Increased tolerance to chlorhexidine following prolonged exposure in staphylococcus species isolated from the skin and mucosae of dogs

This study aimed to investigate if canine staphylococci isolates could develop tolerance to chlorhexidine digluconate after long-term exposure. Staphylococci (Staphylococcus pseudintermedius and coagulase-negative staphylococci, methicillin-susceptible and methicillin-resistant), with and without genotypic chlorhexidine resistance, were investigated for phenotypic chlorhexidine tolerance by determining the MBC (minimal bactericidal concentration) at various time points. The testing was performed as follows: determination of MBC for 30 minutes and 24 hours (MBC 24h-1); exposure of isolates for seven days to concentrations of chlorhexidine equal to 1/2 MBC 24h-1; determination of MBC for 24 h after the first week (MBC 24h-2); exposure of isolates for seven days to concentrations of chlorhexidine equal to 1/2 MBC 24h-2 and rest in Mueller–Hinton broth for seven days; determination of MBC for 24 h after the second week (MBC 24h-3). The MBC for 30 minutes ranged between 16–32 µg/ml compared to the MBC 24h-1 which was between 1–8 µg/ml. The MBCs for 24 h dropped from 8 µg/ml to 0.5 µg/ml for isolate 1, from 2 µg/ml to1 µg/ml for isolate 6 and from 2 µg/ml to 0.5 µg/ml for isolate 8 after being exposed for seven days to concentrations of chlorhexidine equal to 1/2 MBC 24h-1. For one CoNS (coagulase-negative staphylococci), the MBC 24h-2 increased four times from 1 µg/ml (MBC 24h-1) to 4 µg/ml and dropped again to 1 µg/ml after the second week. These results suggest that continuous exposure to chlorhexidine could lead to the selection of chlorhexidine-tolerant staphylococci that could withstand concentrations used during routine decolonisation procedures.