy; means of up to 5 separate determinations on 2 to 3 separate samples. i 2+ Mg plus other cation present during growth.  122. 5 192  b  65  and Mg^" a t high e f f i c i e n c y ) . 1-  EGTA had no measurable  e f f e c t on  2+  c e l l s grown on 0.5 mM  Mg  as the sole d i v a l e n t c a t i o n .  EGTA-  2+  T r i s had some b a c t e r i c i d a l a c t i v i t y on Ca  grown c e l l s , but none  2+ on Mg of  grown c e l l s .  However, the b a c t e r i c i d a l  activity  EGTA was s e v e r a l orders of magnitude lower than that of  EDTA.  P e r m e a b i l i z a t i o n of the outer membrane as a r e s u l t of  c h e l a t o r treatment was a l s o measured, by examining of  a chromogenic  beta-lactam n i t r o c e f i n .  hydrolysis  An i n c r e a s e i n the  r a t e of h y d r o l y s i s i n d i c a t e s i n c r e a s e d permeation of the b e t a lactam through the outer membrane to the p e r i p l a s m i c betalactamase and  (Angus e t a l . , 1982), and thus p r o v i d e s a s e n s i t i v e  specific  technique f o r demonstrating  outer membrane p e r m e a b i l i t y b a r r i e r .  d i s r u p t i o n of the  EGTA- and EDTA-Tris 2+  treatment were of s i m i l a r e f f i c i e n c y i n p e r m e a b i l i z i n g Ca grown c e l l s to n i t r o c e f i n , producing h y d r o l y s i s r a t e s about 30 2+  times higher than those seen i n unbroken c e l l s . Mg grown c e l l s were s i m i l a r l y a f f e c t e d by EDTA, but were not a f f e c t e d by 2+  EGTA.  Furthermore,  i f Ca  grown c e l l s which had been  t r e a t e d with EGTA were subsequently t r e a t e d with EDTA, o n l y a small i n c r e a s e (5-10%) i n the r a t e of h y d r o l y s i s was A similar  i n c r e a s e was seen i n EDTA t r e a t e d Ca  c e l l s subsequently t r e a t e d with EGTA. experiments  grown  A l l of the above  were repeated using an RPl plasmid-encoded  lactamase, with e s s e n t i a l l y  identical  observed.  results.  beta-  TABLE VIII.  E f f e c t s of EGTA-Tris and EDTA-Tris on c e l l s grown i n Mg  Cations during growth (mM)  % Lysis EGTA EDTA  2+  Mg Mg  51  0.5' 2+  2+ 0.02; Ca 0.5  % killing EGTA EDTA  40  36  and Ca  Increase i n ' n i t r o c e f i n hydrolysis^ EGTA ix-Tj EDTA  >99.99 37  >99.99  .  32  30  29  See Table V. Ratio of rate of n i t r o c e f i n hydrolysis i n c e l l s treated f o r 2 min with 10 mM EGTA or EDTA in 10 mM Tris-HCl pH 8.5 to rate of hydrolysis i n untreated c e l l s .  67  5.  P r o t e i n HI  strains  i n d u c t i o n i n other  Pseudomonas s t r a i n s .  in a c o l l e c t i o n representing  types of the  the 17 P_. aeruginosa  17  sero-  I n t e r n a t i o n a l A n t i g e n i c Typing Scheme (IATS) have  been shown to have outer membrane p r o t e i n p a t t e r n s s i m i l a r to that of P. aeruginosa PAO Hancock, 1982).  H103  extremely  (Mutharia,  Nicas  and  A l l these s t r a i n s have a p r o t e i n e q u i v a l e n t  p r o t e i n HI with r e s p e c t weight.  The  to heat m o d i f i a b i l i t y and  When these s t r a i n s and  to  molecular  a type s t r a i n of P. p u t i d a were  2+  grown i n Mg  d e f i c i e n t BM2  succinate  6 s t r a i n , showed l a r g e i n c r e a s e s s i m i l a r to that seen i n H103 6.  Summary.  a l l but one,  the IATS  i n l e v e l s of p r o t e i n Hi  (Fig. 7).  Outer membrane p r o t e i n HI was  present  as  major c e l l u l a r p r o t e i n both i n c e l l s which acquired  the  resistance 2+  to polymyxin B, aminoglycosides and d e f i c i e n t medium and  EDTA by growth in Mg  in polymyxin-resistant  mutants.  -  Those  2+  c a t i o n s which could  s u b s t i t u t e f o r Mg  to these agents a l s o prevented s h i f t experiments the  b i o t i c s and c h e l a t o r s . with a decrease i n c e l l 2+  2+  , or Zn  had  i n d u c t i o n of p r o t e i n HI.  2+  corre-  Increase i n p r o t e i n HI was a s s o c i a t e d 2+ 2+ envelope Mg . C e l l s grown i n Ca ,  l e v e l s of those c a t i o n s  - s u f f i c i e n t medium.  In  i n s u s c e p t i b i l i t y to c a t i o n i c a n t i -  envelopes s i m i l a r to the l e v e l s of Mg^ Mg  resistance  time course of l o s s of p r o t e i n HI  l a t e d with that of i n c r e a s e  Mn  in reversing  in t h e i r  cell  seen in c e l l s grown i n  In c e l l s grown i n Ca  2+  , but not  cells  68  A B 1 2 3 4 5 6 7 8 9 101112 13 141516 F i g u r e 7. I n d u c t i o n o f p r o t e i n HI i n Pseudomonas s t r a i n s grown i n low M g . SDS p o l y a c r y l a m i d e g e l s o f c e l l envelopes o f c e l l s grown i n BM2 s u c c i n a t e with 0.02 mM M g . A. H181; B. Pseudomonas p u t i d a type s t r a i n ; 1-16 IATS serotype s t r a i n s o f P. aeruginosa types 1-16. A l l s t r a i n s were i n d u c i b l e f o r p r o t e i n HI except f o r the type 6 s t r a i n . The type 17 s t r a i n i s not shown. 2+  2+  69  grown i n Mg  9+  , the calcium s p e c i f i c c h e l a t o r EGTA had e f f e c t s  s i m i l a r to those of EDTA.  Both aminoglycosides and polymyxin B 2+  could  be shown to d i s p l a c e Mg  from the c e l l  envelope.  70  CHAPTER THREE ISOLATION AND  CHARACTERIZATION OF A PORIN  DEFICIENT-MUTANT"AND"BACTERIOPHAGE - STUDIES  1.  I s o l a t i o n of outer membrane'protein random heavy'mutagenesis and for  out  by  protein'receptors.  coli  I s o l a t i o n of mutants was  alterations.  to o b t a i n c l a s s e s  is readily available  T h i s method has  selection  adopted a f t e r p r e l i m i n a r y  were g e n e r a l l y  mutagenesis procedure was  mutations i n the  survivors.  I t was  anticipated  inapplicable  slow, l a b o r i o u s  and  designed to generate  that  d e f i c i e n t mutants i s o l a t e d by  any  studies  multiple  outer membrane  protein  t h i s method could then be  phage and  below).  mutagen a f t e r p r e l i m i n a r y  P.  inefficient.  s e l e c t i v e agents such as  (see  protein  to  i d e n t i f y p o t e n t i a l l y useful bacteriocins  NTG  was  In order to avoid s e l e c t i n g  s t r a i n s r e s i s t a n t to the mutagen, the  to  bacterio-  chosen as  experiments showed that  used  the  mutagenesis  with d i e t h y l sulphate to s i m i l a r s u r v i v a l l e v e l s y i e l d e d auxotrophic mutants.  pro-  (Suzuki e_t a l . ,  s e l e c t i o n procedures f o r outer membranes  aeruginosa and  cell  been used  of mutants f o r which no  mutants i n other organisms were o f t e n  The  screening of  c a r d i o l i p i n synthetase  T h i s approach was  showed that  carried  such as mutants i n p e n i c i l l i n binding  t e i n s , l i p o p r o t e i n and 1978).  i s o l a t i o n of phage s p e c i f i c  random heavy mutagenesis followed by  envelopes f o r p r o t e i n i n E.  deficient~strains~by  fewer  mainly  treatment time chosen  was  71  w i t h i n the range where treatment r e l a t e d to s u r v i v a l .  time was  treatment  High frequency  Under the c o n d i t i o n s  with 1 mg/ml NTG)  0.004%, suggesting a high mutation  was  linearly  P r e l i m i n a r y experiments showed a l i n e a r  r e l a t i o n s h i p f o r up to 1 h treatment. chosen (30 min  still  the s u r v i v a l  was  frequency.  of mutation  among the 500 s u r v i v o r s  a l s o i n d i c a t e d by p r e l i m i n a r y screening which showed  unable  to grow on minimal medium, 15% unable  3% unable B39),  to grow at 42°C,  to serve as host f o r p i l u s s p e c i f i c phages (M6  23% unable  to serve as host f o r LPS  44), 6% c a r b e n i c i l l i n r e s i s t a n t , and r a r e mutation  19%  and  s p e c i f i c phage (Phage  0.4%  with the  of r e s i s t a n c e to high l e v e l s  relatively  (0.1 mg/ml) of  streptomycin. Screening of the 500  s t r a i n s f o r membrane p r o t e i n  a l t e r a t i o n s y i e l d e d mutants s e v e r e l y d e f i c i e n t  i n 4 of the 7  major outer membrane p r o t e i n s , F, G,  (Fig. 8).  Hi and  H2  Two  were found which appeared to have p r o t e i n F with a s l i g h t l y a l t e r e d molecular  weight present  i n somewhat reduced  The outer membrane p r o t e i n a l t e r a t i o n s were confirmed p o l y a c r y l a m i d e g e l s of p u r i f i e d outer membranes. deficient  i n p r o t e i n s I, D l , or D2 were  No  amounts. on  SDS  strains  found.  A number of s t r a i n s appeared to have moderately molecular weight  (50,000-80,000) p r o t e i n s present  amounts (e.g., T133  F i g . 8, Gel D).  I t was  high  in large  not e s t a b l i s h e d  whether these were inner or outer membrane p r o t e i n s .  72  A B C D E F G  H I J K L M  Figure 8. Membranes of h e a v i l y mutagenized s t r a i n s with apparent p r o t e i n a l t e r a t i o n s . Arrows i n d i c a t e alterations. Gels A, H, M - H103 (wild type) outer membrane (OM); Gel B T316 c e l l envelope (CE) (Hi d e f i c i e n t ) ; Gel C - T509 CE (G d e f i c i e n t ) ; Gel D - T133 CE (extra p r o t e i n ) ; Gel E - T513 CE (H2 d e f i c i e n t ) ; Gel F - H283 CE ( F d e f i c i e n t ) ; Gel G - T129 (Hi d e f i c i e n t ) ; Gels I and K - T817 OM ( F a l t e r e d ) ; Gels J and L T941 OM ( F a l t e r e d ) ; Gels K, L and M were s o l u b i l i z e d without 2-mercaptoethanol so that F runs i n the F* p o s i t i o n .  73  B a c t e r i o c i n s and bacteriophages receptors. ficient  have outer membrane  S t r a i n s r e s i s t a n t to these agents  i n those r e c e p t o r s , and bacteriophage  are u s u a l l y deand b a c t e r i o c i n s  which use s p e c i f i c outer membrane components can o f t e n be used as a means of s e l e c t i n g outer membrane p r o t e i n  deficient  mutants ( f o r examples see reviews by Osborne and Wu, 1 9 8 0 ; and DiRienzo e_t al.., 1 9 7 8 ) .  An attempt  was made to use 7 s t r a i n s  with a l t e r e d outer membrane p r o t e i n s i s o l a t e d by random mutagenesis to i d e n t i f y agents which could then be used f o r s p e c i f i c outer membrane p r o t e i n mutants.  to s e l e c t  The 7 s t r a i n s  were t e s t e d f o r s e n s i t i v i t y to a s e t o f 2 2 a e r u g i n i c i n s and 3 2 phages.  P a r t i a l c h a r a c t e r i z a t i o n of the r e c e p t o r s of these  phages had shown t h a t 1 9 of them probably had outer membrane p r o t e i n r e c e p t o r s (Table I I ; Nicas and Hancock, 1 9 8 0 ) .  How-  ever, no bacteriophage with obvious p o t e n t i a l u t i l i t y was found i n the c o l l e c t i o n , and none of p r o t e i n d e f i c i e n t mutants was r e s i s t a n t to any a e r u g i n o c i n . A second, more d i r e c t approach attempted l a t i o n of phage of the d e s i r e d s p e c i f i c i t y the enrichment i s based  technique of Verhoef  was the i s o -  from nature using  et a l . ( 1 9 7 7 ) .  T h i s method  on the use of b a c t e r i a l a c k i n g a s p e c i f i c r e c e p t o r to  adsorb out the m a j o r i t y of phages.  A d s o r p t i o n i s followed by  p r o p a g a t i o n , and the process repeated  for several cycles.  Each of the 7 o u t e r membrane p r o t e i n - a l t e r e d i s o l a t e d was used as the adsorbing s t r a i n ments .  strains  i n separate e x p e r i -  74  R e s u l t s of these enrichments are summarized procedure was  successful  or p r o t e i n - a l t e r e d  No attempt was made to e s t a b l i s h  whether  unable to form plaques on s p e c i f i c outer  strains  on  used.  the 3 groups of phages membrane-altered  each represented a s e t of m u l t i p l e  phage or s e v e r a l  The  i n i s o l a t i n g phage unable to p l a t e  3 of the 7 p r o t e i n - d e f i c i e n t  mutants  i n Table IX.  i s o l a t e s of the same  d i f f e r e n t phages with the same host range.  Four phage i s o l a t e s were/ found which d i d not form plaques on the G d e f i c i e n t s t r a i n T509. form plaques on the second G d e f i c i e n t mutants  resistant  not  altered  Nine  apparent on SDS  None  polyacryl-  (V20-V28) were found which d i d  a strain deficient  i n both p r o t e i n  HI  These phages were able to form plaques on o t h e r LPSstrains.  phages had a l t e r e d  However, 19 o f 20 mutants  resistant  LPS as judged by i n a b i l i t y  on LPS r e c e p t o r - s p e c i f i c examined  alterations  Nine phage i s o l a t e s  form plaques on T316,  and LPS.  s t r a i n , T817.  to t h i s group of phages were i s o l a t e d .  had any membrane p r o t e i n amide g e l s .  These i s o l a t e s d i d  phages.  l e v e l s of Hi (data not shown). ( i s o l a t e d a g a i n s t phage V28)  strains  3 had apparently reduced  These were named H317,  and H319  A spontaneous r e v e r t a n t of H318,  to form plaques  Of the 14 r e s i s t a n t  on SDS p o l y a c r y l a m i d e g e l s ,  to these  H318  ( i s o l a t e d a g a i n s t V27) .  named H318b, was  also  found.  A number of phage i s o l a t e s which d i d not form plaques on the p o r i n  deficient  s t r a i n H283 were a l s o obtained (phages  TABLE  IX.  Results protein  Adsorbing Strain  o f enrichment procedure f o r i s o l a t i o n r e c e p t o r - s p e c i f i c phages.  O u t e r Membrane Alteration 3  H283  F-deficient  T817  F-altered;  T941  F-altered  T509  # Isolates Tested A f t e r Enrichment  161 G-deficient  60  o f outer  membrane  # I s o l a t e s Not P l a t i n g on Deficient Strain  15 (V1-V15) 9 (V16-V19)  120  0  G-deficient  60  0  T129  Hl-deficient  60  0  T316  Hl-deficient; LPS altered  120  9 (V20-V28)  T513  H2-deficient  120  0  See  Fig.  Isolate  8. designations.  76  V1-V15). normal  However,  levels  strongly porin  2.  of  revertants  porin  indicating that  (see  F deficient  mutagenesis, strain.  To  revertants isolated H321,  of  strain  circumvent  isolated.  fortuitously selected  for  The  their  repeated  phage  attempted since  of  C),  5  copies/cell  of  the  phages,  something other  isolate.  gel  As  than  these,  for of  expected  strains  revertants  only  with  third,  higher  growing  in  liquid  porin  of  deficiency  cell  P_. a e r u g i n o s a  confin-  Transfer  a v a i l a b l e method of  in  were  The  below).  for  this  spontaneous  and H283 was  (see  electrophoresis  transfer  in  H284 and H324  cells  patterns  the  heavy  3 independent  responsible  the  gene  of  subculture  F has been of  H103.  H 3 2 1 , a n d H324 ( d a t a  levels  of  mutant  strain  F  have  screening  envelopes, tend  to  be  low. Protein  10  these  isolated after  enriching  gene  w a s SDS p o l y a c r y l a m i d e  x  to  g r o w t h o n BM2 g l u c o s e .  sensitivity  s p e c i f i c mutant  and f r e q u e n c i e s  Two  by  isogenicity  quite  to  is  deficient  problem,  after  medium.  was n o t  receptor  H283 was  this  by  the  their  a porin  growth rates  of  also resistant  appear  more t h a n one m u t a t i o n m i g h t be  were  was  ed by  H283 w h i c h  below).  Characterization  protein  were  of  (Fig. proteins  9,  protein  H283 d i d  Gels was  F.  In  the  same i n  The  not  be p r e s e n t  revertants  shown)  contrast,  not  B and E).  shown t o  outer  pattern  the mutant  of  Gel normal  membranes o f  the  levels  other  H283 a n d  9,  1 - 3  apparently  contain observable The  about  H284 ( F i g .  contained the  in  outer  its  of  protein  membrane  revertants.  77  [  •1s  Figure 9. C e l l envelopes o f w i l d type s t r a i n H103, i t s p o r i n d e f i c i e n t mutant H283, and a r e v e r t a n t , H284. G e l A, D. - H103; Gel B, E. - H283; G e l C. - H284. C e l l s envelopes f o r g e l s A, B, and C were s o l u b i l i z e d i n normal r e d u c t i o n mix b e f o r e a p p l i c a t i o n t o the g e l . For g e l s E and D, 2-mercaptoethanol was omitted from the s o l u b i l i z a t i o n mix, so t h a t p r o t e i n F runs i n the F* p o s i t i o n . The amount o f p r o t e i n F d e t e c t a b l e i n H283 i s l e s s than 1% t h a t o f H103 o r H284.  78  When t h e H103 a n d t h e toethanol 1979), larly  electrophoretic  revertants  from the  no o u t e r  other  in  revertant,  although  bands.  parent  inducible porin 3.  strain outer  ing  the  the  can be were  the  the  strain  when t e s t e d  of  piliated.  apparent  of  Dl,  H283 a l s o  of  of  similar  to  two  HI.  membrane  protein  strains.  agents  As  is  bacter-  a  testuseful  No  differences  polymyxin  B-resistant  (Table X).  The in  and 3 3 p h a g e s .  pilus  on  glucose-inducible  the  H103  receptor  H 1 8 1 , H185 a n d H283 h a d of  the  enhancement were  to  F band  a n d 22 a e r u g i n o c i n s  aeruginocins  3 revertants  appeared  protein  showed no d i f f e r e n c e s  smooth LPS o r  that The  strain,  E).  a  these  of  simi-  D and  those  membrane a l t e r a t i o n s .  type  the  H283 was  membrane r e c e p t o r s ,  to  24 p h a g e s  Carey,  produce  and s e r o t y p e outer  from  to  and p r o t e i n outer  F  2-mercap-  and  Gels  to  large  ability  strains  outer  wild  were  concluded  have  9,  revertants  susceptibilities  against  phages  in  1980),  sensitivity  H181 and H185 t o  deficient  the  membrane p r o t e i n s :  revealing  compared t o  of  their  mucoid i s o l a t e s ,  in  of  resulted  and Carey,  Fig.  comparable  H283 and i t s  susceptibility  found  mutants  gels  (Hancock  membrane p r o t e i n s  absence  and b a c t e r i o c i n s  method o f were  the  H103 i n  Bacteriophage mutants,  outer  protein  from s t r a i n  (c.f.  quantities  Strains  (Hancock  iophages  major  H283 i n  SDS p o l y a c r y l a m i d e  modified  of  omission of  buffer  membrane p o l y p e p t i d e  be p r e s e n t  minor  i n c r e a s e d by  solubilization  2-mercaptoethanol All  the  was  mobility  H283 w e r e  when porin  sensitivity Since  specific,  smooth LPS also  some it  and  sensitive  to  79  all  these  with  phages,  pilus  receptors.  suggesting p i l i .  15 p h a g e s  strain,  could type  be  revertant that  to  shown  H103  relatively  poorly H284  strain in  phages  which  serotypes  to  to  but  adsorb  species  porin  Meadow,  1975).  1981).  As b a c t e r i o p h a g e s  Pili  cally  unique  Braun  and K e i g e r - B a u e r ,  form plaques these tivity  sites  also  strains. to  It  protein  since  the  vary are  V4  cells  titre),  but  H283 o r  titre).  This  the  does  not  single  to  surface ability  be n o t e d  and of  the  its suggests for  into LPS  (Brokopp  (Koval  strain  and  (Brinton,  with  chemi-  components  (e.g.,  of  a given  a receptor that  necessarily amino a c i d  17  differences  interact  indicates  however  their  These  from s t r a i n  cell  were  cells.  in  to  H283  H283  whole  LPS 0 s i d e - c h a i n  thought  wild-  on  of  c a n be d i v i d e d  1979).  as  an L P S -  phages,  mutant  phage  the  strains  a bacteriophage  receptor  in  1975),  should  phage  differences  on s p e c i f i c  on s e v e r a l  to  in wild-type  et. a l . ,  chemical variations  and  these  deficient  aeruginosa  reflect  on  l a c k a membrane r e c e p t o r  present  1979; Lanyi  in  in  plate  Revertants of  phages  as w e l l  form plaques  efficiently  b a s e d on a n t i g e n i c  Farmer,  Two  reduction  P.  to  above).  derivatives is  function, to  to  non-motile,  AK1213,  unable  phages.  resistant  also  able  strain,  (see  the  was  flagellar  (2-10% r e d u c t i o n its  The  these  (95-100%  H283 a n d  these  the  H284 w h i c h w a s  (V1-V15)  AK1012,  from nature  resistant  wild  and  This  was d e f e c t i v e of  isolated  also V7,  for  H103, a p i l u s - d e f i c i e n t  deficient was  it  A set  type  except  phage  to  common  to  l a c k of imply  sensi-  absence  substitutions  in  of  80  such p r o t e i n s has  been shown to r e s u l t i n r e s i s t a n c e to phages  which normally use CR63; Braun and used had  Keiger-Bauer, 1977).  allowing  them to  i n t o f i v e groups (see Table I I , Methods).  sensitivity Table XI.  t e s t i n g are l i s t e d The  coli  Receptors of the phages  been p a r t i a l l y c h a r a c t e r i z e d  separated  (e.g., E.  t h i s p r o t e i n as a receptor  in Table X and  be  Results  summarized i n  r e s u l t s with the 13 phages that had  uncharacter-  ized protein receptors  i n d i c a t e d that these receptor  q u i t e w e l l conserved.  For example, phage 7 formed plaques  all  17 £ . aeruginosa s e r o t y p i n g  B9F  and  F116  p l a t e d on 16 and  s i t e s were on  s t r a i n s t e s t e d , while phages  11 s t r a i n s , r e s p e c t i v e l y .  None of the b a c t e r i a studied  appeared rough as  tested  e i t h e r with a bacteriophage which used rough but not smooth as  i t s receptor,  or by examination of gross colony  With the exception p l a t e d one XI).  LPS  morphology.  of IATS type 11 b a c t e r i a , a l l typing s t r a i n s  or more smooth LPS  s p e c i f i c phages (Tables X  Indeed, 9 of the 17 serotypes This  homology ( i . e . , the r e c e p t o r  s i t e ) i n the 0 antigens  d i f f e r e n t serotyping  i n d i c a t e d that there  s t r a i n s despite  s i g n i f i c a n t chemical v a r i a t i o n s .  and  p l a t e d 7 or more of the 9  smooth L P S - s p e c i f i c phages.  i s some  of  the p r i o r o b s e r v a t i o n  I t was  agreement with the f i n d i n g that p i l i  of  f u r t h e r observed  o n l y 3 of the 17 s t r a i n s p l a t e d phages s p e c i f i c in  of  f o r PA01  that pili  d i f f e r from s t r a i n  s t r a i n as demonstrated f o r P. aeruginosa and  other  to  bacteria.  Taken o v e r a l l , the r e s u l t s suggested that the  surfac-  es of P_. aeruginosa c e l l s were moderately w e l l conserved, since  TAB LE X.  Bacteriophage s e n s i t i v i t y o f o u t e r membrane p r o t e i n s e r o t y p i n g s t r a i n s , and mucoid i s o l a t e s .  Phage  119x M6 339 44 E79 F8 109 1214 PBl C3A SI 352 PLS27 2 D3c 1 B5A B7A C7B B9E B9F 7 21 68 C21 F116 G101 B1A A8A B6B D3c~l B6C F10 V1-V15 +  +  +  ABC  Bacterial Strains 1 2 3 4 5 6 7 8 9 10 11 12 13 14 .15 16 17  S S S S S S s s s s s s  s s s s s s s s s s s s  mutants,  3  s s s s s s s  s s s s  s s s  s s s s s S' s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s  s  s s s s s s s s  s s s s s s s s s s  s s s s s s s s s s s s s s s s s s s  s  s s s  s s  s  s  s  s  s  s  s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s  s  s s s  s s s s  s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s  s  s  s  s  s  s  s  s s  s s  s s  s  s  s  s  s s  s  s  s s  s  s s s s s s s s s  s  s s  -  s  s s s s  s s s  s s s  s s s s  s  s s  s s  s  s  s  s s s  s s s 3 s s s s  s s  s  s  s  s  r  s s s  s  s  s  s  s  s s s s s s s s s s s s s s s  s  s s s  D E F G H I J K  s  s s s s s s s. s s s s s s s s s s s s s s - s s - s s - s s - c s  s s s s s s s s  s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s  s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s •s s s s s s s s s s s s s s s s s s 's s s s s s s s s s s s s s s - - s s s - - s s s - - s s s  - - -  s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s  s s s s s s s s s s s  A: H103 (wild t y p e ) ; B: AK1213 ( p i l u s d e f i c i e n t ) ; C: AK1012 ( L P S - d e f i c i e n t ) ; 1-17: IATS serotype s t r a i n s 1-17; D,E: p r o t e i n HI overproducers, H181 and H185; F: H283 ( p o r i n - d e f i c i e n t ) : G, H, I : r e v e r t a n t s o f H283 (H284, H321, H324); J , K: mucoid s t r a i n s , H329 and H325. "S" means s e n s i t i v e ;  "-" means not t e s t e d ;  no e n t r y means r e s i s t a n t .  TABLE XI.  Bacteriophage s u s c e p t i b i l i t y of serotype strains of P_. aeruginosa performed using phages propagated and characterized on P_. aeruginosa PA01 strains. Number of Phages to Which Strain i s Susceptible  Bacterial Strain  Smooth LPS specific phages (9)  H103 Pilus d e f i c i e n t LPS altered 0 Serotype 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17  9 9 0 9 9 9 9 9 7 7 3 1 1 0 1 3 . 7 3 8 1  Nos. of phages reacting with 45% of serotype strains  9/9  Rough LPS specific phage (1)  Pilus specific phages (2 )  Possible LPS receptor phages (6)  Possible protein receptor phages (13)  0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  2 0 2 0 0 0 0 0 2 2 0 0 0 0 0 0 0 0 2 0  6 5 0 4 4 5 5 3 2 3 0 0 3 0 0 2 1 1 2 2  13 13 13 12 12 11 12 13 7 6 4 1 4 2 5 6 8 5 6 4  0/1  0/2  2/6  11/13  Cumulative sus ceptib i l i t y (%)  100% 90% 50% 83% 83% 83% 87% 83% 60% 60% 23% 7% 27% 7% 20% 37% 53% 30% 60% 23% 73%  83  9 of the 17 s t r a i n s p l a t e d  53% or more of the phages screened.  The  s i m i l a r i t y of outer membrane pro-  antigenic  and p h y s i c a l  t e i n s of P. aeruginosa has been f u r t h e r s t u d i e d al.  by Mutharia e_t  (1982). Two mucoid d e r i v a t i v e s of H103 o r i g i n a l l y  selection f o r resistance  i s o l a t e d by  to phage 7 were a l s o t e s t e d .  these was i d e n t i c a l to the parent i n i t s phage  One of  sensitivity,  while the other was s e n s i t i v e to over 60% of the phages, cating ity,  4.  that mucoidy does not n e c e s s a r i l y  impede phage s e n s i t i v -  i n agreement with p r e v i o u s r e s u l t s (Martin,  Summary.  indi-  Random heavy mutagenesis with  1973).  nitrosoguanidine  was used to i s o l a t e mutants d e f i c i e n t i n outer membrane proteins  F, G, HI, and H2.  Three r e v e r t a n t s  d e f i c i e n t s t r a i n were a l s o i s o l a t e d .  of the p r o t e i n F-  Attempts to i s o l a t e phage  with outer membrane p r o t e i n  s p e c i f i c receptors  group of phages which could  be used to s e l e c t f o r mutants  deficient  in protein  y i e l d e d one  Hi and a group of phages unable to form  plaques on the p r o t e i n  F - d e f i c i e n t s t r a i n or i t s r e v e r t a n t s .  Revertants of the p r o t e i n  F - d e f i c i e n t s t r a i n had a p r o t e i n band  on SDS p o l y a c r y l a m i d e g e l s which was i n d i s t i n g u i s h a b l e protein and  from  F i n i t s e l e c t r o p h o r e t i c m o b i l i t y both i n the presence  absence o f 2 mercaptoethanol, while no such p r o t e i n was  detectable  on g e l s of the p r o t e i n  s e n s i t i v i t y of w i l d  type, p r o t e i n  F-deficient strain.  Phage  F - d e f i c i e n t and HI overpro-  ducing mutants were e s s e n t i a l l y the same, i n d i c a t i n g that the  84  mutants were p i l i a t e d , had smooth LPS, and lacked surface a l t e r a t i o n s .  Phage s t u d i e s with  representing  the 17 serotypes  considerable  conservation  i  a set of s t r a i n s  of P. aeruginosa  of phage  any gross  receptors.  indicated  85  CHAPTER  FOUR  MEASUREMENT O F " O U T E R MEMBRANE  O u t e r membrane p r o t e i n porin Benz  on  the  basis  and H a n c o c k ,  although  protein  porins  of  1980),  the  that  only  tional  of  1981).  Such  F forms  enteric  a small  possibility  of  the  protein self.  et  of  a strain  of  this  and  limited  the  severely  strain F)  and  a l . ,  was  and  observed  protein tested  deficient  in  revertants  and  in v i v o could  by  protein  F,  (which  have  The  technique  e m p l o y e d was b a s e d on  the the  This  enough  technique  is  beta-lactamase  is  beta-lactamase activity rate  of  diffusion  of  raises  than  examining  1981).  a  protein the  minor F  it-  permeability  H283.  Isolation  normal  that  levels  of  the  present  in  intact  The  the  to  b a s e d on  of  func-  1982).  be due  above.  (1977).  form  in  aeruginosa  et. a l . ,  F rather  the  low  Hancock, P_.  described  then by  (Angus  than  relatively  wild-type  F _in v i t r o  with  its  of  that  channels  proteins  1 9 8 2 ; Benz  in v i v o  effects  F is  porin  a  1979; Nikaido,  is  providing  plasm,  protein  hypothesis  Rosselet  that,  shown  co-purifying This  protein  been  that  e_t a l . ,  as  e_t a l . , 1 9 7 9 ;  indicated  larger  proportion  also of  have  protein  cells  activity  studies  of  membrane p e r m e a b i l i t y  low  (Hancock  activity  (Angus  characterized  studies  (Hancock  Low o u t e r has  F has been  substantially  bacteria  in v i t r o  channels  iri v i t r o  PERMEABILITY  of  Zimmerman concept  the  cells  beta-lactam across  periwill the  be  86  outer membrane to the p e r i p l a s m i c beta-lactamase. e q u i l i b r i u m r a t e of h y d r o l y s i s of beta-lactam (Vi tact) i  s  n  equal  to the r a t e of d i f f u s i o n  Thus, the  by i n t a c t (Vpjiff).  cells  This  allows  c a l c u l a t i o n of an outer membrane p e r m e a b i l i t y c o e f f i c i e n t C using  the f o l l o w i n g equation V  where S  Q U  int  = Diff V  of Zimmermann and R o s s e l e t :  = < o u t " in> C  £ i s the beta-lactam  S  S  c o n c e n t r a t i o n added and S^  i s the p e r i p l a s m i c c o n c e n t r a t i o n of beta-lactam calculated tam  from the Michaelis-Menten  [which can be  equation].  The b e t a - l a c -  used i n these measurements was the chromogenic  in n i t r o c e f i n  (0'Callaghan,  1972).  cephalospor-  The beta-lactamase used was  the p e r i p l a s m i c TEM-2 enzyme encoded by the plasmid was introduced  n  RPl, which  i n t o each of the s t r a i n s used by c o n j u g a t i o n .  Measurement of t o t a l beta-lactamase a c t i v i t y  i n c e l l s broken by  passage through a French press was c a r r i e d out to confirm p e r i p l a s m i c a c t i v i t y was i n excess. broken c e l l s v a r i e d l i t t l e 500-fold ies  higher  that  Beta-lactamase a c t i v i t y of  from s t r a i n to s t r a i n and was 7 t o  than the a c t i v i t y of whole c e l l s .  Other  stud-  (Angus et. a l . , 1982) have demonstrated that the temperature  c o e f f i c i e n t of n i t r o c e f i n h y d r o l y s i s by i n t a c t c e l l s  (V ^ ) is D  f f  c o n s i s t e n t with n i t r o c e f i n e n t e r i n g v i a a h y d r o p h i l i c pathway. In a d d i t i o n , i t was e s t a b l i s h e d here that n i t r o c e f i n h y d r o l y s i s i n i n t a c t c e l l s was d i r e c t l y p r o p o r t i o n a l to the c o n c e n t r a t i o n of n i t r o c e f i n added  (S  oni  . ) f o r s t r a i n s H103 (RPl) and E. c o l i  87  UB1636 (RPl) over an 8 - f o l d range of s u b s t r a t e  concentrations  ( 0 . 0 2 5 - 0 . 0 2 mg/ml), as p r e d i c t e d by the above d i f f u s i o n equation. The H283 was H103  r e s u l t s shown in Table XII revealed  that  s i g n i f i c a n t l y l e s s permeable than i t s parent  or the r e v e r t a n t H284.  these r e s u l t s were high, nitrocefin  While the standard  d e v i a t i o n s of  H103  or H284 d i d  i s s i g n i f i c a n t l y more permeable than H103  In c o n t r a s t , there was  permeability  than any  this  Z61  revertant  modified  no s i g n i f i c a n t a l t e r a t i o n in  p e r m e a b i l i t y of the aminoglycoside and An E_. c o l i K-12  mutant  or i t s f u l l  (Angus e t a l . , 1982) were confirmed using  mutant H181.  the  As an a d d i t i o n a l c o n t r o l , previous r e s u l t s  demonstrating that the a n t i b i o t i c s u p e r - s u s c e p t i b l e  assay.  not  the means were c l e a r l y d i f f e r e n t as judged by  Student t - t e s t .  H251  strain  the range of r a t e s of h y d r o l y s i s of  i n i n t a c t c e l l s of H283 and  o v e r l a p and  strain  polymyxin r e s i s t a n t  s t r a i n UB1636 had  of the P. aeruginosa  much g r e a t e r  s t r a i n s studied  here .  Summary.  Outer membrane p e r m e a b i l i t y was  measured using  the  r a t e s of h y d r o l y s i s , i n i n t a c t c e l l s , of a chromogenic  beta-  lactam, n i t r o c e f i n , by p e r i p l a s m i c beta-lactamase.  was  It  shown that the p r o t e i n F - d e f i c i e n t mutant H283 had  reduced out-  er membrane p e r m e a b i l i t y  revertant,  r e l a t i v e to i t s parent  or  i n d i c a t i n g t h a t t h i s p r o t e i n i s the major outer membrane p o r i n . P r o t e i n HI overproducing  s t r a i n s were not a l t e r e d in outer  TABLE XII.  Strain  Rate of n i t r o c e f i n h y d r o l y s i s by i n t a c t c e l l s , and outer membrane p e r m e a b i l i t y c o e f f i c i e n t s C, o f P_. aeruginosa s t r a i n H103 (RPl) , i t s RPl p l a s m i d - c o n t a i n i n g d e r i v a t i v e s , Z61 (RPl) and E. c o l i s t r a i n C127 ( R P l ) .  Phenotype  Nos. o f Determinations  Rate o f n i t r o c e f i n hydrol y s i s i n intact cells (pmol n i t r o c e f i n m i n l mg c e l l d r y wt~l) -  a  S i g n i f i c a n c e , by Outer Membrane Student t - t e s t , of permeability d i f f e r e n c e from coefficient C H103 (RPl) ( s e c ~ l mg c e l l dry w t " x 10 ) 1  H103 (RPl)  Parent  H181 (RPl)  4  12  60  ± 17  4.1  GM, PX, EDTA resistant, protein Hi overproducing  9  62  ± 36  4. 2  p>0. 5  H251 (RPl)  Revertant o f Z61  7  58  ± 13  3.9  p>0.5  H284 (RPl)  r e v e r t a n t o f H283  7  59  ± 30  4.0  p>0. 5  H283 (RPl)  protein  5  0. 7  p<0. 01  Z61 (RPl)  a n t i b i o t i c supersusceptible  F-deficient  UB1636 (RPl) E . c o l i  mean ± standard d e v i a t i o n  9.8 ±  7.6  12  360  ±170  24.9  p<0. 001  13  740  ±390  50.6  p<0.001  89  membrane p e r m e a b i l i t y . of J P . aeruginosa  An a n t i b i o t i c  supersusceptible  was found to be s i g n i f i c a n t l y more permeable  than i t s r e v e r t a n t or the w i l d type.  An E.  c o l i s t r a i n had  much g r e a t e r p e r m e a b i l i t y than any of the P_. aeruginosa used  here.  strain  strains  90  DISCUSSION  T h i s study demonstrates that the outer membrane of Pseudomonas aeruginosa p l a y s a major r o l e i n the a n t i b i o t i c r e s i s t a n c e of t h i s organism. studies  The r e s u l t s of the p e r m e a b i l i t y  (Table XII) were c o n s i s t e n t with the hypothesis  a n t i b i o t i c r e s i s t a n c e can be explained permeability  on the b a s i s of low  of the P. aeruginosa outer membrane due to the  p r o p e r t i e s of p r o t e i n F. indicated  Although in v i t r o experiments have  that the area of i n d i v i d u a l p r o t e i n F channels i s up  to 3 - f o l d l a r g e r than the area of E_. c o l i and cell  that  p o r i n channels  (Benz  Hancock, 1981), and that the number of p o r i n molecules per i s about the same i n E. c o l i  and P. aeruginosa  (Benz and  Hancock, 1982; Rosenbusch, 1974), P. aeruginosa was shown to have an outer membrane p e r m e a b i l i t y  coefficient  c a n t l y lower than that of E. c o l i .  This was e s p e c i a l l y s t r i k -  ing  when i t i s considered  daltons)  (C) s i g n i f i -  that the s i z e of n i t r o c e f i n (520  approaches the e x c l u s i o n  l i m i t of E. c o l i p o r i n s .  It  would thus be expected that d i f f u s i o n of n i t r o c e f i n would be slowed by f r i c t i o n a l and s t e r i c the channel as d i s c u s s e d  i n t e r a c t i o n s with the w a l l s o f  by Nikaido  that the d i f f u s i o n constant  and Rosenburg  (1981), so  C would be decreased when compared  to a l a r g e r channel such as p r o t e i n F (Hancock and N i k a i d o , 1978).  In order  to smaller  to c o r r e c t f o r the apparent r e d u c t i o n  channels, the p e r m e a b i l i t y  i n C due  to n i t r o c e f i n of s i n g l e  pores of P. aeruginosa p o r i n compared to those of E. c o l i  91  p o r i n s may be c o r r e c t e d t h e o r e t i c a l l y using F i c k ' s law and the Renkin Lakshminarayaniah, assuming a hydrated  (Nicas and Hancock, 1982)  equations  1969, p.325).  (Renkin, 1954;  T h i s c a l c u l a t i o n can be made  r a d i u s of 0.44-0.53 nm f o r n i t r o c e f i n  ( s i m i l a r to t h a t of a d i s a c c h a r i d e ) and using previous estimates f o r the r a d i i of E_. c o l i  p o r i n l b and P. aeruginosa  p r o t e i n F (0.65 nm and 1.1 nm, r e s p e c t i v e l y ; Benz and Hancock, 1981;  Nikaido and Rosenberg, 1981).  Using such values one  a r r i v e s a t the c o n c l u s i o n that the s t e r i c e f f e c t of i n t e r a c t i o n of  n i t r o c e f i n with E_. c o l i  an apparent  p o r i n channel w a l l s w i l l i n the C value of E.  1 1 - f o l d decrease  result in  coli  pores  r e l a t i v e to the C value f o r s i n g l e pores of P. a e r u g i n o s a . Taken together with the 1 2 - f o l d d i f f e r e n c e t h i s study, the c a l c u l a t e d  i n C value shown i n  total difference  i n outer  pore area a v a i l a b l e f o r the d i f f u s i o n of n i t r o c e f i n aeruginosa was 132-fold lower. aeruginosa channel  membrane i n P.  Since the a c t u a l area of a P.  i s three times that of E.  number of a c t i v e and f u n c t i o n a l p o r i n channels  coli,  the t o t a l  (per c e l l dry  weight) would be l e s s than 1/400 of that of E_. c o l i or about 100 per c e l l  assuming 4 x 1 0  (Rosenbusch, 1974).  4  p o r i n t r i m e r s per c e l l  This difference  in permeability correlates  w e l l with the low p o r i n a c t i v i t y of p r o t e i n F which has been observed  both  iri v i t r o and jjri v i v o (Angus e_t al^., 1982; Benz  and Hancock, 1981) and a l s o c o r r e l a t e s with the known high i n t r i n s i c r e s i s t a n c e of P. aeruginosa to h y d r o p h i l i c antibiotics.  92  It has p r e v i o u s l y 1982)  been demonstrated  (Angus e t a l . ,  that the apparent number of f u n c t i o n a l channels can be  increased  by LPS mutation i n s t r a i n  Z&l, but no e x p l a n a t i o n  has  emerged as to why more than 99% o f p r o t e i n F molecules do not form f u n c t i o n a l pores i n w i l d  type c e l l s .  The evidence  report-  ed here from s t u d i e s with p r o t e i n F - d e f i c i e n t c e l l s argues i n favour of the p o s s i b i l i t y rather  that the a c t u a l p o r i n  than a minor contaminating p r o t e i n .  i s protein F  The p r o t e i n F-  d e f i c i e n t mutant had a s i g n i f i c a n t l y lower outer membrane permeable c o e f f i c i e n t C compared to i t s parent or to r e v e r t a n t strains.  Since s t r a i n H283 does have a measurable C value, i t  may w e l l not be t o t a l l y p o r i n - d e f i c i e n t , but r a t h e r p o r i n  pro-  t e i n F - d e f i c i e n t , i n that other p o r i n p r o t e i n s may be present at lower l e v e l s i n the outer membrane.  Black l i p i d  bilayer  s t u d i e s of f r a c t i o n s from p o r i n d e f i c i e n t mutants of E. (Benz e_t a l . , 1978) have provided s e l e c t i v e channel i s r e s p o n s i b l e these s t r a i n s .  coli  evidence that a more c a t i o n f o r residual porin a c t i v i t y i n  In the case of P. aeruginosa, two other  induc-  i b l e p o r i n p r o t e i n s , P and Dl (Hancock and Carey, 1980; Hancock et a l . , 1982), are p o s s i b l e candidates f o r p r o v i d i n g ual p o r i n a c t i v i t y of the p r o t e i n The  the r e s i d -  F - d e f i c i e n t s t r a i n H283.  low l e v e l of a c t i v e p o r i n  i n wild-type c e l l s  g e s t s that the h y d r o p h i l i c pathway i s a r e l a t i v e l y  sug-  inefficient  means of t r a v e r s i n g the outer membrane of P. a e r u g i n o s a .  It  would thus be expected that h y d r o p h i l i c a n t i b i o t i c s , i n c l u d i n g aminoglycosides, would be r e l a t i v e l y  i n e f f e c t i v e against  93  P. a e r u g i n o s a .  However, t h i s study o f f e r s evidence that  c a t i o n i c a n t i b i o t i c s e f f e c t i v e against aminoglycosides and polymyxins, of an a l t e r n a t e  may  major determinant s i d e s and EDTA.  B.  that the outer membrane i s a aminoglyco-  susceptibility  to c h e l a t o r s ,  In s h i f t experiments,  the decrease  in protein  Hi  by i n c r e a s i n g Mg + i n the growth medium c l o s 2  in s u s c e p t i b i l i t y  to EDTA-Tris  and  When other d i v a l e n t metal c a t i o n s were s u b s t i t u t -  ed f o r Mg +, only 2  c e l l s grown i n those c a t i o n s  which  prevented  i n d u c t i o n of p r o t e i n HI were s u s c e p t i b l e  polymyxin  B and  Nicas and  (Hancock, 1981;  to EDTA,  gentamicin.  I t has been suggested (Brown, 1975;  polymyxin  shown under a v a r i e t y of d i f f e r e n t growth  ely p a r a l l e l e d increase polymyxin  the membrane by means  A c l o s e c o r r e l a t i o n between l e v e l s of outer  B, and gentamicin was  about  cross  of the a c t i v i t y of polymyxins,  membrane p r o t e i n Hi and  brought  P. aeruginosa, i . e . ,  "self-promoted" pathway.  This study demonstrates  conditions.  highly  that polymyxins,  Hancock, 1980), and  Hancock et. a l . , 1981)  the outer membrane.  chelators  aminoglycosides  act at a common s i t e  A model f o r the a c t i o n of these agents  the outer membrane i s i l l u s t r a t e d  i n F i g . 10.  on on  I t i s proposed  that these agents a c t at a s i t e on the outer membrane, p o s s i b l y a polyphosphate  s i t e on the l i p o p o l y s a c c a r i d e  c a t i o n s bound at t h i s s i t e would be r e q u i r e d of the outer membrane.  and  that  f o r the  divalent  stability  Thus EDTA would act to remove d i v a l e n t  c a t i o n s by c h e l a t i o n , while the c a t i o n i c a n t i b i o t i c s , polymyx-  94  SENSITIVITY  °69  QM.  RESISTANCE  QM.  Figure'10. Model i l l u s t r a t i n g the proposed mechanism of r e s i s t a n c e to aminoglycosides, polymyxin B, and EDTA-Tris i n P_. aeruginosa with high l e v e l s of p r o t e i n HI. O.M. outer membrane; R core-the heptose, KDO, rough core r e g i o n of the LPS; O Ag-the somatic antigen of P. aeruginosa LPS; ( P ) - t h e polyphosphate p o r t i o n o f the P. aeruginosa LPS; Hl-major outer membrane p r o t e i n H i , which appears i n l a r g e amounts i n s p e c i f i c mutants and i n c e l l s grown i n Mg2+ d e f i c i e n t medium. I t i s proposed that the c r o s s l i n k i n g o f the n e g a t i v e l y charged polyphosphate r e g i o n s of the LPS by Mg i s important f o r outer membrane s t a b i l i t y i n sensitive c e l l s . EDTA by removing Mg2+, and the h i g h l y c a t i o n i c a n t i b i o t i c s aminoclycosides and polymyxin B by d i s p l a c i n g Mg2+, l e a d t o d i s r u p t i o n of the outer membrane p e r m e a b i l i t y b a r r i e r . I t i s proposed that i n c e l l s with high l e v e l s of p r o t e i n HI, t h i s p r o t e i n r e p l a c e s Mg2+ a t s p e c i f i c s i t e s i n the outer membrane. P r o t e i n H i thus p r o t e c t s the polyphosphate s i t e on the LPS from a t t a c h by aminoglycosides, polymyxin B and EDTA and makes the c e l l r e l a t i v e l y r e s i s t a n t to these agents. n  +  95  ins and aminoglycosides, would a c t by competing f o r the l i p o polysaccharide binding s i t e .  P r o t e i n HI i s proposed  to a c t by  r e p l a c i n g d i v a l e n t c a t i o n s at t h i s s i t e on the l i p o p o l y s a c c h a r i d e , thus p r o t e c t i n g i t from a t t a c k by these Both polymyxin  agents.  and EDTA are known to i n t e r a c t with P.  aeruginosa LPS (Cooperstock,  1974; Michaels and Eagon, 1966).  An e a r l y consequence of polymyxin  B and EDTA a c t i o n i s permea-  b i l i z a t i o n of the outer membrane (Brown and M e l l i n g , 1969; I i d a and Koike, 1974; Laporte e t a l . 1977)  and polymyxin  f  1977; Rosenthal  and Strom,  B has been shown to cause blebbing of the  outer monolayer of the outer membrane, which i s the s o l e t i o n of c e l l u l a r LPS ( G i l l e l a n d Teuber, 1975).  loca-  and Murray, 1976; S c h i n d l e r and  S c h i n d l e r and Osborn  (1979) have demonstrated  that the 2 - k e t o - 3 - d e o x y o c t o n a t e - l i p i d A r e g i o n of Salmonella typhimurium LPS has high a f f i n i t y b i n d i n g s i t e s f o r polymyxin (Kd = 0.3 t o 0.5 uM - approximately polymyxin-phospholipid Ca2+.  B  1 0 - f o l d higher than f o r  i n t e r a c t i o n s ) and f o r Mg2+ and  E a r l i e r s t u d i e s by Newton (1954) demonstrated that  Mg2+ and polymyxin  B competed f o r a P. aeruginosa  s i t e , which he p o s t u l a t e d to be polyphosphate  cellular  i n nature.  P_.  aeruginosa LPS has been demonstrated to have an e s p e c i a l l y high phosphate c o n c e n t r a t i o n (Dewry e t a l . , 1971), and may have up to 8 moles of phosphate per mole of LPS i n the heptose-KDO region  (A. K r o p i n s k i , p e r s o n a l communication).  evidence  There i s  that some of t h i s phosphate i s present as t r i p h o s p h a t e  (Wilkinson, 1981) .  Since phosphoryl  and phosphodiester  groups  96  are n e g a t i v e l y charged the r e l a t i v e l y high M g  2+  highly cationic,  cell  Aminoglycosides  are a l s o  and have been shown to bind to P_. aeruginosa  (Day e t a l . , 1 9 7 8 ) .  interact  content of P. aeruginosa  (Brown and Woods, 1 9 7 2 ) .  envelopes  LPS  at n e u t r a l pH, t h i s could c o n t r i b u t e to  The a b i l i t y of aminoglycosides to  with the outer membrane and promote a s i g n i f i c a n t  alteration  i n i t s p e r m e a b i l i t y was shown by both c e l l l y s i s i n  the presence  of lysozyme and aminoglycosides  (Hancock, R a f f l e  and N i c a s , 1 9 8 1 ) , and i n c r e a s e d h y d r o l y s i s of n i t r o c e f i n presence of g e n t a m i c i n . gram-negative  i n the  Lysozyme i s normally i n a c t i v e on  b a c t e r i a as i t i s unable  to penetrate the outer  membrane to reach i t s s i t e of a c t i v i t y , the p e p t i d o g l y c a n . I t was shown that gentamicin and streptomycin acted to overcome this penetration barrier,  a l l o w i n g lysozyme to a t t a c k the  p e p t i d o g l y c a n and l y s e the c e l l s .  Lysozyme i t s e l f  i s known to  bind to outer membranes (Day e_t a l . , 1 9 7 8 ) , and t h i s a b i l i t y may have c o n t r i b u t e d to the e f f i c i e n c y aminoglycoside-treated c e l l s . relatively  of i t s permeation i n  T h i s i s f u r t h e r suggested  i n e f f i c i e n t gentamicin-mediated  outer membranes to n i t r o c e f i n ,  p e r m e a b i l i z a t i o n of  a chromogenic beta-lactam.  f a c t , t h i s i m p l i e s that aminoglycoside-mediated t i o n has some s p e c i f i c i t y f o r c a t i o n i c lysozyme and a m i n o g l y c o s i d e s . cannot be a t t r i b u t e d  by the  In  permeabiliza-  s u b s t r a t e s such as  The p e r m e a b i l i z a t i o n observed  to aminoglycoside k i l l i n g  as i t was shown  that i t occurs under c o n d i t i o n s i n which aminoglycosides are known not to be t r a n s p o r t e d or l e t h a l  (Bryan and Van Den E l z e n ,  97  1976;  Hurwitz  KCN-treated  and Rosano,  cells  1961)  (Hancock  Aminoglycosides passage seems  of  other  likely  transport. ling  in  is  promotes  It  further  was  with the Mg  site 2 +  of  of  at the  that  Mg  that  less Nicas  at  active  by  of  level  was  able  both the  inducing  of to  results  the  in of  P.  such as  gentamicin,  permeabilization  aminoglycosides 1981).  aminoglycosides  such as  These to  to  promote  outer  a l . , 1971; it  is  exist.  It  has  are highly are  much  lysozyme  also  of  although  streptomycin  results  membrane i s the  et  also  which  that  antagonism  aeruginosa,  aminoglycosides  suggests  of  (Madeiros  competition  This  experiments  outer  high  membrane.  lysozyme.  killing  level  permit-  aminoglycoside  and t o  the  the  interaction  cytoplasmic  strongly  at  k i l -  divalent  the  of  it own  and  periplasm,  unusually  cations  a  This  inhibit  strains,  at  uptake  with  and  their  the  nitrocefin  activity  1973) sites  into  the  membrane,  promoting  membrane.  the  divalent  those  and R a f f l e ,  ability  to  P_. a e r u g i n o s a ,  efficient  2 +  explain  other  b e e n shown t h a t against  outer  Competition  and J a c k s o n ,  probable  at  of  interaction  antibiotic  with  site.  aminoglycosides Zimelis  the  aminoglycoside  membrane may w e l l  outer  promote  aminoglycoside  Hl-overproducing  binding  the  to  that  coupled  protein  through  able  proposed  mediated permeability inhibition,  clearly  requires  or  1981).  capable  transport  observed  chloramphenicol-  also  site  uptake  in  are  P_. a e r u g i n o s a  cation binding  ting  they  al  are  molecules  that It  et  such as  active  more than  are  (Hancock,  suggest  that  permeabilization  of  the the  a  98  outer  m e m b r a n e may i n  activity.  In  this  fact  light,  be a major it  is  interesting  new a n t i - P s e u d o m o n a l a m i n o g l y c o s i d e s compounds 1978)  largely  rather  account  to  for  Studies ing  strains role  i n both  E.  early  slow  latter  their of  coli  phases  or  follow  and l e t h a l i t y  occur  mutants of  197 5 ) . also  resistant  that  the  activity.  (EDP-1), is  energy  onset  inhibited  1975; Hurwitz resistant  affects  of  uptake  uptake  mutants  loss  in  HI  which  in  membrane  shown t o  and o c c u r  by  strains  ribosomal (Ahmad e t  the  in  type  strains  with  overproduce  protein  HI,  phase  presence  coincide  both  and do  EDP-II and  not  and Van  in  a l • , 1980).  an  two  (Bryan  (Bryan  affinity  by  transport  since  1961),  in  uptake  while  chloramphenicol  (strA)  occur  E D P - I I may  viability  uptake  followed  only  plays  1975 and  rapid  independent,  of  overproduc-  Aminoglycoside  binding,  and Rosano,  in wild  a_l. ,  function.  outer  1975 and 1 9 7 6 ) . of  et  membrane c o u l d ,  and a l a t e r  requiring  Alteration  streptomycin  outer  transport  electrostatic  energy  can be  ribosomally  Elzen,  ribosomal  the  parent  (Lee  c y t o p l a s m i c membrane and e l e c t r o n  the  V a n Den E l z e n ,  from t h e i r  ( B r y a n and V a n Den E l z e n ,  rapid  their  some o f  transport  aeruginosa has been  ( B r y a n and V a n Den E l z e n , with  of  in  transport.  evidence  phases  binding  an e n e r g i z e d  the  aminoglycoside  a n d JP.  are  of  increased  uptake phase The  with  aminoglycoside  an i n i t i a l  (EDP-II).  of  in  consecutive  1976):  interact  that  differ  efficiency  inhibition  also provide  a major  three  their  than their  Improved a b i l i t y part,  in  determinant  Den  other Comparison  uptake showed  in that  in  99  the  resistant  delayed  (Fig.  brane has ate  strains  the  2,  It  3).  a critical  EDP-II.  similar  have been observed  for  ribosomal  to  However,  since  polymyxin  the  to  that  mutants  our  given  H185 t o  in  The r e d by This  c o u l d be  aminoglycosides,  sides  for  (1979) for  the  have  sary of  is  in  smaller  to  alternative  activity, proposed  or  EDP-II altered 1980) .  resistance are  1981),  i n i t i -  to usually  it  is  unlikely  Furthermore,  of  strains  H181 and  low  of  protein  HI  levels type  that  the  to  that  on  aminoglycoside different  and  phenotypic  aminoglycosides,  aminoglycosides seen  for  or  fewer  a lower  site.  2  activity  a n t a g o n i s m by  of  (Davis, 2 +  sites  sites of  Mg  1974).  occurs  at  for  and  are  Osborne  affinity  others 2 +  for  aminoglyco-  a higher  while  1  levels  Mg  uptake  Schindler has  confer-  polymyxin.  affinity  polymyxin  high  for  binding  L P S t h a n Mg " ",  moderately  polymyxin  to  binding  LPS b i n d i n g  demonstrated  inhibit  of  alterations.  than that  a requirement  that  to  e t _al_. ,  effects  mem-  revertants  resistance  Salmonella typhimurium  demonstrated  required  (Ahmad  as w i l d  suggesting  outer  mutants w i t h  for  always  m u t a n t s h a d a common b a s i s .  due t o  aminoglycoside  had  the  onset  (Hancock,  step  medium as w e l l  HI  coli  the  ribosomal  sensitivity  that  events in  ribosomal  single  increase  protein  on the  E.  E D P - I I was  appear  selected  and s i n c e  that  the  phase  aminoglycosides  EDTA s u s c e p t i b i l i t i e s , alterations  thus  aminoglycosides  polymyxin  Mg2+-sufficient  certain  a l s o have  was d e m o n s t r a t e d  rapid  delays  m u t a n t was  and EDTA,  specific  would  influence  Somewhat  affinity  late  have  neces-  In quite  the low  case  it  100  Mg  2 +  l e v e l s , and t h i s competition  tends to mask the p r o t e c -  t i v e e f f e c t s of p r o t e i n HI when the p r o t e i n growth i n low M g . 2+  coside  Thus, i n MIC measurements of aminogly-  s u s c e p t i b i l i t y , P. aeruginosa c e l l s  apparently  more s u s c e p t i b l e  the d i f f e r e n t i a l  competitive  than c e l l s  i n low M g  i n high  e f f e c t s of M g  comparison of l o s s of v i a b i l i t y (Tables  i s induced by  2+  2 +  2+  were When  were e l i m i n a t e d by  i n a common assay medium  I I I and V; Hancock, R a f f l e and N i c a s ,  grown i n M g - d e f i c i e n t  Mg .  2+  1981), c e l l s  medium were a c t u a l l y more r e s i s t a n t  to aminoglycoside k i l l i n g  than c e l l s grown i n M g - s u f f i c i e n t 2 +  medium. The  increase  in resistance  seen i n outer membrane  p r o t e i n H i overproducing s t r a i n s cannot be a t t r i b u t e d to a general  decrease i n outer membrane p e r m e a b i l i t y .  pore-forming p r o t e i n  The major  ( p r o t e i n F) was only s l i g h t l y reduced i n  amount i n p r o t e i n HI overproducing s t r a i n s .  Furthermore,  s e n s i t i v i t y to both c a r b e n i c i l l i n and t e t r a c y c l i n e [which use the  so-called hydrophilic  (Nikaido (Table was  and Nakae, 1974)] was not a l t e r e d i n these s t r a i n s  IV) and t h e i r growth r a t e on M g - d e f i c i e n t 2 +  unaffected  normal.  (porin-mediated) pathway i n E_. c o l i  suggesting that the p o r i n  is functionally  The f i n d i n g that the HI overproducing mutants were  unchanged i n t h e i r s e n s i t i v i t y  to phages, i n c l u d i n g LPS r e c e p t -  or s p e c i f i c phages, a l s o suggested that there surface the  media  alterations.  Normal p o r i n  l a c k of s i g n i f i c a n t d i f f e r e n c e  are no major  f u n c t i o n was confirmed by i n permeation of n i t r o c e f i n  101  between w i l d type and p r o t e i n HI overproducing s t r a i n s (Table XII).  This suggests that the h y d r o p h i l i c pathway (Nikaido and  Nakae, 1974) of p a s s i v e permeation pores formed  by p o r i n  (Hancock  through the h y d r o p h i l i c  and N i k a i d o , 1978; Nikaido and  Nakae, 1979) may not be the major route taken by aminoglycoside antibiotics  i n P. a e r u g i n o s a .  P_. aeruginosa (Hancock  Although the l a r g e pore s i z e of  and N i k a i d o , 1978) would not be expected  to o f f e r any b a r r i e r to permeation of such a n t i b i o t i c s , there i s evidence that most of the pores a t any g i v e n time are not i n an a c t i v e , open s t a t e  (Benz and Hancock, 1981; and above).  However, i t i s p o s s i b l e that i n other organisms where a g r e a t e r p r o p o r t i o n of p o r i n s are i n the a c t i v e s t a t e 1980)  (Benz e_t a l . ,  and there are l e s s Mg2+ b i n d i n g s i t e s on the c e l l  surface  (Brown and M e l l i n g , 1969),  the h y d r o p h i l i c pathway o f  a n t i b i o t i c uptake may o f f e r an a l t e r n a t i v e , e f f i c i e n t means of streptomycin permeation.  Evidence f o r t h i s  i s provided by the  f i n d i n g of Foulds and Chai (1978), who demonstrated p o r i n l a d e f i c i e n t mutant of E. c o l i d e f e c t i v e philic  that a  i n the hydro-  uptake pathway was somewhat more r e s i s t a n t to kanamycin  and gentamicin, and s t u d i e s with E. c o l i omp B mutants are  deficient  i n both major p o r i n s ) have shown a 4 - f o l d  t i o n i n aminoglycoside r e s i s t a n c e  and  as would be p r e d i c t e d .  Furthermore,  such as E^.  i s , l e s s a f f e c t e d by Mg2+ antagonism  1971)  reduc-  (V. R a f f l e , E. Buenaventura,  and R.E.W. Hancock, unpublished r e s u l t s ) . tomycin a c t i o n i n other organisms  (which  coli  strep-  should be,  (Madeiros e_t a l . ,  102  An a l t e r n a t i v e e x p l a n a t i o n f o r some of the r e s u l t s presented  here might be that p r o t e i n HI i s a magnesium b i n d i n g  outer membrane p r o t e i n which a l s o binds gentamicin mycin and thus s p e c i f i c a l l y porin.  and s t r e p t o -  l i m i t s access of aminoglycosides to  However, s i g n i f i c a n t d i f f e r e n c e s i n the b i n d i n g of  streptomycin  to mutant s t r a i n H181 with high p r o t e i n HI l e v e l s ,  when compared to our w i l d type s t r a i n H103, could not be demonstrated.  In a d d i t i o n , at a streptomycin  c o n c e n t r a t i o n where we  could demonstrate a l a r g e d i f f e r e n c e i n k i l l i n g and  of s t r a i n s H181  H103 (Hancock, R a f f l e and N i c a s , 1981) l e s s than 0.5% of  the added streptomycin suggesting  become bound to c y a n i d e - t r e a t e d  that b i n d i n g d i d not s i g n i f i c a n t l y a l t e r  t i v e c o n c e n t r a t i o n of streptomycin s t r a i n s had l e s s M g  2+  i n the medium.  the e f f e c A l s o , mutant  i n t h e i r outer membranes, suggesting  that p r o t e i n Hi i s u n l i k e l y to be a s p e c i f i c M g protein.  cells,  2+  binding  F i n a l l y , the above a l t e r n a t i v e does not e x p l a i n  aminoglycoside-mediated the u n s u a l l y high M g  2+  w i l d type P. aeruginosa  p e r m e a b i l i z a t i o n of outer membranes or antagonism of aminoglycosides i n (Madeiros  e t a l . , 1971).  Thus, the  above a l t e r n a t i v e model seems u n l i k e l y , although we cannot r i g o r o u s l y exclude  that b i n d i n g of aminoglycosides  to p r o t e i n  HI c o n t r i b u t e s to the phenotype of the mutants. Resistance to polymyxin acquired during growth under c o n d i t i o n s other than low d i v a l e n t c a t i o n s has been reported by other workers. 1976;  G i l l e l a n d and c o l l e a g u e s ( G i l l e l a n d and Murray,  G i l l e l a n d and L y l e , 1979) and Brown and Watkins (1970)  103  s t u d i e d P_. aeruginosa t r a i n e d to grow on very ug/ml) of polymyxin. alterations  in phospholipid  G i l l e l a n d and  alterations  levels  and  content, r e a d i l y e x t r a c t a b l e  envelope p r o t e i n  L y l e , 1979).  (Brown and  Watkins,  from those of c e l l s grown  i n Mg2+-deficient medium ( G i l l e l a n d and  Murray, 1976).  Thus, t h i s type of r e s i s t a n c e cannot be compared to the here.  P. aeruginosa may  a l s o acquire  b r a n c h e d - c h a i n a c y l d e r i v a t i v e s as s o l e carbon source  and (Conrad  f  a l . , 1979).  resist-  resistance  to polymyxin B by growth on branched-chain amino a c i d s  et  T h i s r e s i s t a n c e appears to be r e l a t e d to  changes i n f a t t y a c i d composition of r e a d i l y e x t r a c t a b l e (Conrad e t a l  M  1981), and  differs  from the r e s i s t a n c e  here i n that i t does not a f f e c t aminoglycoside Alterations reduction  in Mg  2+  i s o l a t e s of P. aeruginosa and  mechanism of acquired  lipid  have a l s o been observed  b a c t e r i a (Brown and  Wood, 1972) .  Yet  other  gram-  been shown i n  t i o n s reduces the amount of membrane p h o s p h o l i p i d s  lipid.  and  in polymyxin  P. f l u o r e s c e n s , where growth under p h o s p h a t e - l i m i t i n g  cationic l i p i d ,  reported  another  polymyxin B r e s i s t a n c e has  about the s y n t h e s i s of a novel  lipids  susceptibility.  content, r e a d i l y e x t r a c t a b l e  in phospholipid  resistant c l i n i c a l negative  lipid,  In a d d i t i o n , u l t r a s t r u c t u r a l  i n these s t r a i n s d i f f e r  ance reported  (750  Such s t r a i n s e x h i b i t a large number of  w a l l phosphorous, LPS 1970;  high  condi-  and  brings  o r n i t h i n e amide  I t i s thus c l e a r that there are a v a r i e t y of mechanisms  for  a c q u i s i t i o n of polymyxin r e s i s t a n c e , as would be  for  an a n t i b i o t i c which i n t e r a c t s with both outer  and  expected inner  104  membrane components to e x e r t i t s l e t h a l e f f e c t s 1977). may  The common f a c t o r i n most of these  (Storm ejt a l . ,  forms of r e s i s t a n c e  be the r e d u c t i o n of the amount or a v a i l a b i l i t y of n e g a t i v -  e l y charged membrane components, e i t h e r on p h o s p h o l i p i d or on LPS,  with which the a n t i b i o t i c may i n t e r a c t .  presented  here f o r p r o t e i n HI mediated r e s i s t a n c e shares  property.  In agreement with  et al.., 1979;  Vaara, 1981)  i n Salmonella  typhimurium  and  have LPS with  information these  The model  t h i s , Vaara and co-workers  have reported  (pmrA) which are polymyxin  reduced binding a f f i n i t y  No  resistance of reported i n  t h i s study may a l s o be r e s p o n s i b l e f o r the gentamicin i s o l a t e s of P. aeruginosa  resistant  f o r polymyxin.  Membrane changes s i m i l a r to those  ance of c l i n i c a l  (Vaara  a c l a s s of LPS mutants  i s a v a i l a b l e on the aminoglycoside  strains.  this  resist-  with reduced amino-  g l y c o s i d e t r a n s p o r t r e p o r t e d by Bryan e_t a l . (1976) and the adaptive  r e s i s t a n c e to gentamicin  and EDTA reported by Pechey  and James (1974 ) . Ca  2 +  , Mn  both i n p r e v e n t i n g to these  agents.  e f f e c t s , implying  2+  and S r  2 +  were able to s u b s t i t u t e f o r M g  HI i n d u c t i o n and i n a l l o w i n g These four c a t i o n s showed very that they are e q u i v a l e n t  regulate protein Hi.  Regulation  susceptibility similar  i n their a b i l i t y to  of p r o t e i n HI p r o d u c t i o n by  c a t i o n s could be mediated by a common r e c e p t o r f o r cations.  these  S i m i l a r r e c e p t o r s p e c i f i c i t y has been seen f o r the  chemotaxis r e c e p t o r (Koshland,  2+  1979).  for Mg  2+  i n S_. typhimurium and E_. c o l i  A l t e r n a t i v e l y , the e x p r e s s i o n  of HI may be  105  s e n s i t i v e to the t o t a l amounts of d i v a l e n t c a t i o n i n the outer membrane i t s e l f , or to d i v a l e n t c a t i o n s bound at s p e c i f i c  sites  i n the outer membrane.  A l t e r a t i o n of outer membrane p r o t e i n  composition  to changes i n other outer membrane  i n response  components (Van Alphen  e_t a l . , 1976; DiRienzo and Inouye, 1979)  i s known to occur i n IS. c o l i .  The a b i l i t y  to respond  to c o n d i -  t i o n s a t the outer membrane by a l t e r a t i o n of outer membrane p r o t e i n i s a l s o seen p r o t e i n s of E. c o l i  i n the modulation i n response  across the outer membrane  isolated  v a l e n t c a t i o n s other than M g  g l y c o s i d e s , polymyxins of M g  pressure g r a d i e n t  2+  from c e l l s grown with d i -  contained some M g , we 2+  that the s i t e of a c t i v i t y of amino-  and EDTA could i n v o l v e t h i s small amount  r a t h e r than a s i t e occupied by other c a t i o n s . The  2+  e f f e c t of EGTA on C a suggested site.  to an osmotic  (Kawaji e_t a l . , 1979).  As c e l l envelopes  considered the p o s s i b i l i t y  of l e v e l s of major outer  that C a  2 +  2 +  grown c e l l s , however, s t r o n g l y  i s replacing Mg  EGTA c h e l a t e s C a  2 +  a t the t a r g e t  e f f i c i e n t l y but M g  ( K f f 10.4 v s . 4.7, Roberts e t a l . , e  c e l l s appeared  2+  1970).  poorly  2+  Ca  2 +  grown  as s e n s i t i v e to the outer membrane e f f e c t s o f  EGTA as they are to those of EDTA, as shown both by the d i r e c t measurement of l y s i s and by i n c r e a s e i n the r a t e of h y d r o l y s i s of n i t r o c e f i n .  EGTA, however, d i d not have the high b a c t e r i -  c i d a l a c t i v i t y o f EDTA, suggesting that a d d i t i o n a l s i t e s a r e involved  i n the b a c t e r i c i d a l a c t i o n of EDTA.  (Boggis e t a l . , 1979) have noted  Other  workers  that s e n s i t i v i t y to l y s i s by  106  EGTA i s growth Mg  dependent  on t h e  medium, but  that  as w e l l  2 +  as  Ca  c a t i o n may o c c u p y The action of may w e l l sites. two  EDTA,  affinities, B were  Ca  envelope  of  Mg  2 +  only  levels  efficiency Zn  2 +  of  grown c e l l s  their  level  found  in  cells  grown i n  2 +  Zn  2 +  2 +  cell  had h i g h  levels  are  not  and the  EDTA.  was  of  grown i n  Ca  or  Mn  It  may b e  cationic  antibiotics sites  which  are  2 +  .  sites  which  protected  c a n be  that  are by  occupied  target also  despite  and  a n d 8% l o w e r  2 +  or  that  lower  only  sensitive  that  than  the  those to  attack  HI,  and  by  (or  indeed  2 +  level  than  protein Zn  is  cell  observed  15-35%  cells  the  high  HI,  was  sites  very  protein  cations  polymyx-  of  could  sensitive  It  least  critical  envelopes  divalent  2 +  10% o f  and g e n t a m i c i n ,  of  cation binding  remaining  a s m a l l number  aminoglycosides  c h e l a t i o n by  divalent  for  with  in  0 . 5 mM M g  sites  of  of  differing  treated  Zn  at  widely  3.5 to  EDTA-Tris,  membrane  lipo-  only  grown i n  of  Zn  on t h e  small proportion  the  number  of  A relatively  cells  outer  total  binding  of  in  demonstrated  sites  affinity  involvement  from c e l l s  polymyxins,  the high  high  displacement  sites  why  2 +  have  either  involved  the  if  chelators.  on t h e  of  the  decreased  two  sites  and g e n t a m i c i n  in  that  the  binding  binding  B.  explain  cation  and M g  polymyxin for  suggesting  (1979)  The  the  EGTA i s  Salmonella typhimurium with  while  by  to  and Osborn 2 +  found.  suggested  of  present  2 +  a small proportion  Schindler of  Ca  a t t a c k e d by  polymyxin  represent  classes  present,  site  number  polysaccharide  in  the  of  sensitivity  is  2 +  amount  the  by  107  any  of the d i v a l e n t c a t i o n s  membrane s t a b i l i t y .  This would suggest that these  d i v a l e n t c a t i o n binding divalent cations, Mn .  studied) are not r e q u i r e d f o r  s i t e s can only be occupied by s p e c i f i c  i . e . , Mg , C a , S r 2+  2 +  2 +  A l t e r n a t i v e l y , p r o t e i n HI could  2+  divalent cations  , or  only  displace  from these s i t e s when induced by l i m i t a t i o n of  these d i v a l e n t c a t i o n s .  Although there  appears to be a l i n e a r  r e c i p r o c a l r e l a t i o n s h i p between the decrease i n M g and  increase  cells,  critical  2+  levels  i n p r o t e i n HI i n the c e l l  envelope of w i l d  i t would appear that r e s i s t a n c e  to c h e l a t o r s and  type  c a t i o n i c a n t i b i o t i c s c o r r e l a t e s more c l o s e l y with the presence and  amounts of p r o t e i n HI than with absolute "levels of d i v a l e n t  cations  i n the c e l l  envelope.  This  i s supported by s t u d i e s of  the mutant s t r a i n s r e s i s t a n t to these agents, which overproduce p r o t e i n HI t o about 7 - f o l d w i l d but Mg  show a r e d u c t i o n 2+  levels.  of l e s s than 2 - f o l d  Protection  to confer  Mg  2+  i n t h e i r envelope  of a r e l a t i v e l y small  of d i v a l e n t c a t i o n binding sufficient  type l e v e l s i n 0.5 mM  proportion  s i t e s would thus appear to be  resistance.  In summary, i t i s concluded that the outer membrane of P. aeruginosa i s a major determinant of the a n t i b i o t i c r e s i s t a n c e of t h i s bacterium. support t h i s c o n c l u s i o n : for  Two separate l i n e s of evidence  1) A l t e r a t i o n of the outer membrane,  example, by growth i n low M g  overproduction, a l t e r s resistance chelators.  2+  o r by mutation to HI to c a t i o n i c a n t i b i o t i c s and  The data presented here suggest that these agents  108  are e f f e c t i v e a g a i n s t P_. aeruginosa because they are able to a c t i v e l y d i s r u p t the outer membrane; and  2) The  outer membrane  of P. aeruginosa has been shown to be r e l a t i v e l y d e s p i t e the g r e a t e r channel  s i z e of i t s p o r i n s , p o s s i b l y due  there being a r e l a t i v e l y small number of a c t i v e , channels.  impermeable to  functional,  Previous s t u d i e s with other b a c t e r i a have  suggested  two major pathways f o r a n t i b i o t i c uptake across the outer membrane. and  These are the h y d r o p h i l i c or porin-mediated  the hydrophobic  pathway which i s apparently r e s t r i c t e d  deep rough mutants of E. c o l i and S a l m o n e l l a . d e s c r i b e d here s t r o n g l y suggests  polycationic antibiotics  to  The work  the e x i s t e n c e of a t h i r d major  pathway i n P. aeruginosa, self-promoted  membrane promoting  pathway  uptake i n which  i n t e r a c t with and d i s r u p t the o u t e r  uptake of f u r t h e r molecules  of  antibiotic.  109 LITERATURE CITED Adelberg, E.A., M. 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