Determination of Virulence Factors and Resistance Profile of Methicillin-Resistant Staphylococcus aureus Strains among Different Types of spa, agr, and SCCmec

In order to restrict the spread of methicillin-resistant S. aureus (MRSA) in hospitals, it is necessary to characterize isolates rapidly and precisely. The objective of this study was to determine virulence factors and resistance profiles of MRSA strains among spa, agr, and SCCmec types. In total, 55 MRSA isolates were collected from clinical specimens. The MRSA isolates were characterized by antimicrobial susceptibility testing, virulence genes, agr typing, spa typing, and SCCmec typing. According to our findings, all MRSA strains were resistant to cefoxitin; 88% and 86.7% of which were resistant to erythromycin and clindamycin, respectively. Type II agr was predominant with 54.54% frequency. Among 27 different spa types, type t030 was most frequently (25.45%). Most MRSA isolates (63.3%) were SCCmec type III. The pvl and tst genes were found in 25.3% and 32.7% of MRSA isolates, respectively. Among the MRSA strains, ermA, ermB, and ermC were present in 50%, 33.3%, and 57.3% of cases, respectively. In addition, 43 of the 55 MRSA strains (78%) harbored aminoglycoside resistance genes. The results of our study revealed that the MRSA rate in our region is dramatically high. Better infection control guidelines in hospitals, as well as ongoing epidemiological surveillance studies, could be strongly suggested for effective prevention of the spread of MRSA to inpatients.


Introduction
Among human pathogens, Staphylococcus aureus (S. aureus) is one of the most common ones [1]. Methicillin-resistant S. aureus (MRSA) strains have emerged as a major problem in hospitals, owing to the increased mortality rate associated with some of these infections. MRSA strain outbreaks have a significant impact on morbidity, mortality, and healthcare costs [2][3][4]. MRSA strains express virulence factors that play a key role in infection progression. The accessory gene regulator (agr) system regulates the expression of numerous virulence factors in S. aureus, and four major agr types have been identified to date. Different agr types have different properties and distributions in various geographic regions; thus, identification of the predominant types in each location would be of benefit [5,6].
It is clear that the spread of MRSA around the world is constantly evolving, with new strains emerging in a variety of geographical regions. The mecA gene, which confers beta-lactam resistance, is found on the staphylococcal cassette chromosome mec (SCCmec) of MRSA strains. MRSA is divided into distinct epidemiological types based on the presence of the SCCmec element. The SCCmec typing method can help distinguish community-associated MRSA (CA-MRSA) from hospital-acquired MRSA (HA-MRSA) infections [7].
Continuous MRSA surveillance in each location necessitates monitoring the epidemiology, host characteristics, and transmission routes of emerging strains [8]. Therefore, clinicians must have a thorough understanding of MRSA's molecular epidemiology in order to assess the efficiency of preventative strategies and provide effective prophylaxis [9]. Prevention of MRSA transmission by screening patients, personnel, and the environment is a critical goal of infection control [9]. However, investigating the origins and routes of transmission of MRSA is possible only through the use of typing approaches, which are necessary for genetic characterization. There is a variety of molecular epidemiological methods used for MRSA surveillance, including multilocus sequencing typing (MLST), pulsed-field gel electrophoresis (PFGE), staphylococcal protein A (spa) gene sequencing, agr, and SCCmec typing [10]. Although each of these methods has a pretty good discriminating power, it has been demonstrated that combining genotyping methods is beneficial and advantageous for distinguishing distinct MRSA clones.
spa typing is a valuable typing instrument owing to its ease of use, cost-effectiveness, and uniform nomenclature, which is based solely on assessment of the repetition space in the X region of the spa gene [11]. The X region polymorphism, which encodes a part of the spa protein, is characterized by variations in tandem repeats as well as variations in base sequences within repetitions. In other words, in any strain of S. aureus, each motif consists of 24 base pairs, which are referred to as unique sequence motif repeats. The order of the repeats determines the spa type for a strain [12]. The spa types are important for identifying S. aureus outbreak isolates and infection control policies around the world. Over the last decade, studies have been conducted on the distribution of spa, agr, and SCCmec types in various geographic areas [13,14]. Therefore, the current research is aimed at determining virulence and antimicrobial resistance profiles of MRSA isolates using spa, agr, and SCCmec typing.

Detection and Isolation of MRSA.
In this cross-sectional study, out of a total of 142 S. aureus isolates, 55 MRSA isolates were identified and included in this study, while the remaining isolates were excluded. The isolates were obtained from clinical samples including blood, urine, wounds, and cerebrospinal fluid collected from different wards (emergency, men, women, children, and intensive care unit) in Al-Zahra Hospital in Isfahan (Iran), then referred to the hospital laboratory. The S. aureuswas identified using growth on mannitol salt agar, showing betahemolysis on 5% sheep blood agar, and being gram positive as well as producing catalase, coagulase, and DNase. The presence of the nucA gene was confirmed by PCR in all S. aureus isolates (Table 1).
2.4. Genomic DNA Extraction. A DNA Mini Kit (Qiagen GmbH, Hilden, Germany) was used for genomic DNA extraction. Fresh colonies harvested from agar plates were washed with 500 μl TE 1x and centrifuged for 10 minutes at 5000 rpm according to manufacturer's protocol. A suspension was then prepared in 200 μl TE 1x with 20 μl lysostaphin (200 μg/ml final concentration) and incubated at 37°C for 20 minutes. Finally, the obtained DNA was dissolved 50 μl RNase-DNase-free water (Sigma). DNA concentration was measured with a spectrophotometer.
2.5. SCCmec Typing. As described previously, multiplex PCR was used to identify various MRSA isolates using genomic DNA as the template [16]. The amplification started with a 3-minute denaturation step at 94°C, then 35 cycles of 30 seconds at 94°C, 1 minute at 55°C, 1 minute at 72°C, and finally 5 minutes at 72°C for final extension.

Detection of agr Types.
Multiplex PCR was performed to detect agr types using a set of primers containing a common forward primer (Pan) and reverse primer (agrI, agrII, agrIII, and agrIV) that are unique to each agr group [19]. The primer sequences are shown in Table 1.

Detection of spa Types.
The identified MRSA strains were subjected to PCR to detect the spa gene ( Table 1). The amplification reaction consisted of an initial denaturation step at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 40 second, hybridization at 56°C for 40 second, and extension to 72°C for 50 second, followed by final extension to 72°C for 5 minutes [11]. Sequencing was then performed on the PCR products. Also, after sequencing, the spa database server (http://spaserver.ridom.de/) was used to determine different types.
2.9. Statistical Analysis. For statistical analysis, SPSS Statistics 22.0 for Windows was used. Data were presented using descriptive statistics (frequency, percentage, mean, and standard deviation).

Discussion
MRSA strains are one of the leading causes of infections in hospitals, but infections from community-related MRSA have become a global public health threat over the recent decades [22]. The widespread occurrence of multidrugresistant (MDR) MRSA augments the cost of antibiotic therapy and limits treatment options. During the last two decades, the widespread use of beta-lactam antibiotics in Iranian hospitals and medical facilities led to increased resistance to these antibiotics [23]. The results of the current study revealed that MRSA strains are resistant to erythromycin (88%), clindamycin (86.6%), tetracycline (68%), rifampicin (57%), and gentamicin (54.6%). Based on these results, linezolid was the most effective drug for MRSA in the study area. In addition, more than 93% of our MRSA isolates were MDR. We did not report the frequency of MRSA strains since the selection criteria of our study were isolation of MRSA strains, and methicillin-sensitive S. aureus (MSSA) strains were not included. As a protein synthesis inhibitor, erythromycin is widely utilized for the treatment of staphylococcal infections [24]. According to Mahdiyoun et al., the frequency of MRSA resistance tested for erythromycin was 84.4% [25]. A study conducted in Taiwan reported that the resistance rates for erythromycin and clindamycin were 94.9% and 86.5%, respectively [26]. High frequency of resistance to erythromycin and clindamycin antibiotics in the present study was in consistent with the findings of previous research in Iran [24] and India [27]. We also found SCCmec type III in a high percentage of MRSA isolates. Similarly, studies in Iran and other Asian 3 BioMed Research International countries have reported the high prevalence of SCCmec type III [28][29][30]. In MRSA isolates, SCCmec mobile genetic factor leads to an expansion of antibiotic resistance determinants as well as virulence factors, which can act as a large reservoir of resistance genes, enterotoxins, and other virulence factor genes. Our results showed that among MRSA with SCCmec type III, sea, hla, and seb were the most frequently found genes encoding virulence factors. Herein, the majority of the isolates with type III were resistant to erythromycin and clindamycin (55% and 54%) while all the isolates were     [31][32][33]. In the present study, among MRSA strains, ermC (63.6%) was the most commonly detected macrolide resistance gene, followed by ermA (20%) and ermB (16.4%). Also, the most frequently identified aminoglycoside resistance gene was aac (6 ′ )-aph (2 ″ ) (54%). These findings are in contrary with the study by Hau et al. [34] conducted with clinical MRSA in the United States in which an incidence of 91.5% for ermA and 12.7% for aac (6 ′ )-aph (2 ″ ) was reported. A similar finding was also reported by Yılmaz and Aslantaş [35] that the ermC and aac (6 ′ )-aph (2 ″ ) genes were detected in 91.5% and 50% of S. aureus isolates, respectively.
In view of the widespread of MRSA isolates, it is imperative that the treating physician encourages the preservation of glycopeptides and linezolid only in the case of MRSA. In Iran, a major MRSA-associated problem is the result of increased incidence and hospitalization rates. Therefore, for screening, epidemiology, surveillance, and infection control, rapid and accurate typing of MRSA isolates is crucial [23]. Several genotyping techniques are available for identifying S. aureus strains in epidemiological studies. Sequence-based typing methods, such as MLST and spa typing, have several obvious advantages; for example, they are easily used, portable, reproducible, and able to provide comparable results compared to tape-based methods, such as small macrorestricted analysis [14]. One of the key regulators of S. aureus, which is involved in the regulation of bacterial virulence factors, is the agr system. There are currently four agr types identified in S. aureus strains (I, II, III, and IV). According to our study, the predominant agr type among the 55 MRSA isolates was type II, with a frequency of 54.54 percent. The majority (69.5%) of the isolates studied by Ghasemian et al., showed agr I, followed by agr III (30.5%) [36]. The agr type III is the most prevalent type of MRSA isolate, according to a study by Goudarzi et al., in Iran [37]. There is a significant relationship between agr types and specific pathogens [38], and the distribution of agr types varies by geographic region. The selected regions of the spa gene are usually short repeats of sequences with enough polymorphisms to allow isolated typing [20]. In the current work, 27 different spa types were found. spa typing analysis indicated that spa type t030 was the most common spa type found in 25.45% of isolates. The second most frequently identified spa type in our study was t037. These results are consistent with those of other studies in Iran and other Asian countries [10]. The spa type t037 was previously reported by Alreshidi et al. in Saudi Arabia [39], Chen et al. in China [40], and Goudarzi et al. in Iran [37]. In agreement with our study, in China, t030 was found to be one of the most common spa types (52.0%) of MRSA isolates [40]. We believed that t030 would result in longer bacterial survival and easier transmission. In this study, we reported that 7.27% of our isolates had spa type t325 and 19 spa types, which were detected, each, in one isolate.

Conclusion
According to recent studies, presence of these common SCCmec (III), spa (t030), and agr (II) types indicates that these MRSA strains are actively circulating in the healthcare setting of Isfahan Province. Despite the high diversity of our spa types (27/55) in this study, most of them are classified as t030 and t037 and are probably phylogenetically related. There is a possibility that these strains may spread to other parts of Iran or the world in relation to the tourism industry in Isfahan Province. Therefore, the current study emphasizes the importance of molecular typing in tracking global trends in the emergence, spread, and persistence of epidemic MRSA strains. Better infection control guidelines in hospitals, as well as ongoing epidemiological surveillance studies,