Meticillin-resistant Staphylococcus aureus (MRSA) is globally recognised as a major causative organism of hospital acquired infection (HAI) and continues to present many challenges for infection prevention and control. Once established within hospitals and healthcare centers, the control of spread of MRSA and therapy is difficult due to resistance to otherwise effective antimicrobials. Government initiatives in the United Kingdom (UK) have led to considerable investments in improving infection control practices, with emphasis on improving hand hygiene compliance of healthcare professionals and hospital environmental cleanliness to control the spread and limit the source of MRSA and other HAIs. This has resulted in the subsequent increase in disinfectant and antiseptic usage containing, quaternary ammonium compounds (QACs), cationic biocides such as chlorhexidine and the bisphenol ether, triclosan, for decontamination of surfaces and disinfection of skin. Thus, there is serious concern that as with antibiotic resistance, continual and intensive exposure of MRSA (and other hospital pathogens) to biocides, may result in the emergence of resistance to these agents with further detrimental consequences and substantial burden for prevention, treatment and control of hospital infections.
MRSA carry a number of plasmid-borne qac genes, predominantly qacA, qacB and smr that encode resistance to commonly used antiseptics and disinfectants in hospitals, nursing homes and other healthcare establishments. The proteins encoded by qacA and qacB mediate efflux via active transport; QacA multidrug exporter mediates resistance to monovalent, divalent cationic and lipophilic antimicrobial compounds, whilst the closely related export protein QacB mediates lower levels of resistance to divalent cations.
In this research a “snapshot” study of hospital strains of MRSA stored at the Hospital Infection Research Laboratory (HIRL), City Hospital, Birmingham, was carried out to determine the prevalence and distribution of qacAB in these isolates and determine a possible association between presence of these genes and biocide resistance. The intercalating dye, ethidium bromide (EtBr) is a substrate for many S. aureus multi-drug resistant (MDR) efflux pumps and was used in the present study as a marker for detection of efflux pump activity. Previous studies have reported that MRSA strains with an MIC of ≥ 64 mg/L to EtBr have qacAB, however, the present study used a lower baseline value of ≥ 32 mg/L resistance to EtBr to capture any isolates with low MICs that may have qacAB and may be missed.
Initially 3,400 MRSA strains collected between October 2002 and October 2006 were screened to identify and select isolates with ≥ 32 mg/L resistance to EtBr. A second MRSA collection stored at the Antimicrobial Chemotherapy Laboratory, City Hospital, Birmingham, comprised 63 isolates that showed MICs of ≥ 64mg/L, were also included in the study. At this stage the study set (Set A) comprised 112 isolates with varying MIC to EtBr ranging from ≥ 32 mg/L to 256 mg/L. At a later date an additional 400 strains were screened from the same stored collection to include strains with lower MICs, i.e. < 32 mg/L. Thus a total of 336 isolates with varying levels of resistance to EtBr were studied.
PCR was carried out on all 336 isolates for detection o qacAB, smr, qacG, qacH and qacJ to determine the presence and prevalence of the genes. Set A isolates positive for qacAB were further investigated to differentiate between qacA and qacB. Restriction digestion using the restriction enzyme Rsa1 was carried out on PCR products followed by PCR using specific primers for detection of the two genes. Urease activity and neomycin sensitivity were used as a means of basic characterization applied to all the study isolates. A select number of samples negative for qacA and qacB were typed using spa typing.
Transfer studies involving, conjugation, plate mating and transformation on selected strains were carried out to attempt transfer of qacAB using the marker EtBr from a strain of MRSA with an MIC of ≥ 256 mg/L to EtBr and qacAB positive to a strain with < 32mg/L MIC to EtBr and lacking qacAB. Unfortunately, conjugation experiments were not successful in this study. Plasmid curing experiments were also carried out to demonstrate loss of plasmid through continual passaging onto selective plates.
A variety of antiseptics and disinfectants are used in hospitals for prevention of HAIs. The present study was limited to carrying out minimum bactericidal concentration (MBC) determinations and MIC of four commonly used hospital biocides against randomly selected strains. The strains reflected ranges of MICs to EtBr and presence or absence of qacAB. These experiments, determined the efficacy of the biocides tested, to effectively destroy MRSA on skin and environment when used in healthcare settings.
The results suggest that in the majority of strains showing high MICs to EtBr i.e. ≥ 64 mg/L, qacAB is present and thus, the mechanism of resistance to biocides may be attributed to an efflux protein pump encoded by these genes. Following restriction digestion of qacAB positive strains, with the restriction enzyme Rsa1, 81 of the 112 qacAB positive strains tested positive for qacA, i.e. 90% and 9 (11%) for qacB. The predominant prevalence of the qacA gene indicates that most of these strains are likely to be resistant to organic cationic biocides and intercalating dyes such as EtBr and acriflavine. However, the results of the MIC and MBC determinations carried out on a selection of biocides commonly used in the healthcare environment implies that the four biocides tested are likely to be 99.9% effective at killing the majority of isolates in this study set. However, five isolates demonstrated MBCs to chlorhexidine of > 32 mg/L. Chlorhexidine is a compound that is widely used in hand hygiene and surgical antisepsis products, and the results suggest that solutions containing this compound would be ineffective in removing MRSA from the hands of healthcare workers and skin sites if used.
Molecular spa typing of selected samples negative for qacAB revealed that Endemic-MRSA (EMRSA) type 15 was the most frequent spa type identified in this study, followed by EMRSA-16 and EMRSA-1. Three strains identified jointly as EMRSA-3 and EMRSA-1. One strain identified as the Berlin clone.
With regards to the challenges presented to infection prevention and control, MRSA has the potential to develop increased tolerance to biocides commonly used in the hospital environment, due to expression of efflux pumps, although currently there is little evidence of this. Further research is required to understand and learn of the various mechanisms of resistance, supported by adherence to control of infection strategies for prevention and spread of infections in healthcare facilities.