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Characteristics of methicillin-resistant Staphylococcus aureus carrying the toxic shock syndrome toxin gene: high prevalence of clonal complex 22 strains and the emergence of new spa types t223 and t605 in Iran

Methicillin-resistant Staphylococcus aureus (MRSA) strains that carry the tst gene are disseminated worldwide with varying regional incidences and different genetic backgrounds. The data on molecular characteristics of these strains is insufficient in Iran. The present study aimed to assess the characteristics and distribution of spa types of tst-positive MRSA strains. We investigated 89 MRSA isolates carrying the tst gene with spa typing, resistance gene detection and in vitro antimicrobial susceptibility. Of the 89 tested isolates, 61 (68.5%) were confirmed as multidrug resistant (MDR). The isolates were distributed across seven clonal complexes (CCs) including CC22 (42.7%), CC8 (28.1%), CC5 (11.2%), CC59 (5.6%), CC30 (4.5%), CC80 (4.5%) and CC15 (3.4%). spa typing identified 11 distinct types, with t223 (16.9%) and t790 (15.7%) being the most prevalent. All high-level mupirocin-resistant strains belonged to t002 (n = 8) and low-level mupirocin-resistant strains belonged to t790 (n = 6) spa types. Fusidic-acid-resistant isolates belonged to t437 (n = 3). iMLSB phenotype was observed in t005 (6.7%), t002 (5.6%), t790 (3.4%), and t030, t044 and t084 (each 2.2%). It was found that in the tst-carrying MRSA strains, there were genetic diversities with a majority of the t223 spa type. Indeed, there is a necessity for more constructive surveillance/infection control strategies to address the prevalence and prevention of the emerging spa types.


Introduction
Staphylococcus aureus is one of the principal nosocomial pathogens with substantial morbidity and mortality. Unfortunately, recent reports described a continuous and heavy burden of hospitalacquired S. aureus infections around the world [1,2]. Currently, S. aureus has become an emerging problem in health-care settings and is becoming a great threat to public health. Infections caused by this bacterium are further exacerbated by the widespread circulation and emergence of drug-resistant strains; particularly, methicillin-resistant S. aureus (MRSA) [2]. Initial reports of MRSA strains indicated an increasing rate of their prevalence in some Asian countries [1]. Recently, a systematic review and metaanalysis reported a 43% prevalence of MRSA strains in Iran [3]. Emerging simultaneous resistance to multiple antibacterial agents among MRSA strains has significantly limited the availability of chemotherapeutic agents for the treatment of staphylococcal infections and leads to deterioration of disease [1][2][3].
Staphylococcus aureus produces cell-wall-associated virulence determinants and a broad spectrum of extracellular proteins, which are related to the severity of the infection [2]. A major virulence factor is toxic shock syndrome toxin-1 (TSST-1, encoded by the gene tst). When TSST, a protein of 29.1 kDa, enters the blood it causes the release of tumour necrosis factor-α, non-specific T-cell proliferation, and interleukin-1 and interleukin-2 production, which can lead to a variety of severe and life-threatening diseases [4,5]. In the absence of proper treatment, a lethal shock could occur within 24 h after the onset of symptoms [2,4,6,7]. Therefore, particular attention should be paid to MRSA strains carrying the tst gene. Although, recent studies have been focused on better understanding of the molecular epidemiology and detection of virulence genes of MRSA isolates carrying the tst gene, epidemiological data on tstcarrying MRSA in Iran is not sufficient. Accordingly, the present study was designed to describe the phenotypic resistance pattern, and the presence of the virulence factors. spa typing which was used to characterize the genotype of the MRSA carrying tst gene.

Materials and methods
Study design, isolation of bacterial isolates and ethics statement In this cross-sectional study, 89 non-duplicated MRSA isolates carrying the tst gene were isolated from various clinical samples from five teaching hospitals in Tehran, Iran. The experiment was carried out from February 2018 to January 2019. Clinical specimens included wound (35.9%), blood (30.4%), pus (10.9%), urine (8.7%), sputum (5.4%), conjunctivitis (5.4%) and body fluids (3.3%) from both genders and different age groups. The Ethics Committee of the Shahid Beheshti University of Medical Sciences in Tehran, Iran certified the protocol of this project (IR. SBMU. MSP.REC. 1397.711). Preliminary detection of S. aureus isolates used standard microbiological and biochemical techniques [8]. Positive isolates were verified by PCR targeting the S. aureus speciesspecific nuc gene [7]. MRSA screening was performed by the disc-diffusion method by using cefoxitin (30 μg) discs in Mueller-Hinton agar (Merck, Darmstadt, Germany) and also by detection of the mecA gene [8,9]. The entire strains were also analysed for the presence of the tst gene by PCR assay according to the previously published method [9].
Criteria for identifying hospital and community-onset Hospital-onset (HO) S. aureus was established if the positive culture of S. aureus was obtained 96 hours or more after admission to a hospital. Community-onset (CO) infection was defined as when the culture was obtained before 4 days of hospitalization by having one or more of the following criteria: (a) a history of hospitalization, surgery, dialysis, or residence in a long-term care facility in 12 months before the culture date, or (b) the presence of a central vascular catheter within 2 days before S. aureus culture [8].

Antimicrobial susceptibility testing
The Kirby-Bauer disc-diffusion method on Mueller-Hinton agar was used to test the susceptibility of the isolates against amikacin, gentamicin, tobramycin, kanamycin, tetracycline, erythromycin, clindamycin, linezolid, teicoplanin, ciprofloxacin, rifampicin, quinupristin-dalfopristin and trimethoprimsulfamethoxazole (Mast Co., Bootle, UK) based on the CLSI guideline. Susceptibility to vancomycin, mupirocin, tigecycline and fusidic acid was assessed by the broth microdilution method to determine the MIC titre. The European Committee for Antimicrobial Susceptibility Testing (EUCAST) breakpoints was used to determine MIC titres of fusidic acid and tigecycline (EUCAST 2018). The results of other antibiotics were interpreted by using the CLSI 2018 breakpoints. Low-level and highlevel mupirocin resistance (LLMUPR, HLMUPR), inducible macrolide-lincosamide-streptogramin group B (iMLS B ) and constitutive (cMLS B ) macrolide-lincosamide-streptogramin group B were identified based on the CLSI guideline. Susceptibility testing was quality controlled using S. aureus ATCC 25923, ATCC 43300 and ATCC 29213 strains. Powders of antibiotics were all obtained from Sigma Chemical Co. (St Louis, MO, USA).

Results
A total of 89 S. aureus strains carrying the tst-encoding gene were recovered from 350 S. aureus isolates, which were all methicillin-resistant and enrolled in the current research. Of the 89 tst-positive MRSA isolates, 16 were from hospital A (18%), 18 from hospital B (20.2%), 19 from hospital C (21.3%), 21 from hospital D (23.6%) and 15 from hospital E (16.9%). The tst-positive MRSA isolates accounted for 64% and 36% of HO (57/89), and CO (32/89) cases, respectively. Fig. 1 summarizes the distribution of HO and CO cases across hospitals.
Our findings revealed that linezolid, teicoplanin and vancomycin had the most desirable antimicrobial activity; whereas gentamicin (78.7%), tetracycline (77.5%) and erythromycin (62.9%) exhibited the poorest antimicrobial activity. Eight isolates indicated HLMUPR and six isolates showed LLMUPR. Of 89 tested isolates, the overall prevalence of cMLS, iMLS and macrolide-streptogramin (MS)-resistant phenotypes were 28 (28.6%), 20 (22.5%) and 8 (9%), respectively. The methicillinresistant and iMLS B phenotypes were distributed in all the tested hospitals with the majority in hospital D (nine isolates); whereas the cMLS B isolates were recovered from hospitals A (12 isolates) and D (16 isolates). Compared with CO-MRSA, the resistance rates of HO-MRSA to tested antibiotics was higher. All the fusidic-acid-resistant isolates belonged to hospital C and the sources for the isolation of them included wound (two isolates) and pus (one isolate). In total, 61 isolates (68.5%) indicated resistance to three or more classes of antibiotics and were confirmed as MDR strains. (see Table 1).
The analysis of resistance-encoding genes among tst-positive MRSA strains indicated that the most prevalent gene was tet(M) in 56 strains (62.  Table 1).

Discussion
Strains of MRSA carrying the TSST-encoding gene are particularly associated with wound and blood infections; in recent years, special attention has been given to these strains [5,9]. A recent systematic review and meta-analysis in Iran indicated that the overall prevalence of TSST-harbouring S. aureus clinical isolates was 21.3%, ranging from 0% to 68% in S. aureus clinical isolates [5]. According to the current findings, among the 350 tested S. aureus, 25.4% (89/350) carried the tst-encoding gene. Different prevalences for tst among S. aureus have been reported: from Turkey (14.2%) [14], Brazil (46.7%) [15], Korea (25.5%) [16] and China (31.4%) [17]. Contrary to Motamedifar et al., they indicated a higher frequency of tst in methicillinsusceptible S. aureus (MSSA) (18.1%) versus MRSA (11.6%) isolates [9]. The present data showed that all tst-harbouring isolates were methicillin resistant. Shams-Abadi et al. [5] showed that the prevalence of tst-harbouring MRSA strains in clinical isolates was 73.9%. The high prevalence of tst-carrying MRSA strains was also reported in studies from Japan (75.7%) [18] and Taiwan (75%) [19]. These differences may be a result of the type of sample and dissemination of specific clones [9,20,21].
In the present study, overall prevalence of resistance to mupirocin was found to be 15.7%. Recent studies in India [22], Jordan [23] and the USA [24] have noted mupirocin-resistance rates of 5%, 2.6% and 13.3%, respectively. Furthermore, we also observed that 9% of examined isolates were HLMUPR, which was lower than the two previous reports in Iran by Shahsavan et al. (25%) [25] and Abbasi-Montazeri et al. (17%) [26]. Differences were observed regarding the resistance to mupirocin, which may arise from the study design, dissemination of specific type among patients and unrestricted policies in taking mupirocin.
In the current investigation, the frequency of iMLS phenotypes was found to be 22.5%. Reports of prevalence of inducible resistance from Iran range from 4.1% to 20.7% [27,28] Despite the discrepancies, our data was similar to previous reports by Moosavian et al. from Iran which had shown the prevalence of 32.3% [29]. Remarkably, a high prevalence of iMLS phenotypes was also reported from Jordan (76.7%) [30].
As illustrated in Table 2, 11 different spa types were detected in this work, which were distributed in seven CCs. In agreement with data that indicated CC22 as the most common type in in Gaza, Palestine [6], the present research reported a prevalence of this CC in 42.7% of isolates. In this study, CC22 had three important spa types (t223 in 16.9% of isolates, t790 in 15.7% of isolates and t005 in 10.1% of isolates). This finding supports previous results from Gaza in which t223 was the predominant spa type, accounting for 16.7% of isolates [6]. A recent report from Kuwait also indicated that t223 was one of the common spa types detected in MRSA strains investigated from 1992 to 2010 (4.7%) [34]. t790 was the other spa type identified in CC22 (15.7%). This spa type is one of the most successful and persistent types reported from Saudi Arabia [35] and Jordan [36]. A contradictory result was reported by Boswihi et al. in Kuwait [34]. A study by Japoni-Nejad et al. in Iran [37] also indicated the low frequency of t790 among their tested isolates. Although virulence determinants and antibiotic resistance profiles in t790 isolates were found to be varied, tst carriage and resistance to mupirocin in t790 strains have been described by several researchers [8,38]. The attained data demonstrated that 10.1% of isolates were related to spa type t005. These findings are similar to those reported in Iran by Khademi et al. [39], that indicated a 7.1% prevalence rate of spa type among S. aureus isolated from clinical samples. Contrastingly, other studies have demonstrated spa type t005 as the most frequent spa type (47.4%) detected among Panton-Valentine leucocidin-positive MSSA strains [40].
Isolates of CC8 corresponded to t037 (14.6%), t030 (7.9%) and t388 (5.6%). According to the evidence, spa type t388 and t037 are associated with health-care-associated MRSA, which was found in Europe, Asia and America [34,38]. Observed frequencies of these spa types were similar to a previous report by Ohadian Moghadam et al. from Iran on 66 S. aureus strains, which recognized 11 different spa types with the most prominent spa types being t037 and t030, and low frequency of t388 [41]. The spa type t030 was previously reported as one of the most common types from different countries [34,38]. Notably, most t030 MRSA isolates were resistant to fluoroquinolones and tetracycline; a finding that was in accordance with Li's study; furthermore, they indicated that all of the tested isolates were resistant to tetracycline, rifampicin and fluoroquinolones [42].
In this study, CC80 and spa type t044 ranked sixth among tst-positive MRSA isolates, accounting for 4.5% of isolates. This observation is supported by a study conducted in Gaza, Palestine during 2008 and 2012, which documented the presence of CC80 and spa type t044 in tst-positive MRSA isolates. This would suggest the potential for greater numbers of isolates harbouring both toxin-encoding genes [6]. Also, several studies have reported the presence of this spa type in S. aureus isolates from Iran. Mirzaii et al. analysed 37 S. aureus strains isolated from different sources and found that only four isolates (10.8%) isolated from the hands and nose of personnel and also from the environment of the intensive care unit belonged to spa type t044 [43]. Asadollahi et al. also demonstrated that the t044 spa type was one of the most common spa types in 11 countries [44]. The other CC detected among tst-positive MRSA strains was CC5 (11.2%) assigned to single spa type t002. As stated in previous documents, CC5 is distributed in both community and hospital environments. Recent studies have shown the presence of CC5/t002 clones in Asian and European countries, such as Iran, Japan, Korea, the United Arab Emirates, Kuwait, Ireland and Australia [34,38]. Based on our analysis, all HLMUPR strains belonged to spa type t002 (n = 8). Similarly, in an experiment conducted in 2016 in Spain, María González-Domínguez et al. revealed resistance to mupirocin in CC55-spa t002 strains. The present survey indicated that all HLMUPR CC5/t002 isolates were positive for the mupA gene. This finding contradicted previous studies stating that CC5/t002 isolates could not carry the mupA gene [45].
Based on the evidence, CC59 (to which t437 belongs) has limited geographical spread. The present study showed that CC59 was present in five isolates, accounting for 5.6%. These findings correspond to previous reports from other countries including Australia, Ireland, the UK, Korea, Kuwait and Taiwan. Notably, resistance to fusidic acid encoded by fusB was also detected in three isolates. This finding is consistent with Shore et al. in Ireland [46].
As mentioned, the frequency of CC15/t084 was found to be 3.4%; in fact, two isolates were confirmed as iMLS B . Sangvik et al. [47] reported that t012 (8.8%), t084 (5.6%) and t065 (5.2%) were the most common spa types in north Norway. It was previously thought that t084 could be detected only in MRSA; nonetheless, there are reports indicating high distribution of this type among MSSA strains [34,38]. To the best of our knowledge, this is the first report regarding the emergence of spa types t223 and 605 in Iran.
In conclusion, this study is the first report regarding the molecular characteristics of tst-positive MRSA strains in Iran. These strains belonged to diverse genetic backgrounds with a predominance of CC22. Our investigation revealed the high prevalence of MDR patterns that highlighted the rational usage of antibiotics to minimize the spread of S. aureus with MDR in Iran; however, some resistance patterns were related to certain  spa types. Our results ought to be further investigated in other health-care settings in Iran to keep track of the emerging spa types.

Funding
This study was supported financially by a grant (No. 16027) from Research Department of the School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. The funder had no role in study design, data collection, and analysis or preparation of the manuscript.