Molecular Characterization of Methicillin- Resistant Staphylococcus aureus in a Tertiary Care hospital in Kuwait

Methicillin-resistant Staphylococcus aureus (MRSA) are a major cause of healthcare and community- associated infections due to their ability to express a variety of virulence factors. We investigated 209 MRSA isolates obtained from 1 January to 31 December 2016 using a combination of phenotypic and genotypic methods to understand the genetic backgrounds of MRSA strains obtained in a General hospital in Kuwait. Antibiotics susceptibility was performed with disk diffusion, and MIC was measured with Etest strips. Molecular typing was performed using SCCmec typing, spa typing, and DNA microarray for antibiotic resistance and virulence genes. The isolates were susceptible to vancomycin, teicoplanin, rifampicin, ceftaroline, and linezolid but were resistant to gentamicin, tetracycline, erythromycin, fusidic acid, chloramphenicol and ciprofloxacin. Molecular typing revealed six SCCmec types, 56 spa types and 16 clonal complexes (CC). The common SCCmec types were type IV (39.5%), type III (34.4%), type V (25.8%) and type VI (3.8%). The dominant spa types were t860 (23.9%), t945 (8.6%), t127 (6.7%), t688 (6.7%), t304 (6.2) and t044 (5.7%). The other spa types occurred sporadically. Genes for PVL was detected in 59 (28.2%) of the isolates. CC8-ST239-MRSA-III + SCCmer (23.3%) was the most prevalent clone, followed by CC6-MRSA-IV (8.3%), CC80-MRSA-IV [PVL+] (5.8%), CC5-MRSA-VI + SCCfus (5.0%), CC30-MRSA-IV[PVL+] (4.1%), CC1-MRSA-V + SCCfus [PVL+] (4.1%), CC5-MRSA-V + SCCfus (4.1%) and CC22-MRSA-IV[PVL+] (4.1%). The study revealed that despite the emergence of MRSA with diverse genetic backgrounds over the years, ST239-MRSA-III remained the dominant clone in the hospital. This warrants reassessment of infection prevention and control procedures at this hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen that can cause mild to invasive, life-threatening infections 1 , MRSA was first reported in the United Kingdom, soon after the use of methicillin in the healthcare system in the 1960's 2 , but have since been reported in many countries [3][4][5] . Methicillin-resistance is mediated by mecA gene which encodes penicillin-binding protein 2a (PBP 2a) that results in resistance to beta-lactam antibiotics such as methicillin, cloxacillin and oxacillin 5,6 . The mecA gene is located on a mobile genomic island known as staphylococcal cassette chromosome mec (SCCmec) and is inserted in the chromosome of methicillin-resistant staphylococci 5 . The SCCmec genetic element differ in size and structural organisation, and on the basis of their size and structural differences, 13 SCCmec types designated types I-XIII have been reported 6,7 .
MRSA isolates have been characterised as either hospital or healthcare-hospital-associated MRSA (HA-MRSA) or community-associated (CA-MRSA) on the basis of their SCCmec types. HA-MRSA harbour SCCmec types I, II and III while the CA-MRSA isolates carry SCCmec types IV, V and VI 6,8 . SCCmec typing 9,10 , and other molecular typing methods including pulsed-field gel electrophoresis, multilocus sequence typing, Staphylococcal protein A (Spa) typing, DNA microarray and whole genome sequencing 5,6,[11][12][13] , have been employed to investigate the clonal distributions of MRSA strains in different countries, and have revealed a diversity in their genetic backgrounds in the different geographical locations.
The epidemiology of MRSA strains is constantly changing and their prevalence and characteristics are known to vary between hospitals in the same country or wards in the same hospital 3,14,15 . Consequently, it is important to study MRSA isolates from local healthcare facilities with unique patient populations to obtain data that can aid empirical treatment of infections and to compare them with those obtained in other healthcare facilities in the country. The Farwaniya Hospital, Kuwait is a university-affiliated hospital with 1,200 beds, which provide services that include medical, surgical, orthopaedic, obstetric and gynaecologic, paediatrics, and two intensive care units 16,17 . A previous study conducted on MRSA obtained at the Farwaniya hospital from 1996 to 2001 using pulsed-field gel electrophoresis, and coagulase gene typing revealed the presence of a dominant and persistent multiresistant MRSA clone 16 , that was later identified as ST239-MRSA-III clone 18 20,21 . This study was conducted on MRSA isolates obtained from patients admitted to the Farwaniya hospital in 2016 to determine their antibiotic resistance patterns and clonal distribution and compare the results to those obtained in 1996-2001 for the purpose of assisting in patients' management and control of infections, and to compare the MRSA clones to those in other Kuwait hospitals.

Distribution of MRSA isolates among clinical sources.
We examined MRSA isolates analysed by DNA microarray according to their clinical sources to establish any specific association between clones and clinical sources and infection types. The most common clinical samples that yielded MRSA isolates were wound (N = 52), blood (N = 26), pus (N = 22), and the respiratory samples, sputum (N = 5) and tracheal aspirates (N = 13). The other specimens yielded fewer MRSA isolates. The well-known HA-MRSA clone, ST239-MRSA-III, was the dominant isolate in axilla, groin, and was the major isolate in nasal and wound samples but was isolated sporadically from blood, skin and respiratory samples (sputum and tracheal aspirates). In contrast, blood, wound and respiratory samples yielded higher proportions of diverse clones carrying CA-MRSA genotypes (that is carrying SCCmec IV, V, VI) with none of the clones dominating in the clinical samples.
Erythromycin resistance was associated with different resistance determinants including ermA (rRNA methyltransferase A), ermC (rRNA methyltransferase C), linA (lincosaminide nucleotidyltransferase), msr(A) (macrolide efflux pump) and mph(C) (macrolide phosphotransferase II). ermA was the most prevalent erythromycin resistance determinant. It was detected in 28 ST239 and one CC5 isolates. This was followed by ermC which was detected in 26 isolates of diverse genetic backgrounds. ermB was detected in a single CC59 isolate. Both  1). linA was detected in three CC15 and one ST239 isolates. A single CC97 isolate was positive for vga(A), an ABC transporter that confers resistance to streptogramins.
Microarray also detected resistance determinants to antibiotics that were not tested phenotypically. These included fosB that is associated with fosfomycin resistance, sat (streptothrin acetyltransferase) that is associated with strepthtothrin resistance, Sdrm, a putative transport protein whose function is unknown. The qacA and qacC (multidrug efflux proteins) that mediate resistance to quaternary ammonium compounds. fosB was detected in 73 of the 120 isolates making it one of the common resistance determinants in a variety of genetic backgrounds.

Discussion
Due to constant changes in the epidemiology of MRSA strains it is necessary to conduct regular surveillance to determine clonal MRSA populations circulating in healthcare facilities to understand the evolution and dissemination of specific MRSA clones causing infections for the application of suitable infection control and preventive measures. The results of the present study have provided updates on the distribution and types of MRSA clones that circulated in the hospital in 2016. The isolates belonged to diverse genetic backgrounds with 60.3% and 39.7% of the MRSA isolates carrying the CA-MRSA and HA-MRSA genotypes respectively which is consistent with global trends 5,6 , and with recent reports that CA-MRSA are increasing in Kuwait hospitals 20,21 . The MRSA isolates belonged to six SCCmec types, 56 spa types and 16 clonal complexes (CC), with CC8, CC5, CC1, CC22, CC6, CC80, CC30 and CC97 as the common CCs highlighting the significant changes in the clonal composition of MRSA in the hospital since the last study in 1996-2001 16 . The reasons for the proliferation of the diverse MRSA clones observed in this study is not fully clear. However, the admission of an increasing number of patients already colonized with community genotypes of MRSA into the hospital may offer partial explanation. It is also worth noting that this observation is consistent with observations in different centres where CA-MRSA clones are overtaking the traditional HA-MRSA clones 5,6,14 .
The study showed that 30 (25%) of the 120 isolates analysed by DNA microarray were ST239-MRSA-III making ST239-MRSA-III the dominant MRSA clone in the hospital in 2016 as was the case in 1996-2001 16  The CC97 (ST97-MRSA-V) isolates were first reported in this hospital in 2007 as a cause of an outbreak in a neonatal intensive care and special care units 28 . However, until now, no further CC97-MRSA-V isolates were detected in the hospital following the termination of the outbreak. Curiously, the current CC97-MRSA-V isolates, except one (t359), belonged to spa t267, and were resistant to gentamicin, kanamycin, and fusidic acid similar to the resistance profile of the outbreak isolates even though they were isolated many years apart. Unfortunately, the spa type for the outbreak strains were not determined which makes it difficult to establish whether the same clone reappeared in the hospital in 2016. However, the spa t267 of the current isolates is different from spa t2297 and t1234 that were reported previously among CC97-MRSA-V in other hospitals in Kuwait 18,20 .
We noticed an isolate belonging to CC59 for the first time in Kuwait in this study. CC59 is a predominant CA-MRSA clone in Taiwan 29 , but has also been isolated in Hong Kong 30 , Japan 31 , and Western Australia 31 . However, it is rare in the Arabian gulf region. Hence, this report of CC59 in Kuwait suggests its recent importation into the country. The isolate in this report shares characteristics with the Taiwan (ST59/952-V T /PVL) and Western Australian (ST59-IV) WA-MRSA-118 clones suggesting a common origin.
We observed that the axilla, groins and nose were colonised mostly by the HA-MRSA clone, ST239-MRSA-III, whereas the other sites were dominated by heterogeneous CA-MRSA clones. This is consistent with previous observations that the groin, axilla and nose are often colonised by ST239-MRSA-III strains which usually serve as sources of MRSA transmission 1,5,14 . Also, the dominance of CA-MRSA isolates in wound, pus and skin samples is consistent with earlier reports that CA-MRSA cause predominantly skin and soft tissue infections 1,4,5,8,14 . Furthermore, the isolation of CA-MRSA isolates from blood, and respiratory samples confirms their capacity to cause different types of infections other than skin and soft tissue infections 1,6,14 .
A high proportion of the isolates were resistant to multiple antibiotics including fusidic acid (63.4%), gentamicin (54.5%), tetracycline (54.5%), erythromycin (52.2%), ciprofloxacin (49.8%) and trimethoprim (21.5%). High prevalence of fusidic acid resistance is common among MRSA obtained in Kuwait hospitals. In recent studies, fusidic acid resistance was detected in 16.5% of MRSA obtained in the Maternity hospital 21 , and in 41.2% of MRSA obtained in Kuwait hospitals between 2011 and 2015 32 . The higher prevalence reported in the present study highlights an increasing trend for fusidic acid resistance among MRSA isolated in Kuwait hospitals.The high prevalence of fusidic acid resistance among MRSA isolates was attributed in part to the emerging chimeric genetic elements comprising SCCmec elements and the fusidic acid resistance determinant, fusC (SCCfusC) 20 as also seen in this study ( Table 1). The chimeric genetic element possibly facilitates the transmission of fusidic acid resistance in isolates belonging to different genetic backgrounds. The increasing prevalence of resistance to gentamicin, tetracycline, erythromycin, ciprofloxacin seen in this study have also been previously reported in MRSA otained from hospitals in Kuwait 16,[18][19][20] .
We detected genes for multiple virulence factors including adhesins, haemolysins, enterotoxins and immune evasion clusters. However, the isolates differed in the prevalence of genes for PVL, enerotoxins, agr and capsular polysacharide (cap).The isolates belonged to cap5 (38.3%) and cap8 (61.7%) in agreement with previous observations that most clinical S. aureus isolates harbour cap5 or cap8 33 . The higher prevalence of cap8 in this study mirrors the results of a previous study that showed that 57% S. aureus obtained from food handlers in Kuwait restaurants carried cap8 34 . Similarly, cap8 was prominent among S.aureus obtained from patients in Gabon 35 . This study also revealed that cap type was associated with defined genetic backgrounds.
The agrI was the leading agr type among the isolates in this study followed by agrII and agrIII. Similarly, most (50%) of S. aureus from food handlers in Kuwait restaurants belonged to agrI 34 . AgrI was also the dominant agr type among MRSA isolates obtained in Egypt 36 , India 37 , Iran 38 , Nigeria 39 , Turkey 40 , and China 41 . The study further revealed that the agr types were linked to specific clonal complexes as have been observed in S. aureus isolated from human and food sources in China 41 .
We identified genes coding for PVL in 30.8% (37/120) of the isolates belonging to different genetic backgrounds. This was higher than the 14.6% prevalence of genes for PVL in S.aureus in the same hospital in 2009-2010 17 . The higher prevalence of PVL-gene-positive isolates in this study may reflect the diversity of current clones in the hospital.
We found genes for staphylococcal enterotoxins (SE) in 85.1% of the isolates with most of the isolates harbouring two to six SE genes.Whereas the prominent SE genes in our study were egc (38.3%), sea (37.5%), seg (37.5%), sei (35.8%), sek (32.5%) and seq (32.5%), sea was the most common SE gene detected in MRSA from patients in Turkey 40 , Iran 38 , and Nigeria 39 . Since the carriage of SE genes as well as genes for agr and cap are genotype specific, the dominance of these factors may reflect the dominance of specific genotypes in the different geographical regions.
In conclusion, the evidence gathered from typing our MRSA isolates have revealed the expansion of the population of MRSA clones over time with the healthcare-associated MRSA, ST239-MRSA-III, as the dominant clone despite the emergence of diverse clones with CA-MRSA genotypes. These findings warrant a review of infection control and prevention procedures for the application of effective control measures that will limit antibiotic usage leading to improved patients' care.

Materials and Methods
Bacterial isolates. A total of 209 single patient MRSA isolates were obtained in the Microbiology laboratory of Farwaniya Hospital during a period of 12 months, from January 1 2016, to 31 December 2016. The isolates were collected from cultures of various clinical specimens as part of routine diagnostic care and submitted for molecular typing at the MRSA Reference Laboratory located at the department of Microbiology, Faculty of Medicine, Kuwait University. Only a single isolate from a patient is submitted for typing. Repeat isolates from the same patient are excluded. The isolates were obtained from wound (N = 52; 24.9%), blood (N = 25; 12%), pus (N = 22; 10.5%), Nasal (N = 6;2.8%), Groins (N = 7;3.3%), axilla (N = 5;2.4%), sputum (N = 5; 2.4%), eye (N = 4;1.9%), ear (N = 3;1.4%), fluids (N = 3;1.4%), high vaginal swabs (N = 2; 0.9%) and an unidentified swab (N = 1; 0.4%). All isolates were identified as S. aureus by their cultural characteristics including growth on blood agar (5% sheep blood), growth on mannitol salt agar, Gram stain, positive tube coagulase test and DNase tests. The isolates were stored at −80 °C in 40% glycerol broth (v/v in brain heart infusion broth) and sub cultured onto brain heart infusion agar for purity prior to investigations.
Sccmec typing and spa typing. All MRSA isolates were characterized using SCCmec typing and spa typing. SCCmec typing was performed as described by Zhang et al. 10 , Spa typing was performed as described by Harmsen et al. 13 . DNA microarray. The S. aureus genotyping Kit 2.0 (Alere Technology, Jena, Germany) was used following protocols provided by the manufacturer. The method detects 334 target sequences including genes encoding species markers, PVL, SCCmec, capsule (cap), and accessory gene regulator (agr) group, enterotoxins, adhesins, and antibiotic resistance 4 . DNA microarray was used to assign the MRSA isolates to clonal complexes (CC) as described previously 11 .