1 Department of Microbiology, Otago School of Medical Sciences, University of Otago, P.O. Box 56, Dunedin, New Zealand; 2 Department of Medical Microbiology, University of Zürich, CH-8028, Zürich, Switzerland
Received 23 March 2004; returned 15 April 2004; revised 2 June 2004; accepted 3 June 2004
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Abstract |
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Methods: Forty-nine MRSA isolates were subjected to passage selection with vancomycin to isolate mutants with reduced susceptibility to vancomycin. One mutant was chosen for detailed molecular and biochemical characterization.
Results: Five vancomycin-resistant mutants (vancomycin MICs, 612 mg/L) were obtained in vitro from five MRSA parent isolates. Upon acquisition of vancomycin resistance, all mutants showed a concomitant decrease in oxacillin resistance. In one particular MRSA strain, selection for vancomycin resistance repeatedly produced deletions and rearrangements, including loss of the mecA gene. Pleiotropic phenotypical changes, such as yellow pigment formation, loss of haemolysis, thickened cell wall, increased resistance to lysostaphin and reduced cell wall turnover were observed in this mutant.
Conclusion: Acquisition of vancomycin resistance in one MRSA strain triggered mecA deletion suggesting that this deletion, coupled to other rearrangements and/or mutations, may be responsible for the increased vancomycin resistance phenotype.
Keywords: MRSA , staphylococci , vancomycin resistance , S. aureus
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Introduction |
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The stability of SCCmec in S. aureus is influenced by environmental factors. For example, spontaneous mecA deletion has been observed during long-term storage in drug-free medium,1 and in cultures that have been aged/starved, grown at high temperatures, or given small doses of UV radiation.2,3 Spontaneous loss of SCCmec has also been reported to occur in some lineages of S. aureus in vivo.4,5
Several reports have implicated a role for mecA in the development of MRSA isolates with decreased susceptibility to vancomycin. These studies have demonstrated a correlation between decreased oxacillin resistance with a concomitant increase in resistance to vancomycin either through the deletion of mecA6 or mecA mutation.7
This study examined 49 clinical MRSA isolates for mutants with decreased susceptibility to vancomycin after passage selection in vancomycin-containing medium. We report here that vancomycin stress repeatedly triggered deletion of mecA in one MRSA strain. This event coincided with a decrease in susceptibility to vancomycin and pleiotropic phenotypic changes.
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Materials and methods |
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The 49 MRSA isolates used in this study were a diverse collection of clinical isolates from the culture collections of this laboratory and the Environmental Science and Research, Communicable Disease Centre, Porirua, New Zealand. S. aureus were defined as MRSA with oxacillin MICs 4 mg/L, and all were vancomycin-susceptible (MIC
2 mg/L). All MICs of vancomycin and oxacillin were determined using Etests (AB Biodisk, Solna, Sweden) on MuellerHinton agar (MHA-Difco, Detroit, MI, USA) containing 2% NaCl. Disc diffusion sensitivity tests were carried out as described by the National Committee for Clinical Laboratory Standards (NCCLS). Haemolytic activity was sought by spotting 10 µL of a 0.5 McFarland Standard suspension on sheep blood agar plates with incubation for 24 h at 37°C. The resulting growth was observed for their surrounding lytic zones. Presence of ß-lactamase was shown with nitrocefin discs (Becton Dickinson, MD, USA).
Selection of mutants with decreased susceptibility to vancomycin
For each of the 49 vancomycin-susceptible MRSA isolates, a single colony was inoculated into 10 mL of tryptic soy broth (TSB) containing 6 mg/L vancomycin and incubated at 37°C until the optical density (OD600, 1 cm light path) was >1.0. The resulting suspension was then diluted to achieve a cell concentration of approximately 100 colonies per 100 µL. Diluted cell suspensions (100 µL) were spread on to tryptic soy agar (TSA) plates containing the same concentration of vancomycin as the broth culture. Plates were then incubated at 37°C until colonies became evident, which took 17 days for positive isolates. Colonies were picked from plates that exhibited good growth, excluding pinpoint colonies, and the cycle of broth and agar growth was repeated three times with medium containing the same concentration of vancomycin. With each repeat passage, the time taken for colonies to appear on plates was reduced to approximately 12 days for all isolates.
Pulsed-field gel electrophoresis
Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA was carried out to confirm that clonal identity was maintained between each parent and the respective passaged mutant. PFGE was carried out by contour-clamped homogeneous electric field (CHEF) electrophoresis using the CHEF-DRIII system (Bio-Rad Laboratories, Richmond, CA, USA) as previously described.8 Gels were routinely run at 6 V/cm, 14°C, at an included angle of 120°, on a 1.2% agarose gel (USB Corporation, Cleveland, OH, USA) with pulse times of 535 s for 22 h. The Low Range PFG Marker (New England Biolabs, Inc., Beverly, MA, USA), containing lambda concatemers and HindIII-digested lambda fragments, was used as a size standard.
All strains were tested for the presence of mecA by PCR and Southern blot hybridization using standard protocols and primers as previously described.8 Primer pair agr S3 (5'-GATTTAAGTCGCAGTATTGGT-3') and agr S4 (5'-ACGCGTCATATTTAATTTTGT-3') were used to amplify a 1.2 kb amplicon containing agrC and agrA.
Cell wall turnover, autolysis assays, lysostaphin susceptibilities and transmission electron microscopy
Cell wall turnover was determined as described by Hanaki et al.9 using tritium-labelled N-acetylglucosamine ([3H]GlcNAc) and measuring the release of radioactive cell wall components. Autolysis activity of whole cells was determined by Triton X-100 treatment as described by Mani et al.10 The cells were incubated at 30°C with shaking and the OD620 was measured at 60 min intervals for 4 h. Lysostaphin (1 mg/L)-mediated cell lysis was carried out as described by Peschel et al.11 The cells were incubated at 30°C with shaking and the OD620 was measured at 10 min intervals for 30 min. The results shown for all experiments are the mean values of two to four independent experiments that were carried out in triplicate. The standard error of the mean did not differ by more than 15%. For electron microscopy, strains were grown in 10 mL of TSB to the late-log phase of growth to an OD600 around 1.0. Cells were pelleted by centrifugation and fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0), with 3 mg/mL Ruthenium Red for 2 h at room temperature. The cells were then washed in 0.1 M sodium cacodylate and mixed for 5 min. This cycle was repeated a further two times. Cells were then post-fixed in 2% osmium tetroxide with 3 mg/mL Ruthenium Red in 0.1 M sodium cacodylate buffer (pH 7.0) for 2 h at room temperature. Three more wash steps were then carried out, and the cells stored at 4°C overnight. Samples were then dehydrated through an ethanol series and propylene-oxide, and embedded in Agar 100 resin (Agar Scientific, Stansted, Essex, UK). The embedded samples were sectioned (80 nm thick) and stained with uranyl acetate and lead citrate. Grids were viewed with a transmission electron microscope Akashi EM-002A at 100 kV accelerating voltage. Mean cell wall measurements were calculated from an average of 30 cells.
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Results |
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To elucidate the factors that contribute to the development of vancomycin resistance in MRSA, we subjected 49 clinical MRSA isolates (all vancomycin-susceptible) to passage selection with vancomycin. These 49 isolates consisted of: 15 isolates (oxacillin MICs, 128 mg/L) with identical SmaI macrorestriction DNA profiles (group I); 24 isolates (oxacillin MICs, 4 to
128 mg/L) with PFGE patterns that differed by only one DNA band (group II); and 10 isolates with distinct (
6 band differences) PFGE patterns (oxacillin MICs, 8 to
128 mg/L) (group III). Based on these results, the 49 MRSA isolates selected for this study represented a relatively diverse genetic group.
Upon passage in liquid medium containing 6 mg/L vancomycin, five of 49 MRSA isolates yielded colonies after 17 days of incubation (Table 1). Two of the vancomycin-resistant mutants, Vr6-1126a and Vr8-1128a, were derived from group I MRSA isolates, and three (Vr6-1130a, Vr6-1132a and Vr12-1134a) from group II MRSA isolates. The vancomycin MICs for these mutants ranged from 6 to 12 mg/L (Table 1). These mutants were termed with the prefix Vr followed by the number indicating their vancomycin MIC. All corresponding parent isolates were mecA-positive, with oxacillin MIC values 128 mg/L, and produced ß-lactamase. The five Vr mutants exhibited decreased resistance to oxacillin upon acquisition of vancomycin resistance, but remained ß-lactamase-positive.
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PFGE analysis of mutants derived from strain 1126, and probing with mecA, revealed that those with vancomycin MICs 4 mg/L had the same PFGE pattern, termed 1a, as the parent strain (Figure 1, lanes 2, 4 and 5). In contrast, mutants with vancomycin MICs >4 mg/L had lost mecA. This loss was associated with large deletions in the SCCmec-carrying SmaIG band (Figure 1, lanes 3, and 69), resulting either in a smaller additional band I' (Figure 1, lanes 3, 6, 7, and 9), or in a larger band F', presumably due to a deletion fusing SmaIG to SmaII in strain Vr10-1126a (Figure 1, lane 8).
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To rule out the possibility that the deletion of mecA from strain 1126 was due to a spontaneous event in the absence of vancomycin, 30 individual cultures of 1126 were incubated for 10 days or more in TSB without daily passage at 37°C. Approximately 200 colonies per culture were tested for oxacillin susceptibility. Of 2000 colonies tested, only three mutants (0.15%) with increased susceptibility to oxacillin were isolated. Two of three mutants were negative for mecA, but their susceptibility to vancomycin remained unchanged. The third mutant was still positive for mecA and susceptible to vancomycin. In contrast, the frequency of mecA deletion was 100% in vancomycin-induced, 1126-derived mutants with vancomycin MICs >4 mg/L. Hence, vancomycin triggered or selected for loss of mecA at a much higher frequency.
A detailed comparative phenotypic analysis was carried out between Vr6-1126a and its partially-isogenic parent 1126. Mutant Vr6-1126a was more resistant to lysostaphin, and cell wall turnover was markedly reduced in the mutant compared with the parent (Table 2). The cell wall of Vr6-1126a was also three-fold thicker than the parent strain (Table 2), but Triton X-100-induced cell autolysis did not differ significantly.
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Discussion |
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The size of the mecA deletion in 1126 varied depending on the mutant, and rearrangements in other parts of the chromosome (e.g. agr locus) were also observed. Some studies have reported that clinical glycopeptide-intermediate S. aureus (GISA) isolates are defective in the accessory gene regulator (agr) locus.12 Whilst compromised agr function is not an absolute requirement for the development of GISA, it appears to be advantageous for the development of vancomycin hetero-resistance.
The phenotypic properties of Vr6-1126a are consistent with abnormalities in cell wall metabolism, as have been reported for other vancomycin-resistant mutants. The results of this study contribute further to our knowledge on the development of vancomycin resistance in MRSA and we have shown that for one MRSA strain, this results in the deletion of mecA at the expense of vancomycin susceptibility. Future work will focus on the apparent incompatibility between oxacillin and vancomycin resistance in this strain.
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Acknowledgements |
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Footnotes |
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References |
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