a Department of Microbiology, Medical School, University of Athens, 115 27 Athens, Greece; b Department of Hygiene and Epidemiology, Medical School, University of Athens, 115 27 Athens, Greece
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Abstract |
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Introduction |
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In order to investigate whether S. aureus with reduced susceptibility to vancomycin were present in Greece, a number of epidemiologically unrelated S. aureus (both methicillin-resistant and -susceptible) were examined.
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Materials and methods |
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Three groups of randomly selected non-replicate nosocomial S. aureus isolates, 56
homogeneously methicillin-resistant (homMRSA), 16 heterogeneously methicillin-resistant
(hetMRSA) and 25 methicillin-susceptible (MSSA) were examined. All were isolated in
19941997 from active infections from nine Athens hospitals, and are presently held in
the collection of the Department of Microbiology, University of Athens.Isolates were considered
homogeneously resistant to methicillin when their inhibition zone diameter in a standard disc
diffusion assay was 9 mm, and heterogeneously resistant when the inhibition zone had a
diameter of >9 mm, but one or more colonies were still growing within a zone of diameter of
9 mm.
Susceptibility tests
The MIC of vancomycin for all isolates was determined by a broth microdilution method, according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS) 6 with cation-adjusted MuellerHinton broth and two-fold dilutions of vancomycin concentrations. The initial inoculum contained 1.5 x 10 5cfu (100 µL of a 1:100 dilution of a McFarland 0.5 cell suspension). For 37 representative homMRSA, all hetMRSA and all MSSA isolates, MICs were also determined by plating 3 x 106 cfu (10 µL of a suspension of bacterial cells corresponding to McFarland standard 1) on brain heart infusion (BHI) agar plates containing 0.5 mg/L increments of vancomycin in the range of 14 mg/L, as previously described. 2 For 16 representative homMRSA isolates, the Etest (AB Biodisk, Solna, Sweden) was also used, following the recommendations of the manufacturers. In order to detect possible sub-populations with reduced susceptibility to vancomycin, a simplified population analysis was also performed. 2 Fifty microlitres of a starting cell suspension (corresponding to McFarland standard 1) and ten ten-fold serial dilutions were spread on BHI agar plates containing concentrations (110 mg/L) of vancomycin increasing by 1 mg/L increments. The plates were incubated at 37°C for 48 h, then colonies were counted; the number of resistant cells contained in 50 µL of the starting cell suspension was calculated.
Pulsed-field gel electrophoresis
Preparation of chromosomal DNA, digestion with SmaI (New England Biolabs, Beverly, MA, USA) and pulsed-field gel electrophoresis (PFGE) (CHEF-DRIII; Bio-Rad, Hercules, CA, USA) were carried out as described previously, 7 using lambda phage DNA concatamers (New England Biolabs) as size markers. Digital pictures (HeroLab, Wiesloch, Germany) of the gels were analysed with the GelCompar software (Applied Maths, Kortrijk, Belgium) for pattern similarity. The Dice coefficient, the UPGMA clustering algorithm and 1% tolerance of band difference were used.
Statistical analysis
Chi-square tests were used for comparison of the MIC values.
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Results |
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PFGE revealed that strain 3716 belonged to one of the major Greek MRSA clones (Kantzanou and Tassios, unpublished observation). Moreover, the PFGE pattern of this strain (Figure 2) was indistinguishable from that of two other homMRSA isolates, nos 3711 and 3715, with agar incorporation MICs of 4 and 3.5 mg/L, respectively. However, these two isolates clearly differed from 3716 in terms of their sub-population structure, since they did not contain sub-populations that could grow in concentrations of vancomycin significantly higher than their MIC.
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Discussion |
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One homMRSA isolate, 3716, for which the MIC of vancomycin was the highest observed (4 mg/L by the broth microdilution method), but still within the susceptible range, contained sub-populations of cells that were able to grow in vancomycin concentrations as high as 8 mg/L, i.e. in the range of intermediate resistance. This pattern of heterogeneous reduced susceptibility was similar to that of a number of Japanese and US isolates. 2,4 Isolate 3716 belongs to one of the major Greek MRSA clones, as defined by PFGE. This was reminiscent of the situation both in Japan 2 and the USA ,4, where strains intermediately resistant to vancomycin have been shown to have arisen within major MRSA clones. However, in the present study, other genotypically indistinguishable isolates belonging to the same clone, and therefore presumably sharing the same mechanisms of homogeneous methicillin resistance, did not display such heterogeneous reduced susceptibility to vancomycin. This could imply that methicillin resistance per se, though a predisposing factor, may not be entirely sufficient for development of reduced susceptibility to vancomycin.
Nevertheless, the existence of clinical isolates that have evolved reduced susceptibility to vancomycin in vivo is important, especially since it has been shown that continuous antibiotic pressure can select for high resistance among an initially heterogeneous population. 2,8 Indeed, when colonies of the 3716 sub-populaiton growing in the presence of 7 mg/L vancomycin were subcultured in increasing concentrations of vancomycin, progeny growing in the presence of 9 mg/L vancomycin could be selected.
Although we were unable to demonstrate intermediate resistance to vancomycin such as has been described in Japan 1,2 or the USA 3,4 (MIC of vancomycin 8 mg/L), strains which display heterogeneous expression of reduced vancomycin susceptibility may be regarded as a preliminary stage towards the development of resistance.
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Acknowledgments |
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Notes |
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References |
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6 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically Fourth Edition: Approved Standard M7-A4 . NCCLS, Wayne, PA.
7 . Tassios, P. T., Vatopoulos, A. C., Xanthaki, A., Mainas, E., Goering, R. V. & Legakis, N. J. (1997). Distinct genotypic clusters of heterogeneously and homogeneously methicillin-resistant Staphylococcus aureus from a Greek hospital. European Journal of Clinical Microbiology and Infectious Diseases 16, 1703.[ISI][Medline]
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Sieradzki, K., Villari, P. & Tomasz, A. (1998). Decreased susceptibilities to
teicoplanin and vancomycin among coagulase-negative methicillin-resistant clinical isolates of
staphylococci. Antimicrobial Agents and Chemotherapy 42 , 1007.
Received 3 August 1998; returned 6 November 1998; revised 30 November 1998; accepted 11 December 1998