Production of capsular polysaccharide does not influence Staphylococcus aureus vancomycin susceptibility

Abstract

Background: Diverse mechanisms (increased cell wall thickness, low cross linking, decreased autolysis, etc.) have been reported for Staphylococcus aureus strains with intermediate vancomycin susceptibility (VISA). This study was conducted to identify common mechanisms responsible for decreased vancomycin susceptibility in a VISA strain pair.

Results: Transcriptional profiling of the clinical heterogeneous VISA isolate SA137/93A and its spontaneous homogeneous mutant strain SA137/93G pointed to an increased capsule production in the strain pair compared to a susceptible control. Furthermore, transcript quantification of the gene cap5E, which is essential for capsule biosynthesis, revealed elevated levels in the VISA strains SA137/93A, SA137/93G and Mu50 in comparison with susceptible strains Reynolds, Newman and SA1450/94. The increased expression was observed in bacteria from exponential as well as stationary growth phase. However, suppression of type 5 capsule formation by expression of antisense RNA did not increase vancomycin susceptibility in the VISA strain SA137/93G. Likewise, construction of inducible mutants of S. aureus Newman or repair of capsule biosynthesis of S. aureus HG001 and S. aureus 1450/94 did not influence resistance to vancomycin. Furthermore, purified type 5 polysaccharide did not protect indicator strains from the action of vancomycin.

Conclusions: The VISA strain tested in this study displayed an increased production of type 5 capsular polysaccharide. However, the production of capsule material did not protect strain SA137/93G and three vancomycin sensitive strains in the presence of vancomycin and thus is not part of the resistance mechanism; however it may represent a by-product of VISA life style that is often characterized by a high sigma factor B activity.

Figure 1

Transcription profiling: comparison of transcriptomes (OD₆₀₀ = 0.8-1.0) of VISA strain SA137/93G and the related VSSA strain SA1450/94. The regulated genes are represented as percentage of all genes constituting a process category. The number of genes per process category is shown in brackets.

Figure 2

Transcript quantification of the essential capsule biosynthesis gene cap5E by real time PCR. a) Transcript amounts of cap5E throughout the growth curve of hVISA SA137/93A (filled square), VISA SA137/93G (filled triangle), VSSA Newman (filled circle) and VSSA SA1450/94 (filled diamond) indicated as copy number per 10⁶ copies of the housekeeping gene gyrB. b) Transcript amounts of cap5E of VSSA strains (R: Reynolds*, N: Newman, 14: SA1450/94) and VISA strains (A: SA137/93A*, G: SA137/93G*, Mu: Mu50) at OD₆₀₀ = 1 and OD₆₀₀ = 4–5 indicated as copy number per 10⁶ copies of gyrB. * Error bars are not visible at OD₆₀₀ = 1 because of minimal data variations.

Figure 3

Comparison of CP5 production in a VISA and two VSSA strains. CP5 was labelled by immunofluorescence (CY3, green). As a control, all cells were stained using DAPI (blue). Cells were grown for 6 h in LB at 37°C. a) VISA SA137/93G; b) control strain SA1450/94; c) S. aureus Newman.

Figure 4

Suppression of capsule formation by expression of cap5D-antisense RNA. CP5 was labelled by immunofluorescence (CY3, green), the cells were stained using DAPI (blue). Cells were grown for 6 h in LB at 37°C. a) S. aureus SA137/93G (control); b) S. aureus SA137/93G harbouring pCapDvorne in the absence of xylose and c) grown in the presence of 50 mM xylose.

Figure 5

Population analyses of different strains in the presence or absence of capsule. a) S. aureus SA137/93G harbouring pCapDvorne grown 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 Newman harbouring an insertion of pMUTIN4 in the capsule promoter grown on MH agar in the absence (□ ) and the presence (▄ ) of 1 mM IPTG.

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.

Figure 7

Induction of capsule production by IPTG in S. aureus Newman-132. CP5 was labelled by immunofluorescence (CY3, green), the cells were stained using DAPI (blue). Cells were grown for 6 h in MH medium at 37°C. a) S. aureus Newman (control) b) S. aureus Newman in the presence of 0.5 mM IPTG; c) S. aureus Newman-132 harbouring pMUTIN4 in the capsule promoter in the absence of IPTG and d) S. aureus Newman-132 harbouring pMUTIN4 in the capsule promoter after induction with IPTG.

Figure 8

Capsule production of different S. aureus SA1450/94 clones. CP5 was labelled by immunofluorescence (CY3, green), the cells were stained using DAPI (blue). Cells were grown for 6 hours in BHI medium at 37°C. a) S. aureus SA1450/94 harbouring pCapAre, which has reconstituted capsule production; b) SA1450/94 (control) and c) SA1450/94 harbouring pCU1 (vector control).