Journal Article

Comparison of assays for detection of agents causing membrane damage in <i>Staphylococcus aureus</i>

Alexander John O'Neill, Keith Miller, Brunello Oliva and Ian Chopra

in Journal of Antimicrobial Chemotherapy

Published on behalf of British Society for Antimicrobial Chemotherapy

Volume 54, issue 6, pages 1127-1129
Published in print December 2004 | ISSN: 0305-7453
Published online December 2004 | e-ISSN: 1460-2091 | DOI: http://dx.doi.org/10.1093/jac/dkh476
Comparison of assays for detection of agents causing membrane damage in Staphylococcus aureus

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Objectives: To develop a novel β-galactosidase leakage assay for Staphylococcus aureus and to evaluate this alongside other simple methods for detection of agents that cause membrane damage in staphylococci.

Methods: Using a PCR-based approach, a derivative of S. aureus RN4220 was constructed carrying the Escherichia coli lacZ gene under the control of the strong staphylococcal promoter, cap1A. Leakage of β-galactosidase (BG) from this strain was examined after exposure for 10 min to various membrane-damaging agents at 4×MIC, using a fluorescence assay and the substrate 4-methylumbelliferyl-β-d-galactoside. Other assays for membrane damage involving protoplast lysis (PL), leakage of material absorbing at 260 nm (OD) and ATP release as well as the BacLight (BL) assay were carried out using established methods.

Results: All the assays, with the exception of the PL assay, detected membrane damage induced by cetyltrimethylammonium bromide, nisin, clofazimine and protegrin IB-367. However, the ability to detect membrane damage induced by these agents differed between the assay systems. The assays also varied considerably in their signal-to-noise ratio, with the ATP assay providing values for nisin approaching 100-fold that of the control.

Conclusions: The PL assay is unsuitable for detection of membrane-damaging agents in S. aureus. The other assays, including the BG assay, detect membrane damage. The OD assay should be sufficient for most purposes since it is effective, rapid and cheap to perform. Studies requiring maximum sensitivity and discrimination should employ the ATP assay.

Keywords: membrane perturbation; protoplasts; fluorescent dyes; ATP; β-galactosidase; components absorbing at 260 nm

Journal Article.  1700 words. 

Subjects: Medical Oncology ; Critical Care

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