Background: Accurate determination of the efficacy of antimicrobial agents requires neutralization of residual antimicrobial activity in the samples before microbiological assessment of the number... Show moreBackground: Accurate determination of the efficacy of antimicrobial agents requires neutralization of residual antimicrobial activity in the samples before microbiological assessment of the number of surviving bacteria. Sodium polyanethol sulfonate (SPS) is a known neutralizer for the antimicrobial activity of aminoglycosides and polymyxins. In this study, we evaluated the ability of SPS to neutralize residual antimicrobial activity of antimicrobial peptides [SAAP-148 and pexiganan; 1% (wt/v) in PBS], antibiotics [mupirocin (Bactroban) and fusidic acid (Fucidin) in ointments; 2% (wt/wt))] and disinfectants [2% (wt/wt) silver sulfadiazine cream (SSD) and 0.5% (v/v) chlorhexidine in 70% alcohol].Methods: Homogenates of human skin models that had been exposed to various antimicrobial agents for 1 h were pipetted on top of Methicillin-resistant Staphylococcus aureus (MRSA) on agar plates to determine whether the antimicrobial agents display residual activity.To determine the optimal concentration of SPS for neutralization, antimicrobial agents were mixed with PBS or increasing doses of SPS in PBS (0.05-1% wt/v) and then 105 colony forming units (CFU)/mL MRSA were added. After 30min incubation, the number of viable bacteria was assessed. Next, the in vitro efficacy of SAAP-148 against various gram-positive and gram-negative bacteria was determined using PBS or 0.05% (wt/v) SPS immediately after 30 min incubation of the mixture. Additionally, ex vivo excision wound models were inoculated with 105 CFU MRSA for 1 h and exposed to SAAP-148, pexiganan, chlorhexidine or PBS for 1 h. Subsequently, samples were homogenized in PBS or 0.05% (wt/v) SPS and the number of viable bacteria was assessed.Results: All tested antimicrobials displayed residual activity in tissue samples, resulting in a lower recovery of surviving bacteria on agar. SPS concentrations at >= 0.05% (wt/v) were able to neutralize the antimicrobial activity of SAAP-148, pexiganan and chlorhexidine, but not of SSD, Bactroban and Fucidin. Finally, SPS-neutralization in in vitro and ex vivo efficacy tests of SAAP-148, pexiganan and chlorhexidine against gram-positive and gram-negative bacteria resulted in significantly higher numbers of CFU compared to control samples without SPS-neutralization.Conclusions: SPS was successfully used to neutralize residual activity of SAAP-148, pexiganan and chlorhexidine and this prevented an overestimation of their efficacy. Show less
Background: We investigated the efficacy of a synthetic antimicrobial peptide SAAP-148, which was shown to be effective against Methicillin-resistant Staphylococcus aureus (MRSA) on tape-stripped... Show moreBackground: We investigated the efficacy of a synthetic antimicrobial peptide SAAP-148, which was shown to be effective against Methicillin-resistant Staphylococcus aureus (MRSA) on tape-stripped mice skin. Unexpectedly, SAAP-148 was not effective against MRSA in our pilot study using rats with excision wounds. Therefore, we investigated factors that might have contributed to the poor efficacy of SAAP-148. Subsequently, we optimised the protocol and assessed the efficacy of SAAP-148 in an adapted rat study.Methods: We incubated 100 mu L of SAAP-148 with 1 cm(2) of a wound dressing for 1 h and determined the unabsorbed volume of peptide solution. Furthermore, 10(5) colony forming units (CFU)/mL MRSA were exposed to increasing dosages of SAAP-148 in 50% (v/v) human plasma, eschar- or skin extract or PBS. After 30 min incubation, the number of viable bacteria was determined. Next, ex vivo skin models were inoculated with MRSA for 1 h and exposed to SAAP-148. Finally, excision wounds on the back of rats were inoculated with 10(7) CFU MRSA overnight and treated with SAAP-148 for 4 h or 24 h. Subsequently, the number of viable bacteria was determined.Results: Contrary to Cuticell, Parafilm and Tegaderm film, < 20% of peptide solution was recovered after incubation with gauze, Mepilex border and Opsite Post-op. Furthermore, in plasma, eschar- or skin extract > 20-fold higher dosages of SAAP-148 were required to achieve a 2-log reduction (LR) of MRSA versus SAAP-148 in PBS. Exposure of ex vivo models to SAAP-148 for 24 h resulted in a 4-fold lower LR than a 1 h or 4 h exposure period. Additionally, SAAP-148 caused a 1.3-fold lower mean LR at a load of 10(7) CFU compared to 10(5) CFU MRSA. Moreover, exposure of ex vivo excision wound models to SAAP-148 resulted in a 1.5-fold lower LR than for tape-stripped skin. Finally, SAAP-148 failed to reduce the bacterial counts in an adapted rat study.Conclusions: Several factors, such as absorption of SAAP-148 by wound dressings, components within wound exudates, re-colonisation during the exposure of SAAP-148, and a high bacterial load may contribute to the poor antimicrobial effect of SAAP-148 against MRSA in the rat model. Show less
