Increased usage of antiseptics is associated with reduced susceptibility in clinical isolates of S. aureus

Hospital acquired infection is a major cause of morbidity and mortality and regimes to prevent infection are crucial in infection control. These include decolonisation of at-risk patients of carriage of MRSA which is commonly achieved by protocols that include the use of chlorhexidine, or octenidine as biocidal agents. There is however no standardised single decolonisation regime agreed upon in the UK or other countries and protocols include a variety of active agents. Antibiotic resistant bacteria cause major problems in hospital medicine and concern has been raised regarding the development of biocide resistance which would cause decolonisation regimes to become unreliable. In this study, we assembled a panel of isolates of S. aureus including isolates collected before the development of chlorhexidine and octenidine through to a contemporaneous panel of isolates from a major hospital trust in the UK during a period when the decolonisation regime was altered. We observed significant increases in the MIC and MBC of chlorhexidine in isolates collected from periods of high usage of chlorhexidine. No isolates had a significantly altered MIC or MBC of octenidine apart from those collected after octenidine was introduced into the trust where isolates with four-fold decreases in susceptibility emerged. There was no suggestion of cross-resistance between the two biocidal agents. A combination of VNTR, PCR for qac genes and whole genome sequencing was used to type isolates and examine possible mechanisms of resistance. The typing data showed no expansion of a single strain was associated with decreased biocide tolerance and isolates with increased chlorhexidine MIC and MBCs were found from different clonal complexes; CC8, CC22 and CC30. Biocide susceptibility did not correlate with carriage of qac efflux pump genes – carriage of qacA and qacB was detected but, with one exception was restricted to isolates of CC8. Analysis of genome sequence data for closely related pairs of strains with differential biocide susceptibility revealed no common mutations or carriage of accessory elements that correlated with biocide tolerance. Mutations with the NorA or NorB efflux pumps, previously associated with chlorhexidine export were identified suggesting this may be an important mechanism of biocide tolerance. The clinical relevance of decreased biocide tolerance in terms of efficacy of decolonisation therapies remains to be established but we present evidence here that isolates are evolving in the face of biocide challenge in patients and that changes to decolonisation regimes are reflected in changes in susceptibility of isolates. More work is needed to assess the impact of these changes to ensure effective and robust decolonisation protocols remain in place.


Abstract 22
Hospital 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 34 isolates 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 36

Introduction 57
Antiseptics, and especially chlorhexidine, have been used widely as one of the key 58 measures implemented in the control of infections caused by methicillin resistant 59 Staphylococcus aureus (MRSA) in hospitals. The most widely used approach has been to 60 use antiseptics as part of a decolonisation protocol for patients who are known to be 61 colonised with MRSA in conjunction with nasal mupirocin. However, in some units, 62 especially intensive care units, antiseptics have been used universally for washing of all 63 patients (Climo et al., 2013). 64 Chlorhexidine, which was first synthesised in 1954 (Davies et al., 1954) five and six of their study, but then decreasing in the following two years (Warren et al., In this study both screening and clinical isolates were included. The isolates were grouped 126 into four panels (Table 1)

Biocide susceptibility of isolates 167
The MIC and MBC of chlorhexidine was significantly different between the four groups. 168 Chlorhexidine MICs ranged from 0.5 µg/ml to 32 µg/ml, with a significant increase in the 169 mean MICs over time from group 1 to 3, with then a slight reduction in group 4 compared to 170 group 3 ( Figure 1). This increase in mean MIC was a result of a shift of susceptibility of most 171 isolates in the population requiring higher MICs rather than being a result of a small sub-172 population of highly resistant isolates. The MBCs of chlorhexidine showed a similar pattern, 173 the percentage of isolates with an MBC >32 µg/ml increased from group 1 to 3, with 0, 5.5 174 and 36.5% of isolates having an MBC >32 µg/ml in groups 1, 2 and 3 respectively (Figure 3). 175 The differences in MIC and MBC distributions between the groups were not likely to be 176 random according to statistical tests (chi-squared and Mann-Whitney tests both returned p 177 values <0.05 comparing groups 3 and 4 and 1 and 2). 178 The MIC and MBC of octenidine dihydrochloride were both lower than chlorhexidine and 179 ranged from 0.09375 µg/ml to 1.5 µg/ml. The MBC of octenidine was stable against isolates 180 in groups 1-3 with all isolates being inhibited by 0.0375-0.75 µg/ml. Isolates with a markedly 181 decreased susceptibility were however isolated in group 4 after introduction of this agent 182 (Figures 1 and 2). The difference between MIC and MBC values for the final group (4) and 183 other groups was statistically significant (P <0.05). 184 Interestingly, there was no correlation between susceptibility to the two agents, i.e. a raised 185 chlorhexidine MIC was not likely to be reflected by a raised octendine dihydrochloride MIC in 186 the same isolate (tested using Pearson's correlation test). 187 The changes in susceptibilty to both agents reflected usage data although it was not 188 possible to statistically analyse these changes.
VNTR analysis of the isolates revealed CC22 to be the most predominant clonal complex, 191 which is the endemic strain within the UK. The other two clonal complexes represented were 192 CC36 and CC8 (Figure 3). 193 The phylogenetic analysis failed to identify a specific clone or lineage which showed reduced 194 susceptibility to chlorhexidine (Figure 3). Interestingly, overlaying susceptibility data onto the 195 phylogeny demonstrated several occasions where isolates with the same VNTR profile 196 differed in MIC for both chlorhexidine and octenidine dihydrochloride suggesting the 197 acquisition of decreased susceptibility is not restricted to one clonal complex and that it may 198 be able to evolve independently from various strains (Figure 3).

Genomic analysis of ST22 isolates 211
Sixteen strains with related VNTR profiles from groups 3 and four and a range of 212 chlorhexidine MBCs were whole genome sequenced and mechanisms which may contribute 213 to decreased susceptiblity to chlorhexidine were identified. Figure 4 shows the phylogeny of 214 these strains based on a whole genome alignment (produced by ROARY). Comparisons of core genes attempted to identify common changes in those isolates with highest MBCs. 217 There were no common accessory genes identified or carriage of a known resistance 218 mechanism that correlates with biocide resistance. To further try and identify a tolerance 219 mechanism two pairs of strains with four fold differences in susceptibilty (by MBC) but which 220 were very closely related according to the phlogeny were compared for changes (strains 3 221 vs 7 and strain 2 vs the reference ST22; HO_5096_041, Figure 5). Analysis of these strain respectively), the two more resistant isolates both had changes in either of two homologous 224 efflux pump systems; NorA and NorB. Both have been shown to export multiple agents 225 including biocides (DeMarco et al., 2007). Strain 2 carried a SNP within norB that results in a 226 change of the NorB protein of Met314Ile. This substitution is adjacent to the predicted 227 translocation pore and may alter the capacity of this strain to export chlorhexidine compared 228 to the reference. Strain 7 had a wild-type norB allele but carried a SNP within norA resulting 229 in a change in NorA of Ala362Val. Substitution at this site has previously been shown to 230 improve efflux capacity of the protein for some substrates (Kaatz et al., 1993). None of the 231 other sequenced strains had changes within either system or in their known regulators. 232 233

Discussion 234
This study has highlighted that the increasing use of chlorhexidine and octenidine 235 dihydrochloride is associated with emergence of reduced susceptibility to each agent in S. 236 aureus. As described in previous studies (Wang et al., 2008;Liu et al., 2015) the number of 237 S. aureus and especially MRSA isolates demonstrating reduced susceptibility to 238 chlorhexidine increased over time, which in the current study was most marked when the 239 MRSA epidemic was at its peak within the UK. suggests that it is unlikely that these isolates will affect clinical efficacy, but they have 260 emerged in a real world situation suggesting there is a significant benefit which has been 261 selected in practice. Interestingly, octenidine dihydrochloride was only in use for one year 262 before there was a marked change in the susceptibility of isolates. 263 systems as a mechanism to increase efficiency of export of substrates. Mutation within the 304 structural pump protein of a multidrug efflux system was also recently shown to increase 305 efficiency of export of some substrates at the expense of others (Blair et al., 2015). The 306 substitutions within NorB (adjacent to the translocation pore) and NorA (known to alter pump 307 efficiency for fluoroquinolone export) identified here may reflect adaptation to increase 308 efficiency of chlorhexidine export. 309 There is an obvious need for more research in this area to provide better surveillance data 310 from larger populations and geographical regimes, and to understand the mechanisms of 311 action and resistance to antiseptics better. The significance and clinical impact of the 312 emergence of isolates with decreased susceptibility to decolonisation regimes remains 313 uncertain. However, the introduction of chlorhexidine and octenidine dihydrochloride has 314 changed the susceptibility of the S. aureus population compared to pre-biocide times and 315 they are therefore having an ecological impact. The consequences of this remain unclear.