In MIC determinations in LB/CM 34, no significant difference in vancomycin resistance was observed after expression of antisense RNA in S. aureus SA137/93G. The value of 1.5 ± 0.4 mg/L vancomycin obtained for encapsulated strains grown in the absence of xylose

was lowered to 1.3 ± 0.3 mg/L vancomycin for capsule-free cells incubated in the presence of xylose. Intermediate vancomycin selleck susceptibility of VISA strains is most easily demonstrated in population analyses on BHI, which is the medium that yields the highest vancomycin MICs and therefore should be the most sensitive medium. Again there was no difference in the population analyses of clones grown in the absence or presence of xylose (Figure 5a). Experiments in TSA-G (TSA without glucose) yielded similar results (Figure 5b). Figure 5 Population analyses of different strains in the presence or absence of capsule. a) S. aureus SA137/93G harbouring pCapDvorne grown OSI-906 concentration on BHI agar in the absence of xylose (capsule; □ ) or in the presence of xylose (no capsule; ▄ ); b) S. aureus SA137/93G harbouring pCapDvorne grown on TSA without glucose in the absence (□ ) or in the presence of xylose (▄ ); c) S. aureus HG001 (□ ) and S. aureus HG001 harbouring pcap5E (▄ ) which leads to reconstitution of capsule biosynthesis on BHI agar; d) S. aureus eFT508 Newman harbouring an insertion of pMUTIN4 in the capsule promoter grown on MH agar in the absence (□ ) and the presence (▄ ) of 1 mM IPTG.

The effect of the capsule on vancomycin resistance in VSSA In addition to the VISA strain, the effect of the capsule on vancomycin resistance in three vancomycin susceptible strains producing CP5 was investigated. All strains of the RN1 (NCTC 8325) lineage harbour a mutation in cap5E that leads to inactivation of capsule biosynthesis. Furthermore a deletion in rsbU leads to a very low activity of sigma B which however is needed for the efficient transcription of the capsule biosynthetic genes [50]. As described Depsipeptide order in [34], capsule production was reconstituted into S. aureus HG001 (rsbU repaired) by introduction of a plasmid carrying a cap5E gene amplified from

S. aureus Newman (Figure 6). Again the population showed a heterogeneous phenotype in immunofluorescence experiments. However, in population analyses no increase in resistance against vancomycin could be detected (Figure 5c). Figure 6 Repair of capsule formation in S. aureus HG001. CP5 was labelled by immunofluorescence (CY3, green), the cells were stained using DAPI (blue). Cells were grown in TSB medium overnight at 37°C. a) S. aureus HG001 (control); b) S. aureus HG001 pCap5E, in which capsule production has been reconstituted. An S. aureus Newman clone with the capsule promoter under control of Pspac was capsule negative in the absence of inducer, but heterogeneous capsule production could be achieved by addition of IPTG to media that did not contain glucose, e.g., MH (Figure 7).