Objective: The aim of this study was to determine antibiotic resistance trends and carriage of staphylococcal cassette chromosome mec (SCCmec) genetic elements in methicillin-resistant Staphylococcus aureus (MRSA) isolated in Kuwait hospitals to ascertain whether they were healthcare associated (HA-MRSA) or community associated (CA-MRSA). Materials and Methods: In total, 6,922 MRSA isolates obtained from different clinical samples were tested for resistance to antibiotics, urease production, and carriage of SCCmec elements. Results: All MRSA isolates were susceptible to linezolid, vancomycin, and teicoplanin. However, some isolates were resistant to kanamycin (2,979; 43%), ciprofloxacin (2,955; 42.7%), erythromycin and clindamycin (2,935; 42.4%), fusidic acid (2,858; 41.2%), gentamicin (2,665; 38.5%), tetracycline (2,652; 38.3%), and trimethoprim (2,324; 33.5%). Whereas the prevalence of resistance to most antibiotics showed annual variations, those resistant to chloramphenicol and rifampicin increased from 2.6 and 0.1% to 9.6 and 1.6%, respectively, and high-level mupirocin resistance declined from 9.3% in 2011 to 3.6% in 2015. In total, 3,244 (53.9%) of the isolates carried SCCmec IV followed by SCCmec III (1,737; 28.8%) and SCCme c V (890; 14.8%). SCCmec I (21; 0.3%) and II (79; 0.8%) occurred sporadically. A total of 3,651 (60.7%) of the isolates belonged to the CA-MRSA genotype and 2,290 isolates (38.1%) were identified as HA-MRSA. Conclusion: This study demonstrates changes in antibiotic resistance patterns of MRSA over time and reinforces the value of surveillance in detecting such changes for the benefit of infection control and patient management.

• This study revealed changes in the resistance patterns of methicillin-resistant Staphylococcus aureus isolates to some antibiotics over time in Kuwait. These findings are significant for empirical antibiotic use and the formulation of antibiotic policy which impacts patient care as well as control and prevention of infections in Kuwait.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare- and community-associated infections worldwide [1]. Since its emergence in UK in 1960s [2], MRSA strains have been isolated from various parts of the world, and the burden of infections caused by them is increasing among different patient populations globally [1,3]. The MRSA isolates were initially associated with hospitals and other healthcare facilities, such as nursing homes and long-term care facilities [4]. However, in the 1990s, MRSA started to be isolated from apparently healthy individuals in communities who had no previous history of hospital admission or medical treatment [5,6]. These types of MRSA strains were described as community-acquired, community-originated, community-associated, or community-onset MRSA (CA-MRSA) [7,8,9,10]. Since then, CA-MRSA isolates have become major causes of infections in the community and healthcare facilities worldwide [1,3,8,9,10].

Methicillin resistance is mediated by the mecA gene, which confers resistance to all beta-lactam antibiotics, located on a mobile genetic island called staphylococcal cassette chromosome mec (SCCmec). SCCmec types differ in size and structural organizations. These differences in SCCmec types have been used to type MRSA for epidemiological purposes and to distinguish HA-MRSA from CA-MRSA [11,12]. Currently, 11 SCCmec types have been described [12]. The CA-MRSA strains harbour SCCmec IV, V, and VI, while HA-MRSA strains usually harbour SCCmec I, II, and III [3,10], except for the UK epidemic MRSA-15 (UK EMRSA-15) which also carries type IV SCCmec, prompting Otter and French [13] to suggest that using SCCmec typing as a marker for CA-MRSA poses a particular problem because the presence of SCCmec IV in successful HA-MRSA lineages such as ST22-SCCmec IV (EMRSA-15) might increase the likelihood that these strains could be misclassified as CA-MRSA. However, EMRSA-15 strains carry a mutation in the UreC gene and therefore do not produce urease [14]. The inability to produce urease is therefore used as a phenotypic marker to distinguish EMRSA-15 isolates carrying SCCmec IV from CA-MRSA [15,16].

A study of MRSA isolated in Kuwait hospitals from 1992 to 2010 revealed that the MRSA isolates belonged to diverse clones that changed in numbers and type over time with ST239-MRSA-III, a healthcare-associated clone as the dominant MRSA clone in conjunction with the emergence of various CA-MRA clones [17]. In this study, MRSA isolated between 2011 and 2015 were characterized for susceptibility to antibiotics to detect changes in the prevalence of resistance to those antibiotics overtime. The isolates were also investigated for their carriage of SCCmec genetic elements to ascertain the distribution of HA-MRSA and CA-MRSA genotypes.

MRSA Isolates

The MRSA isolates were collected as part of routine diagnostic microbiology and submitted to the MRSA Reference Laboratory, located at the Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait, for epidemiological typing. In total, 6,922 MRSA isolates were obtained from patients from January 2011 to December 2015 in 14 public hospitals in Kuwait as shown in Table 1. Most of the MRSA isolates were from patients in Mubarak Al-Kabeer, Al-Amiri, Al-Sabah, Farwaniya, Al-Razi, and maternity hospitals. The isolates were identified at the Diagnostic Microbiology laboratories using cultural characteristics, Gram's stain, and positive tube coagulase and deoxyribonuclease tests. The isolates were preserved in glycerol 15% (v/v) in brain heart infusion broth (BHIB, Oxoid, Basingstoke, UK) at -80°C. The isolates were recovered at the Reference Laboratory by subculturing in BHIB at 37°C for 24 h followed by two further subcultures on brain heart infusion agar.

Susceptibility to Antimicrobial Agents

The disk diffusion method was used [18] to determine susceptibility to antimicrobial agents on Mueller-Hinton Agar (Oxoid, UK) using the following antibiotic disks (Oxoid): benzyl penicillin (2 U), cefoxitin (30 µg), kanamycin (30 µg), mupirocin (200 µg), gentamicin (10 µg), erythromycin (15 µg), clindamycin (2 µg), chloramphenicol (30 µg), tetracycline (10 µg), trimethoprim (2.5 µg), fusidic acid (10 µg), rifampicin (5 µg), ciprofloxacin (5 µg), and linezolid (30 µg). The minimum inhibitory concentration (MIC) of cefoxitin, vancomycin, and teicoplanin were determined with Etest strips (BioMerieux, Marcey L'Etolle, France) according to the manufacturer's instructions [18]. S. aureus strain ATCC 25923 was used as the quality control strain for susceptibility testing. Methicillin resistance was confirmed by detecting PBP 2a using a rapid latex agglutination kit (Denka-Seiken, Japan) according to the manufacturer's instructions.

Urease Production

Urease production was detected on Christensen's urea agar slope after 24 h of incubation at 35°C. All isolates that carried SCCmec IV genetic element were tested for urease production to distinguish between EMRSA-15 and CA-MRSA isolates.

SCCmec Typing

SCCmec typing was performed on all the MRSA isolates obtained from 2012 to 2015 for the carriage of SCCmec types I, II, III, IV, and V using the protocol described by Zhang et al. [19]. SCCmec typing was not performed on MRSA isolates in 2011.

Overall, the number of MRSA isolated annually demonstrated an increasing trend for all the hospitals (Table 1). The skin and soft tissues (2,835; 40.9%) as well as nasal swabs (1,606; 23.2%) were the major sources of MRSA isolates followed by ETS, blood, and throat swabs (Table 2). The clinical sites for 435 (6.2%) of the MRSA isolates were not reported.

Antibiotic Susceptibility of MRSA Isolates

The 5-year cumulative prevalence of resistance to non-beta-lactams antibiotics for the MRSA isolates is shown in Table 3. All the isolates were susceptible to linezolid, while 2,623 (99.4%) of the isolates were susceptible to rifampicin. Overall, 2,979 (43%) of the MRSA isolates were resistant to kanamycin. Resistance to other antibiotics is shown in Table 3. MIC determination for vancomycin and teicoplanin showed that 6,828 (98.7%) of the isolates were susceptible to vancomycin (MIC: ≤2 µg/mL), while 94 (1.3%) had vancomycin with MIC values of 3 µg/mL. None of the isolates expressed vancomycin MIC of ≥4 µg/mL. In total, 6,730 (97.2%) of the isolates were susceptible to teicoplanin (MIC: ≤2 µg/mL) and 180 isolates (2.6%) expressed MIC of 3 µg/mL, while 12 isolates (0.2%) had MIC of 4 µg/mL.

Antibiotic Resistance Trends: 2011-2015

Resistance trends to non-beta-lactam antibiotics in MRSA strains from 2011 to 2015 are presented in Table 3. From 2011 to 2014, the prevalence of resistance to aminoglycosides represented by gentamicin and kanamycin decreased from 44.3% and 53.4% to 31.2% and 36.8%, respectively. This was followed by an increase in resistance to these antibiotics in 2015 to 39.6% for gentamicin and 42.9% for kanamycin. A similar trend was observed for ciprofloxacin, which decreased from 52.2% in 2011 to 33.4% in 2014 with an increase of 51.0% in 2015. The proportion of MRSA isolates that were resistant to erythromycin and clindamycin declined in 2012 (43.8%) compared to 2011 (51.3%), and appeared to stabilize from 2013 (41.2%) to 2015 (40.5%).

The prevalence of resistance to fusidic acid declined in 2012 (424; 35.0%) compared to 2011 (411; 45.3%), but increased gradually from 519 (37.4%) in 2013 to 853 (48.1%) in 2015. The prevalence of resistance to tetracycline declined from 493 (40.8%) in 2012 to 479 (34.5%) in 2013, but showed a slight increase in 2014 (601; 36.4%) and 2015 (629; 35.5%). The proportion of MRSA strains resistant to trimethoprim declined between 2011 (304; 33.5%) and 2013 (417; 30.0%), but increased in 2014 (571; 34.6%) and 2015 (654; 36.9%) to surpass the level of 2011. The number of MRSA strains expressing high-level resistance to mupirocin showed a declining trend from 85 (9.3%) in 2011 to 50 (3.6%) in 2013, and increased slightly to 66 (4.0%) in 2014. In contrast, the proportion of strains that were resistant to chloramphenicol increased steadily from 24 (2.6%) in 2011 to 130 (7.8%) in 2014 and 171 (9.6%) in 2015.

SCCmec Typing of MRSA Isolates

The distribution of the SCCmec elements is presented in Table 4, indicating that 3,244 (53.9%) of the isolates carried SCCmec type IV. A total of 483 (8.0%) isolates were negative for urease production and were classified as EMRSA-15. The proportions of MRSA isolates carrying the CA-MRSA genotype was higher than those carrying HA-MRSA genotypes (Table 4).

This study revealed an increasing trend in the number of MRSA isolates obtained annually in Kuwait public hospitals. This is of concern because MRSA limits the choices of antibiotics available for treating infections caused by them, often warranting the use of more expensive antibiotics [20,21]. The majority (40.9%) of the isolates in this study originated from skin and soft tissue infections concurring with the findings of a previously published study [22]. The results also mimicked the findings of a surveillance study conducted in 2005 in Kuwait hospitals [23] and two studies conducted in the USA and Canada [20,24] where skin and soft tissues were the major sources of MRSA isolates. In contrast to the findings of this study, lower respiratory tract samples were the major sources accounting for 40.6% of MRSA isolates obtained during an Italian national survey conducted in 2012 [25]. These findings highlight the geographical differences in the epidemiology of MRSA colonization and/or infection.

The observation that all MRSA isolates were susceptible to linezolid is similar to reports from other countries that MRSA isolates were susceptible to linezolid [20,22,25]. In addition, the MIC distribution of the glycopeptide antibiotics showed that the majority (∼97%) of the isolates were susceptible to vancomycin and teicoplanin (MIC: ≤2 µg/ml), confirming that vancomycin and teicoplanin remain viable options for treating MRSA infections in Kuwait. Similarly, the low prevalence of high-level mupirocin resistance in this study (3.6-9.3%) is a positive development because it preserves the usefulness of mupirocin for nasal decolonization, treatment of skin infections, and prevention of postsurgical wound infections [26,27]. The results of this study also revealed that the prevalence of resistance to fusidic acid changed from 45.3% in 2011 to 35.0% in 2012 and then increased to 48.1% in 2015, while the prevalence of chloramphenicol resistance increased from 2.6% in 2011 to 9.6% in 2015. These changes in antibiotic resistance patterns probably reflected changes in the genotypes of MRSA circulating in Kuwait hospitals. For example, the increase of resistance to fusidic acid might be due to the increase of the CA-MRSA genotype, ST80-IV-MRSA, characteristically resistant to fusidic acid, in Kuwait hospitals [17].

We observed that 53.9 and 14.8% of the MRSA isolates carrying SCCmec IV and SCCmec V genetic elements, respectively, were identified as CA-MRSA and 28.8% of the isolates carrying SCCmec III were identified as HA-MRSA. These findings are similar to reports in different countries including Switzerland [28], India [29], and Singapore [30], which showed that CA-MRSA strains carrying SCCmec IV and SCCmec V have emerged as dominant MRSA strains in healthcare centers. Until 2010, HA-MRSA strains carrying SCCmec III (belonging to ST239-III-MRSA clone) were the dominant MRSA clone in Kuwait hospitals [17]. Therefore, the presence of CA-MRSA in 60.7% of the MRSA isolates indicates that CA-MRSA has overtaken HA-MRSA isolates as the leading cause of infections and/or colonization in Kuwait hospitals.

In this study, the number of MRSA isolates with CA-MRSA genotypes increased steadily from 2011 to 2015. The isolates were susceptible to linezolid, vancomycin, and teicoplanin with low prevalence of resistance to rifampicin, mupirocin, and chloramphenicol. Whereas the prevalence of resistance to some antibiotics varied between 2011 and 2015, that of fusidic acid and chloramphenicol increased and that of high-level mupirocin resistance declined. These results highlight the importance of regular surveillance in detecting changes in antibiotic resistance. Knowledge of changes in antibiotic resistance pattern has an impact on empiric antibiotic prescription, formulation of antibiotic policy, infection control, and patient management.

We are grateful to the diagnostic microbiology laboratories for submitting the MRSA isolates.

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