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Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Staphylococcus aureus isolates from bacteraemia and nasal colonisation
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JGAR-204; No. of Pages 5 Journal of Global Antimicrobial Resistance xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Short Communication
Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Staphylococcus aureus isolates from bacteraemia and nasal colonisation ˜ oz-Gallego a, Lucia Infiesta a, Esther Viedma a,b, Irene Mun Dafne Perez-Montarelo a,b, Fernando Chaves a,b,* a b
Servicio de Microbiologı´a Clı´nica, Hospital Universitario 12 de Octubre, Madrid, Spain Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 September 2015 Received in revised form 17 November 2015 Accepted 18 November 2015
Chlorhexidine and mupirocin have been increasingly used in healthcare facilities to eradicate methicillin-resistant Staphylococcus aureus (MRSA) carriage. The aim of this study was to determine the prevalence and mechanisms of chlorhexidine and mupirocin resistance in MRSA from invasive infections and colonisation. MRSA isolates obtained from blood and nasal samples between 2012 and 2014 were analysed. Susceptibility to mupirocin was determined by disk diffusion and Etest and susceptibility to chlorhexidine by broth microdilution. The presence of mupA and qac (A/B and C) genes was investigated by PCR. Molecular typing was performed in high-level mupirocin-resistant (HLMR) isolates. Mupirocin resistance was identified in 15.6% of blood isolates (10.9% HLMR) and 15.1% of nasal isolates (12.0% HLMR). Presence of the mupA gene was confirmed in all HLMR isolates. For blood isolates, chlorhexidine minimum inhibitory concentrations (MICs) ranged from 0.125 to 4 mg/L and minimum bactericidal concentrations (MBCs) from 0.125 to 8 mg/L. In nasal isolates, chlorhexidine MICs and MBCs ranged from 0.125 to 2 mg/L. The qacA/B gene was detected in 2.2% of MRSA isolates (chlorhexidine MIC range 0.25–2 mg/L) and the qacC gene in 8.2% (chlorhexidine MIC range 0.125– 1 mg/L). The prevalence of qacC was 18.9% in HLMR isolates and 3.6% in mupirocin-susceptible isolates (P = 0.009). Most of the HLMR isolates (97.1%) belonged to ST125 clone. These results suggest that chlorhexidine has a higher potential to prevent infections caused by MRSA. In contrast, mupirocin treatment should be used cautiously to avoid the spread of HLMR MRSA. ß 2015 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Keywords: Methicillin-resistant Staphylococcus aureus MRSA Mupirocin Chlorhexidine Resistance
1. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) has become a major problem in healthcare facilities. Many strategies can be used to reduce the risk of MRSA transmission and infection. Intranasal mupirocin (MUP) and chlorhexidine (CHX) gluconate baths are widely used to decolonise MRSA carriers [1]. CHX gluconate is a hexamethylene biguanide cationic biocide compound with rapid bactericidal action against a variety of Grampositive and Gram-negative micro-organisms [2]. It has been suggested that decreased susceptibility to CHX is mediated
* Corresponding author. Present address: Servicio de Microbiologı´a Clı´nica, Hospital Universitario 12 de Octubre, Avenida de Co´rdoba, s/n, Madrid 28041, Spain. Tel.: +34 917 792 404. E-mail address: [email protected] (F. Chaves).
primarily through multidrug efflux pumps encoded by the qacA, qacB and smr (qacC) genes [3]. These genes are mainly found on plasmids and are associated with resistance to other biocides [4]. Some studies define CHX resistance on the basis of MRSA isolates possessing qac genes [5–7]. MUP (pseudomonic acid A) is a topical antibacterial agent that interferes with protein synthesis by competitively inhibiting bacterial isoleucyl-tRNA synthetase [8]. High-level MUP resistance is conferred by the mupA gene, which is carried on a plasmid that may also contain resistance determinants to other antimicrobial agents [9]. Recently, a new determinant of high-level mupirocin resistance, mupB, has been identified [10]. Both mupA and mupB are ileS genes imported from other species. A possible association between the presence of qac genes and resistance to MUP has been suggested [6,11]. Since MUP became available in the 1980s, its widespread use has been linked to increasing rates of resistance. Moreover, the
http://dx.doi.org/10.1016/j.jgar.2015.11.005 2213-7165/ß 2015 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
˜ oz-Gallego I, et al. Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Please cite this article in press as: Mun Staphylococcus aureus isolates from bacteraemia and nasal colonisation. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/ j.jgar.2015.11.005
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JGAR-204; No. of Pages 5 I. Mun˜oz-Gallego et al. / Journal of Global Antimicrobial Resistance xxx (2015) xxx–xxx
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increased use of CHX has raised concerns about the possible emergence of CHX-resistant strains. The aim of this study was to determine the prevalence and mechanisms of CHX and MUP resistance in MRSA in two epidemiological scenarios in hospitalised patients, with invasive infections represented by blood isolates and colonisation represented by nasal isolates.
analysis of electropherograms was carried out with BioNumerics software (Applied Maths, Kortrijk, Belgium). A 1.8% tolerance was used for comparison of DNA patterns, and PFGE types were defined using a similarity coefficient of 0.75. Representative isolates were analysed by multilocus sequence typing (MLST), and the sequence types (STs) were assigned using the MLST website (http://www.mlst.net).
2. Materials and methods 2.5. Statistical analysis 2.1. Hospital setting and bacterial isolates This was an observational study conducted at Hospital Universitario 12 de Octubre, a 1300-bed facility serving a population of 550,000 in southern Madrid (Spain). MRSA isolates were collected during 2012–2014 from two groups of adult patients. Group I comprised all hospitalised patients with bacteraemia (n = 64). Group II included all isolates (n = 358) obtained from nasal swab samples used to detect MRSA nasal carriage in patients who were admitted to the hospital with a previous history of MRSA infection/colonisation, or from the surveillance studies at admission to the intensive care units and haemodialysis or surgery wards. 2.2. Chlorhexidine and mupirocin susceptibility testing Isolation and identification of S. aureus were based upon standard microbiological procedures. Identification and antimicrobial susceptibility testing of blood isolates were performed using a MicroScan1 WalkAway1 System (Siemens, West Sacramento, CA). Resistance was defined according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. All isolates were screened for resistance to MUP on Mueller–Hinton agar with a 5 mg disk (Oxoid Ltd., Thermo Scientific, Waltham, MA). A zone of inhibition of 13 mm in diameter was considered to reflect MUP resistance. MUP-resistant organisms underwent minimum inhibitory concentration (MIC) analysis by the Etest strip method (bioMe´rieux, Marcy-l’E´toile, France) in order to classify in highlevel MUP resistance (MIC > 256 mg/L) or low-level MUP resistance (MIC > 1–256 mg/L). CHX susceptibility testing was performed using the broth microdilution method for 134 isolates, comprising all 64 blood isolates and a selection of 70 nasal isolates [30 high-level MUPresistant (HLMR), 10 low-level MUP-resistant (LLMR) and 30 MUPsusceptible]. CHX digluconate (Sigma–Aldrich, St Louis, MO) was prepared in sterile distilled water at a concentration of 100 mg/mL prior to further dilution in broth [12]. The concentration range was 0.125–128 mg/L. CHX resistance was defined as an MIC 4 mg/L [2]. Minimum bactericidal concentrations (MBCs) were determined by subculturing 10 mL from each well without visible bacterial growth on blood agar plates (Soria Melguizo, Madrid, Spain). After 48 h of incubation at 37 8C, the dilution yielding three colonies or fewer was scored as the MBC. S. aureus isolate ATCC 29213 was used for quality control. 2.3. Detection of mecA, mupA and qac genes All isolates were confirmed as MRSA by PCR detection of the mecA gene. PCR was also performed on all HLMR and LLMR isolates to detect the plasmid-associated ileS2 gene (mupA) [13]. The presence of qac (A/B and C) genes was determined by PCR using previously published primers [6]. 2.4. Molecular typing Molecular typing was performed on a selection of HLMR isolates by pulsed-field gel electrophoresis (PFGE). Computer-assisted
Data are represented as percentages. Statistical analysis was performed using IBM SPSS Statistics for Windows v.20.0 (IBM Corp., Armonk, NY).
3. Results MUP resistance was identified in 15.6% (10/64) of blood isolates [3 (4.7%) LLMR and 7 (10.9%) HLMR] and in 15.1% (54/358) of nasal isolates [11 (3.1%) LLMR and 43 (12.0%) HLMR]. Presence of the mupA gene was confirmed in all HLMR isolates; none of the LLMR isolates were positive for the mupA gene. CHX reduced susceptibility was found in 1.6% (1/64) of blood isolates (MIC = 4 mg/L and MBC = 8 mg/L). None of the MRSA nasal isolates showed MICs 4 mg/L. Of the 134 isolates tested, all had CHX MICs 4 mg/L (range 0.125–4 mg/L; MIC50 and MIC90, 1 mg/ L) and MBCs 8 mg/L (range 0.125–8 mg/L; MBC50 and MBC90, 1 and 2 mg/L, respectively) (Fig. 1). Of the 64 blood isolates, all had CHX MICs 4 mg/L (range 0.125–4 mg/L; MIC50 and MIC90, 0.5 and 1 mg/L, respectively) and MBCs 8 mg/L (range 0.125– 8 mg/L; MBC50 and MBC90, 1 and 2 mg/L, respectively) (Fig. 1). Of the 70 nasal isolates, all had CHX MICs and MBCs that were 2 mg/ L (range 0.125–2 mg/L). The MIC50 and MIC90 values were 1 mg/L and the MBC50 and MBC90 values were 1 and 2 mg/L, respectively (Fig. 1). Among the 134 MRSA isolates, 3 isolates (2.2%) harboured the qacA/B gene, all from nasal samples. The CHX MIC ranged from 0.25 to 2 mg/L. The qacC gene was detected in 11 isolates (8.2%), including 1 from blood and 10 from nasal samples. The CHX MIC of these isolates ranged from 0.125 to 1 mg/L. One nasal isolate harboured qacA/B and qacC genes and its CHX MIC and MBC were 1 and 2 mg/L, respectively. The MRSA blood isolate that had a CHX MIC of 4 mg/L did not harbour qacA/B and qacC genes. The possible relationship between MIC and MBC distribution of CHX and the presence of qac genes was analysed (Supplementary Fig. S1). The distribution was similar both in qacpositive and qac-negative MRSA isolates. Furthermore, the relationship between CHX susceptibility, as measured by MIC/ MBC and by the presence of qac genes, and MUP susceptibility was investigated (Table 1). qac genes were detected in 21.6% (8/ 37) of HLMR isolates, 15.4% (2/13) of LLMR isolates and 3.6% (3/ 84) of MUP-susceptible isolates. The presence of qacC was more frequent in HLMR isolates (18.9%; 7/37) than in MUP-susceptible isolates (3.6%; 3/84) (P = 0.009). In addition, the presence of qacC was 14.3% (10/70) in nasal isolates and 1.6% (1/64) in blood isolates (P = 0.009). Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.jgar.2015.11.005. Thirty-five HLMR isolates available for PFGE (30 from nasal samples and 5 from blood) were grouped into two types (Fig. 2). One of them grouped 34 (97.1%) of HLMR isolates (all 5 blood isolates and 29 nasal isolates). Nine representative isolates belonging to this PFGE type were identified by MLST as ST125. The presence of qac genes was observed in seven isolates belonging to the major clone.
˜ oz-Gallego I, et al. Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Please cite this article in press as: Mun Staphylococcus aureus isolates from bacteraemia and nasal colonisation. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/ j.jgar.2015.11.005
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Fig. 1. Distribution of chlorhexidine minimum inhibitory concentrations (MICs) (A, C, E) and minimum bactericidal concentrations (MBCs) (B, D, F) of 134 methicillinresistant Staphylococcus aureus (MRSA) isolates (A, B), 64 blood isolates (C, D) and 70 nasal isolates (E, F).
Table 1 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) distributions of chlorhexidine and the presence of qac genes in methicillinresistant Staphylococcus aureus (MRSA) isolates according to mupirocin (MUP) susceptibility. Type of sample (n)
MUP susceptibility (n)
Chlorhexidine
qac genes
MIC (mg/L)
MBC (mg/L)
Range
MIC50
MIC90
Range
MBC50
MBC90
All samples (134)
HLMR (37) LLMR (13) MUP-susceptible (84)
0.125–1 0.25–2 0.125–4
1 1 1
1 1 1
0.125–2 0.25–2 0.125–8
1 1 1
2 2 2
A/B (1), C (7) A/B (2), C (1) C (3)
Blood samples (64)
HLMR (7) LLMR (3) MUP-susceptible (54)
0.125–1 0.25–0.5 0.125–4
1 0.5 0.5
1 0.5 1
0.125–1 0.25–0.5 0.125–8
1 0.5 1
1 0.5 2
None None C (1)
Nasal samples (70)
HLMR (30) LLMR (10) MUP-susceptible (30)
0.125–1 0.5–2 0.5–2
1 1 1
1 1 2
0.125–2 0.5–2 0.5–2
1 1 1
2 2 2
A/B (1), C (7) A/B (2), C (1) C (2)
HLMR, high-level mupirocin-resistant; LLMR, low-level mupirocin-resistant; MIC50/90, MIC for 50% and 90% of the isolates, respectively; MBC50/90, MBC for 50% and 90% of the isolates, respectively.
˜ oz-Gallego I, et al. Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Please cite this article in press as: Mun Staphylococcus aureus isolates from bacteraemia and nasal colonisation. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/ j.jgar.2015.11.005
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Fig. 2. Dendrogram containing pulsed-field gel electrophoresis (PFGE) patterns of 35 high-level mupirocin-resistant methicillin-resistant Staphylococcus aureus (HLMR MRSA) isolates.
4. Discussion The emergence of MUP and CHX resistance among MRSA isolates causing infections or colonisation has implications for prevention and control efforts. In this study, susceptibility to MUP and CHX in clinical (blood) and colonisation (nasal swab) samples was analysed. HLMR was detected in 10.9% and 12.0% of blood and nasal isolates, respectively. Most of these isolates belonged to a dominant MRSA ST125 clone in our institution. This transmissible mupirocin resistance raises concern about its spread as mupirocin usage becomes more widespread for nasal decolonisation of MRSA and for the treatment of superficial S. aureus skin infections [14]. The widespread use of antiseptics such as CHX in the prevention of dissemination of MRSA between patients and carriers by handwashing and body showers has increased concern about antiseptic resistance. Different methods have been proposed to detect reduced susceptibility to CHX [5,6,12]. One is based on the broth microdilution method [12], and although there is no clear definition of biocide resistance, in staphylococci CHX resistance is often defined as an MIC 4 mg/L [2]. Another method is based on detection of qacA/B and qacC genes as surrogate markers of CHX resistance [5,6]. In the current study, only one isolate (0.7%) was detected by the broth microdilution method as having reduced susceptibility to CHX (MIC = 4 mg/L), and 9.7% of MRSA isolates carried qac genes (2.2% qacA/B and 8.2% qacC). No correlation between the two methods was found. The only isolate with reduced susceptibility to CHX did not carry qac genes and, on the other hand, isolates carrying qac genes did not reveal high MICs and MBCs. These controversial results have also been observed in other works [15–18]. However, other studies have reported reduced susceptibility to CHX in strains carrying qacA/B genes [11] and also in strains carrying qacC genes [19]. A recent report demonstrated that although MICs (measured by broth microdilution) from qac-positive and -negative strains were identical,
qac-positive strains could survive after exposure to 2% CHX for up to 5 min, but not qac-negative strains [17]. A possible explanation was that exposure to low concentrations of CHX over an extended period of time may overwhelm the efflux ability of the Qac pumps to protect the bacterium [17]. This could help to explain the results of two recent clinical studies that used CHX in MRSA carriers for topical decolonisation therapy [5,19]. The authors demonstrated that strains carrying qacA/B [20] and the combination of qacA/B and LLMR in MRSA isolates significantly increased the risk of persistent MRSA carriage after decolonisation therapy [5]. Further investigation is necessary to define the best method to detect CHX resistance and to predict failure of decolonisation. We also investigated the possible relationship between MUP and CHX resistance. In this study, the MIC and MBC distributions of CHX were similar in LLMR, HLMR and MUP-susceptible isolates. However, a statistical association between carriage of mupA and qacC was observed. The qacC gene can be found in either small non-conjugative plasmids or large conjugative plasmids both in S. aureus and coagulase-negative staphylococci [21]. Some reports have also demonstrated a relationship between CHX resistance genes and other antimicrobial resistance genes in staphylococci [2,22]. In addition, a recent study demonstrated an association between carriage of mupA and qacA/B genes [11]. Further studies are needed to prove whether these biocide efflux-mediated resistance genes and the mupA gene are co-located on the same plasmid and whether the presence of these genes can increase the chance of microbe survival. In summary, reduced susceptibility to CHX was not prevalent in MRSA isolates in our hospital. Consequently, these results suggest that CHX has a higher potential to prevent the infection caused by MRSA. Nevertheless, as CHX continues to be widely used, surveillance of CHX resistance and a better understanding of
˜ oz-Gallego I, et al. Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Please cite this article in press as: Mun Staphylococcus aureus isolates from bacteraemia and nasal colonisation. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/ j.jgar.2015.11.005
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resistance mechanisms are required. In contrast, MUP treatment should be used cautiously to avoid the spread of HLMR MRSA. Funding This work was supported by Plan Nacional de I+D+i and Instituto de Salud Carlos III, Subdireccio´n General de Redes y Centros de Investigacio´n Cooperativa, Ministerio de Economı´a y Competitividad, Spanish Network for Research in Infectious Diseases [REIPI RD12/0015 and PI12/01205], co-financed by the European Development Regional Fund (ERDF), ‘A way to achieve Europe’. Competing interests None declared. Ethical approval Not required. Acknowledgment The authors thank Mercedes Murcia for technical assistance. References [1] Ammerlaan HS, Kluytmans JA, Wertheim HF, Nouwen JL, Bonten MJ. Eradication of methicillin-resistant Staphylococcus aureus carriage: a systematic review. Clin Infect Dis 2009;48:922–30. [2] Horner C, Mawer D, Wilcox M. Reduced susceptibility to chlorhexidine in staphylococci: is it increasing and does it matter? J Antimicrob Chemother 2012;67:2547–59. [3] Noguchi N, Hase M, Kitta M, Sasatsu M, Deguchi K, Kono M. Antiseptic susceptibility and distribution of antiseptic-resistance genes in methicillinresistant Staphylococcus aureus. FEMS Microbiol Lett 1999;172:247–53. [4] Noguchi N, Nakaminami H, Nishijima S, Kurokawa I, So H, Sasatsu M. Antimicrobial agent susceptibilities and antiseptic resistance gene distribution among methicillin-resistant Staphylococcus aureus isolates from patients with impetigo and staphylococcal scalded skin syndrome. J Clin Microbiol 2006;44:2119–25. [5] Lee AS, Macedo-Vinas M, Franc¸ois P, Renzi G, Schrenzel J, Vernaz N, et al. Impact of combined low-level mupirocin and genotypic chlorhexidine resistance on persistent methicillin-resistant Staphylococcus aureus carriage after decolonization therapy: a case–control study. Clin Infect Dis 2011;52:1422–30. [6] Lee H, Lim H, Bae IK, Yong D, Jeong SH, Lee K, et al. Coexistence of mupirocin and antiseptic resistance in methicillin-resistant Staphylococcus aureus isolates from Korea. Diagn Microbiol Infect Dis 2013;75:308–12.
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[7] Schlett CD, Millar EV, Crawford KB, Cui T, Lanier JB, Tribble DR, et al. Prevalence of chlorhexidine-resistant methicillin-resistant Staphylococcus aureus following prolonged exposure. Antimicrob Agents Chemother 2014;58:4404–10. [8] Morton TM, Johnston JL, Patterson J, Archer GL. Characterization of a conjugative staphylococcal mupirocin resistance plasmid. Antimicrob Agents Chemother 1995;39:1272–80. [9] Patel JB, Gorwitz RJ, Jernigan JA. Mupirocin resistance. Clin Infect Dis 2009;49:935–41. [10] Seah C, Alexander DC, Louie L, Simor A, Low DE, Longtin J, et al. MupB, a new high-level mupirocin resistance mechanism in Staphylococcus aureus. Antimicrob Agents Chemother 2012;56:1916–20. [11] Lu Z, Chen Y, Chen W, Liu H, Song Q, Hu X, et al. Characteristics of qacA/Bpositive Staphylococcus aureus isolated from patients and a hospital environment in China. J Antimicrob Chemother 2015;70:653–7. [12] Morrissey I, Oggioni MR, Knight D, Curiao T, Coque T, Kalkanci A, et al. Evaluation of epidemiological cut-off values indicates that biocide resistant subpopulations are uncommon in natural isolates of clinically-relevant microorganisms. PLOS ONE 2014;9:e86669. [13] Anthony RM, Connor AM, Power EG, French GL. Use of the polymerase chain reaction for rapid detection of high-level mupirocin resistance in staphylococci. Eur J Clin Microbiol Infect Dis 1999;18:30–4. [14] Chaves F, Garcı´a-Martı´nez J, de Miguel S, Otero JR. Molecular characterization of resistance to mupirocin in methicillin-susceptible and -resistant isolates of Staphylococcus aureus from nasal samples. J Clin Microbiol 2004;42:822–4. [15] Johnson JG, Saye EJ, Jimenez-Truque N, Soper N, Thomsen I, Talbot TR, et al. Frequency of disinfectant resistance genes in pediatric strains of methicillinresistant Staphylococcus aureus. Infect Control Hosp Epidemiol 2013;34: 1326–7. [16] McDanel JS, Murphy CR, Diekema DJ, Quan V, Kim DS, Peterson EM, et al. Chlorhexidine and mupirocin susceptibilities of methicillin-resistant Staphylococcus aureus from colonized nursing home residents. Antimicrob Agents Chemother 2013;57:552–8. [17] Mc Gann P, Milillo M, Kwak YI, Quintero R, Waterman PE, Lesho E. Rapid and simultaneous detection of the chlorhexidine and mupirocin resistance genes qacA/B and mupA in clinical isolates of methicillin-resistant Staphylococcus aureus. Diagn Microbiol Infect Dis 2013;77:270–2. [18] Furi L, Ciusa ML, Knight D, Di Lorenzo V, Tocci N, Cirasola D, et al. Evaluation of reduced susceptibility to quaternary ammonium compounds and bisbiguanides in clinical isolates and laboratory-generated mutants of Staphylococcus aureus. Antimicrob Agents Chemother 2013;57:3488–97. [19] Longtin J, Seah C, Siebert K, McGeer A, Simor A, Longtin Y, et al. Distribution of antiseptic resistance genes qacA, qacB, and smr in methicillin-resistant Staphylococcus aureus isolated in Toronto, Canada, from 2005 to 2009. Antimicrob Agents Chemother 2011;55:2999–3001. [20] Batra R, Cooper BS, Whiteley C, Patel AK, Wyncoll D, Edgeworth JD. Efficacy and limitation of a chlorhexidine-based decolonization strategy in preventing transmission of methicillin-resistant Staphylococcus aureus in an intensive care unit. Clin Infect Dis 2010;50:210–7. [21] Costa SS, Viveiros M, Amaral L, Couto I. Multidrug efflux pumps in Staphylococcus aureus: an update. Open Microbiol J 2013;7:59–71. [22] Oggioni MR, Rosado Coelho J, Furi L, Knight DR, Viti C, Orefici G, et al. Significant differences characterise the correlation coefficients between biocide and antibiotic susceptibility profiles in Staphylococcus aureus. Curr Pharm Des 2015;21:2054–7.
˜ oz-Gallego I, et al. Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Please cite this article in press as: Mun Staphylococcus aureus isolates from bacteraemia and nasal colonisation. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/ j.jgar.2015.11.005
JGAR-204; No. of Pages 5 Journal of Global Antimicrobial Resistance xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Short Communication
Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Staphylococcus aureus isolates from bacteraemia and nasal colonisation ˜ oz-Gallego a, Lucia Infiesta a, Esther Viedma a,b, Irene Mun Dafne Perez-Montarelo a,b, Fernando Chaves a,b,* a b
Servicio de Microbiologı´a Clı´nica, Hospital Universitario 12 de Octubre, Madrid, Spain Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 September 2015 Received in revised form 17 November 2015 Accepted 18 November 2015
Chlorhexidine and mupirocin have been increasingly used in healthcare facilities to eradicate methicillin-resistant Staphylococcus aureus (MRSA) carriage. The aim of this study was to determine the prevalence and mechanisms of chlorhexidine and mupirocin resistance in MRSA from invasive infections and colonisation. MRSA isolates obtained from blood and nasal samples between 2012 and 2014 were analysed. Susceptibility to mupirocin was determined by disk diffusion and Etest and susceptibility to chlorhexidine by broth microdilution. The presence of mupA and qac (A/B and C) genes was investigated by PCR. Molecular typing was performed in high-level mupirocin-resistant (HLMR) isolates. Mupirocin resistance was identified in 15.6% of blood isolates (10.9% HLMR) and 15.1% of nasal isolates (12.0% HLMR). Presence of the mupA gene was confirmed in all HLMR isolates. For blood isolates, chlorhexidine minimum inhibitory concentrations (MICs) ranged from 0.125 to 4 mg/L and minimum bactericidal concentrations (MBCs) from 0.125 to 8 mg/L. In nasal isolates, chlorhexidine MICs and MBCs ranged from 0.125 to 2 mg/L. The qacA/B gene was detected in 2.2% of MRSA isolates (chlorhexidine MIC range 0.25–2 mg/L) and the qacC gene in 8.2% (chlorhexidine MIC range 0.125– 1 mg/L). The prevalence of qacC was 18.9% in HLMR isolates and 3.6% in mupirocin-susceptible isolates (P = 0.009). Most of the HLMR isolates (97.1%) belonged to ST125 clone. These results suggest that chlorhexidine has a higher potential to prevent infections caused by MRSA. In contrast, mupirocin treatment should be used cautiously to avoid the spread of HLMR MRSA. ß 2015 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Keywords: Methicillin-resistant Staphylococcus aureus MRSA Mupirocin Chlorhexidine Resistance
1. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) has become a major problem in healthcare facilities. Many strategies can be used to reduce the risk of MRSA transmission and infection. Intranasal mupirocin (MUP) and chlorhexidine (CHX) gluconate baths are widely used to decolonise MRSA carriers [1]. CHX gluconate is a hexamethylene biguanide cationic biocide compound with rapid bactericidal action against a variety of Grampositive and Gram-negative micro-organisms [2]. It has been suggested that decreased susceptibility to CHX is mediated
* Corresponding author. Present address: Servicio de Microbiologı´a Clı´nica, Hospital Universitario 12 de Octubre, Avenida de Co´rdoba, s/n, Madrid 28041, Spain. Tel.: +34 917 792 404. E-mail address: [email protected] (F. Chaves).
primarily through multidrug efflux pumps encoded by the qacA, qacB and smr (qacC) genes [3]. These genes are mainly found on plasmids and are associated with resistance to other biocides [4]. Some studies define CHX resistance on the basis of MRSA isolates possessing qac genes [5–7]. MUP (pseudomonic acid A) is a topical antibacterial agent that interferes with protein synthesis by competitively inhibiting bacterial isoleucyl-tRNA synthetase [8]. High-level MUP resistance is conferred by the mupA gene, which is carried on a plasmid that may also contain resistance determinants to other antimicrobial agents [9]. Recently, a new determinant of high-level mupirocin resistance, mupB, has been identified [10]. Both mupA and mupB are ileS genes imported from other species. A possible association between the presence of qac genes and resistance to MUP has been suggested [6,11]. Since MUP became available in the 1980s, its widespread use has been linked to increasing rates of resistance. Moreover, the
http://dx.doi.org/10.1016/j.jgar.2015.11.005 2213-7165/ß 2015 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
˜ oz-Gallego I, et al. Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Please cite this article in press as: Mun Staphylococcus aureus isolates from bacteraemia and nasal colonisation. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/ j.jgar.2015.11.005
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increased use of CHX has raised concerns about the possible emergence of CHX-resistant strains. The aim of this study was to determine the prevalence and mechanisms of CHX and MUP resistance in MRSA in two epidemiological scenarios in hospitalised patients, with invasive infections represented by blood isolates and colonisation represented by nasal isolates.
analysis of electropherograms was carried out with BioNumerics software (Applied Maths, Kortrijk, Belgium). A 1.8% tolerance was used for comparison of DNA patterns, and PFGE types were defined using a similarity coefficient of 0.75. Representative isolates were analysed by multilocus sequence typing (MLST), and the sequence types (STs) were assigned using the MLST website (http://www.mlst.net).
2. Materials and methods 2.5. Statistical analysis 2.1. Hospital setting and bacterial isolates This was an observational study conducted at Hospital Universitario 12 de Octubre, a 1300-bed facility serving a population of 550,000 in southern Madrid (Spain). MRSA isolates were collected during 2012–2014 from two groups of adult patients. Group I comprised all hospitalised patients with bacteraemia (n = 64). Group II included all isolates (n = 358) obtained from nasal swab samples used to detect MRSA nasal carriage in patients who were admitted to the hospital with a previous history of MRSA infection/colonisation, or from the surveillance studies at admission to the intensive care units and haemodialysis or surgery wards. 2.2. Chlorhexidine and mupirocin susceptibility testing Isolation and identification of S. aureus were based upon standard microbiological procedures. Identification and antimicrobial susceptibility testing of blood isolates were performed using a MicroScan1 WalkAway1 System (Siemens, West Sacramento, CA). Resistance was defined according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. All isolates were screened for resistance to MUP on Mueller–Hinton agar with a 5 mg disk (Oxoid Ltd., Thermo Scientific, Waltham, MA). A zone of inhibition of 13 mm in diameter was considered to reflect MUP resistance. MUP-resistant organisms underwent minimum inhibitory concentration (MIC) analysis by the Etest strip method (bioMe´rieux, Marcy-l’E´toile, France) in order to classify in highlevel MUP resistance (MIC > 256 mg/L) or low-level MUP resistance (MIC > 1–256 mg/L). CHX susceptibility testing was performed using the broth microdilution method for 134 isolates, comprising all 64 blood isolates and a selection of 70 nasal isolates [30 high-level MUPresistant (HLMR), 10 low-level MUP-resistant (LLMR) and 30 MUPsusceptible]. CHX digluconate (Sigma–Aldrich, St Louis, MO) was prepared in sterile distilled water at a concentration of 100 mg/mL prior to further dilution in broth [12]. The concentration range was 0.125–128 mg/L. CHX resistance was defined as an MIC 4 mg/L [2]. Minimum bactericidal concentrations (MBCs) were determined by subculturing 10 mL from each well without visible bacterial growth on blood agar plates (Soria Melguizo, Madrid, Spain). After 48 h of incubation at 37 8C, the dilution yielding three colonies or fewer was scored as the MBC. S. aureus isolate ATCC 29213 was used for quality control. 2.3. Detection of mecA, mupA and qac genes All isolates were confirmed as MRSA by PCR detection of the mecA gene. PCR was also performed on all HLMR and LLMR isolates to detect the plasmid-associated ileS2 gene (mupA) [13]. The presence of qac (A/B and C) genes was determined by PCR using previously published primers [6]. 2.4. Molecular typing Molecular typing was performed on a selection of HLMR isolates by pulsed-field gel electrophoresis (PFGE). Computer-assisted
Data are represented as percentages. Statistical analysis was performed using IBM SPSS Statistics for Windows v.20.0 (IBM Corp., Armonk, NY).
3. Results MUP resistance was identified in 15.6% (10/64) of blood isolates [3 (4.7%) LLMR and 7 (10.9%) HLMR] and in 15.1% (54/358) of nasal isolates [11 (3.1%) LLMR and 43 (12.0%) HLMR]. Presence of the mupA gene was confirmed in all HLMR isolates; none of the LLMR isolates were positive for the mupA gene. CHX reduced susceptibility was found in 1.6% (1/64) of blood isolates (MIC = 4 mg/L and MBC = 8 mg/L). None of the MRSA nasal isolates showed MICs 4 mg/L. Of the 134 isolates tested, all had CHX MICs 4 mg/L (range 0.125–4 mg/L; MIC50 and MIC90, 1 mg/ L) and MBCs 8 mg/L (range 0.125–8 mg/L; MBC50 and MBC90, 1 and 2 mg/L, respectively) (Fig. 1). Of the 64 blood isolates, all had CHX MICs 4 mg/L (range 0.125–4 mg/L; MIC50 and MIC90, 0.5 and 1 mg/L, respectively) and MBCs 8 mg/L (range 0.125– 8 mg/L; MBC50 and MBC90, 1 and 2 mg/L, respectively) (Fig. 1). Of the 70 nasal isolates, all had CHX MICs and MBCs that were 2 mg/ L (range 0.125–2 mg/L). The MIC50 and MIC90 values were 1 mg/L and the MBC50 and MBC90 values were 1 and 2 mg/L, respectively (Fig. 1). Among the 134 MRSA isolates, 3 isolates (2.2%) harboured the qacA/B gene, all from nasal samples. The CHX MIC ranged from 0.25 to 2 mg/L. The qacC gene was detected in 11 isolates (8.2%), including 1 from blood and 10 from nasal samples. The CHX MIC of these isolates ranged from 0.125 to 1 mg/L. One nasal isolate harboured qacA/B and qacC genes and its CHX MIC and MBC were 1 and 2 mg/L, respectively. The MRSA blood isolate that had a CHX MIC of 4 mg/L did not harbour qacA/B and qacC genes. The possible relationship between MIC and MBC distribution of CHX and the presence of qac genes was analysed (Supplementary Fig. S1). The distribution was similar both in qacpositive and qac-negative MRSA isolates. Furthermore, the relationship between CHX susceptibility, as measured by MIC/ MBC and by the presence of qac genes, and MUP susceptibility was investigated (Table 1). qac genes were detected in 21.6% (8/ 37) of HLMR isolates, 15.4% (2/13) of LLMR isolates and 3.6% (3/ 84) of MUP-susceptible isolates. The presence of qacC was more frequent in HLMR isolates (18.9%; 7/37) than in MUP-susceptible isolates (3.6%; 3/84) (P = 0.009). In addition, the presence of qacC was 14.3% (10/70) in nasal isolates and 1.6% (1/64) in blood isolates (P = 0.009). Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.jgar.2015.11.005. Thirty-five HLMR isolates available for PFGE (30 from nasal samples and 5 from blood) were grouped into two types (Fig. 2). One of them grouped 34 (97.1%) of HLMR isolates (all 5 blood isolates and 29 nasal isolates). Nine representative isolates belonging to this PFGE type were identified by MLST as ST125. The presence of qac genes was observed in seven isolates belonging to the major clone.
˜ oz-Gallego I, et al. Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Please cite this article in press as: Mun Staphylococcus aureus isolates from bacteraemia and nasal colonisation. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/ j.jgar.2015.11.005
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Fig. 1. Distribution of chlorhexidine minimum inhibitory concentrations (MICs) (A, C, E) and minimum bactericidal concentrations (MBCs) (B, D, F) of 134 methicillinresistant Staphylococcus aureus (MRSA) isolates (A, B), 64 blood isolates (C, D) and 70 nasal isolates (E, F).
Table 1 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) distributions of chlorhexidine and the presence of qac genes in methicillinresistant Staphylococcus aureus (MRSA) isolates according to mupirocin (MUP) susceptibility. Type of sample (n)
MUP susceptibility (n)
Chlorhexidine
qac genes
MIC (mg/L)
MBC (mg/L)
Range
MIC50
MIC90
Range
MBC50
MBC90
All samples (134)
HLMR (37) LLMR (13) MUP-susceptible (84)
0.125–1 0.25–2 0.125–4
1 1 1
1 1 1
0.125–2 0.25–2 0.125–8
1 1 1
2 2 2
A/B (1), C (7) A/B (2), C (1) C (3)
Blood samples (64)
HLMR (7) LLMR (3) MUP-susceptible (54)
0.125–1 0.25–0.5 0.125–4
1 0.5 0.5
1 0.5 1
0.125–1 0.25–0.5 0.125–8
1 0.5 1
1 0.5 2
None None C (1)
Nasal samples (70)
HLMR (30) LLMR (10) MUP-susceptible (30)
0.125–1 0.5–2 0.5–2
1 1 1
1 1 2
0.125–2 0.5–2 0.5–2
1 1 1
2 2 2
A/B (1), C (7) A/B (2), C (1) C (2)
HLMR, high-level mupirocin-resistant; LLMR, low-level mupirocin-resistant; MIC50/90, MIC for 50% and 90% of the isolates, respectively; MBC50/90, MBC for 50% and 90% of the isolates, respectively.
˜ oz-Gallego I, et al. Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Please cite this article in press as: Mun Staphylococcus aureus isolates from bacteraemia and nasal colonisation. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/ j.jgar.2015.11.005
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Fig. 2. Dendrogram containing pulsed-field gel electrophoresis (PFGE) patterns of 35 high-level mupirocin-resistant methicillin-resistant Staphylococcus aureus (HLMR MRSA) isolates.
4. Discussion The emergence of MUP and CHX resistance among MRSA isolates causing infections or colonisation has implications for prevention and control efforts. In this study, susceptibility to MUP and CHX in clinical (blood) and colonisation (nasal swab) samples was analysed. HLMR was detected in 10.9% and 12.0% of blood and nasal isolates, respectively. Most of these isolates belonged to a dominant MRSA ST125 clone in our institution. This transmissible mupirocin resistance raises concern about its spread as mupirocin usage becomes more widespread for nasal decolonisation of MRSA and for the treatment of superficial S. aureus skin infections [14]. The widespread use of antiseptics such as CHX in the prevention of dissemination of MRSA between patients and carriers by handwashing and body showers has increased concern about antiseptic resistance. Different methods have been proposed to detect reduced susceptibility to CHX [5,6,12]. One is based on the broth microdilution method [12], and although there is no clear definition of biocide resistance, in staphylococci CHX resistance is often defined as an MIC 4 mg/L [2]. Another method is based on detection of qacA/B and qacC genes as surrogate markers of CHX resistance [5,6]. In the current study, only one isolate (0.7%) was detected by the broth microdilution method as having reduced susceptibility to CHX (MIC = 4 mg/L), and 9.7% of MRSA isolates carried qac genes (2.2% qacA/B and 8.2% qacC). No correlation between the two methods was found. The only isolate with reduced susceptibility to CHX did not carry qac genes and, on the other hand, isolates carrying qac genes did not reveal high MICs and MBCs. These controversial results have also been observed in other works [15–18]. However, other studies have reported reduced susceptibility to CHX in strains carrying qacA/B genes [11] and also in strains carrying qacC genes [19]. A recent report demonstrated that although MICs (measured by broth microdilution) from qac-positive and -negative strains were identical,
qac-positive strains could survive after exposure to 2% CHX for up to 5 min, but not qac-negative strains [17]. A possible explanation was that exposure to low concentrations of CHX over an extended period of time may overwhelm the efflux ability of the Qac pumps to protect the bacterium [17]. This could help to explain the results of two recent clinical studies that used CHX in MRSA carriers for topical decolonisation therapy [5,19]. The authors demonstrated that strains carrying qacA/B [20] and the combination of qacA/B and LLMR in MRSA isolates significantly increased the risk of persistent MRSA carriage after decolonisation therapy [5]. Further investigation is necessary to define the best method to detect CHX resistance and to predict failure of decolonisation. We also investigated the possible relationship between MUP and CHX resistance. In this study, the MIC and MBC distributions of CHX were similar in LLMR, HLMR and MUP-susceptible isolates. However, a statistical association between carriage of mupA and qacC was observed. The qacC gene can be found in either small non-conjugative plasmids or large conjugative plasmids both in S. aureus and coagulase-negative staphylococci [21]. Some reports have also demonstrated a relationship between CHX resistance genes and other antimicrobial resistance genes in staphylococci [2,22]. In addition, a recent study demonstrated an association between carriage of mupA and qacA/B genes [11]. Further studies are needed to prove whether these biocide efflux-mediated resistance genes and the mupA gene are co-located on the same plasmid and whether the presence of these genes can increase the chance of microbe survival. In summary, reduced susceptibility to CHX was not prevalent in MRSA isolates in our hospital. Consequently, these results suggest that CHX has a higher potential to prevent the infection caused by MRSA. Nevertheless, as CHX continues to be widely used, surveillance of CHX resistance and a better understanding of
˜ oz-Gallego I, et al. Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Please cite this article in press as: Mun Staphylococcus aureus isolates from bacteraemia and nasal colonisation. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/ j.jgar.2015.11.005
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resistance mechanisms are required. In contrast, MUP treatment should be used cautiously to avoid the spread of HLMR MRSA. Funding This work was supported by Plan Nacional de I+D+i and Instituto de Salud Carlos III, Subdireccio´n General de Redes y Centros de Investigacio´n Cooperativa, Ministerio de Economı´a y Competitividad, Spanish Network for Research in Infectious Diseases [REIPI RD12/0015 and PI12/01205], co-financed by the European Development Regional Fund (ERDF), ‘A way to achieve Europe’. Competing interests None declared. Ethical approval Not required. Acknowledgment The authors thank Mercedes Murcia for technical assistance. References [1] Ammerlaan HS, Kluytmans JA, Wertheim HF, Nouwen JL, Bonten MJ. Eradication of methicillin-resistant Staphylococcus aureus carriage: a systematic review. Clin Infect Dis 2009;48:922–30. [2] Horner C, Mawer D, Wilcox M. Reduced susceptibility to chlorhexidine in staphylococci: is it increasing and does it matter? J Antimicrob Chemother 2012;67:2547–59. [3] Noguchi N, Hase M, Kitta M, Sasatsu M, Deguchi K, Kono M. Antiseptic susceptibility and distribution of antiseptic-resistance genes in methicillinresistant Staphylococcus aureus. FEMS Microbiol Lett 1999;172:247–53. [4] Noguchi N, Nakaminami H, Nishijima S, Kurokawa I, So H, Sasatsu M. Antimicrobial agent susceptibilities and antiseptic resistance gene distribution among methicillin-resistant Staphylococcus aureus isolates from patients with impetigo and staphylococcal scalded skin syndrome. J Clin Microbiol 2006;44:2119–25. [5] Lee AS, Macedo-Vinas M, Franc¸ois P, Renzi G, Schrenzel J, Vernaz N, et al. Impact of combined low-level mupirocin and genotypic chlorhexidine resistance on persistent methicillin-resistant Staphylococcus aureus carriage after decolonization therapy: a case–control study. Clin Infect Dis 2011;52:1422–30. [6] Lee H, Lim H, Bae IK, Yong D, Jeong SH, Lee K, et al. Coexistence of mupirocin and antiseptic resistance in methicillin-resistant Staphylococcus aureus isolates from Korea. Diagn Microbiol Infect Dis 2013;75:308–12.
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[7] Schlett CD, Millar EV, Crawford KB, Cui T, Lanier JB, Tribble DR, et al. Prevalence of chlorhexidine-resistant methicillin-resistant Staphylococcus aureus following prolonged exposure. Antimicrob Agents Chemother 2014;58:4404–10. [8] Morton TM, Johnston JL, Patterson J, Archer GL. Characterization of a conjugative staphylococcal mupirocin resistance plasmid. Antimicrob Agents Chemother 1995;39:1272–80. [9] Patel JB, Gorwitz RJ, Jernigan JA. Mupirocin resistance. Clin Infect Dis 2009;49:935–41. [10] Seah C, Alexander DC, Louie L, Simor A, Low DE, Longtin J, et al. MupB, a new high-level mupirocin resistance mechanism in Staphylococcus aureus. Antimicrob Agents Chemother 2012;56:1916–20. [11] Lu Z, Chen Y, Chen W, Liu H, Song Q, Hu X, et al. Characteristics of qacA/Bpositive Staphylococcus aureus isolated from patients and a hospital environment in China. J Antimicrob Chemother 2015;70:653–7. [12] Morrissey I, Oggioni MR, Knight D, Curiao T, Coque T, Kalkanci A, et al. Evaluation of epidemiological cut-off values indicates that biocide resistant subpopulations are uncommon in natural isolates of clinically-relevant microorganisms. PLOS ONE 2014;9:e86669. [13] Anthony RM, Connor AM, Power EG, French GL. Use of the polymerase chain reaction for rapid detection of high-level mupirocin resistance in staphylococci. Eur J Clin Microbiol Infect Dis 1999;18:30–4. [14] Chaves F, Garcı´a-Martı´nez J, de Miguel S, Otero JR. Molecular characterization of resistance to mupirocin in methicillin-susceptible and -resistant isolates of Staphylococcus aureus from nasal samples. J Clin Microbiol 2004;42:822–4. [15] Johnson JG, Saye EJ, Jimenez-Truque N, Soper N, Thomsen I, Talbot TR, et al. Frequency of disinfectant resistance genes in pediatric strains of methicillinresistant Staphylococcus aureus. Infect Control Hosp Epidemiol 2013;34: 1326–7. [16] McDanel JS, Murphy CR, Diekema DJ, Quan V, Kim DS, Peterson EM, et al. Chlorhexidine and mupirocin susceptibilities of methicillin-resistant Staphylococcus aureus from colonized nursing home residents. Antimicrob Agents Chemother 2013;57:552–8. [17] Mc Gann P, Milillo M, Kwak YI, Quintero R, Waterman PE, Lesho E. Rapid and simultaneous detection of the chlorhexidine and mupirocin resistance genes qacA/B and mupA in clinical isolates of methicillin-resistant Staphylococcus aureus. Diagn Microbiol Infect Dis 2013;77:270–2. [18] Furi L, Ciusa ML, Knight D, Di Lorenzo V, Tocci N, Cirasola D, et al. Evaluation of reduced susceptibility to quaternary ammonium compounds and bisbiguanides in clinical isolates and laboratory-generated mutants of Staphylococcus aureus. Antimicrob Agents Chemother 2013;57:3488–97. [19] Longtin J, Seah C, Siebert K, McGeer A, Simor A, Longtin Y, et al. Distribution of antiseptic resistance genes qacA, qacB, and smr in methicillin-resistant Staphylococcus aureus isolated in Toronto, Canada, from 2005 to 2009. Antimicrob Agents Chemother 2011;55:2999–3001. [20] Batra R, Cooper BS, Whiteley C, Patel AK, Wyncoll D, Edgeworth JD. Efficacy and limitation of a chlorhexidine-based decolonization strategy in preventing transmission of methicillin-resistant Staphylococcus aureus in an intensive care unit. Clin Infect Dis 2010;50:210–7. [21] Costa SS, Viveiros M, Amaral L, Couto I. Multidrug efflux pumps in Staphylococcus aureus: an update. Open Microbiol J 2013;7:59–71. [22] Oggioni MR, Rosado Coelho J, Furi L, Knight DR, Viti C, Orefici G, et al. Significant differences characterise the correlation coefficients between biocide and antibiotic susceptibility profiles in Staphylococcus aureus. Curr Pharm Des 2015;21:2054–7.
˜ oz-Gallego I, et al. Chlorhexidine and mupirocin susceptibilities in methicillin-resistant Please cite this article in press as: Mun Staphylococcus aureus isolates from bacteraemia and nasal colonisation. J Global Antimicrob Resist (2015), http://dx.doi.org/10.1016/ j.jgar.2015.11.005