Assessing the discriminability of PCR-based open reading frame typing versus single-nucleotide polymorphism analysis via draft whole-genome sequencing of methicillin-resistant Staphylococcus aureus in nosocomial transmission analysis.

Phylogenetic analysis based on single-nucleotide polymorphism (SNP)-based through whole-genome sequencing is recognized as the standard method for probing nosocomial transmission. However, the application of WGS is constrained by the high cost of equipment and the need for diverse analysis tools, which limits its widespread use in clinical laboratory settings. In Japan, the prevalent use of PCR-based open reading frame typing (POT) for tracing methicillin-resistant Staphylococcus aureus (MRSA) transmission routes is attributed to its simplicity and ease of use. Although POT's discriminatory power is considered insufficient for nosocomial transmission analysis, conclusive data supporting this notion is lacking. This study assessed the discriminatory capabilities of SNP analysis and POT across 64 clinical MRSA strains. All 21 MRSA strains of ST5/SCCmec IIa, having more than 16 SNPs, demonstrated distinct clones. Conversely, two strains shared the same POT number and were identified as group A. Among the 12 MRSA strains of ST8/SCCmec IVl with over nine SNPs, five fell into POT group B, and five into POT group C. All four MRSA strains of ST8/SCCmec IVa were classified into POT group D, although they included strains with more than 30 SNPs. Among the 27 MRSA strains of ST1/SCCmec IVa, 14 were classified into POT group E. However, except for two clusters (each comprising two or three strains), all had SNP counts >10 (Fig. 1-D). SNP analysis of MRSA in CC1/SCCmec IV showed that several strains had the same number of SNPs in POT number (106-183-37), even among bacteria with >100 SNPs, indicating POT's limited use in detailed nosocomial transmission analysis.

High Incidence of Metastatic Infections in Panton-Valentine Leucocidin-Negative, Community-Acquired Methicillin-Resistant Staphylococcus aureus Bacteremia: An 11-Year Retrospective Study in Japan.

Panton-Valentine leucocidin (PVL)-negative community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was originally disseminated in Japan and has since replaced healthcare-associated MRSA (HA-MRSA). However, the clinical characteristics of CA-MRSA bacteremia (CA-MRSAB) compared with those of HA-MRSA bacteremia (HA-MRSAB) are unknown. We aim to clarify differences and investigate associations between the clinical manifestations and virulence genes associated with plasma-biofilm formation in PVL-negative CA-MRSA. From 2011 to 2021, when CA-MRSA dramatically replaced HA-MRSA, 79 MRSA strains were collected from blood cultures and analyzed via SCCmec typing and targeted virulence gene (lukSF-PV, cna, and fnbB) detection. The incidence of metastatic infection was significantly higher in CA-MRSAB than in HA-MRSAB. PVL genes were all negative, although cna and fnbB were positive in 55.6% (20/36) and 50% (18/36) of CA-MRSA strains and 3.7% (1/27) and 7.4% (2/27) of HA-MRSA strains, respectively. cna and fnbB carriage were not associated with the development of metastatic infections in MRSAB; however, the bacteremia duration was significantly longer in CA-MRSAB harboring cna. CA-MRSAB may be more likely to cause metastatic infections than HA-MRSAB. Since CA-MRSA is dominant in Japan, suspected metastatic infection foci should be identified by computed tomography, magnetic resonance imaging, and echocardiography when treating MRSAB.

Escalation of antimicrobial resistance among MRSA part 1: focus on global spread.

INTRODUCTION: Staphylococcus aureus produce numerous virulence factors that influence tissue invasion, cytotoxicity, membrane damage, and intracellular persistence allowing them to be very common human pathogens. S. aureus isolates exhibit considerable diversity though specific genotypes have been associated with antimicrobial resistance (AMR) and toxin gene profiles. MRSA is an important pathogen causing both community-acquired (CA) and healthcare-acquired (HCA) infections. Importantly, over the past several decades, both HCA-MRSA and CA-MRSA have spread all over the globe. Even more concerning is that CA-MRSA clones have disseminated into hospitals and HCA-MRSA have entered the community. Factors that enhance spread of MRSA include: poor antimicrobial stewardship and inadequate infection control. The emergence and spread of multidrug resistant (MDR) MRSA has limited therapeutic options. AREAS COVERED: The authors discuss the escalation of MRSA, both HCA-MRSA and CA-MRSA across the globe. A literature search of MRSA was performed via PubMed (up to September 2022), using the key words: antimicrobial resistance; ß-lactams; community-associated MRSA; epidemiology; infection; multidrug resistance; Staphylococcus aureus. EXPERT OPINION: Over the past several decades, MRSA has spread all over the globe. We encourage the judicious use of antimicrobials in accordance with antimicrobial stewardship programs along with infection control measures to minimize the spread of MRSA.

The second nationwide surveillance of antibacterial susceptibility patterns of pathogens isolated from skin and soft-tissue infections in dermatology departments in Japan.

The present study compared trends in antimicrobial resistance patterns in pathogens isolated from skin and soft-tissue infections (SSTIs) in Japan with those of a nationwide survey conducted in 2013. Three organisms that caused most of the SSTIs were collected from 12 dermatology departments in medical centers and 12 dermatology clinics across Japan between April 2019 and August 2020. A total of 390 strains, including 267 Staphylococcus aureus, 109 coagulase-negative staphylococci (CNS), and 14 Streptococcus pyogenes strains were submitted to a central laboratory for antimicrobial susceptibility testing. Patient demographic and clinical information was collated. Methicillin-resistant S. aureus (MRSA) was detected in 25.8% (69/267) of the S. aureus strains. The prevalence of MRSA between the present study and the 2013 survey did not differ significantly. Furthermore, there were no significant differences in MIC values and susceptibility patterns of the MRSA strains to other agents, regardless of a history of hospitalization within 1 year or invasive medical procedures. Methicillin-resistant CNS (MRCNS) was detected in 48.6% (53/109) of CNS isolates, higher than the 35.4% prevalence in the 2013 survey. This difference could be attributed to the heterogeneity in the members of the MRCNS, which comprises multiple staphylococci species, between the 2013 and 2019 surveys. However, it was noted that the susceptibility profiles of the MRCNS to each antibiotic were not significantly different from those identified in the 2013 survey. Most strains of S. pyogenes were susceptible to each antibiotic, similar to the 2013 survey. Continuous monitoring of trends in pathogen and susceptibility profiles is important to advise local public health efforts regarding the appropriate treatment of SSTIs.

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.

A Murine Skin Infection Model Capable of Differentiating the Dermatopathology of Community-Associated MRSA Strain USA300 from Other MRSA Strains.

USA300 is a predominant and highly virulent community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain that is a leading cause of skin and soft tissue infections. We established a murine intradermal infection model capable of demonstrating dermatopathological differences between USA300 and other MRSA strains. In this model, USA300 induced dermonecrosis, uniformly presenting as extensive open lesions with a histologically documented profound inflammatory cell infiltrate extending below the subcutis. In contrast, USA400 and a colonizing control strain M92 caused only localized non-ulcerated skin infections associated with a mild focal inflammatory infiltrate. It was also determined that the dermonecrosis induced by USA300 was associated with significantly increased neutrophil recruitment, inhibition of an antibacterial response, and increased production of cytokines/chemokines associated with disease severity. These results suggest that induction of severe skin lesions by USA300 is related to over-activation of neutrophils, inhibition of host antibacterial responses, and selective alteration of host cytokine/chemokine profiles.

Multiple distinct outbreaks of Panton-Valentine leucocidin-positive community-associated meticillin-resistant Staphylococcus aureus in Ireland investigated by whole-genome sequencing.

BACKGROUND: Panton-Valentine leucocidin (PVL)-positive community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) is increasingly associated with infection outbreaks. AIM: To investigate multiple suspected PVL-positive CA-MRSA outbreaks using whole-genome sequencing (WGS). METHODS: Forty-six suspected outbreak-associated isolates from 36 individuals at three separate Irish hospitals (H1-H3) and from separate incidents involving separate families associated with H2 were investigated by whole-genome multi-locus sequence typing (wgMLST). FINDINGS: Two clusters (CH1 and CH2) consisting of 8/10 and 6/6 PVL-positive t008 ST8-MRSA-IVa isolates from H1 and H2, respectively, were identified. Within each cluster, neighbouring isolates were separated by ≤5 allelic differences; however, ≥73 allelic differences were identified between the clusters, indicating two independent outbreaks. Isolates from the H3 maternity unit formed two clusters (CH3-SCI and CH3-SCII) composed of four PVL-negative t4667 ST5-MRSA-V and 14 PVL-positive t002 ST5-MRSA-IVc isolates, respectively. Within clusters, neighbouring isolates were separated by ≤24 allelic differences, whereas both clusters were separated by 1822 allelic differences, indicating two distinct H3 outbreaks. Eight PVL-positive t127 ST1-MRSA-V+fus and three PVL-negative t267 ST97-MRSA-V+fus isolates from two distinct H2-associated families FC1 (N = 4) and FC2 (N = 7) formed three separate clusters (FC1 (t127), FC2 (t127) and FC2 (t267)). Neighbouring isolates within clusters were closely related and exhibited ≤7 allelic differences. Intrafamilial transmission was apparent, but the detection of ≥48 allelic differences between clusters indicated no interfamilial transmission. CONCLUSION: The frequent importation of PVL-positive CA-MRSA into healthcare settings, transmission and association with outbreaks is a serious ongoing concern. WGS is a highly discriminatory, informative method for deciphering such outbreaks conclusively.

A descriptive analysis of PVL-positive multidrug-resistant Staphylococcus aureus in hospital-associated infections in Saudi Arabia.

Methicillin resistant Staphylococcus aureus infections impose a huge risk to public health in healthcare and community settings worldwide. Therefore, it is of interest to document data on the anti-biogramas and genotypes of isolates from Saudi Arabia. We assessed the antimicrobial susceptibility, determined spa (protein A gene) and analyzed multilocus MLST genotypes, and detected PVL gene in these isolates. We collected 28 clinical MRSA isolates, cultured and determined the minimum inhibitory concentrations of 17 antimicrobial agents using Vitek2 system (BioMerieux, USA) from 3 hospitals in Saudi Arabia during the year 2012. Polymorphic region of the spa and seven housekeeping genes were amplified and sequenced. BioNumerics v.5.1 (Applied Maths) was used for spa typing and MLST. Samples were screened for the presence of PVL and mecA genes using polymerase chain reaction (PCR). Analysis shows that all isolates were susceptible to chloramphenicol, rifampicin, nitrofurantoin, teicoplanin, daptomycin and vancomycin. The T4573/ST22 strains are found to be prevalent in the Saudi Arabia (N=6, 21%). We further noted that three isolates (t363/ST240 strain) were resistant to eight antimicrobial agents. Most of t4573/ST22 strains were PVL positive, resistant to ciprofloxacin and linked to HA-MRSA infections. We document data for the presence of emerging multi drug resistant S. aureus strains carrying the PVL gene circulating within hospitals. This highlights the urgent need for continuous active surveillance and implementation of prevention measures.

Characterization of highly virulent community-associated methicillin-resistant Staphylococcus aureus ST9-SCCmec XII causing bloodstream infection in China.

Previous studies have shown that livestock (LA)-MRSA ST398 evolved from a human-adapted methicillin-susceptible S. aureus (MSSA) clone. However, detailed information regarding ST9 is still unclear. Here, we characterized a community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) ST9-SCCmec XII isolate that has not been previously reported to cause serious disease in China. We obtained whole-genome sequences of one ST9-t899-XII isolate-ZY462471-from a patient with bloodstream infection without livestock contact. The antibiotic susceptibilities of ZY462471 were determined and the clinical information was extracted from medical notes and compared with twenty-seven previously sequenced genomes. Phylogenetic reconstruction was performed to investigate the probable host evolutionary origins of ZY462471, and the difference in resistome and virulence factors were investigated. Virulence assay was performed to evaluate the high virulence potential of ZY462471 and compare the virulence between the closest ST9 MSSA neighbours. Clinical data suggested that ZY462471 is a CA-MRSA. Phylogenetic analysis showed a much closer relationship of ZY462471 with human-associated MSSA ST9 isolates than other LA-MRSA ST9 isolates, suggesting that ZY462471 probably evolved from ST9 MSSA predecessors by acquiring an SCCmec cassette. Importantly, virulence assays indicated that ZY462471 was highly virulent and compared with the MSSA ST9 predecessors, ZY462471 did not show attenuated virulence. Finally, we found that ZY462471 harboured an immune evasion cluster (IEC)-carrying ßC-Φ, which is typically found in human clinical S. aureus rather than LA-MRSA isolates, suggesting that ZY4762471 obtained the IEC-carrying ßC-Φs from human clinical S. aureus strains. Considering its high virulence potential, this strain should be monitored to prevent more widespread dissemination.

Frequent Methicillin-Resistant Staphylococcus aureus Introductions Into an Inner-city Jail: Indications of Community Transmission Networks.

BACKGROUND: Jails may facilitate spread of methicillin-resistant Staphylococcus aureus (MRSA) in urban areas. We examined MRSA colonization upon entrance to a large urban jail to determine if there are MRSA transmission networks preceding incarceration. METHODS: Males incarcerated in Cook County Jail (Chicago) were enrolled, with enrichment for people living with human immunodeficiency virus (PLHIV), within 72 hours of intake. Surveillance cultures assessed prevalence of MRSA colonization. Whole-genome sequencing (WGS) identified preincarceration transmission networks.We examined methicillin-resistant Staphylococcus aureus (MRSA) isolates to determine if there are transmission networks that precede incarceration. A large proportion of individuals enter jail colonized with MRSA. Molecular epidemiology and colonization risk factors provide clues to community reservoirs for MRSA. RESULTS: There were 718 individuals (800 incarcerations) enrolled; 58% were PLHIV. The prevalence of MRSA colonization at intake was 19%. In multivariate analysis, methamphetamine use, unstable housing, current/recent skin infection, and recent injection drug use were predictors of MRSA. Among PLHIV, recent injection drug use, current skin infection, and HIV care at outpatient clinic A that emphasizes comprehensive care to the lesbian, gay, bisexual, transgender community were predictors of MRSA. Fourteen (45%) of 31 detainees with care at clinic A had colonization. WGS revealed that this prevalence was not due to clonal spread in clinic but rather to an intermingling of distinct community transmission networks. In contrast, genomic analysis supported spread of USA500 strains within a network. Members of this USA500 network were more likely to be PLHIV (P < .01), men who have sex with men (P < .001), and methamphetamine users (P < .001). CONCLUSIONS: A large proportion of individuals enter jail colonized with MRSA. Molecular epidemiology and colonization risk factors provide clues to identify colonized detainees entering jail and potential community reservoirs of MRSA.

A Spatial Analysis of Health Disparities Associated with Antibiotic Resistant Infections in Children Living in Atlanta (2002-2010).

BACKGROUND: Antibiotic resistant bacteria like community-onset methicillin resistant Staphylococcus aureus (CO-MRSA) have continued to cause infections in children at alarming rates and are associated with health disparities. Geospatial analyses of individual and area level data can enhance disease surveillance and identify socio-demographic and geographic indicators to explain CO-MRSA disease transmission patterns and risks. METHODS: A case control epidemiology approach was undertaken to compare children with CO-MRSA to a noninfectious condition (unintentional traumatic brain injury (uTBI)). In order to better understand the impact of place based risks in developing these types of infections, data from electronic health records (EHR) were obtained from CO-MRSA cases and compared to EHR data from controls (uTBI). US Census data was used to determine area level data. Multi-level statistical models were performed using risk factors determined a priori and geospatial analyses were conducted and mapped. RESULTS: From 2002-2010, 4,613 with CO-MRSA and 34,758 with uTBI were seen from two pediatric hospitals in Atlanta, Georgia. Hispanic children had reduced odds of infection; females and public health insurance were more likely to have CO-MRSA. Spatial analyses indicate significant 'hot spots' for CO-MRSA and the overall spatial cluster locations, differed between CO-MRSA cases and uTBI controls. CONCLUSIONS: Differences exist in race, age, and type of health insurance between CO-MRSA cases compared to noninfectious control group. Geographic clustering of cases is distinct from controls, suggesting placed based factors impact risk for CO-MRSA infection.

CA-MRSA and HA-MRSA coexist in community and hospital settings in Uganda.

Background: Methicillin resistant Staphylococcus aureus (MRSA) strains were once confined to hospitals however, in the last 20 years MRSA infections have emerged in the community in people with no prior exposure to hospitals. Strains causing such infections were novel and referred to as community-associated MRSA (CA-MRSA). The aim of this study was to determine the MRSA carriage rate in children in eastern Uganda, and to investigate coexistence between CA-MRSA and hospital-associated (HA-MRSA). Methods: Between February and October 2011, nasopharyngeal samples (one per child) from 742 healthy children under 5 years in rural eastern Uganda were processed for isolation of MRSA, which was identified based on inhibition zone diameter of ≤19 mm on 30 µg cefoxitin disk. SCCmec and spa typing were performed for MRSA isolates. Results: A total of 140 S. aureus isolates (18.9%, 140/742) were recovered from the children of which 5.7% (42/742) were MRSA. Almost all (95.2%, 40/42) MRSA isolates were multidrug resistant (MDR). The most prevalent SCCmec elements were types IV (40.5%, 17/42) and I (38.1%, 16/42). The overall frequency of SCCmec types IV and V combined, hence CA-MRSA, was 50% (21/42). Likewise, the overall frequency of SCCmec types I, II and III combined, hence HA-MRSA, was 50% (21/42). Spa types t002, t037, t064, t4353 and t12939 were detected and the most frequent were t064 (19%, 8/42) and t037 (12%, 5/42). Conclusion: The MRSA carriage rate in children in eastern Uganda is high (5.7%) and comparable to estimates for Mulago Hospital in Kampala city. Importantly, HA-MRSA (mainly of spa type t037) and CA-MRSA (mainly of spa type t064) coexist in children in the community in eastern Uganda, and due to high proportion of MDR detected, outpatient treatment of MRSA infection in eastern Uganda might be difficult.

Severe Disease Caused by Community-Associated MRSA ST398 Type V, Australia, 2017.

Using whole-genome sequencing, we identified a community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) sequence type (ST) 398 type V (5C2&5) isolate (typically found in China) in Australia in 2017. This CA-MRSA ST398 variant was highly virulent, similar to other related CA-MRSAs of ST398. This strain should be monitored to prevent more widespread dissemination.

Evidence of latent molecular diversity determining the virulence of community-associated MRSA USA300 clones in mice.

INTRODUCTION: The USA300 clone of community-associated MRSA is reported to be hypervirulent and epidemic in the United States. This clone causes a variety of diseases from lethal pneumonia to mild skin infections. We hypothesized that evolutionary diversity may exist among USA300 clones, which may link virulence traits with host responses and mortality rates. METHODS: USA300 isolates from severe pneumonia (IP) and skin infection (IS) were characterized by pulsed-field gel electrophoresis (PFGE) and next-generation sequencing. Their virulence traits and host responses were compared in a lung infection model. RESULTS: The two USA300 isolates were found to be identical in genomic analysis. Robust IL-6 production, aggregation of bacteria, and hemorrhaging were observed in IP-infected lungs, which were associated with a higher rate of mortality than that observed with strain IS. Few neutrophils were detected in the lungs infected with strain IP, even at high bacterial loads. Massive production of α-toxin and coagulase were evident during the early phase of IP infection, and robust gene expression of hla (α-toxin) and lukS-PV (Panton-Valentine leukocidin), but not coa, agrA, or rnaIII, was confirmed in vitro. Strain IP also induced strong hemolysis in red blood cells. CONCLUSIONS: The present data demonstrated latent diversity in the virulence of USA300 clones. Unknown regulatory mechanisms, probably involving a host factor(s) as a trigger, may govern the virulence expression and resultant high mortality in certain sub-clones of USA300.

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.

Predominance of community-associated sequence type 59 methicillin-resistant Staphylococcus aureus in a paediatric intensive care unit.

PURPOSE: To investigate the distribution of molecular types of methicillin-resistant Staphylococcus aureus (MRSA) in a paediatric intensive care unit (PICU) according to their community-associated (CA) and hospital-associated (HA) source of acquisition, and thus assess the degree to which CA-MRSA has been introduced into the PICU. METHODOLOGY: We implemented an MRSA surveillance in a PICU during 2013-2016 and investigated the genetic diversity of the isolates retrospectively using three genetic typing methods, as well as antibiograms and virulence factor profiles.Results/Key findings. From 2684 specimens, we identified 60 MRSA isolates, 43 of which were ST59 CA-MRSA. These 43 ST59 MRSA isolates could be further subtyped into 2 clusters and 7 sporadic isolates by pulsed-field gel electrophoresis, and 3 spa types, which demonstrated the genetic diversity in ST59 MRSA. Phenotypic diversity was also demonstrated among these ST59 MRSA isolates, with 12 virulence factor profiles and 4 antibiograms being identified. Epidemiological information showed that 43 ST59 MRSA isolates were both community-associated (15 isolates) and hospital-associated (28 isolates) and caused colonization and various types of infections in different age groups of children. CONCLUSIONS: Our results show that a predominant ST59 CA-MRSA has been introduced into the PICU to a significant extent. This has caused the ST59 HA-MRSA and CA-MRSA in the PICU to be indistinguishable. Our results also demonstrate that when we are interpreting situations where the causative agents of infections focus on very limited pathogenic clones, combined typing methods and epidemiological information are needed to investigate isolates' genetic and phenotypic diversity to distinguish an outbreak from endemic cases.

Detection and analysis of methicillin-resistant human-adapted sequence type 398 allows insight into community-associated methicillin-resistant Staphylococcus aureus evolution.

BACKGROUND: Severe infections with highly virulent community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are a global problem. However, the molecular events defining the evolution of CA-MRSA are still poorly understood. MRSA of sequence type (ST) 398 is known to frequently infect livestock, while ST398 isolates infecting humans are commonly methicillin-susceptible or represent MRSA originating from livestock-associated (LA)-MRSA. METHODS: We used whole genome sequencing of newly detected CA-MRSA ST398 isolates, in comparison to geographically matched LA-MRSA and methicillin-sensitive ST398, to determine their evolutionary history. Furthermore, we used phenotypic analyses including animal infection models to gain insight into the evolution of virulence in these CA-MRSA isolates. Finally, we determined methicillin resistance and expression of the methicillin resistance-conferring gene mecA and its penicillin-binding protein product, PBP2a, in a large series of CA-MRSA strains of divergent STs. RESULTS: We report several cases of severe and fatal infections due to ST398 CA-MRSA. The responsible isolates showed the typical genetic characteristics reported for human-adapted methicillin-sensitive ST398. Whole genome sequencing demonstrated that they evolved from human-adapted, methicillin-susceptible clones on several different occasions. Importantly, the isolates had not undergone consistent genetic alterations or changes in virulence as compared to their methicillin-susceptible predecessors. Finally, we observed dramatically and consistently lower methicillin resistance and expression of the resistance gene mecA, as compared to hospital-associated MRSA strains, in a diverse selection of CA-MRSA strains. CONCLUSIONS: Our study presents evidence for the development of highly virulent human-adapted ST398 CA-MRSA isolates from methicillin-susceptible predecessors. Notably, our investigation indicates that, in contrast to widespread notions, the development of CA-MRSA is not necessarily associated with the acquisition of specific virulence genes or other virulence-increasing changes. Rather, our findings emphasize the importance of the CA-MRSA-characteristic staphylococcal cassette chromosome mec types, which provide only low-level methicillin resistance, for that process. Our findings are of particular importance for the diagnosis of CA-MRSA, inasmuch as they indicate that the presence of specific virulence genes cannot generally be used for that purpose.

THE FREQUENCY OF COMMUNITY-ACQUIRED METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (CA-MRSA) AMONG SAMPLES IN INSTITUTE FOR PUBLIC HEALTH IN CANTON SARAJEVO.

BACKGROUND: The increase in the incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections lacking risk factors for exposure to the health care system has been associated with the recognition of new MRSA clones known as community-associated MRSA (CA-MRSA). These strains have been distinguished from health care-associated MRSA (HA-MRSA) strains by epidemiological, molecular and genetic means as well as by antibiotic susceptibility profile, tissue tropism and virulence traits. OBJECTIVE: To assess prevalence and antibiotic susceptibility profile of CA-MRSA in Canton Sarajevo, Bosnia and Herzegovina. RESULTS: Out of 1.905 positive Staphylococcus aureus isolates from various samples of outpatients collected during six months, 279 (14,64%) were MRSA isolates. Out of 279 MRSA samples, 133 (47,67%) were found in nasal swabs, from which 48 (36,09%) were in the age group <1 year and 39 (29,32 %) are in the age 1-5 year. Rate of the positive skin swabs was highest among the subject of age group <1 year (46 or 54,12 %) and 1-5 year (18 or 21,18 %). Predominantly antibiotic types among MRSA strains are resistant to penicillin and cefoxitin (36,90 %) and to penicillin, cefoxitin and erythromycin (61,35 %). CONCLUSION: Continued monitoring of epidemiology and emerging drug resistance data is critical for the effective management of these infections.

The molecular epidemiology of incident methicillin-resistant Staphylococcus aureus cases among hospitalized patients in Alberta, Canada: a retrospective cohort study.

BACKGROUND: Infection Prevention and Control (IPC) surveillance for incident methicillin-resistant Staphylococcus aureus (MRSA) in hospitalized patients is performed in a complete provincial surveillance network of all acute care facilities in Alberta, Canada. IPC surveillance is centralized using a web-based data entry platform so that each patient is counted only once. All diagnostic laboratories submit the first clinical MRSA isolate associated with a patient without previous MRSA positive clinical cultures in the preceding year to the Provincial Laboratory for Public Health (ProvLab) for molecular typing. This study will investigate the relationship between the IPC epidemiological classification based on time of detection following admission to hospital (Hospital Acquired and Community Associated) and the matched laboratory MRSA surveillance data using a retrospective cohort study design. METHODS: Incident IPC MRSA cases were classified according to IPC epidemiologic definitions. DNA sequencing of the Staphylococcus protein A (spa) gene and pulsed-field gel electrophoresis (PFGE) typing was performed. IPC MRSA surveillance data were matched to the ProvLab molecular surveillance data. Univariate comparisons of proportions were performed for categorical variables and the Student's t test for continuous variables. RESULTS: MRSA molecular typing data were available for matching for 46.7 % (2248/4818) of incident IPC cases. There was agreement in definitions for traditional nosocomial clones (USA100/CMRSA2) with Hospital Acquired (HA)-MRSA (65.1 % of all IPC HA-MRSA) and traditional community clones (USA400/CMRSA7 and USA300/CMRSA10) with Community Acquired (CA)-MRSA (62.4 % of CA-MRSA). However, we observed discordance for both traditional nosocomial/CA-MRSA (30.4 % of CA-MRSA) and for traditional community/HA-MRSA (26.9 % of HA-MRSA). CONCLUSIONS: We note agreement between traditional nosocomial clones and HA-MRSA, and traditional community clones and CA-MRSA. However, approximately one-quarter of HA-MRSA are those of traditional community clones while approximately one-third of CA-MRSA are those of traditional nosocomial clones. Collaborative provincial MRSA surveillance is important as the distinction between IPC case attribution in acute care settings and the historical definitions of MRSA clones as community- or healthcare-associated have blurred.

Molecular Characterization of Community-Associated Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Children with Skin Infections in Busan, Korea

The prevalence and molecular characteristics of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and methicillin-susceptible S. aureus (CA-MSSA) from children with skin infection were investigated by staphylocoagulase (SC) typing, multilocus sequence typing (MLST), SCCmec typing and virulent toxins, including Panton-Valentine leucocidin (PVL), and exfoliative toxins (ET). Among 69 cases of CA-S. aureus for a 3 month period from March to June, 2014 at hospital in Busan, 28 (40.6%) were MRSA and 41 (59.4%) were MSSA. Of the 28 CA-MRSA isolates, two major clones were identified as SC type Vb-ST72-SCCmec type IV (53.6%) and SC type l-ST89-SCCmec type II variant (42.8%), and the remaining one (3.6%) was SC type lll-ST8-SCCmec type IV. In CA-MSSA, the prevalent clone was SC type Vb-ST72 (29.3%), followed by SC type Vb-ST188 (21.9%), SC type Va-ST121 (19.5%) and SC type lV-ST30 (9.6%). None was positive for PVL gene, and all of the SC type l-ST89-SCCmec type II variant clones were ETB gene positive. The data suggest that there are significant clonal relatedness with specific SC types, and genetic diversities in both community strains isolated from children with skin infections.