Methicillin-Resistant Staphylococcus aureus (MRSA) in a Tertiary Care Hospital in Kuwait: A Molecular and Genetic Analysis.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen that causes serious infections in healthcare facilities and in communities. The purpose of this study was to investigate MRSA isolates obtained in a tertiary hospital in Kuwait to assess their antibiotic susceptibility profile and clonal composition. Sixty MRSA isolates collected in 2020 were tested through antibiotic susceptibility testing, spa typing, and DNA microarray analysis. All isolates were found to be susceptible to vancomycin (MIC: ≤3 µg/mL), teicoplanin (MIC: ≤3 µg/mL), rifampicin, and mupirocin, but were resistant to fusidic acid (n = 43, 72%), trimethoprim (n = 27, 45%), ciprofloxacin (n = 31, 51.7%), gentamicin (n = 14; 23.3%), kanamycin (n = 20; 33.3%), chloramphenicol (n = 7; 11.7%), tetracycline (n = 17; 28.3%), erythromycin (n = 19; 31.6%), inducible clindamycin (n = 13; 21.7%), and constitutive clindamycin (n = 2; 3.3%). The isolates belonged to 30 spa types and 13 clonal complexes (CCs). The dominant spa types were t304, t442, t311, t688, and t1234, collectively constituting 28.3% of the isolates. The dominant CCs were CC5 and CC6, which together constituted 46.7% of the isolates. This study provides updated research on antibiotic resistance and changes in the clonal composition of MRSA in a Kuwait hospital, including the disappearance of the ST239-MRSA-III clone that was previously the dominant clone in this hospital.

Diversity of clonal complex 22 methicillin-resistant Staphylococcus aureus isolates in Kuwait hospitals.

CC22-MRSA is a major MRSA lineage that is widely reported globally. To characterize CC22-MRSA for trends in antibiotic resistance and emergence of variants, a total of 636 CC22 isolates identified by DNA microarray in 2016 (n = 195), 2017 (n = 227) and 2018 (n = 214) were investigated further using staphylococcal protein A (spa) typing and multilocus sequence typing. The isolates belonged to 109 spa types dominated by t223 (n = 160), t032 (n = 60), t852 (n = 59), t005 (n = 56) and t309 (n = 30) and 10 sequence types (STs) dominated by ST22 (85.5%). Genotypes CC22-MRSA-IV [tst1+]; CC22-MRSA-IV UK-EMRSA-15/Barnim EMRSA variants, CC22-MRSA-IV [PVL+], CC22-MRSA-IV [tst1+/PVL+] and CC22-MRSA-IV + V constituted >50% of the isolates. An increase from 2016 to 2018 were shown in isolates belonging to spa types t223 (43 to 62), t032 (18 to 27) and t309 (10 to 15) and genotypes CC22-MRSA-IV [tst1+] (89 to 102), CC22-MRSA-IV + V (12 to 30) and CC22-MRSA-IV [tst1+/PVL+] (12 to 22). Ninety-nine CC22-MRSA isolates were multi-resistant to three or more antibiotic classes with 76.7% of them belonging to CC22-MRSA-IV [PVL+] and CC22-MRSA-IV [tst1+/PVL+]. The study revealed an ongoing domination of the CC22-MRSA-[tst1+] genotype and the emergence of new clones bearing SCCmec IV + V and multiply resistant variants.

Description of Methicillin-Susceptible Staphylococcus aureus Clonal Complex 30 Related to the Pandemic Phage Type 80/81 Isolated from Patients in Three Tertiary Hospitals in Jos, North Central Nigeria.

OBJECTIVE: The prevalence of phage 80/81 Staphylococcus aureus strains, the pandemic strains that were dominant in the 1950s, had declined in the 1960s and 1970s. However, these strains have reemerged in some countries in recent years. This study investigated the antibacterial resistance, virulence, and the genetic backgrounds of CC30-MSSA isolates obtained from patients in three tertiary hospitals. MATERIALS AND METHODS: Twenty-two CC30-MSSA isolates cultured from different clinical samples were investigated using antibiotic sensitivity testing, spa typing, multilocus sequence typing, and DNA microarray analysis. RESULTS: All 22 isolates were susceptible to vancomycin (MIC ≤2 µg/mL), teicoplanin (MIC ≤2 µg/mL), and cefoxitin but were resistant to penicillin G (n = 22; 100.0%), tetracycline (n = 12; 54.5%), ciprofloxacin (n = 15; 68.2%), cadmium acetate (n = 22; 100%), mercuric chloride (n = 13; 59.1%), and ethidium bromide (n = 3; 13.6%). The isolates belonged to sequence type, ST30, and five spa types: t012 (n = 12; 54.5%), t019 (n = 5; 22.7%), t017 (n = 2; 9.1%), t037 (n = 2; 9.1%), and t318 (n = 1; 4.5%). All 22 isolates were positive for agrIII, cap8, clfA, clfB, icaA, icaC, icaD, cna, and staphylococcal enterotoxin gene clusters (seg, sei, sem, sen, seo, seu). Eight isolates carried lukS-PV and lukF-PV that code for Panton-Valentine leukocidin. CONCLUSION: The current CC30-MSSA isolates share phenotypic and genotypic characteristics with the pandemic phage 80/81 isolates that were common in the 1950s and 1960s. Continued surveillance is recommended to keep abreast of the changing epidemiology of S. aureus causing healthcare and community-associated infections.

Resurgence of Chloramphenicol Resistance in Methicillin-Resistant Staphylococcus aureus Due to the Acquisition of a Variant Florfenicol Exporter (fexAv)-Mediated Chloramphenicol Resistance in Kuwait Hospitals.

Following a surge in the prevalence of chloramphenicol-resistant methicillin-resistant Staphylococcus aureus (MRSA) in Kuwait hospitals, this study investigated the genotypes and antibiotic resistance of the chloramphenicol-resistant isolates to ascertain whether they represented new or a resurgence of sporadic endemic clones. Fifty-four chloramphenicol-resistant MRSA isolates obtained in 2014-2015 were investigated. Antibiotic resistance was tested by disk diffusion and MIC determination. Molecular typing was performed using spa typing, multilocus sequence typing, and DNA microarray. Curing and transfer experiments were used to determine the genetic location of resistance determinants. All 54 isolates were resistant to chloramphenicol (MIC: 32-56 mg/L) but susceptible to florfenicol. Two chloramphenicol-resistance determinants, florfenicol exporter (fexA) and chloramphenicol acetyl transferase (cat), were detected. The fexA-positive isolates belonged to CC5-ST627-VI-t688/t450/t954 (n = 45), CC5-ST5-V-t688 (n = 6), whereas the cat-positives isolates were CC8-ST239-III-t037/t860 (n = 3). While cat was carried on 3.5-4.4 kb plasmids, the location of fexA could not be established. DNA sequencing of fexA revealed 100% sequence similarity to a previously reported fexA variant that confers chloramphenicol but not florfenicol resistance. The resurgence of chloramphenicol resistance was due to the introduction and spread of closely related fexA-positive CC5-ST5-V and CC5-ST627-VI clones.

The Dissemination and Molecular Characterization of Clonal Complex 361 (CC361) Methicillin-Resistant Staphylococcus aureus (MRSA) in Kuwait Hospitals.

Methicillin-resistant Staphylococcus aureus (MRSA) belonging to clonal complex 361 (CC361-MRSA) is rare among patients' populations globally. However, CC361-MRSA has been isolated with an increasing trend among patients in Kuwait hospitals since 2010. This study investigated the molecular characteristics of CC361-MRSA isolated from patients in Kuwait hospitals in 2016-2018 to understand their genetic relatedness and virulence determinants. Of 5,223 MRSA isolates investigated by DNA microarray, 182 (3.4%) isolates obtained in 2016 (N = 55), 2017 (N = 56), and 2018 (N = 71) were identified as CC361-MRSA. The CC361-MRSA isolates were analyzed further using antibiogram, spa typing and multi locus sequence typing (MLST). Most of the isolates were resistant to fusidic acid (64.8%), kanamycin (43.4%), erythromycin (36.3%), and clindamycin (14.3%) encoded by fusC, aphA3, and erm(B)/erm(C) respectively. Nine isolates (4.9%) were resistant to linezolid mediated by cfr. The isolates belonged to 22 spa types with t3841 (N = 113), t315 (N = 16), t1309 (N = 14), and t3175 (N = 5) constituting 81.3% of the spa types, four genotypes (strain types), CC361-MRSA-[V/VT + fus] (N = 112), CC361-MRSA-IV, WA MRSA-29 (N = 36), CC361-MRSA-V, WA MRSA-70/110 (N = 33) and CC361-MRSA-[V + fus] variant (N = 1). MLST conducted on 69 representative isolates yielded two sequence types: ST361 (11/69) and ST672 (58/69). All CC361-MRSA isolates were positive for cap8, agr1, and the enterotoxin egc gene cluster (seg, sei, selm, seln, selo, and selu). The tst1 was detected in 19 isolates. The immune evasion cluster (IEC) genes type B (scn, chp, and sak) and type E (scn and sak) were detected in 20 and 152 isolates, respectively. The CC361-MRSA circulating in Kuwait hospitals consisted of two closely related sequence types, ST361 and ST672 with ST672-MRSA [V/VT + fus] as the dominant genotype. The dissemination of these newly emerged clones and the emergence of linezolid resistance limits therapeutic options, as well as present significant challenges for the control of MRSA infections in Kuwait hospitals.

Characterisation of S. aureus/MRSA CC1153 and review of mobile genetic elements carrying the fusidic acid resistance gene fusC.

While many data on molecular epidemiology of MRSA are available for North America, Western Europe and Australia, much less is known on the distribution of MRSA clones elsewhere. Here, we describe a poorly known lineage from the Middle East, CC1153, to which several strains from humans and livestock belong. Isolates were characterised using DNA microarrays and one isolate from the United Arab Emirates was sequenced using Nanopore technology. CC1153 carries agr II and capsule type 5 genes. Enterotoxin genes are rarely present, but PVL is common. Associated spa types include t504, t903 and t13507. PVL-positive CC1153-MSSA were found in Egyptian cattle suffering from mastitis. It was also identified among humans with skin and soft tissue infections in Saudi Arabia, France and Germany. CC1153-MRSA were mainly observed in Arabian Gulf countries. Some isolates presented with a previously unknown SCCmec/SCCfus chimeric element in which a mec B complex was found together with the fusidic acid resistance gene fusC and accompanying genes including ccrA/B-1 recombinase genes. Other isolates carried SCCmec V elements that usually also included fusC. Distribution and emergence of CC1153-MRSA show the necessity of molecular characterization of MRSA that are resistant to fusidic acid. These strains pose a public health threat as they combine resistance to beta-lactams used in hospitals as well as to fusidic acid used in the community. Because of the high prevalence of fusC-positive MRSA in the Middle East, sequences and descriptions of SCC elements harbouring fusC and/or mecA are reviewed. When comparing fusC and its surrounding regions from the CC1153 strain to available published sequences, it became obvious that there are four fusC alleles and five distinct types of fusC gene complexes reminiscent to the mec complexes in SCCmec elements. Likewise, they are associated with different sets of ccrA/B recombinase genes and additional payload that might include entire mec complexes or SCCmec elements.

Antibiotic resistance and typing of the methicillin-resistant Staphylococcus aureus clones in Kuwait hospitals, 2016-2017.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) belong to diverse genetic backgrounds that differ in antibiotic resistance. Knowledge of the local clonal composition of MRSA strains is important for patients' management and for designing effective control and eradication methods. The aim of this study was to compare the antibiotic resistance patterns and genotypic characteristics of MRSA isolates obtained in public hospitals in Kuwait in 2016 and 2017 for changes in their resistance patterns and clonal composition. METHODS: A total of 4726 MRSA isolates obtained in 2016-2017 from clinical specimens in Kuwait public hospitals were characterized using antibiogram, SCCmec typing, spa typing and DNA microarray. RESULTS: The isolates expressed resistance to fusidic acid (52.9%), kanamycin (41.6%), gentamicin (32.5%) and erythromycin (36.2%). The prevalence of high-level mupirocin resistance decreased from 3.7% in 2016 to 2.4% in 2017, while the proportion of resistance to other antibiotics remained relatively stable. A total of 382 spa types were detected with eight spa types, t688 (N = 547), t304 (N = 428), t860 (N = 394), t127 (N = 306), t044 (N = 230), t311 (N = 243), t223 (N = 184) and t002 (N = 181) constituting 53.1% of the MRSA isolates in 2016-2017. Of the 3004 MRSA isolates obtained in 2016 (N = 1327) and 2017 (N = 1677) selected for DNA microarray analysis, 26 clonal complexes (CCs) were identified. Most of the isolates belonged to CC1 (N = 248), CC5 (N = 833), CC6 (N = 241), CC8 (N = 292), CC22 (N = 421), CC30 (N = 177), CC80 (N = 177) and CC97 (N = 171). The prevalence of CC5 isolates has significantly (p ≤ 0.05) increased from 294 isolates in 2016 to 539 isolates in 2017. Although CC22 increased from 196 isolates in 2016 to 225 isolates in 2017, CC1 increased from 112 isolates in 2016 to 136 isolates in 2017, CC6 increased from 103 isolates in 2016 to 138 isolates in 2017, these changes were not significant (p ≥ 0.05). CONCLUSION: These results revealed the diversity in the genetic backgrounds of MRSA isolates and the stable maintenance of the dominant MRSA clones in Kuwait hospitals in 2016 and 2017 suggesting an on-going transmission of these clones. Novel and creative infection prevention and control measures are required to curtail further transmission.

Evaluating the antibacterial activity of ceftaroline against clinical isolates of methicillin-susceptible and- resistant Staphylococcus aureus in Kuwait hospitals.

Ceftaroline fosamil has been approved by the US Food and Drug Administration for the treatment of skin and soft tissue infections, including those caused by methicillin-resistant Staphylococcus aureus and community-acquired bacterial pneumonia. We evaluated the in-vitro activity of not-in-use ceftaroline against clinical isolates of methicillin- resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus isolates. A total of 453 single-patient S. aureus isolates were tested for susceptibility to ceftaroline. The minimum inhibitory concentration (MIC) was determined using the MIC Evaluator. Disk diffusion test was performed using 30 µg ceftaroline disk according to the Clinical Laboratory Standards Institute criteria. In total, 410 (90.5%) of the 453 strains were susceptible to ceftaroline at MIC of 1 mg/L while 9.8% isolates had MIC of 2-3 mg/L. Among the MSSA, 92.5% of the isolates were susceptible with MIC: 1 mg/L while 36 (7.9%) strains expressed intermediate resistance (MIC range of 2-3 mg/L). None of the strains was resistant (MIC: 4 mg/L). Ceftaroline showed good in-vitro activity against MSSA and MRSA which can serve as an effective alternative to vancomycin in treating infections caused by MRSA.

Antimicrobial resistance and virulence determinants in coagulase-negative staphylococci isolated mainly from preterm neonates.

Coagulase-negative staphylococci (CoNS) are the most common isolates from blood culture in neonates resulting in high mortality and morbidity. This study investigated CoNS obtained from blood cultures of neonates for antibiotic resistance and virulence factors, and possible association with inflammatory response (C-reactive protein). A total of 93 CoNS isolates were collected from 76 blood cultures of neonates at the Maternity hospital in Kuwait in a six-month period and investigated for susceptibility to antibiotics, carriage of staphylococcal cassette chromosome mec (SCCmec), and virulence-associated genes. The 93 CoNS isolates consisted of S. epidermidis (76; 81.7%), S. capitis (12; 12.9%), S. hominis (2; 2.1%), S. warneri (2; 2.1%) and S. haemolyticus (1; 1.0%). Eighty-six (92.4%) of the isolates were resistant to cefoxitin (MR-CoNS) while 49 (52.7%) expressed multi-antibiotic resistance. The methicillin-resistant isolates (MR-CoNS) carried SCCmec III, SCCmec IVa and four combinations of SCCmec types including SCCmec types I+IVa (one S. warneri and 25 S. epidermidis isolates), types I+III (one S. epidermidis isolate), types III+IVa (six S. epidermidis isolates) and types I+III+IVa (one S. epidermidis isolate). The most common virulence-related genes were icaC, seb, arc detected in 69.7%, 60.5%, 40.8% of the isolates respectively. Two isolates were positive for tst1. No association between C-reactive protein and antibiotic resistance or virulence factors was established. This study revealed that S. epidermidis carrying different SCCmec genetic elements, was the dominant CoNS species isolated from neonatal blood cultures with 90.3% and 36.6% of the isolates positive for genes for biofilm and ACME production respectively.

Emergence of Methicillin-Resistant Staphylococcus aureus Belonging to Clonal Complex 15 (CC15-MRSA) in Kuwait Hospitals.

PURPOSE: Methicillin-resistant S. aureus (MRSA) belonging to clonal complex 15 (CC15-MRSA) is rare among clinical isolates with few reports from retail camel meat and human patients. This study investigated the genetic relatedness of CC15-MRSA isolated for the first time from patients in Kuwait hospitals. METHODS: Antibiotic susceptibility was tested by the disk diffusion method. Minimum inhibitory concentration was determined using Etest strips. Molecular typing was performed using spa tying, multilocus sequence tying and DNA microarray. RESULTS: Of 1327 MRSA isolates, 42 (3.1%) were identified as CC15-MRSA. The 42 isolates belonged to sequence type ST1535-harbored SCCmec type V and spa types t084 (36 isolates), t346 (3 isolates) and one of t114, t228 and t7583. All 42 isolates were resistant to gentamicin, kanamycin, fusidic acid and cadmium acetate; 38 isolates were resistant to tetracycline. The isolates harbored aacA-aphD and fusC that codes for gentamicin and fusidic acid resistance, respectively. Tet(K) was present in the tetracycline-resistant isolates. In addition, the 42 isolates carried inu(A) (lincosamide nucleotidyltransferase) that confers resistance to lincomycin and clindamycin although phenotypically susceptible to these antibiotics. The isolates belonged to accessory gene regulator type II and capsular polysaccharide group 8 but lacked genes for Staphylococcus enterotoxins, toxic shock syndrome toxin, collagen-binding adhesins and Panton-Valentine leukocidin. CONCLUSION: This study revealed the emergence and transmission of a previously rare MRSA clone among human patients in Kuwait hospitals and highlights the increasing infiltration of rare MRSA into the human population.

Characterisation of a novel SCCmec VI element harbouring fusC in an emerging Staphylococcus aureus strain from the Arabian Gulf region.

Fusidic acid is a steroid antibiotic known since the 1960s. It is frequently used in topical preparations, i.e., ointments, for the treatment of skin and soft tissue infections caused by Staphylococcus aureus. There is an increasing number of methicillin-resistant S. aureus (MRSA) strains that harbour plasmid-borne fusB/far1 or fusC that is localised on SCC elements. In this study we examined a series of related CC30-MRSA isolates from the Arabian Gulf countries that presented with SCCmec elements and fusC, including a variant that-to the best of our knowledge-has not yet formally been described. It consisted of a class B mec complex and ccrA/B-4 genes. The fusidic acid resistance gene fusC was present, but contrary to the previously sequenced element of HDE288, it was not accompanied by tirS. This element was identified in CC30 MRSA from Kuwait, Saudi Arabia and the United Arab Emirates that usually also harbour the Panton-Valentin leukocidin (PVL) genes. It was also identified in CC8 and ST834 isolates. In addition, further CC30 MRSA strains with other SCCmec VI elements harbouring fusC were found to circulate in the Arabian Gulf region. It can be assumed that MRSA strains with SCCmec elements that include fusC have a selective advantage in both hospital and community settings warranting a review of the use of topical antibiotics and indicating the necessity of reducing over-the-counter sale of antibiotics, including fusidic acid, without prescription.

Livestock-Associated Methicillin-Resistant Staphylococcus aureus in Patients Admitted to Kuwait Hospitals in 2016-2017.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been reported to colonize and cause infections in animals as well as in humans. LA-MRSA isolates have only recently been identified in patients admitted to Kuwait hospitals. This study was conducted to characterize LA-MRSA isolates obtained from patients admitted to Kuwait hospitals. A total of 202 (7.1%) of 2,823 MRSA isolates obtained from clinical samples in 2016 and 2017 in 11 public Kuwait hospitals were assigned to lineages previously known to be associated with livestock. They were characterized using antibiogram, spa typing, and DNA microarray for the assignment of clonal complexes (CCs) and detection of antibiotic resistance and virulence determinants. Identification as putative LA-MRSA clones was based on the molecular definition inferred from DNA microarray. The LA-MRSA isolates consisted of CC96 (N = 31), CC97 (N = 169), and CC398 (N = 2). Isolates belonging to CC96 and CC398 were resistant to erythromycin and clindamycin mediated by erm(A) and erm(C). CC97 isolates were multiresistant to gentamicin, kanamycin, erythromycin, clindamycin, tetracycline, chloramphenicol, fusidic acid, trimethoprim, and ciprofloxacin and harbored aacA-aphD, erm(A), erm(C), msr(A), tet(K), cat, fusC, and dfrS1. In total, 35 spa types were identified among the isolates. CC398 isolates consisted of t899 and t034. Ten spa types were identified among CC96 with t11822 (N = 13) as the most prevalent. CC97 consisted of 26 spa types with most belonging to t267 (N = 73) followed by t359 (N = 39). CC398 was composed of CC398-MRSA-IV and CC398-MRSA-V (PVL+). CC96 belonged to CC96-MRSA-IV and CC96-MRSA-IV (PVL+) Central Asian caMRSA/WA MRSA-119. CC97 consisted of six strains including CC97-MRSA-V (fusC +), CC97-MRSA-IV WA MRSA-54/63, CC97-MRSA-V, CC97-MRSA-(V+fus), CC97-MRSA-(mec VI+fus), and CC97-MRSA (mecV/VT+fus+ccrAB2). Whereas CC96 and CC97 isolates were identified in 2016 and 2017, CC398 isolates were detected only in 2016. This study identified four LA-MRSA clones among MRSA isolated from patients in Kuwait hospitals in 2016-2017 with CC97-MRSA-V (fusC +) as the dominant clone. The presence of LA-MRSA with different genetic backgrounds suggests its independent acquisition from different sources.

Molecular Typing of ST239-MRSA-III From Diverse Geographic Locations and the Evolution of the SCCmec III Element During Its Intercontinental Spread.

ST239-MRSA-III is probably the oldest truly pandemic MRSA strain, circulating in many countries since the 1970s. It is still frequently isolated in some parts of the world although it has been replaced by other MRSA strains in, e.g., most of Europe. Previous genotyping work (Harris et al., 2010; Castillo-Ramírez et al., 2012) suggested a split in geographically defined clades. In the present study, a collection of 184 ST239-MRSA-III isolates, mainly from countries not covered by the previous studies were characterized using two DNA microarrays (i) targeting an extensive range of typing markers, virulence and resistance genes and (ii) a SCCmec subtyping array. Thirty additional isolates underwent whole-genome sequencing (WGS) and, together with published WGS data for 215 ST239-MRSA-III isolates, were analyzed using in-silico analysis for comparison with the microarray data and with special regard to variation within SCCmec elements. This permitted the assignment of isolates and sequences to 39 different SCCmec III subtypes, and to three major and several minor clades. One clade, characterized by the integration of a transposon into nsaB and by the loss of fnbB and splE was detected among isolates from Turkey, Romania and other Eastern European countries, Russia, Pakistan, and (mainly Northern) China. Another clade, harboring sasX/sesI is widespread in South-East Asia including China/Hong Kong, and surprisingly also in Trinidad & Tobago. A third, related, but sasX/sesI-negative clade occurs not only in Latin America but also in Russia and in the Middle East from where it apparently originated and from where it also was transferred to Ireland. Minor clades exist or existed in Western Europe and Greece, in Portugal, in Australia and New Zealand as well as in the Middle East. Isolates from countries where this strain is not epidemic (such as Germany) frequently are associated with foreign travel and/or hospitalization abroad. The wide dissemination of this strain and the fact that it was able to cause a hospital-borne pandemic that lasted nearly 50 years emphasizes the need for stringent infection prevention and control and admission screening.

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.

Antibiotic Resistance Trends in Methicillin-Resistant Staphylococcus aureus Isolated in Kuwait Hospitals: 2011-2015.

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 SCCmec 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.

Characterization of a CC1153 PVL-producing community-acquired methicillin-resistant Staphylococcus aureus from a dog bite wound.

The isolation of a rare community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strain from an infected dog bite wound is here reported. A 27-year old man presented with a deep open wound on his right hand caused by his dog's bite at the outpatient clinic of Infectious Disease Hospital (IDH), Kuwait. A wound swab was obtained for bacteriological culture and susceptibility testing. The wound culture yielded pure heavy growth of an MRSA isolate, designated IDH70, which was susceptible to vancomycin, teicoplanin, erythromycin, clindamycin, trimethoprim, fusidic acid and rifampicin. The patient was successfully treated with a combination of rifampicin and cotrimoxazole twice daily for 10 days. Molecular characterization revealed that IDH70 was positive for genes encoding Panton-Valentine leucocidin. IDH70 also carried the SCCmec V genetic element, belonged to coagulase type XIIIa, spa type t903, and was assigned to clonal complex 1153 and sequence type ST1153 (ST1153-V-t903). The report highlights the increasing burden of CA-MRSA in the community and the risk of its acquisition from bites of companion animals.

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

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.

Diversity of methicillin-resistant Staphylococcus aureus CC22-MRSA-IV from Saudi Arabia and the Gulf region.

OBJECTIVES: CC22-MRSA-IV, UK-EMRSA-15/Barnim EMRSA, is a common and pandemic strain of methicillin-resistant Staphylococcus aureus (MRSA) that has been found mainly in Western Europe, but also in other parts of the world including some Gulf countries. One suspected case of an infection with this strain in a patient who was admitted to the surgical unit in Riyadh, Kingdom of Saudi Arabia (KSA) was investigated in order to check whether this strain has reached KSA. METHODS: Besides the index isolate, 46 additional isolates of CC22-MRSA-IV from patients from KSA, Abu Dhabi, Kuwait, and Germany (patients with a history of travel in the Middle East), were characterized by microarray hybridization. RESULTS: The study revealed a regional presence of as many as six distinct 'strains' of CC22-MRSA-IV that could be distinguished based on carriage of SCCmec IV subtypes and virulence factors. No true UK-EMRSA-15/Barnim EMRSA was identified in Riyadh; all suspected isolates from Riyadh were assigned to other, albeit related strains. However, this strain was identified in Abu Dhabi and Kuwait. CONCLUSIONS: CC22-MRSA-IV from KSA could be linked to other epidemic strains from the Middle East and possibly India, rather than to the Western European UK-EMRSA-15/Barnim EMRSA. High-resolution typing methods, including SCCmec subtyping, might help to differentiate related epidemic strains and to monitor routes of transmission.

Genotypes and virulence genes in group B streptococcus isolated in the maternity hospital, Kuwait.

OBJECTIVE: To characterize group B streptococcus (GBS) isolates obtained from patients at the Maternity Hospital in Kuwait for their genotypes and carriage of virulence genes. MATERIALS AND METHODS: A total of 154 GBS isolates were obtained from July 1 to October 31, 2007, from vaginal swabs (n = 95), urine (n = 46), blood (n = 4) and miscellaneous sources (n = 9). Genotypes were obtained by pulsed-field gel electrophoresis (PFGE), following digestion with SmaI or EagI restriction enzymes. PCR was used to screen for the carriage of virulence genes including: surface protein of group B streptococcus (spb1), secreted fibrinogen-binding protein (fbsB), C5a peptidase (scpB), laminin-binding protein (lmb), α- (bca) and ß-subunits of the C protein (bac), resistance to protease immunity protein (rib), and phage-associated gene (pag); regulatory protein (dltR), and toxins CAMP factor (cfb), hyaluronidase (hylB) and superoxide dismutase (sodA). RESULTS: PFGE defined 14 genotypes differentiating isolates with the same serotypes into different genetic backgrounds. All isolates contained genes for virulence factors. However, cfb (99.4%), scpB (88.3%), lmb (88.3%), bca (57.8%), sodA (55.8%) and dltR (53.9%) were the common virulence genes. In total, 144 (90.3%) of the isolates contained 3 or more virulence genes. However, while cfb, lmb and scpB occurred in all genotypes, others occurred in some but not in all genotypes. CONCLUSIONS: GBS isolates obtained at the Maternity Hospital, Kuwait, belonged to diverse genetic backgrounds with the majority carrying multiple virulence genes.

Serotypes and antibiotic resistance in Group B streptococcus isolated from patients at the Maternity Hospital, Kuwait.

A total of 143 group B streptococcus (GBS) isolates collected from mothers at the Maternity Hospital in Kuwait were investigated for their serotypes and antibiotic resistance, and screened by PCR for the carriage of genes for resistance to tetracycline (tetk, tetM, tetL, tetO), erythromycin (ermA, ermB, ermC, ermTR, ermM, mefA, mefE, msrA) and aminoglycosides (aph3, ant4, ant6). All isolates were serotyped using a latex agglutination test. Most of the isolates belonged to serotypes V (38.5 %), III (20.9 %), Ia (7.7 %) and II (11.2 %). Sixteen isolates (11.2 %) were nontypable. All isolates were susceptible to penicillin, ampicillin and cefotaxime (MICs 0.016-0.094 µg ml(-1)) but were resistant to trimethoprim (92.3 %), tetracycline (89.5 %), minocycline (89.5 %), high-level kanamycin (76.9 %), chloramphenicol (30.0 %), erythromycin (12.6 %), clindamycin (7.0 %), high-level streptomycin (3.5 %) and ciprofloxacin (0.7 %). The tetracycline-resistant isolates contained tetM (94.5 %), tetO (3.9 %), tetL (1.6 %) and tetK (0.8 %). The erythromycin-resistant isolates contained ermB (61.1 %), ermTR (38.9 %), ermA (5.5 %), mefA (5.5 %) and mefE (11 %). All high-level kanamycin-resistant isolates contained aph3. One of the high-level streptomycin-resistant isolates contained ant6. Partial DNA sequencing of aph3 revealed sequences with 99 % similarity to aph3 found in Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis, suggesting that the GBS isolates could have acquired aph3 from other Gram-positive cocci. The high proportion of isolates with resistance to tetracycline, high-level kanamycin and trimethoprim, and the increase in the prevalence of erythromycin resistance, represents an emerging public health concern that needs further surveillance.