JAC Advance Access originally published online on January 28, 2008
Journal of Antimicrobial Chemotherapy 2008 61(3):524-532; doi:10.1093/jac/dkm520
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Original research |
Frequency of biocide resistance genes, antibiotic resistance and the effect of chlorhexidine exposure on clinical methicillin-resistant Staphylococcus aureus isolates
1 Molecular Chemotherapy, Centre for Infectious Diseases, The Chancellor's Building, 49 Little France Crescent, University of Edinburgh, Edinburgh EH16 4SB, UK 2 New Royal Infirmary Edinburgh, Little France Crescent, Edinburgh EH16 4SB, UK
* Corresponding author. Tel: +44-131-2426461; Fax: +44-131-2429375; E-mail: lvali{at}staffmail.ed.ac.uk
Received 1 June 2007; returned 5 December 2007; revised 28 July 2007; accepted 9 December 2007
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Objectives: To detect genes conferring resistance to biguanides, quaternary ammonium compounds, β-lactams and fluoroquinolones in clinical methicillin-resistant Staphylococcus aureus (MRSA) and to demonstrate whether reduced susceptibility is spread clonally and if the presence of any of the detected genes links to a specific epidemic MRSA. Finally, to identify if exposure to chlorhexidine may cause reduced susceptibility to antibiotics and chlorhexidine.
Methods: In total, 120 clinical MRSA isolates were isolated. qacA/B, qacG, qacH, norA, smr and blaZ genes were amplified by PCR. MICs of eight antibiotics were determined and PFGE was used for typing. Surface disinfection and residue tests were performed for chlorhexidine and a selection of isolates.
Results: qacA/B (8.3%), qacH (3.3%), norA (36.7%), smr (44.2%) and blaZ (97.5%) were prevalent within the population but qacG was not detected. EMRSA-15 (19.2%), EMRSA-16 (15%), P3 (15%) and H (12.5%) were the most common PFGE types. Clinical isolates demonstrated various degrees of susceptibility to chlorhexidine in the surface disinfection [mean microbiocidal effect (ME) = 0–1.91] and biocide residue (mean ME = 0.29–3.74) tests. Increases in post-exposure MICs were observed in both EMRSA-16 and the susceptible S. aureus control.
Conclusions: In our study, isolates resembling PFGE type EMRSA-16 harboured more biocide resistance genes than other types. The observed reduction in susceptibility of clinical isolates to chlorhexidine may mean that a selective pressure is being exerted by residues in the clinical environment, and highlights the importance of efficacy testing on clinical strains and good infection control practices. The development of reduced microbial susceptibility to biocides represents a serious cause for concern in the clinical environment.
Keywords: qacA/B , smr , norA , blaZ , PFGE , MRSA
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Staphylococcus aureus is one of the most important pathogens that causes hospital-acquired infections.1,2 These infections include soft tissue abscess, endocarditis and bacteraemia. The fact that S. aureus is able to exhibit antibiotic resistance means that the clinical environment within a hospital may actively select resistant clones.2,3 EMRSA-15 and EMRSA-16 (where MRSA stands for methicillin-resistant S. aureus) are the two main clones that dominate the UK hospitals.4 In clinical practice, decontamination and disinfection are the most important intervention measures to prevent bacterial spread. Over the years, the use of biocides has increased5 including chlorhexidine, probably the most widely used biocide, not only in hand washing and oral products but also as a disinfectant and preservative.6
The effectiveness of chlorhexidine in preventing growth of bacterial pathogens may vary with different organisms.7,8 The efficacy of biocides against standard culture collection strains of S. aureus, Enterococcus hirae, Escherichia coli and Pseudomonas aeruginosa is tested as standard and the products that are considered acceptable are those that achieve equal to or greater than a 5 log reduction (99.999%) in numbers of the challenged organisms after 5 min of contact. However, Payne et al.9 have shown that the margin of pass was higher against laboratory culture collection than clinical strains when a short contact time of 1 min was used, and indeed in some clinical strains, resistance to chlorhexidine may develop.10–12
One of the resistance mechanisms to antiseptic and disinfectants in S. aureus is mediated by an energy-dependent export system encoded by two gene families on the basis of DNA homology and phenotypic properties; qac and smr (staphylococcal multidrug resistance, also known as qacC/D).13–15 smr encodes a protein that functions as a drug pump by an electrogenic drug/proton antiport15,16 and is usually harboured on small plasmids (<3 kb).10 qacA and B genes are closely related and differ at the nucleotide level by seven nucleotides (codon 323). They are usually harboured by large plasmids (>20 kb).14
Concomitant antibiotic and biocide resistance have been previously reported in both Gram-negative and Gram-positive bacteria.5,17 The blaZ β-lactamase gene is one of the mechanisms that confers resistance to β-lactam antibiotics in staphylococci. qacA/B and blaZ usually reside on common plasmids.15 Also, in staphylococci, the norA gene located on the chromosome encodes the fluoroquinolone efflux protein NorA. In addition to fluoroquinolones, it pumps out dyes and quaternary ammonium compounds (QACs) from the bacterial cells to the outer medium.18
At least 12 biocide resistance genes have been identified in staphylococci: qacA-qacJ, smr and norA. Although the qac genes mainly confer resistance to QACs, qacA, qacB, smr and norA confer resistance not only to cationic antiseptics (QACs) but also to biguanides.19,20
The aims of this study were to detect qacA/B, qacG, qacH, norA, smr and blaZ genes in clinical MRSA and examine if the presence of any of these genes is linked to a specific epidemic MRSA, PFGE type or antibiotic resistance pattern, and also to identify if exposure to chlorhexidine may cause reduced susceptibility to disinfectants, particularly chlorhexidine, and antibiotics.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Isolates
One hundred and twenty clinical MRSA were collected from the clinical laboratories of the New Royal Infirmary Edinburgh (NRIE) from February to April 2006 (Table 1).
|
MICs
MICs of a panel of antibiotics including ampicillin (breakpoint >2 mg/L), tetracycline (breakpoint >1 mg/L), vancomycin (breakpoint >8 mg/L), gentamicin (breakpoint >1 mg/L), oxacillin (breakpoint >2 mg/L), cefotaxime (breakpoint >4 mg/L), cefuroxime (breakpoint >4 mg/L) and ciprofloxacin (breakpoint >1 mg/L) were determined by the agar dilution method. Bacterial strains were grown overnight and diluted to 104 cfu/mL. They were inoculated on plates containing different concentrations of antibiotics (0.016–128 mg/L) with a Denley multipoint inoculator (Denley, Billinghurst, UK) to give a final concentration of 2 x 102 cfu per spot. After inoculation, the plates were incubated overnight. The results were interpreted according to recommendations of the BSAC guidelines.21 The susceptible S. aureus standard strain NCTC 6571, an EMRSA-16 strain and the Acinetobacter baumannii standard strain ATCC 19606 were added for comparison.
MICs are inadequate when antiseptics and disinfectants are being considered for the evaluation of antibacterial activity of a biocide.8,10
PCR was performed with HotStar Taq polymerase (Qiagen) according to the manufacturer's instructions and specific primers15,18,20 for mecA, norA, qacA/B, qacG, qacH, smr and blaZ genes are as follows: mecA: MecA1, 5'-GTAGAAATGACTGAACGTCCGATA-3', MecA2, 5'-CCAATTCCACATTGTTTCGGTCTAA-3'; norA+2a: 5'-GTAATACCAGTCTTGCCTGT-3' and norA-5: 5'-GTAATGGCTGGTCGTATCAT-3'; qacG-F, 5'-CAACAGAAATAATCGGAACT-3' and qacG-R, 5'-TACATTTAAGAGCACTACA-3'; qacH-F, 5'-ATAGTCAGTGAAGTAATAG-3' and qacH-R, 5'-AGTGTGATGATCCGAATGT-3'; qacH-F, 5'-CTTATATTTAGTAATAGCG-3' and qacH-R, 5'-GATCCAAAAACGTTAAGA-3'; blaZ-F, 5'-TACAACTGTAATATCGGAGGG-3' and blaZ-R, 5'-AGGAGAATAAGCAACTATATCATC-3'; smr-F, 5'-ATA-AGT-ACT-GAA-GTT-ATT-GGA-AGT-3' and smr-R, 5'-TTC-CGA-AAA-TGT-TTA-ACG-AAA-CTA-3'; and qacA/BF, 5'-GCTGCATTTATGACAATGTTTG-3' and qacA/BR, 5'-AATCCCACCTACTAAAGCAG-3'.
PCR products were sequenced by the School of Biological Sciences Sequencing service (SBCSSS), University of Edinburgh.
Pulsed-field gel electrophoresis
Clinical isolates were typed by PFGE with the CHEF-DR II electrophoresis cell after digestion of the cell lysates with SmaI restriction endonuclease enzyme.22 The running parameters were as follows: initial pulse 5 s, final pulse 40 s, at 6 V/cm for 20 h at 14°C. The gels were stained with ethidium bromide and scanned. The analysis of the gels was performed using BioNumerics software version 4.0 (Applied Maths, Ghent, Belgium). This software facilitates the development of the algorithms necessary for the comparison of profiles of isolates, based on the Dice coefficient and the hierarchic unweighted pair arithmetic average algorithm. Cluster analysis and phylogenetic trees were subsequently prepared. The numbering of MRSA was based on EMRSA-15 and EMRSA-16. The rest of the isolates were alphabetically typed.
Chlorhexidine diacetate hydrate salt (Sigma) was dissolved in sterile distilled water using magnetic stirrers and heat, and filter sterilized through a 0.22 µm filter before use. A stock solution was made and then diluted to the necessary concentrations for each test. The neutralizer solution comprised 0.75% (w/v) azolectin and 5% (v/v) Tween 80 dissolved in sterile distilled water and was autoclave sterilized before use.
Controls testing the effectiveness and toxicity of the neutralizer were performed for each new batch made against the concentrations of chlorhexidine used.8 Neutralizer toxicity was evaluated as follows: 1 mL of neutralizer was added to bacteria and left in contact for 5 min. Cells were resuspended, serially diluted and counted using the drop counting method. The number of survivors was compared with those for a control with sterile distilled water replacing the neutralizer, any difference giving an indication of the toxicity, if any, of the neutralizer. The effectiveness of the neutralizer was tested, to ensure the biocide was being quenched as desired, by addition to a mixture of bacteria and biocide, and counting after 5 min of contact time. The count was compared with a sample without biocide quenching, with sterile distilled water replacing the neutralizer. Significance of the difference between controls with water and tests of the effectiveness and toxicity of the neutralizer was determined using the t-test to establish P values.
Surface disinfection tests were performed to test the efficacy of chlorhexidine upon surface dried bacterial cultures.8 In addition to the controls detailed above, a control was performed for each sample to enable the calculation of microbiocidal effect (ME) while taking into account the effect of drying upon the cells. Washed overnight culture (10 µL; 108–109 cfu/mL) was added to the bottom of a flat-bottomed glass bottle, left to dry for 2 and 24 h and the dried cells were resuspended in 1 mL of sterile water. The mixture was serially diluted and counted using the drop counting method. By comparing counts after the addition of biocide, this control allows for calculation of the log reduction in cell number or ME after exposure to biocide as follows:
ME = number of cfu/mL of the control (biocide and neutralizer replaced by water) – number of cfu/mL after action of the biocide.
For testing the effect of chlorhexidine upon dried bacteria, 10 µL of washed culture was added to the bottom of a 28 mL flat-bottomed glass bottle and left to dry in a laminar air flow cabinet at room temperature for 2 and 24 h. After drying, 0.1 mL of 100 mg/L chlorhexidine was placed over the top of the dried cells and left in contact for 5 min. Neutralizer solution (0.9 mL) was added to stop the reaction and a sterile magnetic stirrer was used to resuspend the cells for 5 min. The neutralized cells were vortexed and serially diluted in sterile distilled water. The number of cells remaining was determined by the drop counting method.
This time the effect of biocide residues on MRSA was determined.8 The control experiments were performed as detailed above. Stock solutions of biocide were prepared in distilled water to give final concentrations of 2.5, 5, 10, 20 and 40 mg/mL. One millilitre of each solution was dispensed into the bottom of a flat-bottomed glass bottle, the excess removed and the bottles left to dry at room temperature for 1, 2, 4, 10, 24, 34 and 48 h. After drying, 20 µL of an overnight culture (108–109 cfu/mL) or 106–107 cfu was added to the bottles containing dried biocide residue. The cells were left in contact with the biocide residue for 5 min at room temperature. Neutralizer (1 mL) was added to the mixture and an aliquot was serially diluted in sterile distilled water and counted by the drop counting method.
Again, the ME was calculated by comparison with a control, where water replaced neutralizer and there was no interaction with biocide residue. The MICs for the exposed cells were also determined against the panel of antibiotics to examine the antibiotic susceptibility profiles after exposure of the cells to dried biocide residue. Aliquots of 10 µL of each of these mixtures were inoculated in 10 mL of nutrient broth and incubated at 37°C overnight. MICs were determined as described before.
Mean MEs for the surface disinfectant and biocide residue tests were calculated from three results, and P values were calculated using the t-test to determine whether differences between ME of chlorhexidine at varying time points and for different isolates were significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Minimum inhibitory concentrations
Table 2 shows the MICs for the isolates of the antibiotics tested. Of the isolates, 99.2% were resistant to ampicillin, 27.5% to gentamicin, 89.2% to oxacillin, 98.3% to cefotaxime, 90% to cefuroxime, 96.7% to ciprofloxacin and 3.3% to tetracycline. All isolates were susceptible to vancomycin.
|
Detection of resistance genes
All isolates were mecA-positive. qacA/B was identified in 10 isolates (8.3%), qacH in 4 (3.3%), norA in 44 (36.7%), smr in 53 (44.2%) and blaZ in 117 (97.5%). However, qacG was not detected (Table 2). qacA/B and smr were detected concomitantly in 4.2% (5/120) of the isolates.
Figure 1 shows there were three distinct clones consisting of 26 PFGE types. The most common PFGE types were EMRSA-15 (19.2%), EMRSA-16 (15%), P3 (15%) and H (12.5%), a close derivative of EMRSA-16.
|
Three isolates were selected for further analysis. LF26 with PFGE pattern I, at 3.3% prevalence, contained only blaZ and was resistant to most antibiotics tested except gentamicin, tetracycline and vancomycin. LF67, PFGE pattern A at 0.8% prevalence, contained the qacH, blaZ and smr genes and it was only resistant to cefotaxime, cefuroxime and tetracycline. LF93 (PFGE type EMRSA-16) at 15% prevalence was positive for norA, blaZ and smr and was resistant to all antibiotics tested excluding tetracycline and vancomycin.
Biocide resistance genes and PFGE types
Table 3 shows the percentage of each biocide resistance gene present in the most common types and demonstrates whether the presence of biocide resistance genes relates to a specific PFGE type. norA, smr and qacA/B genes were detected with higher frequency in PFGE type EMRSA-16 than in EMRSA-15. Only two (11.1%) PFGE type EMRSA-16 isolates harboured qacH.
|
Controls for surface disinfection and biocide residue tests
Comparison of counts with and without the use of a neutralizer showed that the neutralizer successfully quenched the effect of chlorhexidine at all biocide concentrations examined, with no significant difference between the counts with quenched chlorhexidine compared with a control with water alone (P > 0.05). Similarly, comparison of the counts with neutralizer compared with water alone showed that the neutralizer had no significant toxicity to the bacteria (P > 0.05).
Figure 2 shows the mean ME of chlorhexidine when calculated using a drying control, specific to each strain and each time point. This was an attempt to take account of the effect of drying upon bacterial cell survival when calculating the ME. After 24 h, chlorhexidine has a lower impact on the clinical strains (mean ME = 0–0.61) than on the control strains (mean ME = 0.57–1.91) as shown by the lower range of mean MEs observed. Figure 3 shows the mean log cfu/mL of the controls with and without drying, and indicates that drying affects bacterial cell survival at 24 h drying time. After 24 h of drying, the reduction in log cfu/mL was more pronounced for the control strains than the clinical isolates, with mean reductions of 4 log cfu/mL observed in the control strains (EMRSA-16), with a maximum mean reduction of over 2 log cfu/mL in the clinical isolates (LF93).
|
|
Biocide residue test
Figure 4 shows the mean MEs of residues of chlorhexidine (2.5 mg/mL) dried for 2, 24 and 48 h against standard strains and clinical isolates. Generally the efficacy of chlorhexidine against the isolates decreased with longer biocide residue drying times. The residues exerted a similar minimal effect on both the standard strains and clinical isolates.
|
Post-exposure MICs
The MICs for isolates were examined following exposure to chlorhexidine residues. Increases were observed in the MICs of cefotaxime, vancomycin, gentamicin, cefuroxime and oxacillin against the EMRSA-16 standard strain following 48 h of residue drying time (Table 4). There were also increases in the MICs of all tested antibiotics for the NCTC 6571 (S. aureus susceptible) strain following exposure to chlorhexidine residues that had been drying for 48 h (compared with the MICs for the strain before exposure).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
smr, norA and blaZ genes were widely distributed among S. aureus isolated from hospitals in Edinburgh. The presence of these genes provides increased tolerance to disinfecting agents15 and the opportunity for the bacteria to survive antibiotic pressure.23,24 In this study, smr was the predominant resistance gene, not qacA/B,18,25 and also the concomitant presence of qac and smr was higher than previously reported,25 although qacA/B confers resistance to a broader range of biocides than smr.15,26,27 In general, resistance genes were independently detected among different PFGE types suggesting that these determinants are not specific to certain PFGE types and that reduced susceptibility to biocides may occur in any clinical S. aureus isolate. However, isolates harbouring both qacA/B and smr genes were either PFGE EMRSA-16 or closely related. Overall, in this study, strains representing PFGE type EMRSA-16 harboured more biocide resistance genes than EMRSA-15 or other PFGE types. Of interest was LF93 (PFGE type EMRSA-16); it contained norA, blaZ and smr genes, which when expressed may confer less susceptibility to quinolones and β-lactams as well as QACs, but not necessarily to chlorhexidine as one would expect with qacA/B.
Of note is that the MICs of oxacillin, cefotaxime, cefuroxime and ciprofloxacin for the four isolates with the qacH gene were high (
128 mg/L) only when norA was also present. Similar to previous reports,14,20 all the isolates that harboured the qacA/B gene also contained the β-lactamase transposon, blaZ. The close association between qacA/B and blaZ is common and represents a reservoir of resistance genes that can be transmitted to other strains.20,26,28 However, not all isolates with blaZ contained qacA/B. The clinical implications of this are that antibiotic resistance does not necessarily encode biocide resistance; in contrast, reduced susceptibility to biocide is more likely to encode for antibiotic resistance.
There was a low efficacy of chlorhexidine against either standard strains or clinical isolates after 2 h of bacterial drying time. The efficacy was much increased against the standard strains after 24 h of bacterial drying time and increased to a lesser extent against the clinical isolates. It should be noted that drying has an effect on bacterial cell counts even without the presence of chlorhexidine, and attempts were made to take this into account.
The effectiveness of chlorhexidine residues upon bacterial suspensions decreased with longer drying times. However, even after 48 h, the residues still exerted an effect on most isolates. Because this effect was minimal, it therefore may act as a selective pressure and allow the less susceptible strains to persist in the clinical environment by incomplete eradication. Such a situation may occur in the hospital environment where exposure to low or residual concentrations of biocides may persist on surfaces leading to reduced efficacy. Although biocides when used at concentrations instructed by the manufacturers are bactericidal, concentrations that might remain on surfaces after cleaning might provide a selective pressure on microorganisms. In theory, sublethal concentrations of biocide for any given cellular target may occur at some point along this concentration gradient, providing a selective pressure for mutations in a range of cellular targets.29
The varying effect of chlorhexidine upon clinical isolates, as observed in both the surface disinfectant and biocide residue tests, is of importance as it may mean that certain isolates will have an ability to survive chlorhexidine treatment and that the use of biocides could act as a selective pressure to allow these isolates to predominate.
The increases in the MICs of all tested antibiotics for the susceptible control S. aureus strain following exposure to surface dried chlorhexidine residues is of interest as it suggests that the use of chlorhexidine in the hospital environment may be linked to increased resistance to antibiotics in previously susceptible strains. The exposure to subinhibitory doses of biocides selects for up-regulation of efflux pumps capable of transporting these compounds as well as some antibiotics out of the cell and contributes to reduced biocide susceptibility.30 It may be that the long period of surface drying of chlorhexidine leads to reduced efficacy of the biocide, thus allowing the persistence of isolates when the biocide is left as a residue.
Biocides are critical components of intervention strategies used in clinical medicine for preventing the dissemination of nosocomial infections. Reduced susceptibility to biocides and the threat this represents is a serious concern. It is important to determine the susceptibility of clinical MRSA to various biocides to assess the control and preventive measures currently implemented in hospitals.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
This study was funded by the Chief Scientist Office, Scottish Executive, St Andrews House, Edinburgh (grant CZG/2/227) on the effect of biocide usage on clinical MRSA.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
None to declare.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
1 Lowy FD. Staphylococcus aureus infections. N Engl J Med (1998) 339:520–32.
2
Amyes SGB. Treatment of staphylococcal infection. BMJ (2005) 330:976–7.
3
Amyes SGB. The rise in bacterial resistance is partly because there have been no new classes of antibiotics since the 1960s. BMJ (2000) 320:199–200.
4
Moore PCL, Lindsay JA. Molecular characterization of the dominant UK methicillin-resistant Staphylococcus aureus strains, EMRSA-15 and EMRSA-16. J Med Microbiol (2002) 51:516–21.
5 Kõljalg S, Naaber P, Mikelsaar M. Antibiotic resistance as an indicator of bacterial chlorhexidine susceptibility. J Hosp Infect (2002) 51:106–13.[CrossRef][Web of Science][Medline]
6
McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, resistance. Clin Microb Rev (1999) 12:147–79.
7 Pacheco-Fowler V, Gaonkar T, Wyer PC, et al. Antiseptic impregnated endotracheal tubes for the prevention of bacterial colonization. J Hosp Infect (2004) 57:170–4.[CrossRef][Web of Science][Medline]
8 Thomas L, Russell AD, Millard JY. Antimicrobial activity of chlorhexidine diacetate and benzalkonium chloride against Pseudomonas aeruginosa and its response to biocide residues. J Appl Microbiol (2005) 98:533–43.[CrossRef][Medline]
9 Payne DN, Babb JR, Bradley CR. An evaluation of the suitability of the European suspension test to reflect in vitro activity to antiseptics against clinically significant organisms. Lett Appl Microbiol (1999) 28:7–12.[CrossRef][Web of Science][Medline]
10 Russell AD. Bacterial resistance to disinfectants: present knowledge and future problems. J Hosp Infect (1998) 43(Suppl):S57–68.[CrossRef]
11 Kampf G, Jarosch R, Rüden H. Limited effectiveness of chlorhexidine based hand disinfectants against methicillin-resistant Staphylococcus aureus (MRSA). J Hosp Infect (1998) 38:297–303.[CrossRef][Web of Science][Medline]
12 Kampf G, Höfer M, Wendt D. Efficacy of hand disinfectants against vancomycin-resistant enterococcus in vitro. J Hosp Infect (1999) 42:143–50.[CrossRef][Web of Science][Medline]
13 Rouch DA, Cram DS, DiBerardino D, et al. Efflux-mediated antiseptic resistance gene qacA from Staphylococcus aureus: common ancestry with tetracycline- and sugar-transport proteins. Mol Microbiol (1990) 4:2051–62.[CrossRef][Web of Science][Medline]
14
Paulsen IT, Brown MH, Littlejohn TG, et al. Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity. Proc Natl Acad Sci USA (1996) 93:3630–5.
15
Anthonisen I-L, Sunde M, Steinum TM, et al. Organization of the antiseptic resistance gene qacA and Tn552-related β-lactamase genes in multidrug-resistant Staphylococcus haemolyticus strains of animal and human origins. Antimicrob Agents Chemother (2002) 46:3606–12.
16 Grinius LL, Goldberg EB. Bacterial multidrug resistance is due to a single membrane protein which functions as a drug pump. J Biol Chemother (1994) 269:29998–30004.
17
Russell AD, Tattawasart U, Millard J-Y. Possible link between bacterial resistance and use of antibiotics and biocides. Antimicrob Agents Chemother (1998) 42:2151.
18 Noguchi N, Tamura M, Narui K, et al. Frequency and genetic characterization of multidrug-resistant mutants of Staphylococcus aureus after selection with individual antiseptics and fluoroquinolones. Biol Pharm Bull (2002) 25:1129–32.[CrossRef][Web of Science][Medline]
19
Sidhu MS, Heir E, Leegaard T, et al. Frequency of disinfectant resistance genes and genetic linkage with β-lactamase transposon Tn552 among clinical staphylococci. Antimicrob Agents Chemother (2002) 46:2797–803.
20
Bjorland J, Steinum T, Kvitle B, et al. Widespread distribution of disinfectant resistance genes among staphylococci of bovine and caprine origin in Norway. J Clin Microbiol (2005) 43:4363–8.
21
Andrews JM. BSAC standardized disc susceptibility testing method (version 4). J Antimicrob Chemother (2005) 56:60–76.
22 Bannerman TL, Hancock GA, Tenover FC, et al. Pulsed-field gel electrophoresis as a replacement for bacteriophage typing of Staphylococcus aureus. J Clin Microbiol (1995) 33:551–5.[Abstract]
23
Kern WV, Oethinger M, Jellen-Ritter AS, et al. Non-target gene mutations in the development of fluoroquinolone resistance in Escherichia coli. Antimicrob Agents Chemother (2000) 44:814–20.
24
Weber MA, Piddock LJV. The importance of efflux pumps in bacterial antibiotic resistance. J Antimicrob Chemother (2003) 51:9–11.
25
Mayer S, Boos M, Beyer A, et al. Distribution of the antiseptic resistance genes qacA qacB qacC in 497 methicillin-resistant and -susceptible European isolates of Staphylococcus aureus. J Antimicrob Chemother (2001) 47:896–7.
26
Leelaporn A, Paulsen IT, Tennent JM, et al. Multidrug resistance to antiseptics and disinfectants in coagulase-negative staphylococci. J Med Microbiol (1994) 40:214–20.
27 Heir E, Sundheim G, Holck AL. The Staphylococcus qacH gene product: a new member of the SMR family encoding multidrug resistance. FEMS Microbiol Lett (1998) 163:49–56.[CrossRef][Web of Science][Medline]
28
Russell AD. Mechanisms of antimicrobial action of antiseptics and disinfectants: an increasingly important area of investigation. J Antimicrob Chemother (2002) 49:597–9.
29 Bloomfield SF. Significance of biocide usage and antimicrobial resistance in domiciliary environments. J Appl Microbiol (2002) 92:144S–157S.[CrossRef]
30
Kaatz GW, McAleese F, Seo M. Multidrug resistance in Staphylococcus aureus due to overexpression of a novel multidrug and toxin extrusion (MATE) transport protein. Antimicrob Agents Chemother (2005) 49:1857–64.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. S.M. Tom, M. W. Kruse, and R. T. Reichman Update: Methicillin-resistant Staphylococcus aureus screening and decolonization in cardiac surgery. Ann. Thorac. Surg., August 1, 2009; 88(2): 695 - 702. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L.G. Pedreira, D. M. Kusahara, W. B. de Carvalho, S. C. Nunez, and M. A. S. Peterlini Oral Care Interventions and Oropharyngeal Colonization in Children Receiving Mechanical Ventilation Am. J. Crit. Care., July 1, 2009; 18(4): 319 - 328. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Correa, A. De Paulis, S. Predari, D. O. Sordelli, and P. E. Jeric First report of qacG, qacH and qacJ genes in Staphylococcus haemolyticus human clinical isolates J. Antimicrob. Chemother., November 1, 2008; 62(5): 956 - 960. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||