Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 23, Num. 2, 2005, pp. 102-105

Indian Journal of Medical Microbiology, Vol. 23, No. 2, April-June, 2005, pp. 102-105

Original Article

Inhibition of Bacterial Adhesion by Subinhibitory Concentrations of Antibiotics

Vidya K.C., Mallya P.S., Rao P.S.

Department of Microbiology, Kasturba Medical College, Manipal

Correspondence Address:Department of Microbiology, Kasturba Medical College, Manipal - 576 219, Karnataka, psraos82@yahoo.co.in

Code Number: mb05027

Abstract

Background: Urinary Tract Infections (UTIs) due to Escherichia coli is one of the most common diseases encountered in clinical practice. Most common recognised pathogenic factor in E.coli is adhesion. There is accumulating evidence that through subinhibitory concentrations (sub - MICs) of many antibiotics do not kill bacteria, they are able to interfere with some important aspects of bacterial cell function.
Materials and Methods: A study was conducted to investigate the effect of sub MICs (1/2-1/8 MIC) of ciprofloxacin, ceftazidime, gentamicin, ampicillin and co - trimoxazole on E. coli adhesiveness to human vaginal epithelial cells using three strains ATCC 25922, MTCC 729 and U 105.
Results: The 1/2 MIC of all the antibiotics tested produced the greatest inhibition of bacterial adhesion. Morphological changes were observed with ciprofloxacin, ceftazidime and ampicillin at 1/2 MIC and to a lesser extent at 1/4 and 1/8 MIC. Co-trimoxazole caused the greatest suppression of adhesion at 1/2 MIC of E. coli strain MTCC 729 when compared with the controls, followed by ceftazidime.
Conclusion: These results suggest that co - trimoxazole is the most effective antibiotic in the treatment of urinary tract infections caused by uropathogenic E. coli.

Keywords: E. coli, ciprofloxacin, ceftazidime, gentamicin, ampicillin, co - trimoxazole, sub-MICs, adhesion

Urinary tract infections (UTIs) are one of the most common diseases caused by microorganisms and Escherichia coli are the bacteria most frequently responsible for UTIs. The uropathogenic E. coli (UPEC) are of great interest at present and are being widely studied. They are recognized as possessing many pathogenicity factors. Adhesion is by far the best studied and understood mechanism and is also that which acts at the early steps of infection.[1] It is a specific and multifactorial process, the result of compatibility between two cell surfaces, involving bacterial adhesins and cell surface receptors.[2]

It has been observed that subinhibitory concentrations (sub MICs) of various antibiotics are able to modify the molecular architecture of the external surface of bacteria and some bacterial functions, such as the ability to adhere to the host cells, the surface bacterial energy, the susceptibility to host defence mechanisms, motility etc., thus influencing bacterial virulence.[3],[4]

The aim of the present study was to investigate the effects of subinhibitory concentrations of antibiotics on the adhesion of E. coli to human vaginal epithelial cells.

Materials and Methods

Bacterial strains

Three E. coli strains ATCC 25922, MTCC 729 and U 105 (isolated from urine of patient with symptomatic bacteruria) were used for this study.

Antibiotics

Ciprofloxacin (Cipla), ceftazidime (Alkem), ampicillin (Hi media), gentamicin (Hi media) and co - trimoxazole (Wellcome Burroughs) were the antibiotics that were used in the study.

Culture conditions

Bacteria were subcultured in brain heart infusion (BHI) broth at 37°C for 18 hours under static conditions. The organisms were harvested, washed three times in phosphate buffered saline (PBS) and then adjusted to the desired concentration as determined by direct microscopic counts.

Minimum inhibitory concentration

The MICs were evaluated using the microdilution broth method, according to the National Committee for Clinical Laboratory Standards (NCCLS).[5]

Collection of vaginal epithelial cells

Normal healthy females were selected, they were made to lie in the dorsal lithotomic position and the vaginal epithelial cells were collected by gentle scraping of the vaginal mucosa with a cotton tipped swab. The swabs were immersed in 5 mL of 0.01 M PBS (pH 6.4) and processed within 6 hours of collection. The cells were washed three times in PBS (pH 6.8) and finally suspended in PBS (pH 6.8) to get a concentration of 1x10⁵ cells/mL.

Bacterial adherence assay

The effect on adherence properties was studied by the method of Fowler and Stamey.[6]

The bacterial cells were washed thrice in PBS (pH 6.8) and finally suspended in PBS to get a concentration of 1x108 cells/mL. One mL of the bacterial culture was mixed with one ml of vaginal epithelial cells and incubated at 37°C for 30 min. in a shaker water bath. The mixture was then washed three times and filtered, a slide was gently pressed against the filter, air dried alcohol fixed and stained for I min. with dilute carbol fuchsin

The mean number of bacteria adherent to the first 50 epithelial cells was counted and the standard error of mean was calculated for each preparation.

Each test was performed three times. Starting from the observed MIC concentration, serial dilutions of antibiotics containing from 1/2 to 1/8 MIC were prepared in BHI broth. All the strains were grown in medium with or without different antibiotics (control) under static conditions, at 37°C for 18 hours. The bacteria were harvested by centrifugation and resuspended in PBS to a final concentration of 3x108 cells/mL. The adherence assay was then performed as described above.

The statistical analysis was done using the statistical software "SPSS 11.0". The mean and standard deviation of each parameter was obtained. The Kruskal Wallis anova was used to compare between groups.

Results

[Table - 1] denotes the MIC values for the antibiotics in all the three standard strains.

[Table - 2], [Table - 3] and [Table - 4] report the mean (+/- SD) adhesion values for E. coli strains challenged with the five antibiotics. When these strains were grown in the presence of an antibiotic, the adhesive ability of the cells was affected to different extent with different antibiotics. The 1/2 MIC of all the antibiotics tested produced the greatest inhibition of bacterial adhesion. Filamentous forms were observed in ciprofloxacin, ceftazidime and ampicillin at 1/2 MIC and to a lesser extent at 1/4 and 1/8 MIC. Co-trimoxazole caused the greatest suppression of adhesion at 1/2 MIC of E. coli strain MTCC 729 when compared with the controls, followed by ceftazidime. In the case of E. coli strains ATCC 25922 and U 105 ceftazidime showed the greatest suppression at 1/2 MIC. A statistically significant difference was seen between the controls and the three sub-MICs of all the antibiotics (P <0.05). The adherence of all the antibiotics decreased in a dose dependent manner. The [Figure - 1], [Figure - 2] and [Figure - 3] show the comparison of adherence between antibiotics at 1/2, 1/4 and 1/8 MIC.

In the control test, before the challenge with antibiotics the thr ee E.coli strains ATCC 25922, MTCC 729 and U 105 adhered well to human vaginal epithelial cells, with a certain degree of variability in the number of bacteria per cell.

Discussion

Bacterial virulence is a result of the specific properties of both bacterial and host cells and their reciprocal interactions. Adhesion is considered to be the first step in the sequence of events leading to colonization, is an important determinant of virulence and subsequent infection.[7]-[9] The ability to adhere is weakened by exposure of bacteria to sub MICs of antibiotics.[10]-[13] Effect of antibiotics on adhesion may be strain specific and various antibiotics may have varying action on this property. In this study, all antibiotics used generally reduced bacterial adhesiveness. These observations are compatible with the hypothesis that the adhesion of E. coli to eukaryotic cells is mediated by the bacterial proteinaceous adhesions and the synthesis of such membrane associated protein factors can be differentially inhibited by low concentrations of antibiotics.

Sub-MICs of antibiotics can exert their antiadhesive effects by inhibition of synthesis or expression of adhesins on the bacterial cell surface which leads to the formation of functionally aberrant adhesins, or due to release of adhesins from the surface of bacterial cells, or modify bacterial shape in such a way as to interfere with the ability of the microorganisms to approach receptors on animal cell surface.[14]

Ciprofloxacin caused the treated E. coli to elongate and showed reduction in adhesion on vaginal epithelial cells [Table - 1], [Table - 2] and [Table - 3] and produced shortening of adhesive fimbriae following antibiotic exposure. This effect has clinical significance as anti adhesive effect of quinolones has killing effect on bacteria at higher dosages, and low concentrations should effectively reduce the adherent and colonizing ability of these pathogens. Our findings are similar to those observed by others.[15],[16]

Sub MIC of ceftazidime also produced similar effect and such findings were also observed by Braga et al .[17] Low concentrations of ampicillin decreased the attachment of uropathogenic E. coli . As already noted, synthesis and insertion of type 1 fimbriae into the cell envelope of E. coli is inhibited by b lactam antibiotics and a similar explanation may account for the mediation by fimbriae of adhesion of uropathogens. Sanberg et al[18] suggested that elongated bacterial forms (that is induced by b lactam antibiotics) may fit less well into the region of the uroepithelial cell surface that contain fimbrial receptors.

Co - trimoxazole was the most effective antibiotic. It does not directly affect fimbrial synthesis, but may have effect on changes in the cell envelope that interfere with the secretion or assembly of the fimbriae or both. Similar findings were observed by other authors.[4],[19]
The inhibition of adhesiveness provides more complete information than other parameters because it gives not only a simple clue to the alterations in biophysicochemical pathways induced on bacteria by antimicrobial agents, but since it involves human cells mimicking the phenomenon occurring in nature, it shows the real effect of very low concentrations of antibiotics.

The ability of an antibiotic to interfere with many bacterial pathogenicity factors may be one of the main criteria for choosing one drug over another. Our results show that co - trimoxazole is most effective in the treatment of urinary tract infections caused by uropathogenic E. coli.

References

1.	Beachy EH. Bacterial adherence: Adhesin interactions mediating the attachment of bacteria to mucosal surfaces. J Infec Dis 1981;143 :325-45.  Back to cited text no. 1
2.	Caratelli CR, Nuzo I, Galdiero M. Surface properties of E. coli strains for urinary infections. Microbiologica 1987;10 :55-61.  Back to cited text no. 2
3.	Braga PC. Effects of sub inhibitory concentrations of seven macrolides and four fluoroquinolones on adhesion of staphylococcus aureus to human mucosal cells. Chemotherapy 1994;40 :304-10.  Back to cited text no. 3  [PUBMED]
4.	Vaisanen V, Lounatmaa and Korhonan TK. Effects of sublethal concentrations of antimicrobial agents on the hemagglutination, adhesion and ultrastructure of pyelonephritogenic Escherichia coli strains. Antimicrob Agents Chemother 1982;22 :120-7.  Back to cited text no. 4
5.	National committee for clinical laboratory standards 1987. Performance standards for antimicrobial susceptibility testing. Second Informational Supplement M 100 - S2. National Committee for Clinical Laboratory standards, Villanova, PA, USA.  Back to cited text no. 5
6.	Fowler JE (Jr), Stamey TA. Studies of introital colonization in women with recurrent urinary tract infections. Vii The role of bacterial adherence. J Urol 1977;117 :472-6.  Back to cited text no. 6
7.	Abraham SN, Beachy EM. Host defense against adhesion of bacteria to mucosal surfaces. In Gallin JJ, Fauci AS, editors, advances in host defense mechanism , New York: Raven Press; 1985. p. 68-83.  Back to cited text no. 7
8.	Beachy EM, Eisentein BI. Bacterial adherence in infections diseases. Kalamazoo, Mich: Upjohn Company; 1982.  Back to cited text no. 8
9.	Douglas IJ. Adhesion of Candida species to epithelial surfaces. Crit Rev Microbiol 1987;15 :27-43.  Back to cited text no. 9
10.	Costerton JW. Effects of antibiotics on adherent bacteria. In Sabath ID, editor, Action of antibiotics in patients . Vienna, Austria: Hans Huber; 1981. p. 160-75.  Back to cited text no. 10
11.	Schifferli DM, Beachy EM. Bacterial adhesion: Modulation by antibiotics with primary targets other than protein synthesis. Antimicrob Agents Chemother 1988;32 :1609-13.  Back to cited text no. 11
12.	Shibl AM. Effect of antibiotics on adherence of microorganisms to epithelial cell surfaces. Rev Infect Dis 1995;7 :51-6.  Back to cited text no. 12
13.	Lorian V. Some effects of sub inhibitory concentrations of antibiotics on bacteria. Bull NY Acad Med 1975;51 :1046-55.  Back to cited text no. 13  [PUBMED]
14.	Lorian V, Ernst J. Effect of antibiotics on bacterial structure and their pathogenicity. Pathol Biol 1987. p. 1370-6.  Back to cited text no. 14
15.	Balague C, Fernandez L, Perez J, Grau R. Effect of ciprofloxacin on adhesive properties of non - P mannose - resistant uropathogenic E. coli isolates. J Antimicrob Chemother 2003;51 :401-4.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]
16.	Baskin H, Dogan Y, Bahar III, Yulug N. Effect of sub minimal inhibitory concentrations of three fluoroquinolones on the adherence of uropathogenic strains of E. coli . Int J Antimicrob Agents 2002;19 :79-82.  Back to cited text no. 16
17.	Braga PC, Sasso MD, Sala MT. Sub MIC concentrations of cefodizime interfere with various factors affecting bacterial virulence. J Antimicrob Chemother 2000;45 :15-25.  Back to cited text no. 17  [PUBMED]  [FULLTEXT]
18.	Sanberg T, Stenquist K, Svanborg EC. Effects of subminimal inhibitory concentrations of Ampicillin, Chloramphenicoland Nitrofurantoin on the attachment of Escherichia coli to human uroepithelial cells in vitro. Rev Infect Dis 1979;1 :838-44.  Back to cited text no. 18
19.	Schifferli DM, Abraham SN, Beachey EH. Influence of Trimethoprim and Sulfamthoxazole on the synthesis, expression and function of Type 1 fimbriae of Escherichia coli . J Infect Dis 1986;154 :490-6.  Back to cited text no. 19  [PUBMED]

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