Genotyping of methicillin-resistant Staphylococcus aureus in the Sultan Qaboos University Hospital, Oman reveals the dominance of Panton–Valentine leucocidin-negative ST6-IV/t304 clone

The objective of this study was to determine the prevalence and distribution of methicillin-resistant Staphylococcus aureus (MRSA) genotypes circulating at a tertiary hospital in the Sultanate of Oman. A total of 79 MRSA isolates were obtained from different clinical samples and investigated using antibiogram, pulsed-field gel electrophoresis (PFGE), staphylococcal chromosome cassette mec (SCCmec), Spa typing and multilocus sequence typing (MLST). The isolates were susceptible to linezolid, vancomycin, teicoplanin, tigecycline and mupirocin but were resistant to tetracycline (30.4%), erythromycin (26.6%), clindamycin (24.1%), trimethoprim (19.0%), ciprofloxacin (17.7%), fusidic acid (15.2%) and gentamicin (12.7%). Molecular typing revealed 19 PFGE patterns, 26 Spa types and 21 sequence types. SCCmec-IV (86.0%) was the dominant SCCmec type, followed by SCCmec-V (10.1%). SCCmec-III (2.5%) and SCCmec-II (1.3%) were less common. ST6-IV/t304 (n = 30) and ST1295-IV/t690 (n = 12) were the dominant genotypes followed by ST772-V/t657 (n = 5), ST30-IV/t019/t021 (n = 5), ST22-IV/t852 (n = 4), ST80-IV/t044 (n = 3) and 18 single genotypes that were isolated sporadically. On the basis of SCCmec typing and MLST, 91.2% of the isolates were classified as community-associated MRSA and 8.8% of the isolates (consisting of four ST22-IV/t852, one ST239-III/t632, one ST5-III/t311 and one ST5-II/t003) were classified as healthcare-associated MRSA. The study has revealed the dominance of a Panton–Valentine leucocidin-negative ST6-IV/t304 clone and provided insights into the distribution of antibiotic resistance in MRSA at the tertiary hospital in Oman. It also highlights the importance of surveillance in detecting the emergence of new MRSA clones in a healthcare facility.


Introduction
The burden of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is increasing among different patient populations globally [1][2][3]. Following its initial report in 1961 [4], MRSA has remained an important cause of infections in healthcare facilities and in the community globally [1][2][3]. Although previously restricted to healthcare facilities, especially large tertiary-care facilities [5], MRSA has been increasingly identified as a major cause of community-associated infections in previously healthy hosts since the late 1990s [6][7][8]. These new MRSA strains have been described as community-acquired or community-originated MRSA. Community-acquired MRSA can be distinguished from healthcareassociated MRSA isolates on the basis of patient risk factors such as history of previous hospitalization, previous antibiotic treatment, admission to intensive care units, advanced age, location at the time of infection and genetic characteristics [6,7].
Advances in molecular typing techniques, including pulsed-field gel electrophoresis (PFGE) [9], staphylococcal cassette chromosome mec (SCCmec) [10], Spa typing [11,12] and multilocus sequence typing (MLST) [13] have facilitated the study of clonal distributions of MRSA strains isolated in different countries and revealed a diversity in the genetic backgrounds of MRSA isolated in different geographical locations [14]. In addition to the capacity to acquire antibiotic-resistance determinants, some MRSA strains have also acquired the ability to spread rapidly between patients within and between hospitals, thereby causing major problems for infection control. Hence, some epidemic MRSA strains have spread internationally [14]. For example, the epidemic MRSA clones ST239-MRSA-III, ST22-MRSA-IV and ST30-MRSA-IV are widely distributed globally [8] whereas the USA300 MRSA is the dominant MRSA clone in North America and another MRSA clone, the ST80-MRSA-IV clone, is distributed widely in European countries, North Africa, the Middle East and the Gulf Cooperation Council (GCC) countries [15].
Studies on the distribution of MRSA clones in the GCC countries are limited [15][16][17][18]. Although MRSA has been reported in the Sultanate of Oman since 1995 [19], there are no data on the MRSA genotypes prevalent in the country. This study was conducted to determine the prevalence and distribution of MRSA clones in a tertiary hospital in the Sultanate of Oman.

Setting
The Sultan Qaboos University Hospital (SQUH) is a 550-bed teaching hospital of the Sultan Qaboos University. The hospital has 13 different medical departments, which include Surgery, Oral Health, Ophthalmology, Obstetrics & Gynaecology, Medicine, Human Clinical Anatomy, Haematology, Genetics, Family Medicine and Public Health, Emergency Medicine, Child Health, Behavioural Medicine, Anaesthesia and Intensive Care in addition to technical departments.

MRSA isolates
A total of 79 non-repeat MRSA isolates obtained from clinical samples between March and December 2011 at the SQUH were investigated. Isolation and identification of MRSA from clinical samples were performed in the diagnostic microbiology laboratory of SQUH based on cultural characteristics, Gram stain, positive tube coagulase and DNAse tests. Methicillin resistance was confirmed by the amplification of mecA as described previously [20]. The isolates were obtained from samples listed in Table 1. Pure cultures of the isolates were preserved in Cryo-bank vials at À80°C. Molecular typing was performed at the Department of Microbiology, Health Science Centre, Kuwait University, Kuwait.

Detection of genes for Panton-Valentine leucocidin
All isolates were tested for the presence of lukS-PV-lukF-PV, which codes for Panton-Valentine leucocidin (PVL), in PCR assays using previously described primers and protocols [24,25]. PCR products were analysed by agarose gel electrophoresis. Pulsed-field gel electrophoresis The PFGE of SmaI-digested chromosomal DNA was performed as described previously [26]. PFGE patterns were compared using BIOINFORMATICS FPQUEST software version 4.0 software (BioRad, Hercules, CA, USA) and Dice correlation coefficients, with optimization and band position tolerance set at 1.0% and 2.3%, respectively [27].

Spa typing
Spa typing was performed as described by Harmsen et al. [12] for all MRSA isolates. DNA sequencing was performed using a 313091 genetic analyser (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer's protocol. Isolates were assigned to particular Spa types using the Spa typing website (http://www.spaserver.ridom. de).

Multilocus sequence typing
The MLST was performed on all isolates as described by Enright et al. [13]. Isolates were assigned a sequence type (ST) according to the MLST website (http://www.mlst.net).

Results
The 79 MRSA isolates were obtained from 46 male patients and 33 female patients. Forty-three patients were 19-59 years old, 26 patients were ≤18 years old and ten patients were ≥60 years.
Thirty-five (44.3%) MRSA isolates were positive for the presence of lukS-PV-lukF-PV, mostly in isolates that were associated with skin and soft tissue infections and septicaemia but not in isolates recovered from colonization or respiratory tract specimens (Table 1).

Discussion
This study has provided initial data on the prevalence and distribution of MRSA genotypes in the SQUH, a major tertiary hospital in the Sultanate of Oman. The MRSA isolates belonged to diverse genetic backgrounds with ST6-IV/t304 clone, detected in 39.2% of the isolates, as the dominant clone. The dominance of the ST6-IV/t304 clone at the SQUH in Oman was different from the situation in Saudi Arabia [15] and Qatar [18] where ST239-III-MRSA and ST30-IV-MRSA were the dominant MRSA clones, respectively. However, before this report, two ST6-IV/t304 strains were isolated at Tawam Hospital in the United Arab Emirates (UAE) in 2008 [16]. Interestingly, 28 of our ST6-IV/t304 isolates lacked genes for PVL and were susceptible to non-b-lactam antibiotics similar to characteristics of the strains from the UAE hospital [16]. These observations may indicate the expansion of this clone in the GCC countries.
The results also showed that only 8.8% of the isolatesbelonging to ST239-III, ST5-II, ST5-III and ST22-IV cloneswere healthcare-associated MRSA. Therefore, 91.2% of the isolates carrying SCCmec-IV/V genetic elements were community-acquired MRSA. These reports highlight differences in the prevalence of MRSA clones in the GCC countries, strengthening the need for national surveillance for the clonal distribution of antibiotic-resistant pathogens in these countries.
The study also revealed that 44.3% of the isolates carried genes for PVL. This was higher than the 14.6% PVL gene-positive MRSA reported recently in a Kuwait hospital [28] but lower than the 54.2% positive rate obtained in a Saudi Arabian hospital [15], indicating the diversity of MRSA bearing PVL genes in the GCC countries. PVL gene-positive S. aureus have been associated with necrotic skin lesions and commu-nity-acquired necrotic pneumonia [24]. In this study PVL gene-positive MRSA were obtained from skin and soft tissue infections but not from respiratory tract infections. However, the significance of this observation is uncertain because of the small number of MRSA isolated from respiratory tract.
In conclusion, this study has presented the first data on the distribution of MRSA genotypes at the SQUH in Oman. The MRSA isolates belonged to diverse genetic backgrounds with a predominance of CA-MRSA clones comprising ST6-IV/t304 and ST1295-IV/t690, followed by ST772-V/t657, ST30-IV/t019/ t021 and ST80-IV/t044.