Detection of macrolide and disinfectant resistance genes in clinical Staphylococcus aureus and coagulase-negative staphylococci

Background Staphylococcus aureus and Coagulase-negative staphylococci (CoNS) are a major source of infections associated with indwelling medical devices. Many antiseptic agents are used in hygienic handwash to prevent nosocomial infections by Staphylococci. Our aim was to determine the antibiotic susceptibility and resistance to quaternary ammonium compound of 46 S. aureus strains and 71 CoNS. Methods S. aureus (n = 46) isolated from auricular infection and CoNS (n = 71), 22 of the strains isolated from dialysis fluids and 49 of the strains isolated from needles cultures were investigated. Erythromycin resistance genes (ermA, ermB, ermC, msrA and mef) were analysed by multiplex PCR and disinfectant-resistant genes (qacA, qacB, and qacC) were studied by PCR-RFLP. Results The frequency of erythromycin resistance genes in S. aureus was: ermA+ 7.7%, ermB+ 13.7%, ermC+ 6% and msrA+ 10.2%. In addition, the number of positive isolates in CoNS was respectively ermA+ (9.4%), ermB+ (11.1%), ermC+ (27.4%), and msrA+ (41%). The MIC analyses revealed that 88 isolates (74%) were resistant to quaternary ammonium compound-based disinfectant benzalkonium chloride (BC). 56% of the BC-resistant staphylococcus isolates have at least one of the three resistant disinfectants genes (qacA, qacB and qacC). Nine strains (7.7%) among the CoNS species and two S. aureus strains (2%) harboured the three-qac genes. In addition, the qacC were detected in 41 strains. Conclusions Multi-resistant strains towards macrolide and disinfectant were recorded. The investigation of antibiotics and antiseptic-resistant CoNS may provide crucial information on the control of nosocomial infections.


Background
The increasing number of infections caused by oxacillinresistant staphylococci makes glycopeptide antibiotics an important choice [1]. The significant prevalence of nosocomial infections caused by multi-resistant S. aureus and coagulase-negative staphylococci (CoNS) has been documented [2,3]. These species have the ability to survive in medical devices for months [4]. Resistance to erythromycin in staphylococci is usually associated with resistance to other macrolides. Three genes (ermA, ermB, and ermC) encoding methyltransferases responsible of resistance to macrolides, lincosamides and type B streptogramins (MLS B phenotype) by modification of the ribosomal target site have been found in staphylococci [5]. The msrA gene displays another mechanism of inducible resistance to erythromycin by encoding an ATP-dependent efflux pump [6]. On the other hand, macrolide efflux is affected by a membrane protein encoded by the mef gene [7]. Antiseptic agents include various compounds with different chemical structures [8]. The widespread use of quaternary ammonium compounds (QAC) in hospitals actually contributes to the emergence of disinfectant-resistant bacteria [9,10]. Epidemiological data on antiseptic susceptibility and the distribution of resistance genes are both useful for nosocomial infection control. In several staphylococcal species, qac-resistant genes have been identified [11][12][13].
In the present study, we examined the antibiotic susceptibility and resistance to quaternary ammonium compound-based disinfectant benzalkonium chloride (BC) of 46 S. aureus strains isolated from auricular infection and 71 CoNS isolated from dialysis fluids and needles cultures. In order to examine the genetic drug resistance mechanisms, erythromycin resistance genes (ermA, ermB, and ermC) and macrolide efflux genes (msrA and mef) were analysed by PCR multiplex. In addition, quaternary ammonium resistance genes (qacA, qacB, and qacC) were detected by PCR-RFLP.

Biochemical characterization and antimicrobial susceptibility
A total of 117 clinical staphylococcal strains were isolated from Kairouan, in central of Tunisia including 46 S. aureus strains isolated from auricular infection and 71 CoNS strains isolated from dialysis fluid and needles from a dialysis service. All strains were identified using the Api ID32 Staph system (bioMérieux, Marcy l'Étoile, France) according to the manufacturer's recommendations and the results were read using an automated microbiological mini-Api (bioMérieux, Marcy l'Étoile, France). Each strain was tested for 18 antibiotics (penicillin, oxacillin, kanamycin, tobramycin, gentamicin, tetracycline, minocyclin, erythromycin, lincomycin, pristinamycin, fosfomycin, nitrofurantoin, pefloxacin, rifampicin, fusidic acid, vancomycin, teicoplanin and cotrimoxazol) using the ATB Staph system (bioMérieux, France) according to the manufacturer's specifications.

Minimum inhibitory concentration determination
The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of benzalchonium chloride (BC) (Acros organics, USA) (ranging from 0.5 to 256 μg/ml, by serial twofold dilutions) against the tested strains as recommended by the Clinical and Laboratory Standards Institute (CLSI) [23]. An inoculum of 10 4 -10 5 cells ml -1 was used. The lowest concentration of antimicrobial agent totally preventing growth after 24 h was taken to be the MIC.

Multiplex PCR for the detection of macrolide-resistance encoding genes
Macrolide resistance genes ermA, ermB, ermC, msrA, and mef were examined in all strains using multiplex PCR, as previously described [24,25]. Multiplex PCR assays were performed in 25 μl PCR mixture 1 and mixture 2. Mixture 1 contained a DNA template (50 ng), 100 μM concentrations (each) of the four dNTPs, 1 U of Go Taq DNA polymerase (Promega, Lyon, France), 5 μL green Go Taq buffer (5X), 25 pM each of forward and reverse primers ermA, ermC, and msrA ( Table 1). The PCR mixtures were subjected to thermal cycling (3 min at 96°C, followed by 30 cycles of 30 sec at 95°C for denaturation, 30 sec at 55°C for annealing extension, and extension at 72°C for 2 min). A final elongation at 72°C for 10 min was achieved in a DNA thermal cycler (GenAmp PCR system 9700-Applied Biosystem Int., USA). For mixture 2, the forward and reverse primers (25 pM) of genes ermB and mef were used. PCR products were analysed in agarose gel (1%) electrophoresis in 1X Tris-borate-EDTA buffer (TBE) at pH 8.3. The amplification products were photographed and their size was determined using a 100 bp molecular size marker (Bio-Rad, France).
Detection of qacA, qacB, and qacC genes by PCR-RFLP Multiplex PCR-RFLP analysis of the qacA/B and qacC genes was achieved as described by Sekiguchi et al. [26]. For the fragment qacA/qacB, the forward primer (corresponding to nucleotides 924-946) and the reverse primer (corresponding to nucleotides 1143-1124) produced 220 bp. For the qacC gene, the forward primer (nucleotides 73-97) and the reverse primer (nucleotides 321-302) produced 249 bp. PCR was performed in a 25 μl reaction volume containing 50 ng of extracted DNA, 5 μl green Go Taq buffer (5X), 200 μM each of deoxynucleoside triphosphates (dNTP), 25 μM each of qacA, qacB, and qacC forward and reverse primers, 1 U of GO Taq DNA polymerase (Promega, USA). Each PCR was performed in duplicate. The reaction mixtures were heated to 94°C for 5 min and were then subjected to 30 denaturation cycles at 94°C for 1.5 min, annealing at 56°C for 0.5 min and extension at 72°C for 1.5 min, ending with a final extension at 72°C for 10 min.
Following PCR, the product was digested with 5 U of AluI at 37°C for 90 min (Promega, France). Ten microlitres of treated product were analysed by gel electrophoresis in 3% agarose gel in 1X Tris-borate-EDTA buffer (TBE, pH 8.3). The amplification products were photographed and their size was determined using a 25 bp molecular size marker (Promega, USA). The qacB gene was expected to be digested into two fragments: a visible 176 bp fragment and another fragment of 44 bp which is invisible. QacA and qacC genes were not expected to be digested [26].

Statistical analysis
Statistical analysis was performed on SPSS v.13.0 statistics software. Pearson's chi-square -2 test was used to asses inter-group significance. In addition statistical significance was set at P < 0.05.

Resistance to disinfectants agents
The 117 staphylococcus isolates were screened for QAC (BC) resistance. The strains were categorized as BC resistant or sensitive according to the BC MICs. Twenty four (20%) isolates were considered BC highly resistant (BC MICs between 16 and 32 μg/ml), 64 (54%) isolates were resistant to BC (BC MICs between 4 and 8 μg/ml), and 28 (23%) strains were sensitive to BC (BC MICs ≤ 2 μg/ml). 117 staphylococci isolates were analyzed for correlation between BC and antibiotic resistance ( Figure 1). This analysis showed that the frequency of erythromycin resistance 71% and oxacillin resistance 84% was higher among the BC-resistant strains.
Multiplex PCR for the detection of genes encoding macrolide resistance In this study we found that the incidence of the three erythromycin ribosomal methylase genes tested was 9 (7.7%) strains of S. aureus contained ermA, 16 (13.7%) strains harbored ermB gene and 7 (6%) strains were positive for ermC in the total of 117 isolated strains (Figure 2A, B). Furthermore among the CoNS 11 (9.4%) strains contained ermA. In addition, on the total of 117 isolated strains 13 CoNS strains carried the ermB gene, while 32 (27.3%) were positive for ermC. The msrA gene was present in 12 (10.2%) of S. aureus strains and in 48 (41%) CoNS strains ( Figure 2A). In contrast, the mef gene was absent in all the staphylococcal strains tested. Sixteen strains of S. epidermidis, eight strains of S. aureus and eighteen strains of CoNS were positive for the mecA gene, yet were susceptible to oxacillin. Furthermore, only nine strains of CoNS (7.7%) and nineteen strains (16.2%) of S. aureus were susceptible to erythromycin, those isolated did not contain any of the erythromycin-resistance genes tested. However, in five S. epidermidis strains (E7, E20, S23, S27, and S40), and in ten S. aureus stains, the ermA and ermC genes were not detected, although it was resistant to erythromycin (Table 2 and 3).

Discussion
Recently, the increasing numbers of device-related infections associated with methicillin-resistant staphylococci have raised awareness toward the need for alternative agents to prevent these infections. S. epidermidis species represent the CoNS most recovered from clinical specimens [3]. Among the 71 CoNS isolated in this study, the most prevalent were S. epidermidis (n = 32), S. hominis (n = 10), and S. haemolyticus (n = 9). The antibiotic susceptibility of the 117 staphylococal (S.aureus and CoNS) isolated in this study confirmed the multi-resistance of these strains toward the 18 antibiotics cited previously. Oxacillin resistance occurred in 7.7% of S. aureus and in 20.5% of CoNS tested by ATB Staph.
Erythromycin resistance in staphylococci is predominantly mediated by erythromycin-resistant methylase encoded by erm genes [27]. The inducible gene ermA is found on the transposon Tn554 and has a single specific site for insertion into the S. aureus chromosome [28]. The ermB gene is found on the transposon Tn551 of a penicillinase plasmid [29]. The ermC gene is responsible for constitutive or inducible resistance to erythromycin and is generally located on small plasmids [5,27,30]. On the other hand, the investigation of the prevalence of ermA, ermB, ermC and msrA genes in Staphylococci showed that only 28 strains of S. aureus (n = 46) and CoNS (n = 71) were found to be susceptible to erythromycin, yet contained erythromycin-resistant gene, and 44 strains (13 S. aureus and 31 CoNS) have at least one of the four genes (ermA, ermB, ermC and msrA) and were susceptible to erythromycin. Similarly, Sekiguchi et al. [26] found discordance among phenotypic susceptibility and the presence of erm genes. They stated that this discordance might be Table 3 Distribution of disinfectant and macrolide resistance genes in staphylococci (Continued)  Figure 1 Percentages of antibiotic-resistance Staphylococci isolates among BC-resistance and BC-sensitive isolates. Antibiotics used are erythromycin (ERY) and oxacillin (OXA). due to a mutation in the coding or promoter region of the PCR-detected genes. We noted also that eight strains of S. aureus and CoNS strains were found to be resistant to erythromycin but did not carry any erythromycin resistance gene (Table 1 and 2). This result may be explained by the location of these genes in small plasmids, which were occasionally lost. Fluit et al. [31] demonstrated that the ermC gene responsible for erythromycin resistance is located on a small plasmid. In this study, the incidences of ermA in erythromycin-resistant staphylococci were 7.7% for S. aureus and 3.4% for S. epidermidis. These findings are in disagreement with the study by Eady et al. [5] conducted with coagulase-negative staphylococci (CoNS) in the United Kingdom in which an incidence of 5.9% for ermA was reported. In a study performed in Denmark, 16% of S. aureus strains were carrying ermA, while only 3% of CoNS strains had this gene [30]. Regarding ermB, we found that this gene was more frequently encountered than ermA in erythromycin-resistant staphylococci with 24.8% (13.7% for S.aureus and 11% for CoNS) of total strains carrying ermB. In the United Kingdom, an incidence of 7.2% for ermB in CoNS has been reported [5]. Staphylococcal strains resistant to macrolides and type-B streptogramins frequently harbour msrA, which encodes an ATP-dependent efflux pump [5]. Erythromycin resistance may be caused by the msrA or ermB gene, as previously reported with staphylococci [5]. Our results are similar to those of a recent study investigating a high level of ermA and ermC genes in CoNS [32]. Staphylococcal multidrug-resistant gene qacA is generally mediated by plasmids mediated resistance to various toxic organic cations and ethidium bromide, as well 6: E6; 7: S18; 8: E7; 9: S13; 10: S19; 11: S11; 12: S45 and 13: S39. as a number of commonly used antiseptics and disinfectants, such as benzalkonium chloride and chlorhexidine [19]. The qacA gene also encodes resistance to both monovalent and divalent organic cations. In addition, qacB characteristically differs from qacA by conferring lower or no resistance to divalent organic cations [33]. Some investigations have implied that there is disinfectant cross-resistance with antibiotics [34,35]. Among the tested bacteria, 2 strains of S.aureus and 9 strains of CoNS carried the three qac genes (qacA, qacB, and qacC), as shown in Table 1 and 2. QacC gene was the most present in the 117 isolated strains (35%), followed by qacA (24%), and qacB (15. 4%). Of the 117 isolates investigated in this study, 74% were phenotypically resistant to BC. 56% of the BC-resistant staphylococcus isolates have at least one of the three resistant disinfectants genes (qacA/B and qacC). Previous investigators have reported a similar distribution of these three qac resistance genes in clinical S. aureus and CoNS [36,15]. Little is known about the occurrence and possible genetic linkage of qac and antibiotic resistance in staphylococci. Interestingly, we observed that staphylococci resistant to BC were generally more often resistant to antibiotics (Ery and Oxa) than BCsensitive isolates (Figure 1).
Furthermore, among the nine MRSA isolates, two strains (Sa22 and Sa23) were multi-resistant to antibiotic and harboured the qac genes. MRSA isolates resistant to antiseptics and disinfectants have been reported in Australia and in United Kingdom in the last decade [8]. Sekiguchi et al., [26] have found that among the 65 MRSA isolates 32 (49.23%) were positive for qacA, while one isolate was positive for qacB and seven MRSA 10% were positive for qacC. Three strains of S. hemolyticus harboured qacA and qacB genes. In a recent study, S. hemolyticus isolates were shown to contain both qacA and qacB genes [37]. QacA/B genes are typically located on a transposon of transmissible multidrug-resistant plasmids, such as pSK1 [33]. Staphylococci resistant genes to quaternary ammonium compounds (QACs) have been detected in clinical coagulase-negative staphylococci [36]. QAC resistant Staphylococcus spp. hosting qacA/qacB and smr have been isolated from different environments [38]. Results from a recent study in Norway suggest that qac-resistant genes are common in human clinical staphylococci and that a direct link between resistance to QACs and resistance to penicillin occurs in clinical isolates of human and animal origin as well as in foodrelated staphylococci [11,22]. Noguchi et al. [16] reported that when the antiseptic susceptibility and the distribution of antiseptic-resistant genes of MRSA isolated in Japan in 1992 were studied, qacA/B were detected in 10.2% (10/98). However, seven years later, qacA/B genes were detected in 47.9% (198/413) in MRSA isolates in Japan.

Conclusion
It appears that the widespread distribution of staphylococci carrying macrolides and qac-resistant genes found in dialysis biomaterial collected in Tunisia may be due to the transfer of resistance plasmids among species and strains, thereby contributing to the dissemination of staphylococcal resistance. Therefore, a closer investigation of antibiotics and antiseptic-resistant CoNS may provide crucial information on the control of nosocomial infections.