The development of a new three-step protocol to determine the efficacy of disinfectant wipes on surfaces contaminated with Staphylococcus aureus.

We developed a three-step protocol to quantify the efficacy of disinfectant wipes, their ability to remove and prevent microbial transfer from surfaces and their overall antimicrobial activity. Meticillin-resistant (MRSA) or -susceptible (MSSA) Staphylococcus aureus (6-7 log(10)cfu) were inoculated onto stainless steel discs with or without organic load and dried. Grapefruit extract-containing test wipes and unmedicated control wipes were used. In step 1, wipes were mechanically rotated against surfaces for 10s at 60rpm, exerting a weight of 100+/-5g. Bacterial removal was assessed by transferring the steel discs to neutraliser, resuspending and counting remaining bacteria. In step 2, bacterial transfer from wipes was assessed by eight consecutive mechanical adpression transfers to agar/neutraliser plates. Step 3 was the measurement of antimicrobial activity by direct inoculation of the wipes for 10s followed by neutralisation and enumeration. Test wipes achieved a significantly higher bacterial cell removal than control wipes on all surfaces (P<0.05). The low bactericidal activity of the wipes (<1 log(10) reduction when directly inoculated) and the subsequent survival of bacteria on the wipes, however, led to repeated microbial transfer when initially high contamination levels were present. There were no differences between MRSA and MSSA in removal, transfer or antimicrobial activity. The three-step method is a useful tool for developing future guidelines to assess the ability of wipes to disinfect surfaces.

Development of resistance to chlorhexidine diacetate in Pseudomonas aeruginosa and the effect of a "residual" concentration.

Stable resistance in Pseudomonas aeruginosa NCIMB 10421 was obtained by step-wise exposure to gradually increasing concentrations of chlorhexidine diacetate (CHX). Repeated exposure to a proposed "residual" (sub-MIC) concentration of CHX also created stable resistance. Resistance was also developed by a single exposure to the "residual" concentration of CHX, but this was unstable. Similar experiments with Escherichia coli and CHX or cetylpyridinium chloride resulted in no significant increase in resistance. Antibiotic susceptibility profiles of the CHX-resistant P. aeruginosa cultures showed no cross-resistance, although some of the cultures were resistant to benzalkonium chloride.

Antimicrobial efficacy of biocides tested on skin using an ex-vivo test.

An ex-vivo test was used to evaluate the activity of antimicrobials against three microorganisms, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The ex-vivo test is a carrier test using freshly excised animal skin samples maintained in viable conditions for a short period of time. Skin samples came from a veterinary practice and were excised from either dogs or cats. The antimicrobial activity of povidone iodine, chlorhexidine diacetate, cetrimide and benzalkonium chloride was also evaluated with suspension and glass-carrier tests. Generally, the activity of the antimicrobials tested was reduced when applied to the skin surface. Apart from povidone iodine (2%) against S. aureus, the biocides investigated failed to achieve a 5 log10 reduction in bacterial titre when tested with the ex-vivo method. There was no significant difference in reduction of bacterial titres after treatment with antimicrobials between the glass-carrier and the suspension tests. Furthermore, the drying process of bacterial inoculum was less detrimental on skin than on glass surfaces. This study confirmed that the activity of a biocide tested in suspension or on an inanimate surface did not reflect its activity when tested on skin. Further development of the ex-vivo test may be useful, especially for testing the antimicrobial activity of formulations with antiseptic properties.