Department of Microbiology, University of Manitoba, Winnipeg MB R3T 2N2, Canada
Received 15 November 2001; returned 7 March 2002; revised 16 May 2002; accepted 14 June 2002
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Abstract |
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Methods: Thirteen clinical isolates of S. marcescens were screened for resistance to four fluoroquinolones: ofloxacin, ciprofloxacin, norfloxacin and nalidixic acid by determining MICs. The presence of a proton gradient-dependent efflux mechanism was assessed using ethidium bromide accumulation assays. Drug accumulation studies for norfloxacin, ciprofloxacin and ofloxacin were performed to determine the drug specificity of efflux. Western transfer of cellular proteins, followed by immunodetection using anti-AcrA (Escherichia coli) antibodies were used to demonstrate the presence of a resistancenodulationcell division (RND) pump protein. PCR was used to identify a RND pump-encoding gene using primers for two conserved motifs within inner membrane components of RND proteins. A mutant strain of S. marcescens, UOC-67WL, was isolated by culturing the wild-type strain in the presence of ciprofloxacin in T-soy media and was subjected to the same studies as described above for the clinical isolates.
Results: Ethidium bromide accumulation assays confirmed the presence of a proton gradient-dependent efflux mechanism in S. marcescens. One clinical isolate, T-861, and the mutant strain, UOC-67WL, were found to efflux ciprofloxacin and ofloxacin. Western immunoblot results confirmed overexpression of an AcrA-like protein in T-861 and UOC-67WL. Sequencing of the PCR product showed the presence of a mexF-like gene, which is overexpressed in nfxC mutants of Pseudomonas aeruginosa.
Conclusion: This study reports the presence of a proton gradient-dependent efflux mechanism in S. marcescens.
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Introduction |
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Materials and methods |
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Thirteen clinical isolates of S. marcescens (T-849, T-850, T-851, T-852, T-853, T-854, T-855, T-856, T-857, T-858, T-859, T-860 and T-861) were obtained from the Department of Medical Microbiology, Health Sciences Centre, Winnipeg, Manitoba. UOC-67 (S. marcescens strain ATCC 13880) was used as the wild-type strain. UOC-67WL is a mutant strain derived from UOC-67.
Antibiotic susceptibility testing
Susceptibilities of S. marcescens strains to norfloxacin, nalidixic acid, ofloxacin (Sigma-Aldrich, Canada) and ciprofloxacin (a gift from Bayer Inc., Canada), were tested using the two-fold dilution method with an inoculum of 10 cfu.5 Results were reported as MIC, the concentration of antibiotic that inhibited visible growth determined by absence of turbidity in the broth after 18 h of incubation at 37°C.
Fluoroquinolone accumulation
Accumulation of ciprofloxacin, ofloxacin and norfloxacin was measured for clinical isolates showing an MIC of 4 mg/L of each of these antibiotics, following the method of Mortimer & Piddock.2 The fluorescence of antibiotics was measured as follows: ciprofloxacin at excitation and emission wavelengths of 279 and 447 nm; norfloxacin at 281 and 440 nm; and ofloxacin at 292 and 496 nm, respectively. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to a final concentration of 100 µM after 5 min. Accumulation assays for nalidixic acid were not performed, as emission and excitation wavelengths for the drug could not be determined.
The concentrations of antibiotics were calculated using a standard curve for the respective antibiotic (concentration ranging from 100 to 1000 ng) in 0.1 M glycine hydrochloride pH 3.0. The results were expressed as nanograms of antibiotic incorporated per milligram (dry weight) of bacteria.
Selection of S. marcescens mutant strain UOC-67WL
Wild-type S. marcescens, strain UOC-67, was grown on T-soy agar plates and replated after every overnight incubation on plates supplemented with increasing concentrations of ciprofloxacin. One colony capable of growing at 21 mg/L ciprofloxacin was selected and named UOC-67WL. This strain was subjected to the studies described above for clinical isolates.
Whole-cell lysis, urea-SDSPAGE and western immunoblot
A 1.5 mL overnight bacterial culture grown in T-soy broth was pelleted by centrifugation at 5600g, and lysed with 100 µL of cell lysis buffer [2% (w/v) SDS, 4% (v/v) dithiothreitol (DTT), 10% glycerol, 1 M Tris pH 6.8]. Protein samples (150 µg) were analysed on a 13% SDSPAGE gel supplemented with 4 M urea (a modification of the method of Uemura & Mizushima6).
Western immunoblot7 of the protein resolved on SDSPAGE urea gels was performed using antibodies (diluted 1:4000) raised against the AcrA protein of Escherichia coli (a gift from H. Nikaido, University of California, Berkeley, CA, USA).
Genomic DNA preparation, PCR and DNA sequencing
Genomic DNA of S. marcescens UOC-67 was prepared as described by Ausubel et al.8
Two primers, ACRB1236 (GTGGATGACGCCATCGTTGTG) and ACRB2954 (GGTCATCAGGATCGGACGTAA) (Gibco BRL), were used to amplify a 1.7 kb region from the genome of S. marcescens UOC-67. Sequences of primers were derived from two signature sequences of RND proteins as described by Tseng et al.3
DNA sequencing was carried out at the automated sequencing facility of the National Research Council/Plant Biotechnology Institute, Saskatoon, Saskatchewan, Canada. The BLAST 2.0 algorithm of the National Center for Biotechnology Information (NCBI) was used to analyse DNA sequences.
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Results |
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Strains demonstrating an MIC of 4 mg/L of an antibiotic were considered resistant to that particular antibiotic and used for further studies. Strains found to be resistant to ciprofloxacin, norfloxacin and ofloxacin were T-860, T-861 and UOC-67WL. T-856 showed resistance to norfloxacin but not to ciprofloxacin or ofloxacin. All strains, except T-857, T-858 and T-859, were found to be resistant to nalidixic acid.
Fluoroquinolone accumulation
UOC-67WL plus two clinical isolates, T-860 and T-861, were tested for their ability to accumulate ciprofloxacin. T-860 accumulated the drug with time, with no increase in the rate of accumulation upon addition of CCCP. In contrast, T-861 and UOC-67WL showed an increase of up to four-fold in the accumulation of ciprofloxacin after addition of CCCP. Rates of accumulation of ciprofloxacin by T-861 and UOC-67WL were found to be almost identical, whilst T-860 and wild-type, UOC-67, accumulated ciprofloxacin at a similar rate (Figure 1a). Neither the clinical isolates (T-856, T-860 and T-861) nor UOC-67 or UOC-67WL demonstrated any increase in the accumulation of norfloxacin upon addition of CCCP (Figure 1b).
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SDSPAGE and western immunoblot
The western immunoblot from the SDSPAGE urea gel of the whole-cell lysate of UOC-67 (wild-type), and T-861 and UOC-67WL (the two strains capable of accumulating ciprofloxacin and ofloxacin) showed a positive reaction with anti-AcrA antibodies (Figure 2). Two protein bands were visualized, with the intensity of the lower band greatest for T-861 and lowest for UOC-67, suggesting the presence of two different yet related RND pump proteins.
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PCR of the genomic DNA of S. marcescens UOC-67, using primers ACRB1236 and ACRB2954, yielded a product of 1.7 kb. Partial sequence (800 bp) of this PCR product, revealed high homology to the mexF gene of Pseudomonas aeruginosa.
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Discussion |
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To classify the type of pump effluxing fluoroquinolones, western immunoblot experiments were performed. Results demonstrated the presence of at least two AcrA-like proteins in S. marcescens. The AcrAB pump of E. coli belongs to the RND family,3 with the AcrA protein being the periplasmic component of the pump. Western immunoblot experiments showed overexpression of at least one protein in T-861 and UOC-67WL (Figure 2), with the molecular weight of these proteins corresponding to 50 kDa. The presence of two bands in the immunoblot is not surprising considering that seven different RND proteins have been found in E. coli.9 In addition, a portion of the gene encoding the inner membrane component of the RND protein was identified in S. marcescens UOC-67, with the DNA sequence analysis revealing similarity to the mexF gene of P. aeruginosa. Efforts are underway to knock out this gene in S. marcescens to get a clear picture of the role of the gene product in the efflux-mediated resistance and to determine whether more than one product of the same size was obtained as a result of PCR.
This study points strongly towards proton-dependent efflux of fluoroquinolone drugs, possibly via RND pump(s), as a resistance mechanism in S. marcescens.
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Acknowledgements |
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Footnotes |
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References |
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2 . Mortimer, P. G. & Piddock, L. J. (1991). A comparison of methods used for measuring the accumulation of quinolones by Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 28, 63953.[Abstract]
3 . Tseng, T. T., Gratwick, K. S., Kollman, J., Park, D., Nies, D. H., Goffeau, A. et al. (1999). The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins. Journal of Molecular Microbiology and Biotechnology 1, 10725.[Medline]
4 . Berlanga, M., Vazquez, J. L., Hernandez-Borrell, J., Montero, M. T. & Vinas, M. (2000). Evidence of an efflux pump in Serratia marcescens. Microbial Drug Resistance 6, 1117.[ISI][Medline]
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7 . Burnette, W. N. (1981). Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfatepolyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical Biochemistry 112, 195203.[ISI][Medline]
8 . Ausubel, F. M., Bent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. et al. (1989). Current Protocols in Molecular Biology. John Wiley and Sons, New York, USA.
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