Vol. 30, No. 2INFECTION AND IMMUNITY, Nov. 1980, p. 554-5610019-9567/80/1 1-0554/08$02.00/0

Effect of Piliation on Klebsiella pneumoniae Infection in RatBladders

ROBERT C. FADER AND CHARLES P. DAVIS*Department ofMicrobiology, University of Texas Medical Branch, Galveston, Texas 77550

The possible role of pili in the pathogenesis of urinary tract infection caused byKlebsiella pneumoniae was investigated in a rat model of cystitis by utilizingpiliated- and nonpiliated-phase organisms derived from a single parent strain.Bladder surfaces were examined for evidence of infection by scanning electronmicroscopy. In animals infected with piliated-phase organisms, foci of infectionwere evident in the majority of bladders examined. Rat bladders associated withnonpiliated-phase bacteria showed little evidence of infection. The ability of a-methyl-D-mannoside, a known inhibitor of pilus-mediated adherence to mam-malian cells, to protect the bladder surface from colonization was also tested. Theresults showed a significant decrease in the ability of piliated-phase K. pneumo-niae to establish infection in bladders. These observations suggest that pili mayplay an intergral role in the ability of K. pneumoniae to cause urinary tractinfections by mediating the attachment of the bacteria to the uroepithelial surface.

Bacterial adherence to epithelial cell surfacesis believed to be an important first step in theinitiation of certain infections (3, 4, 7, 17). Onemeans of bacterial attachment to mammaliancells is by bacterial surface appendages, referredto as pili or funbriae. Silverblatt has implicatedpili as possibly playing a role in the pathogenesisof pyelonephritis caused by Proteus spp. (13,14). Other investigators have implicated pili asbeing the mediators of Escherichia coli attach-ment to urinary tract epithelial cells under invitro conditions (12, 15). We have previouslyreported that Klebsiella pneumoniae also ap-pears to utilize pili to adhere to rat bladderepithelial cells in vitro (5).Davis et al. investigated the in vivo adherence

of K. pneumoniae to rat bladder epithelium bythe use of scanning electron microscopy (SEM)(1). Their study revealed that K. pneumoniaewas capable of adhering to the bladder surfaceand elicited a host response which consisted ofstrand entrapment of bacteria, epithelial cellexfoliation, and elimination of attached bacteriaby voiding. However, it was not known whetherthe infecting organisms possessed pili. The studyreported here has utilized piliated- and nonpi-liated-phase K. pneumoniae derived from a sin-gle parent strain to determine whether pili influ-ence the alterations that occur when K. pneu-moniae infects bladders. In addition, a-methyl-D-mannoside, a known inhibitor of pilus-me-diated adherence, was tested to determinewhether the saccharide could protect the blad-der surface from K. pneumoniae adherence.

554

MATERLALS AND METHODS

Bacteria. The K. pneumoniae used throughout thestudy was donated by the Clinical Laboratories in theJohn Sealy Hospital, Galveston, Tex., after isolationfrom a human urinary tract infection. The nonpiliatedform of the organism was maintained on nutrient agar(BBL Microbiology Systems, Cockeysville, Md.) andstored at 4°C. The piliated phase was produced byserial passage in broth medium over the course of 1week. Piliation was confirmed by transmission electronmicroscopy (TEM) and by hemagglutination of guineapig erythrocytes. The piliated phase was maintainedin Trypticase soy broth (BBL) with dextrose added ata concentration of 5 g/liter. The organisms were storedat -70°C until use. Serological tests with the organ-isms indicated that, antigenically, the phases differedonly in the presence or absence of pili (5). No biochem-ical differences were noted in the two phases when theorganisms were shifted from the nonpiliated to piliatedphase by serial passage in broth medium (5). To pre-pare bacteria for adherence studies, 24-h broth cul-tures in either piliated or nonpiliated phases werewashed three times in phosphate-buffered saline(PBS, pH 7.2; 5 ml, 10 min, 260 x g) and resuspendedin PBS to a concentration of 108 bacteria per ml.Immediately before catheterization, organisms weretested for hemagglutinating activity to assay the de-gree of piliation in the population (5).Animals and catheterization. Animals were in-

fected by a modification of the technique described byUehling and Wolf (16). Female Wistar rats (TimcoBreeding Laboratories, Houston, Tex.) weighing 151to 175 g were anesthetized by ketamine hydrochloride(Parke, Davis & Co., Detroit, Mich.) and catheterizedwith tapered polyethylene tubing (outer diameter, 0.18cm) attached to a 1-ml syringe with a 19-gauge needle.The bacterial suspension (5 X 107 in 0.5 ml) was

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K PNEUMONIAE INFECTION IN BLADDERS 555

injected, the catheter was removed, and the externalmeatus was sealed with paraffin for 4 h. Control ani-mals were injected with 0.5 ml of sterile PBS. In viewof previously published data (1) that indicated no

surface alterations as a result of injection of PBS intobladders, we used only two rats for controls in eachtime period of the study. In rats sacrificed at 4 h, urinewas removed by syringe after intravesical puncture ofthe bladder wall. In animals sacrificed at 12 h or 5 dayspostinfection, the paraffin was removed after 4 h toallow the animals to urinate.

Tissue preparation for SEM. Animals were sac-rificed at 4 h, 12 h, or 5 days postinfection, and thebladders were removed aseptically. Each bladder wasseparated into two pieces by a longitudinal incision,and the pieces were immediately placed in cold 2.5%glutaraldehyde in 0.1 M sodium cacodylate and keptovernight at 4°C. Specimens were rinsed twice in 0.1M sodium cacodylate and postfixed overnight with 1%osmium tetroxide in 0.1 M sodium cacodylate at 4°C.The bladders were rinsed again in sodium cacodylateand dehydrated through a series of ethanol baths. Thespecimens were critical-point dried, coated with gold-palladium, and examined by SEM.

Electron microscopy of urine. Urine removedfrom rats sacrificed at 4 h was prepared for electronmicroscopy by the following procedure. One drop ofthe urine was placed on a sheet of dental wax (Poly-sciences, Inc., Warrington, Pa.), and a Formvar-coatedcopper grid was floated on the drop for 15 min. Thegrid was transferred to a drop of distilled water for 5min before being floated on a drop of2% uranyl acetatefor 30 min. The grid was then transferred to anotherdrop of distilled water for 5 min, after which excess

liquid was removed from the grid with a pointed pieceof filter paper. The grids were then examined by TEM.

Viable counts of bacteria in bladders. To deter-mine the effect of the 4-h incubation in bladders on

the viability of the bacteria, three rats each were

infected with piliated- or nonpiliated-phase organisms.At the end of 4 h, the abdominal cavity was opened,the ureters and urethra were clamped with hemostats,and the entire bladder was excised and immediatelyplaced in a glass tissue grinder containing 5 ml ofsterile 0.85% saline. The bladders were homogenized,diluted, and plated in duplicate onto nutrient agar

(BBL) and incubated overnight at 37°C. Colonies werecounted on the next morning, and the number of viableorganisms per bladder was calculated. The resultsindicated that no differences in viability between pi-

liated- and nonpiliated-phase organisms existed after4 h in bladders; both groups contained approximately107 bacteria per bladder.Saccharide inhibition. Piliated-phase bacteria

were resuspended to a concentration of 108 bacteriaper ml in PBS containing 50 mg of a-methyl-D-man-noside (Sigma Chemical Co., St. Louis, Mo.) per ml or

50 mg of dextrose (BBL) per ml. The bacterial suspen-sions were injected into the bladders through a cath-eter as described above. Control animals were injectedeither with 0.5 ml of PBS containing 50 mg of a-

methyl-D-mannoside or dextrose per ml or with pi-liated-phase K.pneumoniae resuspended in PBS with-out the saccharides. Animals were sacrificed at 4 h andthe bladders were processed and examined by SEM.

Hemagglutination tests were performed on bacterialsuspensions before injection into the bladders. Pi-liated-phase organisms resuspended in PBS withouta-methyl-D-mannoside were able to hemagglutinatethe guinea pig erythrocytes, whereas piliated-phaseorganisms in PBS containing a-methyl-D-mannosidewere unable to cause erythrocyte agglutination. Dex-trose had no effect on hemagglutination.

RESULTSEffect of piliation on K pneumoniae in-

fection in rat bladders. Animals were sacri-ficed at 4 h, 12 h, or 5 days postinfection, andthe bladders were viewed for evidence of infec-tion. In control animals injected with PBS, noadherence of bacteria, ulceration of the bladdersurface, or swelling of the bladder folds wasevident at any time throughout the study (Table1). In rats infected with piliated- phase K. pneu-moniae and sacrificed at 4 h, all six bladdersexamined showed evidence of severe bladderfold swelling. This swelling often occurred adja-cent to areas of normal bladder folds (Fig. 1).Five of the six bladders examined had bacteriaadherent to the bladder surface and demon-strated areas of ulceration which were charac-terized by tissue disruption with the presence oferythrocytes. Organisms were most often ob-served in areas of necrosis and were usuallyfound entrapped in fibrin-like strands and asso-ciated with exfoliated epithelial cells (Fig. 2).Bacterial entrapment in fibrin-like strands couldalso be observed in the lumen of the bladder

TABLE 1. Effects ofpiliation on K. pneumonriae-induced alterations in rat bladders

Bacte-

*ime' Phase ofb acteria ria on Ulcer- Swell-Time" Phase of bacteria blde ato .ng,bladder ation" ng'surface

4 h Control (PBS)Y 0/2f 0/2 0/2Piliated 5/6 5/6 6/6Nonpiliated 2/6 0/6 2/6

12 h Control 0/2 0/2 0/2Piliated 6/6 6/6 6/6Nonpiliated 1/6 1/6 1/6

5 days Control 0/2 0/2 0/2Piliated 2/6 4/6 4/6Nonpiliated 0/6 1/6 1/6

a Bladders observed by SEM.Time indicates number of hours postinfection

when bladders were removed for observation.'Ulceration indicates areas of histological damage

with erythrocytes present.d Swelling indicates areas where bladder folds are

more tl n two to three times normal width.'Control animals injected with PBS.f Number of positive bladders/total number of blad-

ders observed.

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when urine was examined by TEM (Fig. 3). Inrats infected with nonpiliated-phase K. pneu-moniae, only two of six bladders were observedto have adherent bacteria on the bladder surface,but these few bacteria were not associated withulceration, nor did there appear to be any hosttissue response to their presence (Fig. 4). Inaddition, fibrin-like strands were not associatedwith nonpiliated-phase organisms in the urine(Fig. 5). There was no evidence of tissue damagein the six bladders examined, although two blad-ders exhibited slight swelling of the bladderfolds. No conversions from the piliated to thenonpiliated phase, or vice versa, were noted XAwhen organisms in the urine for 4 h were exam- 0 V-^ined by TEM (Fig. 6).

In the groups of animals sacrificed 12 h post-infection, all six rats infected with piliated-phasebacteria exhibited evidence of cystitis with in-tense bladder fold swelling and ulceration of the abladder surface with numerous organisms pres-ent. Organisms appeared to be penetrating intothe underlying epithelium in areas of severemembrane disruption (Fig. 7). Only one of sixbladders associated with nonpiliated-phase K.pneumoniae demonstrated evidence of infectionwith adherent bacteria in areas of ulceration andswelling of the bladder folds.By day 5 of the study, four of six rats infected

with piliated-phase organisms continued to dem-onstrate evidence of cystitis although organismswere visible on only two of the six bladdersexamined. Organisms observed in areas of necro-sis were again found to be associated with strandformation and exfoliated epithelial cells (Fig. 8).In rats infected with nonpiliated-phase K. pneu-moniae, only one bladder showed surface ulcer-ation and bladder fold swelling, but no organismswere visible on the bladder surface.

FIG. 2. (a) Area of ulceration showing bacteriaentrapped in fibrin-like strands (arrows), altered ep-

_ , 5 =4; 2 ithelium (AE), and an area of normal epithelium5 5 e ~A id (NE). Bar = 30 tum. (b) Higher magnification showing

^ ww bacteria (arrows) and crenated erythrocytes en-trapped in fibrin-like strands. Bar = 15 Aim.

-~iiw} AdEffect of a-methyl-D-mannoside on K.4i i> vpneumoniae-induced cystitis. Results from

our in vitro studies (5) suggested that a-methyl-D-mannoside might block the in vivo adherenceof piliated K.pneumoniae to the bladder surface.To test this hypothesis, rats were injected withpiliated-phase K. pneumoniae resuspended inPBS with a-methyl-D-mannoside added at a

FIG. 1. Rat bladder surface showing normal blad- concentration of 50 mg/ml. Rats were sacrificedder folds (N) and an area of intense bladder fold at 4 h, and the bladders were examined by SEM.swelling (S). Bar = 250 um. To determine what effect saccharides alone

556 FADER AND DAVIS INFECT. IMMUN.

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K PNEUMONIAE INFECTION IN BLADDERS 557

would have on bladder epithelium, D-glucoseand a-methyl-D-mannoside were dissolved inPBS at a concentration of 50 mg/ml, and thesolution was injected into the bladders. After 4h in the bladders, no bacterial adherence wasobserved, nor were any areas of tissue damagepresent (Table 2). One bladder injected withPBS containing a-methyl-D-mannoside ap-peared to have some areas of slight bladder foldswelling.As another control, rats were injected with

piliated-phase organisms in PBS without theaddition of any saccharides. At the end of 4 h,all five bladders examined showed areas of ul-ceration and swelling. Adherent bacteria werenoted on four of the five bladders (Table 2).

FIG. 5. Nonpiliated-phase organisms in the urineofa rat at 4 hpostinfection. Compare with Fig. 3, andnote the reduction of fibrin-like strands associatedwith the bacteria. Bar = 1.5 pum.

FIG. 3. Piliated-phase bacteria entrapped in fi-brin-like strands in the urine of a rat at 4 h postin-fection. Compare with Fig. 5. Bar = 1.5 ,im.

FIG. 4. Nonpiliated-phase organisms (arrows) as-

sociated with normal epithelium. Note the absence offibrin-like strands normally associated with adherentbacteria. Bar = 15 ,um.

FIG. 6. (a) Organism at 4 h after being injectedinto the bladder in the piliated phase. Note the piliprotruding from the bacterial surface. Bar = 0.5 ,tm.(b) Organism at 4 h after being injected into thebladder in the nonpiliated phase. Note the absenceofpili. Bar = 0.5 jim.

Finally, piliated-phase bacteria were resus-pended in PBS containing D-glucose or a-methyl-D-mannoside and injected into the blad-ders. In bladders associated with piliated-phase

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558 FADER AND DAVIS

DISCUSSIONThe attachment of bacteria to the uroepithel-

ium is generally thought to be a requirement forthe successful colonization and subsequent in-fection of the urinary tract. A number of inves-tigators have suggested that pili play an impor-tant role in the pathogenesis of urinary tractinfections; however, the results of these studieshave been inconclusive either because adherencewas investigated with in vitro systems (5, 12, 15)

FIG. 7. (a) Area of severe membrane disruptionwith organisms appearing to penetrate into the un-derlying epithelium (arrows). Bar = 25 pm. (b) Bac-terium penetrating into the underlying epithelium(arrow). Adjacent organisms appear to be lying inmicroplicae on the epithelial surface. Bar = 5 um.

K. pneumoniae in PBS plus D-glucose, bacterialadherence, ulceration of the bladder surface, andswelling of the bladder folds were noted in allfive bladders examined. However, when a-methyl-D-mannoside was injected into the blad-ders with piliated-phase organisms, only onebladder appeared infected, and in this bladderonly one small area of ulceration could be found.Two of the five bladders exhibited slight bladderfold swelling.

FIG. 8. (a) Area ofulceration (U) on day 5 adjacentto an area of normal epithelium (NE). Area in boxshown at a higher magnification in (b). Bar = 25 ,um.(b) Area of necrosis showing an amorphous strand(A) associated with bacterial entrapment (arrows).Bar = 5 um.

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K PNEUMONIAE INFECTION IN BLADDERS 559

TABLE 2. Inhibitory effect of a-methyl-D-mannosideon K. pneumoniae-induced bladder alterations

BacteriaPhase of Treatment on blad- Ulcera- Swell-bacteria der sur- tionb ingC

face

Control a-Methyl- 0/2e 0/2 1/2(PBS) D-man-

nosidedControl D-Glucosef 0/2 0/2 0/2

(PBS)Piliated None 4/5 5/5 5/5Piliated D-Glucoseh 5/5 5/5 5/5Piliated a-Methyl- 1/5 1/5 2/5

D-man-noside'

a Bladders examined by SEM.b Ulceration indicates areas of histological damage

with erythrocytes present.' Swelling indicates areas where bladder folds are

more than two to three times normal width.d Control rats injected with PBS containing 50 mg

of a-methyl-D-mannoside per ml.e Number of positive bladders/total number of blad-

ders observed.f Control rats injected with PBS containing 50 mg

of D-glucose per ml.5Piliated-phase K. pneumoniae resuspended in

PBS with no saccharide.h Piliated-phase bacteria resuspended in PBS con-

taining 50 mg of D-glucose per ml.'Piliated-phase bacteria resuspended in PBS con-

taining 50 mg of a-methyl-D-mannoside per ml.

or because nonpiliated-phase variants of the in-fecting organisms were not obtainable for com-parison. The use of a K. pneumoniae strain thatcan be controlled in its degree of piliation hasenabled us to study the effects of piliation on theability of K. pneumoniae to cause infection inbladders. This is the first study which providesevidence that not only the severity but also theduration of K. pneumoniae infection is signifi-cantly increased after injection of piliated-phaseorganisms into bladders.We have considered the possibility that by

controlling the time of growth in broth mediumwe may be altering other bacterial factors inaddition to the presence or absence of pili. How-ever, extensive serological studies with this or-ganism have indicated that antiserum formedagainst piliated-phase organisms, when ab-sorbed with the nonpiliated-phase bacteria,yields a single band of precipitin to the piliatedorganisms in Ouchterlony plates. In addition,this antiserum also yields a single precipitinband to isolated K. pneumoniae pili. Conversely,when antiserum raised against nonpiliated-phase organisms was absorbed with piliated-phase bacteria, no evidence of antibodies to the

nonpiliated phase remains (5). We interpretedthese results to mean that, antigenically, the twophases differed only in the presence or absenceof pili. In addition, biochemical tests performedwhile the organism was shifted from the nonpi-liated to the piliated phase by daily passage inbroth medium did not result in any biochemicaldifferences between the two phases (5).

Differences in infectivity between the piliated-and nonpiliated-phase organisms became readilyapparent in the rats sacrificed at 4 h. Theseanimals were sacrificed without being allowed tourinate, and the urine was removed from thebladder by syringe. In five of the six rats infectedwith piliated-phase K. pneumoniae, the urineshowed a reddish appearance that, after exami-nation by light microscopy, could be attributedto the presence of erythrocytes in the urine. Thisfinding suggested that piliated-phase organismswere able to cause tissue destruction in thebladder, leading to erythrocyte leakage into thelumen. The urine specimens from animals in-jected with nonpiliated-phase organisms werefree of erythrocytes, as were urine samples takenfrom control animals injected with sterile PBS.When urine was examined by TEM at 4 h, it wasobserved that organisms injected into the blad-der in the piliated phase remained piliated,whereas organisms injected in the nonpiliatedphase remained devoid of pili (Fig. 6).

Further differences in infectivity appearedafter examination of the bladders by SEM. Inbladders infected with piliated-phase bacteria,intense swelling of the bladder folds occurred(Fig. 1), and areas of necrosis with bacteria pres-ent were readily visible. Bacterial penetration ofthe underlying epithelium could be observed(Fig. 7) and may have been responsible for theerythrocytes present in the urine. Similar pene-tration has been observed with Escherichia coli(6, 10). Nonpiliated-phase organisms were rarelyfound on the bladder surface, and these organ-isms did not appear to be penetrating the epi-thelium, nor did they appear to have elicited anyhost tissue response to their presence (Fig. 4).There is a possibility that these adherent organ-isms may have produced pili after injection intothe bladder, and if so, the two bladders demon-strating evidence of cystitis after associationwith nonpiliated-phase K. pneumoniae mayhave actually been colonized by piliated-phasebacteria. In either case, bladder fold swellingand necrosis in bladders associated with nonpi-liated-phase bacteria were much less than thatobserved with piliated-phase organisms.This study also supports the findings of other

investigators concerning the defense system ofthe bladder in response to bacterial infection (1,

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560 FADER AND DAVIS

10). Fibrin-like strands with entrapped bacteriawere noted in areas of ulceration (Fig. 2), over-lying areas of normal epithelium, and in thelumen of the bladder when the urine was ex-amined by TEM (Fig. 3). Amorphous strandformation was also evident (Fig. 8) and wasclosely associated with bacterial entrapment. Inmost areas of necrosis, bacteria were found to beentrapped in strands and associated with exfol-iated epithelial cells. It has been suggested thatentrapment of bacteria and subsequent exfolia-tion of epithelial cells into the lumen may playa role in clearing microorganisms from the urineand bladder surface (1, 10).The results of the saccharide inhibition study

have provided further evidence that the adher-ence of the K. pneumoniae strain is mediated bypili. D-Mannose and its analog, a-methyl-D-man-noside, have long been known to inhibit pilus-mediated adherence to mammalian cells (9). Wepreviously reported that both saccharides in-hibited the adherence of piliated-phase K. pneu-moniae to rat bladder epithelial cells in vitro (5).This study has indicated that the inhibition canoccur in an in vivo system. In the group ofanimals infected with piliated-phase K. pneu-moniae resuspended in PBS with a-methyl-D-mannoside, only one bladder exhibited any tis-sue damage, and only one small area of ulcera-tion with adherent organisms was visible on thebladder surface. We consider this decrease intissue damage as evidence that the saccharideinterfered with the interaction of the bacteriawith the bladder surface. It has been theorizedthat a mannose-containing receptor site for piliis present on certain mammalian cells (8). How-ever, this receptor has not been isolated andcharacterized. No evidence exists that the inhib-itory saccharides bind to the bacteria (2); con-sequently, it is believed that the saccharide in-teracts with the epithelial surface. Whether thesaccharide binds directly to the pilus receptorsite or adjacent to it causing steric hindrance isnot currently known.0rksov et al. have recently suggested that

Enterobacteriaceae possessing type I pili (7 nmin diameter, mannose-sensitive adherence) ad-here to mucin rather than to the actual cellsurface. They state that a new type of pilus,referred to as F7, mediates the adherence of E.coli to uroepithelial cells (11). We have beenable to find only type I pili on the K.pneumoniaestrain used throughout this study, and in viewof our previous report (5) and the results of thisstudy, we can assume that K. pneumoniae isable to adhere to the epithelial cell surface. Theapparent discrepancy between our findings andthose reported by 0rskov et al. may have to dowith the epithelial cells used in the two studies.

0rskov et al. utilized human uroepithelial cellsthat were collected from fresh morning urine.These cells represent the oldest epithelial cellsto the urinary tract and also represent cells fromall areas of the urinary system. We used ratbladder epithelial cells scraped from the bladdersurface, and after three PBS washes of the blad-der cells, we were unable to visualize mucin onany cell surface. Another possible explanationfor the differences observed relates to the use oftwo different genera of bacteria. 0rskov et al.utilized E. coli strains for their study, and al-though K. pneumoniae and E. coli type I pili aremorphologically similar, subtle differences mayexist between the two pilus types and may ac-count for the differences observed in adherence.The results of this study suggest that pili

mediate the attachment of the K. pneumoniaestrain to rat bladder epithelium and that theattachment is a necessary step in the pathogen-esis of cystitis caused by the organism. However,we would like to stress that we do not feel thatpili are the sole virulence factor of this organism.It is likely that adherence of the bacteria to theepithelial surface is only the initial step in theinfectious process. Once the organism is at-tached to the bladder surface, other virulencefactors may produce the tissue alterations ob-served in this study. Nonetheless, piliation ap-parently is necessary for this sequence of path-ogenesis to occur with the K. pneumoniae strainused in our study.

ACKNOWLEDGMENTS

This work was supported in part by Public Health Servicegrant 5 RO1 AI 14508 from the National Institute of Allergyand Infectious Diseases. R.C.F. was supported by the JamesW. McLaughlin Fellowship Fund.We thank Susan Sratzfeld and Andrejs Auots-Auotins for

technical assistance.

LITERATURE CITED1. Davis, C. P., E. Balish, K. Mizutani, and D. T. Uehl-

ing. 1977. Bladder response to Klebsiella infection: ascanning electron microscopy study. Invest. Urol. 15:227-231.

2. Duguid, J. P., and R. R. Gillies. 1957. Fimbriae andadhesive properties in dysentery bacilli. J. Pathol. Bac-teriol. 74:397-411.

3. Ellen, R. P., and R. J. Gibbons. 1974. Parameters af-fecting the adherence and tissue tropisms of Strepto-coccus pyogenes. Infect. Immun. 9:85-91.

4. Evans, D. G., R. P. Silver, D. J. Evans, Jr., D. G.Chase, and S. L. Gorbach. 1975. Plasmid-controlledcolonization factor associated with virulence in Esche-richia coli enterotoxigenic for humans. Infect. Immun.12:656-667.

5. Fader, R. C., A. E. Avots-Avotins, and C. P. Davis.1979. Evidence for pili-mediated adherence of Klebsi-ella pneumoniae to rat bladder epithelial cells in vitro.Infect. Immun. 25:729-737.

6. Fukushi, Y., S. Orikasa, and M. Kagayama. 1979. Anelectron microscopic study of the interaction between

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K. PNEUMONIAE INFECTION IN BLADDERS 561

vesical epithelium and E. coli. Invest. Urol. 17:61-67.7. Jones, G. W., and J. M. Rutter. 1972. Role of the K88

antigen in the pathogenesis of neonatal diarrhea causedby Escherichia coli in piglets. Infect. Immun. 6:918-927.

8. Ofek, I., D. Mirelman, and N. Sharon. 1977. Adherenceof Escherichia coli to human mucosal cells mediatedby mannose receptors. Nature (London) 265:623-625.

9. Old, D. C. 1972. Inhibition of the interaction betweenfimbrial hemagglutinins and erythrocytes by D-man-nose and other carbohydrates. J. Gen. Microbiol. 71:149-157.

10. Orikasa, S., and F. Hinman, Jr. 1977. Reaction of thevestical wall to bacterial penetration: resistance to at-tachment, desquamation, and leukocyte activity. Invest.Urol. 15:185-193.

11. 0rskov, I., F. 0rskov, and A. Birch-Anderson. 1980.Comparison of Escherichia coli fimbrial antigen F7with type 1 flmbriae. Infect. Immun. 27:657-666.

12. Schaeffer, A. J., S. K. Amundsen, and L. N. Schmidt.

1979. Adherence of Escherichia coli to human urinarytract epithelial cells. Infect. Immun. 24:753-759.

13. Silverblatt, F. J. 1974. Host-parasite interaction in therat renal pelvis. A possible role for pili in the pathogen-esis of pyelonephritis. J. Exp. Med. 140:1696-1711.

14. Silverblatt, F. J., and I. Ofek. 1978. Influence of pili onthe virulence ofProteus mirabilis in experimental hem-atogenous pyelonephritis. J. Infect. Dis. 138:664-667.

15. Svanborg-Eden, C., and H. A. Hansson. 1978. Esche-richia coli pili as possible mediators of attachment tohuman urinary tract epithelial cells. Infect. Immun. 21:229-237.

16. Uehling, D. T., and L. Wolf. 1969. Enhancement of thebladder defense mechanism by immunization. Invest.Urol. 6:520-526.

17. Ward, M. E., and P. J. Watt. 1975. Studies on the cellbiology of gonorrhoea, p. 229-242. In D. Danielsson, L.Juhlin, and P. A. Mirdh (ed.), Genital infections andtheir complications. Almqvist & Wiksell International,Stockholm.

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