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Studies of the pathogenesis and treatment of urinary tract infections using a model of the human bladder Eftekhar, Fereshteh 1982

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STUDIES OF THE PATHOGENESIS AND TREATMENT OF URINARY TRACT INFECTIONS USING A MODEL OF THE HUMAN BLADDER by FERESHTEH EFTEKHAR B.Sc., National University of Iran, 1971 M.Sc, Wichita State University, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Microbiology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA 0 F E R E S H T E H E F T E K H A R , 1 9 8 2 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 q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , 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 o r her r e p r e s e n t a t i v e s . 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 n o t 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 cfe> b i 1 0 ( 0 ^ ^ The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT Urinary t r a c t i n f e c t i o n s are generally preceded by transfer of organisms from the d i s t a l urethra to the bladder (20, 148). However, although urinary i n f e c t i o n s are predominantly due to pure cultures of Escherichia c o l i , the d i s t a l urethra contains a mixed f l o r a i n which E. c o l i i s r e l a t i v e l y uncommon and anaerobes predominate (73, 103). This discrepancy between the bladder and d i s t a l u r e t h r a l f l o r a may be due to d i f f e r e n t i a l adhesion or d i f f e r e n t i a l growth rates. In t h i s d i s s e r t a t i o n I have tested the hypothesis that d i f f e r e n t i a l growth rates of u r e t h r a l organisms i n urine explains the predominance of E. c o l i as a pathogen. These experiments showed that the balance between b a c t e r i a l growth and washout may have a p i v o t a l r o l e i n the pathogenesis of i n f e c t i o n and perhaps therefore i n treatment. A model of the human bladder used for'the pathogenesis studies was then used to study the a c t i v i t y of mecillinam and a m p i c i l l i n under conditions simulating human urinary i n f e c t i o n . The model proved r e a l i s t i c e s p e c i a l l y for synergy studies where shortcomings i n conventional i n v i t r o methods are a cause for concern. The following topics were studied. 1. Urine was chosen as a test medium for d e f i n i t i v e experiments because growth rates of organisms other than E_. c o l i were d i f f e r e n t i n broth and i n urine. A method f or s t e r i l i z i n g urine i n bulk was developed which did not a f f e c t growth supporting properties. 2. E. c o l i was shown to grow f a s t e r and to have a shorter lag period than almost a l l other organisms when studied i n shake culture. 3. A continuous culture model of the human urinary bladder was employed for d i f f e r e n t i a l growth studies of organisms i n s t e r i l i z e d human urine. This model reproduced many of the c h a r a c t e r i s t i c s of the human lower i i i u rinary t r a c t and enabled study of the balance between b a c t e r i a l growth and the tendency of urine to wash organisms out of the t r a c t . 4. Mixed cultures of approximately equal numbers of _E. c o l i and a second p o t e n t i a l urinary' pathogen were introduced into the bladder model and quantitative cultures performed at i n t e r v a l s up to 24 h. In 15 experiments _E. c o l i eventually dominated the second pathogen which was sometimes undetectable at 24 h. Similar changes i n b a c t e r i a l populations seen i n infected patients indicate that d i f f e r e n t i a l growth rates may be an important determinant of the pathogenicity of _E. c o l i . 5. The use of the bladder model was then extended to investigations of a n t i b i o t i c a c t i v i t y under r e a l i s t i c conditions. The value of the model for synergy studies with a m p i c i l l i n and mecillinam was assessed by p a r a l l e l conventional ^n. v i t r o tests and an animal i n f e c t i o n protection t e s t * . The bladder model gave s i m i l a r r e s u l t s to mammalian studies and appeared to be far superior to conventional methods. This model may be valuable i n the i n i t i a l assessment of new urinary a n t i b i o t i c s . 6. A representative array of organisms for the above study was selected following a survey of resistance patterns of 2000 c l i n i c a l i s o l a t e s of Enterobacteriaceae. An i n c i d e n t a l by-product of t h i s survey was the establishment of a breakpoint for mecillinam s u s c e p t i b i l i t y i n the Kirby-Bauer a n t i b i o t i c disk t e s t . 7. Work on the e f f e c t of mecillinam and/or a m p i c i l l i n upon b a c t e r i a l v i a b i l i t y was extended to investigations of the r e l a t i v e contribution of permeability b a r r i e r s and 3-lactamases to a n t i b i o t i c s u s c e p t i b i l i t y . Unlike a m p i c i l l i n , mecillinam resistance of 77 c l i n i c a l i s o l a t e s of ba c t e r i a appeared to be independent of i n t r a c e l l u l a r 3-lactamase l e v e l s , Carried out by Dr. R.C. CJeeland. suggesting that the b a r r i e r e f f e c t may be more pronounced i n b a c t e r i a l resistance to mecillinam than to a m p i c i l l i n . K i n e t i c studies using urine as a growth medium, and i n p a r t i c u l a r the use of a bladder model have provided a unifying explanation of many features of both the pathogenesis and treatment of urinary i n f e c t i o n s . V TABLE OF CONTENTS Page INTRODUCTION Obj ectives 1 An Overview of Urinary Tract Infections 2 Epidemiology of Urinary Tract Infections 3 Age and Sex D i s t r i b u t i o n of Urinary Tract Infections 5 Organisms Responsible for Urinary Tract Infections 6 Mi c r o b i a l Factors which Contribute to the Establishment of Urinary Tract Infections 8 Host Factors i n the Pathogenesis of Urinary Tract Infections . . 10 A Review of the Use of "Bladder Models" i n the Pathogenesis and Treatment of Urinary Infections 13 P r a c t i c a l and Theoretical Aspects of the Treatment of Urinary Tract Infections 15 Factors Influencing the Choice of A n t i b i o t i c s f o r Urinary Infections 17 The Properties of Mecillinam 19 An Overview of Beta-Lactamases of Gram Negative Bacteria . . . . 22 Synergy Studies with Mecillinam 23 MATERIALS AND METHODS Organisms 25 Media 26 A n t i b i o t i c s 27 Buffers . 28 Beta-lactamase Preparations 28 A n t i b i o t i c S u s c e p t i b i l i t y Tests 29 Determination of Minimum Inhibitory Concentrations (MIC's) by the Agar D i l u t i o n Technique 30 v i TABLE OF CONTENTS (Continued) Page MATERIALS AND METHODS (continued) Determination of Minimum Inhib i t o r y Concentrations (MIC's) i n Urine and Broth by the M i c r o t i t e r Plate Method • . 30 Assessment of the Reprodu c i b i l i t y of B a c t e r i a l Assays for Mixed Cultures Using D i f f e r e n t i a l Media 31 Determination of B a c t e r i a l Growth Rates i n Shake Cultures 32 The E f f e c t of Antifoam and F i l t r a t i o n on B a c t e r i a l Growth i n Urine 32 Use of the Bladder Model f or Mixed Culture Experiments . . . . 33 Ca l i b r a t i o n of Residual Volume 36 Operation of the Bladder Model f or Mixed Culture Experiments 37 Study of A n t i b i o t i c Synergy i n the Bladder Model 37 Study of Synergy i n the Mouse Model 38 Screening Tests f o r Beta-Lactamase A c t i v i t y 39 Preparation of Crude Extracts of Beta-Lactamase from C l i n i c a l Isolates 40 Microiodometric Technique for Measuring Beta-Lactamase i n Crude Extracts of Bacteria 41 Reagents 41 Test Procedure 41 Preparation of Membrane Fractions from Enterobacteriaceae . . . 42 14 Abortive Studies of C-Mecillinam Binding to Membrane Fractions 42 Abortive Attempts to Label Intact C e l l s with l 4 C - M e c i l l i n a m 43 Polyacrylamide Gel Electrophoresis (P.A.G.E.) 43 Fluorography ( S c i n t i l l a t i o n Autography) 45 v i i TABLE OF CONTENTS (Continued) Page RESULTS Assessment of D i f f e r e n t i a l Media for Use i n Mixed Culture Experiments 46 Ef f e c t s of S t e r i l i z a t i o n Method on the Growth Supporting Properties of Five Samples of Urine 46 Growth Rates of Bacteria in Shake Cultures 48 Mixed Culture Experiments i n the Bladder Model 57 A n t i b i o t i c Resistance Pattern of C l i n i c a l Isolates from Hospitals i n Vancouver 59 1978 Survey 59 1980 Survey 74 Synergy Between A m p i c i l l i n and Mecillinam in v i t r o 78 Study of Synergy Between A m p i c i l l i n and Mecillinam i n the Bladder Model 81 Study of Synergy Between A m p i c i l l i n and Mecillinam i n the Mouse Model 87 Detection of Beta-Lactamases i n C l i n i c a l Isolates of Enterobacteriaceae 91 14 Abortive Attempts to Demonstrate C-Mecillinam Binding to Intact C e l l s and Membranes of 10 C l i n i c a l Isolates of Enterobacteriaceae 102 DISCUSSION Pathogenicity Studies 103 A n t i b i o t i c Disk S u s c e p t i b i l i t y Studies 108 Synergy Studies 110 The Contribution of Beta-Lactamases to Mecillinam Resistance . . . . 114 BIBLIOGRAPHY 118 V l l l LIST OF TABLES Table Page D i s t r i b u t i o n of Organisms Causing Urinary Tract 7 Infections in Community and Hospitalized Patients. Assessment of D i f f e r e n t i a l Media for Use i n Mixed 47 Culture Experiments. E f f e c t of S t e r i l i z a t i o n Methods on the Growth 49 Supporting Properties of 5 Urine Samples for F i f t e e n Aerobic Isolates i n Shake Culture. Viable Counts of Representative Samples of Bacteria 50 Grown i n Urine. Viable Counts of Representative Samples of Bacteria 51 Grown i n Nutrient Broth. Growth C h a r a c t e r i s t i c s of 6 Urinary Isolates of 53 Escherichia c o l i i n Urine and Nutrient Broth. Growth C h a r a c t e r i s t i c s of Urinary Isolates other than E. c o l i i n Urine and Nutrient Broth. 54 8 Growth C h a r a c t e r i s t i c s of Pathogens from Sources 56 other than Urine. 9 Comparison of the Relative Growth Rates of Escherichia 58 c o l i and other Organisms i n Urine. 10 I d e n t i f i c a t i o n of a Sample of 100 Organisms Obtained 61 from Hospitals i n Vancouver. 11 D e t a i l s of A n t i b i o t i c Disk S e n s i t i v i t y Test. 62 12 Antibiograms of 999 Enterobacteriaceae Isolated at 66 Hospitals i n Vancouver (1978). 13 E f f e c t of Choice of Cut-Off Point for I n h i b i t i o n Zone 67 Diameters upon Apparent Levels of Resistance to Mecillinam. 14 I d e n t i f i c a t i o n of 146 C l i n i c a l Isolates of Entero- 71 bacteriaceae Obtained from Hospitals i n New York. 15 Data for Relationship Between Minimum Inhib i t o r y 72 Concentrations of Mecillinam and the Size of the Zone of I n h i b i t i o n Around Mecillinam on Disks. 16 Calculated Diameters of Zones of I n h i b i t i o n in the 73 A n t i b i o t i c Disk S e n s i t i v i t y Test for a Range of MIC Values. LIST OF TABLES (Continued) Identity of a Sample of 100 Enterobacteriaceae from Hospitals i n Vancouver (1980). A n t i b i o t i c Disk S e n s i t i v i t y Pattern of 1000 Enterobacteriaceae Isolated at Hospitals i n Vancouver (1980). Relationship Between Minimum Inhibitory Concen-t r a t i o n s of Mecillinam and the Zone Sizes Around 10 ug Mecillinam Disks for 246 Isolates of Enterobacteriaceae. Minimum Inhib i t o r y Concentrations of A m p i c i l l i n , Mecillinam and a 2:1 Combination Using the Agar D i l u t i o n Technique. Measurement of Minimum Inhib i t o r y Concentrations of A m p i c i l l i n , Mecillinam and a 2:1 Combination i n Mueller Hinton Broth and Urine i n M i c r o t i t r a t i o n Plates. Synergy Between A m p i c i l l i n and Mecillinam by the Disk Test. A c t i v i t y of A m p i c i l l i n , Mecillinam and a 2:1 Combination i n a Model of the Human Bladder. In vivo A c t i v i t y of A m p i c i l l i n , Mecillinam and a 10:1 Combination as Measured by Mouse Protection Tests. Relationship Between Synergistic Responses Observed i n the Urinary Bladder Model and Mouse Protection Tests. Screening Test f o r Beta-Lactamase A c t i v i t y i n C l i n i c a l Isolates of Enterobacteriaceae. Beta-Lactamase A c t i v i t y of Crude Extracts of C l i n i c a l Isolates of Enterobacteriaceae. Relation Between MIC's and A m p i c i l l i n Hydrolysis by Beta-Lactamases for 77 C l i n i c a l Isolates of Enterobacteriaceae. Relation Between MIC's'and Mecillinam Hydrolysis by Beta-Lactamases f o r 77 C l i n i c a l Isolates of Enterobacteriaceae. LIST OF FIGURES Figure Page 1 Flowchart of the continuous culture model of the 34 urinary bladder. 2- Zone s i z e d i s t r i b u t i o n of 999 Enterobacteriaceae 63 using 10 Ug mecillinam disks. 3 Zone s i z e d i s t r i b u t i o n of 999 Enterobacteriaceae 64 using 25 yg mecillinam disks. 4 D i s t r i b u t i o n of mecillinam resistance of 249 68 i s o l a t e s of Enterobacteriaceae (1978 Survey) 5 Correlation of 10 yg mecillinam s u s c e p t i b i l i t y 70 test disk zone diameter with the minimum i n h i b i t o r y concentration (1978 Survey). 6 D i s t r i b u t i o n of mecillinam resistance of 246 77 i s o l a t e s of Enterobacteriaceae (1980 Survey) 7 Correlation of 10 yg mecillinam s u s c e p t i b i l i t y 79 test disk zone diameter with the minimum i n h i b i t o r y concentration (1980 Survey). 8 Relation between MIC's and a m p i c i l l i n hydrolysis 100 by beta-lactamases for 77 c l i n i c a l i s o l a t e s of Enterobacteriaceae. 9 Relation between MIC's and mecillinam hydrolysis 101 by beta-lactamases f o r 77 c l i n i c a l i s o l a t e s of Enterobacteriaceae. ACKNOWLEDGEMENT I express appreciation to the members of my committee for reviewing t h i s t h e s i s . I am e s p e c i a l l y g r a t e f u l to Dr. J.D. Anderson f o r h i s guidance, Dr. J.J.R. Campbell f o r the f a c i l i t i e s placed at my disposal, and S o c i a l Sciences and Humanities Research Council of Canada for t h e i r f i n a n c i a l support. Thanks are also due to Mrs. M.Y. A i r d for technical assistance, Dr. B.J. Morrison for s t a t i s t i c a l help, and Dr. R. Cleeland and associates for animal studies. 1 I N T R O D U C T I O N Obj ectives The majority of i n f e c t i o n s of the urinary t r a c t are i n i t i a t e d by organisms introduced into the bladder from the d i s t a l urethra at catheter i z a t i o n (31, 82), and also i n women, at intercourse (24). The current dominant i n t e r e s t i n the r o l e of b a c t e r i a l adhesion i n pathogenesis has tended to obscure other p o t e n t i a l pathogenic mechanisms which could well be of equal or greater importance. Escherichia c o l i has been given p a r t i c u l a r attention i n t h i s thesis because i t i s the commonest cause of urinary i n f e c t i o n (44, 87, 141, 150). Despite the ascending nature of almost a l l urinary i n f e c t i o n s , E. c o l i i s s u r p r i s i n g l y seldom found at the dis.tal urethra (103) . The main thrust of t h i s thesis has been to test the hypothesis that d i f f e r e n t i a l growth rates of organisms i n t r o -duced into the bladder explains differences between the f l o r a of the d i s t a l urethra and organisms which are commonly found to cause c l i n i c a l l y recognized i n f e c t i o n s . Other investigators have shown that _E. c o l i grows equally well i n broth and urine (33, 60) and may in some in d i v i d u a l s grow s u f f i c i e n t l y r a p i d l y to overcome the tendency of urine to f l u s h organisms out of the urinary t r a c t . Although broth medium may be s a t i s f a c t o r y for growth studies with _E. c o l i , I have shown that other organisms may have quite d i f f e r e n t growth c h a r a c t e r i s t i c s in urine. Despite technical problems and lack of standardization, urine i s c l e a r l y a more r e a l i s t i c medium and should be chosen for growth studies. Much of the d i f f e r e n t i a l growth rate work i n t h i s thesis was c a r r i e d out i n a laboratory model of the human bladder which employed urine as medium and reproduced volume changes and flow c h a r a c t e r i s t i c s of the lower urinary t r a c t . The r e a l i s t i c response of bacteria i n pathogenicity studies i n the bladder model encouraged extension of i t s use to studies with a n t i b i o t i c s A n t i b i o t i c synergy studies present a severe challenge to in v i t r o test systems and the relevance of laboratory studies to therapeutic response i s sometimes uncertain. The bladder model was used to study synergy between a m p i c i l l i n and mecillinam. The value of the model was assessed by p a r a l l e l comparative studies with conventional i n v i t r o methods and a mouse i n f e c t i o n protection test (mouse studies were c a r r i e d out for me by Dr. R.C. Cleeland and h i s colleagues). The bladder model appeared to c l o s e l y mimic response in mouse tests and was far superior to con-ventional laboratory t e s t s . This dynamic model system which employed urine as medium appears to have applications i n the assessment of a n t i -b i o t i c s , or a n t i b i o t i c combinations intended for t r e a t i n g urinary i n f e c t i o n s . The mechanism of the i n t e r a c t i o n between mecillinam and a m p i c i l l i n i n k i l l i n g Enterobacteriaceae was investigated as a c o r o l l a r y to b a c t e r i a l v i a b i l i t y studies. An Overview of Urinary Tract Infections Infections of the urinary t r a c t are-common b a c t e r i a l i n f e c t i o n s which cause considerable morbidity and sometimes may be f a t a l . Urinary tr a c t i n f e c t i o n i s a broad term used to describe both c o l o n i z a t i o n of urine ( b a c t e r i u r i a ) , and invasion of structures i n any part of the urinary t r a c t . Infection may involve the kidneys (pyelonephritis), bladder ( c y s t i t i s ) , prostate ( p r o s t a t i t i s ) , urethra ( u r e t h r i t i s ) or may even s p i l l over into the blood stream (bacteremia, septicemia). Once urine, or any part of the t r a c t i s infected, the e n t i r e system i s at a r i s k of i n f e c t i o n . Infection may be with bacteria, yeasts, mycoplasma, viruses or protozoa, but t h i s thesis i s only concerned with ba c t e r i a . B a c t e r i a l i n f e c t i o n s may have the following manifestations (72, 62): 3. a. Symptomatic b a c t e r i u r i a : a condition i n which urine contains c l i n i c a l l y s i g n i f i c a n t numbers (defined below) of bacteria and where symptoms are present. Symptoms may include, dysuria (pain or a burning sensation on m i c t u r i t i o n ) , urgency, increased frequency, increased volume, nocturia (nocturnal frequency), enuresis (bedwetting), c h i l l s , malaise and fever. b. Asymptomatic b a c t e r i u r i a : a condition i n which urine contains s i g n i f i c a n t numbers of bacteria without symptoms. c. Symptomatic a b a c t e r i u r i a : where symptoms of i n f e c t i o n are present without s i g n i f i c a n t numbers of bac t e r i a . Such symptoms often resumble those associated with u r e t h r i t i s and are then known as the ure t h r a l syndrome. Urine flows over the perinea l and rel a t e d surfaces of the body and may become contaminated when voided. Kass (60) observed that 95% of in d i v i d u a l s with genuine urinary t r a c t i n f e c t i o n had 10^ organisms or more per ml urine i n two consecutive samples of midstream urine. Lesser counts were usually due to contamination. This observation provided a d e f i n i t i o n of b a c t e r i u r i a which has been accepted i n p r a c t i c e . Epidemiology of Urinary Tract Infections In f e c t i o n may be acquired i n two si t u a t i o n s where there are d i f f e r -ences i n both pathogenicity and response to treatment. a. Hospital acquired (nosocomial) i n f e c t i o n s b. Community acquired i n f e c t i o n s . The two groups d i f f e r i n terms of d i s t r i b u t i o n of b a c t e r i a l species, a n t i b i o t i c resistance patterns, and frequency of i n f e c t i o n . The ho s p i t a l environment may be a major source of bacteria i n urinary t r a c t i n f e c t i o n s (41, 134). A number of investigators have shown the importance of cross 4 i n f e c t i o n i n h o s p i t a l wards. B a c t e r i a l contamination of the hands of personnel, hand towels, f l u i d s f or bladder i r r i g a t i o n , b o t t l e s used for c o l l e c t i n g urine, bedpans, r e c t a l thermometers, l u b r i c a n t s for catheter i n s e r t i o n , and also dust and a i r from the ward atmosphere have been im-pl i c a t e d i n h o s p i t a l cross i n f e c t i o n s (38,64,91,104,105). These organisms may either be introduced d i r e c t l y into the urinary t r a c t or i n d i r e c t l y a f t e r c o l o n i z a t i o n of the patients skin or gut. Therefore, nosocomial i n f e c t i o n s are frequently caused by r e l a t i v e l y r e s i s t a n t organisms such as K l e b s i e l l a , Enterobacter, Pseudomonas, Proteus and S e r r a t i a species as well as Escherichia c o l i . These organisms are selected by the wide-spread use of a n t i b i o t i c s i n ho s p i t a l s . Such bacteria r a r e l y cause i n f e c t i o n i n the community. Investigators have also reported a higher prevalence of b a c t e r i u r i a i n h o s p i t a l i z e d patients compared to outpatients which has been a t t r i b u t e d to frequent instrumentation, the existence of a nosocomial f l o r a and susceptible patients (38,59,60,64,91,92,104,105). Community acquired i n f e c t i o n s on the other hand, are generally with les s r e s i s t a n t organisms found i n the community that reside i n the bowel, vagina and skin. Community patients are generally women and the evidence which demonstrates an asso c i a t i o n between sexual intercourse and i n f e c t i o n w i l l be reviewed elsewhere. The commonest organisms causing i n f e c t i o n s i n these women are _E. c o l i and Staphylococcus saprophyticus biotype 3. Individuals with i n f e c t i o n s of the urinary t r a c t may also be divided into two subgroups based on anatomic and physiologic features: a. Those with apparently normal urinary t r a c t who are not p a r t i c u l a r l y susceptible to i n f e c t i o n s . b. Those with functional and s t r u c t u r a l abnormalities who are more l i k e l y to acquire i n f e c t i o n and who tend to be ref r a c t o r y to treatment. 5 These abnormalities are generally associated with an increased r e s i d u a l volume of urine a f t e r the bladder empties. The re s i d u a l volume provides an inoculum of bac t e r i a and i s an important factor i n the establishment and maintenance of i n f e c t i o n . Examples of such abnormalities are: v e s i c o - u r e t h r i c r e f l u x , which i s the r e s u l t of backflow of urine into the ureters during m i c t u r i t i o n , or i n the res t i n g state; presence of renal c a l c u l i ; s p i n a l i n j u r i e s , or other conditions which impair the nerve supply to the bladder, may have the same e f f e c t ; u r e t h r i t i s or inflammation of urethra due to venereal i n f e c t i o n s or trauma; i n f e c t i o n or enlargement of the prostate i n men which obstructs the bladder outflow; deformities of the bladder wall by carcinoma; vaginal prolapse etc. Other conditions such as hypertension or diabetes may also contribute to establishment of urinary t r a c t i n f e c t i o n s . Age and Sex D i s t r i b u t i o n of Urinary Tract Infections Studies have shown that b a c t e r i u r i a i s more prevalent i n male neonates than females (eg. 3.6% vs. 0.9% (72)). However, symptomatic urinary t r a c t i n f e c t i o n s i n preschool c h i l d r e n are more frequent i n g i r l s than i n boys (0.8% vs. 0.2%) (34)) and the frequency increases with age i n g i r l s . Overall based on data drawn from several studies, the prevalence of b a c t e r i u r i a (frequency at any one point i n time) has been shown to be around 0.03% in school boys i n comparison with 1.2% i n g i r l s (65, 66, 68, 69, 71). In adult women b a c t e r i u r i a i s generally accepted to be associated with sexual a c t i v i t y (24). A comparative study with nuns, single and married women has shown that the frequency of b a c t e r i u r i a i n nuns i s s t r i k i n g l y l e s s than the other two groups. B a c t e r i u r i a was found i n 0.4% of nuns, 2.7% of sing l e women and 5.9% i n married women between the age of 15 and 24 (70). The frequency of i n f e c t i o n r i s e s by approximately 1 or 2% per 6 decade for s i n g l e and married women so that up to 10% of women aged 55-65 may be b a c t e r i u r i c whereas, i n nuns i t only reaches 1.6% by the age of 55 (61). In older women factors such as s t r u c t u r a l abnormalities also con-t r i b u t e to i n f e c t i o n . One example i s prolapse, where the bladder f a l l s downward and backwards into the vaginal vault causing an increase i n the re s i d u a l volume. Kass (61) has documented a prevalence of b a c t e r i u i r a between 10 and 15% i n women over the age of 60. Pregnant women are a sub-group of young women at a high r i s k of i n f e c t i o n (5 to 6.5%) (97), perhaps due to s t r u c t u r a l and func t i o n a l changes. During pregnancy the uterus increases i n s i z e and exerts pressure which d i s t o r t s the bladder and i n -creases the re s i d u a l volume. There have been suggestions that hormones released during pregnancy relax smooth muscle and may predispose to u r e t r i c r e f l u x . Furthermore, urine from pregnant women has been shown to permit f a s t e r growth of _E. c o l i than that from non-gravid women (14, 15). Urinary t r a c t i n f e c t i o n s i n adult men are rare u n t i l ; t h e age of 50. The major predisposing factor a f t e r t h i s age i s p r o s t a t i c hypertrophy which again increases the r e s i d u a l volume by d i s t o r t i n g the base of the bladder and r e s t r i c t i n g u r e t h r a l flow. Organisms Responsible for Urinary Tract Infections Most urinary t r a c t i n f e c t i o n s are due to Gram negative aerobic b a c i l l i found i n the gut. The most common agents are members of the Entero- bacteriaceae and of t h i s Family E, c o l i predominates despite the f a c t that i t i s r e l a t i v e l y uncommon i n the area of the urethral o r i f i c e (103). Anaerobic f e c a l f l o r a , although over one thousand f o l d more common in the s t o o l , i s r a r e l y involved (73). Table 1 l i s t s an approximate d i s t r i b u t i o n of organisms involved i n urinary i n f e c t i o n s . The Table was prepared using pooled data from a number of sources (44, 87, 141, 150). Table 1: * D i s t r i b u t i o n of organisms causing urinary t r a c t i n f e c t i o n s i n community and h o s p i t a l i z e d patients. Organism Percent i n Community Hospital Escherichia c o l i 56.0 35.1 Staphylococcus saprophyticus biotype 3 13.4 1.1 Staphylococcus epidermidis 3.0 5.5 Staphylococcus aureus 1.3 1.9 K l e b s i e l l a , Enterobacter spp. 6.5 13.2 Proteus spp. 7.0 14.8 Streptococcus f e c a l i s 5.8 8.75 Pseudomonas spp. 1.4 3.1 S e r r a t i a spp. 0.0 2-7 Others ** 5.6 10.45 Wide v a r i a t i o n s have been observed between surveys. These figures are unweighted means of four published papers (141, 44, 87, 150). * Others include yeasts and non-bacterial urinary t r a c t i n f e c t i o n s . 8. E s c h e r i c h i a c o l i i s found i n a higher percentage of community acquired i n f e c t i o n s than nosocomial i n f e c t i o n s whereas organisms such as Proteus, Pseudomonas, K l e b s i e l l a , Enterobacter and S e r r a t i a spp. are r e l a t i v e l y more common i n h o s p i t a l i z e d p a t i e n t s (38, 64, 91, 104, 105). Gram p o s i t i v e organisms are much l e s s common than Gram negative b a c t e r i a . However, S^. saprophyticus biotype 3 i s the second most common cause of acute u r i n a r y t r a c t i n f e c t i o n s i n the community, e s p e c i a l l y i n younger women (10, 36, 58, 80, 121). By co n t r a s t ^. epidermidis i s the predominant i n f e c t i n g staphylococcus i n h o s p i t a l p a t i e n t s and i s found i n both males and females (58). M i c r o b i a l Factors which Contribute to the Establishment of Urinary Tract  I n f e c t i o n s . Adherence and c o l o n i z a t i o n of host t i s s u e s by the i n f e c t i n g organism i s most l i k e l y the f i r s t step i n the pathogenesis of i n f e c t i o n at most s i t e s (123) . The a b i l i t y of _E. c o l i c e l l s to adhere to e p i t h e l i a l c e l l s may be a d e c i s i v e v i r u l e n c e f a c t o r . In l a b o r a t o r y t e s t s , E. c o l i s t r a i n s which were i s o l a t e d from p a t i e n t s w i t h acute p y e l o n e p h r i t i s were shown to adhere more r e a d i l y to human u r o e p i t h e l i a l c e l l s than b a c t e r i a from p a t i e n t s w i t h asymptomatic b a c t e r i u r i a or the feces from healthy persons (135-137). Two mechanisms have been suggested f o r adherence. F i r s t l y , adherence mediated by type I p i l i which i s i n h i b i t e d by mannose. This has been shown both i n v i t r o and i n a mouse model (13, 100). Secondly, mannose r e s i s t a n t adherence which i s l i k e l y mediated by other than type I p i l i . These p i l i resemble the s p e c i f i c v i r u l e n c e a s s o c i a t e d p i l i such as K88, K99, CFA/II ( c o l o n i z a t i o n f a c t o r antigens) which are r e s p o n s i b l e f o r mannose r e s i s t a n t hemagglutination of erythrocytes of v a r i o u s species (56, 25, 53). 9 Colonization of the d i s t a l urethra may also be a permissive factor i n promoting i n f e c t i o n (133). It has been demonstrated that a p o s i t i v e c o r r e l a t i o n exists between the degree of c o l o n i z a t i o n of the vaginal i n t r o i t u s with _E. c o l i and the b a c t e r i a l count i n urine (81). I t was also shown that women who p e r s i s t e n t l y carry enterobacteria at the u r e t h r a l v e s t i b u l e develop urinary i n f e c t i o n more frequently than those who carry them i n t e r m i t t e n t l y or not at a l l (107). Nevertheless,, a simple r e l a t i o n s h i p does not exist between c o l o n i z a t i o n and i n f e c t i o n . For example _E. c o l i which i s the major cause of urinary t r a c t i n f e c t i o n s , i s uncommon at the d i s t a l urethra, whereas anaerobes which are present i n high numbers r a r e l y cause i n f e c t i o n . A number of other organisms such as Lacto-b a c i l l u s spp., S. epidermidis, Corynebacterium spp., hemolytic Streptococci as well as anaerobes have been cultured from the urethra of healthy females (79) . These organisms have been speculated to be part of the natural defence mechanism by competing with Gram negative rods for adherence to e p i t h e l i a l c e l l s (79) and perhaps for a v a i l a b l e n u t r i e n t s . Organisms which produce urease (eg. Proteus spp. in humans, and Corynebacterium renale i n bovines) s p l i t urea to ammonia which i s t o x i c f o r the tissues and inactivates the fourth component of complement (12, 63). It has been shown that the presence of urease i n h i b i t o r s such as hydroxyurea and thiourea considerably reduces the experimental ascending pyelonephritis by Proteus m i r a b i l i s i n mice (12) . These drugs prevent the establishment of i n f e c t i o n but do not eliminate e x i s t i n g i n f e c t i o n s . It has also been demon-strated that b a c t e r i a l urease plays a prominent r o l e i n the 10 formation of urinary stones by r a i s i n g the urinary pH. S t r u v i t e MgNH^PO^, 6 1^0), and carbonate apatite (Ca-^CPO^)CO^) stones are formed i n i n f e c t i o n s due to Proteus spp., but not _E. c o l i , Pseudomonas, or K l e b s i e l l a i n f e c t i o n s (48). The a b i l i t y of organisms to produce hemolysins may be another factor influencing v i r u l e n c e . Hemolytic _E. c o l i have been found to be responsible for the majority of i n f e c t i o n s in the urinary t r a c t , e s p e c i a l l y the upper t r a c t i n f e c t i o n s (30, 37, 89). Bacteria which are capable of growing r a p i d l y i n urine may have a better chance of causing i n f e c t i o n i n the urinary t r a c t . Urine supports the growth of E. c o l i and other urinary pathogens (14, 15). Relative growth rates of bacteria i n urine could change the dominant population during growth in mixed cultures introduced into the bladder, for example, at intercourse or by c a t h e t e r i z a t i o n . This aspect of the pathogenesis of i n f e c t i o n forms a dominant part of t h i s t h e s i s . Host Factors i n the Pathogenesis of Urinary Tract Infections. It i s generally accepted that urinary t r a c t i n f e c t i o n s a r i s e by the ascending route with organisms from the d i s t a l urethra, bowel, vagina or perineum (20, 148). Bacteria are thought to be introduced into the bladder l a r g e l y by mechanical means during intercourse or possibly by phagocytosis or brownian movement. Descending i n f e c t i o n s are thought to be rare but may s t a r t i n the kidneys which have a r i c h blood supply and receive one quarter of the cardiac output at r e s t . Thus, any systemic b a c t e r i a l i n f e c t i o n can lead to seeding of the kidneys with bacteria, eg. staphylococcal bacteremia (72). Organisms are commonly introduced into the bladder at i n t e r -course (24), by passage of a catheter< (31, 82) or in other mechanical ways. However, c l i n i c a l l y recognizable i n f e c t i o n seldom occurs due to i n t e r n a l clearance mechanisms i n most i n d i v i d u a l s . The clearance mechanisms are: General defence mechansisms S p e c i f i c antibodies towards known serotypes of E. c o l i have been found i n the urine and serum of subjects with urinary t r a c t i n f e c t i o n s (153, 107, 151, 23). Immunofluorescent techniques have shown that immunocytes i n the bladder wall are capable of producing s p e c i f i c antibodies as well as c e l l s from spleen, lymph nodes and renal p a p i l l a (124) . There was also a concurrent increase i n poly-morphonuclear c e l l s i n these patients. Antibody coated bacteria (ACB) have been found in the urine of patients with urinary t r a c t i n f e c t i o n s and a f a i r l y good c o r r e l a t i o n e x i s t s i n adults between the presence of ACB and the c l i n i c a l diagnosis of upper urinary t r a c t i n f e c t i o n (57, 120, 142). Non-specific 0. antibodies ( a n t i -bodies against somatic antigens of Enterobacteriaceae) may also be present i n sera and urine of subjects with urinary t r a c t i n f e c t i o n s (29, 149). Such antibodies have been referred to as natural a n t i -bodies, in contrast with s p e c i f i c antibodies which are active against i n d i v i d u a l b a c t e r i a l serotypes. Phagocytosis by polymorphs has also been observed i n the bladder wall of r a t s infected with _E. c o l i within only two hours of i n f e c t i o n . The death and disappearance of organisms from the bladder wall were at t r i b u t e d to an acute inflammatory response (28). Mucosal a n t i b a c t e r i a l a c t i v i t y has been postulated by a number of i nvestigators (33, 43, 96). Support for t h i s view i s provided i n one study i n which r a t s were rendered leucopenic by i r r a d i a t i o n and b a c t e r i a were then inoculated into the bladder w a l l . Organisms were k i l l e d even though the rats were leucopenic (43) . A further i n v e s t i g a t i o n i n mice showed that bacteria which were not removed by voiding were k i l l e d a f t e r mucosal attachment (96) . Phagocytosis was again not observed i n t h i s study. Hydrokinetic clearance mechanisms These mechanisms can best be understood i f a d i s t i n c t i o n i s made between m u l t i p l i c a t i o n of organisms occuring i n the upper t r a c t (ureter and kidney), or lower t r a c t (bladder and urethra). Conditions i n the upper t r a c t correspond with a conventional laboratory continuous culture system i n which f r e s h medium (urine) i s constantly supplied and the culture i s drained at the same rate to maintain the volume of the system constant (101). Conditions i n the bladder where urine i s introduced continuously but emptying i s intermittent are much more complicated (102, 78, 32, 77, 3, 7, 52). B a c t e r i a l populations i n the bladder are influenced by a number of physical f a c t o r s . The greater the flow rate of urine, the greater the d i l u t i o n e f f e c t on b a c t e r i a l populations. Individuals with a normal bladder have a r e s i d u a l volume of 1 ml or le s s whereas in those prone to urinary t r a c t i n f e c t i o n s the r e s i d u a l volume i s generally higher. An increase in r e s i d u a l volume reduces the e f f i c i e n c y of the f l u s h i n g e f f e c t and provides a higher inoculum each time the bladder i s emptied. This i s a c r u c i a l f a c t o r i n the establishment of urinary t r a c t i n f e c t i o n s (77) . The longer the 13 i n t e r v a l between m i c t u r i t i o n , the greater the opportunity for b a c t e r i a l populations to increase (77). F i n a l l y the growth rate of organisms i n urine may be an important factor i n the establishment of i n f e c t i o n . Faster growing organisms are more l i k e l y able to overcome the f l u s h i n g e f f e c t (3, 7). A primary aim of t h i s d i s s e r t a t i o n i s to prove that d i f f e r e n t i a l b a c t e r i a l growth rates may explain the epidemiology of urinary i n f e c t i o n s . The use of a bladder model has f a c i l i t a t e d exploration of i n t e r a c t i o n s between b a c t e r i a l growth and the tendency of urine flow to f l u s h organ-isms out of the t r a c t . A review of the Use of "Bladder Models" in the Pathogenesis and Treatment  of Urinary Infections. It i s highly desirable to choose an experimental system which re -produces 'in vivo' conditions when in v e s t i g a t i n g i n f e c t i o n processes. This i s p a r t i c u l a r l y so with i n f e c t i o n s of the urinary t r a c t , where a dynamic system e x i s t s and a v a r i e t y of f a c t o r s influence b a c t e r i a l m u l t i -p l i c a t i o n simultaneously. Therefore, a model which simulates conditions i n the urinary bladder i s pertinent to studies of factors which a f f e c t the outcome of i n f e c t i o n by organisms. There have been a number of previous models of the lower urinary t r a c t which are somewhat i n f e r i o r to the model used for these studies. Cox and Hinman (33) used a system i n which a s t e r i l e f l a s k served as a bladder. A healthy male subject voided d i r e c t l y into a f l a s k and the urine was inoculated with E. c o l i . The f l a s k was subsequently incubated at 37°C and was drained each time the volunteer required to void. A r e s i d u a l volume remained i n the f l a s k in the form of a f i l m of l i q u i d containing b a c t e r i a which was s i m i l a r to the r e s i d u a l volume of 14 a normal bladder. Fresh urine was then added to the f l a s k by the same subject and the procedure repeated at i n t e r v a l s . This model was used to study changes of b a c t e r i a l populations as a r e s u l t of the flushing e f f e c t of urine but had disadvantages. The system did not use a continuous flow of urine and a large single volume of urine was added rather than small amounts as i n the bladder. It was inconvenient for the volunteer, and was p o t e n t i a l l y prone to contamination. Also i t could not be used for female subjects because of contamination of urine by perineal and i n t r o i t a l f l o r a . O'Grady and Greenwood (47) produced a model which was more s o p h i s t i -cated. This model consisted of a f l a s k representing the bladder which was maintained at 37°C. Nutrient broth was added to the f l a s k at a con-t r o l l e d rate, and emptying was c o n t r o l l e d by an automatic timer at set i n t e r v a l s . B a c t e r i a l populations were monitored spectrophotometrically. Although the model simulated the hydrokinetics of the human bladder, there were disadvantages. F i r s t l y , n utrient broth was used in place of urine. The growth c h a r a c t e r i s t i c s of _E. c o l i i n urine are very s i m i l a r to i t s growth i n nutrient broth (33, 60), but as i t w i l l be shown i n t h i s t h e s i s , other urinary pathogens show d i f f e r e n t growth c h a r a c t e r i s t i c s in urine than broth. Therefore^ nutrient broth i s not a s u i t a b l e medium to study urinary pathogens other than _E. c o l i . Secondly the turbidometric techniques i s not s e n s i t i v e enough for determination of growth e s p e c i a l l y when small numbers of bacteria are present (<10^). Furthermore, the b a c t e r i a l popu-l a t i o n s do not c o r r e l a t e well-with the t u r b i d i t y of suspensions, e s p e c i a l l y i n the presence of a n t i b i o t i c s . Drugs which i n h i b i t the d i v i s i o n of b a c t e r i a may cause formation of large c e l l s (e.g. mecillinam '54, 74, 96 ), thus a f f e c t i n g the t u r b i d i t y of the culture and producing misleading r e s u l t s . 15 The bladder model, developed by Anderson, et a l (6, 7, 8), has been used for much of the work in t h i s d i s s e r t a t i o n . This model reproduces some of the hydrodynamic features of the lower urinary t r a c t which determines the course of the early stages of establishment of i n f e c t i o n (see Materials and Methods for d e t a i l s ) . Urine was used as culture medium i n t h i s model and b a c t e r i a l populations were determined by a surface plate method which could detect as l i t t l e as 200 cfu's per ml urine. One of the major goals of t h i s thesis was to investigate the contribution of growth rates to b a c t e r i a l pathogenicity. This was f i r s t approached by comparing growth rates of urinary i s o l a t e s i n urine and in broth in shake culture.: Growth c h a r a c t e r i s t i c s of bacteria other than E. c o l i were found to be quite d i f f e r e n t in urine and broth. Despite the p r a c t i c a l problems, urine was chosen as a medium. Patho-genic mechanisms were studied under r e a l i s t i c conditions i n t h i s model using mixed cultures to investigate phenomena such as: d i f f e r e n t i a l growth rates, a b i l i t y to metabolize nutrients i n urine, and production of a n t i b a c t e r i a l substances such as c o l i c i n s , toxins, etc. The bladder model simulates hydrokinetic clearance mechanisms operating i n natural i n f e c t i o n . A n t i b i o t i c concentration and m i l i e u may also be controlled to study in t e r a c t i o n s between bacteria and a n t i b i o t i c s under 'therapeutic' conditions. P r a c t i c a l and Theoretical Aspects of the Treatment of Urinary Tract  Infections. Choice and assessment of the value of a n t i b i o t i c s should take account of the s i t e and nature of i n f e c t i o n since response i s very much dependent on the patient. For t h i s reason, urinary t r a c t i n f e c t i o n s may be divided into three groups (108): 16 a. Uncomplicated acute (medical) Infections which are defined as urinary t r a c t i n f e c t i o n s i n which no underlying s t r u c t u r a l or neurological l e s i o n s are present. These are the commonest in f e c t i o n s seen i n females. The p r i n c i p l e s of therapy are si m i l a r f o r asymptomatic b a c t e r i u r i a , acute c y s t i t i s or acute pyelonephritis. A l l tend to respond well to a short term chemotherapy, e s p e c i a l l y c y s t i t i s which i s li m i t e d to the lower t r a c t . b. Complicated (surgical) acute i n f e c t i o n s , are si t u a t i o n s i n which the t r a c t has been invaded by bacteria repeatedly, leaving r e s i d u a l i n -flammatory changes, or when obstruction, stones or neurological l e s i o n s i n t e r f e r with drainage from the t r a c t . Complicated i n f e c t i o n s commonly p e r s i s t even when the bacteria are se n s i t i v e to a n t i b i o t i c s i n laboratory tests unless the obstruction i s cleared or voiding abnormalities corrected. c. Chronic urinary t r a c t i n f e c t i o n s where i n f e c t i o n has been present for more than a few weeks. The i n f e c t i n g organisms are sometimes very r e s i s t a n t to anti m i c r o b i a l agents. Demonstrable s t r u c t u r a l abnormalities or pathological changes i n tissues may or may not be present (108). One problem i n the management of uncomplicated urinary t r a c t i n f e c t -ions i n females i s the high rate of recurrence that may be due to a relapse or r e i n f e c t i o n . Relapse i s defined as a rapid recurrence of i n f e c t i o n with the same organism present before therapy. Reinfection denotes recurrence with the same or d i f f e r e n t organisms (67, 84). It has been demonstrated that relapses i n women more l i k e l y respond favour-ably to a longer course of chemotherapy whereas the outcome of r e i n f e c t i o n i s not influenced by duration of treatment (143). Furthermore, women with relapsing b a c t e r i u r i a frequently have i n f e c t i o n i n the upper t r a c t , whereas r e i n f e c t i o n s are more common i n patients whose i n f e c t i o n i s confined to the bladder (144, 145). The d i f f e r e n t i a t i o n of renal and bladder i n f e c t i o n s i s also very 17 important for therapeutic purposes. However, i t i s not possible to l o c a l -i z e i n f e c t i o n on c l i n i c a l grounds alone, e s p e c i a l l y in asymptomatic patients (84). Therefore, other diagnostic methods are needed, examples are: a. B i l a t e r a l u r e t e r a l c a t h e t e r i z a t i o n through a cystoscope, which i s a d i r e c t method. However, t h i s method can not be j u s t i f i e d for the routine evaluation of patients with recurrent b a c t e r i u r i a (132, 144). b. Bladder washout method, i n which bacteria are eradicated by i r r i g a t i o n of the bladder with the b a c t e r i c i d a l a n t i b i o t i c such as kanamycin. The kanamycin i s then removed from the bladder by i r r i g a t i o n and urine samples obtained subsequently to detect organisms coming from the upper t r a c t . Urine samples are then cultured at timed i n t e r v a l s to detect b a c t e r i u r i a o r i g i n a t i n g i n the infected upper tr a c t (39). c. I t has been claimed that serum agglutinating antibodies are r a i s e d i n the upper t r a c t i n f e c t i o n s . The test which i s c a r r i e d out using several d i l u t i o n s of the patients serum and organisms i s o l a t e d from the urine has proved u n r e l i a b l e (132). d. An i n d i r e c t study method i s based on the p r i n c i p l e that bacteria often become coated with antibody in upper but not lower t r a c t i n f e c t i o n s . Such bacteria may be detected i n urine by a fluorescent antibody technique. This method i s p o t e n t i a l l y the most convenient screening test but i s u n r e l i a b l e i n p r o s t a t i c i n f e c t i o n and p a r t i c u l a r l y in children (35, 42, 51, 122). Factors Influencing the Choice of A n t i b i o t i c s for Urinary Infections. A n t i b i o t i c s are used to a i d the natural clearance mechanisms by k i l l i n g or reducing the growth rate of b a c t e r i a . Response to an a n t i b i o t i c may d i f f e r i n vivo and i n v i t r o f or the following reasons: 18 Urinary drug concentrations are more important than plasma l e v e l s . A n t i b i o t i c s which are excreted i n urine are l i k e l y to be p a r t i c u l a r l y e f f e c t i v e . For example 60% of an o r a l dose of a m o x i c i l l i n i s excreted to give urinary concentrations i h excess of 500 yg/ml (16). A c t i v i t y of a n t i b i o t i c s i n urine depends upon several factors as well as concentration. The pH of urine i s generally between 4.6 and 7.25 (15) but usually acid and i n h i b i t s anionic drugs such as gentamicin, erythromycin and t e t r a c y c l i n e . Gentamicin i s 40 times l e s s a c t i v e i n urine than at the pH of laboratory media (88). Some a n t i b i o t i c s tend to be more active i n urine than laboratory media (eg. s u l f a drugs). These drugs have a s y n e r g i s t i c e f f e c t with urea which may enhance t h e i r a c t i v i t y i n urine (93). Osmolality of the medium also influences a c t i v i t y . For example with a n t i b i o t i c s which act on the c e l l wall (eg. p e n i c i l l i n s , cephalosporins), a population of susceptible bacteria might be protected when osmolality i s high. E l e c t r o l y t e content may also be important e s p e c i a l l y with amino-glycosides and mecillinam. The growth phase of bacteria may influence response to a n t i b i o t i c s . As an example, p e n i c i l l i n s predominantly act on exponentially growing c e l l s . In such cases, " s e n s i t i v e " bacteria may appear r e s i s t a n t i n the stationary phase of growth. The choice of an a n t i b i o t i c also depends upon pharmacological factors such as the route of administration, adsorption from the gut, urine and serum concentration, t o x i c i t y and side e f f e c t s . Unfortunately no single drug i s i d e a l . Among the a n t i b i o t i c s , p e n i c i l l i n s and cephalosporins (beta-lactam a n t i b i o t i c s ) are considered r e l a t i v e l y safe because of t h e i r minimal toxic e f f e c t s and high therapeutic index. However, these drugs are not usually prescribed for i n d i v i d u a l s who are a l l e r g i c to p e n i c i l l i n s . A l t e r n a t i v e drugs for urinary i n f e c t i o n s include the s u l f a drugs, t e t r a -19 c y c l i n e s , n i t r o f u r a n t o i n , trimethoprim, or a combination of s u l f a drugs and trimethoprim. The Properties of Mecillinam. Mecillinam (previously c a l l e d FL 1060), i s a semisynthetic beta-lactam a n t i b i o t i c . It d i f f e r s from other beta-lactams i n having an amidino linkage instead of an amino l i n k between the side chain and the beta-lactam nucleus (shown below). The alt e r e d structure has resulted in d ifferences i n i t s m i c r o b i o l o g i c a l a c t i v i t y . Mecillinam i s 100 times more ac t i v e against Gram negative bacteria and r e l a t i v e l y i n e f f e c t i v e against Gram p o s i t i v e organisms compared with a m p i c i l l i n (76). Since mecillinam i s poorly absorbed from the gut aft e r o r a l administration, an ester of mecillinam (pivaloyloxymethyl ester or pivmecillinam) i s used for o r a l use. Pivmecillinam i s hydrolyzed by non-specific esterases i n the blood and l i v e r to y i e l d free mecillinam i n the serum (118, 152). Trimethyl a c e t i c a c i d and formaldehyde are also released as the r e s u l t of the reaction. Mecillinam i s mainly excreted i n the urine (118). •COOH B e n z y l p e n i c i l l i n 0 H H .COOH Mecillinam 0 Pivmecillinam 0 H 20 The a c t i v i t y of mecillinam i n v i t r o i s more affected by growth conditions than other beta-lactam a n t i b i o t i c s . Factors such as: inoculum s i z e , osmolality and conductivity of the medium may have a profound influence on the a c t i v i t y of t h i s a n t i b i o t i c . With some organisms, v a r i a t i o n i n these factors have been observed to produce up to a 100-fold change in minimum i n h i b i t o r y concentration measurements (112). Studies have shown that mecillinam converts E_. c o l i c e l l s into large s p h e r i c a l forms which are osmotically r e s i s t a n t . D i v i s i o n i s i n h i b i t e d and l y s i s of c e l l s occurs a f t e r several hours of abnormal growth i n the presence of mecillinam (54, 76, 95). Differences between the p h y s i o l o g i c a l response of E_. c o l i to mecillinam and other beta-lactam a n t i b i o t i c s suggest a d i f f e r e n t mode of action by mecillinam (45, 106). Beta-lactam a n t i b i o t i c s normally i n t e r f e r with b a c t e r i a l growth by i n h i b i t i n g enzymes involved i n the terminal stages of peptidoglycan metabolism (D-alanine carboxypeptidase, peptidoglycan transpeptidase and endopeptidase (21)) by binding to s p e c i f i c proteins. These p e n i c i l l i n binding proteins (PBP's) are located i n the cytoplasmic membrane and 7 PBP's have been detected i n E. c o l i by l a b e l l i n g the membranes with 14 C - p e n i c i l l i n (21, 125-127). Genetic and p h y s i o l o g i c a l studies have i d e n t i f i e d PBP's l b , 2 and 3 as the targets with which beta-lactam a n t i b i o t i c s i n t e r a c t to produce t h e i r l e t h a l e f f e c t s (131). A l l peni-c i l l i n s and cephalosporins previously studied, bind to a l l , or most of the PBP's, whereas mecillinam s p e c i f i c a l l y binds only PBP 2. Mecillinam also f a i l s to i n h i b i t cross l i n k i n g enzymes in the membrane (83, 128, 129). PBP 2 has been shown to be a minor component of the cytoplasmic a, membrane of _E. c o l i where there may be only 2 0 molecules present (130). 14 Furthermore, i t has been demonstrated that when both C - p e n i c i l l i n 14 and C-mecillinam were permitted to simultaneously bind to E. c o l i 21-membranes, l i t t l e competition occurred (129). Mecillinam a c t i v i t y has been studied against a wide v a r i e t y of Gram negative b a c i l l i including Salmonella typhimurium, K l e b s i e l l a pneumoniae and Pseudomonas aeruginosa (128). Mecillinam binding proteins of these bacteria almost c e r t a i n l y corresponded i n function to PBP 2 i n _E. c o l i since s p e c i f i c binding to mecillinam was associated with conversion to round c e l l s . Mecillinam resistance i s less common than a m p i c i l l i n resistance i n c l i n i c a l i s o l a t e s of Enterobacteriaceae, although figures vary i n d i f f e r e n t studies (2, 4, 46,: 76, 83, 146). Slow growing, mecillinam r e s i s t a n t phenotypic variants may a r i s e jm v i t r o when Enterobacteriaceae are grown under c e r t a i n conditions i n the presence of mecillinam. These variants lose t h e i r resistance when subcultured i n the absence of mecillinam (2). However, t h i s does not exclude the p o s s i b i l i t y that they may be a p o t e n t i a l cause of treatment f a i l u r e i n vivo. To date only one f a i l u r e has been f i r m l y shown to be associated with r e s i s t a n t variants (18). These variants grow more slowly in broth and urine and are more susceptible to phagocytosis and antibodies (5, 18). This may explain t h e i r apparent unimportance as a cause of treatment f a i l u r e . However, few investigators have rigorously sought to i d e n t i f y these v a r i a n t s . Gram negative b a c t e r i a become r e s i s t a n t to beta-lactam a n t i b i o t i c s including mecillinam by two main processes acting separately or in com-bination (114): a. Destruction of a n t i b i o t i c by beta-lactamases which can open the beta lactam r i n g of p e n i c i l l i n s and cephalosporins (110, 113, 140, 124) (see below). b. Exclusion of the a n t i b i o t i c s from t h e i r targets within the b a c t e r i a l c e l l s . 22 Resistance i n Gram negative bacteria i s often complex and may r e s u l t from a combination of decreased drug permeability and enzyme i n a c t i v a t i o n . For t h i s reason, i n some Gram negative b a c i l l i , the degree of a n t i b i o t i c resistance correlates with the quantity of beta-lactamases synthesized, while i n others the two show l i t t l e c o r r e l a t i o n (98, 113, 119). An Overview of Beta-Lactamasesof Gram Negative Bacteria P e n i c i l l i n a s e s of E. c o l i were discovered as early as 1941 (1). Two decades l a t e r an enzyme was i s o l a t e d from Enterobacter cloacae, which inact i v a t e d cephalosporin C and was c a l l e d cephalosporinase (40). Today, a wide v a r i e t y of beta-lactamases are known to be present i n many b a c t e r i a l species, probably due to the a b i l i t y of bacteria to exchange genetic information (85, 140). Beta-lactamases were o r i g i n a l l y c l a s s i f i e d into 5 main classes based on the determination of substrate preference and s u s c e p t i b i l i t y to beta-lactamase i n h i b i t o r s (113). The character-i s t i c s of these classes are: Class I Enzymes predominantly a c t i v e against cephalosporins; usually i n h i b i t e d by c l o x a c i l l i n and c a r b e n i c i l l i n and r e s i s t a n t to i n h i b i t i o n by p-chloromercuribenzoate (PCMB). Class II Enzymes predominantly a c t i v e against p e n i c i l l i n s ; usually i n h i b i t e d by c l o x a c i l l i n , but not c a r b e n i c i l l i n or PCMB. Class III Enzymes with approximately equal a c t i v i t y against p e n i c i l l i n s and cephalosporins; usually i n h i b i t e d by c l o x a c i l l i n but not by c a r b e n i c i l l i n or PCMB. Class IV Enzymes with approximately equal a c t i v i t y against p e n c i l l i n s and cephalosporins; usually i n h i b i t e d by PCMB but not with c l o x a c i l l i n . Class V Enzymes predominantly a c t i v e against p e n i c i l l i n s including c l o x a c i l l i n ; i n h i b i t e d by PCMB. Each class has several enzyme types with d i f f e r e n t substrate p r o f i l e s (140). More beta-lactamases are being characterized as new p e n i c i l l i n s and cephalosporins are introduced into c l i n i c a l use and i t i s l i k e l y that other c l a s s i f i c a t i o n schemes w i l l be needed. The majority of beta-lactamases from Gram-negative bacteria are c e l l bound, c o n s t i t u t i v e , and produced in smaller quantities than those produced by Gram p o s i t i v e organisms (140). Although the above scheme i s convenient, simple and widely accepted there appears to be a very wide and varied spectrum of beta-lactams (Sykes and Matthews, 140). Synergy Studies with Mecillinam The term synergy i s derived from the Greek word 'synergos' and l i t e r a l l y means to work together. It i s now commonly used to describe an i n t e r a c t i o n of two or more drugs i n which the e f f e c t produced by the drugs i n combination i s greater than the sum of t h e i r i n d i v i d u a l e f f e c t s (55). Moellering (90) has described synergy as an 'elusive concept' because of the influence of the experimental m i l i e u upon a n t i b i o t i c i n t e r a c t i o n s . Discrepancies are p a r t i c u l a r l y l i k e l y i n extrapolating from jin v i t r o experiments to animal and human studies. Synergy can not be e a s i l y quantified and d e f i n i t i o n s of synergy are a r b i t r a r y . Synergy between mecillinam and other beta-lactam a n t i b i o t i c s has been studied both in v i t r o (94, 147), and i n vivo (27, 49, 50). The mechanisms by which these a n t i b i o t i c s work together are not yet known. It i s possible that mecillinam binds to beta-lactamases and prevents destruction of the second a n t i b i o t i c ( a m p i c i l l i n ) . Mecillinam may 24 penetrate bacteria more e f f e c t i v e l y than a m p i c i l l i n , i n i t i a t e disruption of the c e l l wall and thus enhance the d i f f u s i o n of a m p i c i l l i n to s e n s i t i v e s i t e s (115). A t h i r d mechanism may be that a n t i b i o t i c s with an a f f i n i t y f o r d i f f e r e n t PBPs i n the c e l l membrane could have a better chance of exerting b a c t e r i c i d a l e f f e c t on the organism faster than each .drug alone. The use of the drug combination may i n h i b i t the growth of organisms r e s i s t a n t to either agent used sin g l y . Combination therapy with a m p i c i l l i n and mecillinam may permit reduction i n the dose of each drug, and consequent reduction of side e f f e c t s . Also the p o t e n t i a l wide spectrum of a m p i c i l l i n and mecillinam may spare the use of p o t e n t i a l l y toxic drugs such as aminoglycosides. The problem was investigated by comparing the response of a sample of ten organisms i n a v a r i e t y of conventional ±n v i t r o systems, i n the bladder model and i n a mammalian model. MATERIALS AND METHODS Organisms Most organisms were obtained from in-patients at three ho s p i t a l s i n Vancouver, B r i t i s h Columbia (Vancouver General, Shaughnessy, and St. Paul's H o s p i t a l s ) , and also out-patients at the Health Centre of the University of B r i t i s h Columbia. The i d e n t i f i c a t i o n of Gram negative Enterobacteriaceae was car r i e d out using the API 20E System (API Laboratory Products Ltd., St. Laurent, P.Q.). The system consists of microtubes on a s i n g l e s t r i p which perform 23 standard biochemical tests from a s i n g l e b a c t e r i a l colony. Generally a s i n g l e colony was suspended i n s a l i n e (5 ml) and the suspension was used to r e -constitute the dehydrated media i n the microtubes. The API s t r i p s were then incubated at 37°C f o r 18-24 h and the test r e s u l t s were recorded. An oxidase test was performed on a l l Gram negative lactose non-fermenting organisms to exclude non Enterobacteriaceae Staphylococcus s t r a i n s were tested f o r coagulase reaction by the tube test and coagulase negative s t r a i n s were examined for novobiocin s u s c e p t i b i l i t y using a 5 yg novobiocin disk. Novobiocin r e s i s t a n t , coagulase negative Staphylococci were designated as Staphylococcus saprophyticus biotype 3 (17). Two control organisms were used for beta-lactamase studies, E. c o l i J53-1 which c a r r i e d a plasmid r e s i s t a n t to a m p i c i l l i n and streptomycin (R-tem 1) served as a p o s i t i v e control f o r beta-lactamase production. _E. c o l i J62-1 with a chromosomal marker r e s i s t a n t to n a l i d i x i c acid and highly s e n s i t i v e to beta-lactam a n t i b i o t i c s was used as a negative c o n t r o l . These organisms, were both obtained from Dr. Naomi Datta at the Royal Postgraduate Medical School, Hammersmith, England. A l l b a c t e r i a l cultures were maintained i n nutrient broth con-taining dimethylsulfoxide (8%, v o l / v o l , grade 1 Sigma, No. D-5879) at -80°C. Cultures were transferred d i r e c t l y from the frozen culture to a Mueller-Hinton or blood agar plate using a swab before use. Media C l i n i c a l i s o l a t e s were plated on MacConkey plates with c r y s t a l v i o l e t (0.001 g/1, DF0075-01 Difco Laboratories, Detroit, Michigan, USA), MacConkey without c r y s t a l v i o l e t (Difco, DF0470-01) or Columbia blood agar base (Difco, DF0045-01) containing 5% human blood. Pure cultures were grown in nutrient broth (Difco, DF0003-01) p r i o r to each experiment. A n t i b i o t i c s u s c e p t i b i l i t y tests were ca r r i e d out using Mueller-Hinton broth (Difco, DF0252-02) or Mueller-Hinton agar (Difco, DF0757-02). D i f f e r e n t i a l media were used to enumerate E. c o l i and other organisms i n mixed culture and were: 1. MacConkey agar without c r y s t a l v i o l e t containing 10 yg/ml n a l i d i x i c acid. To prepare n a l i d i x i c a c i d , 500 mg Negram tablets (Winthrop Laboratories, D i v i s i o n of S t e r l i n g Drug Ltd., Aurora, Ontario) were ground up and appropriate amounts of the powder added to agar before autoclaving. 2. MacConkey without c r y s t a l v i o l e t containing 25 yg/ml a m p i c i l l i n . Agar was allowed to cool to 45°C before addition of t h i s a n t i b i o t i c (Penbritin i n j e c t i o n , Ayerst Laboratories, Montreal, Canada) and the plates were dried with covers s l i g h t l y open for 15 minutes. 27 3. Blood agar containing 100 yg/ml sodium azide. Inh i b i t o r y plates were stored at 4°C and were used within 4 days. Urine for each bladder experiment and broth s e n s i t i v i t y t e s t s was pooled from 5-6 healthy male and female adults. These in d i v i d u a l s had not taken alcohol or s i g n i f i c a n t i n h i b i t o r s such as asprin or a n t i b i o t i c s . Urine was stored at 4°C for up to 4 h before being f i l t e r s t e r i l i z e d with a 14 cm asbestose c e l l u l o s e f i l t e r (Carlson-Ford HP/EKS, General F i l t r a t i o n and Engineering Ltd., Scarborough, Ontario). To prevent foaming, one drop of s i l i c o n antifoam (A-5633, Sigma, St. Louis, Miss.) was added to each 2 l i t e r a liquot of urine p r i o r to f i l t r a t i o n . A n t i b i o t i c s The following a n t i b i o t i c disks were used for s u s c e p t i b i l i t y t e s t i n g : a m p i c i l l i n , 10 yg; trimethoprim, 1.25; trimethoprim-sulfamethoxazole, 1.25 + 23.75 yg (BBL Laboratories, Cockeysville, Maryland, D i v i s i o n of Becton* Dickinson and Company) ; cephaloridine, 30; cephalothin, 30; cefamandole, 30; c e f o x i t i n , 30; carbeni-c i l l i n , 50; chloramphenicol, 30; gentamicin, 10; tobramycin, 30; streptomycin, 10; t e t r a c y c l i n e , 30; and n i t r o f u r a n t o i n , 300 yg (Difco Laboratories). Mecillinam 1, 10, and 25 yg disks were puchased from Leo Laboratories Ltd., England. For synergy studies mecillinam, a m p i c i l l i n and combination disks were prepared i n t h i s laboratory (see a n t i b i o t i c s u s c e p t i b i l i t y t e s t s ) . The following a n t i b i o t i c s were also employed: Mecillinam hydrochloride dihydrate (Hoffmann-LaRoche Inc., Nutley, N.J.) and assay grade anhydrous a m p i c i l l i n (A6140, Sigma, St. Louis, Mo., USA). A m p i c i l l i n sodium (Penbritin i n j e c t i o n ) , and p e n i c i l l i n G (Pen K, potassium s a l t i n j e c t i o n ) were purchased from Ayerst 14 Laboratories, Montreal, Canada. C-mecillinam ( s p e c i f i c a c t i v i t y = 2.05 yCi/yg) was a g i f t from Dr. Tybring, Leo Pharmaceutical Products, Ballerup, Denmark. Buffers 1. Phosphate buffered s a l i n e (PBS), pH 7.3. Sodium chloride ( 8 g), dipotassium hydrogen phosphate (1.21 g) and potassium d i -hydrogen phosphate (0.34 g) were dissolved i n water (one l i t e r ) . The pH of the buffer was adjusted to 7.3 by adding sodium hydroxide (1 N) before autoclaving. The so l u t i o n was then stored at 4°C and was used within 2 months. 2. Phosphate buffer (PB), 0.1 M pH 7.0. Stock solutions of 0.1 M dipotassium hydrogen phosphate (17.4 g/1) and 0.1 M potassium dihydrogen phosphate (13.6 g/1) were prepared and the two solutions were mixed at a r a t i o of 2:1 r e s p e c t i v e l y to give a pH of 7.0. It was then autoclaved and r e f r i g e r a t e d f o r up to two months. Beta-Lactamase A broad spectrum mixture of beta-lactamases from B a c i l l u s cereus 569/H9 (Whatman) was purchased from International Enzymes Inc., Fallsbrook, Ca., USA. Each v i a l contained a minimum of 50 units of beta-lactamase II (active against both p e n i c i l l i n s and cephalo-sporins) and 500 units of beta-lactamase I (active against p e n i c i l l i n s alone). Each v i a l was reconstituted with 50 ml of PBS, pH 7.3, and aliquots (10 ml) were frozen (-20 C) and used within a month. A n t i b i o t i c S u s c e p t i b i l i t y Tests Disk t e s t s : The method of Kirby and Bauer (19) was used i n which 4-5 colonies of a pure, overnight plate culture were suspended i n 4 ml of MH broth and then incubated at 37°C f o r 4-6 h. The t u r b i d i t y of the cultures was then adjusted to McFarland standard g number 1 which i s approximately 3 x 10 c e l l s / m l f or _E. c o l i . A s t e r i l e cotton swab was immersed i n the standardized broth culture. A uniform inoculum was obtained by rubbing the swab across the Mueller-Hinton (MH) agar plate i n three planes at 120 degrees. The plates were allowed to dry and a n t i b i o t i c disks were placed on the b a c t e r i a l lawn p r i o r to incubation at 37°C for 18-24 h. The i n h i b i t i o n zones were measured by a plate magnifier giving a 5-f o l d magnification. For synergy studies, s t e r i l e blank disks (Difco) were placed on the b a c t e r i a l lawn on MH plates and 0.02 ml of each a n t i b i o t i c stock s o l u t i o n and also the combination of mecillinam and a m p i c i l l i n were pipetted onto appropriate disks. The f i n a l concentrations of a n t i b i o t i c s on the disks were a m p i c i l l i n , 10 yg; mecillinam, 5 yg; and a combination of a m p i c i l l i n with mecillinam 5 yg and 2.5 yg r e s p e c t i v e l y , or a m p i c i l l i n , 10 yg; mecillinam, 1 yg; and a combin-ation of a m p i c i l l i n with mecillinam 10 yg and 1 yg r e s p e c t i v e l y , per disk. Plates were then incubated for 18-24 h and the i n h i b i t i o n zones were measured. Synergy was defined as an increase of 2 mm or more i n the i n h i b i t i o n zone around the combination disk i n com-parison with the zones surrounding each single drug. 30 Determination of Minimum Inhib i t o r y Concentrations (MIC's) by the Agar D i l u t i o n Technique. S e r i a l two-fold aqueous solutions of each a n t i b i o t i c and a 10:1 or 2:1 combination of a m p i c i l l i n to mecillinam were prepared at 10 X the f i n a l concentration. One part of each a n t i b i o t i c s o l u t i o n was mixed with 9 parts of Mueller Hinton agar (prepared at 1.1 X normal concentration) for preparation of plates. For 2:1 combination experiments, concentrations of a m p i c i l l i n ranged from 1024 to 0.063 yg/ml, and for mecillinam from 512 to 0.031 yg/ml. Corresponding values for experiments with 10:1 combination were 1000 to 0.1, and 100 to 0.01 yg/ml, r e s p e c t i v e l y . r4 A 10 d i l u t i o n of an overnight culture was employed as inoculum with a r e p l i c a t o r d e l i v e r i n g 0.005 ml. The r e p l i c a t o r c a r r i e d 20 cultures simultaneously. The plates were examined a f t e r 18-24 h incubation at 37°C for the presence or absence of growth. The lowest a n t i b i o t i c concentration at which 3 or fewer colonies were observed was considered to be the MIC. The f r a c t i o n a l i n h i b i t o r y concentration (FIC) of a n t i b i o t i c combinations was calculated by d i v i d i n g the MIC for each component i n the combination by the MIC for each component alone and adding the two quotients (27) . A FIG of les s than 0.5 was considered to indicate synergy. In other words, the two a n t i b i o t i c s were s y n e r g i s t i c when the MIC of each drug i n combination was 4 f o l d ; lower than the MIC of each agent alone. Determination of Minimum Inhib i t o r y Concentrations (MIC's) i n Urine  and Broth by the M i c r o t i t e r Plate Method. A m p i c i l l i n , mecillinam and a 2:1 combination of the two drugs were dissolved i n urine or broth at 4 X the f i n a l concentration. Portions (0.1 ml) of each stock solution were placed i n duplicate wells i n the f i r s t row of a m i c r o t i t e r tray (Cooke Engineering Company, Alexandria, Va., type M29 ART). Mueller-Hinton broth or urine (0.05 ml) was added to a l l other wells i n the m i c r o t i t e r trays. The a n t i b i o t i c s were then s e r i a l l y d i l u t e d with the broth or urine (2 f o l d steps) and a further 0.05 ml of medium was added to a l l wells. Overnight broth cultures of organisms were centrifuged, resuspended i n an equivalent volume of s t e r i l e urine or broth and d i l u t e d ten thousand f o l d . Aliquots (0.1 ml) of the d i l u t e d b a c t e r i a l suspensions were then dispensed into appropriate wells. A n t i b i o t i c and c e l l controls were included and plates were sealed with neoprene f i l m and covered. After incubation at 37°C for 18 h, the wells were examined for t u r b i d i t y . The f i r s t well which showed no growth was recorded as the end point (MIC). A FIC of 0.5 was used to indi c a t e synergy as described i n the previous paragraph. Assessment of the Rep r o d u c i b i l i t y of B a c t e r i a l Assays f or Mixed  Cultures Using D i f f e r e n t i a l Media. Overnight suspensions of bacteria were centrifuged and resus-pended i n phosphate buffered s a l i n e , pH 7.3, and d i l u t e d to match McFarland standard #1. An E. c o l i s t r a i n and a second organism were mixed i n 1:1, 1:10, 1:100, 1:500 and 1:1000 r a t i o s . Cultures were s e r i a l l y d i l u t e d and samples (0.1 ml) of the mixed cultures were plated on d i f f e r e n t i a l media containing a n t i b a c t e r i a l agents or a n t i b i o t i c s and also control plates without such i n h i b i t o r s . MacConkey plates containing a m p i c i l l i n were used when the second organism was Pseudomonas, Proteus, S e r r a t i a or K l e b s i e l l a spp. Blood agar plates containing sodium azide (100 yg/ml) were u t i l i z e d to enumerate mixed cultures of _E. c o l i and Streptococcus f e c a l i s and blood agar plates containing n a l i d i x i c acid were used when the second pathogen was Staphylococcus spp. T r i p l i c a t e plates were inoculated at each d i l u t i o n and plates with 20-200 colonies were chosen to provide r e l i a b l e determinations of v i a b l e counts. Determination of B a c t e r i a l Growth Rates i n Shake Cultures Urine was c o l l e c t e d from healthy i n d i v i d u a l s and s t e r i l i z e d by f i l t r a t i o n (see below for d e t a i l s ) . Portions of s t e r i l e urine (25 ml) were d i s t r i b u t e d into f l a s k s (125 ml) which were permitted to e q u i l i -brate i n a waterbath (37°C). Overnight broth cultures of bacteria were d i l u t e d (1:100 v/v), and portions (0.25 ml) of the suspensions were inoculated into f l a s k s containing urine or nutrient broth giving 4 6 a f i n a l c e l l density of about 10 - 10 organisms/ml. Samples were taken at i n t e r v a l s , s e r i a l l y d i l u t e d i n PBS pH 7.3, and 0.1 ml portions were plated on MacConkey agar or blood agar plates using a surface inoculation technique. The regression l i n e of the plot of the populations versus time was calculated using a Texas Instrument S51 c a l c u l a t o r . Generation times and lag periods were derived from t h i s regression l i n e . The E f f e c t of Antifoam and F i l t r a t i o n on B a c t e r i a l Growth i n  Urine. Five batches of urine were divided into two portions. One was f i l t e r e d through a thick s t e r i l i z i n g f i l t e r pad (Carlson-Ford) and antifoam was added as previously described. The second portion was f i l t e r e d through a membrane f i l t e r ( M i l l i p o r e F i l t e r Corporation, Bedford, Mass. Type HA. 0.45 Um). Portions of each batch were d i s t r i b u t e d into duplicate f l a s k s and inoculated with 4 bacteria to an i n i t i a l c e l l density of 10 v i a b l e organisms/ml. F i f t e e n urinary pathogens were tested, lag periods, maximum., growth rates and climax populations were then determined for these shake cultures i n urine f i l t e r e d by both methods. Use of the Bladder Model for Mixed Culture Experiments Figure 1 summarizes the features of the bladder apparatus which f i t t e d together with ground glass and p o l y - t e t r a f l u o r o -ethylene ("Teflon") j o i n t s and could be e a s i l y dismantled and reassembled a f t e r s t e r i l i z a t i o n . The apparatus consisted of a culture v e s s e l which was a c y l i n d r i c a l glass chamber with a capacity of 500 ml. The lower part of the apparatus tapered and i n t e r -changeable glass bases could be f i t t e d to vary the r e s i d u a l volume from 1 to 50 ml. Residual volume was ca l i b r a t e d by a dye d i l u t i o n technique (see below). Urine was pumped into the bladder through a p e r i s t a l t i c metering pump ( P e r i s t a l t i c Pump P3, Pharmacia, Fine Chemicals, Uppsalla, Sweden) and a flow rate of 50 ml/h was chosen for these experiments. An antiflowback device was designed and used to prevent urine from flowing back i n the supply l i n e . Temperature of the apparatus was maintained at 37°C by three photoflood l i g h t s c o n t r o l l e d by a thermistor i n the base of the apparatus. When the contents of the chamber reached 37°C, the current through the lamps was reduced to control the temperature and minimize the 34 F i g . 1: -Flowchart- of-t-he—continuous c u l t u r e model of the u r i n a r y bladder. The Model Bladder Apparatus e f f e c t of strong l i g h t on cultures. Autoclavable p l a s t i c tubing ( S i l a s t i c Medical Grade, 0.104" ID, 0.192" OD, No. HH.4 139., Dow Corning, Midland, Michigan, USA) was used to bring urine from the medium r e s e r v o i r into the bladder through the antiflowback device. A i r (750 ml/min) was s t e r i l i z e d by passage through a cotton f i l t e r p r i o r to entering the culture v e s s e l . This produced aerobic conditions i n the bladder and also helped to s t i r the culture by bubbling through i t . The bladder emptied v i a a s t e r i l i z a b l e e l e c t r o -magnetic t e f l o n valve (Model 800-1132, Mace Corporation, South E l Monta, C a l i f o r n i a ) which was activated every 4 h by an e l e c t r o n i c timer. C a l i b r a t i o n of Residual Volume A s o l u t i o n of methylene blue was prepared i n water to give an o p t i c a l density reading of between 0.3 and 0.7 at i t s maximum a f t e r d i l u t i n g 50 f o l d . The concentrated dye so l u t i o n (200 ml) was added to the bladder, the apparatus was operated for 10-15 min, drained and the O.D. determined. Water (200 ml) was then added to the apparatus and again i t was operated for another 10-15 min, to thoroughly mix the contents p r i o r to draining. The O.D. of the d i l u t e d dye was measured a f t e r drainage (diluted again i f necessary) and the r e s i d u a l volume was calculated using the following formula: O.D. of the d i l u t e d dye X volume of the diluent RV = O.D. of the concentrated dye The apparatus was too cumbersome to determine the r e s i d u a l volume by d i r e c t l y weighing the chamber. 37 Operation of the Bladder Model for Mixed Culture Experiments The bladder culture v e s s e l was s t e r i l i z e d with saturated steam for 15 min. Excess water was drained v i a the solenoid valve and the apparatus was permitted to cool to room temperature. S t e r i l e urine was stored i n the s t e r i l e r e s e r v o i r at room temperature, and pumped through p l a s t i c tubing into the culture v e s s e l . When the temperature s t a b i l i z e d (37°C), the bladder was drained and organisms were introduced through the rubber port i n the base of the apparatus. Overnight nutrient broth cultures were centrifuged and resuspended in PBS pH 7.3 and the t u r b i d i t y adjusted to McFarland standard No. 1. Standardized suspendions of two organisms were d i l u t e d (1:100 v/v) i n s t e r i l e urine and mixed immediately before inoculation into the bladder (10 ml t o t a l ) . A r e s i d u a l volume of 1 ml was chosen for these experiments which simulates the r e s i d u a l volume i n a normal bladder, and the bladder emptied every 4 h. Samples (2 ml) were taken at i n t e r v a l s up to 6 h and at 24 h and used for v i a b l e counts on appropriate d i f f e r e n t i a l and control media. A 1:1 r a t i o of E. c o l i to a second pathogen was intended as the inoculum i n each experiment. Since the v i a b l e count of b a c t e r i a l suspensions could not be accurately predicted by standardizing the t u r b i d i t y , the i n i t i a l r a t i o varied over the range of 0.5:1 to 2:1. Attempts to use a counting chamber to standardize inocula were cumbersome and were not appreciably more accurate. Comparison of r e l a t i v e changes i n populations i n mixed cultures was f a c i l i t a t e d by r e c a l c u l a t i n g the r a t i o s on the basis of an i n i t i a l nominal r a t i o of 1:1 in each case. Study of A n t i b i o t i c Synergy in the Bladder Model The bladder model was set up as described above under "operation 38 of the bladder model" f o r mixed culture experiments. An overnight culture of each test organism was centrifuged, resuspended i n an equivalent volume of s t e r i l e urine and d i l u t e d with urine (1:100 v/v). A suspension (20 ml) of the test bacteria containing approximately 10^ v i a b l e organisms per ml was introduced into the bladder. Two hours a f t e r inoculation, when the apparatus contained 120 ml of culture, urine containing a n t i b i o t i c (either a m p i c i l l i n 500 yg/ml, or mecillinam 250 yg/ml or a m p i c i l l i n 250 yg/ml with mecillinam 125 yg/ml) was substituted for a n t i b i o t i c free urine. The r e s i d u a l volume chosen for these experiments was 4 ml since patients with urinary t r a c t i n f e c t i o n s or i n d i v i d u a l s prone to these i n f e c t i o n s tend to have a higher r e s i d u a l volume than normal i n d i v i d u a l s (77). Urine was withdrawn from the apparatus at i n t e r v a l s shown i n Table 8. Portions (0.1 ml) of each sample were mixed with 0.9 ml of beta-lactamase solution (Whatman Biochemical Ltd.) containing approximately 10 units of beta-lactamase. The tubes were incubated at 37°C for 5 mins to permit the beta-lactamase to destroy the a n t i b i o t i c i n the sample which was then s e r i a l l y d i l u t e d and plated for v i a b l e counts. Study of Synergy i n the Mouse Model : by Dr. R. Cleeland and Associates at Hoffman-La Roche Inc., Nutley, N.J. (This data i s f o r background information only and i s not a d i r e c t part of the t h e s i s ) . For convenience the experimental r e s u l t s are included i n the Results Section rather than the Discussion. Swiss albino mice (CD-I strain) weighing 18-20 g obtained from Charles River Breeding Laboratories, Wilmington, Mass., were used i n a l l experiments. Animals were infected i n t r a p e r i t o n e a l l y with a sus-pension (0.5 ml) containing 100-1000 minimal l e t h a l doses of the organisms prepared i n 5% hog g a s t r i c mucin to enhance t h e i r pathogenicity. The animals were treated subcutaneously immediately a f t e r i n f e c t i o n with 1 ml of graded doses of a m p i c i l l i n , mecillinam or a 1 0 : 1 combin-ation of a m p i c i l l i n + mecillinam. The number of mice surviving 1 4 days a f t e r i n f e c t i o n was used to determine the 5 0 % protection dose ( P D , - Q ) calculated by the method of Reed and Muench ( 1 1 1 ) . In each experimental i n f e c t i o n the s i n g l e agents and the combination of agents were tested simultaneously. Synergy was considered to have occurred i f the FIC was l e s s than 0 . 5 (calculated by d i v i d i n g the P D ^ Q value obtained for each of the com-ponents in the combination by the PD^g value for each component alone and adding the two quotients). Screening Tests for Beta-Lactamase A c i t v i t y The test was based upon detection of beta-lactamase in i s o l a t e d b a c t e r i a l colonies ( 9 9 ) . Regular typing paper containing starch (tested by iodine solution) was cut into 4 x 7 cm s t r i p s . Paper s t r i p s were then soaked i n a so l u t i o n of p e n i c i l l i n G for 1 0 mins. The p e n i c i l l i n G stock was made by d i s s o l v i n g the contents of one v i a l of potassium p e n i c i l l i n G ( i n j e c t a b l e solution) in 0 . 1 M phosphate buffer (pH 7 . 0 ) giving a f i n a l concentration of 1 0 0 , 0 0 0 yg/ml. Each s t r i p was then removed and placed i n a clean p e t r i p late. Single colonies of Enterobacteriaceae were transferred with a 2 mm loop from a MacConkey plate and placed on the paper s t r i p ( 1 . 5 cm apart). The plate was incubated at 3 7 ° C for 3 0 minutes, flooded with Gram's iodine, and drained immediately. Upon contact with iodine, the starch paper turned black in a few seconds except for c l e a r spots present around the b a c t e r i a l colonies capable of producing beta-lactamase. The method i s based on the p r i n c i p l e that p e n i c i l l a n i c acid i s formed from p e n i c i l l i n G by beta-lactamase which i n turn binds iodine. Therefore the starch-iodine complex di s s o c i a t e s , leaving a decolorized spot around the colony. The v a l i d i t y of the test was checked by including p o s i t i v e and negative controls i n every run. Preparation of Crude Extracts of Beta-Lactamase from C l i n i c a l  I solates. Test organisms were grown overnight i n nutrient broth and di l u t e d (1:100 v/v) in f r e s h nutrient broth (50 ml portions i n 125 ml f l a s k s ) . Flasks were incubated i n a shaker bath at 37°C g u n t i l the population density reached between 1 and 5- x 10 c e l l s / m l (usually within 3-5 h). B a c t e r i a l populations were estimated by o p t i c a l density measurements at 660 nm (Spectronic 20, Bausch and Lomb) and subsequently confirmed by a surface plate v i a b l e count on MacConkey without c r y s t a l v i o l e t medium. The cultures were centrifuged 6,000 x g, 10 minutes), and resuspended i n phosphate buffer (0.1 M pH 7.0) to a density of 1 0 1 0 c e l l s / m l . The c e l l s were then disrupted by sonication (Kontes Micro-Ultrasonic C e l l Disrupter, 1200 watt per sq. inch density) at 0°C for 2 minutes, then centrifuged (6,000 x g for 10 minutes) and the supernatant was stored at -80°C. A v i a b l e count was also performed on the sonicated material to determine the e f f i c i e n c y of sonication. 41 Microiodometric Technique f or Measuring Beta-Lactamase i n Crude  Extracts of Bacteria. The p r i n c i p l e of t h i s technique i s based on q u a n t i t a t i v e l y determining the amount of iodine needed to oxidize the product of beta-lactamase action eg. p e n i c i l l a n i c a c i d i n the case of p e n i c i l l i n (140). Reagents. A l l reagents were prepared i n phosphate buffer (0.1 M, pH 7.0). Mecillinam and a m p i c i l l i n (0.2 M) were prepared f r e s h l y each day. Hydrolised starch (0.2% w/v) was dissolved i n the buffer by b o i l i n g for 2-3 minutes and the starch-iodine complex was prepared by adding 0.15 ml of iodine s o l u t i o n (0.08 M i n 3.2 M potassium iodide) to 100 ml of the starch s o l u t i o n . This provided a f i n a l iodine con-centration of 120 ymoles. A l l reagents were c h i l l e d on i c e p r i o r to use. Test Procedure. Starch-iodine (1 ml), a n t i b i o t i c substrate (1 ml) and buffer (0.9 ml) were pipetted into a 1 cm glass cuvette (Canlab, S1941324) and the cuvette was placed i n a spectrophotometer (Beckman DB-GT), where the temperature of the chamber was con t r o l l e d (30°C). Enzyme extract (0.1 ml) was added to the cuvette a f t e r 5 min at 30°C and Q.D. was recorded at 620 nm for 15 minutes. The i n i t i a l o p t i c a l density was 1.2 and the enzyme a c t i v i t y was measured using the following formula: A O.D.£2Q/1-2 x 0.03 x 10 = ymoles substrate destroyed/min/ml enzyme/lO"^ c e l l s at 30°C. The amount of iodine used i n the assay corresponds to 30 ymoles of p e n i c i l l i n (0.03 ml). Preparation of Membrane Fractions from Enterobacteriaceae Organisms were grown in nutrient broth overnight and d i l u t e d (1:100 v/v.) i n f r e s h n u t r i e n t broth and permitted to regrow to y i e l d a population i n the l a t e logarithmic phase of growth (about 5 x 10 ) v i a b l e c e l l s per ml. Cultures were then centrifuged at 6000 x g f o r 10 min, resuspended i n 25 ml of cold sodium phosphate buffer (0.1 M pH 7.0) and 2-mercaptoethanol (0.25 ml). The suspensions were then subjected to sonication (Biosonic, Bronwill S c i e n t i f i c , Rochester, N.Y.) for four periods of 30 seconds between each of which the suspensions were recooled on i c e . Disrupted-cells were centrifuged at 8000 x g for 20 min and the supernatant containing the membrane f r a c t i o n s was centrifuged at 40,000 x g for 40 min at 4°C (SW27 swinging rotor, Beckman L8-80 U l t r a c e n t r i f u g e ) . The p e l l e t was washed twice and resuspended i n buffer to approximately 5-10 mg protein/ml and stored at -80°C u n t i l used. Estimation of protein concentration was based on o p t i c a l density reading at 280 nm ( G i l f o r d Spectrophotometer 250). 14 Abortive Studies of C-Mecillinam Binding to Membrane Fractions. Portions of membrane protein suspensions (0.2 ml) were incubated o 14 at 37 C for 5 min before the add i t i o n of C-mecillinam (100 yg i n 0.02 ml). The mixture was incubated at room temperature for 20 min and the uptake of l a b e l l e d mecillinam was terminated by adding 5 y l of non-radioactive s o l u t i o n of mecillinam (40 mg/ml) and 10 y l of 20% Sarkosyle (sodium l a u r o y l sarcosinate, Geigy I n d u s t r i a l Chemicals, N.Y.). The suspension was then centrifuged (40,000 x g for 40 min) to separate the inner membranes which are soluble in Sarkosyle from the outer membranes. P e n i c i l l i n binding proteins i n the supernatant were then separated i n a polyacrylamide gel electrophoresis system. 14 Abortive Attempts to Label Intact C e l l s with C-Mecillinam. Organisms were grown in nutrient broth (100 ml portions) g to a density of 5 x 10 c e l l / m l , then centrifuged (6000 x g, 10 min) and resuspended i n phosphate buffer (0.1 M, pH 7.0). Portions of c e l l s (0.2 ml) were incubated at 37°C for 5 min followed by addition 14 of 0.02 ml of C-mecillinam (100 yg). The mixture was incubated at room temperature f o r 20 min and 5 y l of unlabelled substrate solution (40 mg/ml) was added to stop the reaction. C e l l s were disrupted by sonication as described above and centrifuged (40,000 x g for 20 min). The p e l l e t was resuspended i n 150 y l of gel sample buffer (0.2 M t r i s pH 6.8, 3% w/v sodium dodecyl s u l f a t e (SDS), 30% v/v g l y c e r o l and 0.003% w/v bromophenol blue). Polyacrylamide Gel Electrophoresis (PAGE). The procedure for sodium dodecyl-sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) was that of Laemmli and Farre (74). The running gel was composed of 10% polyacrylamide (w/v) which was prepared and mixed i n the following proportions: a. Acrylamide/bis acrylamide 10 ml (Bio Rad Laboratories, Richmond, C a l i f o r n i a ) (acrylamide 30% w/v, bis-acrylamide 0.8% w/v). b. Tris-HCl (Bio Rad Laboratories) pH 8.8 (1.5 M), 8.65 ml. c. SDS (sodium dodecyl s u l f a t e , BDH Laboratories Reagents), (10% w/v), 0.3 ml. d. TEMED (Bio Rad Laboratories) (N,N,N',N'-acetomethylethylene-diamine), 0.1 ml. e. Deionized water, 10.9 ml. f. Ammonium persulfate (Bio Rad Laboratories) (100 mg/ml ^ 0 ) was f r e s h l y prepared and 0.1 ml was added to the running gel immediately before pouring the slab. Electrophoresis plates were secured with clamps and 21.5 ml of the running gel was pipetted between the plates. The ge l was overlayed with deionized water for 30 min to allow polymerization. The water was poured o f f and stacking gel was layered on top of the running gel a f t e r i n s e r t i n g a comb with 20 w e l l s ) . Composition of stacking gel was: a. Acrylamide/bis s o l u t i o n (1 ml). b. T r i s , 0.5 M, pH 6.8 (1.25). c. Sodium dodecyl s u l f a t e (SDS), 10% w/v (0.1 ml). d. TEMED (5 U l ) . e. D i s t i l l e d water (7.5 ml). To apply protein suspensions of membrane f r a c t i o n s or intact c e l l s , the comb was c a r e f u l l y removed and the wells covered with 'running' buffer ( T r i s , 4.5 g; SDS, 1.5 g; glycine, 21.6 g dissolved i n 1.5 L of deionized water). Protein samples l a b e l l e d with radioactive mecillinam (100 y l ) were applied to the bottom of the wells with a syringe. The electrophoresis tank was then placed i n the chamber and 'running' buffer was added to the chamber to cover the bottom of the g e l . The cover was placed on the tank and the chamber was connected to the power supply. Electrophoresis was c a r r i e d out at 25 milliamps for 4-5 h u n t i l the 'tracking' dye was within 1 cm of the plates. After electrophoresis was completed, the gel was transferred to a p l a s t i c container and Coomassie blue (0.2% in methanol-acetic acid-water, 5:1:5) added to s t a i n the gel overnight. The s t a i n was then replaced by destaining so l u t i o n (methanol-acetic acid-water, 5:1:5) for 24 h with 3 to 4 changes of sol u t i o n . Fluorography ( S c i n t i l l a t i o n Autography) This method has been developed to increase the s e n s i t i v i t y of autoradiograms for detecting "^ H, "^C and "^S i n polyacrylamide gels (22). The stained gel was prepared for fluorography by soaking i n 200 ml of dimethyl sulfoxide (DMSO) for 30 min. The DMSO solvent was changed and f r e s h DMSO was added for another 30 min. The gel was then immersed f o r 3 h i n 100 ml of 20% (w/v) 2,5-diphenyloxazole (PPO, Baker Chemical Co., Pa.) i n DMSO giving a f i n a l concentration of 22.2% w/v for 3 h and transferred to 200 ml of d i s t i l l e d water (1 h). The gel was then placed on a Whatman 3 MM paper and dried on a gel dryer (Bio Rad Laboratories) for 1 hour. The dried gel was covered with a Kodak RP Royal X-Omat X-ray f i l m , secured between two glass plates and was kept at -70°C for up to 5 months before developing. 45a R E S U L T S Assessment of D i f f e r e n t i a l Media for Use in Mixed Culture  Experiments Table 2 summarizes studies of the v a l i d i t y of methods used to enumerate b a c t e r i a l populations i n mixed cultures. Escherichia  c o l i served both as control and reference organism i n each case, and a range of d i l u t i o n s of the second pathogen was employed. The r e s u l t s show that the d i f f e r e n t i a l media employed permitted enumeration of r e l a t i v e l y small numbers of the second pathogen i n the presence of dense suspensions of _E. c o l i . The greatest errors were noted with Streptococcus f e c a l i s . E f f e c t of S t e r i l i z a t i o n Method on the Growth Supporting Properties  of Five Samples of Urine. Two types of f i l t e r pads were used to study the e f f e c t of s t e r i l i z a t i o n on the growth supporting properties of urine, the thick pads (Carlson Ford) and t h i n M i l l i p o r e F i l t e r pads. Carlson Ford f i l t e r has the advantage of r e s i s t i n g clogging by suspended materials present i n human urine. Carlson Ford f i l t e r s are made of asbestos (a s i l i c a t e of calcium and magnesium) and c e l l u l o s e . The asbestos may act as an ion exchange r e s i n and possibly remove ions and nutrients from the medium. M i l l i p o r e f i l t e r s on the other hand, are made of c e l l u l o s e acetate which i s u n l i k e l y to remove any n u t r i e n t s . Antifoam was also added to urine before s t e r i l i z a t i o n by Carlson Ford f i l t e r pads. Antifoam i s a surfactant and may i n t e r f e r e with c e l l growth. To investigate t h i s p o s s i b i l i t y Table 2: Assessment of D i f f e r e n t i a l Media for Use i n Mixed Culture Experiments Organisms Population of _E. c o l i control Population of the second pathogen for 1:1 r a t i o Escherichia c o l i // B with 3.8 x 10"" Proteus m i r a b i l i s Escherichia c o l i // B with 4.0 x 10 £ Pseudomonas aeruginosa Escherichia c o l i # B with 4.4 x 10 Se r r a t i a marcescens Escherichia c o l i // F with 1.1 x 10^ Providencia s t u a r t i # A Escherichia c o l i # F with 1.2 x 10 £ K l e b s i e l l a pneumoniae c Escherichia c o l i # D with 1.3 x 10 Staphylococcus saprophyticus // A Escherichia c o l i # D with 7.7 x i o ' Providencia s t u a r t i // B Escherichia c o l i // E with 2.4 x 10 Streptococcus f e c a l i s # A Escherichia c o l i # E with 2.5 x 10 Staphylococcus saprophyticus // B 8 9.1 x 10 c 5.8 x 10 £ 5.0 x 10 £ 1.6 x 10 £ 1.1 x 10 £ 1.2 x 10 £ 4.3 x 10 y 5.5 x 10 y 1.5 x 10 £ Determined value for the second pathogen as a percentage of the expected value at d i l u t i o n s : 1:1 1:10 1:100 1:500 1:1000 83.9 ND* 98.7 112.2 86.4 106.2 ND 100.0 ND 83.33 ND 104.6 ND ND ND ND ND ND ND ND ND 80.2 93.1 103.4 87.9 63.8 62.0 100.0 92.0 100.0 108.0 ND ND ND ND 134.5 156.3 161.8 100.0 174.5 93.3 86.6 100.0 116.6 113.3 MacConkey agar plates without c r y s t a l v i o l e t containing 25 yg ampicillin/ml were used for a l l the combinations when Gram negative organisms were mixed with E. c o l i . Blood agar plates with 10 yg n a l i d i x i c acid were used to enumerate jS. saprophyticus biotype 3, and blood agar plates with 0.1% sodium azide used for S^. f e c a l i s . * not done 5 batches of urine were f i l t e r e d by both methods; antifoam was only added to urine f i l t e r e d by Carlson Ford pads and growth character-i s t i c s of 15 urinary i s o l a t e s of Enterobacteriaceae were determined under both conditions. As shown in Table 3, neither the use of Carlson Ford pads, nor the addition of antifoam had any e f f e c t on the lag periods, generation times or the maximum growth rate of the 15 test b a c t e r i a In other words, no e s s e n t i a l nutrients or components appear to have been removed when Carlson Ford pads were used. Therefore, Carlson Ford f i l t e r s were used throughout the experiments and antifoam was added to a l l urine stocks before s t e r i l i z a t i o n . Growth rates of Bacteria in Shake Cultures Growth properties of a s e l e c t i o n of 20 urinary and 8 other i s o l a t e s of bacteria were determined i n shake culture to determine i f growth rates of organisms d i f f e r i n urine and laboratory media. This d i f f e r e n c e obviously may be relevant to pathogenicity. Tables 4 and 5 show representative samples of plate counts of urinary i s o l a t e s in urine (Table 4) and i n nutrient broth (Table 5). The v i a b l e counts were plotted against time and the slopes (K) of the regression l i n e s were computed for l o g a r i t h m i c a l l y growing c e l l s . Generation times were then measured using the following formula: Generation time (minutes) = l o g 1 n 2/K x 60 Lag periods were calculated from the slope, intercept, and -d-Table 3: Ef f e c t of S t e r i l i z a t i o n Method on the Growth Supporting Properties of 5 Urine Samples f or 15 Aerobic Isolates i n Shake Culture. Mean ± Standard Deviation Urine Treatment Method Lag Period Generation Time Population After (hr) (min) 24 hrs A. M i l l i p o r e S t e r i l i z a t i o n 2.14 ± 1.48 35.9 ± 17.5 9.54 x 10 ± 7.7 x 10 B. Carlson Ford F i l t e r pads 2.13 ± 1.45 35.1 ± 17 a f t e r addition of S i l i c o n antifoam C. Significance of 0.999 0.897 0.861 diff e r e n c e of A vs. B by two t a i l e d student t test 8.9 x 10 ± 7 x 10 Table 4: Viable Counts of Representative Samples of Bacteria Grown in Urine. Organism Viable Counts at Time (h) 24 Escherichia co l i isolate B Escherichia coli isolate E Klebsiella pneumoniae Proteus mirabilis Proteus vulgaris 1.8 x 104 3.3 x 10 4 8.0 x 104 3.7 x 106 3.3 x 106 3.1 x 107 1.8 x 108 4 x 109 5.8 x 104 1.8 x 105 8.0 x 105 3.2 x 106 1.2 x 10? 8.4 x 107 4.8 x 108 3 x 109 2.6 x 104 4.0 x 104 1.9 x 105 2.6 x 106 3.0 x 107 1.8 x 108 2.1 x 109 5 x 10 9 2.0 x 104 2.9 x 104 6.4 x 10 4 1.6 x 105 5.5 x 105 1.5 x 105 4.2 x 106 6 x 108 4.1 x 104 3.0 x 104 5.8 x 104 1.3 x 105 4.8 x 105 1.7 x 106 2.6 x 107 Serratia marcescens Providencia stuarti isolate A 3.2 x 10 9 1.9 x 105 1.1 x 10 5 1.2 x 105 6.0 x 105 1.6 x 106 1.2 x 107 1.0 x 108 7.3 x 10 isolate A Pseudomonas aeruginosa 1.6 x l O 4 3.1 x l O 4 7.9 x lo" 1.9 x 10J 7.7 x l O J 2.3x10" 9.4x10  -J i „ i n 4 7 o „ i n A l Q „ i n 5 7 7 v i n 5 ?.3 x 106 9.4 x 10° 5.2 x 10 8 6.1 x 104 6.0 x 104 6.7 x 104 2.7 x 105 3.5 x 105 1.3 x 10° 2.8 x 107 3.0 x 10 8 5 5 . 3 x 1 0 5 3.0 x 106 6.1 x 106 2.0 x 107 1.8 x 10 8 5.0 x 108 Staphylococcus saprophyticus 4.1 x 10 biotype 3 isolate A Streptococcus fecalis 1.8 x 106 2.7x10° 4.4 x l O 7 5.8 x l O 7 7.4 x l O 7 2.0 x l O 8 isolate A , Additional 0.5 h samples were also included for faster growing organisms (.E. c o l i strains). Table 5: Viable Counts of Representative Samples of Bacteria Grown in Nutrient Broth. Viable Counts at Time (h) 0 1 2 3 4 5 6 7 9 24 Escherichia co l i isolate B 8. ,9 X 10 3 3 .1 X i o 4 1. 1 X 10 5 5. ,8 X 10 5 4. 1 X 10 6 4.2 X 10 7 1. 9 X i o 8 8. ,0 X i o 7 Escherichia co l i isolate E 2. .4 X i o 4 3 .2 X 10 4 1. 7 X 10 5 4. ,3 X 10 5 9. 2 X 10 5 2.3 X 10 6 3. ,7 X 10 6 5.7 x 10 7 8, ,0 X i o 8 Klebsiella pneumoniae 1. .0 X i o 4 4 .0 X 10 4 8. 7 X i o 4 8. ,5 X 10 5 5. 9 X 10 6 2.9 X 10 7 2. ,4 X i o 8 5. .0 X 10 9 Proteus mirabilis 2. .1 X i o 5 1 .7 X i o 5 5. 3 X 10 5 1. .3 X 10 6 2. 7 X 10 6 9.0 X 10 6 4. .8 X i o 7 6. .5 X 10 8 Proteus vulgaris 3, .8 X i o 4 2 .3 X 10 4 2. 3 X i o 4 2, .7 X 10 4 4.5 X i o 4 N. .G, 3 X i o 3 Serratia marcescens isolate A 7, .4 X 10 4 1 .3 X 10 5 2. 7 X 10 5 2, .4 X i o 6 1. 3 X i o 7 3.8 X i o 7 5. .1 X i o8 6, .0 X i o 9 Pseudomonas aeruginosa 2. .7 X i o 4 3 .4 X i o 4 1. 3 X 10 5 7. .1 X i o 5 1. 7 X 10 6 3.9 X 10 6 3. .5 X i o 7 8 .6 X i o 8 Providencia stuarti isolate A 1, .5 X i o 5 6 .0 X i o 4 2. 7 X i o 5 4, .2 X 10 5 9. 0 X i o 5 8.0 X i o6 1, .2 X i o 8 2 .7 X i o 9 Staphylococcus saprophyticus biotype 3 isolate A 4 .2 X i o 5 1. 2 X 10 3 N. G N .G. 1.0 x 10 3 1 .0 X 10 3 Streptococcus fecalis 2 .7 X i o 6 3. 2 X i o 6 2. .0 X i o 7 2.3 X 10 7 2.9 x 10 7 4 .9 X i o 7 isolate A See footnotes to Table 3. 2 N.G. = no detectable growth i.e. approx. <10 per ml. observed i n i t i a l v i a b l e count. Table 6 compares calculated growth-c h a r a c t e r i s t i c s of urinary _E. c o l i s t r a i n s i n urine and i n nutrient broth. The growth rates of these st r a i n s were very s i m i l a r i n both media with the exception of E_. c o l i E, which had a longer generation time i n nutrient broth. A l l of the i s o l a t e s i n Table 6 came from infected urine and one may speculate that these s t r a i n s were perhaps well adapted to grow i n urine. Generally, most of the c l i n i c a l i s o l a t e s of Enterobacteriaceae ( e s p e c i a l l y E_. c o l i ) grew s a t i s f a c t o r i l y i n laboratory media but the p o s s i b i l i t y of f i n d i n g organisms such as E. c o l i E-which are better adapted to grow i n urine can not be excluded. Lag periods however, were d i f f e r e n t from s t r a i n to s t r a i n regardless of the culture medium. The range of lag periods i n urine may possibly be accounted for by the v a r i a b l e nutrients in d i f f e r e n t batches of urine. On the other hand, the same kind of r e s u l t s were observed when nutrient broth was used and one may conclude that i n d i v i d u a l b acteria perform d i f f e r e n t l y i n the same medium and there may be u n i d e n t i -f i e d f a c t o rs which influence the length of the lag period. Table 7 i l l u s t r a t e s growth rates of urinary pathogens other than E^ . c o l i i n nutrient broth and urine. A l l Enterobacteriaceae had a f a s t e r growth rate i n nutrient broth than in urine with the exception of a s i n g l e Proteus v u l g a r i s s t r a i n which did not grow in n u t r i e n t broth. In the case of t h i s organism, populations decreased during the f i r s t few hours and by 24 h the b a c t e r i a l counts had returned close to the o r i g i n a l value (see Tables 3 and 4 for v i a b l e counts). In general, the generation times were 5 to 60 times longer i n urine than i n nutrient broth. Table 6: Growth C h a r a c t e r i s t i c s of 6 Urinary Isolates of Escherichia c o l i i n Urine and Nutrient Broth. Isolate Generation Time (min) Lag Period (h) In urine In nutrient broth In urine In nutrient broth Escherichia c o l i i s o l a t e A 21. .1 22. .3 0. .5 0. .82 Escherichia c o l i i s o l a t e B 21. .8 22. .07 0. ,89 0. .92 Escherichia c o l i i s o l a t e C 22. ,1 22. .6 0. ,98 1. .02 Escherichia c o l i i s o l a t e D 20. .9 23. .9 1. ,03 1. .05 Escherichia c o l i i s o l a t e E 22. ,9 41. .37 0. ,57 0. .38 Escherichia c o l i i s o l a t e F 22. ,1 23. .2 1. ,39 1. .11 Table 7: Growth Charac t e r i s t i c s of Urinary Isolates other than Escherichia c o l i i n Urine and Nutrient Broth. Organism Generation Time (min) Lag Period (h) In urine In nutrient broth In urine In nutrient broth S e r r a t i a marcescens i s o l a t e A Ser r a t i a marcescens i s o l a t e B K l e b s i e l l a pneumoniae Proteus v u l g a r i s Proteus m i r a b i l i s Proteus r e t t g e r i Providencia s t u a r t i i s o l a t e A Providencia s t u a r t i i s o l a t e B Pseudomonas aeruginosa Streptococcus f e c a l i s i s o l a t e A Streptococcus f e c a l i s i s o l a t e B Staphylococcus saprophyticus i s o l a t e A biotype 3 Staphylococcus saprophyticus i s o l a t e B biotype 3 Staphylococcus epidermidis 33.1 35.5 22.4 33.47 39.2 37.6 45.5 27.1 50.2 53.65 33.11 51.9 60.6 52.83 23.1 22.4 20.5 N.G. 38.3 31.1 43.2 24.1 31.8 61.7 76.3 65.0 N.G. N.G. 1.68 1.65 0.82 1.60 1.19 2.0 1.79 2.17 1.47 1.3 0.6 0.66 0.35 1.42 0.89 1.04 0.75 N.G. 1.36 1.86 .75 2.80 1.26 0.78 0.68 0.83 N.G. N.G. N.G. = no detectable growth i . e . approximately <10 per ml The lower 5 l i n e s of Table 7 l i s t the growth c h a r a c t e r i s t i c s of Gram p o s i t i v e i s o l a t e s . Unlike Gram negative organisms these bacteria grew much f a s t e r i n urine than in broth, and two of the i s o l a t e s (Staphylococcus saprophyticus i s o l a t e B and Staphylococcus  epidermidis i s o l a t e A) did not even grow in nutrient broth. This fin d i n g was confirmed by repeating each test i n three d i f f e r e n t batches of urine and nutrient broth; mean values are shown in Table 6. The organisms which did not grow i n nutrient broth in shake f l a s k s did however grow overnight i n s t a t i c culture i n tubes of nutr i e n t broth. This was perhaps an inoculum e f f e c t since the i n i t i a l inoculum in the tubes was higher than i n f l a s k s . To i n v e s t i -gate t h i s p o s s i b i l i t y , f l a s k s were inoculated with a range of inocula (10"' to 10^ organisms) and were incubated under s t a t i c conditions. V i s i b l e growth was observed in cultures which had a large o r i g i n a l inoculum (10^) a f t e r 24 h but not i n f l a s k s with smaller inocula. Growth c h a r a c t e r i s t i c s of these organisms were also determined i n shake f l a s k s using 5% CC^. The r e s u l t s showed no enhancement i n the rate of growth or the maximum growth a f t e r 24 h of incubation i n the presence of CC^. Strains were thus not capnophilic. Table 8 describes the growth c h a r a c t e r i s t i c s of a s e l e c t i o n of pathogens i s o l a t e d from s i t e s other than urine. The generation times and lag periods of these organisms were measured i n urine and compared with those of urinary i s o l a t e s . The generation times and lag periods of these organisms are c l e a r l y very s i m i l a r to those of urinary i s o l a t e s i n Tables 5 and 6. Although the number of organisms i n Table 8 i s too few to reach a firm conclusion, Table 8: Growth C h a r a c t e r i s t i c s of Pathogens from Sources other than Urine Organism S i t e of I s o l a t i o n Generation Time Lag Period (min) (h) Escherichia c o l i  E s cherichia c o l i  Proteus m i r a b i l i s  Proteus m i r a b i l i s  Staphylococcus epidermidis  Streptococcus f e c a l i s f e c a l u r e t h r a l i n t r o i t u s f e c a l perineum perineum perineum 22.5 21.0 40.1 38.0 82.0 57.0 1.27 1.17 2.01 1.25 2.19 1.85 i t appears probable that pathogens from other s i t e s may cause urinary t r a c t i n f e c t i o n s . In summary, the r e s u l t s i n d i c a t e that urine i s a good medium for organisms i s o l a t e d from both the urinary t r a c t and other s i t e s , j i . c o l i s t r a i n s and K l e b s i e l l a pneumoniae were the f a s t e s t growing organisms both i n urine and nutrient broth, whereas other pathogens generally grew more slowly i n both media. Slower growth of some organisms i n laboratory broth medium may be important i n diagnostic c l i n i c a l l a b o r a t o r i e s . Mixed Culture Experiments i n the Bladder Model Mixed populations of _E. c o l i and a second pathogen were i n t r o -duced into the bladder model at a r a t i o of 1:1 (approximately). For these p a r t i c u l a r experiments, urine flow into the bladder was 50 ml/h, the r e s i d u a l volume was about 1 ml and empyting frequency was every 4 hours. Changes i n b a c t e r i a l populations were monitored by determining v i a b l e ccunts at the i n t e r v a l s shown i n Table 9. Table 9 shows the r e s u l t s of mixed culture experiments i n the bladder i n which growth rates are expressed as the r a t i o of that of the second pathogen to E. c o l i at each sampling time. The use of a urinary i s o l a t e of _E. c o l i as a reference in each experiment helps compensate for v a r i a t i o n s i n the growth supporting properties of d i f f e r e n t batches of urine. It may be seen that _E. c o l i i s o l a t e s outgrew almost a l l other pathogens at a l l times under conditions which simulate the e f f e c t s of urine flow in the lower t r a c t . Changes i n the r a t i o s of K. pneumoniae to E. c o l i are consistent with r e s u l t s of observed generation times and lag periods (Tables Table 9: Comparison of the R e l a t i v e Growth Rates of E s c h e r i c h i a c o l i and other Organisms i n Urine. P a i r of organisms ino c u l a t e d i n t o bladder R a t i o * population of second u r i n a r y pathogen population of reference E. c o l i i n u r i n e sampled from the bladder model at _E. c o l i reference s t r a i n ( u r i n a r y ) Second organism O r i g i n of second organism 2h 4h 6h 24h A S t r e p t o c o c c u s . f e c a l i s i s o l a t e A u r i n a r y 0.8 0.9 1.2 0.02 B Streptococcus f e c a l i s i s o l a t e B " 1.1 0.4 0.3 0.009 B S e r r a t i a marcescens i s o l a t e A " 0.7 0.1 0.3 0.005 C S e r r a t i a marcescens i s o l a t e B " 0.7 0.2 0.4 0.003 A Pseudomonas aeruginosa " 0.7 0.4 0.1 0.01 B Proteus m i r a b i l i s " 1.2 0.08 0.03 0.002 B. Proteus r e t t g e r i " 0.2 0.01 0.003 0.002 B Proteus v u l g a r i s " 0.4 0.09 0.02 0.002 D Pr o v i d e n c i a s t u a r t i i s o l a t e A " 3.3 0.03 0.03 0.03 D Pr o v i d e n c i a s t u a r t i i s o l a t e B " 0.1 0.01 0.01 0.0002 E Staphylococcus saprophyticus i s o l a t e A „ biotype 3 0.15 0.008 0.006 0.000002 D Staphylococcus saprophyticus i s o l a t e B „ biotype 3 0.37 0.04 0.008 0.000005 F K l e b s i e l l a pneumoniae. " 2.7 4.6 2.7 0.6 F Staphylococcus epidermidis i s o l a t e A " 0.6 0.3 0.001 0.00008 F Staphylococcus epidermidis i s o l a t e B " 0.3 0.04 0.00004 0.000003 * See Methods s e c t i o n f o r method of c a l c u l a t i o n . Nominal r a t i o at the s t a r t of each experiment = 1.0. 59 6 and 7). The increase of the r a t i o during the f i r s t 4 h i s con-s i s t e n t with the longer lag period of E. c o l i than K. pneumoniae; _E. c o l i subsequently gradually outgrew the K l e b s i e l l a . These r e s u l t s support the importance of growth rates i n colonizing a b i l i t y of organisms i n a dynamic system such as the urinary bladder. The bladder model also o f f e r s an opportunity to study a n t i b i o t i c action under r e a l i s t i c conditions. I was p a r t i c u l a r l y interested i n the action of mecillinam and wished to compare the a c t i v i t y of t h i s agent i n vivo (in mice), in the bladder model, as well as in conventional experiments. A n t i b i o t i c Resistance Pattern of C l i n i c a l Isolates of Hospitals  i n Vancouver. This study was c a r r i e d out in order to provide a representative s e l e c t i o n of mecillinam r e s i s t a n t organisms for further in v i t r o and jLn vivo experiments i n mice. Resistance patterns of c l i n i c a l i s o l a t e s to a range of a n t i -b i o t i c s were f i r s t investigated by the a n t i b i o t i c s e n s i t i v i t y disk test i n two independent surveys two years apart. The surveys provided information to e s t a b l i s h a cut-off point for disk s e n s i t i v i t y tests f o r mecillinam since no standards are a v a i l a b l e in North America. The Kirby Bauer technique was used for both surveys (see Materials and Methods Section) and each one was analyzed separately. 1978 Survey The f i r s t survey used 999 Enterobacteriaceae i s o l a t e d from three major hospit a l s i n Vancouver, B.C., (The Vancouver General, St. Paul's and Shaughnessy Hospitals at a r a t i o of roughly 45:45:10). Organisms were l i m i t e d to those from a n t i b i o t i c s e n s i t i v i t y plates which had been f u l l y i d e n t i f i e d . Table 10 l i s t s the i d e n t i t y of a sample of 100 of the 999 organisms. _E. c o l i was the major organism i n the group, followed by Proteus spp., K. pneumoniae., Se r r a t i a spp., Enterobacter spp., Citrobacter spp. and Providencia spp. Disk tests were performed on the 999 c l i n i c a l i s o l a t e s using 8 a n t i b i o t i c s l i s t e d i n Table 11. Mecillinam was used at two concen-tratio n s (10 and 25 yg per disk) i n order to assess which one could be more useful. I n h i b i t i o n zones were then measured using a plate reader (5 x magnification) and the information was transferred to computer cards. Analysis of data was done on an IBM computer, using SPSS language, by Dr. B.J. Morrison of the Department of Health Care and Epidemiology. In some of the s e n s i t i v i t y plates, halos of l i g h t e r growth were formed around the mecillinam disks. In these cases the inner diameter of the halo was a r b i t r a r i l y chosen as a measure of the zone of i n h i b i t i o n . Isolated r e s i s t a n t colonies were also found within zones of i n h i b i t i o n around the mecillinam disks, these colonies proved s e n s i t i v e to mecillinam a f t e r sub-culturing on a n t i b i o t i c free medium and were ignored i n assessing zone diameters. Figures 2 and 3 show the i n h i b i t i o n zone s i z e d i s t r i b u t i o n of the 999 i s o l a t e s of Enterobacteriaceae to mecillinam disks. The i n h i b i t i o n zones with 25 yg mecillinam disks were quite large Table 10: I d e n t i f i c a t i o n of a Sample of 100 Organisms Obtained from Hospitals i n Vancouver (1978). Organism Percentage of Total Escherichia c o l i 46 Proteus m i r a b i l i s 15 Proteus r e t t g e r i 1 Proteus v u l g a r i s 1 Morganella morgani 1 K l e b s i e l l a pneumoniae 14 S e r r a t i a marcescens 7 S e r r a t i a l i q u i f a c i e n s 2 Citrobacter f r e u n d i i 3 Citrobacter spp. 1 Enterobacter cloacae 3 Enterobacter agglomerans 2 Enterobacter aerogenes 1 Enterobacter hafnia 1 Providencia s t u a r t i i 2 Table 11: D e t a i l s of A n t i b i o t i c Disk S e n s i t i v i t y Test A n t i b i o t i c Disk Supplier Organisms adjudged Content s e n s i t i v e i f i n h i -b i t i o n zone _> (mm) A m o x i c i l l i n 10 yg Beckton Dickinson 14 Trimethoprim 1.25 yg Beckton Dickinson 16 Cephaloridine 30 yg Difco Laboratories 18 Cephalothin 30 yg Difco Laboratories 18 Gentamicin 10 yg Difco Laboratories 13 Streptomycin 10 yg Difco Laboratories 15 Tetracycline 30 yg Difco Laboratories 19 Mecillinam 10 yg Leo Laboratories Ltd. see text Mecillinam 25 yg Leo Laboratories Ltd. see text F i g . 2: Zone s i z e d i s t r i b u t i o n of 999 Enterobacteriaceae u s i n g 10 yg mec i l l i n a m d i s k s . 2001 150 (0 E w 'c (0 O) 100 a> E 3 50 8 16 24 Inhibition Zone Diameter (mm) 32 64 F i g . 3: Zone s i z e d i s t r i b u t i o n of 999 Enterobacteriaceae using 25 yg mec i l l i n a m d i s k s . 2001 150 (0 (0 O) O100 «•— o n E 50 8 16 24 32 Inhibition Zone Diameter (mm) 65 (Fig. 3). Comparison of F i g . 2 and 3 showed that large zones (>25 mm) were obtained more than twice as frequently with the 25 yg disks as compared with the 10 yg disks. Such large zones may prove inconvenient i n the c l i n i c a l l a b o r a t o r i e s where several a n t i b i o t i c disks are crowded onto a s i n g l e s e n s i t i v i t y p l ate. Therefore 10 yg mecillinam disks were chosen as more p r a c t i c a l and 25 yg disks were not used i n the subsequent 1980 survey. Table 12 i l l u s t r a t e s antibiograms of the 999 c l i n i c a l i s o l a t e s of Enterobacteriaceae. The highest degree of resistance was observed to t e t r a c y c l i n e followed by a m o x i c i l l i n , cephaloridine, cephalothin, trimethoprim and f i n a l l y gentamicin. Table 13 shows the percentage of mecillinam resistance at d i f f e r e n t i n h i b i t i o n zone s i z e s . Since no standard cut off point e x i s t s f o r mecillinam s u s c e p t i b i l i t y t e s t s , a zone s i z e (16 mm) was a r b i t r a r i l y chosen as the cut-off point f o r a 10 yg mecillinam disk at t h i s stage (see l a t e r f o r evidence which r e t r o s p e c t i v e l y j u s t i f i e d the choice of t h i s value). One hundred and f o r t y nine organisms (14.9%) were found to have i n h i b i t i o n zones of 16 mm or les s when tested with 10 yg mecillinam disks. These bacteria i n addition to a number of randomly picked mecillinam s e n s i t i v e organisms (every 10th sample) were i d e n t i f i e d using API 20E system. Minimum i n h i b i t o r y concentrations of mecillinam were then determined for these bacteria by agar d i l u t i o n method (249 organisms). Figure 4 shows the d i s t r i b u t i o n of the test organisms according to the MIC values. The r e s u l t s indicated that E. c o l i s t r a i n s were mostly s e n s i t i v e to mecillinam (65%) whereas Se r r a t i a s t r a i n s had the highest degree of resistance (70%). Proteus spp., K. 66 Table 12: Antlblograms of 999 Enterobacteriaceae i s o l a t e d at Hospitals i n Vancouver. A n t i b i o t i c Percent Resistant A m o x i c i l l i n 55.0 Cephaloridine 49.5 Cephalothin 33.1 Gentamicin 5.4 Tetracycline 73.1 Trimethoprim 21.8 67 Table 13: E f f e c t of Choice of Cut-off Point for I n h i b i t i o n Zone Diameters upon Apparent Levels of Resistance to Mecillinam. Diameter of Zone Percentage of i n h i b i t i o n (mm) of organisms 're s i s t a n t ' at d i f f e r e n t i n h i b i t i o n zone s i z e c r i t e r i a with 10 ;u.g mecillinam with 25 y,g mecillinam disk disk 1 1 2 1 1 4 < 16 < 18 <_ 20 <_ 22 < 24 < 26 8.9 11.4 14.9 21.1 28.0 39.2 54.6 73.3 6.0 7.9 10.3 12.9 19.0 27.2 35.1 49.2 00 7 3 850f c CO •t-l CO w © o I 2d F i g . 4: D i s t r i b u t i o n of meci l l i n a m r e s i s t a n c e of 249 i s o l a t e s of Enterobacteriaceae (1978 Survey). n I I r E. c o l i [**"! K l e b s i e l l a spp. Proteus spp. S e r r a t i a spp. 1 1 1 I I 1 <o.5 2 4 8 16 32 64 128 Minimum Inhibitory Concentrations (jug/ml ) 256 512 >1024 pneumoniae and other members of Enterobacteriaceae were i n t e r -mediate. Figure 5 i l l u s t r a t e s the semilogarithmic r e l a t i o n s h i p between 10 yg mecillinam disk i n h i b i t i o n zones and MIC values. This f i g u r e i l l u s t r a t e s the r e s u l t s obtained for the 249 organisms i n our laboratory and also an a d d i t i o n a l 146 Enterobacteriaceae i s o l a t e d i n the same manner from hospit a l s i n New York. This provided a wide range of Enterobacteriaceae from North America which may lead to a more r e a l i s t i c i n t e r p r e t a t i o n of the r e s u l t s . The a d d i t i o n a l r e s i s t a n t organisms were c o l l e c t e d by Dr. R. Cleeland (Hoffman-La Roche, Nutley, New Jersey). Table 14 shows the d i s t -r i b u t i o n of the 146 organisms c o l l e c t e d from hospi t a l s i n New York. A semilogarithmic r e l a t i o n s h i p was established between the MICs for mecillinam"and the zone diameter around both 10 and 25 yg mecillinam disks (Table 15). There was a good r e l a t i o n s h i p between MIC values and i n h i b i t i o n zone sizes f or _E. c o l i and K. pneumoniae indicated by c o r r e l a t i o n c o e f f i c i e n t values ( l i n e s 1, 2, 5 and 6, Table 14). Overall a f a i r c o r r e l a t i o n existed for a l l organisms ( l i n e s 4 and 8, Table 15). The information i n Table 15 was then used to compute i n h i b i t i o n zone sizes which correspond to a range of MIC values (Table 16) with the aid of the following formula: Log^ MIC = Intercept + Slope X (diameter of i n h i b i t i o n zone). The zone siz e chosen as a break point i n c l i n i c a l l a b oratories should also r e f l e c t a t t a i n a b l e mecillinam l e v e l s i n serum. It o Fig. 5: Correlation of 10 yg mecillinam susceptibility test disk zone diameter with the minimum inhibitory concentration (1978 Survey) E 1 o : l28r 64 -32 -16 -8 -4 -2 -I -0.5-0.25 -0.12 -:0.06 -A A A ••A A o« A OB A O O O O D O • LEGEND * E.coli o K. pneumoniae • Enterobacter ° Serratia * P. mirabilis « P. rettgeri A P. morganii x P. vulgaris * P. stuartii e> Citrobacter I o 8 s . o 8 i : o 8 So oo _L _L _l_ 10 12 14 16 18 20 22 24 26 28 30 32 Zone diameter: mm Table 14: I d e n t i f i c a t i o n of 146 C l i n i c a l Isolates of Enterobacteriaceae Obtained from Hospitals in New York. Organism Percentage of Total Escherichia c o l i 44 K l e b s i e l l a pneumoniae 30 Enterobacter spp. 22 S e r r a t i a spp. 16 Proteus m i r a b i l i s 17 Proteus v u l g a r i s 17 Table 15: Data f or Relationship Between Minimum Inhibitory Concentrations of Mecillinam (mg/1) and the Size of the Zone of I n h i b i t i o n (mm) Around Mecillinam Disks. Mecillinam Organism Number of Intercept Slope Correlation Significance content of organisms i n c o e f f i c i e n t disks (yg) each group 10 Escherichia c o l i 44 1.107 -0^0701 -0.809 0. 00001 10 K l e b s i e l l a pneumoniae 22 2.386 -0.1127 -0.717 0. 00009 10 Proteus spp. 17 1.322 -0.0448 -0.237 0. 180 10 A l l organisms 99 1.758 -0.0850 -0.591 0. 00001 25 Escherichia c o l i 47 1.697 -0.0834 -0.867 0. 00001 25 K l e b s i e l l a pneumoniae 24 2.698 -0.1113 -0.637 0. 00041 25 Proteus spp. 18 2.881 -0.0958 -0.441 0. 034 25 A l l organisms 109 2.479 -0.1012 -0.661 0. 00001 Figures i n t h i s Table should be applied to the formula log MIC = intercept + slope x (diameter of zone of i n h i b i t i o n ) Table 16: Calculated Diameters of Zones of I n h i b i t i o n i n the A n t i b i o t i c Disk S e n s i t i v i t y Test f o r a Range of MIC Values. MIC : Computed diameter of zone of i n h i b i t i o n around a disk mg/1 containing: 10 yg mecillinam 25 yg mecillinam 1 20.7 24.5 2 17.1 21.5 4 13.6 18.5 8 10.1 15.6 16 6.5 12.6 74 has been shown that the average plasma l e v e l of mecillinam during multiple intravenous infusions of 273 mg every 4 hours reaches 3.2 yg/ml. The corresponding value f o r an o r a l regime of 400 mg of pivmecillinam (equivalent of 273 mg of mecillinam) i s 2.2 yg/ml. The MIC corresponding to an i n h i b i t i o n zone of 16 mm was calculated to be 1.49 yg/ml f o r a l l organisms. In view of the pharmacokinetic data, the 16 mm i n h i b i t i o n zone i n retrospect would appear to be a prudent value to choose f or the break point f o r c l i n i c a l l a b o r a t o r i e s . 1980 Survey One thousand specimens of Enterobacteriaceae were c o l l e c t e d from the same ho s p i t a l s as the 1978 survey (Table 17). C l i n i c a l i s o l a t e s were f i r s t tested for a n t i b i o t i c s u s c e p t i b i l i t y by disk t e s t . As explained above, a zone s i z e of 16 mm was chosen as the cut-off point f o r mecillinam resistance (using a 10 yg d i s k ) . Table 18 shows the resistance patterns of these organisms. The highest degree of resistance was observed with a m p i c i l l i n followed by c a r b e n i c i l l i n , t e t r a c y c l i n e and cephalothin. A high proportion of organisms were susceptible to mecillinam, cefamandole, c e f o x i t i n , trimethoprim, tobramycin and f i n a l l y gentamicin. Levels of resistance to mecillinam i n the two independent surveys (14.9 vs. 16.6%) were s i m i l a r . It must be noted that mecillinam was not used as a therapeutic agent during t h i s 2-year period. As in the 1978 survey i n addition to the organisms r e s i s t a n t by disk t e s t , a number of s e n s i t i v e Enterobacteriaceae ( i n h i b i t i o n zone >16 mm) were also selected and the MIC values measured by the agar d i l u t i o n technique. Figure 6 shows the d i s t r i b u t i o n of resistance Table 17: Identity of a Sample of 100 Enterobacteriaceae from Hospitals i n Vancouver (1980) . Organism Percentage of Total Escherichia c o l i 44 K l e b s i e l l a spp. 21 Proteus spp. 10 S e r r a t i a spp. 10 Enterobacter spp. 12 Citrobacter spp. 2 Providencia spp. 1 Table 18: A n t i b i o t i c Disk S e n s i t i v i t y Pattern of 1000 Enterobacteriaceae Isolated at Hospitals i n Vancouver (1980) . A n t i b i o t i c Disk content Resistant organisms Resistant ug i f i n h i b i t i o n zone organisms l e s s than (mm) % A m p i c i l l i n 10 C a r b e n i c i l l i n 100 Cefamandole 30 C e f o x i t i n 30 Cephalothin 30 Mecillinam 10 Chloramphenicol 30 Gentamicin 10 Tetracycline 30 Tobramycin 10 Trimethoprim 25 14 60.9 23 40.3 18 14.7 18 15.5 18 38.3 16 16.6 18 11.6 13 2.6 19 39.4 14 2.7 16 6.2 77 Percent Resistance 8 / A 2 n> 5" 3 c 3 * S oo o o o <t> 3 0) o' 3 o ro O) > ro 09 ro k O) S2 ro V o ro ^ 01 o •.i.v.v.v.v.sW.'.'. n • ft) 11 cu rt H-Co tn Tt 1 O rr fD C CD CD •a r-1 fD cr CD T> o o I-1 ft o rt CD fD rt H H O f* cr a4 Pi C 0 rt rt H" fD O 1 3 H-cu o O Hi fD 03 S ro ro o H-I— M VO H" 00 H-O 3 CU W 0 C H H < fD fD CD • rt (u 0 D fD CD o ro CD O Hi 78 f o r the 246 organisms tested. As i n the f i r s t survey _E. c o l i s t r a i n s were mainly s e n s i t i v e to mecillinam, and S e r r a t i a spp. showed the highest degree of resistance. The r e l a t i o n s h i p between MIC values and the i n h i b i t i o n zone sizes are i l l u s t r a t e d i n Figure 7. Correlation c o e f f i c i e n t s and other s t a t i s t i c a l parameters of data i n Figure 7 are l i s t e d i n Table 19. The r e s u l t s were s i m i l a r to the previous survey i n which a f a i r l y good c o r r e l a t i o n was observed for the regression l i n e f o r E. c o l i and K l e b s i e l l a spp. but not for Proteus spp. and S e r r a t i a spp. The o v e r a l l calculated MIC for a zone s i z e of 16 mm was 1.7 yg/ml. This value was s i m i l a r to that of 1.49 obtained i n the 1978 survey and confirmed the previous r e s u l t s . In summary, both surveys showed that a cut-off point of 16 mm may be chosen for resistance to mecillinam i n Enterobacteriaceae using 10 yg mecillinam disks. Synergy Between A m p i c i l l i n and Mecillinam i n v i t r o A c t i v i t i e s of a m p i c i l l i n , mecillinam and a 2:1 combination of the two agents were determined against 10 organisms (chosen from mecillinam surveys). The techniques used were agar d i l u t i o n , broth te s t s i n m i c r o t i t r a t i o n trays, and a disk t e s t . Minimum i n h i b i t o r y concentrations of each drug were measured and compared with the MIC of the combination of the two i n each test system. A f r a c t i o n a l i n h i b i t o r y concentration index (FIC) of le s s than 0.5 was taken to denote synergy (see Materials and Methods for a d e t a i l e d explanation of FIC). F i g . 7: Corr e l a t i o n of 10 Mg mecillinam s u s c e p t i b i l i t y test disk zone diameter with the minimum i n h i b i t o r y concentration (1980 Survey). -256 128 64 32 2 16 E ^ 81 ~ 4| O 21 5 1 0.5 0.25 ai2 0.06 11 A E. c o l i • Proteus spp. • K l e b s i e l l a spp. k. Enterobacter spp, Serrat i a spp. • Providencia spp. O Citrobacter spp. 8 10 12 14 16 18 20 22 24 26 28 Inhibition Zone Diameter (mm) 30 32 Table 19: Relationship Between Minimum Inhibitory Concentration of Mecillinam and the Zone Sizes Around 10 yg Mecillinam Disks f o r 246 Isolates of Enterobacteriaceae. Organism Number Intercept Slope Correlation c o e f f i c i e n c y Escherichia c o l i 95 4.06 -0.23 -0.82 K l e b s i e l l a spp. 34 5.68 -0.23 -0.77 Proteus spp. 47 5.16 -0.29 -0.3 Se r r a t i a spp. 39 5.65 -0.23 -0.28 A l l organisms 246 4.55 -0.25 -0.69 Table 20 shows the r e s u l t s obtained by the agar d i l u t i o n method. Synergy was observed f o r 4 out of the 10 organisms. The r e s u l t s of synergy studies i n the m i c r o t i t r a t i o n test i l l u s t r a t e d i n Table 21 show that values are dependent on the test medium. However, the v a r i a t i o n was only observed when mecillinam was used singly or in combination with a m p i c i l l i n and the MICs for a m p i c i l l i n were much less dependent on the medium. Overal l , synergy was observed for IS. c o l i #6, K. pneumoniae and J3. marcescens when tests were c a r r i e d out i n MH broth and no synergy was shown when urine was used as the test medium. The disk s e n s i t i v i t y test technique was applied to a m p i c i l l i n , medillinam, 2:1 combination as well as a 10:1 combination of the two drugs. Synergy was recorded when the i n h i b i t i o n zone of the combination of a m p i c i l l i n and mecillinam was 2 mm larger than each drug alone. Table 22 shows the r e s u l t s obtained by disk t e s t s . Synergy was observed with El. c o l i #2 and P_. m i r a b i l i s when the 2:1 combination was used. With a 10:1 combination, synergy was observed with E. c o l i / / l , //2, #6, _P. m i r a b i l i s and J3. marcescens. The in v i t r o test r e s u l t s f o r synergy were again dependent upon the tes t system and the medium. The c o n f l i c t i n g r e s u l t s for synergy with the same test organisms make i t d i f f i c u l t to choose an 'appropriate' experimental system which can give comparable r e s u l t s with _in vivo conditons. Study of Synergy Between A m p i c i l l i n and Mecillinam i n the Bladder Model. Table 23 shows the r e s u l t s of synergy experiments i n the bladder Table 20: Minimum Inhib i t o r y Concentrations of A m p i c i l l i n , Mecillinam and a 2:1 Combination Using the Agar D i l u t i o n Technique. Organism MIC's (yg/mi) FIC * A m p i c i l l i n Mecillinam A m p i c i l l i n + Mecillinam Escherichia c o l i / / l 2 0.125 0.5 + 0.125 >1 Escheri c h i a c o l i #2 4 0.125 0.25 + 0.125 >1 Escherichia c o l i #3 >1024 4 16 + 8 >1 Escherichia c o l i #4 512 16 0.25 + 0.125 0.08** Escheri c h i a c o l i #5 512 16 32 + 16 >1 Escheri c h i a c o l i #6 4 0.125 0.25 + 0.125 >1 Proteus m i r a b i l i s 1 8 0.25 + 0.125 0.27** Proteus r e t t g e r i 256 256 512 + 256 >1 S e r r a t i a marcescens 128 >1024 32 + 16 0.27** K l e b s i e l l a pneumoniae >1024 512 128 + 64 0.25** * FIC = F r a c t i o n a l i n h i b i t o r y concentration ** = Synergy present Table 21: Measurement of Minimum In h i b i t o r y Concentrations of A m p i c i l l i n , Mecillinam and a 2:1 Combination in Mueller Hinton Broth and Urine i n M i c r o t i t r a t i o n Plates. Organism MIC's i n MH Broth (Ug/ml) FIC MIC's in Urine (yg/ml) FIC * A m p i c i l l i n Mecillinam Amp + Mec Am p i c i l l i n Mecillinam Amp + Mec Escherichia c o l i #1 2 0.5 2 + 1 >0.5 2 2 2 + 1 >0.5 Escherichia c o l i #2 2 0.25 0.5 + 0.25 >0.5 4 0.125 0.5 + 0.25 >0.5 Escherichia c o l i //3 >1024 64 64 + 32 >0.5 >1024 32 64 + 32 >0.5 Escherichia c o l i #4 4 0.125 2 + 1 >0.5 4 0.125 1 + 0.5 >0.5 •Escherichia c o l i //5 >1024 >512 >1024 + >512 >0.5 >1024 256 1024 + 512 >0.5 Escherichia c o l i #6 16 8 0.5 + 0.25 0.06** 4 0.125 0.25 + 0.125 >0.5 Proteus m i r a b i l i s 2 32 1 + 0.5 >0.5 2 64 2 + 1 >0.5 Proteus r e t t g e r i >1024 >512 >1024 + >512 >0.5 >1024 >512 >1024 + >512 >0.5 'Serratia marcescens 128 >1024 32 + 16 <0.5 ** 128 2 4 + 2 >0.5 K l e b s i e l l a pneumoniae >1024 >1024 512 + 256 <0.5 ** >1024 64 64 + 32 >0.5 * FIC = F r a c t i o n a l i n h i b i t o r y concentration (see text for d e t a i l s ) ** Synergy (FIC <0.5) Table 22: Synergy Between A m p i c i l l i n and Mecillinam by the Disk Ti Organism Inhibitory zone (mm) with disks containing Inhibitory zone (mm) with disks containing A m p i c i l l i n Mecillinam A m p i c i l l i n 5 yg + Synergy* Ampiciflin Mecillinam Ampicillin 10 u g + Syngery , n ,,_ t M<wiiHn an. 9 S ut. 10 yg 1 UK Mecillinam 1 yg i o yg Escherichia c o l i #1 20.5 5 yg Mecillin m 2.5 yg 25 25 19.5 yg 20.1 Escherichia c o l i #2 11.25 14 12.8 10.0 18 Escherichia c o l i #3 6.6 Escherichia c o l i #4 8.5 20** 20 Escherichia c o l i #5 Escherichia c o l i #6 18 22.5** 20 18 17.5 22 Proteus m i r a b i l i s 19 16 22.5 18.5 17 23.8 Proteus r e t t g e r i K l e b s i e l l a pneumoniae 6 S e r r a t i a marcescens 12 14 * Synergy was deemed to be present (+) when the zone size of the mixture of drugs was 2 mm larger than each single agent. ** Growth was observed i n the zone. Table 23: A c t i v i t y of A m p i c i l l i n , Mecillinam and a 2:1 Combination in a Model of Human Bladder. Organism A n t i b i o t i c * Percent reduction i n v i a b l e count compared Synergy Rating to o r i g i n a l at: 0.25 h 0.5 h 1 h 3 h 4 h 24 h Escherichia c o l i #1 a m p i c i l l i n 52 99 100 100 100 mecillinam 0 15 100 100 100 amp + mec 95 99.7 100 100 100 + Escherichia c o l i #2 a m p i c i l l i n 29 91.3 100 100 100 100 mecillinam 8.7 0 37 100 100 100 amp + mec 75 99 100 100 100 100 + Escherichia c o l i #3 a m p i c i l l i n 0 0 0 0 0 mecillinam 10 10 99. 6 >99. 9 100 amp + mec 0 0 99. 7 99. 8 100 -Escherichia c o l i #4 a m p i c i l l i n 0 0 0 0 0 mecillinam 0 0 >99. 9 >99. 9 0 amp + mec 32 44 99. 9 >99. 9 98. 7 + Escherichia c o l i #5 amp i c i l l i n 0 0 0 0 0 mecillinam 0 0 99. 8 >99. 9 0 amp + mec 0 0 99. 4 98. 4 91. 2 + Escherichia c o l i #6 a m p i c i l l i n 85 92 100 100 100 mecillinam 50 75 99. 6 99. 7 100 amp + mec 99.5 99.8 100 100 100 + Table 23: Conti'd Organism A n t i b i o t i c * Percent reduction i n v i a b l e count compared Synergy Rating to o r i g i n a l at: 0.25 h 0.5 h 1 h 3 h 4 h 24 h Proteus m i r a b i l i s a m p i c i l l i n 75 97.4 >99.9 >99.9 100 mecillinam 7 0 27 97.8 99. 6 amp + mec 28 58 99.5 99.7 100 -Proteus r e t t g e r i a m p i c i l l i n 91. 2 97.9 >99.9 >99.9 70 mecillinam 0 79.5 92.2 87.5 80. 2 amp + mec 0 44 82.2 85.7 0 — S e r r a t i a marcescens a m p i c i l l i n 80. 6 96.9 >99.9 >99.9 98. 9 mecillinam 0 0 16.3 94.2 90 amp + mec 81. 5 90.8 100 100 100 — K l e b s i e l l a pneumoniae a m p i c i l l i n 0 0 0 0 0 mecillinam 0 0 0 0 0 amp + mec 0 0 64 73 77 + Denotes a m p i c i l l i n 500 yg/ml, mecillinam 250 yg/ml and amp + mec 250 + 125 yg/ml. ** Synergy r a t i n g of + indicates a 25% or greater reduction i n the v i a b l e c e l l count f o r the combination compared to the more ac t i v e single agent. A rating of + indicates a reduction of about 15% and - indicates le s s than 5% reduction i n the v i a b l e c e l l .count. model which are expressed as percent reduction of the o r i g i n a l number of b a c t e r i a . Of the 10 test organisms, synergy was present for _E. c o l i #1, #2, #4 and if6 at 1 h a f t e r being exposed to the mixture of a m p i c i l l i n and mecillinam, and for _E. c o l i //5 and K. pneumoniae a f t e r 3 h or more. No synergy was obtained with the remaining organisms. The r e s u l t s of synergy i n the bladder experiments did not co r r e l a t e with any of the conventional laboratory t e s t s presented above. These r e s u l t s confirm that synergy measurements in d i f f e r e n t systems are very much dependent on the experimental conditions and do not c o r r e l a t e with each other. Study of Synergy Between A m p i c i l l i n and Mecillinam i n the Mouse Model. Table 24 summarizes r e s u l t s of the mouse protection t e s t s . Synergy was observed for _E. c o l i #1, #2, #4, #6 and also P_. m i r a b i l i s . Comparison of these r e s u l t s with those obtained in the conventional tests again showed no agreement. However data obtained i n the bladder model and the mouse protection t e s t s did suggest a c o r r e l a t i o n . This i s i l l u s t r a t e d i n Table 25 where the test organisms are divided into three groups. The f i r s t group consists of organisms which responded to. the s y n e r g i s t i c action of a m p i c i l l i n and mecillinam within the f i r s t hour in the bladder model, and i n the mouse protection t e s t . The second group exhibited synergy in the bladder model at 3 h or l a t e r but no synergy was observed i n the mouse model. F i n a l l y , the t h i r d group where no synergy was detected i n either the bladder' model or the mouse system. In the bladder model a n t i b i o t i c concentrations increased i n the f i r s t 4 h and remained constant thereafter. In the mouse pro-Table 24: In vivo A c t i v i t y of A m p i c i l l i n , Mecillinam and a 10:1 Combination as Measured by Mouse Protection Tests. Organism A n t i b i o t i c * PD 5 Q : mg/kg sc** FIC *** Synergy Escherichia c o l i #1 Escherichia c o l i E scherichia c o l i E scherichia c o l i Escherichia c o l i E s cherichia c o l i a m p i c i l l i n 12 - 37** mecillinam <0.39 - 4.3 amp + mec 1.3 + 0.13 - 3.3 a m p i c i l l i n 9 - 2 8 mecillinam 1 - 3 amp + mec 1 + 0.1 - 4 a m p i c i l l i n >500 mecillinam >250 amp + mec >500 +>250 a m p i c i l l i n >500 mecillinam >250 amp + mec 115 + 11.5 - 156 • a m p i c i l l i n >500 mecillinam >250 amp + mec >500 +>250 a m p i c i l l i n 11.8 - 19.5 mecillinam 2.3 - 6.4 amp + mec <1.95 + <0.195 - 4.7 0.15 >0.43 + 0.4 0.21 - .27 >1 <0.27 - <0.37 >1 + 0.47 <0.24 - 0.31 + + + + Table 24: Cont'd Organism A n t i b i o t i c * PD 5 Q : mg/kg sc ** FIC *** Synergy a m p i c i l l i n mecillinam amp + mec a m p i c i l l i n mecillinam amp + mec a m p i c i l l i n mecillinam amp + mec K l e b s i e l l a pneumoniae a m p i c i l l i n mecillinam amp + mec Proteus m i r a b i l i s Proteus r e t t g e r i S e r r a t i a marcescens 12 - 51 108 - >250 4 + 0.4 - 31 + 3.1 >500 >250 >500 + >250 291 - 500 >250 >250 + >25 >500 >250 >250 + >25 0.33 - <0.61 >1 >1 >1 * Single subcutaneous treatment administered immediately af t e r i n f e c t i o n . ** Represents the range of PD,_q values for two in vivo tests with each organism; One value was given f o r r e s i s t a n t organisms where no change was noted i n the repeat determination. *** F r a c t i o n a l i n h i b i t o r y concentration. Synergy present when FIC <0.5. Table 25: Relationship Between Synergistic Responses Observed i n the Urinary Bladder Model and Mouse Protection Tests. Organism S y n e r g i s t i c * response noted in Synergy* i n the bladder model at: mouse protection • test l h 3 h 4 h 24 h Strains which exhibited synergy i n the bladder model within the f i r s t h Escherichia c o l i #1 + 0 0 0 + Escherichia c o l i #2. + • 0 0 0 + Escherichia c o l i #4 + 0 0 + + Escherichia c o l i //6 + 0 0 0 + Strains which exhibited synergy i Escherichia c o l i #5 0 0 K l e b s i e l l a pneumoniae 0 + Strains which showed no evidence Escherichia c o l i #3 0 0 Proteus r e t t g e r i 0 0 S e r r a t i a marcescens 0 0 Proteus m i r a b i l i s 0 0 the bladder model at 3 or more h 0 + 0 + + 0 f synergy i n the bladder model. 0 0 0 0 0 0 0 0 0 0 0 + * See text f or d e f i n i t i o n s of synergy 91 te c t i o n t e s t , treatment was c a r r i e d out with a si n g l e dose of a n t i -b i o t i c (s). By contrast, the concentration of the drug(s) i n the mouse model was i n i t i a l l y high then gradually decreased. Thus, i f the s y n e r g i s t i c b a c t e r i c i d a l e f f e c t i s rapid i . e . observed within the hour i n the bladder model, i t may be observed i n the ±n vivo test system. Detection of Beta-Lactamase i n C l i n i c a l Isolates of Enterobacteriaceae-Q u a l i t a t i v e Screening Test A representative sample of Enterobacteriaceae (117) was selected and tested f o r beta-lactamase production,; using a screening t e s t . Colonies were placed on starch papers soaked i n p e n i c i l l i n and Gram's iodine added at the end of the incubation period when clear zones around each colony indicated the presence of beta-lactamase (Table 26). The majority of _E. c o l i i s o l a t e s were not beta-lactamase producers. S e r r a t i a s t r a i n s on the other hand, were highly r e s i s t a n t to most a n t i -b i o t i c s including mecillinam (by disk tests and MIC values) but t h i s resistance did not seem to be beta-lactamase mediated (Table 26). K l e b s i e l l a pneumoniae and P_. s t u a r t i i i s o l a t e s were ac t i v e beta-lactamase producers and Enterobacter and Proteus spp. were intermediate. Quantitative Test f o r Beta-Lactamase Production A group of Enterobacteriaceae (77 selected from the mecillinam surveys) were tested for t h e i r beta-lactamase a c t i v i t y and an attempt Table 26: Screening Test f or Beta-Lactamase A c t i v i t y of C l i n i c a l Isolates of Enterobacteriaceae. Organism Total Number Percent beta-lactamase producers Escherichia c o l i 47 27.6 K l e b s i e l l a pneumoniae 22 68.1 Enterobacter spp. 8 37.5 Proteus spp. 17 35 Providencia spp. 5 80 S e r r a t i a marcescens 18 16.6 93 was made to co r r e l a t e these r e s u l t s with the mecillinam and a m p i c i l l i n MIC values of these organisms. The microiodometric technique was used to determine the beta-lactamase a c t i v i t y of crude enzyme extracts of organisms. A m p i c i l l i n and mecillinam were used as substrates. One unit of enzyme was defined as the amount- which destroyed 1 umole of substrate per min at 30°C per 1 0 1 0 c e l l s (Table 27A, B, C). Generally most organisms had lower MIC values f o r mecillinam than for a m p i c i l l i n . However, S_. marcescens, Proteus spp. and Providencia spp. did not follow t h i s general trend. The negative control ( l i n e 2, Table 27A) and p o s i t i v e control ( l i n e 29, Table 21k), are included to demonstrate the r e l i a b i l i t y of the test system. The p o s i t i v e control was an E. c o l i i s o l a t e that c a r r i e d an R-factor mediated beta-lactamase (TEMl). The enzyme from t h i s organism hydrolysed a m p i c i l l i n 100 times f a s t e r than mecillinam. The same r e s u l t was observed with beta-lactamase p o s i t i v e _E. c o l i , K l e b s i e l l a and Enterobacter i s o l a t e s but S e r r a t i a , Proteus and Providencia spp. sometimes had the same or f a s t e r rate of hydrolysis of mecillinam than a m p i c i l l i n . The method could detect preparations with a beta-lactamase a c t i v i t y of >0.01 units of enzyme. The majority of _E. c o l i , K l e b s i e l l a and Enterobacter spp. produced beta-lactamase but the amount of enzyme was not d i r e c t l y r e l a t e d to the MIC values for both a m p i c i l l i n and mecillinam. On the other hand the majority of S e r r a t i a , Proteus and Providencia spp. did not have detectable beta-lactamase a c t i v i t y despite the fact that most had high MIC values. The enzyme l e v e l i n these c e l l s did not thus appear to co r r e l a t e the MIC values. This r e l a t i o n s h i p Table 27A: Beta-lactamase A c t i v i t y of Crude Extracts of C l i n i c a l Isolates of Enterobacteriaceae. Organism MIC'is--'yg/ml Enzyme A c t i v i t y i n Units mecillinam a m p i c i l l i n mecillinam a m p i c i l l i n Escherichia c o l i c o l i 0.125 4.0 0.001 0.0051 " * 0.125 0.125 0.0014 0.0028 I I 0.125 4.0 0.002 0.0022 it 0.125 4.0 0.0012 0.0026 II 0.125 4.0 0.021 0.019 II 0.25 4.0 0.087 0.58 II 0.25 128.0 0.17 0.526 II 1.0 128.0 0.179 0.92 I I 2.0 16.0 0.018 0.045 I I 2.0 128.0 0.006 0.008 I I 2.0 256.0 0.087 2.25 I I 2.0 512.0 0.009 0.16 I I 2.0 512.0 0.087 0.21 I I 2.0 512.0 0.032 0.147 ti 2.0 1024.0 0.288 1.23 II 2.0 1024.0 0.02 0.15 II 4.0 256.0 0.002 0.282 I I 4.0 512.0 0.141 0.425 II 4.0 1024.0 0.033 0.125 II 4.0 1024.0 0.105 0.5 II 4.0 1024.0 0.17 0.44 I I 4.0 1024.0 0.093 0.373 II 4.0 1024.0 0.25 0.523 I I 4.0 1024.0 0.42 0.196 I I 8.0 1024.0 1.46 15:6: ti 8.0 1024.0 0.042 0.196 I I 16.0 512.0 0.063 0.34 I I 16.0 1024.0 0.086 0.284 it 64.0 256.0 0.048 0.414 * Negative control. ** P o s i t i v e control Table 27B: Beta-lactamase A c t i v i t y of Crude Extracts of C l i n i c a l Isolates of Enterobacteriaceae. Organism MIC's ;Ug/ml Enzyme A c t i v i t y i n Units mecillinam a m p i c i l l i n mecillinam a m p i c i l l i n K l e b s i e l l a pneumoniae 0.125 16.0 0.073 0.25 16.0 0.012 0.25 16.0 0.0086 0.5 64.0 0.0127 1.0 256.0 0.26 1.0 256.0 0.113 1.0 512.0 0.25 8.0 1024.0 0.46 8.0 .1024.0 0.3 8.0 1024.0 0.54 512.0 32.0 0.034 512.0 1024.0 0.064 0.139 0.128 0.458 0.14 0.813 0.473 1.61 1.23 1.11 2.86 0.42 0.7 Enterobacter spp, 0.5 1.0 8.0 8.0 1024.0 8.0 32.0 1024.0 1024.0 1024.0 0.0094 0.071 0.452 0.119 0.22 0.004 0.077 3.78 0.657 1.56 Se r r a t i a spp, 32.0 256.0 1024.0 1024.0 1024.0 1024.0 1024.0 1024.0 1024.0 1024.0 512.0 1024.0 - 32.0 32.0 128.0 512.0 1024.0 1024.0 1024.0 1024.0 0.0118 0.20 0.011 0.004 0.01 0.0059 0.0094 0.0016 0.39 0.245 0.0397 0.27 0.0103 0.02 0.002 0.0894 0.012 0.018 1.54 1.08 Table 27G: Beta-lactamase A c t i v i t y of Crude Extracts of C l i n i c a l Isolates of Enterobacteriaceae. Organism MIC's ug/ml Enzyme A c t i v i t y i n Units mecillinam a m p i c i l l i n mecillinam a m p i c i l l i n Proteus spp• Providencia spp • 0.125 1.0 0.046 0.055 0.125 1.0 0.009 0.047 1.0 1.0 0.288 0.066 1.0 2.0 0.129 0.24 1.0 128.0 0.038 0.038 2.0 128.0 0.6 2.43 2.0 512.0 0.015 0.0093 4.0 512.0 1.27 6.16 8.0 1.0 0.0003 0.017 8.0 1.0 0.04 0.033 8.0 2.0 0.133 1.35 16.0 512.0 0.506 2.7 32.0 2.0 0.082 0.096 64.0 1024.0 0.517 2.0 256.0 256.0 0.056 0.678 1024.0 8.0 0.11 1.21 1024.0 128.0 0.0306 0.028 1024.0 256.0 8.018 0.0138 1.0 2.0 0.024 0.073 4.0 128.0 0.279 1.89 1024.0 128.0 0.059 0.22 between beta-lactamase l e v e l s i n the c e l l free extracts, and MICs was examined s t a t i s t i c a l l y by p l o t t i n g enzyme a c t i v i t y against MIC values using l i n e a r / l i n e a r , l i n e a r / l o g a r i t h m i c , logarithmic/ l i n e a r , and logarithmic/logarithmic scales. Correlation c o e f f i c i e n t values were computed for each combination using a Hewlett-Packard c a l c u l a t o r . Ordinate (MICs) and abcissa (enzyme units) were entered and stored i n the c a l c u l a t o r memory p r i o r to c a l c u l a t i o n of the intercept, slope, and the c o r r e l a t i o n c o e f f i c i e n t for each p l o t . Table 28 i l l u s t r a t e s the computed c o r r e l a t i o n c o e f f i c i e n t measures (r) for a l l organisms when a m p i c i l l i n was used as the substrate and Table 29 shows the r e s u l t s with mecillinam as substrate. As shown i n Table 28, the best c o r r e l a t i o n was found when a logarithmic/ logarithmic scale was used. This however, was only true f o r E,. c o l i , K. pneumoniaeand Enterobacter spp. For mecillinam substrate (Table 29), c o r r e l a t i o n s were poor, and o v e r a l l no r e l a t i o n s h i p was observed between the beta-lactamase l e v e l i n crude extract of c e l l s and t h e i r MIC values. Generally, the r e s u l t s from these experiments show that a m p i c i l l i n resistance was to some degree re l a t e d to beta-lactamase l e v e l s present i n the preparations. This however, did not seem to be the case with mecillinam, judged by the slow rate of hydrolysis of the drug by most enzyme extracts and poor c o r r e l a t i o n c o e f f i c i e n t values. Figures 8 and 9 show these logarithmic/logarithmic p l o t s . Table 28: Relation between MIC's and A m p i c i l l i n Hydrolysis by Beta-lactamase for 77 C l i n i c a l Isolates of Enterobacteriaceae. Organism Number of Correlation C o e f f i c i e n t s for the Following Plots Organisms ~ _ — l o g / l i n l i n / l o g log/log l i n / l i n E scherichia c o l i 29 0. 2052 0.5806 0.7304 0.2615 K l e b s i e l l a and Enterobacter spp. 17 0. ,6702 0.5418 0.8120 0.6948 Proteus spp. 18 0. 4920 0.3760 0.3250 0.5000 Se r r a t i a spp. 10 0. 4162 0.6145 0.5529 0.4944 A l l organisms 77 0. ,2581 0.4653 0.4985 0.2793 Table 29: Relation between MIC's and Mecillinam Hydrolysis by Beta-lactamases for 77 C l i n i c a l Isolates of Enterobacteriaceae. Organism Number of Correlation C o e f f i c i e n t s for the Following Plots Organisms l o g / l i n l i n / l o g log/log l i n / l i n E scherichia c o l i K l e b s i e l l a and Enterobacter spp, Proteus spp. Se r r a t i a spp. A l l organisms 29 17 18 10 77 0.2418 0.1383 0.2148 0.0563 -0.0821 0.3143 0.02110 -0.2210 0.0264 -0.1442 0.5238 0.3280 0.3250 -0.0860 0.0881 0.01900 0.0973 -0.2439 -0.1017 -0.1415 F i g . 8: R e l a t i o n between MIC's and a m p i c i l l i n h y d r o l y s i s by beta-lactamases f o r 77 c l i n i c a l i s o l a t e s of Enterobacteriaceae. 101 F i g - 9 : Relation between MIC's and mecillinam hydrolysis by beta-lactamases f o r 77 c l i n i c a l i s o l a t e s of Enterobacteriaceae. 10 co E 3 >> 4-1 > 1 o < CO CO E - 0 1 _co I CO •t-> CD m Q.01 a o o i £ E. c o l i A Klebsiella-Enterobacter spp. • Proteus spp. Ser r a t i a spp. • ^ • ^ i -1 o | Q810 1 MIC's 102 14 Abortive Attempts to Demonstrate C-Mecillinam Binding to Intact  C e l l s and Membranes of 10 C l i n i c a l Isolates of Enterobacteriaceae. Labelled membrane proteins of 10 organisms (those used i n synergy experiments) f a i l e d to show any r a d i o a c t i v i t y on the X-ray f i l m s . The ra d i o a c t i v e mecillinam stock was unstable even though i t was stored as recommended by the manufacturer. The X-ray films were developed one month a f t e r storage at -80°C and due to the negative r e s u l t another f i l m was placed on the gel and was stored for f i v e months. The r e s u l t s were again negative, almost c e r t a i n l y due to the low s p e c i f i c a c t i v i t y of the drug (2.06U Ci/yg), and the unstable nature of the r a d i o a c t i v e preparation. Unfortunately more ac t i v e l a b e l l e d mecillinam preparations were not a v a i l a b l e . DISCUSSION 103 Pathogenicity Studies One of the major objectives was to study the contribution of the growth rate of organisms to the pathogenesis of urinary i n f e c t i o n s . Shake culture experiments demonstrated that a l l Gram negative and Gram p o s i t i v e test bacteria were capable of growth i n urine and most important of a l l , Escherichia c o l i had the f a s t e s t growth rate i n urine. The generation times measured for _E. c o l i i n urine were very s i m i l a r to those found i n nutrient broth (about 20 min). Pathogens other than _E. c o l i had longer generation times (5-60%) i n both urine and nutrient broth. Only K. pneumoniae had a growth rate s i m i l a r to _E. c o l i . S u r p r i s i n g l y a small sample of Gram p o s i t i v e bacteria and also a s i n g l e i s o l a t e of Proteus v u l g a r i s grew more slowly or did not grow at a l l i n nutrient broth under the test conditions. This unexpected r e s u l t led to the conclusion that b a c t e r i a l growth may be a s s i s t e d by dissolved CO2 i n urine. Further experiments i n f l a s k s using 5% CO2 gave the same r e s u l t s for these f i v e organisms. Only when a large inoculum was used (over 10^ cells/ml) was there v i s i b l e growth i n nutrient broth at 24 h. Urine contains very small amounts of glucose together with organic acids, urea as well as vitamins, hormones and a v a r i e t y of metabolic products and has a v a r i a b l e pH (116). Bacteria have to adjust to t h i s environment i n order to grow. Mutation and s e l e c t i o n may lead to the dominance of s t r a i n s that could grow more quickly i n urine. The organisms in the above experiments were c l i n i c a l i s o l a t e s from urine of patients with urinary t r a c t i n f e c t i o n s which may explain the unexpected behaviour of these bacteria in nutrient broth. Relative growth rates i n urine may explain the predominance of _E. c o l i i n f e c t i o n s i n the urinary t r a c t (7). Generally, i n the i n i t i a t i o n of i n f e c t i o n , a mixture of organisms are introduced into the human bladder as described in the Introduction. These organisms compete for growth and even though urine supports most organisms, the f a s t e r growing ones multiply at such a rate that soon they out-number others. In i n d i v i d u a l s with a normal urinary t r a c t , the mixed culture i s then voided to leave a small r e s i d u a l volume. This process i s repeated, and each time f a s t e r growing b a c t e r i a w i l l increase and eventually predominate or even r e s u l t in a pure culture of one organism. The above hypothesis was tested in the bladder model where 15 d i f f e r e n t mixtures of E_. c o l i and a second pathogen were introduced into the apparatus using a predetermined urine flow rate and emptying i n t e r v a l . The r a t i o of the population of the second pathogen to E. c o l i decreased over 24 h i n 14 out of 15 mixed culture experiments with the exception of K l e b s i e l l a pneumoniae. When t h i s organism was mixed with E. c o l i i s o l a t e F, the r a t i o increased up u n t i l 4 h and gradually decreased over-night giving a 24 h r a t i o s i m i l a r to the o r i g i n a l value. The lag period of K. pneumoniae was shorter than E_. c o l i F i n urine (0.82 h vs. 1.39 h respectively) but the generation times of the two organisms were s i m i l a r (22.4 vs. 22.1, Tables 6 and 7). Therefore, one may conclude that due to the d i f f e r e n c e i n lag periods, K. pneumoniae i n i t i a l l y predominated but was then slowly outgrown by jE. c o l i which had a marginally f a s t e r growth rate. Rapid growth rates of _E. c o l i may explain the predominance of 105 t h i s organism i n urinary t r a c t i n f e c t i o n s . On the other hand i n f e c t i o n s caused by Staphylococcus saprophyticus biotype 3 are rather d i f f i c u l t to explain i n these simple terms since t h i s organism does not grow ra p i d l y i n urine (3, 7). Therefore, _S_. saprophyticus biotype 3 must possess a v i r u l e n c e a t t r i b u t e ( s ) other than rapid growth rate in urine. _S. saprophyticus biotype 3 i s known to be urease p o s i t i v e and t h i s may contribute to i t s pathogenicity i n an analogous manner to Proteus spp. and Corynebacterium renale (12, 63). However, urease production does not i n i t s e l f account for the greater frequency of i n f e c t i o n of S^. saprophyticus biotype 3 because many non-pathogenic s t r a i n s are urease p o s i t i v e . Novobiocin resistance in S^. saprophyticus biotype 3 may be associated with virulence, though there i s no known explanation for t h i s a s s o c i a t i o n . Another p o s s i b i l i t y which could account for patho-g e n i c i t y of t h i s s t r a i n could be the a b i l i t y to colonize and attach to u r e t h r a l or v e s i c a l epithelium. However, there i s no experimental evidence l i n k i n g any of the above assumptions to the virulence of JS. saprophyticus biotype 3. This organism grew even more slowly in nutrient broth than i n urine. This slow growth may influence the outcome of conventional laboratory a n t i b i o t i c s u s c e p t i b i l i t y t e s t s . Therefore, one may speculate that i n the studies involving models of the urinary t r a c t , a medium such as urine and preferably from sus-c e p t i b l e patients would be more r e a l i s t i c than other laboratory media. V a r i a t i o n i n the urine batches may influence the lag period of organisms as well as the generation time depending upon the con-s t i t u t i o n of the medium. Also growth conditions of the inocula could not be c l o s e l y standardized. These may a f f e c t the r e l a t i v e performance of b a c t e r i a in mixed cultures from experiment to experiment. However, once the logarithmic growth phase s t a r t s , the major factor determining the dominant population would be the growth rate. In an i n vivo system, a n t i b a c t e r i a l properties of the urothelium, leukocytes, tis s u e i n f e c t i o n , frequency of c o l o n i z a t i o n or contamination of the d i s t a l urethra and virulence of the organisms are a l l factors which determine which organism w i l l become dominant. Relative growth rates i n urine appear, however, to be a major fa c t o r i n explaining the observed frequency d i s t r i b u t i o n of urinary pathogens. Note the r e l a t i o n s h i p between the d i s t r i b u t i o n of species i n c l i n i c a l i s o l a t e s (Table 1) and observed growth rates i n shake culture (Tables 6,7,8) or i n mixed culture i n the bladder model (Table 9). Staphylococcus saprophyticus however, i s an exception-. The a r t i f i c i a l bladder ^ apparatus i s a crude model of the lower urinary t r a c t which simulates conditions i n the early stage of i n f e c t i o n before the occurrence of t i s s u e colonization and upper t r a c t involve-ment . The bladder model has l i m i t a t i o n s as do a l l experimental systems. The p r i n c i p a l shortcomings were as follows: a. Experiments were time consuming. Individual synergy or pathogenicity experiments took from three to f i v e days. In p r a c t i c e the time factor l i m i t e d the number of experiments in each series of tests and prevented deta i l e d exploration of a number of variables such as urine flow rates, r e s i d u a l volumes, and a n t i b i o t i c concentrations. A r e s i d u a l volume of 1 ml was chosen for pathogenicity experiments to simulate values observed in women i n the community. However, in d i v i d u a l s prone to recurrent i n f e c t i o n have been shown to have a r a i s e d r e s i d u a l volume (77) and a value of 4 ml was chosen for a n t i b i o t i c synergy studies. The higher r e s i d u a l volume i s more l i k e l y to represent values found i n infected patients. Furthermore, the denser c e l l populations associated with increased r e s i d u a l volume present greater opportunities for beta-lactamase a c t i v i t y and a greater challenge i n therapy. b. The bladder model merely simulated the hydrokinetic clearance mechanisms of the human bladder and there was no simulation of the actions of the uroepithelium or various defense systems, including phagocytes or s p e c i f i c antibodies. c. No provisions were made to measure oxygen tensions during experiments i n the bladder, mainly due to technical problems. d. No provision was made for s t e r i l i z i n g the urine drain tube to the solenoid valve during experiments. Material at t h i s s i t e could have d i f f u s e d back into the main culture chamber. Furthermore, spray containing organisms which i s swept into the upper part of the apparatus might slowly return to contaminate l a t e r samples. Such contamination i s p a r t i c u l a r l y relevant with slow growing organisms, where the population would be expected to f a l l to very low values. However, despite t h i s reservation, most cultures from the a r t i f i c i a l bladder at 24 h would be reported as a s i g n i f i c a n t growth of a single s t r a i n i f submitted as midstream specimens of urine to conventional procedures i n a routine c l i n i c a l laboratory. C l i n i c a l l a boratories consider populations of >10^ organisms/ml as s i g n i f i c a n t and con-3 ventional techniques cannot usually detect 10 organisms or l e s s . A n t i b i o t i c Disk S u s c e p t i b i l i t y Studies One of the commonest ways to determine the e f f e c t of a n t i b i o t i c s on c l i n i c a l i s o l a t e s of bacteria i s to measure t h e i r s u s c e p t i b i l i t y to a n t i b i o t i c s by the disk t e s t . The i n h i b i t i o n zones around disks are measured and compared with standard controls of known cultures. Results of t h i s semi-quantitative test f a l l into three groups: s e n s i t i v e , intermediately r e s i s t a n t , or r e s i s t a n t to the test a n t i -b i o t i c s . Sensitive bacteria are generally cleared by therapeutic doses of a n t i b i o t i c s and r a r e l y cause problems. D i f f i c u l t i e s a r i s e when the test organism i s r e s i s t a n t or of intermediate resistance. In t h i s s i t u a t i o n , one may have to t r y newly improved a n t i b i o t i c s or a combination of two or more drugs which may act s y n e r g i s t i c a l l y where the i n d i v i d u a l components are not s u f f i c i e n t l y a c t i v e . One aim of t h i s study was to i d e n t i f y an i n h i b i t i o n zone break-point i n the mecillinam disk test to aid i n screening organisms for study and for epidemiologic work (9). To avoid s e l e c t i n g c l i n i c a l l y i r r e l e v a n t contaminants, only specimens which were i d e n t i f i e d i n Vancouver hospita l s were included i n t h i s study. The Kirby Bauer technique was chosen f o r these experiments and Mueller Hinton medium was used throughout. The choice of a test system which i s r o u t i n e l y used i n c l i n i c a l l a boratories i s p r a c t i c a l since mecillinam disks could then simply be included with other a n t i b i o t i c disks for routine t e s t s . An a r b i t r a r y cut off point of 16 mm i n h i b i t i o n zone was i n i t i a l l y chosen for the separation of r e s i s t a n t from s e n s i t i v e organisms using a 10 yg disk. Disks with 25 yg of mecillinam were not p r a c t i c a b l e since the i n h i b i t i o n zones were too large and overlapped other i n h i b i t i o n zones on the agar plate. Figure 5 shows the r e l a t i o n s h i p between MIC's and the i n h i b i t i o n zone sizes for 167 organisms selected i n Vancouver and 146 i s o l a t e s from New York h o s p i t a l s . The a d d i t i o n a l 146 organisms provide a more even d i s t r i b u t i o n among genera and reduce s e l e c t i o n bias due to geographical l o c a t i o n . The r e s u l t s showed that the majority of organisms with an i n h i b i t i o n zone of 16 mm or larger had MIC values of <1 yg/ml. Computation of the MIC value for a 16 mm i n h i b i t i o n zone from the regression l i n e i n Figure 5 gave a value of 1.49 yg/ml which was below the average plasma concentrations of mecillinam. Therefore one may conclude that the choice of 16 mm zone as the cut off point for mecillinam disk s e n s i t i v i t y test i s r e a l i s t i c and may be applied i n c l i n i c a l t e s t s . However, as with other a n t i b i o t i c s which are mainly excreted i n the urine, high concentrations of the drug could sometimes eradicate apparently r e s i s t a n t organisms from the urinary t r a c t . When the 16 mm cut off point was used as the reference i n the second survey (Figure 7), a s i m i l a r r e s u l t was obtained. Over-a l l 16% of the bacteria were r e s i s t a n t to mecillinam by the disk test and the majority of organisms with zone sizes of larger than 16 mm had MIC values of <2 yg/ml. (The computed MIC value f o r a 16 mm zone from the regression l i n e i n Figure 7 was 1.7 yg/ml). The two surveys were ca r r i e d out two years apart, and one may reach the following conclusions: a. There was no apparent increase i n mecillinam r e s i s t a n t population during the two year period. b. The i n h i b i t i o n zone cut o f f point value chosen i n these surveys (16 mm) correlates with a MIC which i s below the at t a i n a b l e plasma l e v e l s of mecillinam. In the i n f e c t i o n s of the urinary t r a c t , however, bac t e r i a are exposed to very high concentrations of t h i s drug and a smaller zone siz e may be appro-p r i a t e . Synergy Studies In v i t r o MIC determinations f o r i n d i v i d u a l a n t i b i o t i c s appeared to c o r r e l a t e with elimination of bacteria from the bladder model, i . e . when the MIC was high organisms were eliminated poorly and v i c e -versa. S i m i l a r l y , i n the mouse protection model which represents an acute systemic i n f e c t i o n , there was generally a good agreement between MICs r e f l e c t i n g c l i n i c a l l y achievable blood l e v e l s and the in vivo response. By contrast there was no s i g n i f i c a n t c o r r e l a t i o n between in v i t r o tests to determine synergy and synergy r e s u l t s i n the bladder model, or the mouse protection model. This lack of c o r r e l a t i o n was not due to s o l e l y defects that might be inherent i n any i n d i v i d u a l ^n v i t r o method',: since m i c r o t i t r a t i o n test and agar d i l u t i o n assay gave equally poor r e s u l t s i n terms of pr e d i c t i o n of synergy i n the two model systems. The a c t i v i t y of mecillinam alone, or i n combination was highly influenced by the media (e.g. urine vs. broth vs. agar) and test conditions. A m p i c i l l i n , on the other hand, I l l was not affected by the medium and MIC values for a m p i c i l l i n i n p a r a l l e l experiments using MH broth and urine were f a i r l y consistent within the experimental error range ( i . e . one d i l u t i o n v a r i a t i o n ) . The bacteria chosen for the study had a range of responses to a m p i c i l l i n and mecillinam _in v i t r o . The degree of s u s c e p t i b i l i t y or resistance of i n d i v i d u a l a n t i b i o t i c s did not appear to be a s i g n i f i c a n t factor i n synergy determinations. As Moellering pointed out, an t i m i c r o b i a l synergism i s d i f f i c u l t to measure i n a consistent and meaningful fashion which w i l l r e f l e c t the c l i n i c a l outcome (90). The synergy r e s u l t s of t h i s thesis c l e a r l y demonstrate that conventional i n v i t r o MIC assays for determination of the s y n e r g i s t i c p o t e n t i a l of the combination can be misleading. One of the biggest problems i n determining synergy by conventional i n v i t r o systems i s that a v a r i e t y of test system factors influence the outcome. For example, i n the agar d i l u t i o n method the b a c t e r i a l inoculum i s spotted on a small area on the agar containing a n t i b i o t i c and the test organism has access to a l i m i t e d amount of d i f f u s a b l e a n t i b i o t i c . In t h i s system, the r e s i s t a n t organisms w i l l produce i n d i v i d u a l colonies (<3 i s considered i n h i b i t i o n ) . In the micro-t i t r a t i o n assay, the l i q u i d phase test system allows free d i f f u s i o n of a n t i b i o t i c s to the bacteria; the t u r b i d i t y of the broth determines the end point of a n t i b i o t i c a c t i v i t y . In such a system, a few r e s i s t a n t bacteria (e.g. mutants selected by a n t i b i o t i c ) may produce high MIC values. Hence, the MIC values may be expected to be higher i n l i q u i d than i n s o l i d media. This was observed i n our experiments (Tables 20, 21) where higher MIC values were obtained i n the micro-t i t r a t i o n system than i n agar d i l u t i o n method. The s i t u a t i o n becomes 112 even more complicated (in both systems) with organisms producing beta-lactamase. Stressed and dead c e l l populations release enzymes which may r a p i d l y i n a c t i v a t e a n t i b i o t i c s . This can bring the a n t i b i o t i c l e v e l below the MIC value and therefore permit b a c t e r i a l gorwth. Therefore, high MICs could be obtained in c e r t a i n circumstances, even i f bacteria are b a s i c a l l y s e n s i t i v e to the test drug. In pus and s i m i l a r s i t u a t i o n s i n the body, b a c t e r i a l populations may contain a high proportion of dead or stressed organisms which not only release beta-lactamase but could also be slow growing and therefore r e l a t i v e l y r e s i s t a n t to beta lactam a n t i b i o t i c s . One may speculate that a n t i b i o t i c s may not neces-s a r i l y act i n a s y n e r g i s t i c manner i n v i t r o even i f i t i s observed i n an jLn vivo system. The checkerboard technique proved even more compli-cated and was abandoned af t e r several preliminary t e s t s . F i n a l l y , mecillinam a c t i v i t y even when acting alone, i s highly influenced by the test medium. This w i l l further confound attempts to c o r r e l a t e in  vivo response and conventional i n v i t r o experiments. Response to a n t i m i c r o b i a l agents in the bladder model at each of the i n i t i a l timed observations (Table 23) i s influenced by pro-g r e s s i v e l y r i s i n g a n t i b i o t i c concentrations. For example, i f no a n t i b i o t i c degradation occurred, introduction of urine containing 250 yg/ml of a n t i b i o t i c would give calculated concentrations of 24 yg/ml at 0.25 h, 75 yg/ml at 1 h, 235 yg/ml at 3 h and almost 250 yg/ml thereafter. By contrast, in the mouse model, a n t i b i o t i c l e v e l s are i n i t i a l l y high and then f a l l progressively. When synergy i n the bladder model occurred early, r e s u l t i n g in a rapid b a c t e r i a l clearance by the ampicillin-mecillinam mixture within one hour of the i n f e c t i o n , synergy was also observed i n the mouse model. In the mouse protection test, the i n f e c t i o n i s acute and overwhelming 113 and must be c o n t r o l l e d within the f i r s t few hours. Thus, i f the s y n e r g i s t i c b a c t e r i c i d a l e f f e c t i s rapid, i t should be observed i n t h i s model. In three out of four cases where no synergy was seen i n the bladder model, synergy was again not seen i n the mouse pro-t e c t i o n model (E. c o l i #3, _P. r e t t g e r i and S_. marcescens) the fourth case was equivocal (P. m i r a b i l i s ) . The three s t r a i n s which did not show synergy i n the mouse model were r e s i s t a n t to i n d i v i d u a l agents i n t h i s model while P_. m i r a b i l i s was susceptible to a m p i c i l l i n and marginally susceptible to mecillinam. Synergy was seen in the bladder model but not in the mouse model with two s t r a i n s (K. pneumoniae and E. c o l i #5) and neither of these s t r a i n s was completely eradicated at 24 h i n the bladder model. Synergy with _K. pneumoniae was f i r s t observed at 3 h, but only at 24 h with J l . c o l i #5 i n the bladder model. In the mouse model, both s t r a i n s were r e s i s t a n t to the i n d i v i d u a l agents and to the combinat ion. Thus, i t appears that the bladder model which measured synergy i n terms of an e f f e c t upon the balance between growth and washout, i s more c l o s e l y p r e d i c t i v e of synergy seen i n a warm-blooded animal than various conventional i n v i t r o assays. Although the bladder model i s valuable i n determining the growth c h a r a c t e r i s t i c s of bacteria, i t i s not p r a c t i c a b l e f or c l i n i c a l l a b o r a t o r i e s . However, a compromise sol u t i o n may be to use f i l t e r e d urine (pooled i f possible) for c e r t a i n selected c l i n i c a l t e s t s of s u s c e p t i b i l i t y to a n t i b i o t i c s . This may be valuable for s u s c e p t i b i l i t y tests with a n t i b i o t i c s such as mecillinam.or sulfonamides which are highly affected by the medium constituents. One such a p p l i c a t i o n may be i n the Abbott MS2 automated a n t i b i o t i c test system. However 114 since urine was seldom superior to broth or agar i n conventional t e s t s , the use of urine as medium in pr a c t i c e i n c l i n i c a l l aboratories i s l i k e l y to be very l i m i t e d . The Contribution of Beta-Lactamases to Mecillinam Resistance. Beta-lactamase production by bacteria plays a s i g n i f i c a n t r o l e i n resistance to beta-lactam a n t i b i o t i c s but i s not the sole factor i n determining beta-lactam resistance i n Gram-negative b a c t e r i a . Permeability b a r r i e r s , a l t e r a t i o n of the p e n i c i l l i n binding targets i n the inner membrane, and h y d r o l y t i c enzymes such as amidases and ac e t y l esterases (which act on d i f f e r e n t s i t e s of p e n i c i l l i n s and cephalosporins) also play important parts. Furthermore, the a c t i v i t y of beta-lactamases i s very dependent upon test conditions and va r i e s f o r i n d i v i d u a l a n t i b i o t i c s . An attempt was made to cor r e l a t e beta-lactamase l e v e l s i n c l i n i c a l i s o l a t e s of Enterobacteriaceae and MIC values. A c o r r e l a t i o n existed f o r a m p i c i l l i n hydrolysis f o r organisms from K l e b s i e l l a -Enterobacter group (r = 0.812) and _E. c o l i (r = 0.73). However, the c o r r e l a t i o n was poor for Se r r a t i a and Proteus spp. The r e s u l t s given i n Table 27 i l l u s t r a t e the beta-lactamase a c t i v i t y of c e l l -f r e e extracts and not in t a c t c e l l s . Therefore, one could only look at the rate of hydrolysis of the a n t i b i o t i c substrate by the enzyme without considering the b a r r i e r e f f e c t i n c e l l s . With t h i s provision, the rate of hydrolysis of mecillinam was much slower than a m p i c i l l i n , and generally the r e l a t i o n s h i p between enzyme l e v e l and MICs was very poor (note the r values shown i n Table 29), for mecillinam MIC values 115 seem to be independent of i n t r a c e l l u l a r beta-lactamase l e v e l s . Hence, the b a r r i e r factor may be more important i n b a c t e r i a l resistance to mecillinam than to a m p i c i l l i n . One may speculate that i f mecillinam could cross the permeability b a r r i e r , i t could act before beta-lactamases can i n a c t i v a t e the drug since i t s rate of hydrolysis was shown to be slower than a m p i c i l l i n (Tables 28, 29). Richmond (115) has shown that p e n i c i l l i n s pass through the outer layers of E. c o l i l e s s f r e e l y than cephalosporins. However i n t h i s same study, he demonstrated that mecillinam, even though more c l o s e l y r e l a t e d to the c l a s s i c a l p e n i c i l l i n s , behaved l i k e a cephalosporin ( s p e c i f i c a l l y cephaloridine) i n t h i s respect. Mecillinam thus seems to have the p o t e n t i a l advantage of both slow beta-lactam i n a c t i v a t i o n and rapid entry into the c e l l . Gram negative bacteria which are r e s i s t a n t to beta-lactam a n t i -b i o t i c s contain beta-lactamases which have been c l a s s i f i e d into 5 main classes (113). Type I l i a beta-lactamase which i s usually determined by an R-factor (R-Tem) i s probably the most important one to consider when evaluating new beta-lactam a n t i b i o t i c s . This p a r t i c u l a r enzyme has a broad spectrum of a c t i v i t y which includes both p e n i c i l l i n s and cephalosporins. Type I l i a beta-lactamase i s widely d i s t r i b u t e d through many Gram negative species and i t i s responsible f o r a m p i c i l l i n resistance i n H. influenzae type B (possessing a Tem type beta-lactamase but l i t t l e permeability b a r r i e r to a m p i c i l l i n (86)). It i s also responsible f o r c a r b e n i c i l l i n resistance i n c l i n i c a l l y serious circumstances among some s t r a i n s of P_. aeruginosa (75, 138). Later, t h i s same enzyme was found to determine p e n i c i l l i n resistance i n gonococcus s t r a i n s i s o l a t e d i n Liverpool and elsewhere (109). 116 Richmond (115) tested the a c t i v i t y of type I l i a beta-lactamase against mecillinam and found a value about 100-fold lower than that for a m p i c i l l i n at low substrate concentrations. This again shows that mec-i l l i n a m follows cephalosporins i n t h e i r s e n s i t i v i t y to type I l i a enzyme. Consequently, mecillinam i s only hydrolysed r a p i d l y by type I l i a enzyme at high concentrations which are well above the therapeutic l e v e l s . The r e s u l t s of beta-lactamase a c t i v i t y of c e l l free extracts of bacteria i n t h i s thesis showed thatmecillinam hydrolysis was several times slower than a m p i c i l l i n when i n t r a c e l l u l a r beta-lactamase was . detectable. There i s thus good agreement with Richmond's findings with a very small sample of organisms and mutants. Although t h i s pattern was observed with the majority of Enterobacteriaceae, two Proteus i s o -l a t e s proved exceptions. Nevertheless, one may make the general s t a t e -ment that mecillinam i s more r e s i s t a n t to the beta-lactamase ( e s p e c i a l l y R-Tem type) of Gram negative c l i n i c a l i s o l a t e s . This property w i l l obviously enhance i t s p o t e n t i a l as a therapeutic agent. The r e l a t i v e s t a b i l i t y and d i f f u s i b i l i t y of mecillinam may explain i t s s y n e r g i s t i c behaviour with a m p i c i l l i n . One may speculate that i t renders the c e l l s more permeable by p a r t l y damaging the d i f f u s i o n b a r r i e r s and permitting entry of a m p i c i l l i n . I t i s conceivable that in some circumstances t h i s p a r t i a l damage i s not s u f f i c i e n t to k i l l organisms when mecillinam acts alone. Another p o s s i b i l i t y i s that mecillinam may i n t e r f e r e with the destruction of a m p i c i l l i n by beta-lactamases and therefore, p a r t i a l l y protect a m p i c i l l i n . The a d d i t i o n a l r o l e of the d i f f e r e n t binding s i t e s for these two a n t i b i o t i c s has already been discussed at length in the Introduction and may be of great importance i n explaining synergy between mecillinam and other beta-lactam a n t i b i o t i c s . 117 K i n e t i c studies using urine as a growth medium, and i n p a r t i c u l a r the use of a bladder model have provided a unifying explanation of many features of both the pathogenesis and treatment of urinary i n f e c t i o n s . Studies of b a c t e r i a l growth rates i n shake cultures and mixed culture experiments i n the bladder model, provided support for the hypothesis that d i f f e r e n t i a l growth rates i n urine tend to determine the dominant population of i n f e c t i n g organism from a mixed population. 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In: Can. J. Public Health J72 (1). Anderson, J.D., Banerjee, M. and Eftekhar, F. 1982. Investigation of a possible dual r o l e of clavulanic aci d i n the treatment of a m o x i c i l l i n r e s i s t a n t urinary i n f e c t i o n s . Manuscript prepared f o r pu b l i c a t i o n . 

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