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JAC Advance Access published online on November 2, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm395
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

The association between biocide tolerance and the presence or absence of qac genes among hospital-acquired and community-acquired MRSA isolates

Karen Smith1, Curtis G. Gemmell2 and Iain S. Hunter1,*

1 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK 2 Department of Immunology, Division of Infection and Immunity, University of Glasgow, UK

* Corresponding author. Tel: +44-141-548-4111; E-mail: i.s.hunter{at}strath.ac.uk

Received 22 May 2007; returned 6 August 2007; revised 21 September 2007; accepted 23 September 2007


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
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 References
 
Objectives: The MBCs of three commonly used hospital biocides [containing quaternary ammonium compounds (QACs), chlorhexidine gluconate and triclosan] were determined for clinical isolates of Staphylococcus aureus, which were also screened for genes encoding Qac efflux pumps.

Methods: MBCs were determined by broth microdilution for 94 clinical isolates of S. aureus, including 38 hospital-acquired methicillin-resistant S. aureus (HA-MRSA), 25 community-associated methicillin-resistant S. aureus (CA-MRSA), 25 methicillin-susceptible S. aureus (MSSA) and 6 with intermediate resistance to vancomycin (VISA). All isolates were screened by PCR for the presence of qacA, B, C, G, H and J.

Results: Biocides had MBCs 10–1000-fold lower than the concentration recommended for use by the manufacturer. HA-MRSA isolates developed significantly enhanced tolerance to QACs following repeat exposure to subinhibitory concentrations. Ten HA-MRSA and four VISA isolates carried qacA. Two HA-MRSA isolates, one MSSA isolate and one VISA isolate carried qacC. One VISA isolate carried qacA and qacC. The CA-MRSA isolates did not carry qac genes. qacG, H and J were not detected in any HA-MRSA. Isolates with qac genes had significantly (P < 0.0001) higher MBCs for biocides containing QACs and chlorhexidine gluconate. These biocides induced expression of qac genes when assayed with a luciferase reporter.

Conclusions: Biocides commonly used in the hospital environment should be effective against clinical isolates of S. aureus if used at concentrations recommended by the manufacturer. However, isolates have the potential to develop increased tolerance to these agents and the expression of Qac efflux pumps results in isolates with a selective advantage when challenged with biocides containing QACs and chlorhexidine gluconate.

Key Words: Staphylococcus aureus , antimicrobials , efflux pumps


   Introduction
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 Introduction
Materials and methods
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Methicillin-resistant Staphylococcus aureus (MRSA) has become established as a major nosocomial pathogen infecting vulnerable individuals in hospitals and healthcare facilities worldwide.1,2 The glycopeptide vancomycin has been vitally important in the treatment of MRSA infections. However, MRSA isolates with reduced susceptibility to vancomycin have now been reported in many countries.3,4 Recently, there has also been an increasing incidence of infection by MRSA in young, otherwise healthy, community-dwelling individuals who have no association with healthcare facilities.5 Community-associated isolates of MRSA (CA-MRSA) can carry an array of genes encoding toxins, such as the Panton-Valentine leucocidin toxin,6,7 which increases the virulence of these strains and intensifies the threat to public health. Therefore, there is an ongoing need to control the spread of MRSA and limit sources of infection.

Considerable efforts have been made in recent years to improve practices of infection control within hospitals, leading to the increased use of disinfectants and antiseptics.8 Quaternary ammonium compounds (QACs), cationic biocides (such as chlorhexidine) and biocides containing the bisphenol ether, triclosan, are used widely in disinfectant preparations in healthcare settings to decontaminate surfaces, disinfect the hands of hospital personnel and treat patients colonized by S. aureus. Despite the implementation of standard practices for infection control, current measures have failed to control the spread of MRSA,9 which has been isolated from numerous locations in hospitals including catheters and disinfectant soap dispensers.10,11 This has raised concerns that, as for antibiotics, intensive exposure of hospital pathogens to biocides may result in the emergence of resistance to these agents.12,13

Efflux-mediated resistance to QACs and cationic biocides has been reported in staphylococcal isolates from numerous sources. Clinical isolates of S. aureus carry a number of plasmid-borne qac genes, qacA, B and C, which encode proton-motive force-dependent export pumps.14,15 The QacA pump confers resistance to a broad range of structurally diverse hydrophobic compounds including QACs, intercalating dyes (including ethidium bromide) and cationic biocides.16 QacB is also able to transport QACs and intercalating dyes from the cell, but mediates limited resistance to divalent cations.17 QacC can expel QACs and ethidium bromide.16–19 Substrate specificity varies between QacA and QacB due to a single amino acid substitution at position 323, where the presence of an acidic residue in QacA is essential for high levels of resistance to compounds containing divalent cations.19 In the absence of substrate, the 188-residue QacR protein represses transcription of qacA/B by binding to a site downstream from the qacA/B promoter and overlapping its transcription start site.19,20 When a substrate for the pump is present, it binds to the QacR protein, causing a conformational change and its release from its binding site, allowing transcription of the gene.21

Other plasmid-borne qac genes, qacG, H and J, have been identified in food-borne and equine and bovine veterinary isolates of S. aureus.22–24 The frequency of these genes in clinical isolates of S. aureus or the effect of expression of the various Qac pumps on tolerance to biocides is not known. The purpose of this study was to determine the frequency of qacA, B and C, G, H and J in CA-MRSA, hospital-acquired MRSA (HA-MRSA) and clinical isolates with intermediate susceptibility to vancomycin (VISA). The effect that carriage of these genes has on tolerance to biocides used commonly in the hospital environment and whether these biocides can induce expression of the qacA/B genes were also investigated.


   Materials and methods
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 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
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Bacterial strains

Bacterial strains were provided by the Scottish MRSA Reference Laboratory (Stobhill Hospital, Glasgow, UK). Ninety-four clinical strains of S. aureus were selected from a large library of clones and subclones based on differences in their PFGE banding patterns. There were 38 HA-MRSA isolates, 25 CA-MRSA isolates, 25 methicillin-susceptible S. aureus isolates (MSSA) and 6 isolates with intermediate resistance to vancomycin (VISA). Two VISA strains were isolated in Scotland, two originated in the USA and two were isolated in Japan. HA-MRSA, CA-MRSA and MSSA isolates were maintained on tryptone soy agar (Oxoid, Basingstoke, UK) and were incubated under aerobic conditions at 37°C for 24 h. VISA isolates were maintained on Mueller–Hinton agar (Oxoid, Basingstoke, UK) containing a subinhibitory concentration of vancomycin. S. aureus strains were stored in 80% (v/v) glycerol at –70°C and freshly subcultured before each experiment.

Biocides

Commonly used hospital biocides were obtained in commercial preparations. These were: Trigene, a product containing a mixture of the QACs (alkyl dimethyl benzyl ammonium chloride and didecyl dimethyl ammonium chloride); Mediscrub, containing 1% (w/v) triclosan; and the cationic biocide MediHex-4, containing 4% (w/v) chlorhexidine gluconate (all supplied by Medichem International, Queensborough, Kent).

MBCs of biocides

MBCs of the three biocides were determined by serial 2-fold dilution of the biocides in Mueller–Hinton broth (Oxoid, Basingstoke, UK) supplemented with 2% (w/v) NaCl.25 Each dilution was inoculated with 2 x 103 cfu of overnight culture in fresh Mueller–Hinton broth. The cultures were incubated at 37°C with shaking for 24 h. An aliquot of 100 µL of each culture was used to inoculate a Petri dish of Mueller–Hinton agar supplemented with 2% (w/v) NaCl. The plates were incubated at 37°C for a further 24 h and colony counts were performed. The MBC was calculated as the concentration of biocide that produced 99.9% killing of cells. The MBC of each biocide for all 94 strains of S. aureus was determined using this method. Each dilution series was performed in triplicate and the entire experiment was repeated once. The results were analysed using Prism® software (GraphPad Software, Inc.) and a one-way ANOVA was used to compare the MBC results for HA-MRSA, CA-MRSA and VISA isolates.

Increased tolerance

A single colony of each strain was cultured for 24 h in Mueller–Hinton broth supplemented with 2% (w/v) NaCl. A dilution of biocide 1/10 MBC was prepared in Mueller–Hinton broth and inoculated with 2 x 103 cfu of overnight culture. The cultures were incubated at 37°C for 24 h with shaking. After 24 h, 100 µL was removed from each culture and used to inoculate a Petri dish of Mueller–Hinton agar [supplemented with 2% (w/v) NaCl]. Plates were incubated at 37°C for 24 h and colony counts were performed. An aliquot of 20 µL was also removed from each culture and added to a dilution of biocide that was 2-fold greater than the original concentration. These cultures were then incubated for a further 24 h at 37°C with shaking. The process was repeated until the cells had been exposed to increasingly higher concentrations of biocides, up to 1000-fold greater than the MBC.

Template for PCR

Two colonies of each strain were inoculated into 500 µL of sterile water, and the suspension was heated to 100°C for 15 min. The tube was then centrifuged at 10 000 g for 2 min and the supernatant containing the bacterial DNA was removed and used in the PCR.

PCR of qac genes

PCR was carried out for detection of qacA, B and C using primer sequences published previously.15 A single primer pair amplified qacA and B: the DNA sequences of these two genes differ in only seven bases. Primer sequences were designed for the detection of qacG, H and J (Table 1).


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  		Table 1. Sequences of PCR primers for qacA/B, qacC, qacG, qacH and qacJ

 
The volume of each reaction in the PCR was 50 µL. Each reaction contained 5 µL of 10x Expand High Fidelity buffer (without MgCl2) (Roche, Lewes, UK), 4 µL of MgCl2 at a final concentration of 2 mM (Roche, Lewes, UK), 8 µL of dNTPs (1.25 mM), 1 µL (50 pmol) of forward primer, 1 µL (50 pmol) of reverse primer and 20 µL of sterile H2O. Template bacterial DNA (10 µL) was added, and a negative control was included containing 10 µL of sterile water instead of template DNA. Finally, 1 U of Expand High Fidelity Enzyme mix (Roche, Lewes, UK), containing thermostable Taq DNA polymerase and Tgo DNA polymerase with proof-reading activity, was added to each reaction. The cycling conditions were as follows: DNA denaturation at 94°C for 3 min, followed by 25 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min. This was completed with an extension step of 72°C for 5 min. PCR products were analysed by electrophoresis on a 2% (w/v) agarose/1x TBE gel.

Cloning and sequencing of PCR products

The amplified qacA/B PCR products were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Southampton, UK) and cloned using the Novagen pT7Blue-3 Perfectly Blunt® Cloning Kit (Merck Biosciences, Nottingham, UK). Insert-containing vectors were isolated using the GFXTM Micro Plasmid Prep Kit (Amersham, Little Chalfont, UK) and sequenced with the BigDye Terminator Kit (ABI Biosystems, Foster City, CA, USA) on an ABI 3730 sequencer (ABI Biosystems) to determine whether isolates carried the qacA or B gene. Sequence analysis and alignments were carried out using BioEdit software (www.mbio.ncsu.edu/BioEdit/bioedit.html) and additional searches for related sequences were performed online via the National Centre for Biotechnology BLAST server (www.ncbi.nlm.nih.gov/BLAST).

Detection of biocide induction of QacR-regulated genes by luciferase assay

The biosensing reporter plasmid, pQacLux, was used to determine whether any of the hospital biocides used in this study induced expression of QacR-regulated genes. pQacLux carries the regulatory region and the divergent coding sequence of qacR from the staphylococcal multiresistance plasmid pSK1, coupled to the modified bacterial luciferase operon luxCDABE from Photorhabdus luminescens.26 Competent cells of the non-pathogenic S. aureus strain RN4220 were prepared and transformed with 0.5 µg of pQacLux DNA by electroporation, as described previously.27 One colony of RN4220/pQacLux was used to inoculate 5 mL of L-broth containing 0.5% (w/v) glucose and 10 µg/mL chloramphenicol (pQacLux carries chloramphenicol resistance) and incubated for 18 h at 37°C with shaking. Following incubation, 100 µL of this culture was diluted in 5 mL of fresh L-broth containing 0.5% glucose (w/v) and 10 µg/mL chloramphenicol and grown until the OD reached 0.06. An aliquot of 190 µL of this culture was then combined with 10 µL of a dilution of Trigene, MediHex-4 or Mediscrub and added to the wells of a white-walled transparent-bottomed 96-well plate (Greiner Bio-one, Gloucestershire). The plate was incubated at 37°C at 200 rpm and light emission and OD400 were measured every 30 min on a luminometer (BMG LUMIstar* Galaxy, Offenberg, Germany) for 6 h. Each reaction was carried out in triplicate. Cells with no biocide (positive control) and a negative control containing media only were included for reference. The entire experiment was repeated once. The maximum relative light value was divided by the OD of the cells at each time point.


   Results
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 Materials and methods
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Discussion
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MBCs of biocides

The MBCs of Trigene, MediHex-4 and Mediscrub for 38 HA-MRSA isolates, 25 CA-MRSA isolates, 6 VISA isolates (exposed to vancomycin) and 25 MSSA isolates were determined by broth microdilution. The MBC varied between isolates and differed between the four groups of CA-MRSA, HA-MRSA, VISA and MSSA isolates.

The manufacturer suggests that a 0.1% (w/v) solution of Trigene is used to disinfect surfaces. In this study, all isolates had MBCs of <0.01% (w/v) Trigene (Figure 1). CA-MRSA isolates showed a mean MBC of 0.0032% Trigene, whereas HA-MRSA isolates had a mean MBC of 0.0069% and VISA strains had a mean MBC of 0.0086%. MSSA isolates had a mean MBC of 0.0036% Trigene. The mean MBC of Trigene for CA-MRSA and MSSA isolates was significantly lower than the mean MBC of HA-MRSA isolates (P < 0.0001), whereas VISA isolates had a mean MBC that was significantly higher than that for HA-MRSA isolates (P < 0.01).


Figure 1
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  		Figure 1. Comparison of the mean MBC of Trigene for HA-MRSA, CA-MRSA, VISA and MSSA isolates. ***P < 0.0001, **P < 0.01. Error bars indicate the standard deviation within each group.

 
MediHex-4 is recommended for use without dilution, giving a final concentration of chlorhexidine gluconate of 4% (w/v). CA-MRSA isolates had a mean MBC of 0.0004%, HA-MRSA isolates had a mean MBC of 0.0033%, VISA strains had a mean MBC of 0.0024% and MSSA isolates had a mean MBC of 0.00037% (Figure 2). The mean MBC for VISA isolates was lower than the mean MBC for HA-MRSA isolates but this difference was not statistically significant. CA-MRSA and MSSA isolates had a significantly lower (P < 0.05 and P < 0.01, respectively) mean MBC of MediHex-4 than HA-MRSA or VISA isolates.


Figure 2
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  		Figure 2. Comparison of the mean MBC of MediHex-4 for HA-MRSA, CA-MRSA, VISA and MSSA isolates. **P < 0.01, *P < 0.05. Error bars indicate the standard deviation within each group.

 
Mediscrub is recommended for use at a final concentration of 1% (w/v) triclosan. CA-MRSA isolates had a mean MBC of 0.00037%, HA-MRSA isolates had a mean MBC of 0.001%, VISA strains had a mean MBC of 0.00055% and MSSA isolates had a mean MBC of 0.0005% Mediscrub (Figure 3). The mean MBC for HA-MRSA isolates was significantly higher than for CA-MRSA and MSSA isolates (P < 0.05). VISA isolates appeared to have a lower mean MBC than HA-MRSA isolates, and CA-MRSA and MSSA isolates seemed to have a lower mean MBC than VISA isolates but these differences were not statistically significant (P = 0.2, P = 0.37 and P = 0.25, respectively).


Figure 3
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  		Figure 3. Comparison of the mean MBC of Mediscrub for HA-MRSA, CA-MRSA, VISA and MSSA isolates. *P < 0.05. Error bars indicate the standard deviation within each group.

 
Detection of qac genes by PCR

qacA/B were found in 10 of the 38 HA-MRSA isolates screened and 4 VISA isolates. qacC was detected in two HA-MRSA isolates, one VISA isolate and one MSSA isolate (Table 2). The HA-MRSA isolates and the MSSA isolate that carried qacC did not carry the qacA/B genes. One VISA isolate carried qacA/B and qacC, although it is not known whether the genes were carried on the same plasmid. None of the clinical isolates of S. aureus produced PCR amplicons with the qacG, H and J primer sets, although control strains carrying qacG, H and J gave positive results. CA-MRSA isolates did not carry any of the qac genes detected in these PCRs.


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  		Table 2. The presence of qac genes in isolates of HA-MRSA, CA-MRSA, VISA and MSSA

 
Sequencing of PCR products

Nucleotide sequencing of the PCR products revealed that all products formed by the qacA/B primers were qacA. Comparison of the qacA gene sequences with published qacA sequence data revealed that two of the qacA-positive HA-MRSA isolates and two VISA isolates carried qacA genes with a base substitution from C-T at position 1103. This alters the amino acid sequence of the protein: an isoleucine replaces a threonine at residue 368. This may have an impact on the function of the protein and more importantly the specificity of the pump. QacA is known to utilize a larger number of substrates and has a higher affinity for divalent cations than QacB. This is due to a difference in amino acid at codon 323 where qacB encodes Ala and qacA encodes Asp.17 Therefore, the proteins encoded by qacA in these four clinical isolates could potentially have an altered specificity to other QacA pumps of isolates carrying ‘wild-type’ qacA. To test this, the MBC of each biocide for these four strains was compared with all other strains that carried qacA, using an unpaired two-tailed t-test. There was no significant difference in MBCs of Mediscrub. The MBCs of Trigene and MediHex-4 were higher for the four isolates that carried qacA with the base substitution but this difference was not statistically significant.

MBC and qac PCR

Isolates were grouped according to the presence/absence of qac genes (as defined by PCR) and results were analysed using the unpaired two-tailed t-test. Isolates carrying qac genes had significantly higher MBCs of Trigene (Figure 4) (P < 0.0001) and MediHex-4 (Figure 5) (P < 0.0001) than those without qac genes. There was no significant difference in the MBC of Mediscrub for isolates carrying qac genes and those without (P = 0.08).


Figure 4
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  		Figure 4. Comparison of mean MBC of Trigene for S. aureus strains carrying qac genes and those without qac genes. Error bars represent the standard deviation between strains. ***P < 0.0001.

 


Figure 5
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  		Figure 5. Comparison of mean MBC of MediHex-4 for S. aureus strains carrying qac genes and those without qac genes. Error bars represent the standard deviation between strains. ***P < 0.0001.

 
Increased tolerance to biocides following exposure to sublethal concentrations

Following contact with low levels of biocide, isolates were gradually exposed to increasing concentrations of the agents. All isolates that carried qac genes showed a significant increase in mean MBC after continued exposure to Trigene (P < 0.05). With MediHex-4, isolates that carried qac genes appeared to show an increase in mean MBC following exposure to increased concentrations of the biocide; however, this increase was not statistically significant (P = 0.08). Following repeated exposure to increasing concentrations of Mediscrub, there was no statistically significant increase in MBC in isolates carrying qac genes (P = 0.1). There was no significant difference in the initial and final MBC, following increased exposure to Trigene, MediHex-4 and Mediscrub, in qac-negative isolates (P = 0.1, P = 0.2 and P = 0.2, respectively).

Detection of induction of QacR-regulated genes by luciferase

pQacLux carries the qacR-qacA intergenic region fused to the luxCDABE operon encoding the luminescent protein luciferase. In the presence of a substrate of QacR, the qacR-qacA intergenic region on pQacLux will be transcribed along with the luciferase protein and light should be emitted. Biocides were added to RN4220/pQacLux cells and the level of light emission was measured. The maximum relative light units at each time point were divided by the OD for each of the biocides tested. The addition of subinhibitory concentrations of Trigene and MediHex-4 produced a significant increase in the level of light emitted from the cells in comparison with a biocide-free control (P < 0.01) (Figure 6). The addition of a subinhibitory concentration of Mediscrub to RN4220/pQacLux cells produced no significant increase in light emission from cells in comparison with the biocide-free control. This would suggest that Trigene and MediHex-4 induce the expression of qacA/B and are likely substrates for these efflux pumps.


Figure 6
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  		Figure 6. Biocide induction of the expression of QacR-regulated genes coupled with luciferase. Error bars represent the standard deviation between triplicate samples. **P < 0.01.

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
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The increasing prevalence of MRSA infections in hospitals worldwide has lead to greater public awareness of the threat of nosocomial infection and increased efforts to control this problem using suitable hygiene measures. Biocides are an essential part of infection control strategies employed in the hospital environment. In this study, the efficacy of three biocides commonly used in hospitals was tested for 38 clinical isolates of HA-MRSA, 25 CA-MRSA, 6 VISA and 25 MSSA isolates. All isolates had MBCs of Trigene, MediHex-4 and Mediscrub of 10–1000-fold lower than concentrations recommended for use by the manufacturers. This would suggest that, if these biocides are used in accordance with the manufacturers’ instructions, 100% of bacteria should be killed. In clinical practice a problem may arise when biocides are used incorrectly, in dirty situations where surfaces are not cleaned of organic matter prior to using a biocide or ‘topping up’ biocides leading to the use of subinhibitory concentrations. In the hospital environment bacteria grow in biofilms on surfaces, which have been shown to afford the cells a 10–1000-fold higher tolerance of antimicrobials, and may be a contributing factor to failure of disinfection.28

The potential for biocide-selected cross-resistance to clinically important antibiotics is the subject of some discussion in the literature.29,30 In this study, HA-MRSA isolates displayed a significantly higher MBC of Trigene, MediHex-4 and Mediscrub than MSSA isolates. This would suggest that there is a correlation between antibiotic resistance and biocide tolerance. Although a definitive link between antibiotic and biocide resistance has not been established, it has been observed that the MBC of QACs and chlorhexidine was significantly higher for clinical isolates of MRSA than MSSA.31 Our study reinforces this link in S. aureus and the issue of biocide–antibiotic co-resistance should no longer be ignored.

Screening of the 94 S. aureus isolates revealed that 10 HA-MRSA and 4 VISA isolates carried the qacA gene. Two HA-MRSA isolates, one MSSA isolate and one VISA isolate carried the qacC gene. One VISA isolate carried qacA and qacC genes. HA-MRSA isolates that carried qacA or qacC had higher MBCs of Trigene and MediHex-4 than isolates without the genes. The isolates carrying qac genes also developed increased tolerance to Trigene following exposure to increasing concentrations of this biocide. This suggests that carriage of qac genes confers a selective advantage on these isolates in the presence of Trigene and MediHex-4 and may contribute to their survival after repeated exposure in the hospital environment to Trigene. Only one MSSA isolate carried qacC. HA-MRSA isolates had significantly higher MBCs of Trigene and MediHex-4 (P < 0.0001 and P < 0.01, respectively) than MSSA isolates and this may be due to the higher frequency of carriage of qac genes in these isolates.

VISA isolates had the highest MBC of Trigene and those carrying qac genes also displayed increased tolerance to this biocide. Such VISA isolates have the potential to survive treatment with diluted biocides and cause infection in the hospital environment. Carriage of two qac genes (qacA and qacC) in the case of one VISA isolate may afford added protection from a wider range of substrates than carriage of a single efflux pump gene. It is imperative that infection control practices restrict the spread of VISA strains as there may be therapeutic failures in clinical practice with vancomycin.

The CA-MRSA isolates included in this study did not carry qac genes. This may be due to the reduced selective pressure from biocides that these isolates experience in the community setting compared with hospital strains.

None of the isolates carried the qacG, H or J genes. These genes have been reported previously in S. aureus isolated from food production areas and animals in Norway, where they have been shown to increase tolerance to QACs.22–24 From the results of this study, the qacG, H and J genes do not seem to pose a problem among Scottish clinical isolates of S. aureus at the moment. However, if carriage of these genes provides isolates with a selective advantage for biocide tolerance, then their appearance is a distinct possibility and must be monitored.

Mayer et al.14 examined the frequency of qac genes in 297 European MRSA isolates and found that 63% carried qacA/B and 6.4% carried qacC. The frequency of qacA/B among European isolates is considerably higher than the frequency found in our study. This may be due to the larger number of isolates screened in the European study, differences in epidemic strains selected for each study or variation in the type of biocides used regularly, applying different selective pressures on MRSA isolates in each region.

Nucleotide sequencing revealed that all qacA/B-positive isolates carried qacA. The multidrug efflux protein QacA is able to utilize more than 30 cationic lipophilic antimicrobial compounds that belong to 11 distinct chemical classes, whereas QacB has a more limited substrate range.16 In the hospital environment, bacteria will encounter multiple classes of antimicrobial agents. Therefore, it is beneficial for isolates to carry the gene that encodes QacA which can extrude a larger number of toxic compounds than QacB, increasing the chances of survival. Four of the isolates carried qacAs that encoded a QacA with an amino acid substitution. The amino acid substitution in the sequences of QacA and QacB imposes a significant effect on the specificity of the efflux pump. Therefore, the altered QacA found in isolates in this study may be expected to alter efflux. Isolates carrying the altered qacA gene displayed no significant difference in MBC of Mediscrub but had higher MBCs of Trigene and MediHex-4 than isolates with the ‘wild-type’ qacA. However, the difference was not statistically significant.

The continued exposure of bacteria to residual levels of biocides in the hospital environment is causing concern.12,13 This study has shown that clinical isolates of S. aureus including HA-MRSA, MSSA, CA-MRSA and VISA strains have MBCs of the commonly used hospital biocides Trigene, MediHex-4 and Mediscrub of 10–1000-fold less than the concentrations recommended for use by the manufacturer. However, HA-MRSA isolates had the ability to develop significantly increased tolerance to Trigene following repeated exposure to this agent. This may suggest that repeated exposure of S. aureus to subinhibitory concentrations of this biocide in the hospital environment could enhance tolerance. HA-MRSA and VISA isolates frequently carried qac efflux pump genes, which significantly increased (P < 0.0001) the MBC of Trigene and MediHex-4 for these isolates compared with isolates that did not carry qac genes.16 Trigene and MediHex-4 were found to induce the expression of the genes encoding the QacA/B efflux pumps, which confirms that these biocides are likely substrates. This suggests that in the presence of these biocides, efflux-mediated increased tolerance has the potential to develop. If biocides are used at concentrations recommended for use by the manufacturer in the hospital environment, then S. aureus isolates should be killed, as even the increased tolerance displayed in isolates failed to develop into complete resistance. However, the presence of qac genes in the clinical S. aureus population and their ability to develop increased tolerance highlights the importance of effective and rigorous infection cleaning and infection control strategies and the use of biocides at concentrations recommended by the manufacturer.


   Funding
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
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 References
 
K. S. received a 3 year scholarship from the Carnegie Trust of the Universities of Scotland.


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None to declare.


   Acknowledgements
 
We would like to thank staff at the Scottish MRSA Reference Laboratory for providing the S. aureus isolates included in this study. K. S. would like to thank the Carnegie Trust for providing a scholarship.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
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 Funding
 Transparency declarations
 References
 
1 . Fluit AC, Wielders CL, Verhoef J, et al. Epidemiology and susceptibility of 3,051 Staphylococcus aureus isolates from 25 university hospitals participating in the European SENTRY study. J Clin Microbiol (2001) 39:3727–32.[Abstract/Free Full Text]

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