INFECTION AND IMMUNITY, June 1994, p. 2178-2186 Vol. 62, No. 60019-9567/94/$04.00+0Copyright ©) 1994, American Society for Microbiology

Effect of Salicylate, Bismuth, Osmolytes, and TetracyclineResistance on Expression of Fimbriae by Escherichia coli

CALVIN M. KUNIN,I* TONG HUA HUA,' RICHARD L. GUERRANT,2 AND LAUREN 0. BAKALETZ3Department of Intemal Medicine' and Otologic Research Laboratories,3 The Ohio State University, Columbus, Ohio

43210, and Department of Medicine, University of Virginia, Charlottesville, Virginia 229082

Received 19 October 1993/Returned for modification 14 December 1993/Accepted 9 March 1994

Adherence of Escherichia coli is facilitated by fimbriae and several outer membrane proteins (OMPs).Hypertonic conditions, salicylate, and Mar mutations are known to reduce OmpF expression. We speculatedthat OMPs involved in export or assembly of fimbrial subunits might be similarly affected. To explore thishypothesis, E. coli expressing P, type 1, S, colonization factor antigen I (CFA/I), or CFA/II fimbriae was grownin the presence of salicylate, bismuth salts, NaCl, and nonfermented sugars. Tetracycline-resistant clones werederived from several P-fimbriated strains. The bacteria were tested for the ability to agglutinate erythrocytes,yeast cells, and a-D-Gal(-4)-,I-D-Gal-bonded latex (Gal-Gal) beads and were examined for fimbriae by electronmicroscopy. Hyperosmolar conditions decreased fimbrial expression for all strains. Expression of P fimbriaeby pyelonephritic strains, all of which were OmpF+, was reversibly repressed by salicylate and bismuth salts.CFA strains were similarly affected. Tetracycline-resistant P-fimbriated strains were OmpF deficient, wereunable to agglutinate erythrocytes and Gal-Gal beads, and lacked fimbriae as observed by electron microscopy.Strains with plasmid-encoded P-fimbrial genes did not demonstrate OmpF on polyacrylamide gel electro-phoresis profiles and were not affected by salicylate. The type 1-fimbriated phenotype was not affected bysalicylate or bismuth unless the strains also expressed P fimbriae. S-fimbriated strains were not affected. Themechanism by which salicylates, bismuth salts, and tetracycline resistance inhibit or modulate the expressionof P fimbriae may be mediated through OmpF and other OMPs.

Fimbriae and other bacterial adhesins facilitate attachmentto epithelial cell surfaces and play an important role in theinitiation of urinary, gastrointestinal, and respiratory tractinfections (1, 13, 20, 25, 34, 38, 43, 44). Escherichia coli strainsbearing P fimbriae are frequently associated with acute, un-complicated pyelonephritis and bacteremia (29, 34) S-fimbri-ated E. coli strains are associated with meningitis and bacte-remia in newborns (24, 35). Type 1 fimbriae appear to have arole in colonization of the bladder (20). The colonizationfactor antigen (CFA) adhesins are important virulence factorsfor enteropathogenic E. coli (13, 19).

Fimbriated E. coli strains undergo phase variation in re-sponse to changes in the growth medium. Type 1 fimbriae arepreferentially expressed in liquid media, and P fimbriae arepreferentially expressed on solid media (5, 25, 30). Phasevariation also occurs in urinary tract infections (36). Urea andother constituents of urine have been shown to modulate theratio of mannose-sensitive Fim+ to mannose-sensitive Fim- E.coli strains (33).

There is a growing body of evidence that in addition tofimbriae, several outer membrane proteins (OMPs) are alsoinvolved in adherence of bacteria to mammalian cells (41, 47)and that their expression is under osmoregulatory control (4,45). Localized adherence of enteropathogenic E. coli has beenshown to be related to the expression of an inducible OMP(47). Invasion of epithelial cells by Shigella fle-xneri is depen-dent on osmoregulated expression of OmpC (4), and adher-ence and invasion of Salmonella typhi requires the high-osmolarity conditions found in the distal ileum (45). Itoccurred to us that since the phenotypic expression of both

* Corresponding author. Mailing address: Department of InternalMedicine, The Ohio State University, Room M110 Starling LovingHall, 320 West 10th Ave., Columbus, OH 43210. Phone: (614)293-8976. Fax: (614) 293-5627.

type 1 fimbriae and several OMPs is responsive to the osmo-larity of the growth medium, these phenomena might belinked. OmpF is repressed by high osmolarity (3, 9, 28, 46), bysalicylate (6, 16, 37, 39), and in multiple-antibiotic-resistant(Mar) strains of E. coli (8). Reduced expression of OmpF isassociated with inhibition of transport of anionic molecules,including beta-lactam antibiotics and other drugs (2, 16).The current study was undertaken to determine whether

hypertonic media, salicylates, and tetracycline resistance,which are known to reduce OmpF expression, might also affectthe expression of fimbriae. Bismuth compounds were included,since bismuth subcitrate and bismuth subsalicylate have beenshown to block adhesion of enteroaggregative E. coli (18) andto enhance opsonophagocytosis of Klebsiella pneumoniae (10).

MATERIALS AND METHODS

Bacterial strains. Strains used were well-characterized E.coli strains expressing P, type 1, S, CFA/I, or CFA/II fimbriaeand several representative clinical isolates (Table 1). Thephenotypes of all strains were confirmed by agglutination testsusing human type 0, guinea pig, and bovine erythrocytes(RBCs) and yeast cells, with or without addition of 1%D-mannose. P fimbriation was also assessed by agglutination oflatex particles covalently bound to 0t-D-Gal(-4)-4-D-Gal-disac-charide (PF TEST; Orion Diagnostica, Espoo, Finland).Strains were grown overnight on Trypticase soy agar (TSA)(BBL, Cockeysville, Md.). Four colonies were harvested, sus-pended in a drop of phosphate-buffered saline (PBS) on thedried latex reagent test strip, and observed for agglutinationafter 2 min.

Strain J 96 (04:K6) was originally isolated from a patientwith pyelonephritis (31, 34). Strains HU849 and JJ48 expressP-fimbrial determinants cloned from strain J 96 (31). Strains1106, 1177, and 845 are pap' pil+ and were isolated from

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TABLE 1. Bacterial strains used in this study

Strain(s) Recombinant Selection antibiotic Adhesin Ompsa Source (reference[s])plasmid phenotype

J 96b None None P, 1 A, C, F J. Johnson (31, 34)1106 None None P, 1 A, C, F C. Svanborg (29)1177 None None P, 1 A, C, F C. Svanborg (29)HU734 None None P, 1 A, F C. Svanborg (43)845 None None P A, C, F C. Svanborg (29)U4 and B12C None None P A, C, F This studyHU849 pRHU845 Tetracycline P A, C S. Hull (31, 34)JJ48 pJJ48 Ampicillin P A, C J. Johnson (21)SC802 pDC1 Chloramphenicol P A, C S. Clegg (7)B24 and U42C None None 1 A, C, F This studyMG1655 None None 1 A, C I. Blomfeld (5)K10 None None 1 A, C, Fd L. T. Smith (27)HB101 pANN801-13 Ampicillin S A, C T. Korhonen (24)U6 None None S A, C, Fd This studyH10407 None None CFA/I A, C, F R. Guerrant (13)1392+ None None CFA/II A, C, F R. Guerrant (13)

a As determined by relative migration in a sodium dodecyl sulfate-PAGE system in this study.b Wild-type pyelonephritis strain from which the P-fimbrial genes were cloned in plasmids pRHU845 and pJJ48.Clinical isolates; B indicates isolation from blood, and U indicates isolation from urine.

d The OmpF band was much less intense than the bands for OmpA and OmpC.

children with their first known episode of acute pyelonephritis(29). Strain HU734 is a lac mutant derivative of an E. colipyelonephritis strain (GR12) derived by S. Hull and R. Hull(43). E. coli H10407 (078:H11) (CFA/I) is an enterotoxigenicstrain isolated in Bangladesh (13). E. coli 1392+ (06:H16)(CFANII) was derived from a strain isolated from a patient withdiarrhea (40). E. coli K10, which is used in studies of osmo-regulation and expresses type 1 fimbriae, was provided byLinda Tombras Smith of the University of California, Davis(27). Strains U4, U6, and U42 are clinical isolates obtainedfrom patients with acute cystitis, and strains B12 and B24 wereisolated from blood. The serotypes of the clinical isolates are02:H7 (U4 and B12), 027:H31 (U42 and B24), and 018:K1:H7 (U6). E. coli U6 is S fimbriated and contains the sfaSlocus in its chromosome (26).

Selection of tetracycline-resistant clones. P-fimbriated E.coli 1177 and U4, which are susceptible to 1.25 ,ug of tetracy-cline per ml, were serially passaged in increasing concentra-tions of tetracycline until clones resistant to 5 and 10 ,ug oftetracycline per ml were obtained. Further studies with thesestrains were conducted in tetracycline-containing media.Growth conditions. Strains expressing P or S fimbriae were

grown overnight at 37°C on TSA. CFA strains were grown onCFA agar (13). Cells were scraped from the plates, washedtwice with 0.15 M PBS (pH 7.2), and prepared as a suspensionof approximately 1010 CFU/ml of PBS containing 1% D-mannose. Strains expressing type 1, mannose-sensitive fim-briae were grown in Trypticase soy broth (TSB) (BBL) for 48h at 37°C. Cells were harvested by centrifugation, washedtwice, and suspended as described above.

Additives to the growth media. Bacteria were incubated inthe appropriate solid or liquid medium alone or with serialtwofold dilutions of 10 mM sodium salicylate (Sigma ChemicalCompany, St. Louis, Mo.), 10 mM colloidal bismuth subcitrate(Proctor & Gamble Company, Cincinnati, Ohio), or 2.5 mMbismuth subsalicylate (Proctor & Gamble). Bismuth subsalicy-late was solubilized from a 9% slurry by adding 1 part slurry to4 parts 0.1 mM sodium tartrate (42). The pH was adjusted to7.0, and the solution was serially diluted in the appropriatemedium. Most strains of E. coli achieved full density afterovernight culture in media containing up to 2.5 mM bismuth

subsalicylate but failed to grow at bismuth subsalicylate con-centrations of 5 mM. The CFA strains failed to grow in 1.25mM bismuth subsalicylate. Colonies or pellets of E. coli grownin the bismuth compounds had a brown coloration, but therewere no obvious morphologic changes noted upon examinationof Gram-stained preparations. The osmolytes used were NaCl,sucrose, adonitol, and cellobiose (Sigma). The sugars were notfermented by the E. coli strains used in this study, with theexceptions that adonitol was fermented by the CFA strains andsucrose was fermented by HU734. The osmolytes were addedto the growth media in 0.1 to 0.2 M stepwise linear dilutions.Growth of the strains was not inhibited at up to 0.8 Mconcentrations of the osmolytes. All experiments were re-peated at least twice.Micro-HA tests. Quantitative hemagglutination (HA) assays

were performed by adding 25 ,tl of serial twofold dilutions ofthe bacterial suspensions, with and without 1% D-mannose, toround-bottom microtiter wells (Corning Assay Plates; CorningGlass Works, Corning, N.Y.) containing 25 ,lI of RBC suspen-sions. Human type 0 RBCs (1%) were used for determinationof expression of P and S fimbriae, guinea pig RBCs (1%) wereused for type 1 fimbriae, and bovine RBCs (2%) were used forCFA. The plates were incubated overnight at 4°C and exam-ined for HA. The HA titer was the highest dilution of bacterialcells grown without additives which produced confluent HA.The end point for inhibition by an additive to the growthmedium was the minimum concentration which completelyprevented agglutination of cells at the HA titer.To determine whether sodium salicylate, bismuth subcitrate,

or bismuth subsalicylate might have a direct effect on HA, thebacteria were suspended in 10 mM concentrations of eachcompound in PBS and serially diluted in the same solutions.Aliquots were examined for HA as described above.

Yeast cell agglutination. Saccharomyces cerevisiae (baker'syeast; Standard Brands Inc., New York, N.Y.) was washedthree times in PBS and diluted to a final concentration of 1%(wt/vol). Agglutination tests were performed on glass slidesand in microdilution plates as described by Korhonen (23).

Preparation of cell extracts for OMPs. OMPs were preparedby differential centrifugation of crude sonicates obtained fromovernight cultures and solubilized by treatment with sodium-

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TABLE 2. Representative experiment in which the P-fimbriatedstrain E. coli HU734 was grown on TSA alone or on TSAcontaining various concentrations of salicylate or NaCl

Addition and concn (mM) HA titer

Salicylate0 1:642.5 1:325.0 1:8

10.0 1:8

NaCl0 1:640.2 1:640.4 1:80.6 1:20.8 1:2

N-lauryl sarcosinate (26). OMPs were resolved by vertical slabgel electrophoresis (12% polyacrylamide) and stained withCoomassie brilliant blue.

Electron microscopy. Twenty-four-hour cultures of isolateswere negatively stained with 2% (wt/vol) ammonium molyb-date-2% (wt/vol) ammonium acetate in distilled water. Thebacteria were suspended in distilled water and mixed with an

equal volume of stain. A Formvar-coated copper grid was

floated on the surface for 5 min. After being stained, grids wereblotted, allowed to air dry, and viewed immediately by trans-mission electron microscopy as previously described (1). Ap-proximately 100 cells were rated for the presence or absence of

fimbriae at a magnification of X42,000. All observations were

made blindly by the same observer.

RESULTS

Direct effect of salicylate or bismuth compounds on bacte-rial HA. Cell suspensions of E. coli 1106 and 1177 (whichexpressed both type 1 and P fimbriae) were obtained followinggrowth either on TSA or in TSB. They were serially diluted andtested for their abilities to agglutinate human 0 or guinea pigRBCs. Coincubation of the bacterial cells in media containing10 mM salicylate, bismuth subcitrate, or bismuth subsalicylatedid not block HA even at the maximum dilution of the cellsuspension.

Effect of growth in salicylate, bismuth compounds, andosmolytes on bacterial HA. A standardized method was used todefine the minimum concentration of each compound whichcompletely inhibited agglutination of RBCs or yeast cells.Results of a representative experiment with the P-fimbriatedstrain E. coli HU734 are shown in Table 2. The effect of growthin salicylate and NaCl on HA became more pronounced as thebacterial suspension was diluted. There was some variation(approximately twofold) among experiments in the HA titerand end points for inhibition by the various compounds. Themedian HA titer and the minimum or inhibitory concentra-tions which prevented agglutination at the HA titer are

reported in Table 3.Salicylate at a concentration of 2.5 mM prevented HA by

P-fimbriated strains isolated from humans with urinary tractinfections and by the CFA/I and CFA/II strains. Similar resultswere obtained with bismuth subcitrate, and a lower concentra-

TABLE 3. Effect of addition of salicylate, bismuth subcitrate, or bismuth salicylate to the growth media on the abilities of various strains ofE. coli to agglutinate RBCs or yeast cells

Agglutination' with:Fimbriae Strain(s) HA titera Bismuth Bismuth

Salicylate subcitrate subsalicylate

P (human 0 RBCs) J96 1:32 S S SS1106 1:128 S S S1177 1:16 S S SSHU734 1:64 S S S845 1:64 S S SSU4 and B12 1:8 S S SSHU849 1:128 R R RRJJ48 1:128 R S RRSC802 1:128 R R RR

Type 1 (guinea pig RBCs or yeast cells) J 96 1:64 R R RR1106 1:64 S R RR1177 1:64 S R RRHU734 1:64 S R RRB24 and U42 1:128 R R NDcMG1655 1:32 R R NDK1O 1:64 R R RR

S (bovine or human 0 RBCs) HB101 1:8 R R RRU6 1:16 R R RR

CFA (bovine RBCs) H10407 1:64 S S NGd1392+ 1:64 S S NG

aMedian HA titer for strain grown in medium alone.b Agglutination at the HA titer. S, inhibited by 2.5 mM (lowest concentration tested); R, not inhibited by 10 mM (highest concentration tested); SS, inhibited by 0.6

mM (lowest concentration tested); RR, not inhibited by 2.5 mM (highest concentration tested).c ND, not done.d NG, did not grow in 1.25 mM bismuth subsalicylate.

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TABLE 4. Effect of growth in Trypticase soy media containing NaCl, sucrose, adonitol, and cellobiose on agglutination of RBCs byrepresentative strains of E. coli

MICb (M)Fimbriae Strain HA titera

NaCI Sucrose Adonitol Cellobiose

P (human 0 RBCs) J 96 1:64 0.6 0.8 0.6 0.6845 1:32 0.3 0.3 0.6 0.4U4 1:8 0.4 0.4 0.6 0.6HU849 1:128 0.4 0.4 0.8 0.4

Type 1 (guinea pig RBCs or yeast cells) J 96 1:64 0.5 0.8 0.8 0.81106 1:64 0.4 0.3 0.4 0.4MG1655 1:32 0.4 0.4 0.4 0.4

S (bovine or human 0 RBCs) HB101 1:16 0.3 0.3 0.4 <0.2U6 1:8 0.4 0.8 0.4 NDC

CFA (bovine RBCs) H10407 1:64 <0.2 0.4 Ferments 0.41392+ 1:64 <0.2 0.6 Ferments 0.4

a Median HA titer for strain grown in unsupplemented medium.b Minimum concentration of osmolyte which prevented agglutination of RBCs or yeast cells.c ND, not done.

tion of bismuth subsalicylate (0.6 mM) prevented HA for somestrains. All of the susceptible strains expressed OmpF. Incontrast, recombinant P-fimbriated strains, which did notdemonstrate a characteristic OmpF band upon electrophore-sis, were not affected by 10 mM salicylate (the highest concen-tration tested). These strains were also insensitive to bismuthsalts, except that there was some effect on strain JJ48.

Salicylate and bismuth subcitrate at concentrations of 10mM had no effect on HA of guinea pig RBCs and yeast cells bystrains which expressed only type 1 fimbriae. They also did notinhibit the S fimbrial phenotype (Table 3). Three strains whichwere both type 1 and P fimbriated (1106, 1177, and HU734)failed to agglutinate RBCs or yeast cells when grown insalicylate (2.5 mM) but were insensitive to the bismuth salts.The type 1 fimbrial phenotype of strain J 96 was inhibited by 10mM salicylate.

Elfect of growth in salicylate and bismuth subcitrate onexpression of P fimbriae as determined by a-D-Gal(-4)-IP-D-Gal-disaccharide latex agglutination test. A positive test wasobtained with all P-fimbriated strains grown on TSA alone. Forstrains grown in salicylate, 1177, 845, U4, and B12 werenegative at 5 mM, weak agglutination was observed for 1106and HU734 at 10 mM, and J 96, HU849, JJ48, and SC802remained positive at 10 mM. For strains grown in bismuthsubcitrate, J 96 and 845 were negative at 5 mM, 1177, HU734,and 1106 were negative at 10 mM, and HU849, JJ48, andSC802 remained positive at 10 mM.

Reversibility of the effect of salicylate on expression of type1 and P fimbriae. E. coli 1106 and 1177, which expressed bothtype 1 and P fimbriae, were grown in TSB for 24 h in thepresence or absence of 5 mM salicylate. The cells wereharvested by centrifugation, washed twice in PBS, and thenadded to fresh medium of the opposite composition. A similarexperiment was conducted by taking cells grown on TSA platesfor 24 h, with or without 5 mM salicylate, and inoculating thecells, without washing, onto plates of the opposite composition.Controls were subcultured on the same medium for 24 and 48h. The effect of salicylate on inhibition of HA was completelyreversed when the cells were transferred to fresh medium andcould be initiated when salicylate was added to cells grown inits absence.

Eflect of addition ofNaCl and sugars to the growth media on

expression ofHA by fimbriated strains of E. coli. The effect ofgrowing cells in various osmolytes is shown for representativestrains in Table 4. Strains varied in sensitivity to the osmolytes,but HA was inhibited regardless of the type of fimbriaeexpressed or the presence or absence of OmpF.

Effect of tetracycline resistance on expression of P fimbriae.The tetracycline-susceptible parent strains of E. coli U4 and1177 agglutinated human 0 RBCs at dilutions of 1:8 and 1:32,respectively. Clones resistant to 5 or 10 ,ug of tetracycline perml were isolated after three passages on tetracycline-contain-ing media. The resistant strains were found to lack OmpF onpolyacrylamide gel electrophoresis (PAGE) and were unableto agglutinate Gal-Gal beads or human 0 RBCs, and fimbriaewere not present as observed by electron microscopy.

Effect of salicylate and bismuth compounds on OMP pro-files. OMP profiles of P-fimbriated strains isolated from hu-mans with acute pyelonephritis, cystitis, or bacteremia werefound to contain OmpF, OmpC, and OmpA (Fig. 1). The

kDa

97.4 -

66.2-

45.0 -

31.0-

2 3 4 5 6 7 8 9 10

FIG. 1. OMP profiles of P-fimbriated E. coli strains isolated frompatients with urinary tract infections and bacteremia and of an

S-fimbriated E. coli strain (HB101) which contains the plasmidpANN801-13. Lanes: 1 and 10, molecular mass markers; 2, 1106; 3,1177; 4, HU734; 5, 845; 6, U4; 7, B12; 8, J 96; 9, HB101. Note theabsence of an OmpC band in E. coli HU734 and of an OmpF band inthe S-fimbriated strain.

-Omp F-Omp C4-Omp A

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kDa

97.4-

66.2--

45.0-_S

31.0-_w

-Omp F-Omp C

4-Omp A

1 2 3 4 5 6

FIG. 2. OMP profiles of P-fimbriated strains of E. coli. Strainsinclude the pyelonephritis strain J 96 (lane 2); strains JJ48 (lane 3),HU849 (lane 4), and SC802 (lane 5), which contain plasmids thatencode P fimbriae (pJJ48, pRHU845, and pDC1, respectively); and theS-fimbriated strain HB101. Lane 1, molecular mass markers. Note thatonly J 96 contains OmpF in the PAGE profile.

observed molecular masses of both OmpF and OmpC in strainJ 96 consistently were greater than those noted for otherstrains. A Lac- derivative of E. coli HU734, from a pyelone-phritis strain (GR12), lacked a characteristic OmpC band, anda strain with a plasmid-encoded S-fimbrial gene (HB101)lacked an OmpF band (Fig. 1). P-fimbriated strains whichcontained plasmids encoding P-fimbrial determinants lackedOmpF at the expected electrophoretic position (Fig. 2).The effect of osmolytes, salicylate, and bismuth on OmpF,

OmpC, and OmpA porins was examined by using the wild-typeP-fimbriated E. coli strain U4. The relative density of theOmpF band was markedly reduced when the cells were grownin .0.4 M NaCl, >0.6 M glucose, or .2.5 mM salicylate. TheOmpF band was slightly less intense when this strain was grownin bismuth subcitrate and was markedly reduced when thestrain was grown in the presence of bismuth subsalicylate (Fig.3).Morphology of cells grown in salicylate and NaCi. E. coli

1177, which was highly responsive to salicylate, and E. coliHU849, which was resistant, were examined after overnight

kDa

97.4-

66.2-

45.0-

41.0-

2 3 4 5

FIG. 3. OMP profiles of P-fimbriated E. coli U4 (02:H7) grown onTSA alone (lane 2), with 0.4 mM sodium tartrate (lane 3), with 2.5 mMbismuth subsalicylate (lane 4), and with 2.5 mM bismuth subsalicylatesolubilized in 0.4 mM sodium tartrate (lane 5). Lane 1, molecular massmarkers.

I-Omp F!--Omp C

i--Omp A

growth on TSA plates with or without 10 mM salicylate (Fig.4). Both strains were heavily fimbriated when grown on TSAalone. Conversely, virtually all cells (96%) of E. coli 1177grown in the presence of salicylate were not fimbriated;however, E. coli HU849 remained heavily fimbriated.

Strain U4 (P fimbriated, 02:H7) was grown on TSA plates inthe presence of increasing concentrations of NaCl. No hollow-cored structures resembling P fimbriae were found on any ofthe cells examined when the strain was grown with 0.6 M NaCl(Fig. 5). The O-D-Gal(-4)-4-D-Gal-disaccharide latex agglutina-tion test was also negative at this concentration of NaCl.

DISCUSSION

The expression of P fimbriae by wild-type strains of E. coliisolated from patients with urinary tract infections was revers-ibly repressed when the strains were grown in hypertonicmedia and in the presence of both salicylate and bismuth salts.Enterotoxigenic E. coli bearing CFA determinants respondedsimilarly. All of these strains were found to express OmpF onPAGE profiles. In contrast, strains containing plasmid-en-coded P-fimbrial determinants lacked OmpF. They were unaf-fected when grown in the presence of salicylate and bismuthsalts but were sensitive to high osmolarity.

Hypertonic conditions (3, 9, 28, 46) and salicylate (6, 16, 37,39) are known to inhibit the expression of OmpF. Growth of E.coli in the presence of salicylate severely depresses the trans-lation of ompF and enhances the transcription of micF andompC independent of the osmolarity of the medium (37). Thefinding that salicylate-resistant strains which contained clonedP-fimbrial genes lacked OmpF on PAGE suggested that thisporin might have a role in susceptibility to salicylate-inducedeffects on fimbrial expression. Further support of the notionthat expression of OmpF and that of P fimbriae are linked wasobtained with tetracycline-resistant (Mar) clones of P-fimbri-ated strains (8). The tetracycline-resistant clones becameOmpF- and were unable to agglutinate human 0 cells orOt-D-Gal(-4)-1-D-Gal-disaccharide-coated latex beads. Theywere no longer fimbriated as observed by electron microscopy.The effect of Mar mutations on bacterial virulence needs to bedetermined.

Strains which expressed only type 1 or S fimbriae wereunaffected by salicylate or bismuth salts regardless of whetherthey expressed OmpF. In contrast, strains isolated from pa-tients with pyelonephritis, which produced both type 1 and Pfimbriae, were inhibited by salicylate. These findings suggestthe possibility that the assemblies of type 1 and P fimbriae maybe linked.The mechanism of action of the bismuth salts is unclear,

since bismuth is a cation and would be expected to be taken upby cells by a mechanism different from that used for salicylate.The finding that bismuth subsalicylate was far more active thansalicylate or bismuth subcitrate suggests that the combinedeffect may be synergistic.The expression of P, type 1, CFA, and S fimbrial phenotypes

was inhibited by growth under hyperosmolarity conditionsinduced by NaCl or nonmetabolized sugars. Hyperosmolarityconditions are known to reduce OmpF expression and enhancethe production of OmpC (3, 9, 28, 46). The mechanism bywhich hyperosmolarity conditions decrease the expression ofseveral fimbrial phenotypes remains to be determined. Onepossibility is that osmolytes affect other outer membrane-associated proteins that are involved in the export or assemblyof fimbriae. Since the osmolarity of human urine may rangefrom 300 to 1,200 mosM/kg, it is possible that the degree of

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FIG. 4. Representative electron micrographs of E. coli strains 1177 and HU849 grown on TSA (A and B, respectively) and on TSA containing10 mM salicylate (C and D, respectively).

fimbrial phase variation observed in patients with urinary tractinfections (33, 36) may be related to the tonicity of the urine.

Salicylates exert a variety of biologic effects in addition totheir well-known anti-inflammatory action. They act as anendogenous signal that induces systemic acquired resistance toinfection in plants (17), and they affect the human heat shockresponse (22). Salicylate also inhibits the production of capsu-lar polysaccharide in K pneumoniae, potentiates the activity ofcationic aminoglycoside antibiotics (10-12), increases resis-tance to antibiotics in Pseudomonas cepacia (6), and reducesthe adherence of E. coli to silastic catheters (14).

Salicylates are often used in combination with sulfonamidesin patients with inflammatory bowel disease. Binding to man-nose and Galoa-*4Gal-containing receptors appears to con-

tribute to the maintenance of E. coli in the large intestine (48).It is possible that salicylate and bismuth compounds mightinhibit the attachment of E. coli to the colonic mucosa.The effect of salicylate on expression of P fimbriae in E. coli

appears to be quite distinct from that of cranberry andblueberry juices (33), since neither salicylate nor bismuth wasfound to directly block RBC agglutination. The fruit juicescontain lectins which directly block the adherence of type 1-and P-fimbriated E. coli to bladder cells and prevent aggluti-nation of yeast cells, RBCs, and a-D-Gal(-4)-13-D-Gal-disaccha-ride-coated latex particles (32).The potential use of salicylates to prevent urinary tract

infections by P-fimbriated E. coli needs to be determined.Urinary excretion of free salicylates is pH dependent (15).

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FIG. 5. Representative electron micrographs of E. coli U4 (02:H7) grown in TSB alone (A), TSB containing 0.5 M NaCl (B), and TSBcontaining 0.6 M NaCl (C).

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About 10% is usually recovered, but as much as 85% of an oraldose may be recovered in alkaline urine, and as little as 5%may be recovered in acid urine. Protection from urinary tractinfections will likely depend upon the concentration of thedrug at bacterial attachment sites.

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