Characterization of resistance genes to macrolides, lincosamides and streptogramins (MLS) among clinical isolates of Staphylococcus aureus in North Lebanon

Objective: Staphylococcus aureus is one of the most significant pathogens causing significant morbidity and mortality. Moreover, the incidence of MLS S. aureus (resistant at least for one macrolide) infections continues to grow globally. The aim of this study is to examine the expression of resistance of S. aureus isolates to MLS and the prevalence of genes involved in this resistance using PCR. Methods: A total 38 strains of S. aureus MLS-resistant were isolated in the Microbiology Labs at Nini Hospital in North Lebanon. The disk diffusion method was used to determine the phenotype of the MLS resistance. The resistance genes involved were detected by PCR using specific gene primers for ermA, ermB, ermC, msrA, linA, mefA, vat and vgb genes. Results: A total of 55.3% of the isolates were positive for inducible phenotype (iMLSB), of these 15.8% were positive for the constitutive phenotype (cMLSB), 23.7% for MSB phenotype and 5.2% for L phenotype. The ermC gene was the most prevalent (52.6%), while ermA, ermB, msrA and linA genes were observed with lower prevalence. A combination of several of these genes was detected,whereas the vgb, vat and mefA genes were not detected in any of the clinical isolates. Conclusion: This study is the first investigated characterization of MLS resistance genes in clinical isolates of S. aureus in Lebanon according to THE INTERNATIONAL ARABIC JOURNAL OF ANTIMICROBIAL AGENTS


Introduction
Macrolides have been known for more than six decades, and, since the introduction of erythromycin molecule into therapy, a number of these antibiotics have been developed for clinical use. For years, these drugs have represented a major alternative of beta-lactams for the treatment of infections caused by gram-positive bacteria such as ß-hemolytic Streptococci, Streptococcus pneumoniae and Staphylococcus aureus. Macrolides, lincosamides and streptogramines (MLS) are related molecules, with similar antibacterial spectrum and mechanisms, but with different chemical structure.The use of these antibiotics has been accompanied by the rapid appearance of resistant strains in staphylococci [1,2]. Various resistance mechanisms have been described to these antimicrobials including ribosomal target modification by single mutation or methylation of 23SrRNA gene, modification of the drug, and efflux pumps [3]. However, the predominant mechanism in staphylococci resistance is target modification mediated by ermA, ermB and ermC (erythromycin ribosome methylase genes) [1,3].
Several common genes responsible for resistance to macrolide, lincosamide and streptogramin B (MLS B ) antibiotics were reported. The erm genes encode enzymes that confer inducible (iMLS B ) or constitutive (cMLS B ) resistance to MLS B antibiotics via methylation of a single adenine in the 23S rRNA gene, thereby reducing binding by MLS B antibiotics to the 50S large subunit of the ribosome [4]. Another fairly common mechanism of macrolide resistance were reported in S. aureus and mediated by two important efflux genes, msrA (macrolide efflux) and mefA (macrolide efflux protein A) genes, which confer only resistance to macrolide and streptogramin B (MS B ) antibiotics [3,5]. The msrA gene encodes a 488-amino-acid ATP-binding cassette (ABC) transporter hydrophilic protein that contains two ATP-binding motifs characteristic of the ABC transporters. However, the mefA gene is a proton motive force-driven efflux pump involved in resistance to MS B antibiotics [3,5,6]. Moreover, hydrolysis of antibiotics through the activity of esterases and/or phosphotransferaseshas only been reported in staphylococci [3].
Resistance to MLS B among S.aureus is an increasing problem. The overlapping binding sites of MLS B in 23S rRNAcauses cross-resistance to the three classes of antibiotics. A wide range of bacterial pathogens that are targets for MLS B express Ermmethylases.The new nomenclature system distinguishes 21 classes of erm genes and as many corresponding Erm proteins with predominance of ermA, ermB and ermC in staphylococcal gene classes [2,3]. In bacterial pathogens, these determinants are mainly carried by plasmids and transposons that are self-transferable. The ermA gene is often harbored on the transposon Tn554 which has been described as predominantly having a single specific site for insertion in the S. aureus chromosome [7,8]. Furthermore, the ermB our knowledge. The study revealed a high prevalence of the inducible resistance to lincosamides (iMLS B phenotype) and the most prevalent resistance determinants was ermC.
Other resistance mechanisms to MLS were reported. The O-nucleotidyltransferases encoded in S. aureus by lincosamide inactivation nucleotidylation (lin)genes, confer resistance to lincomycin but not to clindamycin (L phenotype) [1,11,12]. The staphylococcal vat genes confer resistance to streptogramin A and similar compounds by acetylation of the antibiotics. In addition, the staphylococcal vga genes encode related ATP-binding proteins probably involved in the active efflux of A compounds [13,14].
This study aimed to investigate the epidemiology of MLS resistance in Lebanon, particularly prevalence of MLS resistance phenotypes and molecularly characterize the macrolide resistance genes in clinical strains of S. aureus isolated from patients in North Lebanon.

Sample collection
Thirty-eightS. aureus MLS-resistant isolates were collected from patients in Nini hospital. All isolates were transported immediately to the Health and Environmental Microbiology Laboratory strain bank, in theAZM center for research in biotechnology, doctoral school, Lebanese University in Tripoli.

Antimicrobial susceptibility test
The susceptibility to antibiotics was performed by the disk diffusion method on Muller-Hinton agar (Bio-Rad, France) according to CLSI (Clinical and Laboratory Standards Institute) and CA-SFM 2015 (Comité de l'Antibiogramme de la SociétéFrançaise de Microbiologie 2015) recommendations.

DNA extraction and molecular identification of resistance genes
All S. aureus strains were tested for detection of macrolide resistance genes. DNA was extracted using the GenElute™ Bacterial Genomic DNA (Sigma Aldrich ® ,England), according to the manufacturer's recommended procedures. The DNA was eluted in 200 μl of elution buffer and stored at -20°C until use. All genes known to be responsible for S.aureus resistance to beta-lactams (mecA) and MLS (ermA, ermB, ermC, msrA, mefA, vat, vgb, and linA genes) were detected using PCR and primers specific to each gene (Table 1). Purified bacterial DNA from control strains and deionized water were respectively used as positive and negative control in each PCR run.

Statistical analyses
Statistical analyses were performed with GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA) using the Fisher's exact test to explore the association between resistance to MLS and resistance to beta-lactams. The general significance level was set at a P-value below 0.05.

Results
The percentage of susceptibility of all S.aureus isolates is showed in Table 2. Resistance to betalactams was found in 28/ 38 (73.7%) strains using the disk diffusion method and PCR targeting mecA gene. MLS resistance phenotypes were determined by with D-test showed that among 38MLS-resistant isolates, 27 (71.1%) exhibited the MLS B phenotype: 6 (15.8%) belonged to the cMLS B , and 21 (55.3%) to the iMLS B phenotype. The remaining 11 isolates (28.9 %) were confirmed as MS B (23.7%) and L (5.2%) phenotypes (Table 3). No significant association between resistance to MLS and resistance to betalactams was found (Fisher's exact test, p-value = 0.6).
In addition, PCR analysis targeting macrolide resistance genes showed that all iMLS B and cMLS B strains harbored at least the ermC gene, exceptthree iMLS B isolates that did not carry any of the tested genes.The MS B isolates carried ermA gene (1/9),mrsA gene (5/9), both ermA and ermC genes (1/9) and both ermC and mrsA genes (2/9). The two L isolates carried linA and both linA and mrsA respectively.On the other hand, mefA, vat andvgb genes were not detected in any of the isolates (Table 4).

Discussion
The resistance to antibiotics among S. aureus is an increasing problem, both in hospitals and communities of most Arab Middle East countries. A recent review by Tokajian et al. [19] on the epidemiology of S. aureus in these countries showed wide genetic change due to the introduction of new clones from other countries. Moreover, a previous study conducted in the same geographic region, demonstrated that methicilli-nresistant S. aureus (MRSA) strains colonizing infants carried 1-3 clinically important staphylococcal toxin genes [20]. In Lebanon, a recent study on antimicrobial susceptibility patterns of S. aureus clinical isolates revealed that only 56% were susceptible to all tested antibiotics [21]. In addition, the prevalence of MRSA infection are also increased in Lebanon in the last decade [22,23].These reports have led to renewed interest in the usage of MLS to treat staphylococcal infections [24]. Clindamycin is now a good alternative in the treatment of infections due to S. aureus, particularly MRSA isolates and as an alternative in penicillin-allergic patients. However, clindamycin resistance can be developed rapidly in S. aureus strains, and constitutive resistant mutants have arisen both in vitro testing and in vivo clinical therapy [25,26].
The CLSI and CA-SFM 2015 guidelines for disk diffusion susceptibility recommend the use of D-test to detect the inducible clindamycin resistance (iMLS B phenotype),and suggest that S. aureus isolates with the iMLS B phenotype should be reported as clindamycin-susceptible, but associated with clinical failures possibility related to the selection of clindamycin-resistant mutants.
A total of 38 S. aureus clinical isolates were examined. Firstly, according to the D-test screening, the iMLS B phenotype was the most predominant resistant phenotype (55.3%). These findings were different from the results obtained in a study conducted amongS. aureus isolates in Turkey, whereby the cMLS B phenotype was the predominant resistance phenotype (63%) [27]. Otherwise, we reported a similar distribution of MLS resistance phenotypes to that described in the UK by Hamilton-Miller et al. [28]. However, various studies conducted in Turkey [27], Europe [29], Japan     [30]Colombia [31], and France [17], reported that the iMLS B and cMLS B phenotypes were predominant in methicillin-susceptible S. aureus (MSSA) and MRSA isolates, respectively. However, may be due to the low number of examined S. aureus strains in this study, we could not find any association between MLSresistance phenotypes and resistance to betalactams.
Secondly, it has reported that accurate and rapid determination of antimicrobial resistance genes will help to select the proper the treatment of S. aureus infections and to avoide the spread of resistant genes [32,33]. Overall, 36 / 38 of the examined strains were resistant to erythromycin, of these, 91.6% had at least one of these genes:ermA, ermB, ermC and/ormsrA. These results are similar to those reported by Martinau et al. [34] who demonstrated the same findings among S. aureus strains resistant to erythromycin. However, 3 of those isolates did not carry any of the tested genes.These results were probably associated with the presence of other genes with low frequency in S. aureus, which were not evaluated in our investigation [33]. Also, this study found that efflux genes such as msrA gene was present either alone or in association with other genes (23.7%), while the mefA gene was absent from all tested isolates. These results concur with those described by Zmantar et al. [35], who reported the same observation. Furthermore, we found a predominance of ermC gene (27/38) among MLS resistance genes. The ermC gene was described to be the predominant MLS resistance gene in S. aureus isolates in Greece [36]. On the other hand, the ermA gene was detected alone in one isolate, and in association with ermC gene in 3 isolates of S. aureus, while a Colombian investigation reported that 100% of MRSA resistant to erythromycin had the cMLS B resistance phenotype and harbored the ermA gene [31]. In the same context, recent data from a multicenter study in Europe confirmed the predominance of ermA gene among S. aureus, while ermC and ermB genes were rarely detected [37]. Additionally, two isolates in our study harbored the linA gene. These results are also supported by recent findings, which showed that the linA gene was rarely detected in S. aureus strains [17,38,39].
Finally, all tested S. aureus strains, including methicillin resistant and MLS-resistant were susceptible to linezolid and vancomycin. Our data accord with a previous study conducted by Kanj et al. [40], in which good activity of these antibiotics was described against most Gram-positive pathogens from the Middle East and Africa.
In conclusion, to our best knowledge, this study is the first investigation regarding characterization of MLS resistance genes in clinical isolates of S. aureus in Lebanon. Our data indicates a predominance of iMLS B phenotype and ermC gene in these isolates. Other genes ( ermA, ermB, msrA and linA) were found with lower prevalence. The epidemiological significance of this study remains to be confirmed by further testing large number of S. aureus strains.
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