Protracted transmission and persistence of ST80 vancomycin-resistant Enterococcus faecium clonal complex types CT2933, CT2932 and CT1916 in a large Irish hospital: a 39-month WGS study.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) are significant nosocomial pathogens. Sequence type (ST)80 vanA-encoding VREfm predominate in Irish hospitals, but their transmission is poorly understood. AIMS: To investigate transmission and persistence of predominant complex type (CT) VREfm in two wards of an Irish hospital (H1) using whole-genome sequencing, and their intra- and inter-hospital dissemination. METHODS: Rectal screening (N=330, September 2019-December 2022) and environmental (N=48, November 2022-December 2022) E. faecium were investigated. Isolate relatedness was assessed by core-genome multilocus sequence typing (cgMLST) and core-genome single nucleotide polymorphism (cgSNP) analysis. Likely transmission chains were identified using SeqTrack (https://graphsnp.fordelab.com/graphsnp) using cgSNP data and recovery location. Well-characterised E. faecium (N=908) from seven Irish hospitals including H1 (June 2017-July 2022) were also investigated. FINDINGS: Conventional MLST assigned isolates to nine STs (ST80, 82%). cgMLST identified three predominant ST80 CTs (CT2933, CT2932 and CT1916) (55% of isolates) of related isolates (≤20 allelic differences). cgSNP analysis differentiated these CTs into multiple distinct closely related genomic clusters (≤10 cgSNPs). Parisimonious network construction identified 55 likely inter- and intra-ward transmissions with epidemiological support between patients ≤30 days involving 73 isolates (≤10 cgSNPs) from seven genomic clusters. Numerous other likely transmissions over longer time periods without evident epidemiological links were identified, suggesting persistence and unidentified reservoirs contribute to dissemination. The three CTs predominated among E. faecium (N=1,286) in seven hospitals, highlighting inter-hospital spread without known epidemiological links. CONCLUSION: This study revealed the long-term intra- and inter-hospital dominance of three major CT ST80 VREfm lineages, widespread transmission and persistence, implicating unidentified reservoirs.

Phylodynamic signatures in the emergence of community-associated MRSA.

Community-associated, methicillin-resistant Staphylococcus aureus (MRSA) lineages have emerged in many geographically distinct regions around the world during the past 30 y. Here, we apply consistent phylodynamic methods across multiple community-associated MRSA lineages to describe and contrast their patterns of emergence and dissemination. We generated whole-genome sequencing data for the Australian sequence type (ST) ST93-MRSA-IV from remote communities in Far North Queensland and Papua New Guinea, and the Bengal Bay ST772-MRSA-V clone from metropolitan communities in Pakistan. Increases in the effective reproduction number (Re) and sustained transmission (Re > 1) coincided with spread of progenitor methicillin-susceptible S. aureus (MSSA) in remote northern Australian populations, dissemination of the ST93-MRSA-IV genotype into population centers on the Australian East Coast, and subsequent importation into the highlands of Papua New Guinea and Far North Queensland. Applying the same phylodynamic methods to existing lineage datasets, we identified common signatures of epidemic growth in the emergence and epidemiological trajectory of community-associated S. aureus lineages from America, Asia, Australasia, and Europe. Surges in Re were observed at the divergence of antibiotic-resistant strains, coinciding with their establishment in regional population centers. Epidemic growth was also observed among drug-resistant MSSA clades in Africa and northern Australia. Our data suggest that the emergence of community-associated MRSA in the late 20th century was driven by a combination of antibiotic-resistant genotypes and host epidemiology, leading to abrupt changes in lineage-wide transmission dynamics and sustained transmission in regional population centers.

Genomic analysis of 600 vancomycin-resistant Enterococcus faecium reveals a high prevalence of ST80 and spread of similar vanA regions via IS1216E and plasmid transfer in diverse genetic lineages in Ireland.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) cause a wide range of hospital infections. Ireland has had one of the highest invasive VREfm infection rates in Europe over the last decade, yet little is known about Irish VREfm. OBJECTIVES: To investigate the population structure of Irish VREfm, explore diversity by analysing the vanA transposon region and compare Irish, Danish and global isolates. METHODS: E. faecium (n = 648) from five Irish hospitals were investigated, including VREfm [547 rectal screening and 53 bloodstream infection (BSI)] isolates and 48 vancomycin-susceptible (VSEfm) BSI isolates recovered between June 2017 and December 2019. WGS and core-genome MLST (cgMLST) were used to assess population structure. Genetic environments surrounding vanA were resolved by hybrid assembly of short-read (Illumina) and long-read (Oxford Nanopore Technologies) sequences. RESULTS: All isolates belonged to hospital-adapted clade A1 and the majority (435/648) belonged to MLST ST80. The population structure was highly polyclonal; cgMLST segregated 603/648 isolates into 51 clusters containing mixtures of screening and BSI isolates, isolates from different hospitals, and VREfm and VSEfm. Isolates within clusters were closely related (mean average ≤16 allelic differences). The majority (96.5%) of VREfm harboured highly similar vanA regions located on circular or linear plasmids with multiple IS1216E insertions, variable organization of vanA operon genes and 78.6% harboured a truncated tnpA transposase. Comparison of 648 Irish isolates with 846 global E. faecium from 30 countries using cgMLST revealed little overlap. CONCLUSIONS: Irish VREfm are polyclonal, yet harbour a characteristic plasmid-located vanA region with multiple IS1216E insertions that may facilitate spread.

Exploring the evolution and epidemiology of European CC1-MRSA-IV: tracking a multidrug-resistant community-associated meticillin-resistant Staphylococcus aureus clone.

This study investigated the evolution and epidemiology of the community-associated and multidrug-resistant Staphylococcus aureus clone European CC1-MRSA-IV. Whole-genome sequences were obtained for 194 European CC1-MRSA-IV isolates (189 of human and 5 of animal origin) from 12 countries, and 10 meticillin-susceptible precursors (from North-Eastern Romania; all of human origin) of the clone. Phylogenetic analysis was performed using a maximum-likelihood approach, a time-measured phylogeny was reconstructed using Bayesian analysis, and in silico microarray genotyping was performed to identify resistance, virulence-associated and SCCmec (staphylococcal cassette chromosome mec) genes. Isolates were typically sequence type 1 (190/204) and spa type t127 (183/204). Bayesian analysis indicated that European CC1-MRSA-IV emerged in approximately 1995 before undergoing rapid expansion in the late 1990s and 2000s, while spreading throughout Europe and into the Middle East. Phylogenetic analysis revealed an unstructured meticillin-resistant S. aureus (MRSA) population, lacking significant geographical or temporal clusters. The MRSA were genotypically multidrug-resistant, consistently encoded seh, and intermittently (34/194) encoded an undisrupted hlb gene with concomitant absence of the lysogenic phage-encoded genes sak and scn. All MRSA also harboured a characteristic ~5350 nt insertion in SCCmec adjacent to orfX. Detailed demographic data from Denmark showed that there, the clone is typically (25/35) found in the community, and often (10/35) among individuals with links to South-Eastern Europe. This study elucidated the evolution and epidemiology of European CC1-MRSA-IV, which emerged from a meticillin-susceptible lineage prevalent in North-Eastern Romania before disseminating rapidly throughout Europe.

An epidemic CC1-MRSA-IV clone yields false-negative test results in molecular MRSA identification assays: a note of caution, Austria, Germany, Ireland, 2020.

We investigated why a clinical meticillin-resistant Staphylococcus aureus (MRSA) isolate yielded false-negative results with some commercial PCR tests for MRSA detection. We found that an epidemic European CC1-MRSA-IV clone generally exhibits this behaviour. The failure of the assays was attributable to a large insertion in the orfX/SCCmec integration site. To ensure the reliability of molecular MRSA tests, it is vital to monitor emergence of new SCCmec types and junction sites.

Comparative Microbiological and Whole-Genome Analysis of Staphylococcus aureus Populations in the Oro-Nasal Cavities, Skin and Diabetic Foot Ulcers of Patients With Type 2 Diabetes Reveals a Possible Oro-Nasal Reservoir for Ulcer Infection.

Patients with type 2 diabetes are at higher risk for periodontal disease and diabetic foot ulcer infections (DFUIs), the latter of which are predominantly caused by staphylococcal bacteria. Staphylococci have also been detected in the mouth, nose and gums (the oro-nasal cavity) of patients with periodontal disease and can move between the mouth and nose. The present study investigated if the oro-nasal cavity and/or periodontal pockets (PPs) in diseased gum tissue can provide a microbial reservoir for DFUIs. Eighteen patients with type 2 diabetes and at least three natural teeth (13 patients with ulcers and 5 patients without ulcers) underwent non-invasive microbiological sampling of PP, oro-nasal, skin and ulcer sites. Staphylococci were recovered using selective chromogenic agar, definitively identified and subjected to DNA microarray profiling, whole-genome sequencing and core-genome multilocus sequence typing (cgMLST). Staphylococcus aureus and Staphylococcus epidermidis were recovered from both the oro-nasal and ulcer sites of 6/13 and 5/13 patients with ulcers, respectively. Molecular typing based on the staphylococcal protein A (spa) gene and DNA microarray profiling indicated that for each patient investigated, S. aureus strains from oro-nasal and ulcer sites were identical. Comparative cgMLST confirmed that isolates from multiple anatomical sites of each individual investigated grouped into closely related, patient-distinct clusters (Clusters 1-7). Isolates belonging to the same cluster exhibited an average of 2.9 allelic differences (range 0-11). In contrast, reference genomes downloaded from GenBank selected as representatives of each sequence type identified in the present study exhibited an average of 227 allelic differences from the most closely related isolate within each cluster.

Linezolid resistance in Enterococcus faecium and Enterococcus faecalis from hospitalized patients in Ireland: high prevalence of the MDR genes optrA and poxtA in isolates with diverse genetic backgrounds.

OBJECTIVES: To investigate the prevalence of the optrA, poxtA and cfr linezolid resistance genes in linezolid-resistant enterococci from Irish hospitals and to characterize associated plasmids. METHODS: One hundred and fifty-four linezolid-resistant isolates recovered in 14 hospitals between June 2016 and August 2019 were screened for resistance genes by PCR. All isolates harbouring resistance genes, and 20 without, underwent Illumina MiSeq WGS. Isolate relatedness was assessed using enterococcal whole-genome MLST. MinION sequencing (Oxford Nanopore) and hybrid assembly were used to resolve genetic environments/plasmids surrounding resistance genes. RESULTS: optrA and/or poxtA were identified in 35/154 (22.7%) isolates, the highest prevalence reported to date. Fifteen isolates with diverse STs harboured optrA only; one Enterococcus faecium isolate harboured optrA (chromosome) and poxtA (plasmid). Seven Enterococcus faecalis and one E. faecium harboured optrA on a 36 331 bp plasmid with 100% identity to the previously described optrA-encoding conjugative plasmid pE349. Variations around optrA were also observed, with optrA located on plasmids in five isolates and within the chromosome in three isolates. Nine E. faecium and 10 E. faecalis harboured poxtA, flanked by IS1216E, within an identical 4001 bp region on plasmids exhibiting 72.9%-100% sequence coverage to a 21 849 bp conjugative plasmid. E. faecalis isolates belonged to ST480, whereas E. faecium isolates belonged to diverse STs. Of the remaining 119 linezolid-resistant isolates without linezolid resistance genes, 20 investigated representatives all harboured the G2576T 23S RNA gene mutation associated with linezolid resistance. CONCLUSIONS: This high prevalence of optrA and poxtA in diverse enterococcal lineages in Irish hospitals indicates significant selective pressure(s) for maintenance.

Evolution and Global Transmission of a Multidrug-Resistant, Community-Associated Methicillin-Resistant Staphylococcus aureus Lineage from the Indian Subcontinent.

The evolution and global transmission of antimicrobial resistance have been well documented for Gram-negative bacteria and health care-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. In this study, we traced the recent origins and global spread of a multidrug-resistant, community-associated Staphylococcus aureus lineage from the Indian subcontinent, the Bengal Bay clone (ST772). We generated whole-genome sequence data of 340 isolates from 14 countries, including the first isolates from Bangladesh and India, to reconstruct the evolutionary history and genomic epidemiology of the lineage. Our data show that the clone emerged on the Indian subcontinent in the early 1960s and disseminated rapidly in the 1990s. Short-term outbreaks in community and health care settings occurred following intercontinental transmission, typically associated with travel and family contacts on the subcontinent, but ongoing endemic transmission was uncommon. Acquisition of a multidrug resistance integrated plasmid was instrumental in the emergence of a single dominant and globally disseminated clade in the early 1990s. Phenotypic data on biofilm, growth, and toxicity point to antimicrobial resistance as the driving force in the evolution of ST772. The Bengal Bay clone therefore combines the multidrug resistance of traditional health care-associated clones with the epidemiological transmission of community-associated methicillin-resistant S. aureus (MRSA). Our study demonstrates the importance of whole-genome sequencing for tracking the evolution of emerging and resistant pathogens. It provides a critical framework for ongoing surveillance of the clone on the Indian subcontinent and elsewhere.IMPORTANCE The Bengal Bay clone (ST772) is a community-associated and multidrug-resistant Staphylococcus aureus lineage first isolated from Bangladesh and India in 2004. In this study, we showed that the Bengal Bay clone emerged from a virulent progenitor circulating on the Indian subcontinent. Its subsequent global transmission was associated with travel or family contact in the region. ST772 progressively acquired specific resistance elements at limited cost to its fitness and continues to be exported globally, resulting in small-scale community and health care outbreaks. The Bengal Bay clone therefore combines the virulence potential and epidemiology of community-associated clones with the multidrug resistance of health care-associated S. aureus lineages. This study demonstrates the importance of whole-genome sequencing for the surveillance of highly antibiotic-resistant pathogens, which may emerge in the community setting of regions with poor antibiotic stewardship and rapidly spread into hospitals and communities across the world.

First description of arginine catabolic mobile element (ACME) type VI harboring the kdp operon only in Staphylococcus epidermidis using short and long read whole genome sequencing: Further evidence of ACME diversity.

The arginine catabolic mobile element (ACME) was first described in methicillin-resistant Staphylococcus aureus and is considered to enhance transmission, persistence and survival. Subsequently ACMEs were shown to be more prevalent in the coagulase-negative Staphylococcus epidermidis. Previously, ACME types were distinguished by characteristic combinations of the arc and opp3 operons [I (arc+, opp3+), II (arc+, opp3-) and III (arc-, opp3+)] encoding an arginine deaminase pathway and oligopeptide permease transporter, respectively. Recently two novel ACME types harboring the potassium transporter-encoding operon kdp were described in oral S. epidermidis isolates [IV (arc+, opp3-, kdp+), and V (arc+, opp3+, kdp+)]. This study investigated two independent oral S. epidermidis isolates that yielded amplimers with kdp-directed primers only when subjected to ACME typing PCRs. Hybrid assemblies based on Illumina MiSeq short-read and Oxford Nanopore MinION long-read whole genome sequences revealed that both isolates harbored a sixth, novel ACME type (VI) integrated into orfX. Both ACME VIs lacked the arc and opp3 operons, harbored the kdp operon adjacent to other commonly ACME-associated genes including speG, hsd, sdr, and rep, but the structural organization of the adjacent regions were distinct. These ACMEs were flanked by different direct repeat sequences and the ACME VI-positive isolates belonged to unrelated genetic clusters. Overall these findings are indicative of independent evolution. The identification of ACME type VI further illustrates the diversity of ACME elements in S. epidermidis. The presence of ACMEs harboring kdp may confer a selective advantage on oral S. epidermidis in a potassium-rich environment such as found in dental plaque.

A molecular epidemiological investigation of methicillin-susceptible Staphylococcus aureus causing bloodstream infections in Ireland, 2006-2017.

The prevalence of methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream infections (BSIs) has increased in many countries, including Ireland. This study aimed to investigate the molecular epidemiology of MSSA causing BSIs in Irish hospitals between 2006 and 2017, when MSSA BSIs increased, to identify any potential patient or pathogen contributing factors. A total of 252 MSSA isolates from patients in Irish hospitals in 2006/2007, 2011 and 2017 underwent spa typing and DNA microarray profiling. Each patient's gender, age, 14-day mortality and epidemiological context of infection were recorded. Significant increases in community-onset (CO) MSSA BSIs and the average patient's age and decreases in hospital-onset (HO) MSSA were identified. Although, extensive genetic diversity was detected amongst the isolates, i.e. 24 multilocus sequence type clonal complexes (CCs)/sequence types and 124 spa types, three CCs (CC30, CC45, CC5) dominated, albeit in different proportions, during the study periods. CC30 declined significantly, in particular spa type t021, and was more common amongst HO-MSSA and CC45 and CC8 increased, particularly spa types t015 and t008, respectively, and were more common amongst CO-MSSA. Five of the seven most frequent spa types were more common amongst CO-MSSA. Although overall multidrug resistance decreased, the prevalence of erm(C) increased significantly and virulence genes decreased, mostly notably egc, tst, scn, sep and fnbB. This study highlights the threat posed by the increasing prevalence of CO-MSSA BSIs and suggests an association with the increasing prevalence of CC45 CO-MSSA, decreasing prevalence of CC30 HO-MSSA, an ageing population and an overall decrease in virulence and resistance genes.

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.

Effects of Reforestation on the Structure and Diversity of Bacterial Communities in Subtropical Low Mountain Forest Soils.

Reforestation with different tree species could alter soil properties and in turn affect the bacterial community. However, the effects of long-term reforestation on bacterial community structure and diversity of subtropical forest soils are poorly understood. In the current study, we applied error-corrected barcoded pyrosequencing to characterize the differences in the soil bacterial community in a low mountain, subtropical forest subjected to reforestation. The communities were sampled in the summer and winter from a native broadleaved forest (BROAD-Nat) and two adjacent coniferous plantations, a Calocedrus formosana forest of 80 years (CONIF-80) and a Cunninghamia konishii forest of 40 years (CONIF-40). The soil bacterial communities among three forest types were dominated by Acidobacteria and Alphaproteobacteria. The distribution of abundant genera among communities was different. Based on the Shannon diversity index, the bacterial alpha diversity of CONIF-40 community was significantly higher than that in the CONIF-80 and BROAD-Nat soils. In both of the coniferous plantations, the soil bacterial diversity in summer was also higher than that in winter. Distribution of some abundant phylogenetic groups, K-shuff and redundancy analysis of beta diversity among communities showed that the bacterial structure of three soil communities differed between two seasons. These results suggest that seasonal differences influence the diversity and structure of bacterial soil communities and that the communities remain different even after a long period of reforestation.

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.

Intra-Hospital, Inter-Hospital and Intercontinental Spread of ST78 MRSA From Two Neonatal Intensive Care Unit Outbreaks Established Using Whole-Genome Sequencing.

From 2009 to 2011 [transmission period (TP) 1] and 2014 to 2017 (TP2), two outbreaks involving community-associated clonal complex (CC) 88-MRSA spa types t186 and t786, respectively, occurred in the Neonatal Intensive Care Unit (NICU) of an Irish hospital (H1). This study investigated the relatedness of these isolates, their relationship to other CC88 MRSA from Ireland and their likely geographic origin, using whole-genome sequencing (WGS). All 28 CC88-MRSA isolates identified at the Irish National MRSA Reference Laboratory between 2009 and 2017 were investigated including 20 H1 patient isolates, two H1 isolates recovered from a single healthcare worker (HCW) 2 years apart, three patient isolates from a second hospital (H2) and one patient isolate from each of three different hospitals (H3, H4, and H5). All isolates underwent DNA microarray profiling. Thirteen international isolates with similar microarray profiles to at least one Irish isolate were selected from an extensive global database. All isolates underwent Illumina MiSeq WGS. The majority of Irish isolates (25/28; all H1 isolates, two H2 isolates and the H3 isolate) were identified as ST78-MRSA-IVa and formed a large cluster, exhibiting 1-71 pairwise allelic differences, in a whole-genome MLST-based minimum spanning tree (MST) involving all Irish isolates. A H1/H2, H1/H3, and H1 HCW/patient isolate pair each exhibited one allelic difference. The TP2 isolates were characterised by a different spa type and the loss of hsdS. The three remaining Irish isolates (from H2, H4, and H5) were identified as ST88-MRSA-IVa and dispersed at the opposite end of the MST, exhibiting 81-211 pairwise allelic differences. Core-genome MLST and sequence-based plasmid analysis revealed the recent shared ancestry of Irish and Australian ST78-MRSA-IVa, and of Irish and French/Egyptian ST88-MRSA-IVa. This study revealed the homogeneity of isolates recovered during two NICU outbreaks (despite spa type and hsdS carriage variances), HCW involvement in the outbreak transmission chain and the strain's spread to two other Irish hospitals. The outbreak strain, CC88/ST78-MRSA-IVa, was likely imported from Australia, where it is prevalent. CC88/ST88-MRSA-IVa was also identified in Irish hospitals and was likely imported from Africa, where it is predominant, and/or a country with a large population of African descent.

Significant Enrichment and Diversity of the Staphylococcal Arginine Catabolic Mobile Element ACME in Staphylococcus epidermidis Isolates From Subgingival Peri-implantitis Sites and Periodontal Pockets.

Staphylococcus aureus and Staphylococcus epidermidis are frequent commensals of the nares and skin and are considered transient oral residents. Reports on their prevalence in the oral cavity, periodontal pockets and subgingivally around infected oral implants are conflicting, largely due to methodological limitations. The prevalence of these species in the oral cavities, periodontal pockets and subgingival sites of orally healthy individuals with/without implants and in patients with periodontal disease or infected implants (peri-implantitis) was investigated using selective chromogenic agar and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Staphylococcus epidermidis was predominant in all participant groups investigated. Its prevalence was significantly higher (P = 0.0189) in periodontal pockets (30%) than subgingival sites of healthy individuals (7.8%), and in subgingival peri-implantitis sites (51.7%) versus subgingival sites around non-infected implants (16.1%) (P = 0.0057). In contrast, S. aureus was recovered from subgingival sites of 0-12.9% of the participant groups, but not from periodontal pockets. The arginine catabolic mobile element (ACME), thought to enhance colonization and survival of S. aureus, was detected in 100/179 S. epidermidis and 0/83 S. aureus isolates screened using multiplex PCR and DNA microarray profiling. Five distinct ACME types, including the recently described types IV and V (I; 14, II; 60, III; 10, IV; 15, V; 1) were identified. ACME-positive S. epidermidis were significantly (P = 0.0369) more prevalent in subgingival peri-implantitis sites (37.9%) than subgingival sites around non-infected implants (12.9%) and also in periodontal pockets (25%) compared to subgingival sites of healthy individuals (4.7%) (P = 0.0167). To investigate the genetic diversity of ACME, 35 isolates, representative of patient groups, sample sites and ACME types underwent whole genome sequencing from which multilocus sequence types (STs) were identified. Sequencing data permitted ACME types II and IV to be subdivided into subtypes IIa-c and IVa-b, respectively, based on distinct flanking direct repeat sequences. Distinct ACME types were commonly associated with specific STs, rather than health/disease states or recovery sites, suggesting that ACME types/subtypes originated amongst specific S. epidermidis lineages. Ninety of the ACME-positive isolates encoded the ACME-arc operon, which likely contributes to oral S. epidermidis survival in the nutrient poor, semi-anaerobic, acidic and inflammatory conditions present in periodontal disease and peri-implantitis.

Molecular Characterization of Nasal Methicillin-Resistant Staphylococcus aureus Isolates Showing Increasing Prevalence of Mupirocin Resistance and Associated Multidrug Resistance following Attempted Decolonization.

Sequential methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients following attempted mupirocin nasal decolonization showed an increase in mupirocin resistance (MR) from 6.6% to 20%. MR isolates from patients who failed decolonization yielded indistinguishable spa types and carried multiple antimicrobial and antiseptic resistance genes, which may guide infection control and prevention.

Observational cross-sectional study of nasal staphylococcal species of medical students of diverse geographical origin, prior to healthcare exposure: prevalence of SCCmec, fusC, fusB and the arginine catabolite mobile element (ACME) in the absence of selective antibiotic pressure.

OBJECTIVE: The aim of this study was to investigate co-located nasal Staphylococcus aureus and coagulase-negative staphylococci (CoNS) (mainly Staphylococcus epidermidis), recovered from healthy medical students in their preclinical year, prior to exposure to the healthcare environment, for the carriage of genes and genetic elements common to both species and that may contribute to S. aureus and methicillin-resistant S. aureus (MRSA) evolution. DESIGN: Prospective observational cross-sectional study. Carriage of antimicrobial resistance and virulence-associated genes in the absence of significant antibiotic selective pressure was investigated among healthy medical students from geographically diverse origins who were nasally co-colonised with S. aureus and CoNS. Clonal lineages of S. aureus isolates were determined. SETTING/PARTICIPANTS: Dublin-based international undergraduate medical students. RESULTS: Nasal S. aureus carriage was identified in 137/444 (30.8%) students of whom nine (6.6%) carried MRSA (ST59-MRSA-IV (6/9), CC1-MRSA-V-SCCfus (3/9)). The genes mecA, fusB, ileS2, qacA/qacC and the arginine catabolic mobile element-arc were detected among colonising nasal staphylococci and had a significantly greater association with CoNS than S. aureus. The rate of co-carriage of any of these genes in S. aureus/CoNS pairs recovered from the same individual was <1%. CONCLUSIONS: The relatively high prevalence of these genes among CoNS of the healthy human flora in the absence of significant antibiotic selective pressure is of interest. Further research is required to determine what factors are involved and whether these are modifiable to help prevent the emergence and spread of antibiotic resistance among staphylococci.

First description of novel arginine catabolic mobile elements (ACMEs) types IV and V harboring a kdp operon in Staphylococcus epidermidis characterized by whole genome sequencing.

The arginine catabolic mobile element (ACME) was first described in the methicillin-resistant Staphylococcus aureus strain USA300 and is thought to facilitate survival on skin. To date three distinct ACME types have been characterized comprehensively in S. aureus and/or Staphylococcus epidermidis. Type I harbors the arc and opp3 operons encoding an arginine deaminase pathway and an oligopeptide permease ABC transporter, respectively, type II harbors the arc operon only, and type III harbors the opp3 operon only. To investigate the diversity and detailed genetic organization of ACME, whole genome sequencing (WGS) was performed on 32 ACME-harboring oro-nasal S. epidermidis isolates using MiSeq- and PacBio-based WGS platforms. In nine isolates the ACMEs lacked the opp3 operon, but harbored a complete kdp operon (kdpE/D/A/B/C) located a maximum of 2.8 kb upstream of the arc operon. The kdp operon exhibited 63% DNA sequence identity to the native S. aureus kdp operon. These findings identified a novel, previously undescribed ACME type (designated ACME IV), which could be subtyped (IVa and IVb) based on distinct 5' flanking direct repeat sequences (DRs). Multilocus sequence typing (MLST) sequences extracted from the WGS data identified the sequence types (STs) of the isolates investigated. Four of the nine ACME IV isolates belonged to ST153, and one to ST17, a single locus variant of ST153. A tenth isolate, identified as ST5, harbored another novel ACME type (designated ACME V) containing the kdp, arc and opp3 operons and flanked by DR_F, and DR_B but lacked any internal DRs. ACME V was colocated with a staphylococcal chromosome cassette mec (SCCmec) IV element and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in a 116.9 kb composite island. The extensive genetic diversity of ACME in S. epidermidis has been further elucidated by WGS, revealing two novel ACME types IV and V for the first time.

Range Expansion and the Origin of USA300 North American Epidemic Methicillin-Resistant Staphylococcus aureus.

The USA300 North American epidemic (USA300-NAE) clone of methicillin-resistant Staphylococcus aureus has caused a wave of severe skin and soft tissue infections in the United States since it emerged in the early 2000s, but its geographic origin is obscure. Here we use the population genomic signatures expected from the serial founder effects of a geographic range expansion to infer the origin of USA300-NAE and identify polymorphisms associated with its spread. Genome sequences from 357 isolates from 22 U.S. states and territories and seven other countries are compared. We observe two significant signatures of range expansion, including decreases in genetic diversity and increases in derived allele frequency with geographic distance from the Pennsylvania region. These signatures account for approximately half of the core nucleotide variation of this clone, occur genome wide, and are robust to heterogeneity in temporal sampling of isolates, human population density, and recombination detection methods. The potential for positive selection of a gyrA fluoroquinolone resistance allele and several intergenic regions, along with a 2.4 times higher recombination rate in a resistant subclade, is noted. These results are the first to show a pattern of genetic variation that is consistent with a range expansion of an epidemic bacterial clone, and they highlight a rarely considered but potentially common mechanism by which genetic drift may profoundly influence bacterial genetic variation.IMPORTANCE The process of geographic spread of an origin population by a series of smaller populations can result in distinctive patterns of genetic variation. We detect these patterns for the first time with an epidemic bacterial clone and use them to uncover the clone's geographic origin and variants associated with its spread. We study the USA300 clone of methicillin-resistant Staphylococcus aureus, which was first noticed in the early 2000s and subsequently became the leading cause of skin and soft tissue infections in the United States. The eastern United States is the most likely origin of epidemic USA300. Relatively few variants, which include an antibiotic resistance mutation, have persisted during this clone's spread. Our study suggests that an early chapter in the genetic history of this epidemic bacterial clone was greatly influenced by random subsampling of isolates during the clone's geographic spread.