Molecular Characterizations, Virulence Determinants and Antimicrobial Resistance Proles of Methicillin-Resistant Staphylococcus aureus (MRSA) in the North of Iran

Background: Emergence and prevalence of Methicillin-Resistant Staphylococcus aureus (MRSA) has become a major universal health concern, limiting therapeutic options. Methods: In the North side of Iran, during the years 2015 to 2017, a total number of 37 MRSA isolates, including 19 clinical isolates from hospitalized patients and 18 colonizing isolates from health care workers were identied from three hospitals, in Gorgan, North of Iran. Antimicrobial susceptibility test was performed using the disk diffusion method and E-test. The presence of virulence and antibiotic resistance determinants were evaluated by PCR. The genotypic characterization was further analyzed using multi-locus sequence, spa, SCCmec, and agr typing. Results: The frequency of MRSA among S. aureus isolates was 38.14% (37/97). The most frequent S. aureus resistant isolates were found to be obstinate against penicillin (98%) and gentamicin (82.5%). Additionally, the lowest resistance rates were found against daptomycin (0%), vancomycin (2.7%), and quinupristin-dalfopristin (5.4%). All MRSA isolates were susceptible to daptomycin with MIC 50 /MIC 90 of 0.25/0.5 µg/ml. One isolate belonging to the ST239-SCCmecIII/t037 clone (MIC ≥ 16μg/ml) was resistant to vancomycin. All but one isolate that shares the ST22-SCCmec IV/t790 strain were positive for both tsst and pvl genes. The most predominant MRSA isolates (27%) were associated with the ST239-SCCmec III/t037 clone; and followed by ST239-SCCmec III/t924 (16.2%). Conclusions: In our study, circulating MRSA strains were genetically diverse with a high prevalence of the ST239-SCCmecIII/t037 clone. These ndings emphasize the need for future and continuous surveillance studies on MRSA to prevent the dissemination of multidrug resistance and existing MRSA clones in an effective manner.


Background
Methicillin-resistant Staphylococcus aureus (MRSA) is known to be resistant to various antibiotics and produces many virulence factors, which contribute to high treatment failure [1]. Hence, MRSA is one of the main causes of hospital and community-acquired infections worldwide (HA-MRSA, CA-MRSA) [1][2][3]. MRSA causes infections ranging from skin and soft tissue to deep-seated and severe life-threatening ones (endocarditis, osteomyelitis, necrotizing pneumonia, meningitis, and toxic shock syndrome) [4][5][6][7]. The genetic mobile element of staphylococcal cassette chromosome, mec (SCCmec), is a biomarker that is responsible for resistance of S. aureus to methicillin and other beta-lactam antibiotics [8,9]. Additionally, cell surface adhesive components and exotoxins are signi cant virulence factors of MRSA [9,10]. MRSA is frequently spread by direct contact with an infected wound or contaminated hands [11]. Previous studies suggested that health care workers' (HCWs) nasal colonization of HA-MRSA strains may also play a signi cant role in the pathogenesis and epidemiology of infection in both hospital and community settings [11,12]. There is scarce data regarding MRSA genotypes in Iran.
It is ascertained that the combination of molecular and epidemiological methods in surveillance investigations could be promising for controlling the emergence, colonization, and dissemination of predominant genetic lineages and provide epidemiological data for tracing the source of infection for clinical and treatment purposes [2,13]. The geographical differences in the genotypic characteristic of MRSA have been reported [2]. In Asia, there is signi cant divergence among countries and regions with respect to prevalence of MRSA; in fact, ST22-SCCmec IV/t790 and ST239-SCCmec III/t037 clones are predominant among patients in Iran [2,14], and so is ST239-spa t037 and ST5-spa t002 in China [15]. On the other hand, in many regions in Asia [16,17], sequence type 239 (ST239) is most prevalent, where, in UK, ST36 and ST30 are the most common types [18].
With this background, we are evaluating the molecular characteristics, antibiotic resistance patterns, and virulence genes pro les of MRSA isolates obtained from two kinds of study populations, namely hospitalized patients and health care workers (HCWs) in Gorgan, North of Iran.

Study Design and Sample Collection of S. aureus Isolates
This cross-sectional study was conducted from January 2, 2016 to October 28, 2018 in three hospitals (total of 920-beds) in Gorgan, North of Iran. Written informed consent was obtained from all the patients or HCWs and the study protocol was approved by the Ethics Committee in Golestan University of Medical Sciences (No. 31078693122419), and was conducted in accordance with the Declaration of Helsinki. The demographic pro les of patients and HCWs were recorded. We identi ed S. aureus and MRSA in hospitalized patients and HCWs as well (Table 1). Only the rst sample of each patient was included in the study. In case of HCWs, samples were collected from both anterior nares. 302 unduplicated clinical samples (blood, urine, wound, sputum, and others) were obtained from in-patients, out of which S. aureus and MRSA were identi ed in 53 (17.5%), and 19 specimens (6.29%), respectively. Likewise, 351 unduplicated non-clinical samplings from the anterior nares of HCWs were carried out. All the samples were sent for bacterial culturing and identi cation, using Gram staining, and standard biochemical tests, such as catalase, tube coagulase, DNase test, and mannitol fermentation [19]. The identi cation process of all S. aureus isolates was con rmed by using genotypic methods for the presence of nucA, and femA genes [2,20]. Data on department and period of hospitalization, clinical symptoms, antibiotic usages, and underlying conditions were recorded.

Antimicrobial Susceptibility Test
Antibiotic susceptibility test (AST) was performed using the disc diffusion method based on CLSI guidelines for antibiotics [21]; nitrofurantoin (300 µg), gentamicin (10 µg), rifampin (5 µg Table 6. Results of the agr typing method revealed that 64.86%, 24.3%, and 8.1% of 37 MRSA isolates belonged to agr type I, agr type III and agr type II, respectively. Also, one MRSA isolate was non-typeable. The majority (65.2%) of SCCmec III isolates harbored agr group I. Correspondingly, agr group III was present among SCCmec III (77.8%), and IV (22.2%) isolates and all of agrII group isolates were found in SCCmec IV.
Each of the remaining spa types was characterized in ≤ 3 isolates.

Discussion
S. aureus is one of the most frequent bacterial pathogens in Iran, causing a variety of infections. Genotypic background and antibiotic susceptibility of MRSA strains vary in terms of geographical locations dynamically [2].
In this study, the genotypic characterization, virulence determinants patterns and antimicrobial resistance pro le of 37 MRSA strains isolated from three hospitals in Gorgan, Northern Iran were analyzed. HA-MRSA associated with high morbidity and mortality has developed worldwide, speci cally in Iran [31,32]. A dramatic emergence and expansion of MRSA in different regions of Iran (20.4%-93.3%) leads to an increase in the costs of antibiotic therapy and reduction of treatment choices [2,33,34].
The frequency of MRSA strains diverges in various geographic areas. The relative prevalence (38.14%) of HA-MRSA strains in our study was comparable to the result obtained by Darban et al. (35%) [35]. However, this prevalence was lower than that reported in America, Europe, Africa, and Asia [26,[36][37][38][39][40][41]. The reasons for this discrepancy in the prevalence of MRSA may be related to the dissimilar antibiotic usage patterns, contrary infection control policies, sources of the isolates, and the characteristics of the subjects (HCWs and patients).
Results from the present study showed that the frequency of MRSA was similar in hospitalized patients (51.4%) and HCWs (48.6%). In Iran, the indiscriminate consumption of beta-lactam antibiotics contributes to the spread of resistance of MRSA to these antibiotics [2].
MRSA can arise from MSSA upon speci c site integration of SCCmec into the orfX locus in the chromosome of a susceptible isolate [42].
In the present study, prevalence of MSSA was 61.8% (92.6% among the various health professions and 88.7% in clinical samples), which is in line to the study in Gorgan [43], but was lower compared to Khosravi et al. [44], Heidari et al. [45], and Sepehriseresht et al. [46] surveys.
However, low gentamycin resistance rates in Chinese and Iranian MRSA isolates have been reported [3,15,47]. All of the MRSA isolates in the present study were MDR which is in relative line with previous reports from Iran [2,14] and Taiwan [38]. Our ndings also suggested that vancomycin and linezolid are potent and effective treatment options for MRSA [35,40,41]. The diverse genotypic characteristics of HA-MRSA in distinct geographic regions have been established [2].
Consistent to previously published data [49], MRSA SCCmec type III has been found the most prevalent isolate in our study.
Inconsistent with the previous reports, our research in Iran [50,51] revealed that SCCmec type III was the major SCCmec type among MRSA strains in the present study. Similar to Parhizgari et al. [51] and Zetola's et al. [52] The high frequency of MDR-MRSA in our study belonged to SCCmec type III.
Historically, the expression level of the most virulence factors of S. aureus is regulated by agr locus [3]. In agreement with our previous reports [14,53], the most common MRSA isolates belonged to agr I (64.86%), followed by agr type III (24.3%). According to the previous data, there is a substantial association between agr type and certain bacterial virulence determinants [54]. In addition to that, various agr types disseminate from one geographic area to another. Similar to our results, the frequency of bacterial virulence determinants including toxin and adhesive genes in MRSA isolates with agr type I was higher than type III [2]. The agr type I could have a crucial role in the control of staphylococcal virulence determinants. Nevertheless, in contrast with our ndings, Nowrouzian et al. [54] showed high frequency of toxin coding genes in MRSA isolates belonged to harboring agr type III.
The prevalence of spa types differs with geographic regions, type of samples, and the time of sampling [55]. The most predominant spa types were t032, t008, and t002 in Europe plus t037 and t002 in Asia. In Iran, most MRSA isolates were associated with spa types t701, t12311, t021, t037, and t790. Our study showed that t037 was the most prevalent spa type. However, t030 has been reported as the major spa type in Iran, other than t037 [56], suggesting that t037 has been replaced by t030 spa type in the hospitals in Iran. This nding has been similarly reported in China in the year 1994 to 2008 [57,58].
Sequence type 239 (ST239) in SCCmec III is found to be the most predominant mobile genetic element in Iran. ST239-SCCmec III is characterized into three clades: South American, European, and Asian [2]. The ST239-SCCmecIII/t037 clone, which is the oldest pandemic MRSA strain is a major HA-MRSA clone predominated in Iranian hospitals [59]. It seems that this clone could have been transferred from neighboring countries. This nding suggests that the frequency of ST239 clone may be closely related to MRSA infections. In our study, the MRSA strains which are PVL positive belonged to ST22 and ST15, moreover, MDR-MRSA was detected among the STs and this result is partially consistent with a study conducted in UK [32] disclosing the same matter that MDR-MRSA was found in STs, however, PVL positive MRSA strains belonged to ST772, ST5, ST8, ST22, ST59, and ST8t0 in that paper. Nonetheless, in contrast to the current results, Havaei et al. [60] did not identify any MRSA in ST22 strains. In our research, 62.5% and 37.5% of MRSA strains carrying the pvl gene belonged to ST22-SCCmec IV/t790 and ST15-MRSA IV/t084 clones, respectively. Additionally, all but one isolate that shares the ST22-SCCmec IV/t790 strains were positive for both tsst and pvl genes. This nding is relatively in agreement with recent study conducted in Iran [2] declaring all but one of the ST22-SCCmec IV/t790 strain, harbored the tsst and pvl genes. The antimicrobial resistance pro les frequently differ in ST clones of MRSA [57]. In accordance with results of a study in UK by Ellington et al. [32], in the current study, isolates with ST22-SCCmec IV/t790 were drastically resistant to multiple antibiotic groups.

Conclusions
There is a simultaneous carriage of virulence determinants, multidrug-resistance genes and high genetic diversity among the MRSA strains isolated from patients and HCWs in North of Iran. Sequence typing analysis showed that ST239-SCCmec III/t037and ST22-SCCmec IV/t790 clones have a high expression level of tsst and pvl genes with multidrug resistance genes. Hospital infection control policies and nationwide surveillance efforts are highly demanded to monitor the clonal expansion of MRSA species in the North of Iran.