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 It is still frequently isolated in some parts of the world although it has been replaced by other MRSA strains in, e.g., most of Europe.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

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

Mupirocin-resistant Staphylococcus aureus in Africa: a systematic review and meta-analysis

Background Mupirocin is widely used for nasal decolonization of Staphylococcus aureus to prevent subsequent staphylococcal infection in patients and healthcare personnel. However, the prolonged and unrestricted use has led to the emergence of mupirocin-resistant (mupR) S. aureus. The aim of this systematic review was to investigate the prevalence, phenotypic and molecular characteristics, and geographic spread of mupR S. aureus in Africa. Methods We examined five electronic databases (EBSCOhost, Google Scholar, ISI Web of Science, MEDLINE, and Scopus) for relevant English articles on screening for mupR S. aureus from various samples in Africa. In addition, we performed random effects meta-analysis of proportions to determine the pooled prevalence of mupR S. aureus in Africa. The search was conducted until 3 August 2016. Results We identified 43 eligible studies of which 11 (26%) were obtained only through Google Scholar. Most of the eligible studies (28/43; 65%) were conducted in Nigeria (10/43; 23%), Egypt (7/43; 16%), South Africa (6/43; 14%) and Tunisia (5/43; 12%). Overall, screening for mupR S. aureus was described in only 12 of 54 (22%) African countries. The disk diffusion method was the widely used technique (67%; 29/43) for the detection of mupR S. aureus in Africa. The mupA-positive S. aureus isolates were identified in five studies conducted in Egypt (n = 2), South Africa (n = 2), and Nigeria (n = 1). Low-level resistance (LmupR) and high-level resistance (HmupR) were both reported in six human studies from South Africa (n = 3), Egypt (n = 2) and Libya (n = 1). Data on mupR-MRSA was available in 11 studies from five countries, including Egypt, Ghana, Libya, Nigeria and South Africa. The pooled prevalence (based on 11 human studies) of mupR S. aureus in Africa was 14% (95% CI =6.8 to 23.2%). The proportion of mupA-positive S. aureus in Africa ranged between 0.5 and 8%. Furthermore, the frequency of S. aureus isolates that exhibited LmupR, HmupR and mupR-MRSA in Africa were 4 and 47%, 0.5 and 38%, 5 and 50%, respectively. Conclusions The prevalence of mupR S. aureus in Africa (14%) is worrisome and there is a need for data on administration and use of mupirocin. The disk diffusion method which is widely utilized in Africa could be an important method for the screening and identification of mupR S. aureus. Moreover, we advocate for surveillance studies with appropriate guidelines for screening mupR S. aureus in Africa

High prevalence of toxic shock syndrome toxin–producing epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) strains in Kuwait hospitals

This study characterized EMRSA-15 isolates obtained from patients in Kuwait hospitals for their genotypic relatedness, antibiotic resistance and carriage of virulence genes using pulsed-field gel electrophoresis (PFGE), coagulase serotyping, SCCmec subtyping, spa typing, multilocus sequence typing and DNA microarray. The isolates were resistant to trimethoprim (75.6%), ciprofloxacin (29.7%), erythromycin and clindamycin (24.3%), tetracycline (19.0%), and gentamicin and kanamycin (21.6%). All 37 isolates belonged to sequence type (ST) 22, coagulase type XI, three PFGE types and eight subtypes, ten spa types including t223 (51.3%), t852 (13.5%), t032 (8.1%), t790 (8.1%), t3107 (5.4%) and one each of t309, t2251, t3935, t5708 and t5983. Twenty-six isolates (70.2%) carried SCCmec IVa, eight isolates carried SCCmec IV and three isolates carried SCCmec IVh. All isolates carried agr1, cap5 and egc gene cluster (seg, sei, selm, seln, selo, and selu). tst (toxic shock syndrome toxin) was detected in 23 isolates. Eight isolates (21.6%) were positive for Panton-Valentine leukocidin (PVL). Genotypic analysis revealed that 62.1% of the isolates comprising ST22-IVa-t223 (51.3%) and ST22-IVa-t309/t2251/t3935/t5708 (10.8%) were CC22-[tst1+] UK EMRSA-15/Middle Eastern variant, 21.6% were CC22-PVL+ EMRSA-15 variant and 16.2% were CC22-UK EMRSA-15/Barnim clone. These results show that the tst1 positive-ST22-IVa-t223 (Middle Eastern variant) and the CC22-PVL+ EMRSA-15 variant were the dominant EMRSA-15 variants in Kuwait hospitals

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

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

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

Distribution of MRSA clones among MRSA isolates in 1992–2010.

<p>Distribution of MRSA clones among MRSA isolates in 1992–2010.</p

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