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Peer reviewed: True, INTRODUCTION: International travel has been a major determinant for the introduction of pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) into naïve geographic areas. MRSA clonal complex 239 (CC239) is a highly virulent clone that is predominant in Asia. The objective of this study was to determine the geographic origin of MRSA CC239 isolates recovered from Danish cases with or without a history of international travel during 2004-2016. MATERIALS AND METHODS: Human MRSA isolates with spa types t030 and t037 (n = 60) were obtained from the National Reference Laboratory for Antimicrobial Resistance. For each case, the following data were collected from notification forms: sex, age, isolation year, specimen source (screening swab or clinical sample), infection type, and international travel history. All isolates were whole-genome sequenced, and a comparative genome and phylogenetic analysis was performed. RESULTS: The majority of isolates originated from skin and soft tissue (SST) infections and screening swabs. In 31 out of 60 cases reported international travel to different parts of the world. Fifty-four isolates belonged to CC239, including sequence type 239 (ST239) (n = 43), ST241 (n = 5), ST4377 (n = 2), ST4378 (n = 1), ST1465 (n = 1), ST343 (n = 1), and ST592 (n = 1). The majority of the CC239 MRSA isolates (40/54) belonged to well-known geographic clades, including the Asian (n = 12), Serbian (n = 11), South American (n = 2), and Turkish (n = 15). Most MRSA ST239 isolates belonging to the highly virulent Asian clade carried sasX and were recovered from individuals who had travelled to Asia, Africa and the Middle East. CONCLUSION: Our data reveal multiple introductions of MRSA CC239 into Denmark through international travel, which highlights the importance of continued genomic surveillance of MRSA in persons returning from international travel to areas where MRSA is endemic.

We describe a case of recurrent catheter-related blood stream infections (BSI) with Staphylococcus aureus, in which the first isolate tested susceptible to penicillin, while subsequent isolates were resistant. Phenotypic susceptibility correlated with the absence/presence of the blaZ gene. The in vitro stability of penicillin resistance was investigated by subculturing single colonies. In two out of five colonies, phenotypical resistance was lost after a single subculture, which correlated with loss of the blaZ gene. This in vitro phenomenon probably resulted in a very major error in the microbiology report of the first BSI, where penicillin had been recommended as treatment.

Objective Epidemic methicillin-resistant Staphylococcus aureus (MRSA) clones have been described in Ghana, but so far, no typical livestock-associated MRSA isolates (CC398) have been found. In this study we provide baseline information on antimicrobial resistance, population structure, and virulence gene content of S. aureus isolates from livestock and farm attendants. Methods Nasal samples were collected from cattle, pigs, goats, sheep, and farm attendants from three farms. Staphylococcus aureus was identified by matrix-assisted laser desorption/ionisation time-of-flight and antimicrobial susceptibility testing was performed using VITEK II (Biomerieux, Marcy l'Etoile, France) and interpreted according to EUCAST guidelines. Whole-genome sequencing was performed using the Illumina, San Diego, CA, USA MiSeq Platform. Results In total, 401 nasal swab samples were obtained from 57 farm attendants, 208 pigs, 30 goats, 26 sheep, and 80 cattle. The S. aureus isolates (n = 25) recovered (farm attendants: n = 10; pigs: n = 8; and goats: n = 7) were frequently resistant to penicillin (68%), tetracycline (44%), and ciprofloxacin (32%); two human isolates were MRSA. Twelve isolates (48%) were multidrug resistant (MDR) (>3 classes). Genome sequencing of the isolates revealed ST152-t355, ST9-t1430, and ST133-t8662 as dominant clones among farm attendants, pigs, and goats, respectively. The two MRSA isolates detected belonged to ST8-t334 and ST152-t355. The scn and sak genes associated with human-adaption were detected in 10 isolates; 9 from humans and 1 from a goat. Typing results provided evidence of a single potential transmission event (t861, PVL-, scn+). Conclusion No MRSA was detected among livestock, perhaps because of low intensive farming; however, the relatively high prevalence of MDR isolates may be a result of inappropriate antibiotic usage in Ghanaian livestock production.

Staphylococcus aureus is among the leading causes of bacterial infections worldwide. The pathogenicity and establishment of S. aureus infections are tightly linked to its ability to modulate host immunity. Persistent infections are often associated with mutant staphylococcal strains that have decreased susceptibility to antibiotics; however, little is known about how these mutations influence bacterial interaction with the host immune system. Here, we discovered that clinical S. aureus isolates activate human monocytes, leading to cell-surface expression of immune stimulatory natural killer group 2D (NKG2D) ligands on the monocytes. We found that expression of the NKG2D ligand UL16-binding protein 2 (ULBP2) is associated with bacterial degradability and phagolysosomal activity. Moreover, S. aureus-induced ULBP2 expression was linked to altered host cell metabolism, including increased cytoplasmic (iso)citrate levels, reduced glycolytic flux, and functional mitochondrial activity. Interestingly, we found that the ability of S. aureus to induce ULBP2 and proinflammatory cytokines in human monocytes depends on a functional ClpP protease in S. aureus These findings indicate that S. aureus activates ULBP2 in human monocytes through immunometabolic mechanisms and reveal that clpP inactivation may function as a potential immune evasion mechanism. Our results provide critical insight into the interplay between the host immune system and S. aureus that has evolved under the dual selective pressure of host immune responses and antibiotic treatment. Our discovery of a immune stimulatory pathway consisting of human monocyte-based defense against S. aureus suggests that targeting the NKG2D pathway holds potential for managing persistent staphylococcal infections.

Prosthetic joint infection (PJI) is a feared complication after arthroplasty with Staphylococcus epidermidis as a major pathogen. One diagnostic criteria for PJI diagnosis is the finding of phenotypically identical organisms based on common laboratory tests in two or more periprosthetic microbial cultures. Because of phenotypical variation within a genetic clone, and clonal variation within a phenotype, the criteria may be ambiguous. Here, we investigate the extent of diversity among coagulase-negative staphylococci in PJI and characterize in detail S. epidermidis isolates from these infections.We performed a retrospective cohort study of 62 consecutive patients with PJI caused by coagulase-negative staphylococci (CoNS) from two hospitals in Northern Sweden. From 16 of the patients, two to nine S. epidermidis isolates were available for whole-genome analyses. Hospital-adapted multidrug-resistant genetic clones of S. epidermidis were identified in 40/62 (65%) of the PJIs using a combination of analysis by pulsed-field gel electrophoresis and multiple-locus sequence typing. Whole genome sequencing showed presence of multiple sequence types (STs) in seven (7/16, 44%) PJIs. Among isolates of the same ST, within-patient phenotypical variation in antibiotic susceptibility and/or whole-genome antibiotic resistance gene content was frequent (11/16, 69%).These results highlight the ambiguity of using phenotypical characterization of S. epidermidis as diagnostic criteria in PJI. The results call for larger systematic studies to determine the frequency of CoNS diversity in PJIs, the implications of such diversity for microbiological diagnostics, and for the therapeutic outcome in patients.

Prosthetic joint infection (PJI) is a feared and challenging to diagnose complication after arthroplasty, with Staphylococcus epidermidis as the major pathogen. One important criteria to define PJI is the detection of phenotypically indistinguishable microorganisms with identical antibiotic susceptibility pattern in at least two different samples. However, owing to phenotypical variation within genetic clones and clonal variation within a phenotype, the criteria may be ambiguous. We investigated the extent of diversity among coagulase-negative staphylococci (CoNS) in PJI and characterised S. epidermidis isolates from PJI samples, specifically multiple S. epidermidis isolates identified in individual PJI patients. We performed a retrospective cohort study on 62 consecutive patients with PJI caused by CoNS from two hospitals in Northern Sweden. In 16/62 (26%) PJIs, multiple S. epidermidis isolates were available for whole-genome analyses. Hospital-adapted multidrug-resistant genetic clones of S. epidermidis were identified in samples from 40/62 (65%) of the patients using a combination of pulsed-field gel electrophoresis and multilocus sequence typing. Whole-genome sequencing showed the presence of multiple sequence types (STs) in 7/16 (44%) PJIs where multiple S. epidermidis isolates were available. Within-patient phenotypical variation in the antibiotic susceptibility and/or whole-genome antibiotic resistance gene content was frequent (11/16, 69%) among isolates with the same ST. The results highlight the ambiguity of S. epidermidis phenotypic characterisation as a diagnostic method in PJI and call for larger systematic studies for determining the frequency of CoNS diversity in PJIs, the implications of such diversity for microbiological diagnostics, and the therapeutic outcomes in patients.

These bacteria are capable of adapting to humans, leading to increased spread into the community and healthcare settings., Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) can acquire phage-encoded immune modulators, such as the immune evasion cluster (IEC), which protects bacteria from components of the human innate immune system, and the enzyme TarP, which protects against antibody-mediated immune recognition. We used whole-genome sequencing and epidemiologic investigations to study the effects of IEC- and tarP-harboring phages on household transmission of LA-MRSA in North Denmark Region during 2004–2011. We reviewed information about all patients throughout Denmark who experienced LA-MRSA infection during 2007–2018 to determine whether IEC is associated with increased spread into the general population. Horizontal acquisition of IEC in the human host was associated with increased household transmission of LA-MRSA and spillover into the community and healthcare settings, whereas we found no evidence to suggest that IEC-positive LA-MRSA isolates have become self-sustainable in the general population. By contrast, TarP did not seem to influence household transmission of LA-MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) sequence type 630 (ST630) and spa type t4549 is an emerging lineage in Nordic countries, and some representatives carry the CRISPR-Cas system. Here, the complete genome sequences of two isolates from this lineage are presented, comprising chromosomes of 2,918,239 and 2,877,083 nucleotides, respectively, and a 2,473-nucleotide plasmid carrying erm (C).

Since the late 1990s, changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) were recognized with the emergence of community-associated MRSA (CA-MRSA). CA-MRSA belonging to clonal complex 152 (CC152), carrying the small staphylococcal cassette chromosome mec (SCCmec) type V and encoding the Panton-Valentine leukocidin (PVL), has been observed in Europe. The aim of this study was to investigate its origin, evolution, and dissemination. Whole-genome sequencing was performed on a global collection of 149 CC152 isolates spanning 20 years (93 methicillin-susceptible S. aureus [MSSA] and 56 MRSA isolates). Core genome phylogeny, Bayesian inference, in silico resistance analyses, and genomic characterization were applied. Phylogenetic analysis revealed two major distinct clades, one dominated by MSSA and the other populated only by MRSA. The MSSA isolates were predominately from sub-Saharan Africa, whereas MRSA was almost exclusively from Europe. The European MRSA isolates all harbored an SCCmec type V (5C2&5) element, whereas other SCCmec elements were sporadically detected in MRSA from the otherwise MSSA-dominated clade, including SCCmec types IV (2B), V (5C2), and XIII (9A). In total, 93% of the studied CC152 isolates were PVL positive. Bayesian coalescent inference suggests an emergence of the European CC152-MRSA in the 1990s, while the CC152 lineage dates back to the 1970s. The CA-MRSA CC152 clone mimics the European CC80 CA-MRSA lineage by its emergence from a PVL-positive MSSA ancestor from North Africa or Europe. The CC152 lineage has acquired SCCmec several times, but acquisition of SCCmec type V (5C2&5) seems associated with expansion of MRSA CC152 in Europe.IMPORTANCE Understanding the evolution of CA-MRSA is important in light of the increasing importance of this reservoir in the dissemination of MRSA. Here, we highlight the story of the CA-MRSA CC152 lineage using whole-genome sequencing on an international collection of CC152. We show that the evolution of this lineage is novel and that antibiotic usage may have the potential to select for the phage-encoded Panton-Valentine leukocidin. The diversity of the strains correlated highly to geography, with higher level of resistance observed among the European MRSA isolates. The mobility of the SCCmec element is mandatory for the emergence of novel MRSA lineages, and we show here distinct acquisitions, one of which is linked to the successful clone found throughout Europe today.

Staphylococcus aureus uses several strategies to evade the host immune reaction including expression of the genes on the immune evasion cluster (IEC), which target the innate immune response, and tarP, which alters the structure of the bacterial wall teichoic acids to avoid binding of host antibodies. IEC is carried on an ΦSa3 prophage, while the tarP gene is found located on diverse prophages. Livestock-associated methicillin-resistant S. aureus of clonal complex 398 (LA-MRSA CC398) typically lacks the IEC, but a number of Danish isolates have been found to carry the elements regardless. In this study, whole-genome sequences of 96 isolates from humans and 45 isolates from pigs from the North Denmark Region were used to establish a maximum-likelihood phylogeny from core-genome SNPs and to investigate the prevalence of IEC and tarP. Furthermore, epidemiological and national surveillance data were used to investigate household transmission and the prevalence of IEC in LA-MRSA CC398 isolates from the general population. The study documents several independent acquisitions of IEC on distinct ΦSa3 prophages in humans and an almost 3-fold higher human-to-human transmission rate of LA-MRSA CC398 in households with strains carrying IEC than in households with strains lacking it. No such effect was found for tarP, which is widespread among LA-MRSA CC398 isolates from both humans and pigs. Moreover, IEC also seems to promote spread of LA-MRSA CC398 in the general population. Thus, LA-MRSA CC398 is capable of re-adapting to the human host by acquisition of human-specific immune evasion factors encoded on mobile genetic elements.

Since its emergence in the early 2000s, livestock-associated methicillin-resistant Staphylococcus aureus clonal complex 398 (LA-MRSA CC398) has led to an increasing number of human infections in Denmark and other European countries with industrial pig production. LA-MRSA CC398 is primarily associated with skin infections among pig farm workers but is also increasingly recognized as a cause of life-threatening disease among elderly and immunocompromised people. Pig farm workers may serve as vehicles for the spread of LA-MRSA CC398 and other farm-origin bacteria between farms and into the general population. Yet, little is known about the bacterial community dynamics in pig farm workers and other persons with long- and short-term exposure to the pig farm environment. To gain insight into this, we investigated the nasal microbiomes in pig farm workers during a workweek on four LA-MRSA CC398-positive pig farms, as well as in short-term visitors two hours before, immediately after, and 48 hours after a 1-hour visit to another LA-MRSA CC398-positive pig farm. S. aureus and LA-MRSA CC398 carriage was quantified by means of culture, and the composition of the bacterial communities was investigated through sequencing of the 16S rRNA gene. Pig farm workers often carried LA-MRSA CC398 and other bacteria from the pig farm environment, both at work and at home, although at lower levels at home. In contrast, short-term visitors were subject to a less dramatic and rapidly reversible change in the nasal bacterial community composition. These results suggest that pig farm workers may be an important source of LA-MRSA CC398 and perhaps other pathogens of human and veterinary relevance.

Objectives: We present the results of two European external quality assessments (EQAs) conducted in 2014 and 2016 under the auspices of the Study Group on Staphylococci and Staphylococcal Infections of ESCMID. The objective was to assess the performance of participating centres in characterizing Staphylococcus aureus using their standard in-house phenotypic and genotypic protocols.Methods: A total of 11 well-characterized blindly coded S. aureus (n = 9), Staphylococcus argenteus (n = 1) and Staphylococcus capitis (n = 1) strains were distributed to participants for analysis. Species identification, MIC determination, antimicrobial susceptibility testing, antimicrobial resistance and toxin gene detection and molecular typing including spa typing, SCCmec typing and MLST were performed.Results: Thirteen laboratories from 12 European countries participated in one EQA or both EQAs. Despite considerable diversity in the methods employed, good concordance (90%-100%) with expected results was obtained. Discrepancies were observed for: (i) identification of the S. argenteus strain; (ii) phenotypic detection of low-level resistance to oxacillin in the mecC-positive strain; (iii) phenotypic detection of the inducible MLSB strain; and (iv) WGS-based detection of some resistance and toxin genes.Conclusions: Overall, good concordance (90%-100%) with expected results was observed. In some instances, the accurate detection of resistance and toxin genes from WGS data proved problematic, highlighting the need for validated and internationally agreed-on bioinformatics pipelines before such techniques are implemented routinely by microbiology laboratories. We strongly recommend all national reference laboratories and laboratories acting as referral centres to participate in such EQA initiatives.

Staphylococcus epidermidis is a conspicuous member of the human microbiome, widely present on healthy skin. Here we show that S. epidermidis has also evolved to become a formidable nosocomial pathogen. Using genomics, we reveal that three multidrug-resistant, hospital-adapted lineages of S. epidermidis (two ST2 and one ST23) have emerged in recent decades and spread globally. These lineages are resistant to rifampicin through acquisition of specific rpoB mutations that have become fixed in the populations. Analysis of isolates from 96 institutions in 24 countries identified dual D471E and I527M RpoB substitutions to be the most common cause of rifampicin resistance in S. epidermidis, accounting for 86.6% of mutations. Furthermore, we reveal that the D471E and I527M combination occurs almost exclusively in isolates from the ST2 and ST23 lineages. By breaching lineage-specific DNA methylation restriction modification barriers and then performing site-specific mutagenesis, we show that these rpoB mutations not only confer rifampicin resistance, but also reduce susceptibility to the last-line glycopeptide antibiotics, vancomycin and teicoplanin. Our study has uncovered the previously unrecognized international spread of a near pan-drug-resistant opportunistic pathogen, identifiable by a rifampicin-resistant phenotype. It is possible that hospital practices, such as antibiotic monotherapy utilizing rifampicin-impregnated medical devices, have driven the evolution of this organism, once trivialized as a contaminant, towards potentially incurable infections.

Staphylococcus aureus is a commensal and pathogenic bacterium with impact on public health and livestock industry. The study investigated nasal carriage, antibiotic resistance, and molecular characterization of S. aureus in pigs and pig workers. Nasal swabs from 300 backyard-raised pigs and 101 pig workers were used for the study. Resulting isolates were confirmed using MALDI-TOF MS, tested for antibiotic resistance, and three different multiplex PCRs were used to detect enterotoxin, mecA, spaA, scn, and pvl genes. spa typing was used to annotate the isolates into MLST clonal complexes (CC). Structured questionnaire was used to access possible risk factors for S. aureus carriage. The prevalence of S. aureus in pigs and pig workers were 5.3 and 12.9%, respectively. The isolates were resistant to beta-lactams (97%), tetracycline (62%), sulfonamide (52%), aminoglycoside (20.6%), fluoroquinolone (24%), and mupirocin (3.4%). Twenty seven (93%) of the isolates carried scn, 7(24%) pvl, and 12 (41%) enterotoxin genes, respectively. Questionnaire survey showed medical-related occupation of household members was associated (p

Dust is suspected to be an important factor in transmission of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) between pigs and pig farmers and their families. The aim of this study was to determine the rate of decay for Staphylococcus aureus and LA-MRSA in dust from swine farms. Electrostatic dust fall collectors (EDCs) were used for passive sampling of settling airborne dust in 11 stable sections from six swine farms. Extraction, plating, identification, and enumeration of cultivable S. aureus and LA-MRSA from the EDCs were performed after storage for 0–30 days postsampling. The survival of S. aureus was measured in 196 dust samples from all farms, and data were used to estimate the decay constant λ according to a model for exponential decay: N(t) = N0 × e−λt. The number of S. aureus colonies was up to 600-fold higher than the number of LA-MRSA colonies on MRSA selective agar. The data showed a good fit to the model (λ = 0.13, r2 = 0.86) even with a large difference in initial concentrations of S. aureus between stables. The loads of S. aureus and LA-MRSA in the dust were significantly reduced by storage time, and the half-life was 5 days for both S. aureus and LA-MRSA. In dust samples with high initial concentrations, LA-MRSA and S. aureus could still be cultivated 30 days after sampling. On all farms MRSA isolates belonged to the clonal complex (CC) 398, and at one farm some isolates also belonged to CC30. A screening for other Staphylococcus species in the farm dust revealed 13 different species numerically dominated by Staphylococcus equorum. Based on the exponential decay model, S. equorum had a half-life of 4 days. In conclusion, the presence of MRSA in airborne dust from five of six farms indicates that dust might be an important vehicle for transmission of LA-MRSA. LA-MRSA and S. aureus was found to survive well in farm dust with half-lives of 5 days, and dependent on the initial concentration they could be found in farm dust for weeks. The 99.9% die-off rate was 66 days for LA-MRSA. Thus, farm dust can pose an exposure risk for humans in the farm environment, but also when transported to other environments. On the other hand, the risk will decrease by time. These results provide important knowledge to diminish spread from farm environments to other environments on, e.g., tools or clothing, and in relation to cleaning of emptied LA-MRSA-positive stables.

Methicillin-resistant Staphylococcus aureus (MRSA) is the common name for a heterogeneous group of highly drug-resistant staphylococci. Two major MRSA classes are distinguished based on epidemiology, namely community-associated (CA) and hospital-associated (HA) MRSA. Notably, the distinction of CA- and HA-MRSA based on molecular traits remains difficult due to the high genomic plasticity of S. aureus. Here we sought to pinpoint global distinguishing features of CA- and HA-MRSA through a comparative genome and proteome analysis of the notorious MRSA lineage USA300. We show for the first time that CA- and HA-MRSA isolates can be distinguished by 2 distinct extracellular protein abundance clusters that are predictive not only for epidemiologic behavior, but also for their growth and survival within epithelial cells. This ‘exoproteome profiling’ also groups more distantly related HA-MRSA isolates into the HA exoproteome cluster. Comparative genome analysis suggests that these distinctive features of CA- and HA-MRSA isolates relate predominantly to the accessory genome. Intriguingly, the identified exoproteome clusters differ in the relative abundance of typical cytoplasmic proteins, suggesting that signatures of cytoplasmic proteins in the exoproteome represent a new distinguishing feature of CA- and HA-MRSA. Our comparative genome and proteome analysis focuses attention on potentially distinctive roles of ‘liberated’ cytoplasmic proteins in the epidemiology and intracellular survival of CA- and HA-MRSA isolates. Such extracellular cytoplasmic proteins were recently invoked in staphylococcal virulence, but their implication in the epidemiology of MRSA is unprecedented.

We identified a novel staphylococcal cassette chromosome mec (SCCmec) element in an ST152 methicillin-resistant Staphylococcus aureus (MRSA) isolate by combining Illumina and MinION sequencing. The element contains a new ccrC allotype designated ccrC2. The mec complex resembles mec class A, but with an altered organization of the genes. The element was acknowledged as novel by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) and designated SCCmec type XIII (9A).

Over the last decade, an increasing number of infections with livestock-associated methicillin-resistant Staphylococcus aureus of clonal complex 398 (LA-MRSA CC398) in persons without contact to livestock has been registered in Denmark. These infections have been suggested to be the result of repeated spillover of random isolates from livestock into the community. However, other studies also found emerging sub-lineages spreading among humans. Based on genome-wide SNPs and genome-wide association studies (GWAS), we assessed the population structure and genomic content of Danish LA-MRSA CC398 isolates from healthcare-associated infections from 2014 to 2016 (n = 73) and compared these to isolates from pigs in Denmark from 2014 (n = 183). Phylogenetic analyses showed that most human isolates were closely related to and scattered among pig isolates showing that the majority of healthcare-associated infections are the result of repeated spillover from pig farms, even though cases of human-to-human transmission also were identified. GWAS revealed frequent loss of antimicrobial resistance genes and acquisition of human-specific virulence genes in the human isolates showing adaptation in response to changes in selective pressures in different host environments, which over time could lead to the emergence of LA-MRSA CC398 lineages more adapted to human colonization and transmission.

The possible spillover from pigs into other production animals incites concern for unresolved reservoirs of human exposure. The present investigation was therefore initiated, to elucidate if Danish veal and dairy farms constitute a reservoir of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) CC398 and to potentially identify the source of introduction. We collected nasal swab samples from 17 Danish veal farms, 2 slaughterhouses, and received bulk tank milk samples from 286 dairy farms. All samples were analyzed by culturing and screening on MRSA selective plates and presumed MRSA was verified by MALDI-TOF and PCR. MRSA isolates were subjected to spa typing and whole-genome sequencing. LA-MRSA was found on two veal farms in one and three calves, respectively, with subsequent follow-up samples found negative. Eight of 286 dairy farms (2.8%) were found LA-MRSA positive and follow-up samples, from five farms showed intermittent detection of LA-MRSA. The spa types, t034 and t011, were the most common while a single isolate from a dairy farm belonged to spa type t843 associated to mecC-MRSA CC130 and is the first report of mecC-MRSA in the Danish dairy production. A phylogenetic analysis showed that some of the isolates grouped within or close to the dominant Danish pig clusters, suggesting spillover into cattle farms. Other isolates clustered outside the dominant pig clusters suggesting that other routes of introduction cannot be excluded. Results of the investigation indicated a contamination of veal farms while some dairy farms seemed to be a permanent reservoir. Thus, Danish cattle represent a low prevalence reservoir of LA-MRSA CC398, which at present, is not of major human health concern.

More than 55 million mink skins were produced globally in 2017. As a consequence, a large number of people are employed in mink production worldwide. In Denmark, farmed mink were found to constitute a reservoir of methicillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC) 398 and 6000 mink farm workers in Denmark are potentially exposed to LA-MRSA CC398. The study aim was to elucidate the source of LA-MRSA CC398 in mink farms and to investigate possible transmission to humans. In total, 161 LA-MRSA CC398 isolates from mink (n = 65), mink feed (n = 16) and humans (n = 80) with reported contact to mink, were whole-genome sequenced and compared to 183 LA-MRSA CC398 isolates from Danish pigs and an international collection of 89 S. aureus CC398 isolates. Most of the mink-associated isolates clustered within the predominant LA-MRSA CC398 lineages circulating in the Danish pig production, supporting that pigs are a source of LA-MRSA CC398 in mink feed, mink, and mink farmers.

OBJECTIVES: A recent study from Sweden showed that European hedgehogs may constitute a reservoir for methicillin-resistant Staphylococcus aureus (MRSA), but this host-parasite relationship remains to be investigated in other countries. In this study, we therefore sought to: 1) determine the dissemination of MRSA in European hedgehogs throughout Denmark; 2) investigate determinants of MRSA carriage in hedgehogs; 3) determine the potential for zoonotic transmission of MRSA from hedgehogs to humans; and 4) characterise the detected MRSA on both a phenotypic and molecular level.METHODS: Nasal swabs were taken from 188 dead hedgehogs collected by volunteers throughout Denmark to determine the occurrence of MRSA. Additionally, 16 hedgehog rehabilitators were tested for potential zoonotic transmission of MRSA from hedgehogs to humans. The swabs were incubated in tryptic soy broth supplemented with 6.5% NaCl, followed by spread of 10 μl on Brilliance MRSA 2 agar. One presumptive MRSA colony from each plate was subcultured on 5% blood agar. All S. aureus subcultures were verified by a PCR assay detecting mecA, mecC, lukF-PV, scn, and spa, followed by spa typing.RESULTS: A total of 114 (61%) hedgehogs carried mecC-MRSA, whereas none carried mecA-MRSA. The detected mecC-MRSA belonged to two genetic lineages CC130 (spa-types: t528, t843, t1048, t3256, t3570, t6220, t17133) and CC1943 (spa-types: t978, t2345, t3391, t8835, t16868), 52% of which were spa-type t843 (CC130).The detection rate of mecC-MRSA in the hedgehogs was similar regardless of cause of death, sex, region and habitat type. None of the hedgehog rehabilitators carried MRSA.CONCLUSIONS: This nationwide study confirms a high occurrence of mecC-MRSA in hedgehogs, which could serve as a natural reservoir for this specific type of MRSA. Furthermore, our study did not find signs of zoonotic transmission of mecC-MRSA to hedgehog rehabilitators.

This work was supported by Medical Research Council partnership grants (G1001787/1 and MR/N002660/1) held between the Department of Veterinary Medicine and School of Clinical Medicine at the University of Cambridge, the Moredun Research Institute and the Wellcome Sanger Institute. This publication presents independent research supported by the Health Innovation Challenge Fund (WT098600 and HICF-T5-342)—a parallel funding partnership between the Department of Health and Wellcome Trust. E.M.H. is supported by a UK Research and Innovation Fellowship (MR/S00291X/1). F.C. is supported by the Wellcome Trust (201344/Z/16/Z). X.B. is supported by a UK–China AMR Partnership Grant (MR/P007201/1). Antibiotic resistance in bacterial pathogens threatens the future of modern medicine. One such resistant pathogen is methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to nearly all β-lactam antibiotics, limiting treatment options. Here, we show that a significant proportion of MRSA isolates from different lineages, including the epidemic USA300 lineage, are susceptible to penicillins when used in combination with β-lactamase inhibitors such as clavulanic acid. Susceptibility is mediated by a combination of two different mutations in the mecA promoter region that lowers mecA-encoded penicillin-binding protein 2a (PBP2a) expression, and in the majority of isolates by either one of two substitutions in PBP2a (E246G or M122I) that increase the affinity of PBP2a for penicillin in the presence of clavulanic acid. Treatment of S. aureus infections in wax moth and mouse models shows that penicillin/β-lactamase inhibitor susceptibility can be exploited as an effective therapeutic choice for ‘susceptible’ MRSA infection. Finally, we show that isolates with the PBP2a E246G substitution have a growth advantage in the presence of penicillin but the absence of clavulanic acid, which suggests that penicillin/β-lactamase susceptibility is an example of collateral sensitivity (resistance to one antibiotic increases sensitivity to another). Our findings suggest that widely available and currently disregarded antibiotics could be effective in a significant proportion of MRSA infections. Postprint

This study was aimed to genotyped virulence, toxin and resistance genes of 30 S. aureus isolates from pigs using DNA microarray. The 30 isolates analyzed in this study all belonged to clonal complexes (CC) CC1 (3; 10%), CC5 (3; 10%), CC15 (16; 53.33%) and CC152 (8; 26.66%), respectively. Of antibiotic resistance-associated genes reported; Twenty out of 30 isolates (66.66%) carried the beta-lactamase operon (blaZ/I/R) and the tetracycline resistance gene (tetK), respectively. Also, the macrolide resistance gene (ermA/msrA) were detected in ten isolates (33.33%), the multidrug efflux pump resistance gene (sdrM) was detected in seven isolates (23.33%) and one isolates (3.33%) harboured the chloramphenicol resistance gene (cat). None of the isolates harboured genes conferring methicillin resistance. In terms of genes encoding enterotoxins; seb was detected among all isolates of CC15 while the enterotoxin gene cluster egc and a distinct variant of the enterotoxin A (sea-N315) were detected among isolates of CC5 and CC152, respectively. Two CC152 isolate was positive for genes encoding the Panton-Valentine leukocidin (lukF-PV+lukS-PV). In addition, all the isolates harboured the genes encoding for intracellular adhesion proteins (icaA/C/D) and the biofilm-associated genes (clfA and clfB) except for CC1 isolates. The capsular genes cap5 and cap8 were detected in isolates of CC5 and CC15 while only cap5 was present in CC152 isolates. The study provides detailed genotyping data on the population structure, presence of toxins and antibiotic resistance markers of S. aureus isolates and indicates the importance of the microarray technique in analyzing multiple gene contents of bacteria.

We investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.

OBJECTIVES We unexpectedly identified MRSA isolates carrying mecC (mecC-MRSA) from a Danish swine farm located in eastern Zealand. The objective of the present study was to investigate the origin of these isolates and their genetic relatedness to other mecC-MRSA isolates from Zealand. METHODS WGS was used to infer the phylogenetic relationship between 19 identified mecC-MRSA isolates from the swine farm and 34 additional epidemiologically unrelated human isolates from the same geographical region of Denmark. Variations in the accessory genome were investigated by bioinformatics tools, and antibiotic susceptibility profiles were assessed by MIC determination. RESULTS mecC-MRSA was isolated from a domestic swine farm, but not from cattle reared at the same farm. Phylogenetic analysis revealed that all mecC-MRSA isolates from both farm animals and workers formed a separate cluster, whereas human isolates from the same municipality belonged to a closely related cluster. Analysis of the accessory genome supported this relationship. CONCLUSIONS To the best of our knowledge, this is the first report of mecC-MRSA isolated from domestic swine. The investigation strongly indicates that transmission of mecC-MRSA has taken place on the swine farm between the farmers and swine. The close clustering of farm isolates and isolates from the same municipality suggests a local transmission of mecC-MRSA.

Background and objectives: Widespread use of antibiotics has resulted in selection pressure on genes that make bacteria non-responsive to antibiotics. These antibiotic-resistant bacteria are currently a major threat to global health. There are various possibilities for the transfer of antibiotic resistance genes. It has been argued that animal vectors such as Rattus norvegicus (R. norvegicus) living in hospital sewage systems are ideal for carrying pathogens responsible for fatal diseases in humans. Methodology: Using a metagenomic sequencing approach, we investigated faecal samples of R. norvegicus from three major cities for the presence of antibiotic resistance genes. Results: We show that despite the shared resistome within samples from the same geographic locations, samples from hospital area carry significantly abundant vancomycin resistance genes. Conclusions and implications: The observed pattern is consistent with a selection for vancomycin genes in the R. norvegicus microbiome, potentially driven by the outflow of antibiotics and antibiotic-resistant bacteria into the wastewater systems. Carriage of vancomycin resistance may suggest that R. norvegicus is acting as a reservoir for possible transmission to the human population.

Livestock-associated methicillin-resistant Staphylococcus aureus clonal complex CC398 (LA-MRSA CC398) is resistant to nearly all β-lactams and several non-β-lactam antimicrobials. Over the last decade, it has become widespread in pig farms across Europe and is now an important cause of human infections in countries with previously low levels of MRSA, such as the Netherlands and Denmark. The hitherto uncontrolled spread of LA-MRSA CC398 underscores an urgent need to understand its epidemiology in order to develop evidence-based interventions. This study demonstrates that pig movements between farms in combination with increased bacterial resistance to specific antibiotics and heavy metals were important drivers of the rapid spread of LA-MRSA CC398 in the Danish pig production system. These findings should be taken into consideration when researchers and policy makers evaluate and decide on actions and policies to limit the spread of LA-MRSA CC398 and other pathogens in food animals., The spread of livestock-associated methicillin-resistant Staphylococcus aureus clonal complex 398 (LA-MRSA CC398) within the Danish pig production system has been linked to an increased number of human infections. Yet, the population structure and transmission dynamics of this important pathogen remain poorly understood. In this study, whole-genome sequences from 371 LA-MRSA CC398 isolates collected between 2004 and 2015 were subjected to bioinformatic analyses. The isolates originated from Danish pig farms (n = 209) and people having livestock contact (n = 79). In addition, whole-genome sequence data from 82 isolates representing an international reference collection and 83 isolates from Danish patients were included in the analysis. The results demonstrated that the increasing prevalence of LA-MRSA CC398 in Danish pigs and patients was caused by clonal expansion of three dominant lineages. The results also showed that these lineages were enriched for the tetracycline resistance gene tet(K) and other determinants conferring resistance to some of the most frequently used antimicrobials in Danish pigs. The association between pig movements and the spread of LA-MRSA CC398 was assessed in a Poisson regression analysis of 17,009 pig movements into 273 farms with known LA-MRSA CC398 status. The results demonstrated that animal movements have played a critical role in the dissemination of LA-MRSA CC398 within the Danish pig production system, although other transmission routes may also have contributed. Consistent with this scenario, the genetic relatedness of isolates from different farms was positively correlated with the number of animal movements between the farms.

The role of host genetic variation in the development of complicated Staphylococcus aureus bacteremia (SAB) is poorly understood. We used whole exome sequencing (WES) to examine the cumulative effect of coding variants in each gene on risk of complicated SAB in a discovery sample of 168 SAB cases (84 complicated and 84 uncomplicated, frequency matched by age, sex, and bacterial clonal complex [CC]), and then evaluated the most significantly associated genes in a replication sample of 240 SAB cases (122 complicated and 118 uncomplicated, frequency matched for age, sex, and CC) using targeted sequence capture. In the discovery sample, gene-based analysis using the SKAT-O program identified 334 genes associated with complicated SAB at p, Author summary Complications from bloodstream infection with Staphylococcus aureus (S. aureus) are important causes of hospitalization, significant illness, and death. The causes of these complications are not well understood, but likely involve genetic factors rendering people more susceptible to such infections, differences in the bacteria that cause the infection, and the interactions between them. We examined the parts of the human genome that code for proteins to find variations that were more common in people with complicated S. aureus bacteremia (SAB), and identified one gene, called GLS2, in which variation is more common in complicated SAB cases than uncomplicated cases. Expression of GLS2 is lower in people with SAB than controls and in mouse white blood cells exposed to S. aureus. GLS2 encodes a protein that regulates the metabolism of glutamine, a regulatory process that activates white blood cells. These cells are very important in the immune response to S. aureus infection, and therefore genetic variants that might influence their growth are important potential genetic risk factors for complicated SAB.

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

Objectives Community-acquired (CA) methicillin-resistant Staphylococcus aureus (MRSA) isolates belonging to clonal complex 80 (CC80) are recognized as the European CA-MRSA. The prevailing European CA-MRSA clone carries a type IVc staphylococcal cassette chromosome mec (SCC mec ) and expresses Panton-Valentine leukocidin (PVL). Recently, a significant increase of PVL-negative CC80 MRSA has been observed in Denmark. The aim of this study was to examine their genetics and epidemiology, and to compare them to the European CA-MRSA clone in order to understand the emergence of PVL-negative CC80 MRSA. Methods Phylogenetic analysis of the CC80 S. aureus lineage was conducted from whole-genome sequences of 217 isolates (23 methicillin-susceptible S. aureus and 194 MRSA) from 22 countries. All isolates were further genetically characterized in regard to resistance determinants and PVL carriage, and epidemiologic data were obtained for selected isolates. Results Phylogenetic analysis revealed the existence of three distinct clades of the CC80 lineage: (a) an methicillin-susceptible S. aureus clade encompassing Sub-Saharan African isolates ( n = 13); (b) a derived clade encompassing the European CA-MRSA SCC mec -IVc clone ( n = 185); and (c) a novel and genetically distinct clade encompassing MRSA SCC mec- IVa isolates ( n = 19). All isolates in the novel clade were PVL negative, but carried remnant parts (8–12 kb) of the PVL-encoding prophage ΦSa2 and were susceptible to fusidic acid and kanamycin/amikacin. Geospatial mapping could link these isolates to regions in the Middle East, Asia and South Pacific. Conclusions This study reports the emergence of a novel CC80 CA-MRSA sublineage, showing that the CC80 lineage is more diverse than previously assumed.

Typing of methicillin-resistant Staphylococcus aureus (MRSA) is important in infection control and surveillance. The current nomenclature of MRSA includes the genetic background of the S. aureus strain determined by multilocus sequence typing (MLST) or equivalent methods like spa typing and typing of the mobile genetic element staphylococcal cassette chromosome mec (SCC mec ), which carries the mecA or mecC gene. Whereas MLST and spa typing are relatively simple, typing of SCC mec is less trivial because of its heterogeneity. Whole-genome sequencing (WGS) provides the essential data for typing of the genetic background and SCC mec , but so far, no bioinformatic tools for SCC mec typing have been available. Here, we report the development and evaluation of SCC mec Finder for characterization of the SCC mec element from S. aureus WGS data. SCC mec Finder is able to identify all SCC mec element types, designated I to XIII, with subtyping of SCC mec types IV (2B) and V (5C2). SCC mec elements are characterized by two different gene prediction approaches to achieve correct annotation, a Basic Local Alignment Search Tool (BLAST)-based approach and a k -mer-based approach. Evaluation of SCC mec Finder by using a diverse collection of clinical isolates ( n = 93) showed a high typeability level of 96.7%, which increased to 98.9% upon modification of the default settings. In conclusion, SCC mec Finder can be an alternative to more laborious SCC mec typing methods and is freely available at https://cge.cbs.dtu.dk/services/SCCmecFinder. IMPORTANCE SCC mec in MRSA is acknowledged to be of importance not only because it contains the mecA or mecC gene but also for staphylococcal adaptation to different environments, e.g., in hospitals, the community, and livestock. Typing of SCC mec by PCR techniques has, because of its heterogeneity, been challenging, and whole-genome sequencing has only partially solved this since no good bioinformatic tools have been available. In this article, we describe the development of a new bioinformatic tool, SCC mec Finder, that includes most of the needs for infection control professionals and researchers regarding the interpretation of SCC mec elements. The software detects all of the SCC mec elements accepted by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements, and users will be prompted if diverging and potential new elements are uploaded. Furthermore, SCC mec Finder will be curated and updated as new elements are found and it is easy to use and freely accessible.

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.

The tetracycline resistance gene tet (K) was shown to be integrated within the predominant staphylococcal cassette chromosome mec (SCC mec ) element of Danish livestock-associated methicillin-resistant Staphylococcus aureus CC398 (LA-MRSA CC398). These LA-MRSA CC398 isolates already possessed tet (M), but the acquisition of tet (K) significantly improved their fitness at sublethal concentrations of tetracycline. Because tet (K) is genetically linked to SCC mec , the use of tetracycline in food animals may have contributed to the successful spread of LA-MRSA CC398.

Most Staphylococcus aureus isolates can cause invasive disease given the right circumstances, but it is unknown if some isolates are more likely to cause severe infections than others. S. aureus bloodstream isolates from 120 patients with definite infective endocarditis and 121 with S. aureus bacteraemia without infective endocarditis underwent whole-genome sequencing. Genome-wide association analysis was performed using a variety of bioinformatics approaches including SNP analysis, accessory genome analysis and k-mer based analysis. Core and accessory genome analyses found no association with either of the two clinical groups. In this study, the genome sequences of S. aureus bloodstream isolates did not discriminate between bacteraemia and infective endocarditis. Based on our study and the current literature, it is not convincing that a specific S. aureus genotype is clearly associated to infective endocarditis in patients with S. aureus bacteraemia.

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.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clonal complex (CC) 398 is widespread in the Danish pig production with around 90% of herds being positive. Since 2009, cases of CC398 LA-MRSA infections in Danish mink farmers have been observed. The objective of the study was to examine the presence of LA-MRSA in farmed mink. The investigation comprised three different sample types 1) clinical samples from carcasses submitted to the laboratory for diagnostic examination, 2) paws and pharyngeal swabs from healthy animals collected at pelting, and 3) feed samples from mink feed producers. In clinical samples, LA-MRSA was found in 34% of submissions and was most prevalent in samples from paws (33%) and pharynx (17%), followed by nasal and intestinal samples (each 13%), whereas it was never detected in perineal samples. LA-MRSA was found in healthy animals on 40% of the investigated farms, including paw samples (29%) and pharyngeal samples (16%). Twenty out of the 108 feed samples from feed producers were positive for LA-MRSA. The dominant spa -types were t034 and t011 associated to CC398, corresponding to the dominant spa -types detected in the Danish pig production, from which slaughter offal is used for mink feed. The spa -types, the high prevalence of LA-MRSA on paws and in pharynx, and its detection in feed samples, suggest feed as a possible source of LA-MRSA in mink.

Denmark is a low prevalence country with regard to methicillin resistant Staphylococcus aureus (MRSA). In 2008 and 2014, two neonatal wards in the Copenhagen area experienced outbreaks with a typical community acquired MRSA belonging to the same spa type and sequence type (t015:ST45) and both were PVL and ACME negative. In outbreak 1, the isolates harbored SCCmec IVa and in outbreak 2 SCCmec V. The clinical presentation differed between the two outbreaks, as none of five MRSA positive mothers in outbreak 1 had mastitis vs. five of six MRSA positive mothers in outbreak 2 (p < 0.02). To investigate if whole-genome sequencing could identify virulence genes associated with mastitis, t015:ST45 isolates from Denmark (N = 101) were whole-genome sequenced. Sequence analysis confirmed two separate outbreaks with no sign of sustained spread into the community. Analysis of the accessory genome between isolates from the two outbreaks revealed a S. aureus pathogenicity island containing enterotoxin C and enterotoxin-like L only in isolates from outbreak 2. Enterotoxin C and enterotoxin-like L carrying S. aureus are associated with bovine mastitis and our findings indicate that these may also be important virulence factors for human mastitis.

Rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) isolates is pivotal for the control of their dissemination and for accurate management of infected patients. In 2011, a new mec variant, named mecC , that shows only 70% nucleotide sequence homology with the classical mecA

spa typing of methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been done by PCR amplification and Sanger sequencing of the spa repeat region. At Hvidovre Hospital, Denmark, whole-genome sequencing (WGS) of all MRSA isolates has been performed routinely since January 2013, and an in-house analysis pipeline determines the spa types. Due to national surveillance, all MRSA isolates are sent to Statens Serum Institut, where the spa type is determined by PCR and Sanger sequencing. The purpose of this study was to evaluate the reliability of the spa types obtained by 150-bp paired-end Illumina WGS. MRSA isolates from new MRSA patients in 2013 ( n = 699) in the capital region of Denmark were included. We found a 97% agreement between spa types obtained by the two methods. All isolates achieved a spa type by both methods. Nineteen isolates differed in spa types by the two methods, in most cases due to the lack of 24-bp repeats in the whole-genome-sequenced isolates. These related but incorrect spa types should have no consequence in outbreak investigations, since all epidemiologically linked isolates, regardless of spa type, will be included in the single nucleotide polymorphism (SNP) analysis. This will reveal the close relatedness of the spa types. In conclusion, our data show that WGS is a reliable method to determine the spa type of MRSA.

The aim of this study was to investigate biofilm formation on silicone tubes by genetically diverse methicillin-resistant Staphylococcus aureus (MRSA) strains. Capacity of biofilm formation on dacryocystorhinostomy silicone tubes was tested on 30 MRSA strains. Identification and methicillin resistance were confirmed by PCR for nuc and mecA genes. Strains were genotypically characterised (SCCmec, agr and spa typing). Biofilm formation was tested in microtiter plate and on silicone tubes. Tested MRSA strains were classified into SCCmec type I (33.3 %), II (3.3 %), III (20.0 %), IV (26.7 %) and V (16.7 %), agr type I (56.7 %), II (36.7 %) and III (6.6 %), and eight spa clonal complexes (CCs). All tested MRSA strains showed ability to form biofilm on microtiter plate. Capacity of biofilm formation on silicone tubes was as follows: 33.3 % of strains belonged to the category of low biofilm producers, and 66.7 % to moderate biofilm producers. There was statistically significant correlation between spa CC and the category of biofilm production on silicone tubes (p = 0.01): CC5 and CC45 with moderate amount of biofilm, and CC8 with low amount of biofilm. A moderate amount of biofilm formed on silicone tubes correlated with agr type II MRSA strains (p = 0.008). Biofilm formation by MRSA on silicone tubes is highly dependent on genetic characteristics of the strains. Therefore, MRSA genotyping may aid the determination of the possibility of biofilm-related ocular device infections. Genotyping and biofilm quantification may be helpful in determining when decolonisation and cohort isolation are required to prevent device-related infections.

The antibiotic susceptibility and molecular epidemiology of Panton-Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) isolates reported from 17 countries in the Americas, Europe and, Australia-Asia were analysed. Among a total of 3236 non-duplicate isolates, the lowest susceptibility was observed to erythromycin in all regions. Susceptibility to ciprofloxacin showed large variation (25%, 75% and 84% in the Americas, Europe and Australia-Asia, respectively). Two vancomycin-intermediate PVL-positive MRSA isolates were reported, one from Hong Kong and the other from The Netherlands. Resistance to trimethoprim/sulfamethoxazole and linezolid was

The implementation of routine whole-genome sequencing (WGS) promises to transform our ability to monitor the emergence and spread of bacterial pathogens. Here we combined WGS data from 308 invasive Staphylococcus aureus isolates corresponding to a pan-European population snapshot, with epidemiological and resistance data. Geospatial visualization of the data is made possible by a generic software tool designed for public health purposes that is available at the project URL (http:// www.microreact.org/project/EkUvg9uY?tt=rc). Our analysis demonstrates that high-risk clones can be identified on the basis of population level properties such as clonal relatedness, abundance, and spatial structuring and by inferring virulence and resistance properties on the basis of gene content. We also show that in silico predictions of antibiotic resistance profiles are at least as reliable as phenotypic testing. We argue that this work provides a comprehensive road map illustrating the three vital components for future molecular epidemiological surveillance: (i) large-scale structured surveys, (ii) WGS, and (iii) communityoriented database infrastructure and analysis tools. IMPORTANCE The spread of antibiotic-resistant bacteria is a public health emergency of global concern, threatening medical intervention at every level of health care delivery. Several recent studies have demonstrated the promise of routine wholegenome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control. However, as this technology becomes more widely adopted, the key challenges of generating representative national and international data sets and the development of bioinformatic tools to manage and interpret the data become increasingly pertinent. This study provides a road map for the integration of WGS data into routine pathogen surveillance. We emphasize the importance of large-scale routine surveys to provide the population context for more targeted or localized investigation and the development of open-access bioinformatic tools to provide the means to combine and compare independently generated data with publicly available data sets., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

Background The recent discovery of a mecA homologue (mecA(LGA251)) with a high level of variability between the two gene variants suggested that Staphylococcus aureus harbouring mecA(LGA251) could be wrongly identified as methicillin-susceptible S. aureus (MSSA), in the absence of antimicrobial susceptibility testing. Methods In this context we designed a real-time quadruplex PCR assay to distinguish unequivocally between mecA and mecA(LGA251), alongside the nuc gene (a species-specific marker) and detection of the lukS-PV gene [encoding the Panton-Valentine leucocidin (PVL) toxin]. Results and discussion The assay was validated using a collection of (i) PVL-positive and PVL-negative MSSA and methicillin-resistant S. aureus (MRSA) and (ii) known MRSA harbouring mecA(LGA251) from the UK, Denmark and France. When applied to a retrospective collection of oxacillin-non-susceptible, mecA-negative human isolates, three were found to encode mecA(LGA251), including one from blood, representing the first hitherto recognized case of bacteraemia due to S. aureus possessing the mecA(LGA251) in England. Finally, the assay was introduced into the routine Staphylococcus Reference Unit (HPA Microbiology Services, London, UK) workflow in August 2011, and, during the first 5 months of use, 10 isolates harbouring the mecA homologue were identified out of 2263 S. aureus tested, suggesting a low but continuous circulation within the human population in England.

The recent finding of a new mecA homologue, mecA(LGA251) , with only 70% nucleotide homology to the conventional mecA gene has brought the routine testing for mecA as a confirmatory test for methicillin-resistant Staphylococcus aureus (MRSA) into question. A multiplex PCR was designed to differentiate mecA(LGA251) from the known mecA together with detection of lukF-PV and the spa gene fragments, enabling direct spa typing by sequencing of the PCR amplicons. The PCR analysis and subsequent spa typing were validated on a large collection (n=185) of contemporary MRSA and methicillin-sensitive S. aureus isolates, including 127 isolates carrying mecA(LGA251) . The mecA(LGA251) gene was situated in staphylococcal cassette chromosome mec type XI elements, and sequence variation within a 631-bp fragment of mecA(LGA251) in 79 isolates indicated a very conserved gene sequence. Following a successful validation, the multiplex PCR strategy was implemented in the routine testing of MRSA for national surveillance. Over a 2-month period, among 203 samples tested, 12 new MRSA cases caused by isolates carrying mecA(LGA251) were identified, emphasizing the clinical importance of testing for these new MRSA isolates.

Summary Background Animals can act as a reservoir and source for the emergence of novel meticillin-resistant Staphylococcus aureus (MRSA) clones in human beings. Here, we report the discovery of a strain of S aureus (LGA251) isolated from bulk milk that was phenotypically resistant to meticillin but tested negative for the mecA gene and a preliminary investigation of the extent to which such strains are present in bovine and human populations. Methods Isolates of bovine MRSA were obtained from the Veterinary Laboratories Agency in the UK, and isolates of human MRSA were obtained from diagnostic or reference laboratories (two in the UK and one in Denmark). From these collections, we searched for mecA PCR-negative bovine and human S aureus isolates showing phenotypic meticillin resistance. We used whole-genome sequencing to establish the genetic basis for the observed antibiotic resistance. Findings A divergent mecA homologue ( mecA LGA251 ) was discovered in the LGA251 genome located in a novel staphylococcal cassette chromosome mec element, designated type-XI SCC mec . The mecA LGA251 was 70% identical to S aureus mecA homologues and was initially detected in 15 S aureus isolates from dairy cattle in England. These isolates were from three different multilocus sequence type lineages (CC130, CC705, and ST425); spa type t843 (associated with CC130) was identified in 60% of bovine isolates. When human mecA -negative MRSA isolates were tested, the mecA LGA251 homologue was identified in 12 of 16 isolates from Scotland, 15 of 26 from England, and 24 of 32 from Denmark. As in cows, t843 was the most common spa type detected in human beings. Interpretation Although routine culture and antimicrobial susceptibility testing will identify S aureus isolates with this novel mecA homologue as meticillin resistant, present confirmatory methods will not identify them as MRSA. New diagnostic guidelines for the detection of MRSA should consider the inclusion of tests for mecA LGA251 . Funding Department for Environment, Food and Rural Affairs, Higher Education Funding Council for England, Isaac Newton Trust (University of Cambridge), and the Wellcome Trust.

We characterized all of the Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus isolates collected between 2005 and 2008 in the Bilbao, Spain, area. For the first time, the USA300 clone is reported as predominant among PVL-positive clones in a European autochthonous population, requiring active monitoring of the incidence of USA300 in Spain and throughout Europe.

Analysis of methicillin-resistant Staphylococcus aureus (MRSA) characterized as USA300 by pulsed-field gel electrophoresis identified two distinct clones. One was similar to community-associated USA300 MRSA (ST8-IVa, t008, and Panton-Valentine leukocidin positive). The second (ST8-IVa, t024, and PVL negative) had different molecular characteristics and epidemiology, suggesting independent evolution. We recommend spa typing and/or PCR to discriminate between the two clones.

Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCC mec ) types I to V and 103 different staphylococcal protein A ( spa ) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCC mec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCC mec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.

In Europe, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have been caused predominantly by isolates belonging to the “European CA-MRSA” clone (sequence type 80, staphylococcal cassette chromosome mec type IV). In this study, the epidemiology of European CA-MRSA was investigated on a nationwide scale, covering the period from 1993 to 2004. Denmark has been a low-prevalence country regarding MRSA since the mid-1970s but has experienced an increase in the number of new MRSA cases in recent years. Our results show that European CA-MRSA contributed to this increase. The isolates primarily caused skin and soft tissue infections (SSTIs) in patients outside hospitals, and transmission between household members was the predominant mode of spread. Although some of the isolates were found in hospitalized patients, nosocomial transmission seemed likely in only one instance, pointing to endogenous infections as an important factor. Compared to the CA-MRSA clone most common in the United States (USA300), the European CA-MRSA clone seems less well adapted to persist in hospital environments. Patients with a recent history of travel or family relation to the Mediterranean or Middle East were highly overrepresented. The epidemiological data indicated that the European CA-MRSA isolates were introduced into Denmark on multiple occasions, paralleled by an increasing level of genetic diversity of the isolates found during the study period. European CA-MRSA has previously been described as a rather uniform clone. However, we found pronounced, diverse pulsed-field gel electrophoresis subtypes, staphylococcal protein A gene ( spa ) types, and susceptibility patterns.

Resistance to the antibiotic fusidic acid in European strains of Staphylococcus aureus causing impetigo has increased in recent years. This increase appears to have resulted from clonal expansion of a strain we have designated the epidemic European fusidic acid-resistant impetigo clone (EEFIC), which carries the fusidic acid resistance determinant fusB on its chromosome. To understand better the properties of the EEFIC responsible for its success, we have performed detailed phenotypic and genotypic characterization of this clone. Molecular typing revealed the EEFIC to be ST123, spa type t171, and agr type IV and therefore unrelated to earlier prevalent fusB + strains found in the United Kingdom. EEFIC strains exhibited resistance to fusidic acid, penicillin, and, in some cases, erythromycin, which are all used in the treatment of impetigo. PCR analysis of the EEFIC and complete DNA sequencing of the 39.3 Kb plasmid it harbors identified genes encoding several toxins previously implicated in impetigo (exfoliative toxins A and B and EDIN-C). The location of fusB was mapped on the chromosome and found to be associated with a novel 16.6-kb genomic island integrated downstream of groEL . Although this element is related to classical staphylococcal pathogenicity islands, it does not encode any known virulence factors and consequently has been designated SaRI fusB (for “ S. aureus resistance island carrying fusB ”).