A Theoretical and Experimental Analysis of Bacterial Growth in the Bladder

Motecutar Microbiotogy (1992) 6(4), 555-562

A theoretical and experimental analysis of bacterial growth in the bladder

David M. Gordon''' and Margaret A. Svanborg-Eden, 1978). There are severat different Department of Zoology, University of f\/lassachusetts. adhesin proteins expressed in E. coli. such as those Amherst. Massachusetts 01003, USA. encoded by the pap (eyetonephritis-associated Qiti), prs (eap-retated sequence), pilC, and afa/operons (Arthur et ai, 1989; Katlenius et ai,, 1982; Labigne-Roussett and Summary Falt^ow, 1988; O'tHantey etai. 1985; Ptos etai. 1989). A mathematical model of human micturition dyna- The potentiat rote of btadder hydrodynamics on the mics and bacterial growth predicts the population etimination of bacteria from the btadder has tong been growth rate required for a bladder infection to recognized (Boen and Sytwester, 1965; Cox and Hinman, become established in the absence of adhesin-medi- 1961; Dugdale, 1969; Hinman. 1968; Hinman and Cox. ated surface growth. Escherichia co//strains isolated 1966; Mact^intosh et ai, 1975a,b; O'Grady and Cattelt. from the urinary tract have significantly higher in 1966). tt has been suggested that without adhesin-medi- vitro growth rates in urine than strains isolated from ated surface growth E. co//coutd not overcome the tosses the intestinal flora. The results suggest that, for E. due to micturition, and hence could not estabtish poputa- coli isolated from the urinary tract, adhesin-mediated tions in the urinary tract (Arthur ef ai, 1989; Reid and surface growth may not be required for infections to Sobet. 1987; Svanborg-Eden, 1978). tmpticit in this become established and persist. The growth-rate dif- hypothesis is the suggestion that the growth rate of E. coti ferences observed between urinary tract and Intesti- in urine is insufficient to overcome these tosses. nal isolates suggests that the ability to survive and The purpose of this study was to determine if E. coii is efficiently utilize the resources available in urine is an abte to cotonize the human bladder in the absence of important adaptation for E. coli inhabiting the urinary adhesion. First, we present a mathematicat modet of tract. • - human micturition dynamics and bacteriat growth. The modet represents a 'worst-case scenario'; it assumes that there is no surface growth. The model predicts the growth Introduction rate required for the estabtishment and maintenance of Escherichia coii is the species responsible for most bacte- bacterial poputations in the btadder in the absence of rial urinary tract infections in humans (Svanborg-Eden, adhesion. We then determine the in vitro growth rates, in 1978). Atthougti the commensat intestinat flora have been urine, of E. coii isotated from the intestinal ftora and uri- impticated as the prinnary reservoir of infection (Boltgren nary tract infections. and Winberg. 1976; Caugant. 1983; Turck et ai, 1962; Vosti etai, 1964), certain properties are observed among Results urinary tract strains of E. co//that are not prevalent among intestinat isotates. tjrinary tract isotates have higher fre- A modei of bacterial grov\nh in the biadder quencies of O and K antigens, haemotysin production, The modet predicts the fate of a smatl number of bacteria aerobactin retease. serum resistance and adhesins that have succeeded in reaching the btadder. We assume (Arthur et ai., 1989; Doofson et ai, 1973; Glynn e^ ai, that the bladder fitis at a constant rate A, (mt h"^). fvlicturi- 1971; Kaijserefa/., 1977;Orsf

The establishment condition states that the number of bacteria produced between micturition events must exceed the number that are tost due to micturition. The number of bacteria produced is a function of their growth rate and the time availabte for growth to occur. The dura- tion of the growth period is determined by the rate of urine production and the amount of urine that accumulates in the btadder between micturitions. Figure 1 presents growth rate (y) extinction-establishment boundaries as a function of ftow rate {X), and maxi- 0 15 30 45 60 75 90 105 128 135 IS Urine Flow Rate (mt/h) mum (V^) and residuat (VJ btadder votumes. If ttie bacteriat poputation has a growth rate which ties above a boundary it witt estabtish a poputation. When the poputation growth rate ties betow a boundary the bacterial poputation witt not increase its numbers and witt eventuatty be 03 wastied out of the btadder. To infer the growtti rate B Establishment required for estabtishment of an E. coii poputation in ttie IT human btadder requires a t^nowtedge of typicat micturition dynamics in humans. IO tn heatthy adutts, normat urine production ranges from Extinction 1-2 titres per day, resulting in ftow rates (X.) that span

"0 0.0025 0005 00075 O01 00125 0015 00175 0 OS" 40-80 mt h"^ (Mundy etai., 1984). tWicturition resutts in tVlinimum / Maximum Btadder Votume the retease of 200-400 mt of urine (V^) (tVlundy et ai., Fig. 1. The growth rate extinction-establishment boundary for bacterial 1984). The votume (V^) of urine remaining in the bladder populations as a function of human micturition dynamics. If the bacterial foltowing micturition is about 1 mt (Shand etal, 1968). growth rate lies below the boundary the baderia will fail to establish; if Figure la presents ttie growth-rate boundary for a above, the population will estabtish and be maintained. A, Effect of the rate of urine production (/,) on the boundary. Residual range of ftow rates white assuming that 300 mt of urine is bladder volume (V,,) was 1 ml and maximum btadder volume (I/,) was released at micturition (V,) and 1 mt remains (V^). This 300 ml. figure ittustrates that the growth rate required for estab- B. Effect of the ratio of residual :maximum bladder volume on the boundary. The flow rate (X) was 60 ml h~'. tishment is a tinear function of the rate of urine production (k). Figure 1b demonstrates the effect of residuat votume (expressed as ttie fraction V^fV^) on the growth-rate grows at a constant rate \\> (h ^). The vatidity of this boundary. The ftow rate was 60 ml h^' and V^ was 300 ml. assumption in ttie context of the modet is demonstrated in This figure shows that, except for very smalt vatues of V^. a tater section of this paper, tn the time between micturi- a change in residuat volume has tess effect on the growth tions, ttie number of bacteria in the btadder is given by rate required for establishment than an equivatent per cent change in the urine ftow rate (X). Rates of urine production (X) and the amount of urine where NQ is the number of bacteria present in the residuat produced at micturition (V^) normatty vary for a number of urine. Ttiese two equations specify our modet. and with reasons. To investigate the effect of this variation on the these we can determine the condition for estabtishment. estabtishment condition, modifications were made to the That is, given the dynamics of urine ftow through the blad- basic modet. The modifications consisted of atlowing vari- der, we can ast^ what growth rate the bacteriat poputation ation in either the amount of urine voided at micturition must exhibit in order to estabtish and persist in the blad- (V^), or ttie rate of urine production (X) during the intervals der, assuming no adhesion. between micturitions. Vatues of I/, or X were setected ran- The intervat T(h) between micturition events is domly from a normat distribution with a given mean and T={V,-V,)/X. standard deviation. The simutations were initiated witti Let Nh be the number of bacteria remaining in the btadder 100 cetts present in the btadder. Estabtishment occurred after the k[U micturition. Then if the number of bacteria grew to exceed 10'' cetls. and extinction occurred if the poputation dectined to less ttian 10 celts. Ttie parameter vatues used were: Vn=^ mt, The bacterial poputation is growing if, and onty if, l/;,= 300 mt and ?. = 60 ml h"V In simulations examining ^^;>Nh. that is when the effect of variation in X, 60 mt h"' represented ttie or mean ftow rate, while V^ was hetd constant at 300 mt. The Bacteriat growth in the bladder 557

or 120 mt, a growth rate of 1.2 h '' was required for more than 50% of the simulations to result in estabtishment. The resutts show that random variation in X or I/, can resutt in estabtishment even when the growth rate is tess than required and that this occurs more often as the variation in X or V^ increases. tHowever, in att cases the proba- bitity of estabtishment increases as the growth rate increases. Variation in the amount of urine voided has a greater effect on the frequency of estabtishment than does variation in the rate of urine production, tn these BO 100 120 140 160 180 200 Time (h) simulations the rate of urine production affects the time avaitabte for bacteriat growth (7"), whiie variation in the Fig. 2. Simulations of the modei to illustrate the effect of variation in the volume of urine voided at micturition on the change, through ttme. in the amount voided affects both the time avaitabte for growth, number of bacteria {Nt,) in the bladder. In these simulations the amount of and the proportion of the bacteriat poputation tost at mic- unne produced at micturition (I/,) was different for every micturition event. turition {l/^/VJ. The volumes were randomly selected from a normal distribution having a mean of 300 ml and a S.D. of 60 ml. Other parameter values were: V„=^ tn addition to random variation, the rate of urine produc- ml, ^ = 60 ml h ', (|i = 1.0 h"'. Symbols represent the results of the tion or the frequency of micturition vary in a periodic man- simulations and the solid straight line the solution of the deterministic modet. ner. A series of modifications were made to the basic modet that mimicked these periodic effects. For exampte, micturition often does not occur during the night. Simuta- Opposite was true in simulations investigating ttie effect of tions were done ttiat assumed a constant rate of urine variation in V^. The effects of three levels of variation production (?t = 60 mt h"') and mimimum btadder volume (S.D.) in X or V^ on estabtishment success were exam- (Vn=1 mt). The intervats between micturitions were ined. For each tevet of variation, simutations were carried varied such that there was one tong interval (7) between out for different vatues of a constant bacteriat growth rate. voidings. followed by severat shorter intervals — for For every growth rate/standard deviation combination, exampte, one 8 h intervat foltowed by four 4 h intervals. 1000 simulations were run, and ttie number of times the By hotding the flow rate constant and varying the fre- bacteriat population estabtished or went extinct was quency of micturition we are varying the volume of urine recorded. voided at micturition {V^). At! of these simutations had Figure 2 presents the resutts of two simutations in which I/, was attowed to vary, and ittustrates the change in the number of bacteria (W;,) through time retative to the Table 1. The results ol simulations exploring the effect of variation in the predictions of the deterministic modet. In these simula- rate of urine production [X.) or the amount of urine voided (V,) on the tions, bacteriat numbers fluctuated considerably but in establishment success of bacteria in the bladder. both cases the populations met the establishment crite- Establishment Success (%) rion. The resutts of the complete simulation anatysis are presented in Table 1. Given the parameter vatues used in Rate of urine oroduction these simutations, the modet predicts that the bacteria >., Mean±S.D. shoutd estabtish and maintain a poputation in the btadder Growth rate 60±6 60±12 60±24 1.00 0.0 0.0 28.9 provided that the growth rate exceeds 1.145 h"\ 1.10 0.2 23.7 50.3 Variation in the rate of urine production (k) had no sys- 1.15 75.9 67.0 59.6 tematic effect on the growth rate required for estabtish- 1.20 99.6 88.3 68.6 1.25 100.0 97.0 77.6 ment as defined by the deterministic modet. Poputations 1.30 100.0 99.3 83.8 were more tikely to establish when the growth rate exceeded the rate specified by the model (1.145 h"'), and Volume of urine voided were more likety to become extinct when the growth rate V,, Mean±S.D. was less than that required. For exampte, when the Growth rate 300±30 300+60 300+120 1.00 0.0 0.1 0.1 growthi rate was 1.15 h"'. the majority of simutations 1.10 0.0 0.6 10.4 resutted in the poputation becoming estabtished, regard- 1.15 62.1 35.0 28.9 tess of the degree of variation in the rate of urine produc- 1.20 99.6 84.3 51.8 1.25 100.0 95.5 63.3 tion. 1.30 100.0 98.7 71.1 Variation in the amount of urine voided (V^) had ttie The frequency of establishment was based on the results of lOOO effect of stightty increasing the growthi rate required for simulations. Parameter values used in the simulations were: V,, = l ml, establishment. When the standard deviation in ^, was 60 U, =300 ml, and >. =60 ml h"'. 558 D. M. Gordon and M. A. Riiey

density of about 10^ cells ml ', and similar results were obtained. In urine, the growth rates of 12 E. co/; clones isolated from urinary tract infections ranged from 1.4-2.2 h"' and lag-phase durations spanned 1-3 h (Table 2). The growth rates of 10 intestinal clones ranged from 0.9-1.6 h"^ and their lag-phase durations spanned 2.0-4.5 h (Table 2). The average growth rate (1.91 h~') of the urinary tract clones was significantly higher than the average growth rate (1.2 h^^) of the intestinal isolates (F,i 64) = 143.2, 3 4 5 6 89 p<0.001). This difference in growth rate was not obtained Time (h) when the growth medium was Luria broth (Table 2); the average growth rate of both the urinary tract and intestinal clones was 1.9 h~\

Discussion We have presented a mathematical model of micturition dynamics and bacterial growth. For bacteria to successfully establish a population, without surface growth, they must grow at a rate determined by the rate of urine production (X), the amount of urine released at micturition 1 2345676 (V^), and the volume of urine remaining in the bladder fol- Time (h) lowing micturition {V^). Fig. 3. The growth of urinary tract isolates under different culture The model is a deterministic one; in reality, rates of conditions. A. Growth of UTI strain 18 in urine (•) and in broth (•). The growth rate in urine was 1.93 h"', and in broth, 2.27 h^'. B. Growth of UTI strain 14 under conditions of good agitation and aeration (•) (growth rate = 1.64 h"'), and no agitation and poor aeration Table 2. Growth parameters of E. coliin human urine and Luria broth. (D) {growth rate =, 1.57 h~'). Each point represents the average of three replicates. Urine Luria Broth Growth Lag Growth rate phase rate strain (h-') (h) (h-') Source simitar outcomes {results not presented}. There was i either no systematic effect on the growth rate (v) required un Type' 14 for establishment, or establishment occurred when the C 1.99 1.0 1.78 Arthur efa/. (1989) 17 C 2.16 2.0 2.11 Arthur efa/. (1989) growth rate was smaller than the rate predicted by the 18 P 1.93 1.5 2.02 Arthur efa/. (1989) basic model. 23 P 1.93 1.5 2.07 Arthur efa/. (1989) 36 C 2.08 1.0 1.99 Arthur efa/. (1989) 45 c 1.91 1.5 1.77 Arthur e(a/. (1989) E. coli growth rates 175 s 2.04 1.5 1.86 Arthur efa/. (1989) 475 s 2.23 3.0 1.93 Arthur efa/. (1989) 70 A Strains isoiated from urinary tract infections grow weli in 1.92 1.5 1.81 Caugant (1983) 72 A 1.91 1.5 1.93 Caugant {1983) human urine. Figure 3a shows the change in ceii density 52 C 1.53 1.5 1.81 Caugant {1983) of strain 18 in urine and Luria broth. The exponentiai- 67 P 1.36 1.5 1.80 Caugant (1983) phase growth rate in urine was 1.93 h~^ and in broth was Intestinal 2.27 h~\ In urine, the stationary-phase density averaged 4 1.23 2.0 1.82 Caugant (1983) 9.9x10^ceils mi"\ and 1.2x10^ceils mr^ in broth. 10 1.04 2.5 1.89 Caugant (1983) 14 1.55 3.5 2.07 Caugant (1983) No difference in the exponential phase growth rate of 20 1.41 2.0 2.07 Caugant (1983) UTi strain 14 could be detected when the cells were 29 1.16 4.5 1.84 Caugant (1983) grown in well-aerated urine (1.69 h"^), relative to cultures 42 1.16 2.0 1.67 Caugant (1983) 51 that were poorly aerated (1.57 h"^) (Fig. 3B). The cell- 1.45 3.0 1.85 Caugant (1983) 55 0.98 3.0 1.64 Caugant (1983) density curves diverged as stationary phase was 140 0.92 4.5 1.87 Arthur efa/. (1989) approached, suggesting that the growth rate declines in 141 1.23 3.0 1.95 Arthur efa/. (1989) the poorly aerated cultures once high cell densities are a. Patient diagnosis: C, cystitis; P, pyelonephritis; S, urosepsis; A, asypm- achieved. This experiment was repeated, using an initial tomatic. Bacteriai growth in the biadder 559

growth rate and resource concentration. Establishment success is determined by the growth rate during the early phase of colonization. During this stage, bacteriai density is low and resources enter the bladder at a rate greater than the rate at which the bacteria can consume them. Once the bacteria are at a density sufficient to cause significant resource depletion, thereby reducing their growth rate, successful establishment has occurred. Our assumption that the bacterial growth rate is constant during the early phase of colonization is supported by the 0 15 30 45 60 75 90 105 IM 135 1! Urine Flow Rate (ml/h) empirical evidence. When urine is inoculated with about 10'' cells ml"^ the growth rate remains constant until cell density has increased by more than four orders of magni- tude, an increase that requires about 4 h (Fig. 3B). Previous studies have demonstrated the importance of bladder hydrokinetics on clearing bacterial infections from the bladder (Boen and Sylwester, 1965; Cox and Hinman, 1961; Dugdale, 1969; Hinman, 1968; Hinman and Cox, 1966; Mackintosh et ai. 1975a,b; O'Grady and Cattell, 1966). However, our interest was in determining if the growth rate of E. coli in urine, is sufficient to overcome

0.0025 0005 0.0075 0.01 00125 0.015 0.017S 0.02 the mortality associated with micturition. In normal adults, Minimum / Maximum Bladder Volume typical values for the parameters of the model (X = 60 ml h~\ l/^= 300 ml, \/n= 1 ml) suggest that E. co//attempting Rg. 4. Growth rates of urinary tract and intestinal strains in relation to the extinction-establishmeni boundary. Micturition parameter values defining to establish a population in the human bladder must grow Ihe boundary are as in Fig. 1. The parallel lines bound the mean growth at a rate that exceeds 1.2 h"^, Is the growth rate of E. coli rate ±1.0 S.D. of the unnary tract and intestinal isotates. The shaded sufficient to allow it to colonize the bladder in the absence region indicates the range of ftow rates (A) or minimum/maximum bfadder volumes {B) where the model predicts the bacteria would fail to establish of surface growth? a population in the bladder. The empirical results suggest that the E. co//clones isolated from urinary tract infections would be successful in urine production and the volume of urine released at mic- establishing a population in the bladder. Figure 4 pre- turition vary for many reasons. Simulation results demon- sents the extinction-establishment boundaries assuming strate that incorporating random or periodic variation in typical micturition parameter values. The parallel lines the rate of urine production {X) or the voiume of urine encompass the range defined by the average growth rate voided at micturition (V^) has a minor effect on the estab- ±1.0 S.D. Given the observed growth rates, these strains lishment condition defined by the deterministic model. would fail to establish only if normal daily urine production Our model does not predict the equilibrium number of exceeded 2 I d"^ (Fig. 4a), or if the residual volume of bacteria in the bladder, i.e. the value of W^^i, when N^^.^ = urine was unrealistically low (Fig. 4b). Wfc. The system described by the model is not self-regulat- Less clear is the fate of the clones isolated from the ing. No combination of growth rate, flow rate or bladder intestinal flora attempting to colonize the bladder. Their volumes will result in a stable population. The model pre- growth rates result in significant regions of extinction (Fig. dicts that the number of bacteria in the bladder will either 4). Although it cannot be concluded that the E. co//clones increase without limit, or decline until extinct. This result isolated from intestinal flora would fail to establish stable demonstrates that to achieve an equilibrium population populations in the bladder, their low growth rates and long requires some form of resource limitation. The equilibrium lag phases do indicate that the clones from this source density would depend on the dynamics of micturition and would be less likely to become established than strains the functional relationship between the growth rate and isolated from the urinary tract. resource concentration. Further empirical work is Our model represents a 'worst-case scenario' in that it required to describe the relationship between the popula- assumes no surface growth. Any surface growth, regard- tion growth rate and the concentrations of the resources less of its nature, would result in a smaller fraction of the upon which the bacteria are growing. population being lost during micturition and. as a result, Although the equilibrium density will depend on the growth rate required for establishment would be resource concentration, the establishment condition is lower. However, even in the worst-case situation, the essentially independent of the relationship between the empirical results suggest that the growth rates of E. coii 560 D. M. Gordon and M. A. Riiey clones isolated from urinary tract infections are more than growth-rate differences between these two groups of sufficient to overcome losses due to micturition even with- strains. When grown in Luria broth, the average growth out adherence to the bladder wall. E. coli isolated from rates of strains isolated from the two habitats were the faecal flora are significantly less likely to successfully same, Kaye (1968) also observed that faecal isolates of establish infections in the absence of surface growth. E coii tended to grow more poorly in urine than did uri- Although it is possible that our in vitro growth-rate esti- nary tract isolates. mates may not accurately reflect the growth rate of E coii E co//strains isoiated from urinary tract infections nor- under in vivo conditions, such a discrepancy does not mally originate from the intestinal flora (Bollgren and Win- seem to result from the physical conditions found in the berg, 1976; Caugant, 1983; Svanborg-Eden etai, 1989; bladder. The growth-rate estimates were made under cul- Turck ef ai, 1962; Vosti et ai, 1964). Therefore, the ture conditions quite different from those in the bladder, growth-rate differences do not so much reflect the habitat i.e. well agitated and aerated. However, when the ceils from which they were isolated, but instead whether they were cultured under conditions of very poor aeration and had experienced urine as a growth medium prior to being no agitation, the estimated exponential-phase growth rate isoiated. The intestinal strains had perhaps never, or at was less than 8% lower than when the cultures were well least not recently, been exposed to urine. We do not know aerated. Successful establishment depends on the the nature of the adaptation that permits E. coti strains growth rate observed when cell density is low. As a result, previously exposed to urine as a growth medium to grow the observed divergence in the cell-density curves as the 150% faster than intestinal isolates. However, there are at cultures approached stationary phase is not significant ieast two possible explanations for these results — one (Fig. 3b). physiological and one genetic. The urine samples used for the growth-rate estimates The urinary tract isolates may grow better in urine were collected on different days, at different times, and because, having been previously been exposed to urine, from both sexes. Differences between urine samples did they have been induced in some manner that permits not appear to have a substantial effect. The growth rate of faster growth in urine. However, it is unlikely to be a case urinary tract strain 14 was measured on three occasions, of conventional induction. The effect would have had to namely in urine from a female, and in two samples col- persist during the procedures involved in isolating and lected from the same male; the average growth rates identifying the strains, their long-term storage in stabs or were 1.98, 1.69, and 1.78 h"\ respectively. The growth at -70°C, subsequent subculturing, and, finally, several rate of strain 36 was also estimated twice, in urine from a growth cycles on or in Luria broth prior to our growth-rate female (1.96 h"') and a male (2.08 h"'). Urine osmolality experiments. Alternatively, the clones that grow well in and pH can influence E. coli growth rates, factors which urine may form a genetic subpopulation of the intestinal depend on sex, diet, and the time at which the urine is E coli flora. The superior growth rate of this subpopula- produced (Asscher et ai. t966; Kaye, 1968). However, tion would result in these strains being selected for in the the normal ranges of pH and osmolality in healthy adults, bladder, and they would then become urinary tract iso- particularly women, are such that they do not significantly lates. If these strains represent a subpopulation of the influence E. co//growth rates (Asscher etai. 1966; Kaye, intestinal flora, then in a large sample of intestinal iso- 1968). lates, we would expect to find strains that grow well in There have been several studies investigating E. coli urine. growth in urine (Asscher etai, 1966; Kaye, 1968; Stamey The results of our theoretical and empirical studies sug- and Mihara, 1980). For a number of reasons, meaningful gest that the growth rates of E coii isolated from urinary comparisons of the results reported here and those of tract infections are sufficient to overcome the losses due previous studies are not possible. In some studies the to micturition and that these clones have the ability to urine was filter-sterilized, centrifuged, or stored prior to establish a population in the bladder without adhesin- use. Often, a single urinary tract or laboratory strain of E. mediated surface growth. The growth rates of the clones coli was used in the study. Another problem with some isolated from faecal flora, strains which may have never studies was that the experiments were started at a high experienced urine as a growth medium, are sufficiently cell density (10^), a density close to the point at which the low that the ability to adhere to the wall of the bladder growth rate starts to decline (Fig. 3a). However, rough would increase their likelihood of establishing popula- qualitative comparisons suggest that the growth rates tions. reported in previous studies fall within the range of growth What role do adhesins play in urinary tract infections? rates we observed for the urinary tract strains. Adhesin-mediated surface growth is required for colo- The difference observed between the average growth nization of the upper urinary tract (O'Hanley et ai, 1985) rates, in urine, of E coli isolated from urinary tract and and may also play an important role in allowing E coii to intestinal habitats does not appear to reflect intrinsic ascend the urethra. In addition, adhesion is responsible Bacteriai growth in the biadder 561

for some of the symptoms associated with infection. The conditions; tow and high aeration. To achieve good aeration, results of this study do not allow us to conclude that sur- the flasks were shaken at 200 r.p.m. and closed with foam face growth is unneccessary for the establishment and stoppers. For the low-aeration treatment, the flasks were persistence of E. co//populations in the human bladder. closed with rubber stoppers and not shaken. In addition, to However, the predictions of the model together with the minimize the amount of atmospheric oxygen dissolving in the urine, an alcohol flame was introduced into these flasks and observed growth rates of clones isolated from urinary allowed to extinguish itself. This step was performed prior to tract infections do suggest that surface growth is not the start of the experiment, and at each sampling time. The required to explain the maintenance of E co//populations flasks were briefly vortexed prior to being opened to remove a in the bladder. sample, as the cells had to be uniformly suspended in order for There have been many studies describing the nature cell density to be estimated. and frequency of virulence determinants in E co//causing The growth rates reported are based on the average of three urinary tract infections. Our results suggest that the ability replicates. Statistical analyses were performed on untrans- formed data using analysis of variance. to survive and efficiently utilize the resources available in urine is an important adaptation found in E coii clones inhabiting the urinary tract. Further in vivo experimental Acknowledgements studies are required in order to fully understand the role of We wish to thank Frank Stewart for his suggestions regarding adhesins in the dynamics of urinary tract infections. the mathematical model- We thank Bruce Levin. Lone Simon- sen, and Richard Goldstein for their comments on the manuscript. The technical assistance of Sonia Bobekova and Experimental procedures Petra Levin is gratefully acknowedged. Richard Goldstein and Strains and media Caterina Svanborg-Eden generously allowed us the use of their strain collections. This research was supported by the The E co/f strains used in this study were isolated from the uri- Medical Research Council of Canada (D.M.G.). the Sloan nary and intestinal tracts of humans as part of epidemiological Foundation (M.A.R.) and by an NIH grant GM33782 to B. R. surveys (Arthur et ai, 1989; Caugant, 1983). From these two Levin. large collections 22 strains were selected- The urinary tract strains chosen represented isolates from patients diagnosed with asymptomatic infections, cystitis, urosepsis, and pyelone- References phritis. Cells were cultured in urine or Luria broth. Urine was sup- Arthur, M., Johnson, C.E., Rubin. R.H., Arbeit, R.D., Campan- plied by the authors and a colleague, and was from the first or elli, C, Kim, C, Steinbach, S,, Agarwal, R.W., and Goldstein second micturition of the day; it was collected under aseptic R. (1989) Molecular epidemiology of adhesin and hemolysin conditions and used within the hour. The absence of bacteria in virulence factors among uropathogenic Escherichia coli. the urine samples was confirmed by plating 0.1 ml of the sam- Infect immun 57: 303-313. ple on a Luria-broth plate. Asscher, A.W., Sussman, M., Waters, W.E., Davis. R-H-, and Chick, S. (1966) Urine as a medium for bacterial growth. Lancet \i: 1037-1041. Growth-rate determinations Boen, J.R., and Sylwester. D.L. (1965) The mathematical relationship among urinary frequency, residual urine and bacte- The growth rates, in urine, of the 22 strains were estimated in ria! growth in bladder infections- Invest Urol2: 468-473- vitro by monitoring the change in the density of colony-forming Bollgren, I,, and Winberg, J. (1976) The periurethral aerobic units over time. Fifty-millilitre Erienmeyer flasks containing 20 flora in girls highly susceptible to urinary infections. Acta ml of urine were inoculated with cells from overnight Luria- Paediatr Scand65: 81-87. broth cultures at an initial density of about 10"* cells m\'\ The Caugant. D.A. (1983) Enzyme polymorphism in Escherichia cultures were incubated at 37'=C and shaken at 150 r.p,m. The coti: genetic structure of intestinal populations; reiationships flasks were sampled after 1 h, and then at half-hourly intervals. with urinary tract infection strains and with Shigelta. Ph.D. Cell densities were estimated by plating on Luria broth agar fol- Thesis, Department of Clinical Immunology, Institute of Med- lowing serial dilution. For comparative purposes, the growth ical Microbiology, University of Goteborg, Goteborg, Swe- den. rate of one of the strains in Luria-broth was estimated in the same manner. The growth rates of all 22 strains were esti- Cox, C.E., and Hinman, Jr, F, (1961) Experiments with induced mated in Luria broth by measuring the change in the optical bacteriuria. vesical emptying and bacterial growth on the mechanism of biadder defense to infection, J Urot 86: density of cultures (675 nm). The initial density of these cul- 739-748, tures was about 10^ cells ml"'. The cultures were incubated at Dootson, P.H-, MacLaren, D.M., and Titcombe, D.H.M. (1973) ST^C and shaken at 150 r.p.m. The distribution of urinary 0-groups of Escherichia co/iin uri- Under the culture conditions used in our growth-rate experi- nary tract infections and in the normal fecal flora. In Urinary ments the cells are thoroughly suspended and the cultures well Tract infections. Brumfitt, W., and Asscher, A. (eds). Oxford: aerated. Cells growing in the bladder may not be so well mixed Oxford University Press, and the concentration of dissolved oxygen will be lower. To Dugdale, A,F. (1969) The defenses of the urinary tract against confirm the validity of our growth-rate estimates, the growth infection: a mathematical model, invest UrotG: 371-379. rate of one strain was measured, in urine, under two culture Giynn, A.A,, Brumfitt, W-, and Howard, C.J. (1971) K antigens 562 D. M. Gordon andM. A. Riley

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