Susceptibility of capsular Staphylococcus aureus strains to some antibiotics, triclosan and cationic biocides

Paul Seaman1, Martin Day1,*, A. Denver Russell2 and Dietmar Ochs3

1 Cardiff School of Biosciences and 2 Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3TL, UK; 3 Ciba Spezialitätenchemie Grenzach GmbH, Postfach 1266, D-79630 Grenzach-Wyhlen, Germany

Keywords: S. aureus , capsule polysaccharides , antimicrobial susceptibility

Sir,

Approximately 90% of Staphylococcus aureus isolates produce one of 11 serotypes of capsular polysaccharides. Serotypes CP5 and CP8 account for ~25% and 50%, respectively, of isolates recovered from humans, offering support for their pathogenic significance.1 The importance and relevance of these capsule types is confirmed by the development of a conjugate vaccine, StaphVAX that includes type 5 and 8 capsule polysaccharides.2 CP5 and CP8 serotypes are considered as microcapsules because they are much smaller than those produced by the mucoid serotypes 1 and 2. The microcapsules of CP5 and CP8 are extracellular, uronic acid-containing polysaccharides that are too small to be visualized by negative stains such as India ink. These two capsules are very similar and differ only in the position of O-acetyl groups and the linkages between the amino sugars. The function of CP5 and CP8 in S. aureus virulence has been investigated in great depth, especially with regard to their role in impeding phagocytosis.1 However, few authors have reported upon the potential for a capsule polysaccharide to present a permeability barrier to antimicrobial agents. Gram-positive bacteria possess a cell wall that is usually permeable and does not limit the incursion of antimicrobials. However, resistance through reduced penetration has been shown to occur, for example vancomycin-intermediate resistant S. aureus (VISA) strains produce a distinctly thickened cell wall.3 Furthermore, in 1984 Kolawole4 discussed the effect of a mucoid capsule upon disinfectant and antiseptic susceptibility in S. aureus. He concluded that the thick capsule, as associated with serotypes CP1 and CP2, does provide a permeability barrier to common biocides but fell short of testing the more common and clinically important serotypes. We wish to report how CP5 and CP8 microcapsules affect the susceptibility of S. aureus to several antibiotics and three widely used biocides: triclosan, chlorhexidine gluconate and cetylpyridinium chloride.

A range of 14 antibiotic discs were purchased from Oxoid (Basingstoke, UK) and used to analyse S. aureus Reynolds and two isogenic capsule mutants. Antibiotic susceptibility was established as per the BSAC standardized disc susceptibility testing methodology5 (Table 1). The MICs of nine of these clinically relevant antibiotics were elucidated by Etest strips (AB Biodisk, Sweden) according to the manufacturer's recommendations. MICs for the three biocides were calculated using Iso-Sensitest agar (Oxoid, Basingstoke, UK), multipoint inoculator (Denley; Mast Diagnostics, Bootle, UK) and incubation in air at 37°C for 18–20 h, in accordance with BSAC guidelines.6 Triclosan (Irgasan DP300) was a gift from Ciba Speciality Chemicals; chlorhexidine gluconate and cetylpyridinium chloride were purchased from ICN Biomedicals Inc. (Ohio, USA). Of the three S. aureus Reynolds strains, one was wild-type, expressing a serotype CP5 capsule, one a mutant expressing CP8 and the third a second mutant, lacking a capsule (CP–).7 The acapsular Reynolds strain was constructed by replacing the serotype-specific capsule genes cap5HIJK on the bacterial chromosome with an erm(B) gene, conferring erythromycin resistance. S. aureus NCTC 6571 (Oxford) was included alongside the capsule strains as a control. The MICs were defined as the lowest concentration of antimicrobial with which there was no visible growth of the organism and are shown in Table 2, along with the Etest data. In addition, all strains were investigated for ß-lactamase production by nitrocefin stick (Oxoid, Basingstoke, UK; Table 2).


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Table 1. Antibiotic susceptibility of the three S. aureus capsule strains alongside the control strain NCTC 6571 for which the antibiogram is known

 

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Table 2. MICs for S. aureus Reynolds expressing either capsule serotype CP5, CP8 or CP– (acapsular)

 
The antibiogram for NCTC 6571, as deduced by antibiotic disc susceptibility testing, was as expected. No difference in antibiotic susceptibility was observed between the capsule strains other than for erythromycin, resistance to which was found in CP–. This was as expected due to the construction of the CP– strain by disruption of the serotype-specific capsule genes with an erm(B) gene. The nitrocefin test implies that penicillin and ampicillin resistance is conferred by ß-lactamase production in S. aureus Reynolds. MICs for NCTC 6571 were within plus or minus one two-fold dilution of the expected MIC.6 The capsule strains all demonstrated MICs for each antimicrobial within plus or minus one two-fold dilution of each other. The only exception to this was—once again—erythromycin, confirming the results of the susceptibility disc test. From this we deduced that there is no significant difference between the antibiotic and biocide susceptibilities of the strains investigated herein. Consequently, we conclude that the capsule polysaccharide serotypes CP5 and CP8 do not present a permeability barrier to the antibiotics used in this investigation, or to triclosan, chlorhexidine gluconate or cetylpyridinium chloride.

These results indicate that whereas S. aureus capsule polysaccharides are implicated in virulence, they are not involved in conferring reduced antibiotic or biocide susceptibility. Clinical implications are pertinent through the choice of clinically relevant antibiotics and biocides regularly used in the hospital setting.

Acknowledgements

This work was supported by Ciba Speciality Chemicals Inc. We are grateful to Dr Jean Lee of the Channing Laboratory, Harvard University, Boston, MA, USA, who supplied the S. aureus Reynolds strains.

Footnotes

* Corresponding author. Tel: +44-29-20875768; Fax: +44-29-20-874305; Email: day{at}cardiff.ac.uk

References

1 . O'Riordan, K. & Lee, J. C. (2004). Staphylococcus aureus capsular polysaccharides. Clinical Microbiology Reviews 17, 218–34.[Abstract/Free Full Text]

2 . Fattom, A. I., Horwith, G., Fuller, S. et al. (2004). Development of StaphVAXTM, a polysaccharide conjugate vaccine against S. aureus infection: from the lab bench to phase III clinical trials. Vaccine 22, 880–7.[CrossRef][ISI][Medline]

3 . Lambert, P. A. (2002). Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria. Journal of Applied Microbiology 92, 46S–54S.[CrossRef][ISI][Medline]

4 . Kolawole, D. O. (1984). Resistance mechanisms of mucoid-grown Staphylococcus aureus to the antibacterial action of some disinfectants and antiseptics. FEMS Microbiology Letters 25, 205–9.[CrossRef][ISI]

5 . Andrews, J. M. (2001). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 43–57.[Abstract/Free Full Text]

6 . Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 5–16.[Abstract/Free Full Text]

7 . Nilsson, I. M., Lee, J. C., Bremell, T. et al. (1997). The role of staphylococcal polysaccharide microcapsule expression in septicemia and septic arthritis. Infection and Immunity 65, 4216–21.[Abstract]





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