Fitness cost of staphylococcal cassette chromosome mec in methicillin-resistant Staphylococcus aureus by way of continuous culture.

We examined the effect of introducing type I or IV staphylococcal cassette chromosome mec (SCCmec) elements on the growth yield of Staphylococcus aureus in glucose-limited continuous culture. Type I showed increased glucose consumption and ATP demand per gram of cells synthesized and decreased cell yield compared to those of the parent strain. In contrast, type IV SCCmec elements had no adverse energetic effect.

Persistence of vancomycin-resistant enterococci in New Zealand broilers after discontinuation of avoparcin use.

Large amounts of tylosin, zinc-bacitracin, and avilamycin are currently used as prophylactics in New Zealand broiler production. Avoparcin was also used from 1977 to 2000. A total of 382 enterococci were isolated from 213 fecal samples (147 individual poultry farms) using enrichment broths plated on m-Enterococcus agar lacking antimicrobials. These isolates were then examined to determine the prevalence of antimicrobial resistance. Of the 382 isolates, 5.8% (22 isolates) were resistant to vancomycin, and 64.7% were resistant to erythromycin. The bacitracin MIC was > or =256 microg/ml for 98.7% of isolates, and the avilamycin MIC was > or =8 microg/ml for 14.9% of isolates. No resistance to ampicillin or gentamicin was detected. Of the 22 vancomycin-resistant enterococci (VRE) isolates, 18 (81.8%) were Enterococcus faecalis, 3 were Enterococcus faecium, and 1 was Enterococcus durans. However, when the 213 fecal enrichment broths were plated on m-Enterococcus agar containing vancomycin, 86 VRE were recovered; 66% of these isolates were E. faecium and the remainder were E. faecalis. Vancomycin-resistant E. faecium isolates were found to have heterogenous pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested DNA, whereas the PFGE patterns of vancomycin-resistant E. faecalis isolates were identical or closely related, suggesting that this VRE clone is widespread throughout New Zealand. These data demonstrate that vancomycin-resistant E. faecalis persists in the absence and presence of vancomycin-selective pressure, thus explaining the dominance of this VRE clone even in the absence of avoparcin.

Acquired bacitracin resistance in Enterococcus faecalis is mediated by an ABC transporter and a novel regulatory protein, BcrR.

Bacitracin resistance (bacitracin MIC, >/=256 microg ml(-1)) has been reported in Enterococcus faecalis, and in the present study we report on the genetic basis for this resistance. Mutagenesis was carried out with transposon Tn917 to select for E. faecalis mutants with decreased resistance to bacitracin. Two bacitracin-sensitive mutants (MICs, 32 microg ml(-1)) were obtained and Tn917 insertions were mapped to genes designated bcrA and bcrB. The amino acid sequences of BcrA (ATP-binding domain) and BrcB (membrane-spanning domain) are predicted to constitute a homodimeric ATP-binding cassette (ABC) transporter, the function of which is essential for bacitracin resistance in E. faecalis. The bcrA and bcrB genes were organized in an operon with a third gene, bcrD, that had homology to undecaprenol kinases. Northern analysis demonstrated that bcrA, bcrB, and bcrD were transcribed as a polycistronic message that was induced by increasing concentrations of bacitracin but not by other cell wall-active antimicrobials (e.g., vancomycin). Upstream of the bcrABD operon was a putative regulatory gene, bcrR. The bcrR gene was expressed constitutively, and deletion of bcrR resulted in a bacitracin-sensitive phenotype. No bcrABD expression was observed in a bcrR mutant, suggesting that BcrR is an activator of genes essential for bacitracin resistance (i.e., bcrABD). The bacitracin resistance genes were found to be located on a plasmid that transferred at a high frequency to E. faecalis strain JH2-2. This report represents the first description of genes that are essential for acquired bacitracin resistance in E. faecalis.

Vancomycin-induced deletion of the methicillin resistance gene mecA in Staphylococcus aureus.

OBJECTIVE: To elucidate factors that contribute to the development of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA). 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, 6-12 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.

Characterization of a vancomycin-resistant Enterococcus faecalis (VREF) isolate from a dog with mastitis: further evidence of a clonal lineage of VREF in New Zealand.

We report here on the characterization of a vancomycin-resistant Enterococcus faecalis (VREF) isolated from a dog with mastitis. The isolate was positive for the vanA, ermB, and tet(M) genes, with vanA and ermB carried on the same transferable plasmid. Comparison of this isolate with VREF from poultry and human sources in New Zealand demonstrated identical SmaI macrorestriction patterns and Tn1546-like elements. This is further evidence of a clonal lineage of VREF in New Zealand.

A clonal lineage of VanA-type Enterococcus faecalis predominates in vancomycin-resistant Enterococci isolated in New Zealand.

Avoparcin was used as a feed additive in New Zealand broiler production from 1977 until June 2000. We report here on the effects of the usage and discontinuation of avoparcin on the prevalence of vancomycin-resistant enterococci (VRE) in broilers. Eighty-two VRE isolates were recovered from poultry fecal samples between 2000 and mid-2001. VRE isolates were only obtained from broiler farms that were using, or had previously used, avoparcin as a dietary supplement. Of these VRE isolates, 73 (89%) were VanA-type Enterococcus faecalis and nine (11%) were VanA-type Enterococcus faecium. All E. faecalis isolates were found to have an identical or closely related pulsed-field gel electrophoresis (PFGE) pattern of SmaI-digested DNA and were susceptible to both ampicillin and gentamicin. The PFGE patterns of the nine E. faecium isolates were heterogeneous. All VRE contained both the vanA and ermB genes, which, regardless of species or PFGE pattern, resided on the same plasmid. Eighty-seven percent of the VRE isolates also harbored the tet(M) gene, while for 63 and 100%, respectively, of these isolates, the avilamycin and bacitracin MICs were high (>or=256 microg/ml). Five of eight vancomycin-resistant E. faecalis isolates recovered from humans in New Zealand revealed a PFGE pattern identical or closely related to that of the E. faecalis poultry VRE isolates. Molecular characterization of Tn1546-like elements from the VRE showed that identical transposons were present in isolates from poultry and humans. Based on the findings presented here, a clonal lineage of VanA-type E. faecalis dominates in VRE isolated from poultry and humans in New Zealand.

Phenotypic and molecular characterization of community occurring, Western Samoan phage pattern methicillin-resistant Staphylococcus aureus.

In New Zealand, it is estimated that greater than half of the methicillin-resistant Staphylococcus aureus (MRSA) strains recovered from patients belong to what has been termed Western Samoan phage pattern types 1 and 2 (WSPP1, WSPP2). These strains differ from classical MRSA isolates in terms of their lack of multiresistance and community occurrence, suggesting that such strains possess properties and/or characteristics different from those of other MRSA. To address this hypothesis, 10 WSPP1 and WSPP2 isolates from Western Samoa, New Zealand and Australia were compared with common hospital MRSA isolates. All WSPP isolates were identical with regard to pulsed-field gel electrophoretic pattern of SmaI-digested DNA, coagulase gene restriction fragment length polymorphism pattern and localization of mecA to a 194 kb SmaI digestion fragment. The WSPP strains were no more resistant/sensitive to various environmental stresses (e.g. skin fatty acids, UV light, desiccation) compared with hospital epidemic MRSA strains, except for their higher tolerance to salt. In terms of virulence, the WSPP MRSA were quantitatively better at attaching to the epithelial cell line HEp2, were uniformly egg-yolk opacity factor negative and produced higher levels of haemolytic toxins compared with non-WSPP MRSA isolates.