Emerging variants of methicillin-resistant Staphylococcus aureus genotypes in Kuwait hospitals

Background Frequent changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) occurring worldwide demand regular surveillance to study their composition and distribution in healthcare facilities. We investigated the genotypic characteristics of MRSA obtained in Kuwait hospitals to better understand their clonal distribution. Materials and methods A total of 1,327 MRSA isolates obtained from clinical samples in 13 Kuwait hospitals from 1 January to 31 December 2016 were investigated using antibiogram, SCCmec typing, spa typing and DNA microarray. Results The isolates belonged to six SCCmec types with the majority belonging to type IV (658; 49.5%) and type V (355; 26.7%). Two hundred and sixty-one spa types were identified with spa types t688, t304, t860, t127, t044, t311, t002, t223, t267, t019, t3841, t005, t084, t852, and t657 constituting 51.0% (n = 677) of the isolates. Among the 1,327 MRSA isolates, 102 (7.68%) isolates were identified as novel variants of internationally recognized MRSA clones. These 102 isolates were investigated further and belonged to 14 clonal complexes (CCs) with CC361 (32; 32.3%), CC30 (15; 14.7%), CC22 (13; 12.7%) and CC1 (11, 10.7%) as the dominant CCs. Eighty-one (79.4%) of the novel isolates harbored SCCmec IV or V+fusC composite genetic elements. Four isolates (3.9%) harbored unusual combinations of ccr and mec complexes comprising of CC6-MRSA [IV+fusC+ccrC], CC97-MRSA [V/VT+fusC+ccrAB2], CC121-MRSA [V/VT+fusC+ccrB4] and CC1-MRSA-pseudoSCCmec [class B mec+fusc+ccrAB1]. Forty-six (45.1%) of these isolates were positive for PVL and 89 (87.2%) were resistant to fusidic acid mediated by fusC. Conclusions The study showed the emergence of novel variants of previously recognized MRSA genotypes with unusual genetic characteristics including high prevalence of PVL and fusidic acid resistance in Kuwait hospitals. This has added to the dynamic lists of known variations in MRSA genomes which can impose serious challenges for infection control and treatment of MRSA infections.


Conclusions
The study showed the emergence of novel variants of previously recognized MRSA genotypes with unusual genetic characteristics including high prevalence of PVL and fusidic acid resistance in Kuwait hospitals. This has added to the dynamic lists of known variations in MRSA genomes which can impose serious challenges for infection control and treatment of MRSA infections.

Background
Methicillin-resistant Staphylococcus aureus (MRSA) can spread rapidly in hospitals and other healthcare settings resulting in an increased workload for healthcare workers and economic burden [1]. Prior to the 1990s, MRSA was reported mostly in elderly patients admitted to healthcare facilities with previous history of hospitalization and antibiotic treatment. However, beginning in the early 1990s MRSA strains were reported in patients in the community with no previous history of admission to healthcare facilities in Western Australia and elsewhere [2,3]. These strains were different from the multiply-resistant epidemic MRSA (EMRSA) that were prevalent at the time by being susceptible to most non -beta-lactam antibiotics and were subsequently designated non-multiresistant MRSA or community-originated / communityassociated MRSA (CA-MRSA) [3,4]. One of the early CA-MRSA strains reported belonged to ST30, a clone known as the Southwest Pacific clone (SWP) [5]. Since then, several CA-MRSA belonging to diverse clones have been reported worldwide [6,7]. The composition of MRSA clones is changing in different healthcare facilities in different countries. For example, the CA-MRSA clone, ST30-MRSA-IV replaced the multiresistant clone, ST239-MRSA-III, in Singapore and Malaysian hospitals [8]. Studies from the United Arab Emirates [9], Portugal [10], India [11] and Germany [12] have also reported the replacement of ST239-III-MRSA by CA-MRSA clones. Similarly, ST22-MRSA-IV and ST772-MRSA-V have replaced ST239-MRSA-III as the dominant clones in Indian hospitals [13].
Furthermore, a recent study which investigated changes in the epidemiology of MRSA strains from 1992 to 2010 in Kuwait hospitals revealed that most of the MRSA strains obtained in the 1990s belonged to the well-studied healthcare -associated MRSA genotype, ST239-MRSA-III. However, since 2010 the prevalence of ST239-MRSA-III strains declined accompanied by an increase in the number and diversity of CA-MRSA clones including ST772, a clone widely reported in India and Bangladesh and in other countries [14].
In furtherance of the need to obtain current data on the epidemiology of MRSA strains in Kuwait hospitals, MRSA obtained from1 January to 31 December 2016 were investigated using a combination of molecular typing techniques. Results of molecular typing revealed the presence of a mixed population of MRSA composed of internationally recognized clones and variants of those clones that were not previously reported in Kuwait. The purpose of this study was to characterize the novel variants of these MRSA clones for antimicrobial resistance and carriage of virulence-related genes.
December 2016 were investigated. Isolates were obtained from clinical samples collected from patients and identified using standard bacteriological methods including growth on Mannitol Salt Agar, Gram stain, tube coagulase and DNase testing. Isolates were preserved in 40% glycerol (v/v) in brain heart infusion broth at -80˚C. Isolates were subcultured twice on brainheart infusion agar (BHIA) before processing.

Molecular typing
SCCmec typing was performed on all MRSA isolates as described previously [17]. Spa typing was performed using protocol and primers published previously [18]. DNA for PCR amplification was isolated and purified as described previously [19].

DNA microarray
The 1,327 MRSA isolates representing each spa type detected in each hospital were selected for DNA microarray analysis. The S. aureus genotyping Kit 2.0 (Alere Technology, Jena, Germany) was used following protocols provided by the manufacturer [7]. DNA microarray was performed to assign the MRSA isolates to clonal complex (CC). The method detects 334 target sequences including genes encoding species markers, SCCmec, capsule and agr group typing markers, exotoxins, adhesins, and antibiotic resistance [7].

CC5
The novel variant of CC5-MRSA genotype, CC5-MRSA-[V/V T +fusC], carried genes for both TSST1 and PVL ( Table 1). The isolate also carried a composite genetic element, SCCmec V T plus fusidic acid resistance gene, fusC. It belonged to spa type t1588 and was only resistant to fusidic acid in contrast to the CC5-SCCmec V-t688 isolate previously reported in Kuwait that was resistant to tetracycline mediated by tet(K) and tet(M) [14]. However, the isolate carried agrII and cap5 which are typical of CC5-MRSA isolates [7].

CC8
The six isolates belonging to the CC8-MRSA [V/V T +fusC] variant were unique because they carried a composite SCCmecV+fusC element which is not common in this lineage. These isolates belonged to spa types t008 (5 isolates) and t211 (1 isolate), harbored sea and seb, and were resistant to fusidic acid (Table 1) and differed from the CC8-V-MRSA isolate that was obtained in 2010 in a Kuwait hospital [14]. The 2010 CC8-V-MRSA was resistant to erythromycin, gentamicin and kanamycin, belonged to spa type, t064 and harbored agrI, cap5, and enterotoxins genes sea, seb, sek, seq.

CC22
Twelve of the 13 variant isolates of CC22-MRSA consisted of isolates belonging to spa types t005 (7 isolates), t223 (1 isolate), t309 (3 isolate) and t10659 (1 isolate) were identified as CC22-MRSA-IV (tst1 + / PVL + ) ( Table 1). These isolates were unique because they carried the unusual combination of PVL and TSST1. In addition, an isolate identified as CC22-MRSA-[VI+fusC] belonged to spa type t16578 and carried the SCCmec VI genetic element not previously identified in CC22-MRSA. This isolate was resistant to fusidic acid, was PVL-negative but was positive for tst1, and carried egc, agrI and cap5.

CC45
The CC45-MRSA variant genotype, C45-MRSA-[VI+ fusC], was identified in two isolates belonging to spa type t362. The isolates carried a composite element, SCCmec VI + fus, which is rare in this lineage.

CC88
The two CC88 -MRSA variants belonged to spa types t786 and t2622. Both isolates belonged to genotype, CC88-MRSA- [IV+fusC]. These isolates carried a composite genetic element consisting of SCCmec IV and fusidic acid resistance gene fusC. Both isolates were resistant to tetracycline mediated by tet(K) but differed in the carriage of enterotoxin genes. While the t2622 isolate carried sea, the t786 isolate lacked enterotoxin genes. In addition, the t786 isolate was resistant to trimethoprim mediated by dfrS1. Both carried agrIII and cap8.

CC97
Two CC97-MRSA variants belonging to spa types t2297 and t359 were identified as CC97-MRSA-[V/V T +fusC+ccrA2B2] and CC97-MRSA-[VI+fusC] respectively ( Table 1). The CC97-MRSA-[V/V T +fusC+ccrA2B2]-t2297 isolate may harbor a new SCCmec element because it carries an additional ccrA2B2 element which is usually found in SCCmec IV isolates [7]. It was negative for genes encoding PVL, TSST1 and enterotoxins, but was positive for agrI, cap5 and to the fusidic acid resistance determinant, fusC. The t359 isolate was unique because it carried SCCmec VI which is novel in this clonal complex. The isolate lacked genes for PVL, TSST1 and enterotoxins but carried fusC and dfrS1 which mediate resistance to fusidic acid and trimethoprim respectively.

CC121
Six isolates were identified as variants of CC121-MRSA. Five of the isolates were identified as CC121-MRSA-V/V T +fusC [PVL + ] ( Table 1). These isolates carried a unique composite genetic element consisting of SCCmec V and the fusidic acid resistance gene, fusC and differed from the CC121-IV-MRSA isolates reported previously in Kuwait which were susceptible to fusidic acid [14]. The sixth isolate was identified as CC121-MRSA-[V/V T +fusC+ccrB4] (PVL + ). This isolate was unique because it carried an additional ccrB4 but ccrA4 was not detected. The ccrA4B4 allotype is usually found in isolates carrying SCCmec VI or VIII [21]. It was resistant to fusidic acid mediated by fusC and contained seb and egc. All six isolates were positive for agrIV and cap8.

CC913
Three isolates were identified as variants of CC913-MRSA. All three isolates, identified as CC913-MRSA-[VI+fusC], belonged to spa type t991. The isolates were unique because they carried SCCmec VI which is new in this lineage. All three isolates lacked genes for PVL, TSST1and enterotoxins, but carried agrII, cap8, etA and etD encoding exfoliative toxins A and D respectively. All isolates carried erm(C), dfrS1 and fusC encoding resistance to erythromycin and clindamycin, trimethoprim, and fusidic acid respectively. One isolate carried an additional determinant, vgaA, that mediates streptogramin-A resistance (Table 1).

CC1153
The CC1153/t504 isolate identified in this study, CC1153-MRSA [I+fusC] PVL + was unique because it carried a novel composite genetic element composed of SCCmec I and a SCCfus/ ccrA1B1 element. It lacked enterotoxin genes but was positive for PVL and fusC that mediates fusidic acid resistance.

ST2867
One isolate was identified as ST2867-MRSA-V/V T . The variant carried the SCCmec V variant V T which was not reported in this singleton before.The isolate lacked genes for PVL,TSST1 and enterotoxins but carried agrII and cap5 (Table 1).

Discussion
The application of molecular typing and DNA microarray analysis facilitated the discovery of novel MRSA variants within existing clonal complexes in Kuwait hospitals. Of the 102 MRSA isolates, 88 carried composite genetic elements comprising SCCmec and fusidic acid resistance gene fusC ( Table 1). The carriage of these elements may be beneficial for the survival of the strains. The co-residence of fusC and SCCmec genetic element may ensure the stability of fusidic acid resistance and enhance its spread. Although fusC-mediated fusidic acid resistance has previously been reported in ST239-III-MRSA clone in Kuwait [14] the presence of fusC in isolates belonging to different genetic backgrounds suggests that it was acquired independently by the different clones probably involving a common bacteriophage. It is also possible that the composite SCCmec IV+fusC and SCCmecV+fusC elements have started wandering across different lineages that are favored by certain selective pressures such as the use of over-thecounter fucidin ointments, or a drive to replace mupirocin use with fucidin as currently observed in New Zealand [22,23]. While investigating fusidic acid-resistant MRSA in the UK, Ellington et al., [24] observed that the fusidic acid resistance determiminant, fusC, was located on multiple SCCmec elements including chimeric cassette elements that conferred resistance to beta-lactams and fusidic acid. Therefore, the composite elements involving SCCmec and fusC oberved in this study may represent chimeric elements similar to the report by Ellington et al [24]. The emergence of different chimeric genetic elements involving SCCmec, antibiotic resistance and virulence genes in MRSA may signal new adaptive mechanisms for their survival in the presence of antibiotics. Curiously, the majority (32 of 102) belonged to CC361-MRSA which have been observed in small numbers in previous reports from human [14,25,26] and animal [7,27,28] sources. Furthermore, 28 of the 32 isolates belonged to a single spa type, t3841,which were isolated from wound, nasal, respiratory, ear, vaginal, groins and axilla in seven hospitals signifying the capacity of the clone to spread, colonize as well as cause infections in humans. However, the apparent high prevalence of CC361-MRSA in this study was due to the detection of a single CC361-MRSA-t3841 clone in different hospitals. Continuous surveillance is needed to monitor future expansion of this clone in the country.
The study also revealed the presence of probable novel SCCmec (3 isolates) and pseudo SCCmec (1 isolate) elements (Table 1). DNA Microarray analysis revealed the presence of either ccrA2B2 or ccrC elements in isolates belonging to CC6-MRSA-[IV+fusC+ccrC]and CC97-MRSA-[V/V T +fusC+ccrA2B2]. Additionally, an isolate identified as CC121-MRSA-[V/ V T +fusC+ccrB4] (PVL+) contained ccrB4 that is not usually associated with SCCmec type V suggesting that this may be a new SCCmec element. Also, an isolate with a novel genotype, CC1-MRSA-PseudoSCCmec [class B mec+fusC+ccrA1B1]/t127, was detected among the CC1-MRSA ( Table 1). The ccrC gene is usually found in isolates carrying SCCmec V and the ccrA2B2, has been associated with isolates carrying SCCmec II or SCCmec IV [7]. The carriage of these novel elements in non-traditional backgrounds may represent the emergence of novel SCCmec elements. These composite genetic elements probably evolved by recombinations between genetic elements native to the host and acquired foreign DNA as has been reported in other MRSA backgrounds [20]. Previously, ccrA4B4 which shared 100% identity to similar elements in S. epidermidis was detected in ST8-MRSA-t190 isolates obtained in Irish hospitals [29] suggesting that ccrA4B4 was acquired from S. epidermidis [30]. Additionally, a novel pseudo SCCmec element carrying mecA with a novel mec class region and fusidic acid resistance gene (fusC) was detected in ST779-MRSA also in Irish hospitals [31]. The genetic backgrounds of our isolates are different from those isolated in Irish [29] and Indian [32] hospitals suggesting that these events are probably more widespread than currently appreciated and highlights the value of DNA Microarray in detecting these events. However, Whole genome sequencing of these isolates will be required to clearly understand the organization of these novel composite elements. SCCmec-associated genes.
This study also revealed the carriage of an unusual combinations of genes encoding TSST1 and PVL in the same cell in isolates belonging to CC22-MRSA and CC30-MRSA (Table 1). Previously, some CC22-MRSA subtypes had either carried genes for PVL as in ST22-IV-t852/ t790 [33,34] or tst1 as in the Middle East variant of EMRSA-15), CC22-IV-MRSA/ tst1+] [33,35,36]. However, recently, Khairalla et al., [37] reported CC22-IV-MRSA isolates obtained from three healthcare workers in a dental practice in Egypt that carries both tst1 and PVL suggesting that this may be an emerging trait in CC22-IV-MRSA.Their three isolates belonged to spa types, t14339 (2 isolates) and t8506 (1 isolate) and were resistant to gentamicin and clindamycin. In contrast, the isolates in this study belonged to spa types, t005, t309 and t223 but were also resistant to gentamicin and clindamycin. It is not clear whether the carriage of both determinants act synergistically to elevate the virulence capacity of these isolates. This is the first report of MRSA with SCCmec V subtype V T in Kuwait. Isolates that carrry SCCmec V T were first reported in 2005 in Taiwan by Boyle-Vavra et al., [38]. Whereas, SCCmec V element harbor ccr complex class C1 and mec complex class C2 [36], SCCmec V T contains a ccrC2 which is a ccrC recombinase gene variant and mec complex C2. All of the Taiwaness SCCmec V T strains belonged to ST59 [38], In contrast, the isolates in this study with SCCmec V T element belonged to different lineages including CC1, CC5, CC8, CC97, CC121 and CC361. Similarly, MRSA isolates carrying SCCmec V T have been reported in different clonal complexes including CC7, CC8, CC30, CC45, CC59, CC152, and CC398 in Germany [39] suggesting the independent acquisition of SCCmec V T .
The study further revealed the occurence of SCCmec type I and type VI in lineages such as CC22, CC30, CC45, CC97, CC152, CC913, and CC1153 that usually carry SCCmec types IV or type V [8,21]. Reports of CC1153-MRSA are rare in the literature, and a previously reported isolate in Kuwait carried SCCmec V element and was susceptible to fusidic acid [40]. Therefore the carriage of SCCmec type 1 and fusC in an isolate of CC1153-MRSA in this study represents a novel development.
The SCCmec type VI element was detected in 15 CC30-MRSA and three CC913-MRSA isolates. Until now, only two isolates of CC30-MRSA-[VI+fusC] (PVL+) was previously reported in Saudi Arabia [41]. The 15 CC30-MRSA-[VI+fusC] (PVL+) isolates belonged to five spa types including t012, t018, t021 that are usually associated with CC30-MRSA suggesting that the compositive genetic element was recently acquired by CC30-MRSA. The other spa types, t253 and t318 represent sporadic isolates. Although epidemiologic relationship could not be established between the isolates from Kuwait and Saudi Arabia, their identification in 15 isolates in this study may signal the expansion of this lineage in the region.
CC152 isolates are rare and those reported earlier carried SCCmec V [7], which is different from the SCCmec VI in the isolate in this study. In addition, the detection of spa type t362 in SCCmec VI that was not detected previously in CC45 isolates obtained in Kuwait [14] indicates the emergence of new CC45 variants in Kuwait hospitals. The emergence of unusual SCCmec types in genetic backgounds that they are not normally associated with may cause difficulty in classifying these MRSA isolates on the basis of their SCCmec elements.
The main limitation of this study is the absence of whole genome sequencing data of the isolates to further analyze these variant strains. Whole genome sequencing will be required to understand the orgnization of the SCCmec encoding genes which may help identify new SCCmec elements.

Conclusion
This study has revealed the emergence of novel variants of known MRSA genotypes in Kuwait hospitals. The new isolates all belonged to CA-MRSA genotypes and highlights the growing diversity of CA-MRSA strains globally [1,3,7,41]. It also points to a shift from an era where a small number of highly prevalent epidemic strains with small numbers of SCCmec elements to a high number of very diverse strains and very diverse SCCmec elements that both recently evolved or continues to evolve. While this makes molecular typing more interesting, it spells trouble for infection control. Therefore, conducting molecular surveillance is important at regular intervals to prevent the establishment and trasnmission of these strains in Kuwait healthcare facilities.