Effects of Low Concentrations of Antibiotics on Escherichia

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1982, p /82/ $02.00/0 Vol. 21, No. 6 Effects of Low Concentrations of Antibiotics on Escherichia coli Adhesion KLAUS VOSBECK,* HELMUT METT, URSULA HUBER, JACQUELINE BOHN, AND MARCELLE PETIGNAT Pharmaceuticals Division, Research Department, Ciba-Geigy Limited, CH-4002 Basel, Switzerland Received 4 January 1982/Accepted 22 March 1982 We have previously shown that subinhibitory concentrations of antibiotics may infliience the adhesion of Escherichia coli SS142 to human epithelioid tissue culture cells. This report shows that these effects are not limited to E. coli SS142 or to our tissue culture system. Most of the 10 E. coli strains studied showed decreased adhesion to Intestine 407 tissue culture cells after growth in 25% of the minimum inhibitory concentration of streptomycin, tetracycline, trimethoprimsulfametrole, chloramphenicol, and clindamycin. Nalidixic acid at 25% of the minimum inhibitory concentration caused an increase of adhesion. The hemagglutinating activity of the five hemagglutinating strains and the adhesiveness of E. coli SS142 to human buccal cells were similarly affected by low concentrations of the above-mentiqned antibiotics. We conclude that E. coli adhesion to human epithelioid tissue culture cells is a valid model of bacterial adhesion because of its high accuracy and reproducibility. We have recently described and characterized an in vitro assay for measuring bacterial adhesion to the human epithelioid tissue culture cell line, Intestine 407 (2, 9, 10). The adhesiveness of Escherichia coli SS142 was found to be reduced in this assay system after growth in subinhibitory concentrations of tetracycline, trimethoprim-sulfametrole, and clindamycin (9). A reduction of E. coli adhesion was also observed by others in different model systems after growth in the presence of several antibiotics (1, 3, 7). In this report, we compare the effects of various antibiotics on the adhesion to tissue culture cells of several E. coli strains. We demonstrate that E. coli adhesion is affected in a strain-specific manner, but that some antibiotics are more likely to change bacterial adhesiveness than others. Furthermore, we show a general correlation between the effects on adhesiveness observed in our tissue culture model, in a hemagglutination assay, and in human buccal cell adhesion. These correlations underline the usefulness of our tissue culture model for studies on bacterial adhesion. Our results also provide further evidence for a participation of bacterial protein structures in the process of E. coli adhesion, thus confirming existing concepts of the mechanism of E. coli adhesion (6). MATERIALS AND METHODS Medi and solutions. Minimal essential medium and calf serum were from GIBCO Laboratories, Glasgow, Scotland. Brain heart infusion (BHI) broth and agar were obtained from BBL Microbiology Systems, 864 Cockeysville, Md. HEPES (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid)-buffered Hanks solution (HEPES-Uanks) and phosphate-buffered saline were composed as described previously (9). Bacterial sdran. Ten E. coai strains of different pathogenic origin which adhered to Intestine 407 tissue culture cells Were selected fbr this study (Table 1). The strains were maintained on brain heart infusion (BHI) agar plates at 4C after overnight growth at 37 C and were subcultured weekly. Tissue culture cells. The cell line Intestine 407 (ATCC CCL6) was obtained from Flow Laboratories, Irvine, Scotland, and were maintained in minimal essential medium containing 10%o calf serum in a 5% CO2 atmosphere at 370C as described previously (9). Antibiotics. Tetracycline (Achromycin, Lederle Laboratories, Pearl River, N.Y.), clindamycin (Dalacin, The Upjohn Co., Kalamazoo, Mich.) nalidixic acid (Negram, Winthrop Laboratories, New York, N.Y.), trimethoprim-sulfametrole (Lidaprim, Ciba- Geigy/Chemie Linz, Linz, Austria), ampicillin (Penbritin, Beecham Research Laboratories, Ltd., Brentford, England), chloramphenicol (Chloromycetin, Parke, Davis & Co., Detroit, Mich.), and streptomycin (Streptomycin Novo, Novo Industri A/S, Copenhagen, Denmark) were obtained commercially. Determination of MIC. The minimum inhibitory concentration (MIC) of each drug was determined under conditions identical to those used for culture of bacteria in the adhesion assays. Serial twofold dilutions of each test substance were prepared in BHI broth (5 ml each), inoculated with 5 x 106 E. coli cells, and incubated statically at 370C for 18 h. The MIC was defined as the lowest concentration of antibiotic that resulted in complete inhibition of bacterial growth. Determination of bacterial viabilty. Tenfold serial dilutions of bacterial suspensions in physiological sa-

2 VOL. 21, 1982 line were prepared, and 0.1-ml samples were streaked onto BHI agar plates. Colonies were counted after overnight incubation at 37 C, and the codcentration of viable bacteria was calculated as the average number of colony-forming units for two consecutive dilutions. Estimation of morphological alterations. Gramstained smears of bacterial suspensions were examined microscopically. The percentage of bacteria elongated more than fivefold was determined by counting 100 bacteria in randomly chosen microscopic fields. Monolayer adhesion assay. The adhesion of bacteria to tissue culture monolayers was measured as described previously (9). Briefly, Intestine 407 cell monolayers were prepared in multiwell tissue culture dishes (2 cm2 each) with 3 x 105 cells as an inoculum per well. Confluent monolayers were obtained after 20 h of growth. Bacteria were grown statically at 37 C in BHI broth (5 ml) containing 10 FCi of sodium [14C]acetate (specific activity, 40 mci/mmol) per ml. For adhesion assays with bacteria grown in the presence of subinhibitory concentrations of antibiotics, the antibiotic was added to the growth medium to give the appropriate fraction of the MIC at the beginning of the incubation period. After 18 h of growth, the bacteria were washed three times with 10 ml of HEPES-Hanks (9), thus removing the antibiotic, if present, and the bacterial concentration was adjusted photometrically to an optical density of 0.2 at 650 nm (corresponding to 10" bacteria per ml). The monolayers were washed twice with HEPES-Hanks and incubated with 5 x 10' bacteria in a total volume of 1.0 ml. The monolayer plates were shaken at 120 rpm on a reciprocating shaker at 37 C for 40 min. Thereafter, unbound bacteria were removed by repeated washing with HEPES- Hanks, and the monolayers with the adherent bacteria were solubilized by further incubation in 0.5% (wt/vol) sodium dodecyl sulfate. The number of bound bacteria was determined by liquid scintillation counting of a sample of the solubilized mixture. Hemagglutinaton. Bacteria were grown as described above, washed twice with 10 ml of phosphatebuffered saline, and adjusted photometrically to a concentration of 1010 bacteria per ml. Serial twofold dilutions were prepared in phosphate-buffered saline, using microtiter plates, and an equal volume of a 3% washed human type A erythrocyte suspension was added to each well. The agglutination was read visually after a 2-h incubation at room temperature. The number of wells agglutinated by each culture was recorded and compared with the number of wells agglutinated by a control culture grown in the absence of antibiotics. Bacterial adhesion to exfoliated buccal cells. Buccal cells obtained by scraping the buccal epithelium of one person with the rim of a plastic test tube were suspended in a 0.5 mm solution of EDTA adjusted to ph 7.4 and incubated for 60 min at 370C. This treatment prevented clumping of the cells. Thereafter, the cells were sedimented by centrifugation at 200 x g for 10 min, the supernatant was removed, and the cells were washed twice with cold (4 C) HEPES-Hanks and adjusted to 5 x 104 cells per ml by hemocytometer counting. Bacteria were grown in the presence or absence of antibiotics as described above. They were suspended at a density of 109 bacteria per ml (optical density of 2.0 at 650 nm) in HEPES-Hanks. One milliliter each of the buccal cell suspension and the EFFECTS OF ANTIBIOTICS ON E. COLI ADHESION 865 bacterial suspension were mixed and incubated on a roller drum for 40 min at 37 C. Thereafter, the mixture was filtered through a Nuclepore filter (Nuclepore Corp., Pleasanton, Calif.; pore diameter, 8,m). The residue was washed five times with 5 ml of HEPES- Hanks. The filter was then fixed with rubber bands to a microscopic slide, and the cells were stained for 25 min in an aqueous 0.05% crystal violet (Merck AG, Darmstadt, West Germany) solution. The filter was washed with distilled water, transferred to a new microscopic slide, dried, and examined microscopically. The average number of bacteria adhering to each cell was determined by counting the bacteria associated with 20 randomly chosen cells. RESULTS Sensitivity to antibiotics. Table 1 summarizes MICs of the antibiotics used in this study for the 10 adhesive E. coli strains. It is to be noted that the MIC of trimethoprim-sulfametrole was determined in BHI broth as previously described (9). The MIC of trimethoprim-sulfametrole in minimal essential medium ranged from 0.25 to 2 i.gwml for all strains except strains 7 (32 gxg/ml) and 8 (16,ug/ml). Adhesiveness and hemagglutinating capability of strains. The 10 selected strains exhibited different adhesiveness to Intestine 407 cells (Table 2). Nonadhesive strains show less than 0.4% adhesion per hour in this assay. Five of the strains agglutinated human erythrocytes (group A) to various extents. There was no quantitative correlation between adhesiveness to the monolayers and hemagglutinating capability. Strain 7 exhibited guinea pig erythrocyte agglutination inhibitable by 50 mm a-methyl mannoside. Strain 5 also agglutinated guinea pig erythrocytes, but this hemagglutination could not be inhibited by 50 mm a-methyl mannoside. None of the other strains agglutinated guinea pig erythrocytes. Effects of subinhibitory concentrations of antibiotics. When the strains were grown in the presence of the arbitrarily chosen concentration of 25% of the MIC of the antibiotics listed in Table 1, their adhesion to Intestine 407 monolayers and their hemagglutinating capability were changed. In these experiments trimethoprimsulfametrole was used at 4,ug/ml, regardless of the MIC value, since it reduced bacterial adhesiveness nearly maximally even at this low level. Streptomycin, tetracycline, trimethoprim-sulfametrole, and chloramphenicol caused a decrease of the adhesion of most strains (Fig. 1). Single strains remained unaffected by one or the other antibiotic or exhibited even an increased binding of radioactivity. Strain 5 was an exception, since its adhesiveness was decreased by only one antibiotic, chloramphenicol. Clindamycin was the least effective inhibitory compound, and nalidixic acid caused a consistent enhance-

3 866 VOSBECK ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Origin of bacterial strains and MIC of antibiotics MIC (>Lg/ml) E. coli Strain b strain no.a Origin Streptomycin Tetracycline Trimethoprim- Chloramphenicol Clindamycin Nalidixic acid B499 1 UTId 32 1 > B495 2 UTId B488 3 UTI' B476 4 LAB' B413 5 UTIf 16 2 > B528 6 DIAM > B524 7 DIA' 8 16 > B523 8 DIAM > B498 9 UTId 32 2 > SS UTIh a See Fig. 1. b UTI, Urinary tract infection; LAB, laboratory strain; and DIA, diarrhea. c MIC measured in BHI broth. d Obtained from C. Svanborg Eden, Goteborg, Sweden. ' Obtained from K. Jann, Freiburg, West Germany. f Obtained from G. Chabanon, Toulouse, France. 8 Obtained from H. U. Bertschinger, Zurich, Switzerland (porcine K88 strains). h Obtained from M. Richmond, Bristol, England. ment of binding of bacteria. An apparent stimulation of adhesion was also observed with a few strains after growth in subinhibitory concentrations of clindamycin or chloramphenicol. The hemagglutinating activity of the five hemagglutinating strains decreased consistently after growth in subinhibitory concentrations of trimethoprim-sulfametrole, chloramphenicol, and tetracycline and less with streptomycin and clindamycin. Nalidixic acid caused a slight increase of the hemagglutinating activity (Table 3). There was a good correlation between the effect of the different antibiotics on the adhesion of E. coli SS142 (strain 10) to exfoliated buccal cells and to Intestine 407 monolayers (Table 4). TABLE 2. Adhesion to tissue culture cells and hemagglutinating capability of bacterial strains Adhesion to Hemagglutination Strain no. intestine 407 cellsa (twofold steps) (% of inoculum/h) ob a Based on the radioactivity associated with tissue culture cells after 40 min of incubation with 14Clabeled bacteria. bnonagglutinating at 1010 bacteria per ml (see Materials and Methods). Adhesion was greatly reduced after growth in 25% of the MIC of tetracycline, trimethoprimsulfametrole, and clindamycin and was only marginally affected by chloramphenicol and not at all by streptomycin. After growth in 25% of the MIC of nalidixic acid, its adhesiveness was increased, however. Effects on bacterial growth. The effects of subinhibitory concentrations (25% of the MIC) of antibiotics on bacterial growth were studied by comparing the number of colony forming units after growth in the presence and absence of antibiotics (data not shown). Cell numbers were decreased by about 50% in a few cultures, but were not affected in most cases. There was no correlation between the slight reduction of growth of some strains in the presence of low concentrations of some antibiotics and the effects on bacterial adhesion described above. After adjusting the bacterial density photometrically to an optical density of 0.2 at 650 nm, no significant differences in the bacterial viability were detectable. Bacteria grown in the presence of nalidixic acid exhibited moderate filamentation; 10 to 50% of the bacteria were more than fivefold elongated compared with the control (data not shown). There was, however, no correlation detectable between the degree of filamentation and the increase of adhesiveness. In fact, the strain showing the most pronounced increase of adhesion (strain 8) exhibited the least degree of filamentation. DISCUSSION The data presented in this report confirm and extend our previous observation that certain

4 VOL. 21, 1982 Adhesion %of Control > Strer EFFECTS OF ANTIBIOTICS ON E. COLI ADHESION 867 A B A B A B FIG. 1. Effect of subinhibitory concentrations of antibiotics on the adhesion of E. coli strains to intestine 407 monolayers. Ten E. coli strains (numbers 1 to 10; see Table 1) were grown in the presence or absence of streptomycin, tetracycline, trimethoprim-sulfametrole, chloramphenicol, clindamycin, or nalidixic acid as indicated. The antibiotics were used at 25% of the MIC, except trimethoprim-sulfametrole, which was used at 4.ug/ml. The adhesiveness to intestine 407 tissue culture monolayers of bacteria grown in the presence of antibiotics (B) was expressed as the percentage of the adhesiveness of control bacteria (A). The slope of the connecting lines indicates an inhibition or enhancement of bacterial adhesion. Streptomycin, tetracycline, and trimethoprim-sulfametrole caused a trend toward decreased adhesiveness, whereas nalidixic acid caused a trend toward increased adhesiveness. Chloramphenicol and clindamycin exhibited less clear trends. See the text for details. antibiotics at subinhibitory concentrations influence bacterial adhesion (9). These effects are not limited to E. coli strain SS142 or to our tissue culture system, but they occur with many different E. coli strains and also in different assay systems. In addition similar antiadhesive effects caused by low antibiotic concentrations have been reported by other authors, using different assay systems (3, 7). The assay of bacterial adhesion in our tissue culture system therefore represents a valid adhesion model, and data obtained in this system can largely be generalized. The measurement of bacterial adhesion to tissue culture cells offers several experimental advantages, such as technical simplicity, high accuracy, and reproducibility and ease of manipulation, but its biological relevance may be criticized. The present data demonstrate that effects of low concentrations of antibiotics on bacterial adhesion to tissue culture cells correlate with the effects observed in at least two other assay systems, adhesion to human buccal epithelial cells and hemagglutination. Preliminary evidence suggests that tetracycline, trimethoprim-sulfametrole, and clindamycin also decrease adhesion of E. coli SS142 to human uroepithelial cells, whereas chloramphenicol and streptomycin do not exhibit such an activity (K. Vosbeck, manuscript in preparation). Therefore, with E. coli SS142 there is quite good agreement between the effects seen in the tissue culture model and those observed in buccal cell adhesion, hemagglutination, and uroepithelial cell adhesion. Furthermore, our data from the tissue culture system essentially confirm the

5 868 VOSBECK ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. Effects of subinhibitory concentrations of antibiotics on the hemagglutinating activity of E. coli strains' Change in hemagglutinating activity' after growth in 25% of the MIC of: Strain no. Srpoyi Teaclne Trimethoprim- Streptomycin Tetracycline sulfametrole Chloramphenicol Clindamycin Nalidixic acid a Strains agglutinating human erythrocytes at 1010 bacteria per ml. b Change in the number of twofold dilution steps causing agglutination compared with a control culture. See the text for details. TABLE 4. Adhesion of E. coli SS142 to human buccal cells and to intestine 407 cells after growth in subinhibitory concentrations of antibiotics Adhesion to human Adhesion to buccal cellsb intestine 407 Antibiotica cellsc Bacteria % of % of control per cell control None (control) 32 ± 9d Streptomycin Tetracycline Trimethoprim sulfametrole Chloramphenicol Clindamycin Nalidixic acid a The bacteria were grown in the presence of 25% of the MIC of the respective antibiotic (see the text for details). b The number of adhering bacteria was determined microscopically using a filtration assay (see the text). c Binding of radioactivity. d Mean ± standard deviation of six experiments. Other values are means of duplicate experiments. antibiotic effects on bacterial adhesiveness described in other systems (1). It is not surprising in view of the apparent complexity and the strain specificity (2, 5) that there is no absolute quantitative correlation between different systems. It seems quite clear that some antibiotics are more likely to cause a decrease of bacterial adhesiveness than others, although the effects on adhesion appear to be strain specific. All of the antibiotics used in this investigation, except nalidixic acid, are inhibitors of translation, and each reduced bacterial adhesiveness in general. Nalidixic acid, an inhibitor of DNA synthesis, in contrast, caused an increase of adhesiveness. Several antibiotics acting by other mechanisms did not show such effects (9). These observations are compatible with the hypothesis that the adhesion of E. coli to eucaryotic cells is mediated by bacterial proteinaceous adhesins (6) and that the synthesis of such membrane-associated protein factors can be differentially inhibited by low concentrations of antibiotics (4). This contention is further substantiated by the fact that E. coli may lose their pili, which are thought to mediate bacterial adhesion, after growth in low concentrations of antibiotics that inhibit adhesiveness. (1, 8; unpublished observations). A strain-specific inhibition of bacterial virulence factors, such as toxins and extracellular enzymes, by low concentrations of some antibiotics has been described by C. G. Gemmell, P. K. Peterson, D. Schmeling, and P. G. Quie, Program Abstr. Int. Congr. Chemother. 12th, Florence, Italy, abstr. no. 670, 1981). The inhibition of adhesion reported here may be another special case of such an inhibitory effect on the synthesis of bacterial extracellular protein factors. LITERATURE CITED 1. Beachey, E. H., B. I. Ehenstein, and I. Ofek Sublethal concentrations of antibiotics and bacterial adhesion, p In K. Elliott, M. O'Connor, and J. Whelan (ed.), Adhesion and microorganism pathogenicity. Ciba Foundation Symposium 80, Pitman Medical Ltd., London. 2. Cohen, P. S., A. D. Elbein, R; Solf, H. Mett, J. Regis, E. B. Menge, and K. Vosbeck Michaelis-Menten kinetic analysis of Escherichia coli SS142 adhesion to Intestine 407 monolayers. FEMS Microbiol. Lett. 11: FElemsae, B. I., I. Ofek, and E. H. Beaciey Interference with the mannose binding and epithelial cell adherence of Escherichia coli by sublethal concentrations of streptomycin. J. Clin. Invest. 63: Hrhhna, A., G. Childs, and M. Inouye Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia coli. J. Mol. Biol Jann, K., B. Jmnn, and G. Schmidt SDS polyacrylamide gel electrophoresis and serological analysis of pili from Escherichia coli of different pathogenic origin. FEMS Microbiol. Lett. 11: Ofek, I., and E. H. Beachey General concepts and principles of bacterial adhesion in animals and man, p In E. H. Beachey (ed.), Bacterial adherence, recep-

6 VOL. 21, 1982 EFFECTS OF ANTIBIOTICS ON E. COLI ADHESION 869 tors and recognition, series B, vol. 6. Chapman & Hall, Ltd., London. 7. Sandberg, T., Stenqvist, K., and C. Svanborg Eden Effects of subminimal inhibitory concentrations of ampicilin, chloramphenicol and nitrofurantoin on the attachment of Escherichia coli to human uroepithelial cells in vitro. Rev. Infect. Dis. 1: Vosbeck, K., J. Bohn, and U. Huber Adhesiveness, hemagglutination and piliation of Escherichia coli grown in sub-mic concentrations of antibiotics. Experientia 36: Vosbeck, K., H. Handschin, E. B. Menge, and 0. Zak Effects of subminimal inhibitory concentrations of antibiotics on adhesiveness of Escherichia coli in vitro. Rev. Infect. Dis. 1: Vosbeck, K., and U. Huber An assay for measuring specific adhesion of an Escherichia coli strain to tissue culture cells. Eur. J. Clin. Microbiol. 1:22-28.