Hospital-acquired infection is a major cause of morbidity and mortality, and regimes to prevent infection are crucial in infection control. These include the decolonization of vulnerable patients with methicillin-resistant Staphylococcus aureus (MRSA) carriage using antiseptics, including chlorhexidine and octenidine. Concern has been raised, however, regarding the possible development of biocide resistance. In this study, we assembled a panel of S. aureus isolates, including isolates collected before the development of chlorhexidine and octenidine and isolates, from a major hospital trust in the United Kingdom during a period when the decolonization regimes were altered. We observed significant increases in the MIC and minimum bactericidal concentration (MBC) of chlorhexidine in isolates from periods of high usage of chlorhexidine. Isolates with increased MICs and MBCs of octenidine rapidly emerged after octenidine was introduced in the trust. There was no apparent cross-resistance between the two biocidal agents. A combination of variable-number tandem repeat (VNTR) analysis, PCR for qac genes, and whole-genome sequencing was used to type isolates and examine possible mechanisms of resistance. There was no expansion of a single strain associated with decreased biocide tolerance, and biocide susceptibility did not correlate with carriage of qac efflux pump genes. Mutations within the NorA or NorB efflux pumps, previously associated with chlorhexidine export, were identified, however, suggesting that this may be an important mechanism of biocide tolerance. We present evidence that isolates are evolving in the face of biocide challenge in patients and that changes in decolonization regimes are reflected in changes in susceptibility of isolates.

22Hospital acquired infection is a major cause of morbidity and mortality and regimes to 23 prevent infection are crucial in infection control. These include decolonisation of at-risk 24 patients of carriage of MRSA which is commonly achieved by protocols that include the use 25 of chlorhexidine, or octenidine as biocidal agents. There is however no standardised single 26 decolonisation regime agreed upon in the UK or other countries and protocols include a 27 variety of active agents. Antibiotic resistant bacteria cause major problems in hospital 28 medicine and concern has been raised regarding the development of biocide resistance 29 which would cause decolonisation regimes to become unreliable. In this study, we 30 assembled a panel of isolates of S. aureus including isolates collected before the 31 development of chlorhexidine and octenidine through to a contemporaneous panel of 32 isolates from a major hospital trust in the UK during a period when the decolonisation regime 33 was altered. We observed significant increases in the MIC and MBC of chlorhexidine in 34isolates collected from periods of high usage of chlorhexidine. No isolates had a significantly 35 altered MIC or MBC of octenidine apart from those collected after octenidine was introduced 36into the trust where isolates with four-fold decreases in susceptibility emerged. There was no 37 suggestion of cross-resistance between the two biocidal agents. A combination of VNTR, 38 PCR for qac genes and whole genome sequencing was used to type isolates and examine 39 possible mechanisms of resistance. The typing data showed no expansion of a single strain 40 was associated with decreased biocide tolerance and isolates with increased chlorhexidine 41 MIC and MBCs were found from different clonal complexes; CC8, CC22 and CC30. Biocide 42 susceptibility did not correlate with carriage of qac efflux pump genes -carriage of qacA and 43 qacB was detected but, with one exception was restricted to isolates of CC8. Analysis of 44 genome sequence data for closely related pairs of strains with differential biocide 45 susceptibility revealed no common mutations or carriage of accessory elements that 46 correlated with biocide tolerance. Mutations with the NorA or NorB efflux pumps, previously 47 associated with chlorhexidine export were identified suggesting this may be an important 48 mechanism of biocide tolerance. The clinical relevance of decreased biocide tolerance in 49 terms of efficacy of decolonisation therapies remains to be established but we present 50 evidence here that isolates are evolving in the face of biocide challenge in patients and that 51 changes to decolonisation regimes are reflected in changes in susceptibility of isolates. More 52 work is needed to assess the impact of these changes to ensure effective and robust 53 decolonisation protocols remain in place. 54 55 56 not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.or...