However, the widespread use of vancomycin to treat MRSA infections has resulted in the increased frequency of isolation of vancomycin intermediate-level-resistant S. aureus (VISA) strains, from both clinical

and community sources (Walsh & Howe, 2002). These data underscore the need for a better understanding of the molecular underpinnings of how resistance may arise to PF-562271 existing, and in particular, investigational antimicrobials (Mangili et al., 2005). The generation of an S. aureus strain with reduced susceptibility to ramoplanin provides a model system to gain greater insight into the mechanisms of ramoplanin action and the evolution of resistance mechanisms in Gram-positive bacteria. Staphylococcus aureus strain NCTC 8325-4 (also known as NRS135; Novick, 1967) was obtained from the repository maintained by the Network on Antimicrobial Resistance in S. aureus (http://www.narsa.net). To generate ramoplanin-resistant S. aureus, a step pressure method was used. Isolated colonies of S. aureus NCTC 8325-4 were inoculated into 5-mL aliquots of cation-adjusted Muller–Hinton broth II supplemented with 0.02% Fraction V bovine serum albumin (CAMHB2+BSA) containing ramoplanin at concentrations of 0.1–10 μg mL−1. The cultures were incubated at 37 °C with aeration for 48 h. At 48 h, growth was observed in the culture containing 0.1 μg mL−1 ramoplanin. This culture was used to inoculate

5 mL CAMHB2+BSA containing ramoplanin at concentrations of 0.1–5 μg mL−1 at a cell density of ∼106 CFU mL−1. These cultures selleck compound were incubated for 24–72 h at 37 °C with aeration. The culture with growth in the highest concentration of ramoplanin was used to inoculate another series at a cell density of ∼106 CFU mL−1. Passage of the culture was continued in this manner through the 16th series. In the 16th series, growth was observed in a culture containing 5 μg mL−1 ramoplanin. A sample from this culture was plated on tryptic soy agar (TSA) with no antibiotic and incubated at 37 °C overnight. An isolated colony was selected and streaked onto TSA and grown overnight at 37 °C twice, and then an isolated colony was selected and named RRSA16.

Oligonucleotide primers 16s_fw_sa (5′-CGTGCCTAATACATGCAAGTC-3′) and 16S_univ_rv (5′-ACGGGCGGTGTGTACAAG-3′) were used to amplify Phosphoglycerate kinase a portion of the genes encoding the 16s rRNA from genomic DNA prepared from each NCTC 8325-4 and RRSA16. The nucleotide sequences obtained from reactions performed with primers 16s_fw_sa and 16S_univ_rv on the amplified sequences from NCTC 8325-4 and RRSA16 were identical to each other and the published sequence of NCTC 8325-4. An overnight culture of RRSA16 was subcultured into CAMHB2+BSA containing no antibiotics at a cell density of ∼104 CFU mL−1 and incubated overnight at 37 °C with shaking. The overnight growth was used to inoculate a fresh CAMHB2+BSA culture containing no antibiotic at a cell density of ∼104 CFU mL−1 and incubated overnight at 37 °C with shaking.