Shifts in the Clonal Distribution of Methicillin-Resistant Staphylococcus aureus in Kuwait Hospitals: 1992-2010

PLOS ONE, Sep 2016

Background As the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is constantly changing globally, determining the prevailing MRSA clones in a local healthcare facility is important for better management of infections. This study investigated clonal composition and distribution of MRSA isolates in Kuwait’s hospitals using a combination of molecular typing methods. Materials and Methods In total, 400 non-repeat MRSA isolates were obtained between 1992 and 2010 in 13 public hospitals and were characterized using antibiogram, SCCmec typing, spa typing, and multilocus-sequence typing. Clonal assignment and detection of virulence factors and antibiotic resistance genes were performed by DNA microarray. Results The isolates were resistant to kanamycin (74.2%), erythromycin (69.5%), tetracycline (66.7%), gentamicin (61%), ciprofloxacin, (61%), fusidic acid (53.5%), clindamycin (41.5%), high-level mupirocin resistance (5.2%) and carried aphA3, aacA-aphD, ermA, ermC, mupA, tetK, tetM, fusC and far1. Molecular typing revealed 31 different MRSA clones consisting of ST239-MRSA-III (52.2%), ST22-MRSA-IV (9.2%), ST80-MRSA-IV (7.5%), ST5-MRSA-II/IV/V/VI (6.5%), ST30-MRSA-IV (3.5%), ST241-MRSA-III (2.7%), ST6-MRSA-IV (2.2%), ST36-MRSA-II (2%) and ST772-MRSA-V (1.75%). The isolates differed in the carriage of genes for enterotoxins, Panton–Valentine leukocidin (PVL), toxic shock syndrome toxin (tst-1), arginine catabolic mobile element (ACME) and exfoliative toxins. The number of clones increased from one (ST239-III-t037) in 1992 to 30 in 2010 including ST8-IV-t008 [PVL+] [ACME+] (USA300), ST772-V (Bengal Bay clone) and ST2816 identified for the first time in Kuwait. Conclusion The study revealed that the MRSA isolates belonged to diverse clones that changed in numbers and diversity overtime. Although ST239-MRSA-III, a healthcare-associated clone remained the dominant MRSA clone overtime, the newly emerged clones consisted mostly of community-associated.

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Shifts in the Clonal Distribution of Methicillin-Resistant Staphylococcus aureus in Kuwait Hospitals: 1992-2010

September Shifts in the Clonal Distribution of Methicillin- Resistant Staphylococcus aureus in Kuwait Hospitals: 1992-2010 Samar S. Boswihi 0 1 Edet E. Udo 0 1 Noura Al-Sweih 0 1 0 Department of Microbiology, Faculty of Medicine, Kuwait University , Jabriya , Kuwait 1 Editor: Karsten Becker , Universitatsklinikum Munster , GERMANY As the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) is constantly changing globally, determining the prevailing MRSA clones in a local healthcare facility is important for better management of infections. This study investigated clonal composition and distribution of MRSA isolates in Kuwait's hospitals using a combination of molecular typing methods. In total, 400 non-repeat MRSA isolates were obtained between 1992 and 2010 in 13 public hospitals and were characterized using antibiogram, SCCmec typing, spa typing, and multilocus-sequence typing. Clonal assignment and detection of virulence factors and antibiotic resistance genes were performed by DNA microarray. - OPEN ACCESS Data Availability Statement: All relevant data are within the paper. Funding: This study was supported by grant YM 02/ 12 and Research Core Facility project no. SRUL02/ 13 from Kuwait University Research Sector. S. Boswihi received a graduate student fellowship from the College of Graduate Studies, Kuwait University, Kuwait. Competing Interests: The authors have declared that no competing interests exist. Background Results The isolates were resistant to kanamycin (74.2%), erythromycin (69.5%), tetracycline (66.7%), gentamicin (61%), ciprofloxacin, (61%), fusidic acid (53.5%), clindamycin (41.5%), high-level mupirocin resistance (5.2%) and carried aphA3, aacA-aphD, ermA, ermC, mupA, tetK, tetM, fusC and far1. Molecular typing revealed 31 different MRSA clones consisting of ST239-MRSA-III (52.2%), ST22-MRSA-IV (9.2%), ST80-MRSA-IV (7.5%), ST5-MRSA-II/ IV/V/VI (6.5%), ST30-MRSA-IV (3.5%), ST241-MRSA-III (2.7%), ST6-MRSA-IV (2.2%), ST36-MRSA-II (2%) and ST772-MRSA-V (1.75%). The isolates differed in the carriage of genes for enterotoxins, Panton–Valentine leukocidin (PVL), toxic shock syndrome toxin (tst-1), arginine catabolic mobile element (ACME) and exfoliative toxins. The number of clones increased from one (ST239-III-t037) in 1992 to 30 in 2010 including ST8-IV-t008 [PVL+] [ACME+] (USA300), ST772-V (Bengal Bay clone) and ST2816 identified for the first time in Kuwait. Conclusion The study revealed that the MRSA isolates belonged to diverse clones that changed in numbers and diversity overtime. Although ST239-MRSA-III, a healthcare-associated clone remained the dominant MRSA clone overtime, the newly emerged clones consisted mostly of community-associated. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen causing a wide range of infections [ 1 ]. MRSA was initially confined to healthcare systems and were known as healthcare-associated MRSA (HA-MRSA). However, the epidemiology of MRSA changed with the evolution of community-acquired or community-associated MRSA (CA-MRSA) strains among healthy individuals with no previous exposure to healthcare facilities [ 2 ]. Characteristically, HA-MRSA are usually multi-resistant and carry SCCmec types I, II or III, while CA-MRSA are usually more susceptible to non-beta-lactam antibiotics and carried SCCmec types IV, V or VI [ 3 ]. Molecular typing has provided tools for surveillance and outbreak investigation, which has enhanced the detection of closely related strains [ 3, 4, 5 ]. MRSA constitutes a major drug resistance problem in many hospitals and healthcare settings worldwide leading to limited choices of therapeutic options for the treatment of multi-resistant strains [ 1, 4 ]. By using epidemiological typing methods such as multilocus-sequence typing (MLST) and SCCmec typing, it is possible to group MRSA strains into different clones [ 5, 6 ] and has contributed to the understanding of MRSA transmission in healthcare facilities. The nomenclature of MRSA clones is based on sequence type (ST) and SCCmec types [ 5 ]. Most of the epidemic MRSA isolates belong to MLST clonal complexes (CCs) including CC5, CC8, CC22, CC30, and CC45 [ 5, 6 ]. Other reported clones include CC6, CC7, CC9, CC12, CC15, CC20, CC59, CC75, CC80, CC88, CC93, CC96/ST154, CC97, CC130, CC121, CC152, CC188, CC361, CC395/ST426, CC398, CC509, CC779 and CC913 [ 5,6 ]. Strains belonging to CC97 and CC398 have been associated with livestock (livestock-associated MRSA) [ 7 ]. Some MRSA clones are spread globally while others are restricted to specific geographical regions. For example, while ST239-III and ST22-IV are widespread globally, isolates belonging to ST59 and ST93 have limited geographical spread [ 8, 9, 10, 11 ]. The ST239-III-MRSA is a well-known pathogen that has been associated with healthcare-associated infections. It is a pandemic clone which has been widely reported from Asia, Europe, Middle East, South and North America [5]. DNA sequence analysis of ST239 isolates obtained from early 1980s by Harris et al., [ 12 ] has revealed several variants corresponding to Brazilian, Portuguese, Hungarian and Viennese clones. The ST22-IV-MRSA, also known as epidemic MRSA-15, is another healthcare-associated pathogen that has been reported from different parts of the world [ 5 ]. ST22-IV-MRSA was initially isolated in the UK in 1991 [ 13 ]. Since then it has been reported in other European countries, Australia, Asia, and the Middle East [ 5, 14, 15 ]. Using phylogenomic methods to analyze the genome sequence of 193 S. aureus isolates, Holden et al., [ 14 ] showed that the current pandemic population of EMRSA-15 descends from a healthcare-associated MRSA epidemic that spread throughout England in the 1980s. In recent years, ST22-IV-MRSA has diversified into variants, based on SCCmec subtypes, acquisition of PVL and TSST genes, known as UK EMRSA-15/Barnim, UK EMRSA-15/Middle Eastern variant and CC22-MRSA-IV-PVL+, variants [ 5, 15 ] 2 / 21 The number of MRSA isolates obtained in Kuwait hospitals has increased over the years [ 16 ]. Previous studies revealed that MRSA constituted 32% of S. aureus isolated from clinical samples in Kuwait and consisted of both healthcare-associated and community-associated strains [ 16, 17 ]. Despite these studies, there has been no systematic characterization of MRSA isolated in Kuwait hospitals overtime to catalogue changes in their clonal distribution. The purpose of this study was to investigate the number and type of MRSA clones circulating in major Kuwait hospitals and determine their genotypic characteristics. The strains were investigated using a combination of molecular typing techniques including Staphylococcal chromosome cassette mec (SCCmec) typing, spa typing and multilocus-sequence typing (MLST). Additionally, the study aimed to determine if there were changes in the distribution of the MRSA clones in Kuwait hospitals over time. Tracking the emergence and spread of MRSA and the introduction of new clones will provide information needed to guide policy for appropriate therapy and infection control. In addition, it will provide information about the composition of MRSA clones in Kuwait, which is necessary for epidemiological purposes. Materials and Methods Collection of MRSA strains In total 400 non-duplicate MRSA, isolates representing isolates obtained from different clinical (Table 1) samples in 13 government Kuwait hospitals between 1992 and 2010 were * Wound (53 samples), Pus (26 samples), bed sore (18 samples), abscess (nine samples), Skin (seven samples). ** MRSA screening swab (11 samples), ear (five samples), Urine (five samples), eye (four samples), catheter tips (four samples), foot swab (three samples), Gastro (three samples), perianal (two samples), fluid (two sample), ulcer (two samples), buttocks (one sample), bronchial lavage (one sample), carbuncle (one sample), central venous pressure (CVP) catheter tip (one sample), femoral vein catheter (FVC) tip (one sample), hairline (one sample), laparoscopy (one sample), percutaneous endoscopic gastrostomy (PEG) site (one sample), peritoneal fluid (one sample), tongue swab (one sample), tracheal secretion (TS) (one sample). 3 / 21 investigated. The isolates were selected from a collection of MRSA isolates archived at the MRSA Reference Laboratory in Kuwait. The isolates were previously characterized by pulsedfield gel electrophoresis (PFGE) and interpreted according to the criteria prescribed by Tenover et al., [ 18 ]. Those selected for this study represented different PFGE patterns obtained in 1992– 2010. During the 90’s and early 2000’s, the number of PFGE patterns were small due to the presence of an endemic clone [ 19, 20 ] which is reflected in the small numbers of isolates chosen for this study from these years. The distribution of MRSA and numbers of isolates per year is as follows: 1992 (N = 15), 1996 (N = 20), 1997 (N = 32), 1998 (N = 16), 1999 (N = 21), 2001– 2002 (39), 2005 (N = 52), 2010 (205). Antimicrobial susceptibility testing Antibiotic susceptibility testing was performed by disc diffusion method according to the Clinical Laboratory Standards Institute (CLSI); [ 21 ] with the following antimicrobial disks (Oxoid): benzyl penicillin (10U), cefoxitin (30 μg), amikacin (30 μg), kanamycin (30 μg), tobramycin (10 μg), mupirocin (200 μg and 5 μg), gentamicin (10 μg), erythromycin (15 μg), clindamycin (2 μg), Spectinomycin (25 μg), chloramphenicol (30 μg), tetracycline (10 μg), trimethoprim (2.5 μg), fusidic acid (10 μg), rifampicin (5 μg), ciprofloxacin (5 μg), teicoplanin (30 μg), and linezolid (30 μg). Minimum inhibitory concentration (MIC) for cefoxitin, vancomycin and teicoplanin were determined with Etest strips (AB BioMerieux, Marcy l’Etoile, France) according to the manufacturer's instructions. S. aureus strain ATCC25923 was used as a quality control strain for susceptibility testing. The D-test was used to test for inducible resistance to clindamycin. Susceptibility to fusidic acid was interpreted according to the British Society to Antimicrobial Chemotherapy (BSAC) [ 22 ]. Etest was used to determine the MIC for vancomycin and teicoplanin. The interpretation of the MIC values was based on the antibiotic breakpoint concentration recommended by the CLSI; [ 21 ]. Detection of Panton-Valentine leukocidin (PVL) PVL was performed for all MRSA isolates using primers and PCR protocol published by Lina et al., [ 23 ]. SCCmec typing and Staphylococcus protein A (spa) typing All MRSA isolates were characterized using SCCmec typing and spa typing. SCCmec typing was performed as described by Zhang et al., [ 24 ]. Detection of SCCmec-IV subtypes IVa, IVb, IVc, IVd, IVg and IVh was determined using multiplex PCR described by Zhang et al., [ 24 ] and Milheiriço et al., [ 25 ]. Spa typing was performed as described by Harmsen et al., [ 26 ]. Multilocus sequencing typing (MLST) MLST was performed as described by Enright et al., [ 27 ] on 103 isolates representing different spa types in 1992–2010. Electronic based upon related sequence types (eBURST) (http://eburst. mlst.net) [28] analysis was performed and assigns each ST that shares at least five of seven identical alleles into a single clonal complex (CC). DNA Microarray DNA microarray was performed for 110 MRSA isolates representing each spa type and sequence type isolated from 1992 to 2010. Genes encoding virulence factors and antibiotic resistance were determined using the ArrayMate Reader DNA Microarray platform with 4 / 21 Identibac S. aureus genotyping Kit 2.0 (Alere Technology, Jena, Germany) following protocols provided by the manufacturer. Results Detection of genes for PVL among MRSA isolates The result showed that 62 (15.5%) isolates were positive for PVL, while 338 isolates (84.5%) of the isolates was negative. Molecular typing of MRSA isolates The result of SCCmec typing revealed five SCCmec types including types II (N = 18; 4.5%), III (N = 221; 55.2%), IV (N = 127; 31.7%), V (N = 33; 8.2%), and VI (N = 1; 0.2%). There were 60 spa types with the dominant ones being t421 (N = 86; 21.5%) and t037 (N = 76; 19%). Other common spa types were t945, t044, t223 and t860 which were detected in 27 (6.7%), 23 (5.7%), 19 (4.7%) and 18 (4.5%) isolates, respectively. Spa types t002 and t030 were each detected in 11 (2.7%) isolates; t019 was detected in 10 (2.5%) isolates. The other spa types occurred sporadically. A population snap shot of the relationship of the spa types is presented in Fig 1. It divided the isolates into eight spa clonal complexes (Spa-CC) with Spa-CC421 as dominant followed by Spa-CC223. Twenty-seven sequence types (STs) were identified among the 103 selected for MLST. For the remaining 297 isolates, ST was predicted based on their spa types. The majority of the isolates (209; 52.2%) belonged to ST239. This was followed by ST22 (37; 9.2%), ST80 (30; 7.5%), ST5 (N = 26; 6.5%), ST30 (N = 14; 3.5%), ST241 (N = 11; 2.7%), ST6 (N = 9; 2.2%), ST36 (N = 8; 2%), and ST772 (N = 8; 2%). ST8 was detected in seven (1.7%) isolates, while ST97 was Fig 1. Spa clonal complex (Spa-CC) of MRSA isolates. 5 / 21 detected in six (1.5%) isolates. Four (1%) isolates, each belonged to ST1, ST88, ST121 and ST913. ST113 and ST225 were detected in three (0.7%) isolates each, whereas ST105, ST149 and ST361 were each detected in two (0.5%) isolates. The other sequence types (STs) consisting of ST46, ST72, ST508, ST1289, ST1465, ST1637 occurred in single isolates (0.2%). A new sequence type, ST2816 was described in this study. Characterization of MRSA clones in Kuwait hospitals The application of eBurst identified 13 clonal complexes and one singleton, while the combination of MLST and SCCmec typing had identified 32 different MRSA clones. The distribution of MRSA clones and their resistance and virulence genes profiles are shown in Tables 2–5. All of the tested MRSA isolates carried combination of genes encoding virulence factors including hemolysins, leukocidins, Staphylococcal enterotoxins, Staphylococcal superantigen/enterotoxin-like genes (ssl/set), epidermal cell differentiation inhibitors (edinA, edinB, edinC). In addition, they carried at least one of the genes encoding proteases such as aureolysin encoding genes (aur), serine protease (splA, splB, splE), glutamylendopeptidase (sspA), staphopain B protease (sspB), staphopain A protease (sspP). Furthermore, they carried adhesion factors/ MSCRAMM encoding genes including bone sialoprotein-binding protein (bbp), collagenbinding adhesion (cna), clumping factors (ClfA, ClfB), cell surface elastin binding protein (epbs), enolase (eno), fibrinogen binding protein (fib), fibronectin-binding protein A (fnbA), S. aureus surface protein G (sasG), Ser-Asp rich fibrinogen-/bone sialoprotein-binding proteins (sdrC, sdrD), van Willebrand factor binding protein (vwb), surface protein involved in biofilm formation (bap), and at least one of the biofilm encoding gene, ica (icaA, icaC, icaD). In addition, nearly all strains carried hyaluronate lyase (hysA). Clonal complex 1 (CC1). The seven isolates that belonged to CC1 were obtained in 2010. They consisted of two sequence types; ST1-V (three isolates) and ST772-V (four isolates). The ST1-V isolates corresponds to the ST1-MRSA-V SCCfus clone, while the ST772-V isolates correspond to the Bengal Bay/WA MRSA-60 clone. The ST1-V isolates consisted of three spa types, t127, t321, and t6811, which were positive for agrIII and cap8 but varied in their carriage of PVL and antibiotic resistance genes. ST1-V-t6811 isolate was negative for PVL but resistant to erythromycin and clindamycin mediated by ermC, whereas t127 and t321 isolates were positive for PVL, and were multiresistant to gentamicin, kanamycin, tetracycline, fusidic acid, ciprofloxacin, tobramycin and carried aacA-aphD, aphA3, fusC, sdrM and sat determinants. The ST772-V isolates corresponds to the Bengal Bay/WA MRSA-60 clone. The ST772-V isolates belonged to four spa types, t345, t657, t10795 and a novel spa type t12211 described during this study. All ST772-V isolates were positive for agrII, cap5, sea, egc gene cluster (seg, sei, selm, seln, selo, selu) and PVL but differed in the carriage of sec and sel (Table 2). All isolates were multiresistant and carried msrA, mphC, aphA3, tetK, sat, fosB and sdrM. In addition, ST772-V-t657/t12211 isolates were resistant to gentamicin and carried aacA-aphD (Table 2). Clonal complex 5 (CC5). The CC5 isolates were detected from 2001 to -2010. They belonged to six clones consisting of (i) ST105-MRSA-II-t002 [tst+] isolates which corresponds to the New York-Japan clone, (ii) the ST5-II-003/t105/t242 and ST225-II-t045 [tst-] isolate which correspond to the New York-Japan/Rhine-Hesse EMRSA clone, (iii) The ST5-IV-t306/ t2164 isolates which correspond to the Pediatric clone, (iv) The ST149-IV-t1154/t2164 (fusC) isolates which corresponds to the Maltese clone, (v) ST5-V-t688 and ST1637-V-t5258 isolates carrying sed, sej, ser which corresponds to WA-MRSA-11/34/35 /90/108, and (vi) the ST5-VIt688 isolate which corresponds to the New Pediatric clone. All CC5 isolates harbored agrII, and cap5 and the genes for virulence determinants and antibiotic resistance summarized in Table 2. 6 / 21 *the isolate also carried sed. **the isolate also carried TSST, sec and sel. Abbreviations: Ak, amikacin; C, chloramphenicol; CN, gentamicin; CIP, ciprofloxacin; CC, clindamycin; E, erythromycin; FD, fusidic acid; K, kanamycin; HLR-Mup, high-level mupirocin resistance; RF, rifampicin; TE, tetracycline; TOB, tobramycin; SH, spectinomycin; W, trimethoprim. ST105-II-t002 isolate was PVL-negative, but was positive for tst gene in addition to genes, sea and egc gene cluster. The ST5-II-003/t105/t242 and ST225-II-t045 isolates were PVL- and tst-negative but carried enterotoxins genes sed, sej, ser, egc as shown in Table 2. In addition, t003 and t045 isolates carried an additional gene, sea. The ST5-II-t003/t105/t242 and ST225-II-t045 isolates were resistant to erythromycin and clindamycin, and carried ermA. However, each spa type showed differences in the carriage of aacA-aphD, aadD, dfrS1, and mupA (Table 2). The ST5-IV clone consisted of two spa types, t306 and t2164, which were PVL-positive and carried similar enterotoxins genes, sea, sed, sej, ser and egc, and contained ermC mediated erythromycin and clindamycin resistance (Table 2). The ST149-IV was PVL-negative but carried sea and egc. In addition, ST149-IV-t1154 isolate was positive for sec, sel and tst. All ST149-IV-t1154/t2164 isolates were resistant to erythromycin, clindamycin and fusidic acid and carried ermC, fusC, fosB and sdrM. The ST5-V-t688 isolate carried sea, sed, sej, ser and egc and was sensitive to the majority of the antibiotics tested in this study except tetracycline mediated by tetK and tetM. In addition, the isolate harbored fexA, fosB and sdrM determinants. The ST1637-V-t5258 isolate was PVLnegative, but was positive for sea, sed, sej, ser and egc and was resistant to the aminoglycosides, gentamicin, kanamycin, amikacin and tobramycin and harbored aacA-aphD, fosB and sdrM determinants. The ST5-VI-t688 clone harbored sed, sej, ser and egc and were multiresistant carrying XylR, vga, dfrA, tetM, fexA, fosB and sdrM. Clonal complex 6 (CC6). The two representative CC6 isolates selected for microarray analysis carried sea, agrI, cap8 and the virulence and antibiotic resistance genes summarized in 7 / 21 MRSA clones CC8 ST8-IV ST8-IV ST8-IV ST113-IV aOne of the isolates obtained in 2001 lacked LukF-PV (P83). bST239-III isolates carried sea, sak, chp, scn, ermC, lnuA aphA3, qacA, qacC, Sat, tetK, cat, merA, merB, mupA variably. cOne t037 isolate obtained in 2005 lacked ermA. dST241-III isolates carried fnbB, ermC, tetK, qacA, qacC, sat, cat variably. and 2010 carried ermC and one isolate obtained in 2005 lacked ermA and ermC. The ST239-III-t037 isolates, except three isolates obtained in 1999, 2005 and 2010 harbored quaternary ammonium compound resistance genes, qacA or qacC. Two isolates obtained in 1996 and 2010 were resistant to chloramphenicol and carried cat (Table 3). The ST8-IV-t064 (two isolates) and ST113-IV-t064 (one isolate) clones that were isolated in 2001–2002 and 2010, respectively, resembled USA500 clone. All three isolates were PVL- and ACME-negative but carried enterotoxin genes, sea, seb, sek and seq. ST113-IV-t064 carried an additional enterotoxin gene, sed. All three isolates were resistant to gentamicin, kanamycin, tobramycin, erythromycin, clindamycin, tetracycline, trimethoprim and ciprofloxacin and carried ermC, aacA-aphD and fosB. Although, both clones were resistant to tetracycline, ST8-IVt064 isolates carried tetM, while ST113-IV-t064 isolate carried tetK. ST8-IV-t064 and ST113-IV-t064 isolates differed in their resistance to amikacin, chloramphenicol and highlevel mupirocin. Whereas, ST8-IV-t064 isolates were resistant to amikacin and high-level mupirocin resistance and carried aadD, mupA and qacC, the ST113-IV-t064 isolate was resistant to chloramphenicol and carried cat gene. One isolate belonging to ST8-IV-t064 was resistant to teicoplanin (MIC: 24 μg/L) but lacked vanZ. The ST8-IV-t008 isolate, resembling the Lyon/UK EMRSA-2 clone was isolated in 2005. It was negative for PVL and ACME but positive for sea. The ST8-IV-t008 [PVL+] [ACME+] that resembled the USA300 was isolated in 2010. The isolate carried enterotoxin genes, sek and seq. ST113-IV-t064 isolate that resembled UK EMRSA-14/WA MRSA-5 clone obtained in 2005 was negative for PVL, ACME and enterotoxin encoding genes. The ST8-V-t064 isolate yielded negative results for PVL and ACME but carried sea, seb, sek and seq. Clonal complex 22 (CC22). CC22 consisted of 37 isolates, all of which belonged to ST22. Thirteen isolates obtained in 2005 (three isolates) and 2010 (10 isolates) were selected for microarray analysis. The ST22 isolates belonged to 10 spa types including t032, t223, t309, t790, t852, t2251, t3107, t3935, t5708 and t5983. All representative ST22 isolates were positive for agrI and cap5. The ST22-IV isolates were positive for virulence genes and antibiotic resistance genes summarized in Table 4. Microarray analysis classified the ST22-IV isolates into three groups comprising (i) t032-SCCmec IVh and t790-SCCmec IVa obtained in 2005 and 2010, respectively, were related to UK EMRSA-15/Barnim-EMRSA clone; (ii) the t223 (2005 and 2010), t309 (2010), t2251 (2005), t3935 (2010) and t5708 (2010) isolates were related to UK EMRSA-15/Middle Eastern variant; and (iii) the t852, t3107 and t5983 isolates obtained in 2010 were related to CC22-MRSA-IV [PVL+] clone. Clonal complex 30 (CC30). Isolates belonging to CC30 were first isolated in 1996 and continued to be isolated until 2010. The 11 CC30 isolates selected for microarray analysis belonged to two sequence types, ST30-IV (seven isolates) and ST36-II (four isolates). The ST30-IV isolates belonged to spa types, t019, t318, t345 and t1130, whereas the ST36-II isolates belonged to spa types t018 and t605. Microarray analysis revealed that the ST30-IV isolates were similar to the CC30-IV [PVL+] Southwest Pacific clone, whereas the ST36-II isolates were similar to the UK EMRSA-16 clone. The ST30-IV-t019/t318/t345/t1130 isolates which resembled the Southwest Pacific clone were positive for PVL, agrIII, cap8 and egc. The ST30-IV-t345 carried an additional enterotoxin gene, sea. The ST30 isolates expressed different antibiotic resistance determinants. While the ST30-IV-t1130 isolate expressed resistance to erythromycin, clindamycin, spectinomycin and trimethoprim and harbored ermA, sdrM, dfrS1 and fosB, the ST30-IV-t019/t345 isolates were susceptible to the non-beta-lactam antibiotics tested but harbored sdrM and fosB. The 9 / 21 a One of the isolates obtained in 2005 also carried seb, sec, and sel. b the isolate also carried sec. c the isolate also carried sea. d the isolate also carried sea. *t318 isolate was resistant to trimethoprim but lacked dfrS1 and lacked chp-encoding gene. **t018 isolate obtained in 1999 carried aphA3, sat, tetK, TSST but lacked sea, while the isolate obtained in 2010 carried ermC, aacA-aphD, dfrS1, and qacC. ***the isolate carried seb, sek, seq. §t042 isolate lacked tetK. ¥ one of the t044 isolates obtained in 2001 lacked tetK, while the isolate obtained in 2010 carried qacC. egc TSST, egc TSST, egc TSST, egc TSST, egc PVL, egc PVL, egc PVL, egc PVL, egc sea, egc TSST, sea, egc egc seb, egc, ACME sec, sel, egc etD PVL, etD ST30-IV-t318 isolate was also resistant to erythromycin, clindamycin and spectinomycin and carried ermA (Table 4). The ST36-II isolates consisted of ST36-II-t018 (N = 3) and ST36-II-t605 (N = 1). All ST36-II were positive for agrIII, cap8 and egc gene cluster. All isolates were PVL-negative. Two isolates obtained in 1999 (t018 and t605) were tst-positive. The ST36-II isolates were resistant to kanamycin, erythromycin, clindamycin, spectinomycin and ciprofloxacin, and carried ermA, aadD, sdrM and fosB. Of the three ST36-II-t018 isolates, one isolate obtained in 2010 was resistant to gentamicin and carried additional aacA-aphD and additional erythromycinresistance determinant, ermC. Another isolate obtained in 1999 carried aphA3. Two isolates obtained in 1999 were resistant to tetracycline and carried tetK, while two isolates obtained in 1999 and 2010 were resistant to trimethoprim and carried dfrS1 (Table 4). Clonal complex 45 (CC45). CC45 was represented by two isolates; ST46-IV-t370 and ST508-IV-t050 detected in 2001–2002 and 2005 respectively. Microarray analysis revealed that clones ST46-IV-t370 resembled the Berlin EMRSA clone, whereas ST508-IV-t050 resembled 10 / 21 the CC45-MRSA-IV [ACME+]. Both clones were positive for agrI and cap8. The ST46-IVt370 isolate was PVL and ACME-negative and carried egc and aphA3 encoding kanamycin resistance (Table 4). The ST508-IV-t050 isolate was PVL-negative but was positive for ACME, seb and egc, and was sensitive to all non-beta lactams antibiotics (Table 4). Sequence types 72 (ST72). One isolate, ST72-V-t4000 which resembled WA MRSA-91 was negative for PVL and ACME but was positive for sec, sel and egc. The isolate was resistant to gentamicin, kanamycin, tetracycline, tobramycin and fusidic acid and carried aacA-aphD, tetkK and fusc (Table 4). Clonal complex 80 (CC80). Isolates belonging to ST80-IV were obtained in 1997 (one isolate), 1998 (one isolate), 1999 (two isolate), 2001 (three isolate), 2005 (one isolate) and 2010 (four isolate). The isolates belonged to six spa types including t018, t042, t044, t376, t1154, t8154. However, the majority (seven isolates) of the isolates obtained in 1997, 1998, 1999, 2001, 2005 and 2010 belonged to t044. All the ST80-IV representative isolates were positive for agrIII and cap8. The PVL-positive ST80-IV isolates were similar to the CC80-IV [PVL+] European CA-MRSA clone, while the PVL-negative ST80-IV isolates defined a different CC80-MRSA variant. Genes for virulence and antibiotic resistance of CC80 are summarized in Table 4. Clonal complex 88 (CC88). Four CC88 representative isolates, consisting of ST88 (three isolates) and ST1289 (one isolate) were selected for microarray analysis. The three ST88 isolates were ST88-IV-t690, ST88- IV-t4067 and ST88-IV-t5041. The ST1289-IV isolate belonged to spa type t5562. All CC88 isolates were positive for agrIII and cap8. Based on microarray analysis, the [PVL+] ST88-V isolate correspond to the CC88-MRSA-IV [PVL+] group, while the [PVL-] ST1289-IV resembled WA MRSA-2 (Table 5). The ST88-IV isolates differed in their carriage of enterotoxin encoding genes. The ST88-IVt4067/t5041 isolates carried sea, while the ST88-IV-t690 isolate carried sea, sek and seq. One t690 isolate carried lukF-PV and lacked lukS-PV. The isolates were also non-multiresistant to antibiotics and carried few antibiotic resistance genes. All the ST88-IV isolates were resistant to ermC, tetK dfrS1 dfrS1, fosB fosB aphA3, Sat, fosB ermC tetK PVL, sea Sak, scn PVL, seb, egc PVL, seb, egc TSST, sel, egc Sed, etA, etD MRSA clones CC88 ST88-IV ST1289 CC97 ST97-V CC121 ST121-IV ST121-V CC361 ST361-IV CC913 ST913-IV New sequence type ST2816-V *t690, **t4067, t5041 t5562 t1234, t13204 t314 t314 t991 t605 ***t315, ****t1427 E, CC, TE W W K TE, CIP *the t690 isolate carried sek, seq and LukF-PV and lacked LukS-PV. ** The t4067 isolate was sensitive to tetracycline and lacked tet gene. *** The t315 isolate also carried sec. ****the t1427 isolate also carried seb. 11 / 21 erythromycin and clindamycin and carried ermC. In addition, the two ST88-IV isolates belonging to t690 and t5041 were resistant to tetracycline and carried tetK. The ST1289-IV-t5562 (WA MRSA-2 clone) isolate lacked enterotoxin and PVL genes, and was only resistant to trimethoprim and carried dfrS1 (Table 5). Clonal complex 97 (CC97). All four CC97 isolates were obtained in 2010. The isolates belonged to ST97-V and spa types t1234 and t13204. All CC97 isolates were positive for agrI and cap5,sak, scn, but were negative for genes encoding staphylococcal enterotoxins, PVL, TSST-1, ET and ACME (Table 5). The CC97 isolates were susceptible to non-beta-lactam antibiotics tested, although they carried sdrM (Table 5). Clonal complex 121 (CC121). The three representatives of CC121 isolates belonged to ST121-IV-t314 (one isolates) and ST121-V-t314 (two isolates) obtained in 2005 and 2010, respectively. The ST121 isolates were positive for PVL, seb, egc, agrIV and cap8 (Table 5). The isolates were sensitive to most of the antibiotics tested but harbored fosB and sdrM. One ST121-IV-t314 isolate was resistant to trimethoprim and carried dfrS1 (Table 5). Clonal complex 361 (CC361). CC361 consisted of two isolates, ST361-IV-t315 and ST361-IV-t1427, that were obtained in 2010. Microarray analysis showed that ST361-IV-t315/ t1427 isolates resembled WA MRSA-29. The isolates were positive for tst, agrI, cap8, sel and egc but differed in their carriage of seb and sec. The ST361-IV-t1427 isolate carried seb, while the ST361-IV-t315 isolate carried sec determinant (Table 5). The ST361-IV isolates were resistant to kanamycin and carried aphA3. All isolates carried sat, fosB and sdrM (Table 5). Clonal complex (CC913). CC913 isolates were all isolated in 2010 and were represented by four isolates that belonged to ST913-IV-t991. A representative isolate selected for microarray analysis was positive for agrII, cap8, sed, etA, etD and the resistance determinants ermC and sdrM (Table 5). New sequence type (ST). A new sequence type, ST2816, was identified during this study. The isolate carried SCCmec-V and belonged to spa type t605. The isolate was negative for enterotoxins, PVL, TSST-1, ET and ACME but was positive for ebh, fnbB, map, sak and scn (Table 5). It was resistant to tetracycline and ciprofloxacin and carried tetk. Changes in the prevalence of MRSA clones from 1992 to 2010 *CA-MRSA clones (aN) ST30-IV-t019 (2) ST80-IV-t044 (1) ST80-IV-t044 (1) ST30-IV-t019 (2) ST80-IV-t044/ t1154 (2) ST5-IV-t306 (1) ST8-IV-t064 (2) ST30-IV-t318 (2) ST80-IV-t044/t018/t852 (6) ST508-IV-t050 (1) ST6-IV-t304 (1) ST8-IV-t008 (1) ST30-IV-t019/t345 (2) ST46-IV-t370 (1) ST72-V-t4000 (1) ST80-IV-t044 (5) ST113-IV-t064 (2) ST121-V-t314 (1) *CA-MRSA clones (aN) ST2816-V-t605 (1) aNo. of MRSA isolates. bMRSA clones detected in the 90’s and the early 2000’s. cMRSA clones detected only in 2010. *MRSA strains were classified as HA-MRSA or CA-MRSA based on the carriage of SCCmec types. HA-MRSA carries SCCmec I, II and III+ST22-IV. CA-MRSA carry SCCmec IV (except ST22), V, VI and others [ 5, 61 ]. (Table 6). Additionally, it was observed that clones detected in previous years were associated with different spa types in 2010. For example, the ST5-IV clone previously detected in 2001– 2002 belonged to spa type t306, but ST5-IV that were isolated in 2010, were associated with spa types, t002, t688 and t2164. Similarly, ST5-II clone that was detected in 2001–2002 was associated with spa type t105, but in 2010, the ST5-II was detected in four isolates that were associated with spa types, t002, t003 and t242. Similarly, the 33 ST22 isolates obtained in 2010 contained new spa types, t852, t790, t3107, t309, t3935, t5708 and t5983 in addition to spa types, t223 and t032 that were detected in 2005. The ST239-III-t037/t421/t945 clone was found in 69 isolates in 2010 including spa type, t860 that was not detected in previous years. The 18 new clones that appeared in 2010 consisted of three HA-MRSA clones, ST225-IIt045, ST105-II-t002, and ST1465-III-t459, and 15 CA-MRSA clones consisting ST5-V-t688/ t2164, ST772-V-t345/t657/t10795/t12211, ST97-V-t1234/t13204, ST1-V-t127/t321/t6811, ST88-IV-t690/t4067/t5041, ST913-IV-t991, ST8-V-t064, ST121-IV-t314, ST149-IV-t1154/ t2164, ST361-IV-t315/t1427, USA300/ST8-IV-t008, ST5-VI-t688, ST1289-IV-t5562, ST1637-V-t5258 and ST2816-V-t605. Discussion This study revealed a striking diversity in the numbers and types of MRSA clones obtained in Kuwait hospitals from 1992 to 2010. Although, several MRSA clones were identified, the majority (64.2%) corresponded to the well-established healthcare-associated ST239-III and ST22-IV MRSA clones [ 5 ]. Microarray analysis distinguished two types of ST239-III isolates in this study. One type, consisting of spa types, t037/t421/t860/t945, resembled the well characterized pandemic clone also known as the Hungarian/Brazilian clone [ 8, 9 ] which is one of the most successful MRSA lineages reported in many parts of the world [ 29, 30, 31, 32 ] including the Middle East [ 33, 34, 35 ]. The isolates in this group were positive for sea, sek and seq but lacked PVL and ACME. These were similar to ST239 strains reported in Saudi Arabia [ 33 ], Turkey [ 34 ], Russia [ 36 ] and Germany [ 37 ]. The second type consisted of ST239-III-t030 and ST1465-III-t459, characterized by their carriage of ACME genes. ACME has been widely reported among ST8-IV (USA300) [ 38 ] clone but less commonly in other MRSA clones [ 39, 40 ]. However, ACME-positive ST239-III MRSA isolates have been reported in Australia [10], Singapore [ 41 ], Malaysia [ 42 ] and strains belonging to other genetic backgrounds such as ST764-SCCmec-IIa obtained from cases of acute otitis media in Japan [ 43 ] indicating that ACME is spreading among MRSA with different genetic backgrounds. This report of ACME in MRSA isolates obtained in Kuwait hospitals signifies the emergence of ACME producing MRSA clone in our region. Microarray analysis of antibiotic resistance genes in these isolates yielded two interesting results. First, it showed that none of the fusidic acid resistant ST239-MRSA-III isolates carried 14 / 21 fusB or fusC determinants included in the microarray panel suggesting the involvement of an additional mechanism of fusidic acid resistance such as fusA [ 44 ]. A previous study in Kuwait showed that fusidic acid resistant MRSA isolates expressed chromosomally-mediated high levels of fusidic acid (MIC: 256 μg/L) suggesting the presence of fusA [ 45 ]. Therefore, the isolates in this study may harbor fusA. Secondly, the microarray results revealed the presence of fosB encoded fosfomycin resistance that was not previously reported in MRSA isolates in Kuwait. Fosfomycin susceptibility testing is not performed routinely in Kuwait, which explains why the resistance was missed even though a substantial number of the isolates were resistant. The detection of this resistance by microarray highlights the benefits of this technology over traditional antibiotic susceptibility testing in detecting novel resistance determinants. ST22-IV clone was the second most common clone isolated in Kuwait hospitals. It is a wellknown epidemic MRSA clone that emerged in the United Kingdom in the early 1990s [ 13 ] and has become prevalent in Europe since then [ 31, 46, 47, 48 ]. The results of this study showed that ST22-IV MRSA corresponded to three groups including CC22-UK EMRSA-15/Barnim EMRSA, CC22-MRSA-IV-PVL+ and CC22-tst+ UK EMRSA-15/Middle Eastern variant clones. Interestingly, the majority of the isolates were related to the tst+ UK EMRSA-15/Middle Eastern variant [15]. The diversity of the ST22-IV variants circulating in Kuwait hospitals may reflect diverse sources of their acquisition. The other major MRSA clones detected during this study included ST80-IV, ST30-IV, ST6-IV and ST772-V. ST80-IV- MRSA was previously reported as the most common CA-MRSA clone in Kuwait hospitals in 2005–2006 [ 16, 17 ] and was postulated to consist of European and some indigenous clones due to differences in their antibiotic resistance phenotypes [17]. The present results showed that the ST80-IV MRSA were heterogeneous, belonged to different spa types including t044, t042, t8154 and t376 which confirms previous suggestions of their diverse route of acquisitions [ 17 ]. Surprisingly only 2.9% (6/205) of the isolates obtained in 2010 were ST30-IV-MRSA. Whereas in our previous studies ST30-IV-MRSA was the second dominant CA-MRSA in Kuwait hospitals with prevalence of 30% and 22% of MRSA isolated in 2001–2003 and 2006 respectively [ 16,17 ]. The reduced number of ST30-IVMRSA obtained in 2010 may reflect changes in the MRSA population in Kuwait hospitals. ST6-IV-t304 MRSA was recently reported as the dominant MRSA clone in a hospital in Oman [ 35 ]. Spa type t304 has also been reported in MRSA isolates in UAE [ 49 ], MSSA obtained from patients in Lebanon [ 50 ] and in MSSA from Camels in UAE [ 5 ] suggesting that the strains may be transmitted between humans and Camels. It would be interesting to undertake further investigations into the prevalence of this clone in the region. The ST772-V MRSA, known as the Bengal Bay clone because of its origin in Bangladesh and India [ 51 ], is becoming the dominant MRSA clone in India [ 52 ] but has also been reported in Australia where it is called WA60 [ 53 ] and in the United Kingdom [ 54 ]. Although ST772-V clone was reported previously from patients in Saudi Arabia [ 33 ], UAE [ 49 ], Qatar [ 55 ] and Oman [ 35 ], this is the first report in Kuwait hospitals. ST772 isolates were usually associated with spa type t657. However, other spa types including t10795, t345 and t12211 were detected in small numbers in this study signaling the evolution of variants of the Bengal Bay clone. The ST8-IV-t008, also known as USA300, was also detected for the first time in Kuwait hospitals. The USA300 is a multiresistant, PVL+ CA-MRSA clone that has been recognized as the leading cause of community-associated as well as healthcare-associated infections in North American hospitals [ 56, 57, 58 ]. The USA300 MRSA clone is also increasingly being reported in hospitals outside North America [ 34, 43, 59 ]. The detection of the USA300 CA-MRSA clone in Kuwait highlights its growing importance as an international epidemic MRSA clone. The ST5 MRSA isolates were heterogeneous in the carriage of SCCmec elements. ST5 isolates carried SCCmec II (1.2%), SCCmec IV (2.7%), SCCmec V (2.2%) and SCCmec VI (0.2%). 15 / 21 Isolates carrying SCCmec II genetic element are classified as HA-MRSA whereas those carrying SCCmec IV, V and VI are classified as CA-MRSA. Therefore, the ST5 isolates in this study consisted of HA MRSA and CA-MRSA clones. The low prevalence of ST5-IV MRSA in this study (2.7%) mirrors the situation in Asian countries such as Japan, China, Taiwan [ 60 ] and Sri Lanka [ 32 ] where ST5-IV-MRSA have been rare. Other members of CC5 identified in this study included the ST5/ST1637-V, which was related to WA MRSA 11/34/35/90/108 [ 53 ], the ST5-II, related to the New York/Japan MRSA clone, ST149-IV, related to the Maltese clone, and the ST5-VI, which was related to the New Pediatric clone. All of the CC5 isolates carried the egc gene cluster and all CC5 isolates, except those related to the Maltese (ST149-IV) and [tst+] New York/Japan clones (ST5-II) carried an additional enterotoxin, ser [ 61 ]. The egc gene cluster was also common in CC5 isolates reported from Western Australia [ 53 ], Malta [ 61 ], Germany [ 37 ], Ireland [ 62 ] and USA [ 63 ]. In addition, this study and studies from Malta and Ireland reported the presence of sea and sej among the CC5 isolates [ 61,62 ]. Whereas, ST5-IV (Pediatric clone) were PVL-positive, tst was found in ST105-II (New York/Japan clone) and ST149-IV (Maltese clone) isolates. ST97-V isolates were detected in six MRSA isolates in this study. ST97-MRSA-V isolates were previously reported to cause an outbreak in a neonatal unit of a Kuwait hospital in 2007 [64]. However, the ST97-V isolates obtained in 2010 in this study were all susceptible to non-beta-lactam antibiotics; whereas the 2007 outbreak isolates were resistant to gentamicin, kanamycin and fusidic acid indicating that the two ST97-V isolates were unrelated. The ST97 isolates carried sak and scn encoding genes (immune evasion cluster/IEC type E) suggesting that they are of human origin [ 7 ]. In addition to revealing the diversity of MRSA clones that circulated in Kuwait hospitals from 1992 to 2010, the study also demonstrated changes in the composition of the MRSA clones during the same period. The distribution of the MRSA clones in Kuwait hospitals remained largely unchanged from 1992 to 1999, as the clonal composition of MRSA was mostly those of healthcare-acquired MRSA represented by ST239-III, the Brazilian/Hungarian MRSA clone. Despite the increase in the number of MRSA clones from 1996 onwards, the ST239-III clone continued to be the dominant clone in Kuwait hospitals. Similar to the result of this study, ST239-III has remained the predominant MRSA clone in Saudi Arabia [ 33 ] and Malaysia [ 42 ]. Other studies have reported the replacement of ST239-III MRSA as the dominant MRSA clone by CA-MRSA clones in UAE [ 49 ], Singapore [ 65 ] and Portugal [ 66 ], India, ST239-III was replaced by CC22-MRSA-IV and ST772-MRSA-V [ 52 ]. Similarly, displacement of ST239-III MRSA by ST22-MRSA-IV has been reported in Germany [ 67 ] and Czech Republic [ 47 ]. These results highlight the differences in the distribution of MRSA clones in different countries. The emergence of CA-MRSA in the mid 90's changed the epidemiology of MRSA globally [ 68 ]. The present study has shown that CA-MRSA appeared in Kuwait hospitals in 1996 with the detection of ST30-MRSA-IV, the Southwest Pacific clone (SWP), followed by ST80-IV in 1997. The other CA-MRSA clones appeared in 2000 and beyond. It is interesting that the ST30-IV MRSA isolates appeared in Kuwait hospital in 1996 a year after it was reported in New Zealand [ 69 ]. It is possible that a South West Pacific national or a returning Kuwaiti tourist to that region introduced it to Kuwait. The prevalence of ST80-IV, the European CA-MRSA clone, increased in from its introduction in 1997 until 2010. Curiously, ST80-IV-MRSA has not been reported as extensively in Asian countries as in European countries. A surveillance conducted by Song et al., [ 32 ], in Asian countries including Korea, Taiwan, Hong Kong, Thailand, Vietnam, India, Sri Lanka and the Philippines) did not detect ST80-IV-MRSA, and only few studies in Malaysia [ 70 ] and Singapore [ 71 ] reported the presence of ST80 in small numbers of their MRSA isolates. In contrast, ST80-IV has become the most common CA-MRSA clones in the Middle East and North 16 / 21 Africa [ 16, 17, 33, 49, 72, 73 ]. The presence of ST80 isolates in the Middle East and not in other parts of Asia highlights the yet unexplained observations whereby certain clones flourish in certain places but not in others. The number of MRSA clones increased from one in 1992 to 30 in 2010 including 18 new ones. The 18 new clones that appeared in 2010 included three HA-MRSA clones, ST105-II, ST225-II, ST1465-III, and 15 CA-MRSA clones consisting of USA300, ST1-V, ST5-V, ST5-VI, ST8-V, ST88-IV, ST97-V, ST121-IV, ST149-IV, ST361-IV, ST772-V, ST913-IV, ST1289-IV, ST1637-V and ST2816-V, highlighting the growth of CA-MRSA isolates in 2010. Two of these clones, ST772-V and ST8-IV-t008 [PVL+] (USA300) are recognized epidemic MRSA clones. Although ST239-III was the dominant clone, it decreased in prevalence from 59.6% in 2005 to 33.6% in 2010 as the prevalence of diverse CA-MRSA clones increased. In conclusion, the results of this study has shown that MRSA isolated in Kuwait hospitals belonged to diverse genetic backgrounds suggesting multiple routes of their acquisition. The majority of the isolates were related to the established healthcare-associated MRSA clones, ST239-III and ST22-IV. However, spa typing revealed that the isolates were heterogeneous representing imported and local clones. 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Samar S. Boswihi, Edet E. Udo, Noura Al-Sweih. Shifts in the Clonal Distribution of Methicillin-Resistant Staphylococcus aureus in Kuwait Hospitals: 1992-2010, PLOS ONE, 2016, DOI: 10.1371/journal.pone.0162744