Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The effect of streptomycin on the induction of Penicillinase in Bacillus cereus 1958

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
UBC_1958_A6_7 M4 E3.pdf [ 2.42MB ]
UBC_1958_A6_7 M4 E3.pdf
Metadata
JSON: 1.0106182.json
JSON-LD: 1.0106182+ld.json
RDF/XML (Pretty): 1.0106182.xml
RDF/JSON: 1.0106182+rdf.json
Turtle: 1.0106182+rdf-turtle.txt
N-Triples: 1.0106182+rdf-ntriples.txt
Citation
1.0106182.ris

Full Text

THE EFFECT OF STREPTOMYCIN ON THE INDUCTION OF PENICILLINASE IN BACILLUS CEREUS by C. ANN LORNA MITCHELL B . A . , Un iver s i ty of B r i t i s h Columbia, 1955 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of BIOCHEMISTRY We accept t h i s thes i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1958 i ABSTRACT The effect of dihydrostreptomycin on the growth and induct ion of the enzyme, p e n i c i l l i n a s e , i n B a c i l l u s cereus has been s tudied . Two B. cereus var iants were used: a s ens i t ive c u l t u r e , the growth of which was arrested by approximately 0.3 uni t s of dihydrostreptomycin per m i l l i l i t e r of medium; and a re s i s t ant type which would grow i n the presence of 2,000 uni t s per m i l l i l i t e r of d ihydro- streptomycin. This re s i s t ant s t r a i n was developed from the parent s ens i t ive organism by successive t r a n s f e r r i n g and p l a t i n g techniques . The enzyme, p e n i c i l l i n a s e , was induced with p e n i c i l l i n and assayed manometrically. In the a n t i b i o t i c - sens i t ive B. cereus. i t was found that the formation of p e n i c i l l i n a s e , and not p e n i c i l l i n a s e ac t ion , was i n h i b i t e d by dihydrostreptomycin. Further , t o t a l i n h i b i t i o n of p e n i c i l l i n a s e induct ion occurred with a concentrat ion of a n t i b i o t i c that i n h i b i t e d growth of the organism. This i n h i b i t i o n of p e n i c i l l i n a s e formation was found to f i t the mass law equation, xy = C, where x i s the dihydrostreptomycin concentrat ion, y i s the f r a c t i o n a l enzyme synthes i s , and C i s a constant. In the a n t i b i o t i c - r e s i s t a n t B. cereus. ne i ther i i growth nor p e n i c i l l i n a s e formation was i n h i b i t e d by much higher concentrations of dihydrostreptomycin. A s l i g h t " p a r t i a l dependence" on the a n t i b i o t i c was noted. When a n t i b i o t i c - r e s i s t a n t cultures, which had been grown i n the absence of dihydrostreptomycin, were induced with p e n i c i l l i n i n the absence and i n the presence of streptomycin, there was, i n the absence of the a n t i b i o t i c , a longer l a g period f o r the formation of p e n i c i l l i n a s e . That i s , the r e s i s t a n t organism showed a s l i g h t dependence on streptomycin i n the early stages of growth and enzyme induction. It was found that short periods of sonic treatment of suspensions of B. cereus produced an increase i n the rate of p e n i c i l l i n a s e induction. Longer periods of sonic treatment, however, decreased the rate of enzyme induction. The r e s u l t s of t h i s study «* that streptomycin i n h i b i t s the formation of p e n i c i l l i n a s e i n sen s i t i v e B. cereus but does not i n h i b i t the action of t h i s enzyme - were speculatively correlated with the known synergism between streptomycin and p e n i c i l l i n . I n 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 o f t h e r e a u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f B i o c h e m i s t r y The U n i v e r s i t y o f B r i t i s h Columbia, Vancouver 8, Canada. Date A p r i l 22, 195& i i i TABLE OF CONTENTS Acknowledgment • • • . • . v I Introduction 1 1. The Chemistry of Streptomycin . . . . . . . 1 2 . The Action of Streptomycin on Bacteria • • • • 4 3 . The Inducible P e n i c i l l i n a s e of B a c i l l u s cereus . . 9 4 . The Importance of Enzyme Induction 12 5 . Objectives of t h i s Study 13 I I Experimental . 14 1. Methods and Materials 14 A. Growth Experiments 14 B. Induction and Determination of P e n i c i l l i n a s e . 16 C. Sonic Disrupted Preparations • 20 2 . Results 21 A. Growth Experiments • . . . . 2 1 Figure I: The Ef f e c t of Dihydrostreptomycin on the Growth of Sensitive B a c i l l u s cereus . . to face page 21 Figure I I (Data of Table I ) : The E f f e c t of Dihydrostreptomycin on the Growth of Resistant B a c i l l u s cereus ( i ) . . . . . . . to face page 22 Figure III (Data of Table I I ) : The E f f e c t of Dihydrostreptomycin on the Growth of Resistant B a c i l l u s cereus ( i i ) to face page 22 B. Induction of P e n i c i l l i n a s e . . . . . . . 2 3 Figure IV: The Determination of P e n i c i l l i n a s e A c t i v i t y . . . . to face page 23 i v Figure V: The Induction of P e n i c i l l i n a s e i n Dihydrostreptomycin-sensit ive B a c i l l u s cereus . . to face page 21+ Figure V I : The Ef fect of Varying Concentrations of Dihydrostreptomycin on P e n i c i l l - inase Induction i n Sens i t ive B a c i l l u s cereus . . to face page 25 Figure VI I : The Induction of P e n i c i l l i n a s e i n Dihydrostreptomycin-resistant B a c i l l u s cereus ( i ) to face page 26 Figure V I I I : The Induction of P e n i c i l l i n a s e i n Dihydrostrept omyc i n - r e s i s t a n t B a c i l l u s cereus ( i i ) to face page 27 Figure IX: The Induction of P e n i c i l l i n a s e under Aerobic Conditions i n Dihydrostreptomycin-sensi t ive B a c i l l u s cereus . . to face page 28 Figure X: The Ef fect of Dihydrostreptomycin on the Act ion of P e n i c i l l i n a s e ' . to face page 29 C. Sonic Disrupted Preparations 30 Table I I I : The P e n i c i l l i n a s e A c t i v i t y of Suspensions of Dihydrostreptomycin- sens i t ive B a c i l l u s cereus exposed to Sonic O s c i l l a t i o n s for various times . • • • . to face page 30 I I I Discuss ion . . • • • . 3 1 IV Conclusions . • • • • • • • • • . . . • . 3 7 V Bibl iography 40 ACKNOWLEDGMENT The interes t shown and advice so ably- given by Dr . W.J. Polglase during the course of the research and w r i t i n g of t h i s thes i s are g r a t e f u l l y acknowledged. The f i n a n c i a l assistance which was received from the Defence Research Board of Canada was grea t ly appreciated. 1 . I INTRODUCTION 1. The Chemistry of Streptomycin Streptomycin belongs to a group of compounds, known as a n t i b i o t i c s , which are produced by micro-organisms and which possess the property of i n h i b i t i n g the growth of other micro-organisms. The i s o l a t i o n of streptomycin was the culminating point of a painstaking search for a n t i m i c r o b i a l agents produced by actinomycetes, a group of organisms c l o s e l y re l a ted to the b a c t e r i a . The streptomycin-producing s t r a i n of Streptomyces griseus was f i r s t i so l a t ed i n September 1 9 4 3 and was found to be ac t ive against a v a r i e t y of the gram-posit ive, gram-negative and ac id- fas t b a c t e r i a , i n p a r t i c u l a r , Mycobacterium tuberculos i s ( 1 ) . Since t h i s t ime, the production and c l i n i c a l a p p l i c a t i o n of t h i s a n t i b i o t i c have had a phenomenal r i s e . Within a year i t was i s o l a t e d i n the form of pure c r y s t a l l i n e s a l t s ( 2 ) and w i t h i n three years af ter the announcement of the i s o l a t i o n of streptomycin came the almost complete e l u c i d a t i o n of i t s chemistry. Streptomycin has been hydrogenated, c a t a l y t i c a l l y , to y i e l d dihydro- streptomycin which has a n t i b i o t i c a c t i v i t y equal to that of streptomycin. Further , the p r a c t i c a l p o t e n t i a l i t i e s of streptomycin and dihydrostreptomycin for disease cont ro l have been d e f i n i t e l y e s tab l i shed . The streptomycin molecule embodies a subst i tuted cyclohexane r i n g to which i s attached, by g l y c o s i d i c l inkage , 2 . a complex sugar residue (3K It i s a strong base, with three basic funct iona l groups. On hydro ly s i s , i t s p l i t s in to two compounds, s t rep t id ine and streptobiosamine. C21H39N7°12+ H 2 ° * C 8 H 1 S N6 ° 4 * C 13 H 23 N 0 9 Streptomycin S t rept id ine Streptobiosamine On further hydrolys i s with strong mineral ac ids , the streptobiosamine y i e l d s N-methyl-L-glucosamine. The s i x carbon sugar, streptose, i s decomposed during hydro lys i s and therefore has not been i s o l a t e d . C 1 3 H 2 3 N 0 9 H 2 0 > C 6 H 1 0 0 5 4- G ? H 1 5 N 0 5 St re pt ob i o samine Streptose N-methyl-L-glucosamine The s tructure of the streptomycin molecule i s as fo l lows : NH I. NHCNHp /\ HOHC CH - I I HoNCHNHC CHOH 2 « \ / NH CHOH STREPTIDINE 0 -CH I CH - I 0-CHCOH I CH I CH, CHoNHCH i I CHOH I CHOH I •CH STREPTOSE^ CH2OH ^N-METHYL-L-GLUCOSAMINE STREPTOBIOSAMINE 3. Neither s t rep t id ine nor streptobiosamine have a n t i b i o t i c a c t i v i t y . Contro l led hydro lys i s of dihydrostreptomycin removes two amidine groups from the s t rep t id ine moiety and r e s u l t s i n a loss of a c t i v i t y . Therefore, both the i n t e g r i t y of the t r i s acchar ide and the b a s c i c i t y of t h i s t r i s a c c h a r i d e are necessary fo r a n t i b i o t i c a c t i v i t y . Various modif icat ions of streptomycin are produced by d i f ferent species of Streptomyces, notably S. b i k i n i e n s i s . mannosidostreptomycin; and S. griseocarneus, hydroxystreptomycin (4). The dihydro der iva t ives of these compounds have been obtained by c a t a l y t i c hydrogenation which reduces the aldehyde group of the streptose moiety (5). OCHCOH I K) CH I CH20H S 0 -G 0 HYDROXYSTREPTOMYCIN DIHYDROSTREPTOMYCIN S = S t rept id ine G = N-methyl-L-glucosamine Mannosidostreptomycin has a mannose group attached to the fourth carbon of the glucosamine group (6) . The spectra of a c t i v i t y of a l l of the streptomycins i s the same and the quant i ta t ive di f ferences i n a c t i v i t y are sma l l . 2. The Act ion of Streptomycin on Bacter ia 4 Although rap id advances were made i n the chemistry and therapeutic app l i ca t ions of streptomycin, the mode of ac t ion of t h i s a n t i b i o t i c remains u n c l a r i f i e d . The ant imicrob ia l ac t ion of streptomycin i n v i t r o has been described as b a c t e r i o s t a t i c i n low concentrations and as ^ b a c t e r i c i d a l i n high concentrat ions; the former t « r m being used to ind ica te only the prevention of m u l t i p l i c a t i o n and the l a t t e r to ind ica te death of the exposed micro-organisms (7) . The extent of these act ions i s dependent on the organism used and the spec i f i c condit ions of the t e s t . The mechanisms by which t h i s agent i n h i b i t s m u l t i p l i c a t i o n or causes death of the b a c t e r i a l c e l l are not known. Although our present knowledge i s somewhat fragmentary and contrad ic tory , many observations seem pert inent to the o v e r a l l p i c t u r e . The effects of streptomycin on the oxidat ion of various carbohydrate intermediates have been a f i e l d of ac t ive research. A metabolic pathway present i n s ens i t ive micro- organisms which was i n h i b i t e d by streptomycin was sought and, fur ther , a corresponding a l ternate metabolic pathway i n re s i s t ant organisms which was unaffected by streptomycin. Umbreit found that streptomycin produced ef fects on the metabolism of r e s t i n g c e l l s of Escher ich ia c o l i and that t h i s drug i n h i b i t e d the oxaloacetate pyruvate-condensation at a concentrat ion which a l so i n h i b i t e d growth ( 8 , 9 ) . Umbreit and 5 . his colleagues postulated a pathway other than acetate- oxalacetate condensation as a means of entry for pyruvate into the terminal r e sp i ra tory system. They concluded that a pyruvate-oxalacetate reac t ion not forming c i t r a t e was present i n the organisms and i t was t h i s r eac t ion that streptomycin i n h i b i t e d ( 1 0 ) * In a l a t e r communication, Umbreit reported the i s o l a t i o n and i d e n t i f i c a t i o n of a seven carbon condensation product of pyruvate and oxalacetate, 2 -phospho - 4 -hydroxy - 4 - carboxy-adipic a c i d , whose formation was i n h i b i t e d markedly by streptomycin ( 1 1 ) . Expermental studies by other workers, however, do not substantiate these r e s u l t s . Geiger, working with E . c o l i , found that streptomycin had no effect on the oxidat ion of glucose, l a c t a t e , g l y c e r o l , succinate , malate, fumarate, oxaloacetate, and pyruvate ( 1 2 ) . Further , Henry and h i s colleagues found that streptomycin i n high concentrations d id not i n h i b i t such enzyme systems as catalase , carbonic anhydrase, cytochrome, cytochrome-oxidase, succinoxidase, carboxylase, urease or t r y p s i n ( 1 3 ) . F i t z g e r a l d and coworkers reported that streptomycin i n a concentrat ion of ten micrograms per m i l l i l i t e r i n h i b i t e d the ox idat ion of benzoic ac id by a suscept ible s t r a i n of mycobacteria ( 1 4 , 1 5 ) . C e l l s grown i n a benzoic ac id medium d id not show such i n h i b i t i o n . Stanier has a t t r ibuted t h i s to the i n h i b i t i o n of formation of the enzyme required i n t h i s ox idat ion ( 1 6 ) . Indeed, t h i s i n t e r p r e t a t i o n of adaptive enzyme i n h i b i t i o n by streptomycin may enl ighten the c o n f l i c t i n g reports of the 6. effect of streptomycin on oxidat ion of carbohydrate substrates . Cohen observed that streptomycin combines with desoxyribonucleic acids (DNA) to produce polymeric compounds which p r e c i p i t a t e and that t h i s reac t ion could be reversed by the add i t ion of sodium chlor ide ( 1 7 , 1 8 ) . -He suggested that t h i s phenomenon might be re l a ted to the a n t i b i o t i c a c t i v i t y of streptomycin, which i s a lso reversed by sodium ch lor ide and other s a l t s . Other workers, however, found that the a c t i v i t y of the s a l t s i n revers ing the DNA-streptomycin complex d i f fe red from that which reversed the i n h i b i t i o n of m u l t i p l i c a t i o n ( 1 9 ) . Peretz and Polglase have noted that while nucleases can disperse p rec ip i t a t ed streptomycin- mammalian nuc le ic ac id complexes, they cannot d i s so lve complexes formed with streptomycin and nuc le ic ac id containing mater ia l from extracts of E . c o l i ( 2 0 ) . In view of the current hypothesis of pro te in synthes i s , that i s , that nuc le ic acids act as templates accomplishing the organizat ion of amino ac id residues wi th in prote in s tructures ( 2 1 ) , i t seems reasonable that the b a c t e r i a l nuc le ic acids may be the s i t e of ac t ion of streptomycin. I t has a l so been suggested that streptomycin reacts with the su l fhydry l groups on enzymes thereby i n a c t i v a t i n g them ( 2 2 ) . Other workers, however, obtained no i n h i b i t i o n by streptomycin of the a c t i v i t y of urease, succinoxidase or carboxylase, enzymes which require free su l fhydry l groups for 7. t h e i r a c t i v i t y (13). I f streptomycin i n h i b i t s the formation or u t i l i z a t i o n of a substance e s sent i a l for b a c t e r i a l m u l t i p l i c a t i o n , then add i t ion of t h i s substance should antagonize the ac t ion of streptomycin. Many workers have sought to i s o l a t e an antagonist of streptomycin (23,14). The presence of a substance i n the cul ture f i l t r a t e s of Pseudomonas pyocyanea which antagonized the a n t i b a c t e r i a l ac t ion of dihydrostreptomycin was reported e a r l i e r (24). Recently t h i s substance has been i s o l a t e d and character ized by Cornforth and James (25). I t was found to be a mixture of 2-n-heptyl-4-hydroxy quinol ine N-oxide and 2-n-nonyl-4- hydroxy quinol ine N-oxide ( i n a two:one r a t i o ) with traces of other s i m i l a r compounds. I t was found that the oxidat ion of reduced cytochrome b by cytochrome c i n heart muscle preparations was i n h i b i t e d by the heptyl n-oxide . Any modi f ica t ion of the quinol ine ni trogen oxide molecule which re su l ted i n los s or reduct ion of the dihydro-streptomycin antagonist a c t i v i t y was accompanied by los s or reduct ion of the effect on the cytochrome e lec t ron t ranspor t . Thi s p a r a l l e l i s m suggests to these workers the p o s s i b i l i t y of a r e l a t i o n s h i p between these two a c t i v i t i e s . As previous ly mentioned, F i t z g e r a l d , et a l , f i r s t observed that streptomycin i n h i b i t e d adaptive enzyme formation i n mycobacteria (14). Creaser a lso observed that i n a 8. streptomycin-susceptible strain of Staphylococcus aureus, /2-galactosidase formation was inhibited by streptomycin (26). A study of the effect of streptomycin on the formation of /3-galactosidase in E. c o l i has recently been pursued by Polglase (27,28,29). /^-galactosidase i s produced adaptively by Escherichia c o l i i n the presence of a suitable inducer (lactose). Three E. c o l i mutants were used i n this work: variants which were sensitive to, resistant to, and dependent on, the antihiotic. It was found that the concentration of dihydrostreptomycin which inhibited multiplication of antibiotic-sensitive E. c o l i (thirty units per m i l l i l i t e r ) also inhibited the formation of the adaptive enzyme (28). Further, the formation of this enzyme was not inhibited in resistant or dependent E. c o l i by higher concentrations of dihydrostreptomycin. In the latter organisms, enzyme production did not occur to any appreciable extent in the absence of the antibiotic and the concentration of dihydro- streptomycin which affected c e l l multiplication was similar to that which affected the adaptive enzyme formation. This type of concomitant study of resistant and dependent strains, derived from the susceptible strain used, would seem a reasonable guide to whether the response to streptomycin which is measured i s related to the bacteriostatic activity of streptomycin. 9. 3. The Inducible P e n i c i l l i n a s e of B a c i l l u s cereus The a b i l i t y of various b a c t e r i a l species to inac t iva te p e n i c i l l i n was f i r s t observed by Abraham and Chain i n 1940 (30) . The ac t ive agent was extracted from crushed' c e l l s of E . c o l i and the name p e n i c i l l i n a s e proposed. In 1944, Duthie observed that p e n i c i l l i n a s e behaved as an adaptive enzyme. Using a s t r a i n of B a c i l l u s s u b t i l i s , he found that the add i t ion of p e n i c i l l i n to the cul ture media produced a t h i r t y f o l d increase i n the amount of enzyme (31) • La te r , the p e n i c i l l i n a s e of B a c i l l u s cereus was shown to have t h i s c h a r a c t e r i s t i c (32), and a p u r i f i e d h igh ly ac t ive p e n i c i l l i n a s e preparat ion was obtained (32,33)• Under a l k a l i n e condi t ions , the p e n i c i l l i n s are inac t iva ted to y i e l d p e n i c i l l o i c acids i n which the /3-lactam linkage i s ruptured while the t h i a z o l i d i n e r i n g i s unaffected (34). I * S — C — C H o C H , 3 I 3 ^NH-CH-CH R-C C — ^ N — C - C O O H + HoO * R-C-NH-CH-CH I I I I I \ 0 0 H 0 C O NH-C-COOH ^ S - C - C H ^ I I OH H PENICILLIN PENICILLOIC ACID R = <C3>-CH2 • f o r BENZYL PENICILLIN 10. P e n i c i l l i n a s e catalyzes the same transformation. Foster i n 1945 observed that a c e l l - f r e e preparation from B. s u b t i l i s inac t iva ted p e n i c i l l i n with the formation of an a c i d (35). This formation of an a c i d i c group with pK 4.7 was confirmed by Benedict , et a l . us ing p e n i c i l l i n a s e from a s t r a i n of B . cereus (36). La ter , Henry and Housewright showed conc lus ive ly by measuring carbon dioxide evolut ion from a bicarbonate buf fer , manometrically, that the p e n i c i l l i n a s e i n a broth cu l ture f i l t r a t e of B. cereus destroyed p e n i c i l l i n by quant i ta t ive hydrolys i s to p e n i c i l l o i c ac id with the formation of one ac id equivalent (37). The production of p e n i c i l l i n a s e by B a c i l l u s cereus has been studied by Po l lock . The enzyme i s mainly exoce l lu la r with only 10$ of t o t a l a c t i v i t y remaining attached to the organisms af ter centr i fugat ion (38). This d i s t r i b u t i o n i s constant and does not vary markedly with condit ions of c u l t u r e , phase of growth, or l e v e l of i n d u c t i o n . The general c h a r a c t e r i s t i c s of p e n i c i l l i n a s e production are s i m i l a r to those of /3-galactosidase and other enzyme adaptations i n micro-organisms. However, several d i s t i n c t l y d i f fe rent features are involved which make t h i s enzyme of spec i a l i n t e r e s t . Unlike a l l other induct ive systems s tudied, a high rate of enzyme synthesis (about t h i r t y times the basal rate) i s maintained a f ter complete removal of the inducer from the external environment. Thi s a b i l i t y to form p e n i c i l l i n a s e i n a p e n i c i l l i n - f r e e medium i s acquired 11. merely by t r e a t i n g c e l l s at 0 ° C . for a few minutes with a small amount of p e n i c i l l i n which i s then removed. This high rate of enzyme a c t i v i t y per s i s t s for at leas t seven c e l l generations (39) . When a treated populat ion i s placed i n a growth medium, the t o t a l number of c e l l s increases exponentia l ly during enzyme synthes i s . P e n i c i l l i n a s e formation, however, a f ter a short l a g phase, proceeds l i n e a r l y with respect to t ime. This would seem to imply that the enzyme- forming capacity of the t o t a l populat ion maintains a constant l e v e l , f ixed by the p e n i c i l l i n pretreatment. A study of t h i s phenomenon has been further pursued by Pol lock (40,41j42). The induct ion of p e n i c i l l i n a s e i s s p e c i f i c . The only known inducer i s p e n i c i l l i n . The product of the enzyme a c t i v i t y , p e n i c i l l o i c a c i d , does not st imulate adaptat ion. The pronounced effect of minute concentrations i n s t imula t ing adaptation i s i n t e r e s t i n g . Less than .004 uni t s per m i l l i l i t e r of p e n i c i l l i n was found to be e f fec t ive while maximal enzyme production occurred with 1 un i t per m i l l i l i t e r of p e n i c i l l i n . Higher concentrations were found to be les s e f f e c t i v e . 12. 4. The Importance of Enzyme Induction The question ar i ses of the importance of adaptive enzyme formation i n the l i f e of a b a c t e r i a l c e l l . There i s evidence to indicate that induced and cons t i tu t ive enzymes are formed by b a s i c a l l y the same mechanism. Cohn and Monod observed that the development of enzyme a c t i v i t y corresponded to the production of an equivalent amount of some d i s t i n c t enzymatica l ly-act ive prote in (43). Recently (44), severa l workers have shown that the induced and cons t i tu t ive p e n i c i l l i n a s e of B. cereus are i d e n t i c a l i n the fo l lowing respects : s p e c i f i c a c t i v i t y , sedimentation r a te , d i f f u s i o n constant, e lec trophoret ic m o b i l i t y and sa l t s o l u b i l i t y . They conclude that the induct ion of p e n i c i l l i n a s e corresponds to the formation of a s p e c i f i c physico-chemical ly d i s t i n c t pro te in and that induced and cons t i tu t ive enzymes are formed by b a s i c a l l y the same mechanism. Further , Stanier has stated that " there are probably few induc ib le enzyme systems whose a c t i v i t y (given a s u f f i c i e n t l y sens i t ive assay) i s completely undetectable before induct ion" (45). He bel ieves that the effect of induct ion i s to cause an acce le ra t ion i n the d i f f e r e n t i a l rate of synthesis of the enzyme i n quest ion. In accordance with these hypotheses, i t seems reasonable that any knowledge gained of the ac t ion of streptomycin on adaptive enzyme synthesis may be appl icab le to the ac t ion of streptomycin on any enzyme synthesis i n a sens i t ive organism. 13 5. Objectives of this Study The purpose of the work reported herein was to observe the effect of dihydrostreptomycin on the induction of penicillinase in Bacillus cereus. The consistent effects of streptomycin on /3-galactosidase in Escherichia c o l i had already been demonstrated (19,28). The extension of such studies to penicillinase was therefore logical. Further, the synergism between p e n i c i l l i n and streptomycin might find some rational explanation in a study of this nature. II EXPERIMENTAL 1 4 . 1 . METHODS AND MATERIALS A . GROWTH EXPERIMENTS B a c i l l u s cereus var iant number 79 was obtained from D r . Stock of the Department of Bacter io logy , U n i v e r s i t y of B r i t i s h Columbia. The s e n s i t i v i t y of t h i s organism to streptomycin was determined. Cultures were grown i n nutr ient broth medium (Difco) at pH 7 i n Roux f lasks at 3 7 ° C The c e l l s were harvested a s e p t i c a l l y a f ter twenty-one hours growth and washed twice with s t e r i l e p h y s i o l o g i c a l s a l ine ( . 8 5 $ ) . A l l centr i fugat ion was c a r r i e d out at low temperatures (approximately 5 ° C ) . The c e l l s were resuspended i n broth to 7 a concentration equivalent to 4 . 4 x 10 v i ab le c e l l s per m i l l i l i t e r . S t e r i l e streptomycin was added i n varying concentrations from 0 to 5 uni t s per m i l l i l i t e r and the t o t a l volume equalized with the add i t ion of b r o t h . O p t i c a l densi ty readings were recorded a f ter twenty-four hours growth, us ing the Beckman Model B Spectrophotometer at a wave length of 420 m i l l i m i c r o n s . The streptomycin used was an aqueous s o l u t i o n of dihydrostreptomycin sulphate . There i s no evidence that the b a c t e r i a l spectrum or the a n t i b i o t i c a c t i v i t y of dihydro- streptomycin and streptomycin d i f f e r s i g n i f i c a n t l y and there i s adequate evidence that organisms which develop res i s tance to streptomycin have also developed res i s tance 1 5 . to dihydrostreptomycin ( 6 , 4 6 ) . One uni t of dihydro- - 6 streptomycin i s equivalent to one microgram (1 x 10 gram) of the c r y s t a l l i n e streptomycin base. Solut ions of streptomycin were s t e r i l i z e d by u l t r a f i l t r a t i o n i n a u l t r a f ine glass f i l t e r . A streptomycin re s i s t ant B a c i l l u s cereus var iant was developed. B a c i l l u s cereus cultures were innoculated in to medium containing varying amounts of dihydrostreptomycin (0 to 256 uni t s per m i l l i l i t e r ) and incubated for 72 hours at o 37 C . No v i s i b l e growth could be detected at the higher concentration of a n t i b i o t i c . These cultures were plated on s o l i d broth-agar medium. Among those cul tures which produced growth on the p la te s , the highest streptomycin concentrat ion was 128 uni t s per m i l l i l i t e r . This p la te was incubated for nine days at which time a l l growth was scraped from the agar and innoculated into nutr ient broth containing one uni t per m i l l i l i t e r of dihydrostreptomycin. Af te r 4$ hours incubat ion, t h i s cu l ture was re t rans ferred to broth containing two uni t s per m i l l i l i t e r of dihydrostreptomycin. Growth was evident a f ter 24 hours incubat ion . By successive t ransfers to higher dihydrostreptomycin l e v e l s and by p l a t i n g on s o l i d medium, a pure cul ture of a B a c i l l u s cereus var iant re s i s t ant to 2,000 uni t s of streptomycin per m i l l i l i t e r was i s o l a t e d . The culture was t rans ferred into broth containing no streptomycin and also in to broth containing 2,000 uni t s of streptomycin per m i l l i l i t e r u n t i l there was no percept ib le 16. difference in the 24 hour growth of the variant whether in the absence or presence of streptomycin. Microscopically, this resistant variant resembled the sensitive parent organism: a darkly staining gram-positive rod occasionally appearing slightly elongated, occurring singly, in pairs, and in chains. The rate of growth of resistant Bacillus cereus with varying concentrations of streptomycin was determined. Duplicate tubes were set up containing nutrient broth and dihydrostreptomycin to a constant total volume. The dihydrostreptomycin concentrations were varied from 0 to 100 units per m i l l i l i t e r . Two types of innocula were used: cells which had been grown in the presence of 20 units per m i l l i l i t e r of dihydrostreptomycin and cells similarily grown in the absence of the antibiotic. Twelve hour cultures were used and in the case of the former innoculum, the antibiotic was diluted -2 out to a f i n a l concentration of 4 x 10 units per m i l l i l i t e r . The assay was incubated at 37°G. Growth was followed by measuring the optical densities of the cultures hourly. B. INDUCTION AND DETERMINATION OF PENICILLINASE Antibiotic-sensitive Bacillus cereus cells were grown in Roux flasks as described for the growth experiments. The cells were harvested (non-aseptically), washed twice with saline and suspended in M/50 phosphate buffer, pH 7.0, containing M/600 magnesium and M/50 glucose concentrations. 1 7 . The addi t ion of magnesium and glucose has been shown to increase enzyme a c t i v i t y ( 3 9 ) . The c e l l s were suspended to g a standard concentrat ion of 8 .75 x 10 c e l l s per m i l l i l i t e r . When the rate of p e n i c i l l i n a s e induct ion was to be measured, the c e l l suspension containing one uni t of p e n i c i l l i n per m i l l i l i t e r ( f i n a l concentration) was incubated i n a water bath at 3 5 ° C . According to the experimental observations of Pol lock ( 3 9 ) , t h i s p e n i c i l l i n concentrat ion and temperature y i e l d s maximum p e n i c i l l i n a s e product ion. Three m i l l i l i t e r a l iquot s were removed at hal f-hour i n t e r v a l s for 3 i hours . To each a l iquot was immediately added a so lu t ion of 8-hydroxy quinol ine (oxirae) to a f i n a l concentrat ion of M /1200 and the samples stored at 2 ° C . u n t i l assayed. These preparations were usua l ly assayed wi th in four hours, and never l a t e r than 1& hours. Oxiree has been shown to i n h i b i t further enzyme formation for at leas t 18 hours at 2 ° C . ( 3 9 ) . The p e n i c i l l i n used was an aqueous so lu t ion of the potassium sa l t of benzyl p e n i c i l l i n ( p e n i c i l l i n G- E l i L i l l y & C o . ) . One mi l l i g ram i s equivalent to 1 ,585 u n i t s . Solut ions of p e n i c i l l i n deter iorate rapidly , at 2 ° C . (47) and therefore f r e sh ly prepared solut ions were used for each experiment. When the effect of dihydrostreptomycin was to be s tudied , the c e l l suspensions were prepared s i m i l a r l y . Dihydrostreptomycin i n varying concentrations from 0 to 25 uni t s per m i l l i l i t e r was added to the suspensions twenty 18. minutes p r i o r to the add i t ion of one uni t of p e n i c i l l i n per m i l l i l i t e r . The ac t ion of dihydrostreptomycin i n i n h i b i t i n g enzyme formation i s rap id when i t reaches the c e l l before the enzyme inducer (48) • The suspensions were incubated i n a water bath at 3 5 ° C for 3 a hours before the add i t ion of M/1200 oxime. Enzyme preparations were stored as before at 2 ° C . P e n i c i l l i n a s e was analyzed manometrically under anaerobic condit ions by the procedure of Pol lock (39). Each Warburg f la sk contained: i n the main compartment; 2 m i l l i l i t e r s of enzyme preparation and .5 m i l l i l i t e r s of .043 Molar sodium bicarbonate, and i n the side arm; .1 m i l l i l i t e r s of .043 Molar sodium bicarbonate, .1 m i l l i l i t e r s of p e n i c i l l i n of concentrat ion 100,000 uni t s per m i l l i l i t e r , and .3 m i l l i l i t e r s of water. The f la sks and manometers were f lushed three times with a 5% carbon dioxide i n nitrogen gas mixture. The f la sks were incubated i n a Warburg bath, with constant shaking, at 30 ° C . Af ter e q u i l i b r a t i o n for ten minutes, the so lu t ion i n the side arm was t ipped into the main compartment and measurements were taken of the production of carbon d iox ide . The amount of p e n i c i l l i n a s e induced was measured i n terms of the number of m i c r o l i t e r s of carbon dioxide produced as ca lcu la ted from the f l a sk constant; a r e l a t i o n s h i p f i r s t shown by Henry and Housewright (37). The p e n i c i l l i n a s e induct ion of the dihydro- streptomycin-resistant var iant was s i m i l a r l y ca r r i ed out and 19. measured. The c e l l s were grown e i ther i n the presence of 100 uni t s per m i l l i l i t e r of dihydrostreptomycin or i n the absence of the a n t i b i o t i c . The c e l l s were harvested and resuspended i n buffer as descr ibed. The adaptive enzyme production by the non-pro l i f e ra t ing c e l l s was followed i n the absence or i n the presence of 100 uni t s per m i l l i l i t e r of dihydrostreptomycin. P e n i c i l l i n a s e induct ion was a l so measured under aerobic condit ions by a modi f ica t ion of t h i s procedure. B a c i l l u s cereus c e l l s were grown, harvested, and resuspended i n buffer as previous ly descr ibed . In the main compartment of each Warburg cup, there were 2 m i l l i l i t e r s of c e l l suspension and .5 m i l l i l i t e r s of .043 Molar sodium bicarbonate. In the side bulb there was a mixture of bicarbonate, p e n i c i l l i n and water as previous ly recorded. Unless i n d i c a t e d , the c e l l suspensions were not induced with p e n i c i l l i n p r i o r to the manometric measurement. Induction occured during the assay i n the Warburg f l a s k . When the effect of dihydrostreptomycin was being studied 50 uni t s were added to the main compartment and the t o t a l f l a sk volume adjusted to three m i l l i l i t e r s . After a twenty minute e q u i l i b r a t i o n period i n the Warburg bath at 30 °C. with constant shaking, the p e n i c i l l i n so lu t ion i n the s ide arm was t ipped in to the main compartment. The rate of evolut ion of carbon dioxide i n the presence of a i r was a measure of the amount of enzyme produced. 20. C. SONIC DISRUPTED PREPARATIONS A n t i b i o t i c - s e n s i t i v e B a c i l l u s cereus c e l l s were grown and harvested as descr ibed. F ive m i l l i l i t e r a l iquots of the c e l l suspension i n buffer (that had not been i n contact with p e n i c i l l i n ) were disrupted by treatment i n a 9 K i l o c y c l e Raytheon sonic o s c i l l a t o r . The length of sonic treatment was v a r i e d . P e n i c i l l i n a s e production was measured a e r o b i c a l l y , enzyme induct ion occuring i n the Warburg f l a s k . .5 F I G U R E I 2 1 . 2 . RESULTS A. GROWTH EXPERIMENTS FIGURE I: THE EFFECT OF DIHYDROSTREPTOMYCIN ON THE GROWTH OF SENSITIVE BACILLUS CEREUS The effect of varying concentrations of dihydrostreptomycin on the growth, as measured by o p t i c a l densities, of sensitive B a c i l l u s cereus i s shown i n Figure 1 . At a concentration of 0 .3 units per m i l l i l i t e r of dihydrostreptomycin, there was no increase i n t u r b i d i t y of suspensions at 420 millimicrons. However, at a concentration of a n t i b i o t i c very much lower than t h i s ( 0 . 0 1 units per m i l l i l i t e r ) , there i s greater than 50% i n h i b i t i o n of growth. TABLE I TABLE I I HOURS OF GROWTH 1 OPTICAL DENS DIHYDROSTREPTOMYCIN CC 0.1 1.0 10.0 ITY VALUES NCENTRATION UNITS/ML 0.1 1.0 10.0 0 .002 .002 .002 .001 .002 .002 2 .005 .01 .015 .005 .005 .01 4 .01 .015 .025 .06 .07 .11 5 .04 .075 .125 •13 .135 .15 6 .13 .17 .20 .22 .22 .26 7 .19 .20 .25 .24 .23 .27 $ .26 .29 .33 .29 .30 .31 9 .29 .32 .36 .32 .33 .33 10 .32 .33 .35 .35 .35 .35 11 .34 .34 .35 .34 .35 .35 FIGURE II FIGURE II I 22. FIGURE II (DATA OF TABLE I ) : THE EFFECT OF DIHYDROSTREPTOMYCIN ON THE GROWTH OF RESISTANT BACILLUS CEREUS ( i ) A n t i b i o t i c - r e s i s t a n t B a c i l l u s cereus c e l l s were grown i n the absence of dihydrostreptomycin and used as the innoculum for a ser ies of tubes containing varying amounts of dihydro- streptomycin as described on page 16. The o p t i c a l density of the cultures was measured hourly (Table I ) . Figure II shows the rate of growth of the cultures when no dihydrostreptomycin i s present (Curve A) and when 100 uni t s per m i l l i l i t e r of the a n t i b i o t i c are present (Curve B ) . When the a n t i b i o t i c i s present, there i s a s i g n i f i c a n t increase i n the rate of growth although, as previous ly mentioned, the t o t a l 24 hour growth of the cultures i s constant. FIGURE III (DATA OF TABLE I I ) : THE EFFECT OF DIHYDROSTREPTOMYCIN ON THE GROWTH OF RESISTANT BACILLUS CEREUS ( i i ) A n t i b i o t i c - r e s i s t a n t B a c i l l u s cereus c e l l s were grown i n the presence of dihydrostreptomycin (20 uni t s per m i l l i l i t e r ) and used as the innoculum for a ser ies of tubes (Table II) as i n the above f i g u r e . Unlike the re su l t s shown i n Figure I I , the add i t ion of dihydrostreptomycin (Curve B) produced only a s l i g h t increase i n the rate of growth. 2 0 0 H M I N U T E S FIGURE IV B. INDUCTION OF PENICILLINASE FIGURE IV: THE DETERMINATION OF PENICILLINASE ACTIVITY P e n i c i l l i n a s e converts p e n i c i l l i n to p e n i c i l l o i c acid so that i n a bicarbonate buffer solution, measurement of the rate of production of carbon dioxide by a suspension of B a c i l l u s cereus i s an i n d i c a t i o n of the amount of p e n i c i l l i n a s e present. For induction of the enzyme, a suspension of sensitive B a c i l l u s cereus was incubated at 35°C. f o r 3s hours i n the presence of p e n i c i l l i n , one unit per m i l l i l i t e r . At the end of t h i s time, oxime was added to i n h i b i t further enzyme formation and the rate of carbon dioxide production measured under the conditions referred to on page 18. The production of carbon dioxide i s l i n e a r with time when excess p e n i c i l l i n i s present. The slope of the l i n e (Figure IV) i s thus a measure of induced p e n i c i l l i n a s e . 1 1 1 1 1 — i 1 r 0 1 2 3 HOURS INDUCT ION WITH P E N I C I L L I N FIGURE V 24. FIGURE V: THE INDUCTION OF PENICILLINASE IN DIHYDROSTREPTOMYCIN SENSITIVE BACILLUS CEREUS A suspension of a n t i b i o t i c - s e n s i t i v e B a c i l l u s cereus was incubated with p e n i c i l l i n (one uni t per m i l l i l i t e r of suspension) as described on page 17. Samples were removed at various time i n t e r v a l s , t reated with oxime, and the p e n i c i l l i n a s e content assayed. In the fo l lowing Figures (V- VIII ), enzyme a c t i v i t y i s expressed as the number of m i c r o l i t e r s of carbon dioxide produced per minute during the assay per m i l l i l i t e r of enzyme preparat ion t e s t ed . Fol lowing an i n i t i a l l ag of 0.5 hours, induct ion proceeded at an increas ing rate for the durat ion of the experiment. -I 1 1 1 r 0 1 2 3 D I H Y D R O S T R E P T O M Y C I N C O N C E N T R A T I O N U N I T S P E R M I L L I L I T E R FIGURE V I FIGURE V I : THE EFFECT OF VARYING CONCENTRATIONS OF DIHYDRO- STREPTOMYCIN ON PENICILLINASE INDUCTION IN SENSITIVE BACILLUS CEREUS A n t i b i o t i c - s e n s i t i v e B a c i l l u s cereus cultures were induced with one uni t per m i l l i l i t e r of p e n i c i l l i n for 3i hours i n the presence of dihydrostreptomycin from 0 to 25 unit s per m i l l i l i t e r of suspension. The dihydrostreptomycin was always added p r i o r to the p e n i c i l l i n as previous ly descr ibed. The c e l l concentration was twenty times that used i n the growth experiment. Figure VI shows that 50% i n h i b i t i o n of enzyme induct ion occurs at a concentrat ion of 0.6 un i t s of dihydro- streptomycin per m i l l i l i t e r , w h e r e a s no p e n i c i l l i n a s e a c t i v i t y could be detected at concentrations of a n t i b i o t i c above ten times t h i s va lue . FIGURE VII 26. FIGURE VII : THE INDUCTION OF PENICILLINASE IN DIHYDRO- STREPTOMYCIN RESISTANT BACILLUS CEREUS (i) Antibiotic-resistant Bacillus cereus cells were grown in the absence of dihydrostreptomycin and assayed for penicillinase activity at various times during the induction with one unit of p e n i c i l l i n per m i l l i l i t e r of suspension as described on page 18. Curve A represents the induction of penicillinase activity when 100 units per m i l l i l i t e r of dihydrostreptomycin were added 0.5 hours prior to the addition of p e n i c i l l i n . Curve B represents the induction of penicillinase activity by a similar suspension when no dihydrostreptomycin was present. There is a significantly greater rate of induction of penicillinase in the presence of dihydrostreptomycin (A) than in i t s absence. 3 FIGURE VIII 27 FIGURE VIII: THE INDUCTION OF PENICILLINASE IN DIHYDRO- STREPTOMYCIN RESISTANT BACILLUS CEREUS ( i i ) A n t i b i o t i c - r e s i s t a n t B a c i l l u s cereus c e l l s were grown i n the presence of dihydrostreptomycin. The a n t i b i o t i c was removed by several washings with s a l i n e . As described on page IS, suspensions were assayed f o r p e n i c i l l i n a s e a c t i v i t y at various times during the induction with one unit of p e n i c i l l i n per m i l l i l i t e r . Curve A represents the induction of p e n i c i l l i n a s e a c t i v i t y when 100 units per m i l l i l i t e r of dihydrostreptomycin were added 0.5 hours p r i o r to the addition of p e n i c i l l i n . Curve B represents the induction of p e n i c i l l i n a s e a c t i v i t y by a s i m i l a r suspension when no dihydrostreptomycin was present. There i s no increase i n the rate of induction of the enzyme i n the presence of dihydrostreptomycin; indeed, there i s a s i g n i f i c a n t i n h i b i t i o n of induction aft e r 3h hours incubation'with p e n i c i l l i n and dihydrostreptomycin. FIGURE IX 28. FIGURE IX: THE INDUCTION OF PENICILLINASE UNDER AEROBIC CONDITIONS IN DIHYDROSTREPTOMICIN-SENSITIVE BACILLUS CEREUS In the experiments described on the previous pages (pp.23-27) , p e n i c i l l i n a s e was induced with one unit of p e n i c i l l i n per m i l l i l i t e r of b a c t e r i a l suspension at 35°C. aer o b i c a l l y . Enzyme formation was then stopped with oxime and the l e v e l of p e n i c i l l i n a s e determined under anaerobic conditions i n the Warburg apparatus. In the following experiments, Figures IX and X, p e n i c i l l i n a s e was induced i n the respirometer as described on page 19. At zero time (Figure IX), p e n i c i l l i n (10,000 units) was added to a c e l l suspension of a n t i b i o t i c — sensitive B a c i l l u s cereus i n the Warburg cup. There had been no previous induction with p e n i c i l l i n . Carbon dioxide pro- duction was measured d i r e c t l y during enzyme induction. Curve A i s the p e n i c i l l i n a s e induction curve obtained by t h i s method for dihydrostreptomycin-sensitive B a c i l l u s cereus. When dihydrostreptomycin (50 units) i s added to the c e l l suspension 20 minutes p r i o r to the addition of p e n i c i l l i n (that i s , 20 minutes pr i o r to zero time), an i n h i b i t i o n of p e n i c i l l i n a s e production r e s u l t s (Curve B). i 1 1 1 1 1 1 i r~ 0 20 40 60 80 M I N U T E S FIGURE X 29. FIGURE X: THE EFFECT OF DIHYDROSTREPTOMYCIN ON THE ACTION OF PENICILLINASE Suspensions of dihydrostreptomycin-sensi t ive B a c i l l u s cereus were treated for 2 hours at 35 °C . with p e n i c i l l i n (one unit per m i l l i l i t e r ) . A l iquot s were assayed for p e n i c i l l i n a s e a c t i v i t y a e r o b i c a l l y . P e n i c i l l i n (10,000 units) was added to the c e l l suspension i n the Warburg cup at zero time (Figure X ) . Curve C shows the effect of dihydrostreptomycin (50 uni t s per m i l l i l i t e r ) added p r i o r to the pre- induct ion with p e n i c i l l i n . Curve B shows the effect of dihydrostreptomycin added 20 minutes before zero t ime; that i s , a f ter the 2 hour induct ion per iod . No dihydrostrepto- mycin was present i n the suspensions producing Curve A . * When dihydrostreptomycin reaches the c e l l before the p e n i c i l l i n , no p e n i c i l l i n a s e i s produced. However, Curve B shows that , once induced, p e n i c i l l i n a s e i s not i n h i b i t e d by dihydrostreptomycin i n i t s ac t ion on p e n i c i l l i n . Curve A represents the combined a c t i v i t y of the p e n i c i l l i n a s e produced during the pre- induct ion and during the assay i n the respirometer . TABLE III TIME OF SONIG TREATMENT (MINUTES) OPTICAL DENSITY :1 x 10" PENICILLINASE ACTIVITY 0 . 3 0 1 . 0 0 1 . 2 4 1.08 2 . 2 2 1 . 1 5 3 .18 . 6 6 3 5 . 1 5 . 2 4 4 10 .13 . 1 6 5 3 0 . C. SONIC DISRUPTED PREPARATIONS TABLE I I I : THE PENICILLINASE ACTIVITY OF SUSPENSIONS OF DIHYDROSTREPTOMYCIN-SENSITIVE BACILLUS CEREUS EXPOSED TO SONIC OSCILLATIONS FOR VARIOUS TIMES Suspensions of s ens i t ive B a c i l l u s cereus (5 m i l l i l i t e r s ) were t reated i n a sonic o s c i l l a t o r as described on page 2 0 . P e n i c i l l i n ( 10 ,000 uni t s ) was then added to 3 m i l l i l i t e r s of t h i s b a c t e r i a l suspension i n the Warburg cup and the ra te of evolut ion of carbon dioxide measured. There had been no previous induct ion with p e n i c i l l i n before the sonic treatment. The a c t i v i t y of the whole c e l l preparat ion as m i c r o l i t e r s of carbon dioxide produced per minute, per m i l l i l i t e r of suspension i s a r b i t r a r i l y assigned the value 1 . 0 0 . The o p t i c a l densi ty values a f ter the sonic treatment were a l so recorded. Table I I I shows that the p e n i c i l l i n a s e a c t i v i t y i s increased by short periods of sonic treatment (up to two minutes) but then decreases to a low value as the sonic treatment i s continued. 31. I l l DISCUSSION It has been observed that streptomycin re s i s t an t s t ra ins of bacter ia develop with as tonishing r a p i d i t y from cultures of sens i t ive organisms exposed to the a n t i b i o t i c (49,50). I t i s thought that t h i s development o f res i s tance to streptomycin can occur exc lus ive ly by natura l s e l ec t ive processes although adaptation to streptomycin may be i n v o l v e d . In the presence of re s i s t ant bac te r i a , streptomycin i s not destroyed, nor i s a s i g n i f i c a n t amount removed from the medium during growth (51). The metabolic di f ferences between the sens i t ive and re s i s t ant var iants are not known. Lam and Sevag found, when working with a thymine-requir ing s t r a i n of E . c o l i . (15T ), that incubation with streptomycin but without thymine produced an increase i n the frequency of streptomycin- re s i s t ant mutants. They concluded that t h i s increased rate of mutation was a r e s u l t of a s i g n i f i c a n t l y a l t e red DNA metabolism (52). The attempt to develop a s treptomycin-res is tant B. cereus was success fu l . The o r i g i n a l B . cereus s t r a i n was i n t r i n s i c a l l y very s ens i t ive to streptomycin. Growth was completely i n h i b i t e d by approximately .3 un i t s of streptomycin per m i l l i l i t e r of medium, while 50% i n h i b i t i o n was observed at .01 u n i t s . The var iant developed was re s i s t an t to concentrations of streptomycin i n the order of 2,000 uni t s per m i l l i l i t e r of medium. 32. P e n i c i l l i n a s e induct ion was followed by two methods as previous ly descr ibed, both producing s i m i l a r r e s u l t s . A n o n - p r o l i f e r a t i n g suspension of B. cereus was t reated with p e n i c i l l i n (one uni t per m i l l i l i t e r ) and a l iquot s tes ted for enzyme a c t i v i t y at h a l f hour i n t e r v a l s . Figure V i s the t y p i c a l enzyme adaptation curve obtained. Enzyme production was a l so followed d i r e c t l y i n the Warburg apparatus with B. cereus c e l l s that had not previous ly been induced. A s i m i l a r curve i s obtained (Figure IXA). It was found that dihydrostreptomycin i n h i b i t s the formation of p e n i c i l l i n a s e i n the a n t i b i o t i c - s e n s i t i v e B a c i l l u s cereus (Figure I X ) . Further , that i t i s the formation and not the ac t ion of t h i s enzyme which i s i n h i b i t e d by streptomycin, i s shown i n Figure X. When B a c i l l u s cereus c e l l s are pre-induced with p e n i c i l l i n before the add i t ion of dihydrostreptomycin, the already-formed p e n i c i l l i n a s e i s not affected (Curve B ) . However, the formation of further enzyme i s i n h i b i t e d . Where dihydrostreptomycin i s not present (Curve A) add i t iona l enzyme continues to be synthesized. As previous ly mentioned, when dihydrostreptomycin i s added before the inducer , no p e n i c i l l i n a s e i s formed (Curve C ) . The re su l t s of the study on the effect of varying concentrations of dihydrostreptomycin on p e n i c i l l i n a s e induct ion i n sens i t ive B a c i l l u s cereus are i n t e r e s t i n g (Figure V I ) . T o t a l i n h i b i t i o n occurred when the concentrat ion of 33. dihydrostreptomycin was of the same magnitude as that required for i n h i b i t i o n of growth. Thi s type of curve seems to ind ica te that the reac t ion of dihydrostreptomycin i s r e v e r s i b l e . When enzyme i n h i b i t i o n i s p lo t ted against the r e c i p r o c a l of the dihydrostreptomycin concentrat ion a l i n e a r graph i s obtained. That i s , i n h i b i t i o n of p e n i c i l l i n a s e formation f i t s the mass law equation, xy=C, where x i s the dihydrostreptomycin concentrat ion and y i s the f r a c t i o n a l enzyme synthes i s . From Figure V I , i t may be seen that at 0.5 uni t s per m i l l i l i t e r of dihydrostreptomycin, the f r a c t i o n a l enzyme synthesis i s 0.58. Therefore: G = 0.5 x 0.58 = 0.29 When the dihydrostreptomycin concentrat ion i s 1.0 un i t per m i l l i l i t e r , the f r a c t i o n a l enzyme synthesis i s 0.32. C = 1.0 x 0.32 = 0.32 When the dihydrostreptomycin concentrat ion i s 2.0 uni t s per m i l l i l i t e r , the f r a c t i o n a l enzyme synthesis i s 0 .15. C = 2.0 x 0.15 = 0.30 Thus, i t appears that the reac t ion of dihydrostreptomycin with some c e l l component required for enzyme synthesis i s an equi l ibr ium reac t ion i n v o l v i n g presumably, because of the basic character of streptomycin, some a c i d i c c e l l component. 34 A most remarkable observation concerning the ef fects of streptomycin was that bac ter ia could become dependent on streptomycin for growth. Thi s was f i r s t reported by M i l l e r and Bohnhoff working with meningococci (53). Further , the phenomenon of p a r t i a l dependence of re s i s t ant organisms has been described by Bolglase (20). In contrast to h i s observations with E . c o l i , r e s i s t ant B a d i l l u s cereus showed dependence only to a very s l i g h t degree. Figure II and I I I show the growth curves for the re s i s t ant organism a f ter t rans fer from media containing no dihydrostreptomycin and 20 uni t s per m i l l i l i t e r dihydrostreptomycin r e s p e c t i v e l y . In Figure I I , the add i t ion of 100 un i t s of dihydrostreptomycin per m i l l i l i t e r causes a s i g n i f i c a n t increase i n the rate of growth. Further , Table I shows that the add i t ion of increas ing l e v e l s of dihydrostreptomycin causes a consistent increase i n the rate of growth. This increase i s much le s s when the c e l l s had been previous ly grown on dihydrostreptomycin (Figure I I I ) . Thus, although t h i s var iant of B a c i l l u s cereus i s re s i s t ant to dihydrostreptomycin as shown by a twenty-four hour growth cu l ture , i t exhib i t s a s l i g h t dependence on the a n t i b i o t i c i n the ear ly hours of growth. S imi l a r condit ions with respect to p e n i c i l l i n a s e production are shown i n Figures VII and V' l I I . When the r e s i s t an t organism has been grown on a dihydrostreptomycin-containing media, there i s a shorter l ag per iod for the formation of p e n i c i l l i n a s e . Again, t h i s 35. effect i s s l i g h t . These r e su l t s may be consistent with the fact that there was no evidence for the development of a dependent mutant during the i s o l a t i o n of the re s i s t ant v a r i a n t . It would appear that the B a c i l l u s cereus organism i s l e s s prone to develop dihydrostreptomycin dependence than i s E . c o l i . Adaptive enzyme synthesis i n c e l l free preparations was f i r s t reported by Gale and Folkes (54). Staphylococcus aureus c e l l s were disrupted by supersonic v ib ra t ions i n an o s c i l l a t o r for twenty to t h i r t y minutes. Using t h i s preparat ion, they showed a s t imulatory effect of RNA and DNA on amino ac id residue exchanges. At the same time, Kramer reported enzymatic adaptation i n c e l l - f r e e extracts of B a c i l l u s cereus (55). In a l a t e r communication (56), i t was shown that the phenomenon of induct ion occurs through factors present only i n in t ac t c e l l s , s ince only c e l l s pretreated with p e n i c i l l i n before homogenisation showed an increase i n p e n i c i l l i n a s e content. They concluded that induct ion i s not simply due to the d i rec t effect of the substrate on the s i t e of pro te in synthes i s . Because of these report s , an attempt was made to determine i f b roken-ce l l suspensions of B a c i l l u s cereus could form p e n i c i l l i n a s e . Table I I I shows the r e s u l t s of t h i s attempt. I t w i l l be noted that for short periods of sonic treatment (one and two minutes) there i s an increase i n the . 36. rate of p e n i c i l l i n a s e product ion. It was d i f f i c u l t to a scer ta in the extent of d i s rupt ion i n these preparations by microscopic examination. These r e s u l t s can probably be explained i f sonic v ibra t ions for a l i m i t e d time increase the permeabi l i ty of the c e l l w a l l e i ther to p e n i c i l l i n or to p e n i c i l l i n a s e without damaging the enzyme-synthesizing s i t e . On continued sonic treatment however, t h i s s i t e i s damaged and there i s an apparent decrease i n e i ther p e n i c i l l i n a s e induct ion or i n p e n i c i l l i n a s e a c t i v i t y . 37 IV CONCLUSIONS I f streptomycin blocks the formation of a substance e s sent i a l to the b a c t e r i a l c e l l , there w i l l be a r e l a t i o n s h i p between the b a c t e r i o s t a t i c a c t i v i t y and the i n h i b i t i n g a b i l i t y of streptomycin. In t h i s study, the i n h i b i t i o n of p e n i c i l l i n a s e a c t i v i t y i n the a n t i b i o t i c - sens i t ive B a c i l l u s cereus was brought about by a concentration of dihydrostreptomycin which caused bac ter io s ta s i s of the organism. Furthermore, the a b i l i t y of the a n t i b i o t i c - r e s i s t a n t B a c i l l u s cereus to form p e n i c i l l i n a s e was not af fected by much higher concentrations of dihydrostrept omyc i n . The phenomenon of prote in synthesis involves many complex and as yet unknown r e a c t i o n s . The p a r t i c i p a t i o n of nuc le ic acids i n pro te in synthesis has been hypothesized. Reiner and Goodman (57) have suggested that the rate l i m i t i n g step i n induced enzyme synthesis i s the formation of some polynucleot ide mater ia l wi th in the c e l l s . E a r l i e r , Pardee had demonstrated an absolute u r a c i l requirement for adaptive v enzyme formation i n E . c o l i (58). I t i s thought that the s i t e of ac t ion of streptomycin may u l t imate ly prove to be i n t h i s u n c l a r i f i e d f i e l d of nucle ic ac id p a r t i c i p a t i o n i n prote in synthes i s . It i s admitted, therefore , that t h i s study offers no further c l a r i f i c a t i o n of the ac t ion of streptomycin. The r e su l t s merely extend the knowledge of the effect of dihydrostreptomycin on enzyme synthesis to the induc ib le p e n i c i l l i n a s e of B a c i l l u s cereus. However, there may be ce r t a in advantages to u t i l i z i n g the p e n i c i l l i n a s e system for a study of streptomycin a c t i v i t y . Unl ike the induct ion of /^-galactosidase with l ac tose , i n which other enzymes (for example, galactozymase) are induced, the induct ion with p e n i c i l l i n produces only one enzyme, p e n i c i l l i n a s e . Thus, quant i ta t ive studies of streptomycin effects can be made more r e a d i l y . One might also speculate regarding the observed synergism between p e n i c i l l i n and streptomycin. The concept - of the " b l o c k i n g " of metabolic pathways by an a n t i b i o t i c i s widespread. Resistance may occur by the development of an a l t e rna t ive metabolic r e a c t i o n . This work has shown that streptomycin i n h i b i t s the formation of an enzyme i n s ens i t ive micro-organisms, and i t might be speculated that i n v i v o , i t would i n h i b i t the formation, i n the i n f e c t i n g organism, of the enzymes required for t h i s a l ternate metabolic pathway. Secondly, a n t i b i o t i c s become i n e f f e c t i v e when the b a c t e r i a l c e l l can destroy them. P e n i c i l l i n i s rendered i n e f f e c t i v e as an a n t i b i o t i c by hydro lys i s to p e n i c i l l o i c a c i d . Some b a c t e r i a l c e l l s can adapt ively form an enzyme, p e n i c i l l i n a s e , which can accomplish t h i s . Streptomycin, however, prevents 3 9 the synthesis of p e n i c i l l i n a s e and thus would prevent the development of res i s tance to p e n i c i l l i n . I t has been observed ( 5 9 ) that there i s p r a c t i c a l l y no synergism between p e n i c i l l i n arid streptomycin i f streptomycin i s administered a few hours a f ter the i n j e c t i o n of p e n i c i l l i n . This can be explained on the basis of the r e s u l t s here in recorded on the i n h i b i t o r y effect of streptomycin on p e n i c i l l i n a s e formation. During the few hours p r i o r exposure i n v ivo to p e n i c i l l i n , p e n i c i l l i n a s e formation could occur and t h i s enzyme, once formed, would not be i n h i b i t e d by streptomycin. However, i f streptomycin i s present f i r s t or simultaneously with p e n i c i l l i n , the formation of p e n i c i l l i n a s e would be i n h i b i t e d and the synerg i s t i c effect of the combined a n t i b i o t i c s would be explained. Indeed, the hindrance of the development of r e s i s t an t s t ra ins i s a part of the bas i s of synergism. 40. V BIBLIOGRAPHY 1. Schatz, A . , Bugie, E . , Waksman, S. , P roc . Soc. E x p t l . B i o l . Med. , 55:66 (1944). 2. F r i e d , J . , Winters te iner , 0., Science, 101:613 (1945). 3 • P ra t t , R . , Dufrenoy, J . , e d s . , A n t i b i o t i c s , P h i l a d e l p h i a , J . B . L ipp ineot t C o . , 1953, p . 157. 4. Waksman, S . A . , A n t i b i o t i c s and Chemotherapy, 3:333 (1953). 5. Reference 3., p . 160. 6. Waksman, S . A . , e d , , Streptomycin. Bal t imore, The Wil l iams &Wilkins C o . , 1949, p . 69. 7. Wayson, N . E . , McMahon, M . C . , J . Lab. C l i n . Med. , 31:323 (1946). 8. Umbreit, W.W., J . B i o l . Chem., 177:703 (1949). 9. Oginsky, E . L . , Smith, P . H . , Umbreit, W.W., J . B a c t . , 53:747 (1949). 10. Umbreit, W.W., Smith, P . H . , Oginsky, E . L . , J . Bac t . , 61:595 (1951). 11. Umbreit, W.W., J . B a c t . , 66:74 ( 1953) . 12. Geiger, W . B . , A r c h . Biochem., 15:227 (1947). 13. Henry, J . , Henry, R . J . , Berkman, S . , Housewright, R . D . , J . B a c t . , 56:527 (194&1). 14. F i t z g e r a l d , R . J . , Bernheim, F . , F i t z g e r a l d , D . B . , J . B i o l . Chem., 175:195 (1948). 15 . F i t z g e r a l d , R . J . , Bernheim, F . , J . B a c t . , 55:765 (194&1). 16. S tan ier , R . Y . , J . B a c t . , 54:339 (1947). 17. Cohen, S .S . , J . B i o l . Chem., 168:511 (1947). 18. Cohen, S .S . , J . B i o l . Chem., 166:393 (1946). 41. 19. Donovick, R. , Bayan, A . P . , Canales, P . , Pansy, F . , J . B a c t . , 56:125 (1948). 20. Peretz , S . , Polg lase , W . J . , A n t i b i o t i c s Annual. 1956-1957, Medical Encyclopedia , I n c . , New York, N . Y . , p . 533. 21. Gale, E . F . , Adv. i n Prote in Chem., 8:287 (1953). 22. C a v a l l i t o , C . J . , J . B i o l . Chem., 164:29 (1946). 23. Rhymer, I . , Wallace, G . I . , Byers , L . W . , Car ter , H . E . , J . B i o l . Chem., 169:457 (1947). 24. Lightbrown, J . W . , Nature, 166:356 (1950). 25. Cornforth , J . W . , James, A . T . , Biochem. J . , 63:124 (1956). 26. Creaser, E . H . , J . Gen. M i c r o b i o l . , 12:288 (1955). 27. Roote, S . M . , Polg lase , W . J . , Can. J . Biochem. P h y s i o l . , 33:792 (1955) . 28. Polg lase , W . J . , Can. J . Biochem. P h y s i o l . , 34:554 (1956) . 29. Polg lase , W . J . , Peretz , S . , Roote, S . M . , Can. J . Biochem. P h y s i o l . , 34:558 (1956). 30. Abraham, E . P . , Chain, E . , Nature, 146:837 (1940). 31. Duthie , E . S . , B r i t . J . Exp. P a t h . , 25:95 (1944). 32. Lepage, G . A . , Morgan, J . F . , Campbell, M . E . , J . B i o l . Chem., 166:465 (1946). 33. Housewright, R . D . , Henry, R . J . , J . B i o l . Chem., 167:553 (1947). 34. Committee on Medical Research, Science, 102:627 (1945). 35. Fos ter , J .W. , Science, 101:205 (1945). 36. Benedict , R . G . , Schmidt, W . H . , C o g h i l l , R . D . , A r c h . Biochem., 8:377 U945) . 42. 37. Housewright, R . D . , Henry, R . J . , J . B i o l . Chem., 167:559 (1947). 38.. P o l l o c k , M.R . , J . Gen. M i c r o b i o l . , 15:154 (1956). 39. Po l lock , M . R . , B r i t . J . Exp. P a t h . , 31:739 (1950). 40. Po l lock , M . R . , B r i t . J . Exp. P a t h . , 32:387 (1951). 41. Po l lock , M . R . , B r i t . J . Exp. P a t h . , 33:587 (1952). 42. P o l l o c k , M . R . , B r i t . J . Exp. P a t h . , 34:251 (1953). 43. Cohn, M . , Monod, J . , Biochim. et Biophys. Ac ta , 7:153 (1951). 44. Kogut, M . , Po l lock , M . R . , T r i d g e l l , E . J . , Biochem. J . , 62:391 (1956). 45. S tan ier , R . Y . , i n Nei lands, J . B . , Stumpf, P . K . , eds . , Outl ines of Enzyme Chemistry. New York, John Wiley & Sons, I n c . , 1955, p . 285. 46. Bar tz , Q .R . , Cont rou l i s , J . , Crooks, H . M . , Rebstock, M . C . , J . Am. Chem. S o c , 68:2163 (1946). 47. Reference 3., p . 106. 48. Polg lase , W . J . , Un iver s i ty of B r i t i s h Columbia, personal communication. 49. Reference 3., p . 177. 50. M i l l e r , C P . , Bohnhoff, M . , Science, 105:620 (1947). 51. Reference 6., p . 198. 52. Lam, G . T . , Sevag, M . G . , J . B a c t . , 69:184 (1955). 53. M i l l e r , C . P . , Bohnhoff, M . , J . B a c t . , 54:467 (1947). 54. Gale, E . F . , Folkes , J . T . , Nature, 173:1223 (1954). 55. Kramer, M . , Acta P h y s i o l . Hung., 62 Suppl.:90 (1954).

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
United States 4 0
Japan 1 0
City Views Downloads
Mountain View 2 0
Tokyo 1 0
Redmond 1 0
Ashburn 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}

Share

Share to:

Comment

Related Items