Molecular epidemiological typing of emerging methicillin-resistant Staphylococcus aureus strains in the community, among livestock and in healthcare facilities in Ireland, 2001-2015

THESIS 11335Methicillin-resistant Staphylococcus aureus (MRSA) has been endemic in Irish hospitals for four decades and displacement of predominant clones has occurred several times. Sequence type (ST) 22 carrying staphylococcal cassette chromosome (SCC) mec type IV has predominated in Irish healthcare facilities (HCF) since 2002. In recent years, an increase in sporadically-occurring non-ST22-MRSA-IV from bloodstream infections (BSIs) was observed indicating that strain displacement may occur again. Elsewhere, sporadically-occurring strains recognised in Ireland have emerged as predominant strains in HCFs. The first part of this study undertook an in-depth molecular characterisation of 276 sporadically-occurring MRSA isolates from patients attending Irish hospitals, general practitioners (GP) and a veterinary laboratory from 2001-2015. All isolates underwent antimicrobial susceptibility testing (AST), spa typing and DNA microarray profiling to investigate the genetic backgrounds, SCCmec-associated genes and antimicrobial resistance and virulence genes. The 276 isolates were assigned to 86 spa types, 14 multilocus sequence typing (MLST) clonal complexes (CC), 11 STs and 18 combinations of SCCmec genes. Combining the CCs and SCCmec types resulted in 48 different type combinations. Community associated (CA), livestock associated (LA) and healthcare-associated (HCA) MRSA lineages were identified including CC1-MRSA-IV (22.5%, 62/276) & CC5-MRSA-V (8.3%, 23/276), CC398-MRSA-V (3.6%, 10/276) & CC130-MRSA-XI (0.4%, 1/276) and ST239-MRSA-III (1.8%, 5/276), respectively. Common virulence genes included the immune evasion cluster (IEC) (86.6%, 239/276), the enterotoxin gene cluster (egc; 46.7%, 129/276) and enterotoxin genes sek/q (13.0%, 36/276) and seh (26.8%, 74/276). The isolates harboured multiple resistance genes including those encoding resistance to beta-lactams (blaZ/blaZxi, 96%, 264/276), macrolides (erm(A), erm(B), erm(C), lnu(A) and/or mph(C), 59.8%, 165/276), aminoglycosides (aacA-aphD, aadD and/or aphA3, 43.2%, 119/276) and tetracycline (tet(K) and/or tet(M), 25.0%, 69/276), as well as the SCCmec/SCC-encoded fusidic acid resistance gene fusC (19.2%, 53/276). Several nosocomial outbreaks caused by CA-MRSA lineages were investigated, including two by pvl-positive ST772-MRSA-IV, one by CC1-MRSA-IV and one by CC5-MRSA-V. These isolates harboured multiple resistance and virulence genes. In the case of one ST772-MRSA-IV outbreak, a healthcare worker (HCW) who had recently been hospitalised in India where this strain predominates subsequently transmitted the strain to babies in a neonatal intensive care unit in Ireland. This highlights the need for MRSA screening of HCWs and of the threat posed by the spread of pvl-positive CA-MRSA into hospitals. Livestock-associated CC398 MRSA were also identified (n = 12) which along with an additional nine CC398 MRSA and 10 CC398 methicillin susceptible S. aureus (MSSA) were further investigated. The CC398 MRSA harboured SCCmec IVa or VT, exhibited spa type t011 or t034 and carried different combinations of multiple resistance genes including those encoding resistance to erythromycin, tetracycline, spectinomycin and aminoglycosides. Four distinct incidents of CC398 MRSA were identified, including the transmission of CC398 from a veterinarian with recent travel to Belgium to a horse and between pigs (n = 9) and farm workers (n = 9) on two farms, one of which had been restocked with gilts from Germany. These findings have significant implications for human and animal health and the Irish agricultural industry

Whole-genome sequencing identifies highly related Pseudomonas aeruginosa strains in multiple washbasin U-bends at several locations in one hospital: evidence for trafficking of potential pathogens via wastewater pipes

Background: Hand washbasin U-bends have increasingly been associated with nosocomial outbreaks by Gram-negative bacteria, including Pseudomonas aeruginosa which is virtually ubiquitous in U-bends. Wastewater networks servicing U-bends are potential highways for trafficking pathogenic bacteria. Aim: To use P. aeruginosa to investigate trafficking of bacteria between hospital washbasin U-bends. Methods: Twenty-five washbasin U-bends in five locations in Dublin Dental University Hospital (DDUH) were investigated for trafficking of P. aeruginosa: 10 in Clinic 2 (C2), 10 in the Accident & Emergency Department (A&E) and five in three other locations. In addition, washbasin tap samples (N=80) and mains and tap water samples (N=72) were cultured for P. aeruginosa. Selected P. aeruginosa isolates recovered over 29 months underwent whole-genome sequencing, and relatedness was interpreted using whole-genome multi-locus sequence typing and pairwise single nucleotide polymorphism (SNP) analysis. Findings: P. aeruginosa was recovered from all U-bends but not from taps or water. Eighty-three U-bend isolates yielded 10 sequence types (STs), with ST560 and ST179 from A&E, C2 and two other locations predominating (70%). ST560 was also recovered from a common downstream pipe. Isolates within ST560 and ST179 were highly related regardless of source. ST560 was divided into Cluster I (N=25) and Cluster II (N=2) with average allelic differences and SNPs of three and zero, and two and five, respectively. The 31 ST179 isolates exhibited an average allelic difference and SNPs of three and 12, respectively. Conclusion: Highly related P. aeruginosa strains were identified in multiple U-bends in several DDUH locations, indicating trafficking via the wastewater network

A novel multidrug-resistant PVL-negative CC1-MRSA-1 IV clone emerging in Ireland and Germany likely originated in South-Eastern Europe

This study investigated the recent emergence of multidrug-resistant Panton-Valentine leukocidin (PVL)-negative CC1-MRSA-IV in Ireland and Germany. Ten CC1-MSSA and 139 CC1-MRSA isolates recovered in Ireland between 2004 and 2017 were investigated. These were compared to 21 German CC1-MRSA, 10 Romanian CC1-MSSA, five Romanian CC1-MRSA and two UAE CC1-MRSA, which were selected from an extensive global database, based on similar DNA microarray profiles to the Irish isolates. All isolates subsequently underwent whole-genome sequencing, core-genome single nucleotide polymorphism (cgSNP) analysis and enhanced SCCmec subtyping. Two PVL-negative clades (A and B1) were identified among four main clades. Clade A included 20 German isolates, 119 Irish isolates, and all Romanian MRSA and MSSA isolates, the latter of which differed from clade A MRSA by 47?130 cgSNPs. Eighty-six Irish clade A isolates formed a tight subclade (A1) exhibiting 0?49 pairwise cgSNPs, 80 of which harboured a 46?kb conjugative plasmid carrying both ileS2, encoding high-level mupirocin resistance, and qacA, encoding chlorhexidine resistance. The resistance genes aadE, aphA3 and sat were detected in all clade A MRSA and the majority (8/10) of clade A MSSA isolates. None of the clade A isolates harboured any enterotoxin genes other than seh, which is universally present in CC1. Clade B1 included the remaining German isolate, 17 Irish isolates and the two UAE isolates, all of which corresponded to the Western Australia MRSA-1 (WA MRSA-1) clone based on genotypic characteristics. MRSA within clades A and B1 differed by 188 cgSNPs and clade-specific SCCmec characteristics were identified, indicating independent acquisition of the SCCmec element. This study demonstrated the existence of a European PVL-negative CC1-MRSA-IV clone that is distinctly different from the well-defined PVL-negative CC1-MRSA-IV clone, WA MRSA-1. Furthermore, cgSNP analysis revealed that this newly defined clone may have originated in South-Eastern Europe, before spreading to both Ireland and Germany

A novel multidrug-resistant PVL-negative CC1-MRSA-IV clone emerging in Ireland and Germany likely originated in South-Eastern Europe

This study investigated the recent emergence of multidrug-resistant Panton-Valentine leukocidin (PVL)-negative CC1-MRSA-IV in Ireland and Germany. Ten CC1-MSSA and 139 CC1-MRSA isolates recovered in Ireland between 2004 and 2017 were investigated. These were compared to 21 German CC1-MRSA, 10 Romanian CC1-MSSA, five Romanian CC1-MRSA and two UAE CC1-MRSA, which were selected from an extensive global database, based on similar DNA microarray profiles to the Irish isolates. All isolates subsequently underwent whole-genome sequencing, core-genome single nucleotide polymorphism (cgSNP) analysis and enhanced SCCmec subtyping. Two PVL-negative clades (A and B1) were identified among four main clades. Clade A included 20 German isolates, 119 Irish isolates, and all Romanian MRSA and MSSA isolates, the latter of which differed from clade A MRSA by 47-130 cgSNPs. Eighty-six Irish clade A isolates formed a tight subclade (A1) exhibiting 0-49 pairwise cgSNPs, 80 of which harboured a 46 kb conjugative plasmid carrying both ileS2, encoding high-level mupirocin resistance, and qacA, encoding chlorhexidine resistance. The resistance genes aadE, aphA3 and sat were detected in all clade A MRSA and the majority (8/10) of clade A MSSA isolates. None of the clade A isolates harboured any enterotoxin genes other than seh, which is universally present in CC1. Clade B1 included the remaining German isolate, 17 Irish isolates and the two UAE isolates, all of which corresponded to the Western Australia MRSA-1 (WA MRSA-1) clone based on genotypic characteristics. MRSA within clades A and B1 differed by 188 cgSNPs and clade-specific SCCmec characteristics were identified, indicating independent acquisition of the SCCmec element. This study demonstrated the existence of a European PVL-negative CC1-MRSA-IV clone that is distinctly different from the well-defined PVL-negative CC1-MRSA-IV clone, WA MRSA-1. Furthermore, cgSNP analysis revealed that this newly defined clone may have originated in South-Eastern Europe, before spreading to both Ireland and Germany