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Healthcare workers (HCWs) are proposed as the potential source of transmission of Staphylococcus aureus to hospitalized patients, especially in burn units. This study aimed to investigate S. aureus from burn wound infections and those from the nose of HCWs in terms of antibiotic resistance, the presence of Panton-Valentine leucocidin-encoding gene (pvl) and the arginine catabolic mobile element (ACME), and the ability for biofilm formation. Also, the genetic diversity of isolates was assessed using staphylococcal protein A (spa) typing and staphylococcal cassette chromosome mec (SCCmec) typing. Overall, regarding the studied factors, significant differences were found neither between isolates from patients and HCWs nor between methicillin-resistant and methicillin-susceptible isolates (except for multidrug resistance which was significantly higher in MRSA). The most frequent SCCmec types were type I and III. ACME-arcA was only detected in isolates from patients and similarly the presence of ACME-opp3 was the most prevalent in this group. The presence of common clonal complexes among patient isolates and more importantly between isolates from patients and HCWs is warning. The high prevalence of virulence factors, both in MRSA and MSSA, emphasizes the importance of MSSA in burn centers. Finding no significant difference in the presence of virulence-associated factors between isolates from patients and HCWs demonstrates the need to take HCWs into account as important reservoirs.

Background USA300 [ST8-staphylococcal cassette chromosome mec type IVa (ST8-IVa)/arginine catabolic mobile element (ACME) positive] is a major Panton–Valentine leucocidin (PVL)-positive community-acquired MRSA (CA-MRSA) clone. In Japan, we identified USA300-like strains with characteristics (ST8-IVc/ACME negative) similar to those of USA300. Objectives To reveal the evolution of the USA300-like strains. Methods The whole-genome sequence of a USA300-like strain was determined and genome analysis was performed using Type Strain Genome Server, MUSCLE and progressiveMauve. Results Genome-based phylogenetic analysis showed that the USA300-like strain is more similar to the USA300-Latin American variant (USA300-LV), which is a PVL-positive CA-MRSA clone identified in South America, than to USA300. Instead of the ACME, copper and mercury resistance mobile elements were located on the genome of the USA300-like strain. In addition, the USA300-like strain possessed a unique mobile genetic element, ICE6013. Therefore, we named this novel USA300-LV variant identified in Japan as USA300-LV/J. Conclusions Our findings strongly suggest that a PVL-positive CA-MRSA USA300-LV/J clone originating from abroad has uniquely evolved and disseminated in Japan.

Background: Staphylococcus epidermidis is a commensal of human skin flora and a frequent causative micro-organism in prosthetic joint infections (PJIs). To date, no single marker has been identified to distinguish infecting strains from commensal S. epidermidis populations. Aim: To find possible genetic markers to distinguish between the two populations. Methods: Fifty S. epidermidis strains from patients with PJIswere analysed, 50 fromthe skin of healthy individuals (commensal strains) and 17 from the surgical field of patients undergoing primary arthroplasty. In these three groups the antimicrobial susceptibility profile, sequence type, biofilm formation, and virulence factors were studied. Strains from the surgical field have not been compared previously with strains from the other two groups. Findings: S. epidermidis strains from PJI patients were significantly more antibiotic resistant than commensal strains and surgical field strains. A wide variety of sequence types was found in commensal and surgical field strains. The predominant sequence type was ST2 and it was only present in PJI strains (44%). Differences in biofilm production did not differ between populations. Virulence genes sdrF and bhp, the complete ica operon, and the insertion sequence IS256 were significantly predominant in PJI strains. By contrast, embp and hld genes and the arginine catabolic mobile element (ACME) were more prevalent in commensal strains. Surgical field strains could be a valid control group to discriminate between infecting and commensal strains. Conclusion: A combination of characteristic features can differentiate between infecting and commensal S. epidermidis strains in PJI, whereas a single marker cannot. (C) 2020 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Panton-Valentine leukocidin (PVL) is a pore-forming toxin encoded by genes on bacteriophages distributed to Staphylococcus aureus, associated with its increased virulence to humans. In this study, molecular epidemiological characteristics were investigated for 239 clinical isolates of S. aureus collected in a tertiary care hospital in Yangon, Myanmar, particularly with regard to methicillin resistance and PVL genes. Methicillin-resistant S. aureus (MRSA) accounted for 13.8% (33/239) and possessed mostly types IV- and V-SCCmec, while types III- and IX-SCCmec were identified in a few isolates. PVL genes were detected in 66.7% and 28.6% in MRSA and methicillin-susceptible S. aureus (MSSA), respectively. Among PVL-positive MRSA, ST772/SCCmec-V isolates (i.e., Bengal Bay clone) were predominant (73%, 16/22), and harbored PVL gene-encoding bacteriophage ΦSa119. Furthermore, two ST8-MRSA-SCCmec-IVa isolates harbored type-I arginine catabolic mobile element and ΦSa2usa: these isolates were considered the USA300 clone first identified in Myanmar. ΦPVL was the most frequent PVL phage among MSSA (56%, 33/59), and distributed to various genotypes, with ST88 and ST121 being dominant. In contrast, ΦSa2usa and ΦSa119 were also detected in MSSA with genotypes other than ST8 or ST772, suggesting the spread of these PVL phages to MSSA. The present study revealed potentially high prevalence of PVL phages among diverse clones of MRSA and MSSA in Myanmar.

Molecular epidemiological characteristics were investigated for 1,041 isolates of methicillin-resistant Staphylococcus aureus (MRSA) collected in a tertiary care hospital in northern Japan for a 4-year period (2011-2014). Genotypes (staphylococcal cassette chromosome mec [SCCmec], sequence type, spa, coa, etc.) and the presence of drug resistance/virulence factor genes in the isolates were analyzed by multiplex/uniplex PCR, and PCR-direct sequencing as needed. Among these MRSA, predominant SCCmec type was IIa (87.2%), followed by IV (10.1%) and V (1.2%). The SCCmec IIa-MRSA belonged to coagulase genotype (coa) IIa and ST5/ST764, which are known as major health care-associated-MRSA (HA-MRSA) in Japan (New York/Japan clone) and its variant. Panton-Valentine leucocidine (PVL) genes were detected in only five isolates (0.5%) with genotypes ST8-SCCmec IVa/spa-t008/coa-IIIa (USA300 clone), ST6-SCCmec IVb, and ST59-SCCmec V (Taiwanese clone). Arginine catabolic mobile element (ACME) type I and II' were identified in three and five isolates of ST8-SCCmec IVa and ST764-SCCmec IIa MRSA, respectively. PVL-/ACME- isolates were classified into various STs/clonal complexes (CCs), with CC1, CC5, CC8, CC89, and CC121 being common. It was notable that SCCmec IVl was the most common among SCCmec IV subtypes, and was carried by almost half of coa-IIIa isolates (47%, 34/72) without PVL genes, which represented the novel ST8 MRSA clone spreading in Japan (i.e., "ST8/CA-MRSA/J"). Uncommon MRSA clones in Japan, ST72-SCCmec IV (South Korean clone), ST398 livestock-associated clone, and ST20 bovine-associated MRSA, were identified. Furthermore, we isolated PVL-negative ST8-SCCmec I/coa-IIIa and ST81-SCCmec V/coa-VIIa MRSA, which were considered presumptive novel clones. The present study revealed the genetic diversity of HA-MRSA, including potentially emerging clones of putative different origins.

The acquisition of drug resistance and virulence by staphylococcal species colonizing humans is a growing public health concern. The present study was conducted to investigate the prevalence, antimicrobial resistance and genetic characteristics of Staphylococcus isolates from the oral cavity and skin (hand) of systemically healthy subjects with dental disease and dental staff in northern Japan. Among a total of 133 subjects (91 patients and 42 staff), 87 coagulase-positive Staphylococcus (83 S. aureus/4 S. argenteus) and 162 coagulase-negative Staphylococcus (CoNS) isolates were recovered from 59 (44.4%) and 95 (71.4%) subjects, respectively. Three oral isolates were methicillin-resistant S. aureus (MRSA) (3.6%, 3/83) that were genotyped as ST8-SCCmec-IVl, ST4775(CC1)-SCCmec-IVa and ST6562(CC8)-SCCmec-IVa. Remarkably, the ST6562 isolate harbored PVL genes on ΦSa2usa and type I ACME (arginine catabolic mobile element). Four methicillin-susceptible isolates were identified as S. argenteus belonging to ST1223 and ST2250, which harbored enterotoxin genes egc-2 and sey, respectively. Among the fourteen CoNS species identified, methicillin-resistant (MR) isolates were detected in five species (11 isolates, 13.3% of CoNS), with S. saprophyticus and S. haemolyticus being the most common. ACME was prevalent in only S. epidermidis and S. capitis. These findings indicated the potential distribution of USA300 clone-like MRSA, toxigenic S. argenteus and MR-CoNS in the oral cavity of dental patients.

Staphylococcus epidermidis has become an important nosocomial pathogen. Multidrug resistance makes S. epidermidis infections difficult to treat. The study aims to describe the genomic characteristics of methicillin-resistant S. epidermidis (MRSE) isolated from clinical sources, to comprehend the genetic basis of antibiotic resistance, virulence, and potential pathogenicity. Sixteen MRSE underwent whole-genome sequencing, and bioinformatics analyses were carried out to ascertain their resistome, virulome, mobilome, clonality, and phylogenomic relationships. In all, 75% of isolates displayed multidrug resistance and were associated with the carriage of multiple resistance genes including mecA, blaZ, tet(K), erm(A), erm(B), erm(C), dfrG, aac(6′)-aph(2′′), and cat(pC221) conferring resistance to β-lactams, tetracyclines, macrolide–lincosamide–streptogramin B, aminoglycosides, and phenicols, which were located on both plasmids and chromosomes. Their virulence profiles were evidenced by the presence of genes involved in adherence/biofilm formation (icaA, icaB, icaC, atl, ebh, and ebp), immune evasion (adsA, capC, and manA), and antiphagocytosis (rmlC, cdsA, and A). The community-acquired SCCmec type IV was the most common SCCmec type. The CoNS belonged to seven multilocus sequence types (MLSTs) and carried a diversity of mobile genetic elements such as phages, insertion sequences, and plasmids. The bacterial anti-phage defense systems clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) immunity phage system and restriction-modification system (R-M system) and the arginine catabolic mobile element (ACME) involved in immune evasion and transport of virulence genes were also found. The insertion sequence, IS256, linked with virulence, was found in 56.3% of isolates. Generally, the isolates clustered according to STs, with some similarity but also considerable variability within isolates. Whole-genome sequencing and bioinformatics analysis provide insights into the likely pathogenicity and antibiotic resistance of S. epidermidis, necessitating surveillance of this emerging pathogen.

The methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 8 Panton-Valentine toxin (PVL)-positive USA300 clone has a worldwide distribution. The USA300 North American (NA) variant, harbouring the arginine catabolic mobile element (ACME), is predominant in the USA while the Latin American (LV) variant is predominant in Northern South America. Both variants have failed to become endemic in Europe. We examined here the epidemiology of the USA300 clone in Belgium from 2006 to 2019. A total of 399 clonal complex 8 PVL-positive MRSA isolates received between 2006 and 2019 by the Belgian National Reference Laboratory for S. aureus were investigated for the presence of ACME. Selected ACME-positive (n=102) and ACME-negative (n=16) isolates were sequenced, characterized for the presence of several resistance and virulence molecular markers and subjected to phylogenetic analysis. A total of 300 isolates were USA300-NA (ACME-positive), while only 99 were ACME-negative. Most USA300-NA interspersed in the phylogeny analysis with isolates from other countries, suggesting multiple introductions. However, two big clades were maintained and spread over a decade, peaking between 2010 and 2017 to finally decrease. Few ACME-negative isolates, mainly related to trips to South America, were identified as USA300-LV. The remaining ACME-negative isolates were ST8 SCCmec IVb or ST923 SCCmec IVa (COL923). Two clades of the USA300-NA clone have successfully spread in Belgium, but seem to currently decrease. Related South American variants have been detected for the first time in Belgium, including the emerging COL923 clone., SCOPUS: ar.j, info:eu-repo/semantics/published

In Poland artisanal cheese production is an important local economic activity. Artisanal cheese is usually produced using raw cow's milk, animal rennet and salt, without the addition of starter cultures. Coagulase negative staphylococci (CoNS) are often present in artisanal cheeses. Pathogenic potential of some CoNS species, especially S. epidermidis, suggests that they could correspond to emerging pathogens. The identified risk factors correspond to virulence, antibiotic resistance and biofilm formation. Therefore, we aimed to characterize S. epidermidis isolated along the artisanal raw milk production chain. Seventy artisanal cheeses samples from unpasteurized cow milk purchased in Podlasie and Warmia and Mazury region in Poland, were included in this study. A total of 26 S. epidermidis isolates were obtained. Most of them were antimicrobial resistant, such as to penicillin (84,6%), clindamycin (46,2%), tetracycline (42,3%), erythromycin (42,3%) and cefoxitin (26,9%). Only one isolate was susceptible to all antibiotics used in the study. All methicillin resistant S. epidermidis strains (26,9%) harbored mecA gene. Isolates, phenotypic resistant to tetracycline, harbored at least one tetracycline resistance determinant on which tet(M) was most frequent. Moreover, all tetracycline resistant strains harbored Tn916-Tn1545-like integrase family gene. In the erythromycin resistant isolates, the macrolide resistance genes ermC, ermB or msrA/B were present. Seven strains demonstrated a strong ability to form biofilm and moderate and weak biofilm was demonstrated by 4 strains, whereas 11 of S. epidermidis isolates were found to be unable to form a biofilm. All strains producing strong biofilm harbored the icaD gene which occurred independently or in combination with the icaA. Insertion element IS256, was identified in 15,4% of S. epidermidis strains, all of which were multidrug resistant. Arginine Catabolic Mobile Element (ACME) was identified in 13 of the 26 examined strains (50%). Most common was ACME type I (26,9%), followed by type III (15,4%) and type II (7,7%). Our data indicate that S. epidermidis are widely present in artisanal cheeses from raw whole cow milk in Poland. Many isolated strains containing more virulence factors and antibiotic resistant and carry mobile genetic elements which represent a potential source of resistance transmission to bacteria in humans.

Objectives: Reports of USA300 methicillin-resistant Staphylococcus aureus (MRSA) strain were still scarce in neonatal intensive care units (NICUs) and the relationship of USA300 MRSA to clinical infections is still controversial. The primary outcome was the incidence of MRSA infections caused by the USA300 and non-USA300 strains at a NICU in Japan. Methods: This retrospective cohort study was conducted between November 2011 and October 2016 at Tokyo Metropolitan Children’s Medical Center in Japan. All MRSA isolated after 48 h of hospitalization were included for analysis by pulsed-field gel electrophoresis (PFGE) using the standard USA300 strain. Genes were tested for Panton-Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME). A whole genome sequence was performed for representative isolates of USA300. Results: In total, 109 MRSA isolates were included for analysis. PFGE classified 34 and 75 isolates of USA300 and non-USA300 MRSA, respectively. Both PVL and ACME genes were detected in USA300 and non-USA300 strains at rate of 100% (34/34) and 5.3% (4/75), respectively (P

Difficult-to-treat infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are of concern in people living with HIV infection as they are more vulnerable to infection. We aimed to identify molecular characteristics of MRSA colonizing newly diagnosed HIV-infected adults in Tanzania. Individuals newly diagnosed with HIV infection were recruited in Dar es Salaam, Tanzania, from April 2017 to May 2018, as part of the randomized clinical trial CoTrimResist (ClinicalTrials.gov identifier: NCT03087890). Nasal/nasopharyngeal isolates of Staphylococcus aureus were susceptibility tested by disk diffusion method, and cefoxitin-resistant isolates were characterized by short-reads whole genome sequencing. Four percent (22/537) of patients carried MRSA in the nose/nasopharynx. MRSA isolates were frequently resistant towards gentamicin (95%), ciprofloxacin (91%), and erythromycin (82%) but less often towards trimethoprim-sulfamethoxazole (9%). Seventy-three percent had inducible clindamycin resistance. Erythromycin-resistant isolates harbored ermC (15/18) and LmrS (3/18) resistance genes. Ciprofloxacin resistance was mediated by mutations of the quinolone resistance-determining region (QRDR) sequence in the gyrA (S84L) and parC (S80Y) genes. All isolates belonged to the CC8 and ST8-SCCmecIV MRSA clone. Ninety-five percent of the MRSA isolates were spa-type t1476, and one exhibited spa-type t064. All isolates were negative for Panton-Valentine leucocidin (PVL) and arginine catabolic mobile element (ACME) type 1. All ST8-SCCmecIV-spa-t1476 MRSA clones from Tanzania were unrelated to the globally successful USA300 clone. Carriage of ST8 MRSA (non-USA300) was common among newly diagnosed HIV-infected adults in Tanzania. Frequent co-resistance to non-beta lactam antibiotics limits therapeutic options when infection occurs.

S. epidermidis is an opportunistic bacterium that has established itself as a successful nosocomial pathogen. The modern era of novel therapeutics and medical devices has extended the longevity of human life, but at the same time, we also witness the evolution of pathogens to adapt to newly available niches in the host. Increasing antibiotic resistance among pathogens provides an example of such pathogen adaptation. With limited opportunities to modify the core genome, most of the adaptation occurs by acquiring new genes, such as virulence factors and antibiotic resistance determinants present in MGEs. In this study, we describe that the sesJ gene, encoding a recently discovered cell wall-anchored protein in S. epidermidis, is present in both ACME and the SCC element. The presence of virulence factors in MGEs can influence the virulence potential of a specific strain. Therefore, it is critical to study the virulence factors found in MGEs in emerging pathogenic bacteria or strains to understand the mechanisms used by these bacteria to cause infections., Staphylococcus epidermidis is a leading cause of nosocomial infections in patients with a compromised immune system and/or an implanted medical device. Seventy to 90% of S. epidermidis clinical isolates are methicillin resistant and carry the mecA gene, present in a mobile genetic element (MGE) called the staphylococcal cassette chromosome mec (SCCmec) element. Along with the presence of antibiotic and heavy metal resistance genes, MGEs can also contain genes encoding secreted or cell wall-anchored virulence factors. In our earlier studies of S. epidermidis clinical isolates, we discovered S. epidermidis surface protein J (SesJ), a prototype of a recently discovered subfamily of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) group. MSCRAMMs are major virulence factors of pathogenic Gram-positive bacteria. Here, we report that the sesJ gene is always accompanied by two glycosyltransferase genes, gtfA and gtfB, and is present in two MGEs, called the arginine catabolic mobile element (ACME) and the staphylococcal cassette chromosome (SCC) element. The presence of the sesJ gene was associated with the left-hand direct repeat DR_B or DR_E. When inserted via DR_E, the sesJ gene was encoded in the SCC element. When inserted via DR_B, the sesJ gene was accompanied by the genes for the type 1 restriction modification system and was encoded in the ACME. Additionally, the SCC element and ACME carry different isoforms of the SesJ protein. To date, the genes encoding MSCRAMMs have been seen to be located in the bacterial core genome. Here, we report the presence of an MSCRAMM in an MGE in S. epidermidis clinical isolates.

USA300 is a predominant community-associated methicillin-resistant Staphylococcus aureus strain which carries an arginine catabolic mobile element (ACME). ACME contains potential virulence factors including an arginine deiminase (arc) pathway and an oligopeptide permease (opp-3) system, which are proposed to play a role in bacterial virulence and transmission. However, the role of ACME in evolution and pathogenicity of USA300 remains to be elucidated. ACME and arcA deletion mutants were created by allelic replacement from a USA300 clinical isolate. By comparing wild type and isogenic ACME deletion USA300 strains, ACME was shown not to contribute to bacterial survival on plastic surfaces, and mouse skin surfaces. ACME did not contribute to bacterial virulence in cell invasion and cytotoxicity assays, invertebrate killing assays and a mouse skin infection model. Wild-type ACME negative USA300 clinical isolates showed similar associations with invasive anatomic sites as ACME positive isolates. Our experiments also demonstrated that ACME can spontaneously excise from the bacterial chromosome to generate an ACME deletion strain at a low frequency. Our results do not support that the ACME element alone is a significant factor in the transmission and virulence of USA300 strain, and ACME may have been coincidently incorporated into the genome of USA300.

Background Staphylococcal cassette chromosome mec (SCCmec) elements are highly diverse and have been classified into 13 types. The arginine catabolic mobile element (ACME) is an SCC-like element harbouring an arginine deiminase pathway gene cluster (ACME-arc). ACME type I (ACME I), additionally including a spermidine/spermine-N1-acetyltransferase gene (speG), is considered to have contributed to the rapid spread of the most successful MRSA clone, USA300. Objectives To characterize the SCC composite islands (SCC-CIs) in ST5 MRSA positive for both ACME-arc and speG. Methods Three ST5 MRSA strains (SC640, SC792 and SC955) collected in Hokkaido, Japan were subjected to WGS and the SCC-CIs were determined. Results The SCC-CIs consisted of four (SC640 and SC792) or three (SC955) SCC/SCC-like elements and commonly harboured both an ACME type II′ and an SCC encoding speG. These SCC-CIs appear to mimic ACME I in USA300, in that they are equipped with ACME-arc and speG. The SCC-CIs of SC640 and SC792 contained novel SCCmec/SCCmec-like elements at the 3′ end, whereas SC955 contained SCCmec type V. The SCCmec of SC792 carried mec complex A and ccrC1, which was determined to be novel and designated as SCCmec type XIV (5A). SC640 harboured an SCCmec-like element derived from SCCmec type XIV. It lacked most of the downstream region of the mec complex, including the left chromosomal attachment site (SCCmec XIV Δkdp/DR-L), and lost its capability for chromosomal excision, suggesting that the mecA gene is immobilized on the chromosome. Conclusions These findings provide evidence for increasing complexity of SCC-CIs.

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.

The prevalence of Panton-Valentine leukocidin gene (pvl)-positive community-acquired methicillin-resistant Staphylococcus aureus USA300 clone, which is designated as the ST8-staphylococcal cassette chromosome (SCC) mec type IV (ST8-IV) lineage, is a major public health concern worldwide. Thus, to elucidate the prevalence and characteristics of pvl-positive community-onset MRSA in Japan, we conducted a molecular epidemiological analysis for 854 S. aureus isolates obtained from outpatients with skin infections during 2013 and 2014. The isolation rate of MRSA was 25.6% (219 isolates), and the ratio of pvl-positive MRSA was 13.2% (29 isolates). Notably, the proportion (93.8%) of pvl-positive isolates was particularly high among MRSA isolates from Ishigaki island in Okinawa. Pulsed-field gel electrophoresis and multilocus sequence typing showed that the pulsotype C isolates (11 isolates) were typical USA300 clones with arginine catabolic mobile element (ACME) type I-CC8-IV lineages and prevalent on the main island of Japan (Honshu). Pulsotypes A (11 isolates) and B (four isolates) consisted of ACME-negative CC8-IV clones and were specific for Ishigaki island. Both USA300 and Okinawa-Ishigaki specific clones were associated with deep-seated skin infections, such as furuncle and cellulitis. Pulsotypes D (two isolates) and E (one isolate) were ACME-negative clonal complex (CC) 59-IV clones and were related to superficial skin infections, such as impetigo. Our findings revealed that pvl-positive MRSA associated with deep-seated skin infections are spreading in Japanese communities, particularly in Ishigaki, Okinawa.

Staphylococcus epidermidis has been recently recognized as an emerging nosocomial pathogen. There are concerns over the increasing virulence potential of this commensal due to the capabilities of transferring mobile genetic elements to Staphylococcus aureus through staphylococcal chromosomal cassette (SCCmec) and the closely related arginine catabolic mobile element (ACME) and the copper and mercury resistance island (COMER). The potential pathogenicity of S. epidermidis, particularly from blood stream infections, has been poorly investigated. In this study, 24 S. epidermidis isolated from blood stream infections from Oman were investigated using whole genome sequence analysis. Core genome phylogenetic trees revealed one third of the isolates belong to the multidrug resistance ST-2. Genomic analysis unraveled a common occurrence of SCCmec type IV and ACME element predominantly type I arranged in a composite island. The genetic composition of ACME was highly variable among isolates of same or different STs. The COMER-like island was absent in all of our isolates. Reduced copper susceptibility was observed among isolates of ST-2 and ACME type I, followed by ACME type V. In conclusion, in this work, we identify a prevalent occurrence of highly variable ACME elements in different hospital STs of S. epidermidis in Oman, thus strongly suggesting the hypothesis that ACME types evolved from closely related STs.

Background: The CDC has performed surveillance for invasive Staphylococcus aureus (iSA) infections through the Emerging Infections Program (EIP) since 2004. SCCmec and spa typing for clonal complex (CC) assignment and genomic markers have been used to characterize isolates. In 2019, whole-genome sequencing (WGS) of isolates began, allowing for high-resolution assessment of genomic diversity. Here, we evaluate the reliability of SCCmec typing, spa typing, and CC assignment using WGS data compared to traditional methods to ensure that backwards compatibility is maintained. Methods:S. aureus isolates were obtained from a convenience sample of iSA cases reported through the EIP surveillance system. Overall, 78 iSA isolates with diverse spa repeat patterns, CCs, SCCmec types, and antimicrobial susceptibility profiles were sequenced (MiSeq, Illumina). Real-time PCR and Sanger sequencing were used as the SCCmec and spa typing reference methods, respectively. spa-MLST mapping (Ridom SpaServer) served as the reference method for CC assignment. WGS assembly and multilocus sequence typing (MLST) were performed using the CDC QuAISAR-H pipeline. WGS-based MLST CCs were assigned using eBURST and SCCmec types using SCCmecFinder. spa types were assigned from WGS assemblies using BioNumerics. For isolate subtyping, previously published and validated canonical single-nucleotide polymorphisms (canSNPs) as well as the presence of the Panton-Valentine leukocidin (PVL) toxin and arginine catabolic mobile element (ACME) virulence factor were assessed for all genome assemblies. Results: All isolates were assigned WGS-based spa types, which were 100% concordant (78 of 78) with Sanger-based spa typing. SCCmecFinder assigned 91% of isolates (71 of 78) SCCmec types, which were 100% concordant with reference method results. Also, 7 isolates had multiple cassettes predicted or an incomplete SCCmec region assembly. Using WGS data, 96% (75 of 78) of isolates were assigned CCs; 3 isolates had unknown sequence types that were single-locus variants of established sequence types. Overall, 70 isolates had CCs assigned by the reference method; 100% (70 of 70) concordance was observed with WGS-based CCs. Analysis of canSNPs placed 42% (33 of 78) of isolates into CC8, with 17 (52%) of these isolates classified as USA300. PVL and ACME were not accurate markers for inferring the USA300 subtype as 24% (4 of 17) of isolates did not contain these markers. Conclusions:S. aureus CCs, SCCmec, and spa types can be reliably determined using WGS. Incorporation of canSNP analysis represents a more efficient method for CC8 assignment than the use of genomic markers alone. WGS allows for the replacement of multiple typing methods for increased laboratory efficiency, while maintaining backward compatibility with historical typing nomenclature.Funding: NoneDisclosures: None

International audience; Intravenous drug users are at increased risk of Staphylococcus aureus infections. Most cases are related to clones prevalent in the community. We report an outbreak of community-acquired methicillin-resistant Staphylococcus aureus infections that occurred from 2007 to 2009 in intravenous drug users and their close contacts in Northwestern France. Clinical and molecular investigations suggested that the clones were more similar than those usually isolated in the American continent although none of the patients traveled abroad or had contact with individuals who had traveled to the Americas. Then, a retrospective whole genome sequencing and phylogenetic analyses demonstrated that the strains isolated from the first case belong to the USA300 Latin-American variant clone, based on the absence of arginine catabolic mobile element (ACME), and the presence of copper and mercury resistance mobile element (COMER), a distinctive feature of the South American variant. Our study shows genetic evidence for introduction of this clone as early as 2007 in France. This report also illustrates the importance of genome sequencing to finely characterize and monitor the emergence of unexpected S. aureus clones among high-risk populations, especially when living in promiscuity.

Sequence type 88 community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strain SR434, isolated from an outpatient with skin and soft tissue infection, was subjected to whole genome sequencing, antimicrobial susceptibility testing, mouse skin infection model and hemolysis analysis to identify its virulence and resistance determinants. MRSA strain SR434 is resistant to clindamycin, erythromycin and fosfomycin. Four plasmids with resistance genes were identified in this strain, including a 20,658bp blaZ-carrying plasmid, a 2473bp ermC-carrying plasmid, a 2622bp fosB7-carrying plasmid (86% identity with plasmid in a ST2590 MRSA strain) and a 4817bp lnuA-carrying plasmid (99% identity with pLNU4 from bovine coagulase-nagetive Staphylococci). This strain contains staphylococcal cassette chromosome mec type IV and does not contain arginine catabolic mobile element or Panton-Valentine-Leukocidin. SR434 harbors genomic islands νSaα, νSaβ, νSaγ and ΦSa3 and pathogenicity islands νSa2 that carries genes encoding toxic shock syndrome toxin 1, superantigen enterotoxin C and superantigen enterotoxin L. Mouse skin infection model results show that SR434 had similar virulence potential causing invasive skin infection as a PVL-negative epidemic Korea clone HL1 (ST72). CA-MRSA strain of ST88 lineage might be a great concern for its high virulence. PVL has limited contribution to virulence phenotype among this lineage.

Colonization of methicillin resistant Staphylococccus aureus (MRSA) can occur more commonly in healthy people who live in close together or are in close physical contact with each other. Having knowledge about the molecular characteristics of these strains provides considerable discernment into the epidemiology of this important microorganism. A total of 806 nasal swabs were collected from healthy workers of an automaker company in the southeast of Iran and were analyzed to detect MRSA isolates. Multilocus sequence typing (MLST), spa typing, and detection of staphylococcal cassette chromosome mec (SCCmec) were performed. The presence of genes encoding Panton-Valentine Leukocidin (PVL) and Arginine Catabolic Mobile Element (ACME) were also investigated. Carriage rate of S. aureus was 20%. Among 10 identified MRSA, no acme was found while high prevalence of pvl (60%) was of great concern. Seven different spa types including five new ones were identified. The most frequent sequence type was the novel one; ST 3373 (n = 3), followed by each of ST22, ST88, ST859 (n = 2) and ST1955 (n = 1). MRSA isolates were clustered into two main clonal complexes; CC22 (n = 6) and CC88 (n = 4). Low genetic diversity with the dominance of CC22, SCCmecIV was found. Distribution of previously found hospital-associated MRSA was demonstrated among our isolates.

Arginine catabolic mobile element (ACME) is a genomic island of staphylococcus and is considered to confer enhanced ability to survive and growth on host bacterial cells. ACME has been typically identified in Panton-Valentine Leukocidin (PVL)-positive ST8 methicillin-resistant Staphylococcus aureus (MRSA) with SCCmec type IVa (USA300 clone), and it is also found in other lineages at low frequency. Prevalence and molecular characteristics of PVL+and/or ACME+ MRSA were investigated for 624 clinical isolates collected from outpatients in northern Japan from 2013 to 2014. Both PVL genes and ACME type I were detected in nine isolates (1.4%), which were ST8-MRSA-SCCmec IVa/spa type t008/agr-I; whereas solely PVL genes were positive in two isolates, ST30-MRSA-SCCmec IV and ST59-MRSA-SCCmec V. ACME type II' (previously referred to as ACME ΔII) was detected in 36 isolates (5.8%) with SCCmec II and V (32 and 4 isolates, respectively), exhibiting an increased rate within SCCmec II-MRSA (7.1%) compared with our previous studies (0.86-4.5%, 2008-2011). ACME II'-positive MRSA strains were classified into ST5-SCCmec IIa/V or ST764-SCCmec IIa belonging to five different spa types, with t002 being dominant. They harbored mostly enterotoxin gene clusters (seg-sei-sem-sen-seo-seu) and some more enterotoxin genes (seb1, seb2, sec3, sel, sep), showing resistance to more antimicrobials than ST8-MRSA-SCCmec IVa. ACME-SCCmec composite island (CI) of the 36 ACME II'-positive MRSA was classified into five types (ii)-(vi), among which type (ii) (orfX-ΨSCCΔJ1 SCCmec I-ACME II'-SCCmec II) was dominant and subdivided into the A3 variant and the less common A2 variant. CI types (v) and (vi) were considered novel genetic organizations having speG (acetyltransferase genes for polyamines) in inserted SCC4610/SCC266-like genetic elements. The present study revealed increased prevalence and genetic diversity of the ST5/ST764-MRSA-SCCmec II with ACME II' in northern Japan.

FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais Introdução: As infecções de corrente sanguínea (ICS) hospitalares são responsáveis por frequências significativas de morbidade e mortalidade em Unidades de Terapia Intensiva Neonatal (UTIN), resultando em hospitalização prolongada e aumento nos custos hospitalares, principalmente em neonatos de baixo peso. Objetivos: O presente estudo propôs determinar a patogênese de infecções de corrente sanguínea relacionada ao cateter (ICS-RC) causadas por Staphylococcus epidermidis utilizando a técnica de PFGE (“Pulsed Field Gel Electrophoresis”) bem como avaliar os fatores de risco para mortalidade entre os neonatos com pesos ≤ 1500 gramas e >1500 gramas. Adicionalmente, foram determinados os fatores de risco para desenvolvimento de ICS causadas por S. epidermidis utilizando estudo caso x controles pareados. Material e métodos: O estudo incluiu neonatos da UTI do Hospital de Clínicas da Universidade Federal de Uberlândia durante o período de Outubro de 2010 a agosto de 2012. Para o estudo da patogênese foram utilizadas amostras de S. epidermidis recuperadas do sangue, pele no sítio de inserção, canhão do cateter, assim como das mucosas da narina e intestino, a partir de 48 horas até 14 dias da utilização do Cateter ou até obtenção de cultura positiva para S. epidermidis e da ponta do cateter obtidas após sua remoção. Foi verificado a presença dos genes mecA e o ACME (Arginine Catabolic Mobile Element) por reação em cadeia da polimerase (PCR), em S. epidermidis. Resultados: No total, 125 neonatos desenvolveram ICS (27,0%), com 98 (78,4%) definidos como de natureza hospitalar, totalizando 124 episódios. A sepse precoce acometeu 20% dos neonatos com ICS com 48% de mortalidade, principalmente entre os neonatos com peso ≤ 1500 gramas. A presença de ventilação mecânica e sepse clínica foram os fatores associados com mortalidade entre os neonatos de maior peso. No estudo caso x controle pareados, todas as variáveis analisadas [Tempo de hospitalização (P 1500 grams. Methods: The study included neonates from the ICU of Hospital de Federal University of Uberlândia during the period from October 2010 to August 2012. For the study of the pathogenesis, were used samples of S. epidermidis recovered from blood, skin at the insertion site, hub, as well as the mucosa of the nostril and gut, from 48 hours to 14 days after the use of the Catheter or until obtaining a positive culture for S. epidermidis and catheter tip obtained after removal. In addition, were determined the risk factors for the development of BSI caused by S. epidermidis using case study x paired control. In addition to verifying the presence of mecA genes and ACME (Arginine Catabolic Mobile Element) by polymerase chain reaction (PCR), in S.epidermidis. Results: In total, 125 neonates developed BSI (27.0%), with 98 (78.4%) defined as hospital-type, totaling 124 episodes. Sepsis preterm affected 20% of newborns with BSI with 48% mortality, mainly in neonates weighing ≤ 1500 grams. The presence of mechanical ventilation and clinical sepsis were the factors associated with mortality among the heaviest neonates. In the case study x paired control, all variables analyzed [Hospitalization time (P

This work aimed to determine the prevalence, diversity, antibiotic-resistance phenotype/genotype and virulence factors in staphylococci of farm-animals. Nasal samples of 117 farm-animals (calve: 72; lamb: 37; goat: 8) were collected from one slaughterhouse in La Rioja/Spain and cultured for staphylococci and methicillin-resistant Staphylococcus (MRS) recovery. Identification was performed by MALDI-TOF. Antimicrobial resistance phenotype/genotype was determined by susceptibility testing and specific PCRs. Molecular typing (spa-typing, multilocus-sequence-typing, agr-typing, SCCmec), and detection of 12 virulence genes and human Immune-evasive-cluster (IEC) genes were performed by PCR/sequencing in S. aureus. Two marker genes of arginine catabolic mobile element (ACME) were determined by PCR (USA300-MRSA detection). Staphylococci were identified in 50%, 54% and 21% of goat, lamb and calve samples, respectively. Among the 13 S. aureus isolates recovered, 11 were susceptible to all antimicrobials tested, and two were multidrug-resistant-MRSA [beta-lactams (blaZ, mecA), macrolides [(msr(A)/msr(B)] and fluoroquinolones]. The MSSA harboured either tst or enterotoxin genes, while the MRSA harboured the lukF/lukS-PV genes. Five sequence-types were detected. The two MRSA strains (from lamb and goat) were typed as t5173/ST8/agr-I/SCCmec-IVa/ACME-positive, corresponding to USA300 clone, and were IEC-B-positive. Among the 47 coagulase-negative staphylococci (CoNS), six species were identified, predominating S. simulans (n = 25) and S. sciuri (n = 11). Fifty-three percent of CoNS showed resistance to at least one antimicrobial agent (six multidrug-resistant strains), and the following resistance phenotypes/genotypes were detected: streptomycin [27.6%; ant(6)-Ia, str], tetracycline [23.4%; tet(M), tet(L), tet(K)], clindamycin [19.1%; lnu(A), vgaA], erythromycin [10.6%; erm(C), msr(A)/msr(B)], chloramphenicol (8.5%; fexA), tobramycin (6.4%), penicillin-cefoxitin (4.3%; blaZ, mecA), and SXT (2.1%). The detection of the MRSA-USA300 lineage in food animals is worrisome and should be further monitored.

Limited information is available on antimicrobial susceptibility and clonal distribution of Staphylococcus aureus in the Caribbean region. The aims of this study were to determine the prevalence of antimicrobial resistance among S. aureus isolates and to reveal the frequency and population structure of methicillin-resistant S. aureus (MRSA) in St. Kitts and Nevis, a small island country in the West Indies. A total of 152 S. aureus isolates were collected from consecutive samples submitted to the clinical microbiology laboratory of the main referral hospital from March 2017 to January 2018. Samples came from all units in the hospital and a small number came from external submissions, and comprised a total of 119 clinical specimens and 33 nasal swabs collected from staff and patients. All S. aureus isolates were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Minimal Inhibitory Concentrations (MICs) of clinically relevant antimicrobials were determined by broth microdilution, and diversity of MRSA isolates was assessed by whole genome sequencing (WGS) analysis. MRSA accounted for 45% (69/152) of the isolates. The highest rates of resistance to non-β-lactam agents were observed for erythromycin (55%), moxifloxacin (41%), and levofloxacin (40%), whereas resistance to the other drugs tested was .6%. All isolates were susceptible to ceftaroline, linezolid, teicoplanin, telavancin, and vancomycin. WGS-based multilocus sequence typing (MLST) showed that approximately 88% of the MRSA isolates belonged to ST8. Phylogenetic analysis on 801 single-nucleotide polymorphisms (SNPs) identified among the MRSA ST8 isolates indicates a large degree of genetic diversity. However, all ST8 strains clustered within the distinct clade that defines the USA300 North American Epidemic lineage (Panton-Valentine Leukocidin (PVL) +, arginine catabolic mobile element (ACME) +, Staphylococcal cassettes chromosome mec IVa (SCCmec IVa)). Our data show high levels of methicillin, macrolide, and fluoroquinolone resistance among S. aureus on St. Kitts and Nevis. The USA300 North American epidemic lineage is responsible for the vast majority of MRSA infections on this Caribbean island.

This study investigated the molecular characterizations of 80 methicillin resistant Staphylococcus epidermidis (MRSE) collected during 2012-2013 in Tehran Children's Medical Center, Iran. About 90% of MRSE isolates were multi-drug resistant (MDR) and the highest resistance was observed to cotrimoxazole and they were quite sensitive to quinupristin-dalfopristin and linezolid. Though vanA gene was not detected, the majority of isolates showed intermediate resistance to vancomycin (MIC90 16 μg/ml). Resistance to mupirocin was observed in 18 isolates. Staphylococcal cassette chromosome mec (SCCmec) types V, III, IV and II were detected in 23.75%, 7.5%, 6.25% and 5% of isolates respectively, in some of which the additional parts of mec or ccr complexes were observed. In 57.5% MRSE isolates SCCmec types were not classified. 41.2% of MRSE isolates were carrying intercellular adhesion (ica) operon and 40% had strong or intermediate biofilm. The types of arginine catabolic mobile element (ACME) were limited to type I and II. Nine sequence types (STs) were seen in mupirocin resistant MRSE isolates. The common STs were ST2, ST5 and ST22 with 27.7% (5/18), 22.2% (4/18) and 16.6% (3/18) frequencies, respectively. ST23, ST54 and ST179 plus three novels STs 580, 581,588 were also observed. The majority of STs, 83.3% (15/18) belonged to clonal complex 2 (CC2). The spread of antibiotic resistance and virulence factors among MRSE species is an alarming sign in Children's Hospitals. The combination of these two issues leads to increase the chance of successfully establishing of common STs in hospital environments, and promotes the device-related infections and bacteremia.

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.

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-SCC fus (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 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.

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.

Significance USA300 is a hypervirulent, community-acquired, multidrug-resistant Staphylococcus aureus clone that started to spread in the United States around 17 years ago. Many studies detected it also in South America, Europe, and the Asia-Pacific region. In this study, we show that USA300 is also circulating in sub-Saharan Africa. Locating the temporal and spatial origin of clonal lineages is important with respect to epidemiology and molecular evolution of pathogens. We show that USA300 evolved from a less virulent and less resistant ancestor circulating in Central Europe around 160 years ago. Constant surveillance of pathogen transmission routes is vital to prevent and control potential outbreaks. Whole genome sequencing proved to be a useful tool for epidemiological surveillance., USA300 is a pandemic clonal lineage of hypervirulent, community-acquired, methicillin-resistant Staphylococcus aureus (CA-MRSA) with specific molecular characteristics. Despite its high clinical relevance, the evolutionary origin of USA300 remained unclear. We used comparative genomics of 224 temporal and spatial diverse S. aureus isolates of multilocus sequence type (ST) 8 to reconstruct the molecular evolution and global dissemination of ST8, including USA300. Analyses of core SNP diversity and accessory genome variations showed that the ancestor of all ST8 S. aureus most likely emerged in Central Europe in the mid-19th century. From here, ST8 was exported to North America in the early 20th century and progressively acquired the USA300 characteristics Panton–Valentine leukocidin (PVL), SCCmec IVa, the arginine catabolic mobile element (ACME), and a specific mutation in capsular polysaccharide gene cap5E. Although the PVL-encoding phage ϕSa2USA was introduced into the ST8 background only once, various SCCmec types were introduced to ST8 at different times and places. Starting from North America, USA300 spread globally, including Africa. African USA300 isolates have aberrant spa-types (t112, t121) and form a monophyletic group within the clade of North American USA300. Large parts of ST8 methicillin-susceptible S. aureus (MSSA) isolated in Africa represent a symplesiomorphic group of ST8 (i.e., a group representing the characteristics of the ancestor), which are rarely found in other world regions. Isolates previously discussed as USA300 ancestors, including USA500 and a “historic” CA-MRSA from Western Australia, were shown to be only distantly related to recent USA300 clones.

The type III arginine catabolic mobile element (ACME) was detected in three Staphylococcus epidermidis oral isolates recovered from separate patients (one healthy, one healthy with dental implants, and one with periodontal disease) based on ACME- arc -operon- and ACME- opp3 -operon-directed PCR. These isolates were subjected to whole-genome sequencing to characterize the precise structural organization of ACME III for the first time, which also revealed that all three isolates were the same sequence type, ST329.

Widespread infections with community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) have occurred in the United States with the dissemination of the USA300 strain beginning in 2000. We examined 105 isolates obtained from children treated at the University of Chicago from 1994 to 1997 (75 methicillin-susceptible S. aureus [MSSA] and 30 MRSA isolates) in order to investigate for possible evidence of USA300 during this period. Infections were defined epidemiologically based on medical record review. The isolates underwent multilocus sequence typing (MLST), as well as assays for the Panton-Valentine leukocidin (PVL) genes, the protein A gene ( spa ), and arcA and opp3 , proxy markers for the arginine catabolic mobile element (ACME), characteristic of USA300 MRSA. MRSA isolates also underwent staphylococcal cassette chromosome mec (SCC mec ) typing and pulsed-field gel electrophoresis (PFGE) subtyping. MSSA isolates belonged to 17 sequence type (ST) groups. The 12 epidemiologically defined CA-MRSA infection isolates were either ST1 ( n = 4) or ST8 ( n = 8). They belonged to 3 different PFGE types: USA100 ( n = 1), USA400 ( n = 5), and USA500 ( n = 6). Among the CA-MRSA infection isolates, 8 (67%) were PVL + . None of the MRSA or MSSA isolates contained arcA or opp3 . Only one MRSA isolate was USA300 by PFGE. This was a health care-associated (HA) MRSA isolate, negative for PVL, that carried SCC mec type II. USA300 with its characteristic features was not identified in the collection from the years 1994 to 1997.

Data on community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) in Israel are scarce. The objective of this study was to characterize the major CA-MRSA clones in Israel. All clinical MRSA isolates detected in the community during a period of 2.5 years (2011–2013) from individuals insured by a major health maintenance organization in Israel were collected, with additional data from medical records. Antibiotic susceptibility patterns and staphylococcal chromosomal cassette mec (SCCmec) typing were determined. SCCmec IV and V isolates were further typed by pulsed-field gel electrophoresis (PFGE), spa typing, and detection of a panel of toxin genes. MRSA were detected in 280 patients, mostly from skin infections. Patients with SCCmec IV (n = 120, 43 %) were younger (p

Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, but there are few data from Central Africa. The objective of our study was to characterise S. aureus colonisation isolates from healthcare-exposed professionals in the Democratic Republic of the Congo (DRC). Healthcare workers and medical students (n = 380) in Kisangani, DRC were screened for S. aureus nasal carriage in a single-centre cross-sectional study in the University Hospital of Kisangani. The isolates were identified and characterised using phenotypic and genotypic methods. The nasal carriage rate of S. aureus was 16.6 % and 10 out of 63 isolates (15.9 %) were MRSA. We found 28 different spa types. Most MRSA isolates belonged to ST8-spa t1476-SCCmec V. The majority of MRSA were multidrug-resistant to non-beta-lactam antibiotics. Overall, 28.5 % of S. aureus carried Panton-Valentine leucocidin (PVL)-encoding genes (all methicillin-sensitive) and 17.5 % carried toxic shock syndrome toxin-1 (TSST-1)-encoding genes. The finding of MRSA carriage among healthcare workers in a setting with limited access to diagnostic microbiology and appropriate therapy calls for improved education on infection control practices and supports the introduction of surveillance programmes.

The objective of this study was to provide baseline information on circulating methicillin-resistant Staphylococcus aureus (MRSA) clones in Ghana. Thirty MRSA isolates collected between 2010 and 2013 from patients and healthy carriers were characterised by DNA microarray analysis, staphylococcal protein A (spa) typing, multilocus sequence typing (MLST) and minimum inhibitory concentration (MIC) determination to 21 antimicrobial agents. Phenotypic resistance was detected to tetracycline (67%), norfloxacin (40%), moxifloxacin (37%), erythromycin (37%), clindamycin (33%), gentamicin (30%), kanamycin (30%) and ceftaroline (20%), whereas no resistance was observed for glycopeptides, linezolid, daptomycin and tigecycline. DNA microarray analysis showed that tet(M) (43%), tet(K) (33%), aphA3 (23%), aacA-aphD (17%) and erm(C) (13%) were the most prevalent resistance genes. ST88-IV (WA MRSA-2) (n=8), ST8-IV (USA300) (n=5) containing arginine catabolic mobile element (ACME) and Panton-Valentine leukocidin (PVL), and ST247-I (North German/Iberian EMRSA) (n=4) were the most frequent clones detected. All MRSA contained sak and scn genes, one isolate (ST36-II) harboured the gene encoding the toxic shock syndrome toxin (TSST) and none contained exfoliative toxin genes. In conclusion, the relatively high levels of resistance to easily accessible non-β-lactam agents further complicate the treatment of MRSA infections in Ghana. The occurrence of USA300 and other epidemic multidrug-resistant MRSA clones in this African country is a matter of public health concern due to the lack of adequate infrastructures for MRSA surveillance and control in this geographical setting.

This study aimed to investigate the presence of arginine catabolic mobile element (ACME) and its associated molecular characteristics in methicillin-resistant Staphylococcus pseudintermedius (MRSP). Among the 72 S. pseudintermedius recovered from various infection sites of dogs and cats, 52 (72.2%) were MRSP. ACME-arcA was detected commonly (69.2%) in these MRSP isolates, and was more frequently detected in those from the skin than from other body sites (P=0.047). There was a wide genetic diversity among the ACME-arcA-positive MRSP isolates, which comprised three SCCmec types (II-III, III and V) and 15 dru types with two predominant clusters (9a and 11a). Most MRSP isolates were multidrug-resistant. Since S. pseudintermedius could serve as a reservoir of ACME, further research on this putative virulence factor is recommended.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pervasive healthcare-acquired (HA) pathogen with recent emergence as a community-acquired (CA) pathogen. To elucidate whether meat mediates MRSA transmission between animals and humans in Japan, this study examined MRSA isolates from retail meat (n = 8), cows with mastitis (n = 7), and humans (HA-MRSA = 46 and CA-MRSA = 54) by molecular typing, virulence gene analyses, and antimicrobial susceptibility testing. MRSA isolates from retail meat were classified into sequence type (ST) 8/spa type t1767 (n = 4), ST8/t4133 (n = 1), ST59/t3385 (n = 1), ST88/t375 (n = 1), and ST509/t375 (n = 1). All seven MRSA isolates from cows with mastitis were ST8/t1767. 46 HA-MRSA were clonal complex (CC) 5, divided into t002 (n = 30), t045 (n = 12), and t7455 (n = 4). 54 CA-MRSA were classified into 6 different CCs: CC1 (n = 14), CC5 (n = 7), CC8 (n = 29), CC45 (n = 1), CC89 (n = 1), CC509 (n = 1), and into 16 different spa types including newly identified t17177, t17193, and t17194. The majority were CC8/t1767 (n = 16). CC of one CA-MRSA isolate (spa type t1767) was not classified. Among 41 CC8 MRSA (five from meat, seven from cows with mastitis, and 29 CA-MRSA), 14 ST8/SCCmec IVl isolates (three from meat, one from a cow with mastitis, and 10 CA-MRSA) had identical pulsed-field gel electrophoresis patterns and similar spa type (t1767, t4133, and t17177), and were typed as CA-MRSA/J (ST8/SCCmec IVl, positive for sec + sel + tst but negative for Panton–Valentine leukocidin and the arginine catabolic mobile element). These results suggest that there is a transmission cycle of CA-MRSA/J among meat, cows, and humans in Japan, although it is unclear whether the origin is cow.

Severe skin lesions caused by Staphylococcus aureus infection are associated with production from bacterial cells of Panton-Valentine leukocidin (PVL), a typical virulence factor of community-acquired methicillin-resistant S. aureus (CA-MRSA), as well as other toxins represented by exfoliative toxins. Through a retrospective study of 26 S. aureus strains isolated from skin lesions of diabetic patients admitted to a hospital in Bangladesh, 2 PVL-gene-positive MRSA-IVa strains and 8 PVL-negative, exfoliative toxin D (ETD) gene (etd)-positive MRSA-IVa strains were isolated. A PVL-positive MRSA-IVa strain had a type I arginine catabolic mobile element (ACME), belonged to ST8/agr-type I/spa-type t121 (a variant of t008), and harbored blaZ, tet(K), msrA, and aph(3')-IIIa, which are mostly typical characteristics found in USA300, a predominant CA-MRSA clone in the United States. Another PVL-positive MRSA strain, belonging to ST1929 (CC88)/agr-type III/spa-type t3341, was negative for ACME, but possessed blaZ and tet(K). The etd-positive MRSA-IVa strains possessed the epidermal cell differentiation inhibitor B (EDIN-B)-encoding gene (edinB) and belonged to ST1931 (CC80)/agr-type III/spa-type t11023 (a variant of t044), which was genetic trait similar to that of the European CA-MRSA ST80 clone. However, unlike the European ST80 strains, the etd-positive MRSA strains detected in the present study harbored seb, sek, and seq, while they were negative for tet(K), aph(3')-IIIa, and fusB, showing susceptibility to fusidic acid. These findings suggested that etd-positive ST1931 MRSA strains belong to the same lineage as the European ST80 MRSA clone, evolving from a common ancestral clone via acquisition of a different pathogenicity island. This is the first report of a USA300-like MRSA-IV strain, PVL-positive ST1929 (CC88) MRSA-IV, and European ST80 CA-MRSA-like etd-positive ST1931 (CC80) MRSA-IV strains isolated in Bangladesh.

Several oligopeptide permease (Opp) systems have been found in staphylococcal species, including Opp1-4, Opp3' and the arginine catabolic mobile element (ACME)-encoded Opp system (ACME-Opp). They confer upon bacteria the increasing fitness, but their evolutionary histories remain unclear. In this work, we performed a genome-wide identification of Opp systems in staphylococcal species. Novel Opp systems were identified, including the duplicate of Opp4 in Staphylococcus pseudintermedius and the ACME-Opp-like systems in coagulase-negative staphylococci (CoNS). Phylogenetic analysis revealed that all of the identified Opp systems were derived from Opp3 system by operon duplication during species divergence, while lateral gene transfer might also confer to the dissemination of Opp in staphylococci. In addition, we proposed an improved theory on evolution of ACME: the Opp and arginine-deiminase systems were firstly transferred from Staphylococcus haemolyticus to Staphylococcus epidermidis independently; in S. epidermidis they were assembled together and then transferred to Staphylococcus aureus.

Objectives In Spain, despite the high rates of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA), the incidence of community-associated (CA) MRSA seems to be low on the basis of a small number of studies. We analysed the evolution of CA-MRSA in Spain from 2004 to 2012, and identified the clonal lineages and population structure. Methods The study included 8326 MRSA strains. Susceptibility to 18 antimicrobials was determined. Isolates were tested for the presence of mecA, Panton-Valentine leucocidin (PVL) and the arginine catabolic mobile element (ACME) by PCR, and typed by staphylococcal cassette chromosome mec, PFGE, spa, multilocus sequence typing and agr. Results Among the 8326 isolates, 246 (2.9%) were CA-MRSA. We identified genotypically 226 PVL-positive CA-MRSA isolates (88% agr type I, 10.2% agr type III and 1.8% agr type II) and 20 PVL-negative CA-MRSA isolates (all agr type I) from children and adults (82.1% from wounds) from 13 different geographical areas. A significant increase in the rates of CA-MRSA was observed when comparing 2004-07 (0.43%) with 2008-12 (5.44%). Resistance rates were as follows: only β-lactams, 84.5%; erythromycin, 12.8%; tetracycline, 8.8%; clindamycin, 4.9%; ciprofloxacin, 3.1%; fusidic acid, 2.0%; others, 0.4%; and multiresistant, 6.2% (six isolates USA300). The strains belonged to the PVL-positive clones ST8-IVc (69.9%), ST8-IVa-ACME-positive (USA300, 8.9%), ST8-IVa-ACME-negative (0.8%), ST30-IVc (4.5%), ST80-IVc (2.0%), ST5-IVc (1.2%) and others (ST59, ST72, ST88, ST642, ST1472 and ST1829; 4.5%) and to the PVL-negative ST398-V (8.1%). Conclusions We confirm an increase in CA-MRSA in Spain, the predominance of the ST8-IVc clone, the emergence of the USA300 clone, a high genetic diversity among PVL-positive CA-MRSA isolates and the recent emergence of the pig-associated ST398-V clone.

Allelic replacement mutants were constructed within arginine deiminase ( arcA1 and arcA2 ) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ∼5.0 to ∼5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ΔADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 Δ arcA1 (native) or Δ arcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis . Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ΔADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is genetically heterogeneous and various genotypes are spreading worldwide. We herein report a case of systematically disseminated Japan-intrinsic CA-MRSA infection that was successfully treated. A genetically identical single strain was isolated from a total of 11 different parts of the patient's body, and the pathogen was found to be multilocus sequence type 8, staphylococcal cassette chromosome mec IV, coagulase type III and negative for both Panton-Valentine leukocidin and arginine catabolic mobile element. The epidemiology and pathogenicity of the Japan-intrinsic CA-MRSA strain remain unknown, and further investigation of this life-threatening organism is warranted.

Panton–Valentine leukocidine (PVL) is a distinctive virulence factor of community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA), and arginine catabolic mobile element (ACME) is a staphylococcal genomic island that enhances fitness and the ability of bacterial cells to colonize on skin and mucous membranes. ACME is characteristically found in USA300, which is a predominant CA-MRSA clone [sequence type (ST) 8] in the USA and is spreading globally, and has also been detected in non-ST8 MRSA at low frequency. In Japan, spread of MRSA with PVL and/or ACME and their genetic traits have not yet been well characterized. In the present study, the prevalence and genetic diversity of PVL+/ACME+ MRSA were investigated for 422 MRSA clinical isolates collected from outpatients in northern Japan over a period of 1 year. All the isolates were genotyped for the staphylococcal cassette chromosome mec (SCCmec) and coagulase genes (coa), and screened for PVL and ACME genes. The PVL+/ACME+ isolates were studied further by genetic analysis, including single-nucleotide polymorphism (SNP) analysis based on PVL genes (lukS-PV-lukF-PV), ACME (arc and opp3 clusters) and the sarU promoter region. Among all the isolates examined, PVL genes and ACME were detected in eight (SCCmec-II, n = 1; SCCmec-IV, n = 6; SCCmec-V, n = 1) and 20 (SCCmec-II, n = 14; SCCmec-IV, n = 5; SCCmec-V, n = 1) isolates, respectively. Five isolates were found to have both PVL genes and ACME (type I), and were classified into ST8/spa-t008/agr-I/coa-IIIa, which is the same genetic traits as USA300. Fifteen PVL−/ACME+ isolates had type ΔII-ACME, belonging to either ST5 or ST764 [clonal complex (CC) 5], and spa-t001, -t002 or -t3557. All the ST8 PVL+/ACME-I+ MRSA had identical sequences of PVL genes (haplotype R) and ACME arc/opp3 clusters as those of USA300. In contrast, in the CC5 PVL−/ACME-ΔII+ MRSA, SNPs in the arc cluster were detected in 11 sites (four haplotypes), with some different profiles of virulence/resistance factors. These results indicated single clonality of ST8 PVL+/ACME-I+ MRSA and heterogeneity of CC5 PVL−/ACME-ΔII+ MRSA, and suggest their potential spread in northern Japan.