Comparison of assays for detection of agents causing membrane damage in Staphylococcus aureus.
OBJECTIVE
To develop a novel beta-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 beta-galactosidase (BG) from this strain was examined after exposure for 10 min to various membrane-damaging agents at 4 x MIC, using a fluorescence assay and the substrate 4-methylumbelliferyl-beta-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.
O'Neill
Alexander John
AJ
Antimicrobial Research Centre and School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK.
Miller
Keith
K
Oliva
Brunello
B
Chopra
Ian
I
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001433:Bacteriolysis; D002462:Cell Membrane / Q000187:drug effects*; D002463:Cell Membrane Permeability / Q000187:drug effects; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009561:Nisin / Q000494:pharmacology; D011523:Protoplasts / Q000187:drug effects; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000900:Anti-Bacterial Agents; D002593:Cetrimonium Compounds; D009561:Nisin; D001616:beta-Galactosidase; D000077286:Cetrimonium