Department of Microbiology, Rosario University School of Biochemistry and Pharmacy, and Institute for Molecular and Cellular Biology (IBR-CONICET), Suipacha 531, Rosario 2000, Argentina
Received 22 March 2002; returned 13 August 2002; revised 3 October 2002. accepted 18 October 2002
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Abstract |
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Introduction |
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Materials and methods |
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E. coli strains were isolated from hospitalized patients with urinary infection at the Hospital Provincial del Centenario (Rosario, Argentina). During this screening, only ciprofloxacin-susceptible (cips) strains were considered to avoid the possibility that ciprofloxacin resistance mechanisms interfered with the interpretation of the results. A total of 100 cips E. coli strains were recovered, and 25 of them were able to haemagglutinate human group A erythrocytes in the presence of mannose but failed to agglutinate guinea pig erythrocytes coated with the specific P fimbriae receptor. These 25 isolates constituted the pool of strains selected for the present study.
Determination of MICs
MuellerHinton broth (Difco Laboratories, Detroit, MI, USA) was used to determine MICs of ciprofloxacin by the tube dilution method.4 MICs of ciprofloxacin were in the range 0.030.06 mg/L, and for the following experiments we describe essentially the effects of a dose of one quarter the MIC because this concentration was the highest tested that did not produce measurable alterations of the cultures (data not shown).
Haemagglutination (HA)
Cultures were grown in colonization factor antigen (CFA) broth containing casein hydrolysate (Merck, Darmstadt, Germany) at 37°C for 18 h. Mannose-sensitive and -resistant haemagglutination (MSH and MRH, respectively) were characterized using fresh guinea pig and human type A erythrocytes in the absence or presence of 1% mannose.5 Characterization of MRH was made with guinea pig erythrocytes coated with a specific globoside harbouring the receptor for P fimbriae.5
Hydrophobicity (PP), surface proteins and adherence assay
Cellular PP was measured with the solvent/water partition test of Chapman & Georgeopapadakou.6 Extraction and quantification of surface proteins were carried out following an adaptation of the method of Hoschutzky et al.7 For the bacterial adherence assay, cultures were grown on CFA agar plates with and without ciprofloxacin at 37°C for 18 h. Each culture was resuspended in HEPES-buffered Hanks solution to an optical density of 0.8 at 540 nm. Each bacterial suspension was fractionated into tubes and contacted with a segment of latex-silicone urinary catheter (1 mm thick) and incubated at 37°C for 2 h. The catheters were stained with Methylene Blue, washed, and the bacteria adhering to the internal surface of the catheter were counted by light microscopy.
Electron microscopy
Bacterial suspensions were prepared after overnight growth on CFA agar plates with and without ciprofloxacin. Electron microscopy was carried out on copper grids coated with collodion-carbon membrane. Preparations were negatively stained with phosphotungstic acid (PTA) for 1 min.
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Results and discussion |
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The effect of one quarter the MIC of ciprofloxacin on HA and PP was variable from strain to strain. Whereas 12 isolates did not show any statistically significant modification of the analysed properties (data not shown), 13 strains showed a noticeable enhancement of HA and/or PP after antibiotic treatment (Figure 1a). We interpreted that this increase in adhesive properties could be due to a higher expression of surface proteins, which include appendages such as fimbriae or fibrils. To examine this idea, we selected seven isolates with increased HA and/or PP, and in agreement with our hypothesis all of them showed a significant elevation in the levels of surface proteins when grown in the presence of antibiotic (Figure 1b).
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The ciprofloxacin-induced modifications of surface properties must correlate with changes at a structural level. This presumption prompted us to analyse by electron microscopy individual bacterial cells of two representative strains (RM9015 and RM10387) after overnight exposure to sub-MIC ciprofloxacin (Figure 1c). Cultures of RM9015 grown in the absence of ciprofloxacin showed essentially cells with no fimbriae. In contrast, when the culture was exposed to the antibiotic, it was possible to observe numerous peripherally located fimbriae for most of the cells. These fimbriae were thin and irregularly shaped, with the majority being <1 µm in length but some >2 µm. On the other hand, after several attempts for strain RM10387, it was not possible to observe the presence of defined fimbriae under any condition. This result could indicate the presence of other types of adhesin without a regular shape and morphology (afimbrial adhesins), which could impair their visualization by electron microscopy.1,2
Effect of ciprofloxacin on the adherence to urinary catheters and the importance of PP
In order to shed some light on the clinical significance of the positive variation of the studied surface-related properties that should render microbial cells more adhesive, we evaluated whether sub-MIC of ciprofloxacin might affect the ability of these strains to adhere to urinary catheters. We chose this medical device because of its common use in hospitalized patients and the reported risk of urinary tract infections initiated by catheter infiltration.8 The study with ciprofloxacin-treated strains was carried out in parallel with non-treated and trimethroprim-treated cultures as controls. The inclusion of trimethroprim as a control is based on a previous report indicating that this antibiotic does not increase bacterial adherence.9 In Table 1, we summarize the values of UPEC cells adhering to the inner surface of urinary catheters of the same seven strains shown in Figure 1b after 18 h of exposure to ciprofloxacin. There was a significant stimulation by ciprofloxacin of the number of cells adhering to the urinary catheter for strains of groups I and II. These strains share in common an increase in average hydrophobicity after antibiotic treatment (Figure 1a), which suggested the importance of this property for the attachment under the experimental conditions. This conclusion was reinforced with the result obtained with strains of group III (increased HA, but unaffected PP after ciprofloxacin treatment). Strains of this group did not show any significant variation of their adhesiveness to the catheter, with or without ciprofloxacin treatment (Table 1). These results indicate that the increase in PP, due to the antibiotic, was the cause for the enhanced binding ability of the bacteria to the catheter. Moreover, it was previously reported that urinary catheters are susceptible to colonization by UPEC strains without using fimbriae but exhibiting high PP.10 Accordingly, strain RM10387, which has a significant increase in its binding to the catheter (Table 1) without using fimbriae (Figure 1c), also showed a tendency of the PTA stain to precipitate along the periphery of the cell during the electron microscopy preparation, something previously observed for cells with elevated PP.11
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Acknowledgements |
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Footnotes |
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References |
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