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High frequency of SCCmec type IV and multidrug-resistant SCCmec type I among hospital acquired methicillin-resistant Staphylococcus aureus isolates in Birjand Imam Reza Hospital, Iran

Background and Objectives: The ever-increasing of antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) has become a major threat to public health worldwide. Molecular typing is used to determine the source of MRSA infections as well as to control and prevent the spread of these pathogens. The present study aimed to investigate the characteristics of staphylococcal cassette chromosome mec (SCCmec) types and antibiotic resistance of community- acquired (CA-) and hospital acquired (HA-) MRSA isolates. Materials and Methods: In this cross-sectional study, the antibiotic susceptibility patterns of 109 clinical S. aureus isolates were determined by the Kirby-Bauer disk-diffusion and microdilution broth methods. MRSA isolates were confirmed using the polymerase chain reaction (PCR) method for the detection of the mecA gene. SCCmec typing was performed by a multiplex PCR assay among MRSA isolates. Results: The prevalence of MRSA isolates was 39.4%. Linezolid, vancomycin, and ceftaroline were the most effective agents against MRSA isolates. The incidence of multidrug-resistant (MDR) and resistance to most antibiotics were significantly higher in MRSA than methicillin-susceptible S. aureus (MSSA) isolates (P<0.05). SCCmec types I, III, and IV were identified in 27.9%, 23.3%, and 37.2% of MRSA isolates, respectively. SCCmec type I and IV were the most prevalent SCCmec types in HA-MRSA isolates (each was 32.4%). While SCCmec type IV (66.7%) was the most frequently SCCmec type associated with CA-MRSA isolates. Conclusion: Our findings demonstrated a high rate of MDR among MRSA isolates. The high existence of SCCmec type IV was reported among the HA-MRSA isolates, which can indicate the spread of MRSA community isolates to hospital settings. Therefore, appropriate antibiotic stewardship plans and microbiological surveillance initiatives must be implemented in healthcare facilities to monitor and limit the spread of these resistant bugs.


INTRODUCTION
Staphylococcus aureus is one of the most frequent pathogens in community and health care facilities and is considered as a serious threat to human health. This pathogen is responsible for a wide range of diseases from folliculitis to food poisoning, as well as causing life-threatening infections such as bacteremia, endocarditis, necrotizing pneumonitis, and osteomyelitis (1,2).
The emergence of methicillin-resistant S. aureus (MRSA) strains has become an increasing health concern worldwide. The potential for genetic adaptation and the remarkable ability of MRSA strains to acquire resistance to multiple antimicrobials complicated the treatment of related infections. Therefore, a major public health concern still remains with respect to the high morbidity and mortality of infections caused by MRSA, along with increased hospitalization and health care costs (3,4).
Although MRSA infections were originally acquired only from hospital settings (HA-MRSA), outbreaks of infection in the community were first reported in the 1990s. However, CA-MRSA infections are now increasingly spreading in hospital settings and are replacing traditional HA-MRSA strains (2,5). Since antibiotic management and virulence properties of CA-MRSA strains are different from HA-MRSA, it can be important to identify and differentiate the bacteria to reduce unnecessary suffering, the length of hospital stay, and healthcare costs for affected patients (6,7).
The staphylococcal cassette chromosome mec (SCCmec) mobile element carries both the mecA or mecC genes that mediate resistance to methicillin in S. aureus (8,9). To date, thirteen different types of SCCmec elements (SCCmec I-XIII) have been identified based on structural organization and genetic content, and each SCCmec type has individual characteristics (4,10). Noteworthy, SCCmec types I, II, and III are the most seen types found in hospital acquired MRSA (HA-MRSA), whereas types IV and V are prominent SCCmec types among community-acquired MRSA (CA-MRSA) strains (11,12).
Given the importance of global surveillance studies on resistance profiles and epidemiological types of MRSA strains, along with the current challenges in the treatment of infections caused by these pathogens, the present study aimed to investigate the characteristics of SCCmec types and antibiotic resistance of CA-and HA-MRSA isolates in Birjand Imam Reza hospital, Iran. S. aureus isolates were identified using conventional microbiological methods, such as evaluation of colony morphology on sheep blood agar, Gram-staining, catalase activity, production of coagulase, DNase test (Merck, Germany), and mannitol fermentation on mannitol salt agar (Merck, Germany).
Screening of methicillin-resistant S. aureus (MRSA). MRSA strains were identified phenotypically using the cefoxitin disk diffusion method (30 μg; MAST, UK) according to the CLSI guidelines (13). S. aureus isolates with an inhibition zone diameter of ≤21mm around the cefoxitin disk were confirmed as MRSA strain. Detection of mecA gene. The existence of the mecA gene in all MRSA isolates was determined employing PCR assay with specific primers described in Table 1. Genomic DNA was extracted from pure cultures of the isolates using a High Pure PCR Template Preparation Kit (Roche, Germany) according to the manufacturer's instructions. The PCR amplification for the mecA gene was carried out as described previously (14). The amplified products were electrophoresed on 1% agarose gel containing 1× RedSafe DNA stain (Intron, USA).
SCCmec typing. In this study, a multiplex PCR assay with specific primers ( Table 1) described by Boye and colleagues (15) was developed to SCCmec typing (SCCmec type I-V) among MRSA isolates harboring the mecA gene. Amplification of SCCmec genes was performed in a final volume of 25 mL consisting of 12.5 μl of 2× Hot Star Taq Master Mix (Amplicon, Denmark), 3 μl of the DNA template, an optimized amount of each primer with a concentration of 10 pmol/μL (0.5 μl of each β, α3, ccrCF, and ccrCR; 0.3 μl of each 1272F1, 1272R1, 5RmecA, and 5R431), and 6.3 μl of ddH2O. DNA amplification was performed in a thermocycler (PEQLAB, Erlangen, Germany) with an initial denaturation step at 94°C for 4 minutes; 30 amplification cycles each for 45 seconds at 94°C, 30 seconds at 55°C, and 1 minute at 72°C; and followed by an additional extension step of 5 minutes at 72°C. The amplified products were electrophoresed on 1.5% agarose gel containing 1× RedSafe DNA stain (Intron, USA). Noteworthy, the SCCmec types were determined based upon the results of the obtained band pattern comparing to ATCC 10442 (SCCmec type I), N315 (SCCmec type II), 85/2082 (SCCmec type III), MW2 (SCCmec type IVa), and Table 1. Target genes and their primers used in this study.
WIS (SCCmec type V), as reference strains. Isolates with no visible bands, or with a band pattern that was not in agreement with one of the five predicted band patterns, were classified as non-typeable (NT).

Statistical analysis.
The data were analyzed with the Pearson chi-square and Fisher's exact tests, using SPSS software (version 21), to evaluate the statistical significance of associations between potential variables. P-values of less than 0.05 were regarded as statistically significant.
Antibiotic resistance characteristics. The results of the antimicrobial resistance determinations of S. aureus isolates are reported in Table 2. The findings showed a high susceptibility of the isolates to linezolid (100%), vancomycin (100%), ceftaroline (99.1%), and quinupristin/dalfopristin (88.1%). In the present study, 48 S. aureus isolates (44%, 48/109) were identified as multidrug-resistant (MDR) that these MDR SCCmec typing patterns. In the present study, the mecA gene was found in all MRSA isolates. Out of 43 mecA positive isolates, SCCmec types I, III, and IV were identified in 12 (27.9%), 10 (23.3%), and 16 (37.2%) of MRSA isolates, respectively, and five (11.6%) isolates were not typeable. The PCR amplification of products obtained from SCCmec typing of MRSA isolates is shown in Fig. 1. Noteworthy, SCCmec type I and IV were the most prevalent SCCmec types in HA-MRSA isolates (each was 32.4%). While SCCmec type IV (66.7%) was the most frequently SCCmec type associated with CA-MRSA isolates (Table 3). In this study, statistical analysis did not show a significant difference in the frequency of SCCmec types between HA-MRSA and CA-MRSA isolates (P>0.05).
The antimicrobial resistance patterns of MRSA isolates grouped by SCCmec types are summarized in Table 4. The findings showed high resistance to most Table 2. Resistance pattern of S. aureus and MRSA isolates to different antimicrobial agents.     antibiotics in MRSA with type III SCCmec. So that, the MRSA SCCmec type III strains were significantly more resistant to rifampin (P=0.025), gentamicin (P=0.007), tetracycline (P=0.043), and ciprofloxacin (P=0.01) than strains with other types of SCCmec. Furthermore, all MRSA SCCmec type III strains were identified as MDR (100%). In contrast, compared with SCCmec I/III MRSA strains, a greater proportion of SCCmec IV strains were susceptible to most antibiotics tested. The results indicated that the MRSA SCCmec type IV strains were more multidrug-susceptible compared to the other SCCmec types.

DISCUSSION
Today, the emergence of MRSA isolates has become a major challenge in public health. MRSA isolates are commonly MDR strains, and this issue can lead to limited therapeutic options for the control of infections, causing high morbidity and mortality, especially in hospitalized patients (3,10).
The findings revealed that resistance to tetracycline (58.1%), clindamycin (55.8%), erythromycin (44.2%), and ciprofloxacin and rifampin (each was 39.5%) was the most common resistance pattern among MRSA isolates. Moreover, 81.4% of MRSA isolates were identified as MDR. This pattern of antibiotic resistance is in line with the results of many studies. Japoni et al. demonstrated a reduced gradient of MRSA susceptibility to rifampin, co-trimoxazole, clindamycin, tetracycline, ciprofloxacin, and erythromycin (31). In another study, the highest resistance of MRSA isolates was related to levofloxacin, ciprofloxacin, erythromycin, and clindamycin (9). In the study of Rossato and colleagues, the highest levels of resistance among MRSA isolates were reported against erythromycin, ciprofloxacin, and clindamy- cin (4). Nevertheless, in many studies in line with our study, vancomycin, linezolid, and ceftaroline were introduced as the most effective antibiotics against MRSA isolates (16,20,(31)(32)(33).
In this study, the rates of antibiotics resistance in MRSA isolates were higher in comparison with MSSA isolates. The results indicated that resistance to most antibiotics such as erythromycin, rifampin, quinupristin/dalfopristin, clindamycin, doxycycline, gentamicin, tetracycline, and ciprofloxacin was significantly higher in MRSA than MSSA isolates. Noteworthy, the incidence of MDR in MRSA isolates was significantly higher than MSSA isolates. Many studies in line with our study have reported high antibiotic resistance of MRSA isolates to MSSA (10,(33)(34)(35). Hence, the accurate identification and reporting of MRSA isolates would help select the appropriate antibiotic therapy and, control and minimize the spread of these MDR isolates.
In humans, bacterial infections from MRSA have been acknowledged for several years as either HA-MRSA or CA-MRSA depending on the source of infection. CA-MRSA strains have been found to have distinctive genetic composition, antimicrobial characteristics, and virulence properties that set them apart from HA-MRSA strains. Clinically, HA-MRSA strains are usually MDR and the infections caused by them are associated with high morbidity and mortality (2,7,17). In the present study, the infection rate of MRSA isolates (51.4% vs. 16.2%, P=0.0001) was shown to be significantly higher in in-patients (HA) compared with out-patients (CA). A similar result was reported by Preeja et al. where the prevalence of MRSA among in-patients and out-patients was 96.1% and 55.6%, respectively. Furthermore, a significant difference was observed between the isolation of HA-MRSA and CA-MRSA from the inpatient and outpatient groups (2). In another study, the incidence of HA-MRSA and CA-MRSA infection was reported to be 73% and 37%, respectively (23). In the study of Tsige and colleagues, the frequency of MRSA isolates in in-patients (19.5%) was higher as compared to out-patient (5.4%) (36). It is noteworthy that in our study, the resistance to most antibiotics in HA-MRSA strains was higher than CA-MRSA so that this difference was significant for quinupristin/ dalfopristin (29.7% vs. 0%, P=0.018) and clindamycin (62.2% vs. 16.7%, P=0.037). Moreover, among CA-and HA-MRSA, 66.7% and 81.1% were found to be MDR, respectively. These findings are in line with the results of many studies that have reported high antibiotic resistance in HA-MRSA strains compared to CA-MRSA (2,11,18,35). Overall, it should be noted that the high occurrence of MRSA isolates with high rates of resistance to commonly used antimicrobials among hospitalized patients is not surprising due to various hospital-related factors such as long-term hospitalization, the use of various antibiotics for treatment, and underlying immunodeficiency conditions, which predispose patients to acquire MRSA.
Nowadays, applying a simple, rapid and accurate typing method can help identify the source of antibiotic-resistant infections. SCCmec typing provides useful information about antimicrobials resistance, and to determine the epidemiological relationship between various MRSA strains and origin of infection (3,10). In the current study, the most prevalent SCCmec types among MRSA isolates was SCCmec IV (37.2%), followed by SCCmec I (27.9%), and SCCmec III (23.3%). Similar results in some studies indicate the predominance of type SCCmec IV in MRSA isolates (3,4,7,10,37), which can be explained by the fact that the small size of this SCCmec type may facilitate its spread among MRSA isolates collected from hospitals and communities (10,38). Noteworthy, SCCmec type I and IV were the most prevalent SCCmec types in HA-MRSA isolates (each was 32.4%) in our study. While, SCCmec type IV (66.7%) was the most frequently SCCmec type associated with CA-MRSA isolates. In this regard, some studies have shown a high prevalence of SCCmec type IV among the HA-MRSA isolates (2,10,11,23), which can indicate that the MRSA community isolates has spread to the hospital settings. Therefore, applying an action plan with appropriate antibiotic stewardship and the implementation of strict aseptic techniques can help control colonization and the spread of CA-MRSA isolates to health care facilities. Finally, the findings of current study showed a high resistance to most antibiotics in MRSA with types I and III SCCmec, while a greater proportion of SCCmec IV strains were susceptible to most antibiotics. Similar results have been found in many previous studies (4,16,17,39), and this is because SCCmec types I and III is often observed in HA-MRSA isolates, which generally has a high antibiotic resistance.

CONCLUSION
Our findings demonstrated a high rate of MDR among MRSA isolates. In this study, linezolid, vancomycin, and ceftaroline were still effective antibiotics to treat MRSA infections. The present results showed the high existence of SCCmec type IV among the HA-MRSA isolates, which can indicate the spread of MRSA community isolates to hospital settings. It is imperative that appropriate antibiotic stewardship plans and microbiological surveillance initiatives are implemented in healthcare facilities to monitor and limit the spread of these resistant bugs.