antibacterial mechanisms of bladder€¦ · the antibacterial action of bladder mucosa, as...

Antibacterial Mechanisms of the Urinary Bladder

CARLW. NORDEN,GARETHM. GREEN, and EDWARDH. KASS

From the Thorndike Memorial and Channing Laboratories, Department ofMedical Microbiology, and Harvard Medical Unit, Boston City Hospital;and the Department of Medicine, Harvard Medical School,Boston, Massachusetts 02118

A B S T R A C T The disappearance of bacteriafrom the normal urinary bladder is apparently afunction of two host defense mechanisms: themechanical clearance of organisms by voiding, andthe antibacterial activity of the bladder wall. Thisstudy quantified the relative contribution of eachof these mechanisms to the resistance of thebladder to bacterial infection.

32Phosphorus-labeled E. coli, S. aureus, andP. mirabilis were each injected into the urinarybladders of unanesthetized female guinea pigs. Atintervals after voiding, the bladders were removed,washed, homogenized, and assayed for residualradioactivity and viable bacteria. Mechanical clear-ance was measured by the changes in total radio-active count. Antibacterial activity was quantifiedby comparing the bacterial to radioactive ratiosof the original bacterial inoculum with similar ra-tios in the bladder homogenates.

MNore than 99.9% of the bladder inoculunm1 wasrapidly excreted and about 0.1%7 (104-105) orga-nisms remained attached to the bladder wall. Ofthose E. coli attached to the bladder, rapid sequen-tial reduction in viability occurred and reached alevel of 85% loss at 30 min after inoculation. 4 hrafter challenge, less than 10% of those organismsstill attached to the bladder mucosa remained viable.P. nzirabilis was handled with equal facility, butS. auireuts showed a reduction in viability of only467c at 1 hr and 677c at 4 hr after inoculation. 6

Part of this work appeared in abstract form in 1968Clin. Res. 16: 333.

Address requests for reprints to Dr. Edward H. Kass,Channing Laboratory, Boston City Hospital, 818 Har-rison Avenue, Boston, Mass. 02118.

Received for publication 9 May 1968 and in revisedform 24 Auguist 1968.

hr after infection with S. aureus, 6 of 12 guineapig bladders showed multiplication of the organ-isms still attached to the bladder wall; only 1 of 12animals challenged with E. coli had comparablemultiplication.

The mechanism whereby the bladder wall killsbacteria is unclear, but it did not appear to be re-lated to an antibacterial activity of urine, clumpingof organisms on bladder mucosa, phagocytosis byrleukocytes, or serum levels of bactericidal anti-body. Although it is clear that the bladder ex-hibits intrinsic antibacterial properties, the role ofthis defense mechanism in the pathogenesis of uri-nary tract infection requires further clarification.

INTRODUCTION

Multiplication of bacteria within the bladder maylead to ascending infection of the urinary tractby spread of bacteria up the ureters and invasionof the kidneys (1, 2). Clinical observations andlaboratory studies in man (3) and experimentalanimals (4) have confirmed that when bacteriaare introduced into the bladder, they often disap-pear from the urine. Those instances in which thegenitourinary tract fails to rid itself of bacteriamay be viewed as instances of a failure of hostmechanisms of resistance to bacterial infection.

A significant part of bladder defense is the ca-pacity to excrete large numbers of organisms inthe urine (5). However, the quantity of bacteriaremaining in the bladder after voiding is sufficient,if its growth is uninhibited, to perpetuate infection.The fact that the bladder does become sterile sug-gests that there is an additional mechanism whichdeals with those residual organisms. The experi-

The Journal of Clinical Investigation Volume 47 1968 2689

ments of Vivaldi, Mufioz, Cotran, and Kass (6)and of Cobbs and Kaye (7) indicate that thebladder exerts a bactericidal action on bacteria,although this concept has been disputed by Paquin,Perez, Kunin, and Foster (8) and by Mulholland,Foster, Gillenwater, and Paquin (9).

In all previous studies the experimental animalswere anesthetized and manipulated surgically. Fur-thermore, since such studies were concerned withthe antibacterial action of bladder mucosa, as dis-tinct from voiding mechanisms, there has beenno study of the interactions between voiding andantibacterial defense systems within the bladder.

A simple model has been developed in thislaboratory that permits the use of normally voidinginfected animals. Radiolabeled bacteria are intro-duced into the bladder and the relative numbers oforganisms removed mechanically by voiding andrendered nonviable by the bladder wall are deter-mined. With this model, evidence of rapid andefficient killing of bacteria by the bladder was ob-tained, and the interaction of this mechanism withthe voiding mechanism was assessed. A series ofexperiments attempted to identify the nature ofthis antibacterial bladder activity.

METHODSGuinea pigs and rats were inoculated intravesically witha radiotracer-labeled overnight broth culture of E. coli,P. mirabilis, or S. aureus. At intervals after the inocula-tion the animals were killed and the bladders removed.Quantitative measurements of radioactivity and of viablebacteria were made on the original inocula and bladderhomogenates. From these data, the antibacterial effectsof voiding and of bladder mucosa were calculated.

Preparation of radioactive inocuitui. The strain ofE. coli used in these experiments was obtained from theurine of a patient with acute pyelonephritis; the strains ofP. wirabilis and S. autreus used were laboratory strains(10). In some experiments, a strain of E. coli 055 wasused, and this is noted in the text. In preparation foreach experiment, a loopful of bacteria was transferredfrom a fresh agar slant culture to a 125 ml Erlenmeyerflask containing 20 ml of culture medium. The mediumconsisted of casamino acids, proteose peptone, and dex-trose, prepared as previously described (11). The bac-teria were grown overnight at 37'C in a shaker waterbath and labeled with 32P during growth (11). The la-beled bacterial suspension was centrifuged, washed threetimes in phosphate buffer at pH 7.4 to remove all un-attached label, and resuspended in neutral phosphate buf-fer. This suspension was used as the bacterial inoculum.

Infection of animals. Female guinea pigs (275-300 g)were tied on their backs, and a No. 90 polyethylenecatheter was inserted per urethra into the bladder. A No.

20 needle was fitted securely into the catheter and 1 mlof the radioactive inoculum was injected into the bladder.The catheter, which remained in place for about 20 sec,was then removed. The animal was returned to its cageand allowed to void spontaneously.

A similar procedure was used for the infection of fe-male rats (175-200 g, Charles River Laboratories, CDstrain). The technique differed only in that a No. 26 1Y2in. blunted needle was used instead of a catheter, theinoculum was only 0.1 ml, and ether anesthesia lastingabout 1 min was utilized during catheterization.

Preparation of tissue. The peritoneal cavity was en-tered under aseptic technique, the bladder was exposed,and the vesical neck clamped. The bladder was removedalong with any residual urine within the lumen. Witha No. 27 needle, the urine was aspirated, and a 1 ml ali-quot of sterile neutral phosphate buffer was injected intothe bladder and removed by aspiration. This procedurewas repeated three times with separate volumes of buffer.The total aspirate was combined with residual urine andlabeled "washout." The clamp was then removed, andthe bladder incised. With the mucosa exposed, the tissuewas passed through three baths, each containing 2-ml ofsterile phosphate buffer. The washed bladder was thenhomogenized in a ground glass homogenizer tube con-

taining 2.5-ml of trypticase soy broth until a homogeneoussuspension was obtained. Pour plates were made in tripli-cate from serial dilutions of the bladder homogenates andthe inoculum.

Radioactive counting. Samples of inoculum, thebladder homogenate, and washout fluid were preparedfor radioactive counting. 1-ml aliquots of the specimenswere mixed with 4-ml of hyamine in disposable s-rew

cap vials and the mixture was digested at 60'C overnight.The next day, 5-ml of 95% alcohol and 10-ml of toluenescintillation solution 1 were added to each vial to bringthe total volume to 20-ml. All samples were counted for10 min in a Packard-Tricarb model 3003 liquid scintilla-tion counter. Correction for the quench effect of bladdermucosa was achieved in control vials containing 1 ml ofbladder homogenate, obtained from uninfected animals,and appropriate dilutions of the original inoculum.

Inmmni-ationi of aiinimals and determiniationi of antibodytiters. Guinea pigs received 0.1 ml of a heat-killed (560Cfor 30 min) overnight culture of P. niirabilis or S. atrensintraperitoneally at 3-day intervals for four doses andthen 0.5 ml of a live overnight culture of the same or-

ganism 3 days later. After 2 wvk, the animals were ex-

sanguinated by cardiac puncture. The blood was allowedto clot and the serum was separated and stored at- 20'C. For determination of antibody titer, an 18 hrbroth culture of the test organism was centrifuged, re-

suspended in an equal volume of neutral phosphate buffer,and heated for 90 min at 600C. To serial twofold dilu-tions of serum was added an equal volume of bacterialsuspension, diluted until it was barely turbid. This mix-

1 This solution contains 5 g of 2,5-diphenyloxazole(Packard) and 0.1 g of 1,4-bis-[2-(5-phenyloxazolyl) ]-benzene (Packard) dissolved in 1000 ml of toluene.

2690 C. W. Norden, G. M. Green, and E. H. Kass

ture was incubated at 370C for 1 hr, refrigerated for 18hr, and read macroscopically for agglutination the fol-lowing morning. The reciprocal of the highest dilutionshowing bacterial agglutination was read as the titer forthat serum.

Calculation of bacterial killing. The bactericidal ac-tivity of urinary bladder mucosa was determined by themethod of Green and Goldstein (11). The radiotracerserved as an inert marker to enumerate the total numberof organisms in the original inoculum and the bladder.The bacterial killing in each bladder was calculated fromthe ratio (RI) of viable bacterial count to radioactivecount in the original inoculum (I), compared with theratio (RB) found in the homogenate of bladder (B) ob-tained from the infected animal. The ratio (RB) dividedby the ratio found in the inoculum (R,) gives the percent change in the proportion of viable bacteria attachedto the bladder wall, during the elapsed time intervalafter infection. Thus:

(1) Per cent bacteria remaining viable on bladder wall

proportion of bacteriaviable in bladder (B) Xproportion of bacteriaviable in inoculum (1)

bacterial coulnt B132PB

100 = bacterial count I

X 100 = B X 100.RIIt then follows that:(2) Per cent bacteria killed on bladder wall = 100 - per

cent bacteria remaining viable on bladder wallRB

= 1 - X 100.

Fig. 1 presents hypothetical applications of this tech-nique. If, for example, the original inoculum of 1 X 10'bacteria contained 1 X 106 radioactive counts (column1), and after a certain time in the bladder, 1 X 10' radio-active counts, but only 1 X 107 bacteria were recovered, a10-fold loss of viability would have occurred with novoiding (column 2). On the other hand, after voiding itmight be found that 1 X 10' bacteria with 1 X 103 radio-active counts remained, indicating a 1000-fold loss ofbacteria due to discharge of labeled bacteria, but no lossof viability (column 3). If the experiment showed 1 X 104bacteria remaining with 1 X 103 radioactive counts, itwould indicate a 1000-fold loss of bacteria due to voiding

LS OFCOUNTS

10 _98 -76

4

3

2

M Bacterial Count (BC)0 3"p Count (P)

BC/P. 100BC/P- 10

BC/P-.10

KILLING3 4

FIGURE 1 Schematic representation of method.

and an additional 10-fold loss of viability within thebladder (column 4).

RESULTS

Number of organisms removed by voidbig.Animals infected with 1 ml containing about 109bacteria voided within 10 min after challenge.The total number of bacteria remaining in thebladder after micturition was calculated from theradioactive counts present in the bladder ho-mogenate and residual urine (Table 1). The dif-ference between this figure and the total radio-activity instilled into the bladder represents theradioactivity removed from the bladder by voiding.This assumes that all labeled bacteria either re-main in the bladder or are voided in the urine.The assumption seems valid since samples ofliver, spleen, kidneys, and serum showed eithertrace or no radioactivity 1 hr after inoculation oforganisms into the bladder. Table I shows datafrom groups of animals infected with E. coli orS. aureus and indicates the number of organismsremoved by voiding. More than 99.9%7 of the

TABLE IEffect of Voiding on Removal of Bacteria from the Bladder

Bacteria in- Bacteria in Bacteria in Total bacteria % injected bac-jected into bladder bladder in bladder teria removed

No. bladder homogenate washout after voiding by voidingOrganism animals X 109 X 106 X 105 X 105

E. coli 30 2.7 ±40.2* 2.0 ±0.7* 6.3 ±2.0* 8.3 ±2.1* > 99.9

S. aureus 6 2.3 ±0.1 0.9 ±0.3 1.0 ±40.7 1.9 ±0.8 > 99.9

P. mirabilis 6 2.0 ±-0.1 0.6 ±0.2 0.9 ±0.6 1.5 ±0.6 > 99.9

* 1Xleani ±SE.

Antibacterial Mechanisms of the Urinary Bladder 26;91

TABLE I IKilling of E. coli by Guinea Pig Bladder in 1 hr

Bacterial count %bacteriaRatio killed by

Sample Bacterial count 32p count 32p count bladder wall*

No. bacteria/ml counts/JO min per ml1

Inoculum 2.5 ±0.1 X 109§ 6.0 ±0.2 X 108§ 4.1 ±0O.1§

Homogenates of bladdersA 1.1 X 103 6.1 X 103 0.18 95.6B 1.5 X 102 5.1 X 103 0.03 99.3C 7.8 X 102 1.1 X 10, 0.07 99.3D 2.1 X 102 1.1 X 104 0.02 99.5

* Calculated from formula (2) in Methods.X Corrected for background.§ Mean 4SE of determinations performed in triplicate.

total bacteria placed in the bladder were removedby voiding, and less than 0.1%o remained in thebladder. This 0.1%o residuum containing 104_106organisms was the subject of detailed investiga-tion, since it represented that portion of the infec-tive inoculum with which antibacterial mechanismsof the bladder had to contend.

Antibacterial activity of bladder. Table IIshows the method of calculating bacterial killingin an experiment in which guinea pigs were in-oculated with E. coli and killed 1 hr later. Threealiquots of the inoculum were counted, and the ra-tio of viable bacteria to radioactive count was de-termined for each aliquot. The mean of the threealiquot ratios was calculated and served as themean ratio for the inoculum. It is clear that theratios obtained from each bladder homogenate aremuch lower than the mean values of the originalinoculum, showing that there has been a markedloss of viability among those organisms attachedto the bladder wall.

Fig. 2 shows the mean reduction in viability ofthose E. coli or S. aureus attached to the bladderwall at each time interval. E. coli were killed rap-idly on the bladder wall; half of the organisms onthe bladder wall 4 min after inoculation were nolonger cultivable, and 85% were nonviable 30 minafter infection. At 4 hr after infection, the meanreduction in viability was 93%o, and at 6 hr, 91%o.1 animal of 12 showed bacterial multiplication onthe bladder wall at 6 hr after infection.

S. aureus, when introduced into guinea pigbladder, was not killed as rapidly as E. coli. At 1hr after challenge, the mean reduction in viable

organisms on the bladder wall was only 46%, andthere was greater variation in the rate of killingfrom animal to animal than was the case withE. coli. At 4 hr after infection, there was a meanreduction of 67% in viability of those S. aureusremaining attached to the bladder wall. Of the12 animals killed 6 hr after infection with S. aureus,six showed multiplication of the organisms at-tached to the bladder wall, so that the mean per-centage killed was reduced to only 22%c.

The ability of S. aureus and E. coli to multiplyin the bladder urine differed markedly (TableIII). 4 hr after infection, multiplication of E. coliin the urine was significantly greater (P < 0.05)than the degree of multiplication seen for S. aureus.This is shown in Table III by the increase in ra-tio (R17) of bacterial to radioactive counts in theurine compared with the ratio in the original inoc-

PER CENTBACTERIAONBLADDERWALLREMAININGVIABLE

100Noof AnlimVs

80

60 o -e

40 2

20 _ 4 13

IiI I EEcoli t

0 40 80 120 160 200 240TIME (MINUTES)

FIGURE 2 Bacterial killing by bladder wall.

2692 C. W. Norden, G. Al. Green, and E. H. Kass

ulum (RI). However, at the same time that E.coli were multiplying in the residual bladder urine,the bladder wall continued to exert a bactericidaleffect on those organisms attached to its surface;the bladder homogenates at this time showed a93% reduction in viable organisms. In contrast,although S. aureus did not multiply vigorously inthe urine, there was only moderate killing of thisorganism on the bladder wall 4 hr after infection.

That the antibacterial activity of the bladder wasnot dependent on the species of animal used wasshown in a series of experiments with rats. Inaddition to E. coli and S. aureus, a strain of P.mirabilis that readily causes pyelonephritis in ratswas tested in both guinea pigs and rats to deter-mine if it was handled comparably by the bladder.The ability of the bladder mucosa to inactivatedifferent bacterial species is shown in Table IV foranimals killed 60 min after infection. The bladdersof guinea pigs and rats kill these strains of E. coliand P. nuirabilis with equal facility, and both ani-mal species exert a lesser bactericidal effect onS. aureus.

Methodologic controls. A series of experimentswas carried out to exclude several potential sourcesof error in methods or interpretation of results.

(1) The method depends upon a firm bindingof radiotracer and bacterium. Any significantseparation would invalidate the meaning of theratios of bacterial to radioactive counts. Firmbinding of radioactive label and organism was dem-onstrated. Upon recentrifugation of the labeledbacterial inoculum, only 3-5% of the total radio-activity was found in the supernatant phase. 4 hrlater, 5-7% of radioactive counts was present inthe supernatant phase. There is thus a slow lossof label from viable organisms which does notaffect the interpretation of the results.

TABLE IIIViability of Bacteria on Bladder Wall and in Bladder

Urine 4 hr after Infection

No.Organism animals Ri RB Ru

E. coli 6 10 0.7 +0.3* 360 +173*

S.aureus 8 10 3.3 a1.0 28-+7

R, ratio bacterial to radioactive counts; 1, inoculurm; B,bladder homogenate; U, bladder urine.* Mean 4SE.

TABLE IVAntibacterial Activity of the Bladder Wall

No. %bacteriaanimals killed in

Animal Organism tested 1 hr

Guinea pig E. coli 42 85 +3.8*P. mirabilis 15 91 +3.4E. coli 055 8 92 +4.5S. aureus 30 46 +6.4

Rat E. coli 10 88+3.6P. mirabilis 11 89 +4.2S. aureus 8 58 +8.2

* Mean +SE.

(2) Preferential attachment of killed bacteriaor free tracer to the bladder wall might producemisleading results. To determine if nonviable or-ganisms or free label had a greater affinity for thebladder wall than did labeled viable organisms,32P-labeled viable E. ccli, 32P-labeled heat-killedE. coli, and trypticase soy broth containing 32P asNa2HPO4but no bacteria, were injected into thebladders of three groups of guinea pigs (TableV). One-half of each group was killed within 4min after inoculation, and the residual inoculumwas removed by aspiration. The bladder waswashed, homogenized, and assayed for residualradioactivity. The remaining guinea pigs werekilled 1 hr after voiding had occurred, and thebladder was handled identically. At 4 min afterinoculation and again at 60 min after inoculation,there were no significant differences in the meanamounts of radioactivity attached to the bladderwall among the three groups of animals receivingviable bacteria, nonviable bacteria, or free label.60 min after infection, the levels of radioactivityhad fallen by 91, 89, and 88%7 in the animals re-ceiving viable bacteria, nonviable bacteria, andfree radiotracer, respectively. This clearly showsthat there was no preferential attachment of non-viable organisms or free label to the bladder wall,nor was there increased excretion of either of thesein the urine.

(3) The demonstration of an antibacterial ac-tivity of the bladder requires that the process ofinjecting the labeled inoculum or of homogenizingthe bladder tissue does not alter the bacterial toradioactive ratio by exerting an antibacterial effect.

When the radioactive inoculum was aspirated

Antibacterial Mechanisms of the Urinary Bladder 2693

TABLE VUptake of Radioactivity by Bladder Wall

Time after Radioactive countinjection No. Radioactive present on

Material injected into bladder into bladder animals count injected* bladder wall:

min

Viable, labeled E. coli 4 6 1.0 X 108 6.0 A4.7 X 105Heat-killed, labeled E. coli 4 6 1.0 X 108 3.0 42.4 X 10632p in sterile broth 4 6 1.0 X 108 2.5 41.2 X 105

Viable, labeled E. coli 60 6 1.0 X 108 5.3 ±2.2 X 10IHeat-killed, labeled E. coli 60 6 1.0 X 108 3.2 ±2.2 X 10432p in sterile broth 60 6 1.0 X 108 3.1 i2.5 X 104

* Mean No. counts/10 min per ml.t Mean No. counts in bladder homogenates/10 min per ml ±SE.

from the bladder immediately after injection soy broth and an uninfected guinea pig bladder.through the catheter, no change was noted in the Comparable studies were also done using an in-ratio of bacterial to radioactive counts from that oculum of about 105 E. coli. The mixture wasof the original inoculum. The neutral phosphate ground together for the same duration of time asbuffer used to wash the bladders exhibited no in experiments with bladders from infected ani-antibacterial activity. The final wash of the bladder, mals, until a homogeneous suspension was ob-before homogenization, contained less than 0.01 % taimed. The results of eight such experiments areof the total radioactivity present in the ho- shown in Table VI. No significant change in bac-mogenate. This finding suggested that there was a terial to radioactive ratio was noted in homogenatesfirm attachment of residual bacteria to the bladder when compared with the original inoculum.wall and that this attachment was not disturbed Homogenates of bladders from uninfected guineaby the process of washing. pigs or rats were made in 1 ml of trypticase soy

To exclude the possible artifact of bacterial kill- broth and incubated with 103 E. coli. There was noing or clumping due to the process of homogeniza- antibacterial activity. Fig. 3 shows that the growthtion, these experiments were performed. The same curve of these organisms in bladder homogenatesinoculum of 32P-labeled E. coli as was injected into over a 4 hr period closely paralleled that in trypti-the living animal (about 109 bacteria) was placed case soy broth alone.in an homogenizer containing 2.5 ml of trypticase These experiments demonstrate that neither the

TABLE VIEffect of Homogenization on Bacterial-Radioactive Ratio

Inoculum Homogenate

Bacterial count Bacterial countRatio Ratio

Bacterial count 32p Count 32P count Bacterial count 32p count 2p count

No. bacteria/ml counts/1O min per ml No. bacteria/l coants/)o min per ml

1.1 X 109 4.7 X 107 23.4 1.4 X 108 6.0 X 106 23.31.1 X 109 4.7 X 107 23.4 1.9 X 108 8.1 X 106 23.51.6 X 109 7.4 X 107 21.6 1.9 X 108 8.8 X 106 21.61.6 X 109 7.4 X 107 21.6 1.8 X 108 8.2 X 106 22.0

1.4 X 105 6.0 X 103 23.3 2.6 X 104 1.1 X 103 23.61.4 X 105 6.0 X 103 23.3 1.9 X 10, 8.1 X 102 23.51.8 X 105 7.2 X 103 25.0 2.2 X 104 8.8 X 102 25.01.8 X 105 7.2 X 103 25.0 2.1 X 104 8.3 X 102 25.3


lo" ° Broth+ E.coli

06 -

BACTERIAL 10 -

COUNTS/ML 4

10 _

10'

0 1 2 3TIME (HOURS)

FIGURE 3 Growth of E. coli in nutrienthomogenates of bladder.

process of injection of bacteria intonor the homogenization of the bladdbacterial to radioactive ratio. No antitivity of bladder homogenates couldstrated.

Antibacterial activity of guineaGuinea pig urine might have antibactties that would explain the loss of vikganisms introduced into the bladder.such a mechanism, urine was obtainedzation from 19 uninfected guinea pi~of urine was added 1 ml of bacteriacontaining approximately 103 E. coli.was incubated at 37°C for 24 hr. Onsample showed any bactericidal activ24 hr. The other 18 supported bacteriktion with final concentrations at 24from 2 x 108 to 4 x 108 organisms/case soy broth incubated 24 hr with Eably yielded final concentrations of 1-teria/ml.

Studies aith excised bladders.were carried out with excised bladd(tempt to learn if the bactericidal prop

bladder demonstrated in intact animals could betE.coli reproduced in vitro. Guinea pigs were killed by

intraperitoneal administration of pentobarbital.The bladders were clamped, removed aseptically,and held in a moistened Petri dish. Radioactiveinoculum was injected into the bladder lumenthrough a No. 27 needle. After an appropriateinterval, the inoculum was removed by aspiration,and the bladder was washed, homogenized and ex-

4 5 amined as previously described. When labeledinocula of E. coli were removed 1 min after in-

broth and in jection into the excised bladder, there was a meanreduction of 55% in viable organisms remainingattached to the bladder wall (Table VII). This

the bladder was comparable with the results obtained whener alters the the bladder was left in situ and the inoculum wasbacterial ac- removed within 3 min after infection. However,

be demon- if the inoculum was removed from the excisedbladder 1 min after injection, and the bladder held

pig urine. at room temperature or 370C for 1 hr before ho-erial proper- mogenizing, the mean reduction in viability ofability of or- these organisms attached to the wall remained atTo exclude 55%. The excised bladder did not exhibit the

by catheteri- progressive killing of attached bacteria that wasgs. To 1 ml seen in the intact animal (Table VII).1L suspension The mean reduction in viability of 55 /c 1 hrThe mixture after challenge was unchanged even when thely one urine bladder was held at 4 or 370 for 2 hr before chal-,ity at 1 and lenge, or when the actual inoculation of theal multiplica- bladder was carried out at 4°C (Table VIII).

hr ranging Placing killed E. coli into the bladder for 1 hr/ml. Trypti- before to challenge with the same strain of live

coli invari- E. coli did not alter the rate of killing of these-3 x 109 bac- viable bacteria attached to the bladder wall.

Vigorous washing of the bladder wall with sterileExperiments phosphate buffer before inoculation of bacteriaers in an at- had no effect on bacterial killing (Table VIII).)erties of the Effect of immunization. Guinea pigs were im-

TABLE VIIBacterial Killing in Intact and Excised Bladders

%bacterial %bacterialOrganism killing 4 min No. killing 60 min No.

Condition of bladder injected after inoculation animals after inoculation animals

Intact animal E. coli 49 ±2.4* 18 85 ±3.8 42

NS P < 0.01

Excised, in vitro E. coli 55 i2.8 14 55 ±4.9 20

* Mean ±SE.


*- Bladder Homogenote-

munized with P. mirabilis or S. aureus to learn ifproduction of antibody played any role in the anti-bacterial activity of the bladder. P. mirabilis wasused, since this laboratory strain has been shownto be an effective immunizing agent (10). S.altreus was employed since it would not be ex-pected to be affected directly by antibody. Serafrom guinea pigs immunized with P. mirabilisgave antibody titers of 160 or greater, whereascontrol nonimmunized animals had no detectableantibody at 1: 2 dilutions of serum. At 20 minafter infection, immunized and nonimmunizedguinea pigs were killed and their bladders exam-ined. The nonimmunized guinea pigs showed amean bacterial killing of 69%o and the immunized76%, a difference that is not statistically signifi-cant (Table IX).

Comparable experiments were performed withS. aureus as the immunizing agent. No increasein killing of this organism by the bladder was seenin immunized guinea pigs as opposed to controls(Table IX).

Clhtiipiiig of bacteria. Were bacteria to clumptogether on the bladder wall, they would grow as asingle colony when pour plates were made frombladder homogenates. The falsely lowered totalbacterial count produced in this way could ac-count for a reduction in the bacterial to radioac-tiv e ratio, and suggest a killing action. To examine

TABLE VIIIEffect of Various Conditions on Bacterial Killing

by Excised Bladder

%bacterial killingNo. in excised bladder

Condition of bladder animals 1 hr after infection

I nfected immediately afterexcision 12 55 +2.8*

Held at room temperaturefor 1 hr before infection 9 52 +3.1

Held at 40C for 2 hr beforeinfection 10 54 +2.9

Held at 37'C for 2 hr beforeinfection 10 51 +3.0

Washed with phosphatebuffer 2 hr before in-fection 10 52 -+3.2

Killed E. coli placed inbladder for 1 hr beforeinfection with viableE. coli 11 52 +2.8

* Mean 4SE.

TABLE I XEffect of Immunization on Bacterial Killing by Bladder Wall

%killing of bacteriaImmune state No. attached to bladder

Organism of animal animals wall

P. mirabilis Control 11 69 +4.0* P > ()1Immunized 8 76 +3.6

S. aureus Control 30 46 ±6.4 P (. IImmunized 19 35 +7.4

* Mean +sE.

this possibility, bladders from guinea pigs infectedwith E. coli 055 2 were removed 1 hr after infec-tion. Impression smears of the exposed mucosawere stained with specific immunofluorescent anti-body. If clumping were responsible for the reduc-tion in colony-forming units, 99% reduction wouldmean that bacteria were clumping in groups of ap-proximately 100. Instead, although few organismswere seen on the slides, the bacteria were observedsingly or occasionally in pairs. At no time whensignificant killing of organisms had occurred werelarge clumps of bacteria seen. No bacterial clumpswere seen in frozen tissue sections stained withGiemsa stain, but single and paired organismswere seen in the lumen (Fig. 4).

Cell counts and sections of the bladder. Blad-ders were removed from control animals, from ani-mals catheterized and challenged with E. coli, andfrom animals catheterized and given sterile phos-phate buffer into the bladder. Cell counts per-formed on the "washouts" obtained 1 hr after in-fection revealed red blood cells and epithelial cells.Only a rare lymphocyte and no polymorphonuclearleukocytes were seen. Fixed tissue sections of un-washed bladder from comparably challenged ani-mals sacrificed 1 hr after infection showed no in-flammatory response and only a rare polymorpho-nuclear leukocyte in the bladder lumen.

Effect of varying inoculum size. It was ob-served in early experiments that those bladderswith high radioactive counts often exhibited lowerrates of bacterial killing than did bladders withfewer retained radioactive counts. To determineif the nulaber of bacteria attached to the bladder

2 This organism was selected because it had been shownto be killed by the bladder (Table IV) and becausecommercially prepared specific fluorescent antiserum wasavailable.


Lumen

FIGURE 4 (a) Histological frozen section from mucosa of guinea pig bladder 1 hr after inoculation with 1 x 109E. coli. Note that the mucous membrane is intact and that there is no evidence of acute inflammation in this section.Stained with Giemsa stain. X 130. (b) and (c) Higher magnification of two areas from the same section as (a)to show single (c) and probably paired (b) organisms adherent to the bladder mucosa. x 1180.

wall affected the rate of bacterial killing, guineapigs were given into the bladder undiluted, 1: 10,and 1: 100 dilutions of a radiotracer-labeled bac-terial inoculum and were sacrificed immediately.The mean reduction in viability of those bacteriaattached to the bladder wall 4 min after inoculationwas determined for eight animals with greater than104 radioactive counts, and for 10 with less than104 radioactive counts. The mean killing was 52%±3.2 for the group with a greater residual activityand 69% ±3.7 for those animals with fewer or-ganisms attached to the wall, a difference that isstatistically significant (P < 0.01).

DISCUSSION

These experiments have demonstrated the follow-ing: (1) More than 99.9% of bacteria injectedinto the bladder are removed by voiding, and lessthan 0.1 % ( 104_106 organisms) remain in theresidual urine or attached to the bladder wall.These remaining bacteria provide a sufficient in-oculum to perpetuate infection within the bladderif their growth is uninhibited. (2) Guinea pig andrat bladders exhibit rapid killing of E. coli and

P. mirabilis that become attached to the mucosa.In contrast, S. aureus is not killed as rapidly or asefficiently by the bladder. (3) Multiplication ofE. coli within the residual bladder urine occursat a time when the bladder mucosa is still exertingan antibacterial effect on organisms attached to itssurface. (4) Theoretical objections to the experi-mental method have been studied and excluded.(5) The excised bladder exerts an initial rapidantibacterial effect, but it does not exhibit theprogressive killing over time seen in the intactanimal. (6) No evidence of bacterial clumping orof phagocytosis is seen.

It is clear that the bladder possesses intrinsicantibacterial activity. Because the experimentalmethod allowed voiding to occur naturally, the si-multaneous study of both bladder antibacterial ac-tivity and mechanical clearance of bacteria waspossible under physiologic conditions. The relia-bility of the radiotracer method depends upon firmbinding of the radioactive label to the live bac-teria. The stability of the label used in the experi-ments, and the viability of these organisms overthe course of experiments has been demonstrated


previously for 1'. ntirabilis (10) and in the presentstudies for E. coli.

Vivaldi et al. (6), using an exteriorized rabbitbladder to which E. coli was applied in minutevolumes, showed significant reductions in viabilityof the microorganisms 1 hr after application to themucosa. They further showed, using radiolabeledbacteria, that the label persisted in the bladder wallwhen viability had decreased, a finding that sug-gests rapid and efficient killing of bacteria by thebladder mucosa.

Cobbs and Kaye (7), using rats with bilaterallyligated ureters, showed striking antibacterial ac-tivity of the rat bladder against two strains ofE. coli and lesser activity against a third strain ofE. coil and against P. mirabilis. Antibacterial ac-tivity was totally abolished if the bladder outflowtract was obstructed.

In contrast, Mulholland et al. (9) were unableto demonstrate any inhibition of bacterial growthin rabbits in which only ureters were ligated andE. coli instilled into the bladder in minute volume.This negative finding resulted despite the fact that8 of 20 normally voiding rabbits sterilized theirurine within 72 hr after introduction of one millionE. coli.

The reasons for the discrepancies in resultsamong various investigators are not entirely clear.It is possible that the rabbit bladder possesses noantibacterial activity or that the bacterial strainsused were resistant to the antibacterial propertiesof the bladder. Each of the experimental modelswas artificial to some extent, in that each involvedsurgical manipulation and prevention of voidingin order to study the action of the bladder wall.The system reported herein avoided these diffi-culties, since a catheter was inserted only brieflyand the unanesthetized animals were allowed tovoid freely. Further, unless the bladder wall isthoroughly washed before homogenization, it mayremain contaminated with residual urine. Urinehas been shown in our system to support bacterialmultiplication at a time when the bladder mucosa isstill exerting a bactericidal effect. Therefore, re-ports that examine only the total bacterial count inthe bladder at 4-6 hr after infection may, if multi-plication has occurred in the residual urine, totallyoverlook any antibacterial activity of the badderwall itself.

The mechanisms of the antibacterial activity of

the bladder are as yet unclear. Clumping of bac-teria, such that many organisms form a unit thatwill grow as a single colony on agar, has been sug-gested as a means of accounting for the reductionin viability seen in experimental models. How-ever, histologic sections and impression smearsfailed to demonstrate clumping in experiments inwhich clumps as large as 100 bacteria would haveto be invoked to explain the reduction in viablenumbers of organisms.

A possible mechanism for reduction in viablenumbers of bacteria in the bladder could be phago-cytosis and killing of organisms by leukocytes, orby bladder mucosa. Cobbs and Kaye (7) demon-strated an inflammatory response that occurred inthe bladder after bacteria were introduced. Theseinvestigators demonstrated histologic evidence ofinflammation 2 hr after introduction of bacteriainto rat bladder and an increase in polymorphonu-clear leukocytes in the bladder "washout" 4 hrafter infection. In the present study, reduction inviability occurred within a few minutes and noevidence of an inflammatory reaction was seeneven 1 hr after infection, when only a rare leuko-cyte could be recovered by washing the bladderwall. Thus, at a time when 85-90%/c reduction inviability of the bacteria attached to the bladder wallhad occurred, no evidence of inflammation or ofpolymorphonuclear leukocyte mobilization wasseen. It seems likely that phagocytosis and kill-ing of bacteria do occurs in the bladder over aperiod of time, but it is difficult to invoke this toexplain the extremely rapid reduction in viabilityseen in the 1st hr after infection. 0. Kunii and E.H. Kass (unpublished data) were unable to showany evidence that bladder mucosal cells couldphagocytize bacteria.

The rapid reduction in viable bacteria demon-strated in our model makes antibody a possiblemechanism for the antibacterial activity of thebladder. Against this is the evidence that activeimmunization of guinea pigs with P. mirabilis,which produced significant titers of antibody inthe serum, did not enhance the degree of killingof P. mirabilis by the bladder. Further, prior in-stillation of killed E. coli into the excised guineapig bladder and subsequent challenge with thesame strain of viable E. coli did not alter thereduction in viability of bacteria produced by thebladder. S. aitreus is an organism against which


antibody should not be effective, yet a certain de-gree of killing of the bacteria occurred on the blad-der wall, a finding that suggests again that anti-body is not of great significance in this phenome-non. These data indicate that antibody does notplay a major role in the antibacterial activity ofthe bladder.

An alternative possibility might be the presenceof organic acids or other substances that mightcreate an environment at the bladder mucosal sur-face unfavorable to bacterial growth (12). Theantibacterial action of organic acids is related tothe number of undissociated acid molecules pres-ent. If a critical pH were to be achieved at theinterface of bladder mucosa and urine, organicacids (present as end products of cellular metabo-lism) could exert an antibacterial effect. At pres-ent no direct evidence exists to support or refutesuch a hypothesis.

Although the mechanism for the antibacterialactivity of the bladder remains unclear, it can besuggested that a substance (as yet unidentified)exists in the bladder wall which is used and re-plenished as the intact bladder exerts its antibac-terial activity. The following evidence can be citedas support.

When E. coli is placed into the bladder of theintact animal, viability of the bacteria attached tothe bladder wall begins to be lost promplty andcontinues to decrease with time. In contrast, inthe excised bladder an immediate reduction inviability ocurs (as in the intact animal), but nofurther bacterial killing occurs with time. Thus,in the intact bladder the mechanism for bacterialkilling is not exhausted, whereas in the excisedbladder, after an initial effect, there is a lack offurther antibacterial activity.

Secondly, it has been shown that decreasing thesize of the inoculum applied to the bladder wallsignificantly increases the per cent of organismskilled by the wall. This "inoculum effect" againsuggests a substance that has a finite capacity tohandle a certain number of organisms and whoseantibacterial activity may be overcome by increas-ing the numbers of organisms with which it mustcontend.

The significance of the antibacterial activity ofthe bladder in handling an infecting inoculum isstill unclear. It is reasonable to think that the anti-

bacterial activity of the bladder acts to inhibit mul-tiplication of organisms between voidings, and byso doing hastens the clearance of bacteria fromthe bladder. However, in this experimental systemS. aureus, which is not a usual urinary tract patho-gen in rats (when inoculated into the bladder),was poorly killed by the bladder mucosa, whereasP. mirabilis, a known urinary pathogen in rats,was killed rapidly by the bladder mucosa. It isdifficult presently to reconcile these experimentalresults with the theoretical possibility that the sus-ceptibility of some individuals to urinary tract in-fection may be due to relatively decreased anti-bacterial activity of the bladder. Despite thesedifficulties, it is clear that the bladder possessesintrinsic antibacterial activity. The role and signifi-cance of this mechanism in the pathogenesis of uri-nary tract infections clearly warrants furtherinvestigation.

ACKNOWLEDGMENTS

We are grateful to Miss Anne Gillespie and Miss RoseMary Bacina for excellent technical assistance. Wethank Dr. Ramzi Cotran for his assistance with the his-tologic material and for his encouragement and advice.We are indebted to Dr. Paul Levy for statistical advicegiven during this study.

This work was supported by grants T1-AI-00068 andAI-06577 from the National Institute of Allergy andInfectious Diseases, and grant 176B from the New YorkTuberculosis and Health Association, Inc.

REFERENCES

1. Vivaldi, E., R. Cotran, D. P. Zangwill, and E. H.Kass. 1959. Ascending infection as a mechanism inthe pathogenesis of experimental non-obstructivepyelonephritis. Proc. Soc. Exptl. Biol. Aled. 102: 242.

2. Cotran, R. S., E. Vivaldi, D. P. Zangwill, and E. H.Kass. 1963. Retrograde Proteus pyelonephritis inrats. Amn. J. Pathol. 43: 1.

3. Cox, C., and F. Hinman. 1961. Experiments with in-duced bacteriuria, vesical emptying and bacterialgrowth on the mechanism of bladder defense to in-fection. J. Urol. 86: 739.

4. Cotran, R. S., L. D. Thrupp, S. N. Halj, D. P.Zangwill, E. Vivaldi, and E. H. Kass. 1963. Retro-grade E. coli pyelonephritis in the rat: a bacterio-logic, pathologic and fluorescent antibody study. J.Lab. Clin. Med. 61: 987.

5. Cox, C., and F. Hinman. 1965. Factors in resistanceto infection in the bladder. I. The eradication of bac-teria by vesical emptying and intrinsic defense mecha-nisms. In Progress in Pyelonephritis. E. H. Kass,editor. F. A. Davis Company, Philadelphia. 563.


6. Vivaldi, E., J. Mufioz, R. S. Cotran, and E. H. Kass.1965. Factors affecting the clearance of bacteriawithin the urinary tract. In Progress in Pyelone-phritis. E. H. Kass, editor. F. A. Davis Company,Philadelphia. 531.

7. Cobbs, C., and D. Kaye. 1967. Antibacterial mecha-nisms in the urinary bladder. Yale J. Biol. Med. 40:93.

8. Paquin, A., J. Perez, C. Kunin, and E. Foster. 1965.Does the bladder possess an intrinsic antibacterialdefense mechanism? J. Clin. Invest. 44: 1084.

9. Mulholland, G., E. Foster, J. Gillenwater, and A.Paquin. 1966. Effect of vesical mucosa on bacterialgrowth. Clin. Res. 14: 341.

10. Green, G. M., and E. H. Kass. 1964. The role of thealveolar macrophage in the clearance of bacteriafrom the lung. J. Exptl. Med. 119: 167.

11. Green, G. M., and E. Goldstein. 1966. A method forquantitating intrapulmonary bacterial inactivation inindividual animals. J. Lab. Clin. Med. 68: 669.

12. Kass, E. H. 1960. Bacteriuria and the pathogenesisof pyelonephritis. Lab. Invest. 9: 110.


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