Molecular characterization of staphylococcal cassette chromosome mec and virulence encoding genes in methicillin-resistant staphylococci at a medical center in Lebanon

Background: Methicillin-resistant staphylococci (MRS) are major human pathogens accounting for most hospital-acquired (HA) and community acquired (CA) infections worldwide. The recent increase inMRS in a medical center in Lebanon elicited the determination of SCCmec types, genotypes, and prevalence of Panton-Valentine leucociden (PVL) and toxic shock syndrome toxin-1 (TSST-1) among the MRS isolates. Methods: Thirty-six MRS isolates collected between October 2010 and September 2011 at a medical center, Lebanon were typed using phenotypic and genotypic methods. Antimicrobial susceptibility was determined using the disk diffusion agar method. SCCmec typing was performed by multiplex PCR and sequence analysis. The prevalence of the genes encoding PVL and TSST-1 virulence factors and their transcription levels, were determined respectively by PCR and semiquantitative real-time PCR. The genomic relatedness of the isolates was assessed by random amplified polymorphic DNA (RAPD) analysis. Results: Antimicrobial susceptibility revealed three distinct antibiotypes. The predominant SCCmec type found among the MRS isolates was type IVa (51%). Twenty-nine percent harbored SCCmec type III and 14% harbored SCCmec type II. One isolate harbored SCCmec type IVc, and another harbored SCCmec type I. All methicillin-resistant Staphylococcus aureus (MRSA) isolates were negative for the gene encoding for PVL, and two were positive for the gene encoding for TSST-1. RAPD analysis demonstrated high genomic diversity among the MRS isolates. THE INTERNATIONAL ARABIC JOURNAL OF ANTIMICROBIAL AGENTS


Introduction
Staphylococcus aureus is considered a leading cause of serious and sometimes life-threatening infections.S. aureus is frequently found on the skin, nasal and other mucous membranes of humans, and in various food products.In England in 1961, the first methicillin-resistant S. aureus (MRSA) was reported, only one year following the introduction of methicillin.Since then, MRSA has been recognized as a major cause of health-care associated infections.Recently, MRSA has emerged as an important community pathogen, and this was followed by the recognition of methicillin-resistant coagulase negative staphylococci (MRCNS) as important nosocomial pathogens [1,2] A study by Tokajian S, 2014, showed that the local epidemiology within the Middle East (western Asia) region is fluctuating, owing to the introduction of new strains with the global exchange of several clones [3].Another study performed by Shehabi et al. demonstrated the presence of 1-3 clinically important staphylococcal toxin genes more frequently colonizing Jordanian outtpatient infants [4].Furthermore, another study by Nazeer A et al., 2012 retrieved 73 studies through PubMed using the search terms "methicillin-resistant S. aureus" (MRSA), and Saudi Arabia.They reported MRSA incidence or prevalence in patients and/or percentage among S. aureus isolates and evaluated these metrics against the closest matching SHEA (Society for Healthcare Epidemiology of America) /HICPAC (Healthcare Infection Control Practices Advisory Committee metrics) [5] Methicillin resistance occurs in Staphylococcus species due to the expression of an altered penicillin-binding protein termed PBP2a or PBP2', that is encoded by the mecA gene [2] The mecA gene is located on a mobile staphylococcal cassette chromosome mec (SCCmec) element.This element exists in MRSA; as well as, MRCNS isolates.To date, at least five SCCmec (I-V) types have been described with sizes ranging between 21 to 67 kb.The SCCmec types (I-V) have aided in the differentiation between hospital-acquired (HA) and community-acquired (CA) MRS strains.The larger Sccmec types (I-III) have been associated with HA infections; whereas, the smaller SCCmec types (IV and V) have been associated with CA infections [6][7][8][9].SCCmec includes the mec gene complex which is comprised of the mecA gene and its regulators and the ccr gene complex that encodes site-specific recombinases.These recombinases are responsible for the mobility of the SCCmec [ 10].
Strains of S. aureus isolated from human sources can produce a variety of virulence factors.Panton-Valentine leucociden (PVL) is a pore-forming cytotoxin that is associated with severe skin and soft tissue infections and necrotizing pneumonia.PVL with SCCmec type IV is considered as a marker for CA-MRSA infections; however, exceptions exist [11].Another main virulence factor includes the toxic shock syndrome toxin-1 (TSST-1), which is a superantigen having a size of 29.1 kDa.TSST-1 is encoded by the tst gene.TSST-1 causes toxic shock syndrome (TSS), a severe and potentially fatal manifestation when it enters the bloodstream [12].
Infections due to hospital-acquired methicillin resistant staphylococci (HA-MRS) must be controlled otherwise they may become permanently present in a hospital environment.These endemic MRS are very challenging to overcome.In most cases, they increase the number of infections, lengthen the duration of hospital stay, and raise the amount of health-care costs.Since MRS strains are generally resistant to a broader range of antimicrobial agents, the chances of treatment become limited and the spectrum of antibiotic prescriptions changes.Therefore, the adequate identification of such resistant bacteria is a crucial and necessary obligation for the selection of the proper antimicrobial treatment as well as for the implementation of appropriate infection control measures [13].
In Lebanon and the Middle East there are numerous molecular characterization and epidemiological studies on MRSA especially during the last decade, highlighting their increased prevalence in the region [4,[14][15][16][17][18][19].However, there is scarce data available concerning the antibiotic susceptibility profiles, the SCCmec types and the genotypes of MRS strains that could be circulating in Lebanon.
Accordingly, the objective of this study was to perform phenotypic and genotypic characterization of MRS isolates from clinical specimens at a medical center in Lebanon.

Bacterial isolates, identification and confirmation of methicillin-resistance by phenotypic methods
Thirty-six non-duplicated methicillin resistant staphylococci were isolated from clinical specimens (blood, pus, urine, bone, catheter, soft tissue, wound swabs, respiratory samples, and exudates) of in and out-patients at a medical center in Lebanon from October 2010 to September 2011.These isolates were identified as staphylococci based on their Gram stain, colonial morphology, as well as their catalase properties.Rapid agglutination and coagulase tests were done to distinguish between S. aureus and coagulase negative staphylococci (CNS).Methicillin resistance was confirmed using an oxacillin disk on Mueller-Hinton agar (Oxoid, England) according to Clinical and Laboratory Standards Institute (CLSI)guidelines [20].

Antimicrobial susceptibility testing of staphylococcal isolates
Antimicrobial susceptibility testing was performed by disk diffusion agar method on Mueller-Hinton agar against a panel of antimicrobial agents disks (Oxoid, England) according to CLSI guidelines [20].

DNA extraction
DNA was extracted from the staphylococcal isolates using illustra bacteria genomic Prep Mini Spin kit (GE Healthcare, UK) in accordance with the manufacturer's protocol for purification of genomic DNA from Gram-positive bacteria.

Molecular characterization by multiplex PCR amplification and DNA sequencing
Three multiplex PCR assays carried out using the PCR Sprint Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA) were used for characterization of the methicillin-resistant staphylococcal isolates.

Polymerase Chain Reaction
SCCmec-typing Multiplex-PCR 1for typing and sub-typing of the SCCmec element with the simultaneous detection of the mecA gene The PCR mixture contained nine pairs of primers, specific for typing and sub-typing the SCCmec element; as well as, an internal control primer for confirming the presence of the mecA gene, were used in this multiplex polymerase chain reaction (M-PCR) assay.The various types and sub-types detected by the M-PCR included: I, II, III, IVa, IVb, IVc, IVd, and V. M-PCR was performed in a 25 µl final reaction mixture with 5 µl of the prepared DNA template having a final concentration 10 μg/ml, 2.5 µl of 10x Taq DNA polymerase buffer with KCl (Fermentas, USA), 2.5 µl of MgCl 2 (25mM) (Fermentas, USA), 0.2 µl of Taq DNA polymerase (5U/μl) (Fermentas, USA), 1 µl of 5 mM dNTPs (Fermentas, USA), 13.4 µl of nuclease free distilled water (Amresco, USA) and 0.2 µl of each primer (Thermo Scientific Inc., USA) [13].

Mec gene complexcharacterization M-PCR 2
Two pairs of primers were used in this M-PCR assay to characterize the mec gene complex as either class A or B. The M-PCR was performed as described in 2.5.1 with thefollowing modification: 13.64 µl of nuclease free distilled water (Amresco, USA) and 0.08 µl of each primer, (Thermo Scientific Inc., USA) [13].

Ccr gene complex characterization M-PCR 3
Six primers were used in this M-PCR assay to characterize the ccr gene complex into four allotypes (types 1, 2, 3, and 5).The M-PCR was performed as described in 2.5.2Amplification was performed using the same cycling conditions for all three sets of M-PCR reactions with denaturation at 94°C for 5 min; 35cycles of 94°C for 45 s, 56°C for 45 s, and 72°C for 1.5 min; and extension at 72°C for 10 min.

MRSA-PVL PCR.
Was performedto detect the PVL toxin gene.The PCR was performed as described in Amplification was performed with denaturation at 94°C for 5 min; 35 cycles of 94°C for 10 s, 52°C for 20 s, and 72°C for 40 s; and extension at 72°C for 5 min.

Gel electrophoresis
The amplicons from M-PCR1, 2, and 3 were separated on 2% agarose gels using a suitable DNA ladder; either, a 50 bp DNA ladder (Fermentas, USA) or a 123 DNA ladder (Sigma-Aldrich, USA).Gels were visualized under a UV transilluminator(Haakebuchler Instruments Inc, USA) and captured using Olympus digital camera, and the DigiDoc-ItProgram (UVP, UK).The amplicons were purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, California) and sequenced by the ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA,USA).

RNA extraction& reverse transcription
RNA extraction was performed using the illustra RNAspin Mini RNA Isolation kit (GE healthcare, UK) for all MRSA isolates (n=13).Subsequently,reverse transcription was performed using the QuantiTect® Reverse Transcription Kit (QIAGEN, Germany) to synthesize cDNA from the extracted RNA.

Real Time Semi-Quantitative qRT-PCR
qRT-PCRwas carried out using the Bio-Rad CFX96 Real Time System C1000 Thermal Cycler (Germany) to determine the transcription levels of the gene encoding TSST-1.The qRT-PCR was performed in a 20 µl final reaction mixture with 10 µl of QuatiFast SYBR Green PCR master mix, 4 µl of RNase free H 2 O, 2 µl of forward primer having a concentration of 5μM, 2 µl of reverse primer having a concentration of 5μM, and 2 µl of synthesized cDNA.The following cycling conditions were used: denaturation at 94°C for 5 min; 40 cycles of 94°C for 10 s, 56°C for 20 s, and 72°C for 40 s; and extension at 72°C for 5 min.Data analysis was performed using the Bio-Rad CFX Manager software (BioRad, USA).

Random Amplified Polymorphic DNA analysis (RAPD)
RAPD analysis was performed in order to detect the genomic polymorphisms of the thirty-six MRSA isolates.RAPD was done using the Ready-To-Go RAPD Analysis Beads kit (GE Healthcare, UK Limited) as specified by the manufacturer, using the PCR Sprint Thermal Cycler (Thermo Fisher Scientific, Waltham MA, USA).

SCCmec typing
The SCCmec typing M-PCR1 assay included specific primers for the various SCCmec types, as well as an internal control primer for the detection of the mecA gene.This single M-PCR assay determined SCCmec types and subtypes I, II, III, IVa, IVb, IVc, IVd, and V with the simultaneous discrimination between MRS and methicillin sensitive staphylococci (MSS) isolates (Figure 1).
M-PCR1 showed that 35/36 (97.2%) of the MRS isolates were positive for the mecA gene confirming methicillin resistance.Isolate 1 was negative for

MRSA-PVL PCR
A total of 13 cultured MRSA isolates were subjected to MRSA-PVL PCR and none were PVL gene positive.

Real Time semi-quantitative qRT-PCR
Assay for detecting the gene encoding for the TSST-1 virulence factor showed that 2/13 MRSA isolates (isolates 2 and 3) were positive for the tst gene.
The predominant RAPD type C included 9 methicillin resistant coagulase-negative staphylococci (MRCNS) isolates, having 58.5% genomic similarity; five of them were CA and four were HA strains.All of these isolates were resistant to erythromycin, and all except three were resistant to clindamycin.Moreover, all were sensitive to the glycopeptides class of antimicrobial agents (vancomycin and teicoplanin).Isolates 12, 13 and 15 of RAPD type C were genetically identical, all three containing SC-Cmec type IVa, indicating that they are community acquired strains.Isolates 12 and 13 were derived from the ICU and isolate 15 from patients' suites.
The RAPD type E included 7 MRS isolates (6 MRSA and 1MRCNS), having 65.1% genomic similarity; four were HA and three were CA strains.These isolates were all susceptible to the glycopeptides.All but three were sensitive to erythromycin, and only two strains were resistant to clindamycin.
The RAPD type B included 6 MRCNS isolates, having 55% genomic similarity; three were HA and three were CA strains.The vast majority of these isolates were resistant to clindamycin and erythromycin except for one isolate which was sensitive to both.All were susceptible to the glycopeptides.Isolates 21 and 22 of RAPD type B were genetically identical.Both were hospital acquired strains having SCCmec type II.Isolate 21 was isolated from an outpatient while isolate 22 was isolated from the NICU.
The RAPD type D included 5 methicillin resistant S. aureus (MRSA) isolates, having 55.3% genomic similarity; four were CA and one was a HA strain.These isolates were all sensitive to clindamycin, erythromycin, and the glycopeptides.Isolates 2 and 3 of RAPD type D were genetically identical containing SCCmec type IVa; and hence, were CA strains.Isolate 2 was isolated from an out-patient whereas isolate 3 was isolated from patients' suites.These 2 MRSA isolates were positive for the TSST-1 toxin gene.
The RAPD type G included 4 MRS isolates (2 MRSA and 2 MRCNS), having 30.2% genomic similarity; three were HA and one was a CA strain.These isolates were all susceptible to the glycopeptides; however, they were resistant to erythromycin.All but one isolate were resistant to clindamycin.
The RAPD type A included 3 MRCNS isolates, having 45.6% genomic similarity; two were CA and 1 was a HA strain.All three isolates were resistant to erythromycin and sensitive to glycopeptides, and one was sensitive to clindamycin.
The RAPD type H included only 1 MRCNS isolate.This isolate was HA, for it contained SCCmec type III.It was resistant to clindamycin and erythromycin and sensitive to the glycopeptides.
The RAPD type F also included only 1 MRCNS isolate which did not demonstrate the presence of the mecA gene; it was only phenotypically regarded as a methicillin resistant strain.This isolate was sen-sitive to clindamycin, erythromycin, and glycopeptides (Figures 2 & 3).

Discussion
This study highlighted the antimicrobial-resistance profiles of the collected MRS isolates, which can serve as a useful reference for assessing the antibiotic resistant trends of the MRS strains in Lebanon.
Antimicrobial susceptibility testing showed that MRCNS are inclined to be more multi-drug resistant    [21].The percentage of resistance to erythromycin and clindamycin in MRCNS isolates in this study was 91% and 65% respectively; whereas, the percentage of resistance to erythromycin and clindamycin in MRSA isolates was 38% and 31% respectively.The high resistance rates to erythromycin and clindamycin renders the use of these antimicrobial agents inefficient in the treatment of MRS infections.Currently, the glycopeptides (vancomycin and teicoplanin) are considered the drugs of last resort against MRS infections; however, the emergence of vancomycin-intermediate and resistant staphylococcal strains has imposed a serious threat [22].Moreover, all of the Staphylococcus isolates in this study were sensitive to vancomycin and teicoplanin.Molecular characterization of the studied MRSA isolates by performing staphylococcal cassette chromosome mec (SCCmec) typing using M-PCR revealed that 53.8% of these MRSA isolates were SCCmec type IVa, 38.5% were SCCmec type III and 7.7% were SCCmec type IVc.
None of the MRSA isolates were pvl positive, but 15.4% of MRSA isolates were tst positive by qRT-PCR.The presence of the tst gene, encoding for TSST-1, in isolates 2 and 3 is alarming because it can contribute to the invasiveness and severity of the S. aureus infection.Therefore, routine investigation for genes encoding virulence factors is recommended in order to predict potential diseases that may be associated with these virulence factors.This may lead to the application of the appropriate measures if possible to manage such diseases.
Furthermore, SCCmec typing by M-PCR allowed for the differentiation between HA-MRSA and CA-MRSA [23].Established molecular criteria classified isolates of MRSA harboring SCCmec type I-III as HA-MRSA and those harboring SCCmec type IV-V as CA-MRSA.The majority (61.5%) of the collected MRSA isolates were CA.The existence of these isolates carrying SCCmec type IV in the hospital indicates the presence of a community reservoir or possibly the entrance of these isolates into the hospital environment; hence, CA-MRSA isolates are no longer confined to the community [24][25][26].This change in the epidemiology of CA-MRSA has raised speculations concerning the origin of CA and HA MRSA.The basic structure of the SCCmec genetic element can provide an insight about the origin of CA and HA-MRSA.As noted earlier, the various SCCmec types share some basic structural features, including direct repeats, the ccr gene complex and the mec gene complex.The structural differences between the community-associated SCCmec types IV-V and the hospital-associated SCCmec types I-III rely mainly on the larger size and the presence of additional resistance determinants associated with HA-MRSA strains.Therefore, a possible explanation is that CA-MRSA has entered the hospital setting, acquired additional fragments and resistance determinants rendering them HA.
CA-MRSA isolates have been known to be susceptible to non-beta-lactam antimicrobial agents.The antimicrobial susceptibility results obtained in this study confirm it; while 80% of HA-MRSA isolates showed multidrug-resistant phenotypes, all CA-MRSA isolates presented with a multi-drug susceptible pattern.Even though the CA-MRSA isolates were susceptible to multiple classes of antimicrobial agents as compared to HA-MRSA, 54% of the patients were hospitalized, indicating the severity of infections caused by CA-MRSA.
SCCmec typing and RAPD analysis revealed that the staphylococcal isolates 2 and 3 which were characterized as CA-MRSA belonging to cluster D, isolates 12, 13 and 15 which were characterized as MRCNS carrying SCCmec type IVa belonging to cluster C, and isolates 21 and 22 which were characterized as MRCNS carrying SCCmec type II belonging to cluster B, were genomically identical, and they were isolated within the same time period, hence, indicating possible outbreak strains [27].
The SCCmec genetic elements were highly preva-lent among MRCNS in this study.Moreover, there were strains of MRCNS and MRSA that showed close or possible genomic relatedness by RAPD (isolates 29 & 30 and isolates 7 & 35 showed 85.7% and 50% genomic similarity respectively).This indicates that MRCNS constitute potential reservoirs of the SCCmec that provide S. aureus with antimicrobialresistant determinants [28][29][30][31][32]. RAPD analysis revealed the presence of multiple SCCmec types within a single RAPD cluster.This indicates that staphylococcal clones having similar genomes may have developed methicillin-resistance upon the acquisition of the SCCmec genetic element by methicillin-sensitive strains of staphylococci.
The MRS isolates were diversified as defined by RAPD analysis, and there was a significant genomic heterogeneity particularly among staphylococcal isolates carrying the SCCmec type IV, which clustered into six RAPD types.This observation points out to the enhanced ability of SCCmec type IV genetic element to become mobilized, which is followed by its insertion into the chromosome of Staphylococcus species.Further, SCCmec types I, II and III were more commonly associated with HA isolates, whereas types IVa and IVc were more commonly associated with CA isolates.The small size associated with SCCmec type IV might aid in the transfer of this element on a plasmid or bacteriophage and hence, contributing to the enhanced spread of methicillin-resistance [33].

Figure 1 .
Figure 1.Gel electrophoresis of the M-PCR assay for the characterization of the SCCmectypes in MRS isolates.Lane 1 of the upper and lower gels represents the molecular weight marker (L: 50 bp ladder); lane 2 of the upper and lower gels represents the negative control (NC)

Figure 3 .
Figure 3. Dendrogram obtained from RAPD analysis, depicting clonal relationship (eight clusters: A, B, C, D, E, F, G, & H) of the 36 MRS clinical isolates obtained from a Medical Center, Lebanon.

Table 1a .
Clinical data and sources of MRSA isolates

Sample # Clinical Ward Date of Isolation Source Nature of Specimen Sex/Age of Patient (years)
*MRSA: methicillin resistant S. aureus, NA: not available, M: male, F: femaleTHE INTERNATIONAL ARABIC JOURNAL OF ANTIMICROBIAL AGENTS

Table 1b .
Clinical data and sources of MRCNS isolates

Table 2 .
Characteristics of MRS isolates