Genetic makeup of Shiga toxin-producing Escherichia coli in relation to clinical symptoms and duration of shedding: a microarray analysis of isolates from Swedish children.

Shiga toxin (Stx)-producing Escherichia coli (STECs) cause non-bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome, and are the primary cause of acute renal failure in children worldwide. This study investigated the correlation of genetic makeup of STEC strains as revealed by DNA microarray to clinical symptoms and the duration of STEC shedding. All STEC isolated (n = 96) from patients <10 years of age in Jönköping County, Sweden from 2003 to 2015 were included. Isolates were characterized by DNA microarray, including almost 280 genes. Clinical data were collected through a questionnaire and by reviewing medical records. Of the 96 virulence genes (including stx) in the microarray, 62 genes were present in at least one isolate. Statistically significant differences in prevalence were observed for 21 genes when comparing patients with bloody diarrhea (BD) and with non-bloody stool (18 of 21 associated with BD). Most genes encode toxins (e.g., stx2 alleles, astA, toxB), adhesion factors (i.e. espB_O157, tir, eae), or secretion factors (e.g., espA, espF, espJ, etpD, nleA, nleB, nleC, tccP). Seven genes were associated with prolonged stx shedding; the presence of three genes (lpfA, senB, and stx1) and the absence of four genes (espB_O157, espF, astA, and intI1). We found STEC genes that might predict severe disease outcome already at diagnosis. This can be used to develop diagnostic tools for risk assessment of disease outcome. Furthermore, genes associated with the duration of stx shedding were detected, enabling a possible better prediction of length of STEC carriage after infection.

Staphylococcus aureus colonization related to severity of hand eczema.

Knowledge on Staphylococcus aureus colonization rates and epidemiology in hand eczema is limited. The aim of this study was to clarify some of these issues. Samples were collected by the "glove juice" method from the hands of 59 patients with chronic hand eczema and 24 healthy individuals. Swab samples were taken from anterior nares and throat from 43 of the 59 patients and all healthy individuals. S. aureus were spa typed and analysed by DNA-microarray-based genotyping. The extent of the eczema was evaluated by the hand eczema extent score (HEES). The colonization rate was higher on the hands of hand eczema patients (69 %) compared to healthy individuals (21 %, p < 0.001). This was also seen for bacterial density (p = 0.002). Patients with severe hand eczema (HEES ≥ 13) had a significantly higher S. aureus density on their hands compared to those with milder eczema (HEES = 1 to 12, p = 0.004). There was no difference between patients and healthy individuals regarding colonization rates in anterior nares or throat. spa typing and DNA-microarray-based genotyping indicated certain types more prone to colonize eczematous skin. Simultaneous colonization, in one individual, with S. aureus of different types, was identified in 60-85 % of the study subjects. The colonization rate and density indicate a need for effective treatment of eczema and may have an impact on infection control in healthcare.

Molecular characterization of Staphylococcus aureus isolates causing skin and soft tissue infections in patients from Malakand, Pakistan.

Comparatively few studies have been published describing Staphylococcus aureus/MRSA epidemiology in Central Asia including Pakistan. Here, we report the genotyping of Staphylococcus aureus strains (that include both methicillin-susceptible and methicillin-resistant Staphylococcus aureus) from community- and hospital-acquired skin and soft-tissue infections in a tertiary care hospital in the Malakand district of the Khyber Pakhtunkhwa Province of Pakistan. Forty-five isolates of Staphylococcus aureus were characterized by microarray hybridization. Twenty isolates (44 %) were MRSA, whereas 22 (49 %) were PVL-positive. Fourteen isolates (31 %) harboured both mecA and PVL genes. The dominant clones were CC121-MSSA (n = 15, 33 %) and the PVL-positive "Bengal Bay Clone" (ST772-MRSA-V; n = 13, 29 %). The PVL-positive CC8-MRSA-IV strain "USA300" was found once. The pandemic ST239-MRSA-III strain was absent, although it has previously been observed in Pakistan. These observations require a re-assessment of schemes for initial antibiotic therapy to cover MRSA and they emphasise the need for a rapid and non-molecular test for PVL.

Multiple cases of methicillin-resistant CC130 Staphylococcus aureus harboring mecC in milk and swab samples from a Bavarian dairy herd.

The discovery of a new mecA homolog, mecC, necessitates a modification of diagnostic procedures for the identification of methicillin-resistant Staphylococcus aureus (MRSA), as most assays used for the genotypic and phenotypic mecA detection cannot currently recognize mecC. Although the prevalence, distribution, and importance of mecC are not yet completely understood, an exchange of mecC-MRSA between humans and animals seems possible. All previously reported observations of mecC-positive strains have been sporadic. To the best of our knowledge, this is the first report about multiple cases of mecC-positive Staph. aureus in 1 dairy herd. Clonal complex 130 Staph. aureus harboring mecC were found in milk samples from 16 of 56 lactating cows kept in a herd in Bavaria, Germany. Almost all quarter milk samples positive for mecC-MRSA had the lowest possible California Mastitis Test score; composite somatic cell counts obtained from monthly milk recordings showed a mean of 51,600 cells/mL in mecC-MRSA affected cows. Additionally, mecC-positive clonal complex 130 Staph. aureus were detected in swab samples from the mammary skin and a teat lesion of 1 cow from this herd. This report suggests that mecC-carrying strains are able to spread among livestock, and that they have the ability to cause multiple cases in single herds. Therefore, future studies targeting MRSA in dairy cows need to consider mecC.

An emerging Panton-Valentine leukocidin-positive CC5-meticillin-resistant Staphylococcus aureus-IVc clone recovered from hospital and community settings over a 17-year period from 12 countries investigated by whole-genome sequencing.

BACKGROUND: A novel Panton-Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC)5-MRSA-IVc ('Sri Lankan' clone) was recently described from Sri Lanka. Similar isolates caused a recent Irish hospital outbreak. AIM: To investigate the international dissemination and diversity of PVL-positive CC5-MRSA-IVc isolates from hospital and community settings using whole-genome sequencing (WGS). METHODS: Core-genome single nucleotide polymorphism (cgSNP) analysis, core-genome multi-locus sequence typing (cgMLST) and microarray-based detection of antimicrobial-resistance and virulence genes were used to investigate PVL-positive CC5-MRSA-IVc (N = 214 including 46 'Sri Lankan' clone) from hospital and community settings in 12 countries over 17 years. Comparators included 29 PVL-positive and 23 PVL-negative CC5/ST5-MRSA-I/II/IVa/IVc/IVg/V. RESULTS: Maximum-likelihood cgSNP analysis grouped 209/214 (97.7%) CC5-MRSA-IVc into Clade I; average of 110 cgSNPs between isolates. Clade III contained the five remaining CC5-MRSA-IVc; average of 92 cgSNPs between isolates. Clade II contained seven PVL-positive CC5-MRSA-IVa comparators, whereas the remaining 45 comparators formed an outlier group. Minimum-spanning cgMLST analysis revealed a comparably low average of 57 allelic differences between all CC5/ST5-MRSA-IVc. All 214 CC5/ST5-MRSA-IVc were identified as 'Sri Lankan' clone, predominantly spa type t002 (186/214) with low population diversity and harboured a similar range of virulence genes and variable antimicrobial-resistance genes. All 214 Sri Lankan clone isolates and Clade II comparators harboured a 9616-bp chromosomal PVL-encoding phage remnant, suggesting both arose from a PVL-positive meticillin-susceptible ancestor. Over half of Sri Lankan clone isolates were from infections (142/214), and where detailed metadata were available (168/214), most were community associated (85/168). CONCLUSIONS: Stable chromosomal retention of pvl may facilitate Sri-Lankan clone dissemination.

Molecular epidemiology and characterisation of MRSA isolates from Trinidad and Tobago.

Eighty methicillin-resistant Staphylococcus aureus (MRSA) isolates from three hospitals in Trinidad and Tobago were collected and genotyped using microarray hybridisation. They were found to belong to three distinct MRSA strains. Of the 80 isolates, 76 were assigned to ST239-MRSA-III. They were largely homogeneous, although some variations affected the presence of the enterotoxin A gene, as well as of resistance markers (mercury resistance operon, aadD, tet(K), qacA). The mupA gene conferring mupirocin resistance was found in 7.3% of isolates. One isolate was identified as CC5-MRSA-II and three isolates belonged to the Panton-Valentine leukocidin (PVL)-positive ST8-MRSA-IV strain USA300. While community-acquired MRSA strains are rare in Trinidad and Tobago, the vast majority of MRSA cases can be attributed to healthcare-associated strains. Thus, infection control procedures within medical facilities need to be revised and enforced. This could substantially reduce the burden of MRSA to healthcare in Trinidad and Tobago.

Dogs as carriers of virulent and resistant genotypes of Clostridioides difficile.

While previous research on zoonotic transmission of community-acquired Clostridioides difficile infection (CA-CDI) focused on food-producing animals, the present study aimed to investigate whether dogs are carriers of resistant and/or virulent C. difficile strains. Rectal swabs were collected from 323 dogs and 38 C. difficile isolates (11.8%) were obtained. Isolates were characterized by antimicrobial susceptibility testing, whole-genome sequencing (WGS) and a DNA hybridization assay. Multilocus sequence typing (MLST), core genome MLST (cgMLST) and screening for virulence and antimicrobial resistance genes were performed based on WGS. Minimum inhibitory concentrations for erythromycin, clindamycin, tetracycline, vancomycin and metronidazole were determined by E-test. Out of 38 C. difficile isolates, 28 (73.7%) carried genes for toxins. The majority of isolates belonged to MLST sequence types (STs) of clade I and one to clade V. Several isolates belonged to STs previously associated with human CA-CDI. However, cgMLST showed low genetic relatedness between the isolates of this study and C. difficile strains isolated from humans in Austria for which genome sequences were publicly available. Four isolates (10.5%) displayed resistance to three of the tested antimicrobial agents. Isolates exhibited resistance to erythromycin, clindamycin, tetracycline and metronidazole. These phenotypic resistances were supported by the presence of the resistance genes erm(B), cfr(C) and tet(M). All isolates were susceptible to vancomycin. Our results indicate that dogs may carry virulent and antimicrobial-resistant C. difficile strains.

Diversity of methicillin-resistant coagulase-negative Staphylococcus spp. and methicillin-resistant Mammaliicoccus spp. isolated from ruminants and New World camelids.

Information about livestock carrying methicillin-resistant coagulase-negative staphylococci and mammaliicocci (MRCoNS/MRM) is scarce. The study was designed to gain knowledge of the prevalence, the phenotypic and genotypic antimicrobial resistance and the genetic diversity of MRCoNS/MRM originating from ruminants and New World camelids. In addition, a multi-locus sequence typing scheme for the characterization of Mammaliicoccus (formerly Staphylococcus) sciuri was developed. The study was conducted from April 2014 to January 2017 at the University Clinic for Ruminants and the Institute of Microbiology at the University of Veterinary Medicine Vienna. Seven hundred twenty-three nasal swabs originating from ruminants and New World camelids with and without clinical signs were examined. After isolation, MRCoNS/MRM were identified by MALDI-TOF, rpoB sequencing and typed by DNA microarray-based analysis and PCR. Antimicrobial susceptibility testing was conducted by agar disk diffusion. From all 723 nasal swabs, 189 MRCoNS/MRM were obtained. Members of the Mammaliicoccus (M.) sciuri group were predominant (M. sciuri (n = 130), followed by M. lentus (n = 43), M. fleurettii (n = 11)). In total, 158 out of 189 isolates showed phenotypically a multi-resistance profile. A seven-loci multi-locus sequence typing scheme for M. sciuri was developed. The scheme includes the analysis of internal segments of the house-keeping genes ack, aroE, ftsZ, glpK, gmk, pta1 and tpiA. In total, 28 different sequence types (STs) were identified among 92 selected M. sciuri isolates. ST1 was the most prevalent ST (n = 35), followed by ST 2 (n = 15), ST3 and ST5 (each n = 5), ST4 (n = 3), ST6, ST7, ST8, ST9, ST10 and ST11 (each n = 2).

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

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

Molecular fingerprinting of Staphylococcus aureus from bone and joint infections.

The objective of the study was to determine if a clonal complex (CC) of Staphylococcus aureus or certain virulence and adhesion factors were associated with infections of bones and prosthetic implants. One hundred and nineteen isolates were characterised using microarrays. There was no evidence for a single virulence factor or CC being causative for bone and implant infections. Isolates belonged to 20 different CCs, with CC8 (19.33%), CC45 (17.65%) and CC30 (12.61%) being dominant. Population structure and the relative abundances of virulence genes was similar to previously described isolates from healthy carriers. Differences to carrier isolates included a higher proportion of CC45, a lower proportion of CC15, as well as a higher abundance of sak (staphylokinase) among patient isolates. For 23 patients with infections of total knee or hip prosthetics, it was possible to simultaneously obtain nasal swabs. Fifteen (65.2%) carried S. aureus in their anterior nares. In nine of them (39.1%), isolates from the infection site were identical to carriage isolates. This suggests an elevated risk of infection for S. aureus carriers and the possibility of endogenous infection in a high proportion of them. Therefore, the pre-operative screening and eradication of S. aureus in patients receiving total joint prosthetics should be considered.

Characterisation of MRSA from Malta and the description of a Maltese epidemic MRSA strain.

Malta has one of the highest prevalence rates of methicillin-resistant Staphylococcus aureus (MRSA) in Europe. However, only limited typing data are currently available. In order to address this situation, 45 MRSA isolates from the Mater Dei Hospital in Msida, Malta, were characterised using DNA microarrays. The most common strain was ST22-MRSA-IV (UK-EMRSA-15, 30 isolates). Sporadic strains included ST36-MRSA-II (UK-EMRSA-16, two isolates), PVL-positive ST80-MRSA-IV (European Clone, one isolate), ST228-MRSA-I (Italian Clone/South German Epidemic Strain, one isolate) and ST239-MRSA-III (Vienna/Hungarian/Brazilian Epidemic Strain, one isolate). Ten MRSA isolates belonged to a clonal complex (CC) 5/ST149, spa type t002 strain. This strain harboured an SCCmec IV element (mecA, delta mecR, ugpQ, dcs, ccrA2 and ccrB2), as well as novel alleles of ccrA/B and the fusidic acid resistance element Q6GD50 (previously described in the sequenced strain MSSA476, BX571857.1:SAS0043). It also carried the gene for enterotoxin A (sea) and the egc enterotoxin locus, as well as (in nine out of ten isolates) genes encoding the toxic shock syndrome toxin (tst1) and enterotoxins C and L (sec, sel). While the presence of the other MRSA strains suggests foreign importation due to travel between Malta and other European countries, the CC5/t002 strain appears, so far, to be restricted to Malta.

Molecular epidemiology of Staphylococcus aureus in asymptomatic carriers.

Microarrays were used to extensively characterise 155 Staphylococcus aureus isolates obtained from asymptomatic carriers from Saxony, Germany, in order to determine clonal complex affiliation, as well as the carriage of clinically relevant genes. Isolates belonged to 20 different clonal complexes (CCs). The most common CC was CC8 (18.71%), followed by CCs 15, 30 and 45. Three isolates (1.94%) were methicillin-resistant S. aureus (MRSA). Beta-lactamase was common (70.97%), but other resistance genes were found only sporadically. Genes encoding superantigens were abundant. The enterotoxin cluster egc was found in 45.81% of isolates. The toxic shock syndrome toxin gene tst was detected in 14.84% of isolates and 17.42% harboured enterotoxin A alleles (sea, sea-N315). Contrarily, Panton-Valentine leukocidin (lukS/F-PV) was rare, being found in only one methicillin-susceptible CC30 isolate. Its low prevalence in asymptomatic carriers might emphasise a pathogenetic significance in patients with skin and soft tissue infections. Most microbial surface components recognising adhesive matrix molecules of the host (MSCRAMMs) genes were nearly ubiquitously present. However, two MSCRAMM genes, cna (collagen adhesion) and sasG (surface protein G), were detected in only some CCs. These data provide an insight into its pathogenesis, especially when compared to isolates from patients with defined clinical conditions. They might also be helpful for the design of a future vaccine.

Intra-strain variability of methicillin-resistant Staphylococcus aureus strains ST228-MRSA-I and ST5-MRSA-II.

Isolates belonging to two major epidemic strains of methicillin-resistant Staphylococcus aureus (MRSA) from clonal complex 5 were characterised using diagnostic microarrays in order to detect and analyse intra-strain variability. Isolates were sampled from hospitals scattered all over Germany. The study included 56 isolates of ST228-MRSA-I, which is also known as the South German Epidemic Strain, and 40 isolates of ST5-MRSA-II (UK-EMRSA-3, Rhine-Hesse Epidemic Strain, New York/Japan Clone), as well as, for comparison, some control strains and overseas isolates of ST5-MRSA-II. Both strains showed a remarkable variability. This affected plasmid-borne resistance genes (tetK, blaZ/R/I, aacA-aphD, qacA), genes from SCCmec elements (aadD, ermA, merA/B/R/T), toxin gene clusters on pathogenicity islands (sec/l, tst1) or, probably, on plasmids, (sed/j/r), the presence or absence of beta-haemolysin-converting phages (sea, sea-N315, sak, chp, scn), deletions of single chromosomal genes (bbp, clfA) or, occasionally, of rather large clusters of neighbouring genes (seg, sei, sem, sen, seo, seu, lukD/E). Both strains could be split into four major clusters each, based on the presence of a mercury resistance operon (merA/B/R/T) and lukD/E in ST228-MRSA-I or of tst1 and enterotoxin genes seD/J/R in ST5-MRSA-II. The use of this variability for typing purposes as well as its phylogenetic significance are discussed.

A DNA microarray facilitates the diagnosis of Bacillus anthracis in environmental samples.

AIMS: In order to improve the diagnosis of Bacillus anthracis in environmental samples, we established a DNA microarray based on the ArrayTube technology of Clondiag. METHODS AND RESULTS: Total DNA of a bacterial colony is randomly biotinylated and hybridized to the array. The probes on the array target the virulence genes, the genomic marker gene rpoB, as well as the selective 16S rDNA sequence regions of B. anthracis, of the Bacillus cereus group and of Bacillus subtilis. Eight B. anthracis reference strains were tested and correctly identified. Among the analysed environmental Bacillus isolates, no virulent B. anthracis strain was detected. CONCLUSIONS: This array clearly differentiates B. anthracis from members of the B. cereus group and other Bacillus species in environmental samples by chromosomal (rpoB) and plasmid markers. Additionally, recognition of B. cereus strains harbouring the toxin genes or atypical B. anthracis strains that have lost the virulence plasmids is feasible. SIGNIFICANCE AND IMPACT OF THE STUDY: The array is applicable to the complex diagnostics for B. anthracis detection in environmental samples. Because of low costs, high security and easy handling, the microarray is applicable to routine diagnostics.

Differences in molecular epidemiology of Staphylococcus aureus and Escherichia coli in nursing home residents and people in unassisted living situations.

BACKGROUND: The usefulness of colonization pressure as a working model and proxy for infection transmission is limited due to the inability to grade or quantify the specific risk within environments that are subject to change. AIM: To elaborate on the colonization pressure model by comparing the molecular epidemiology of two bacteria, Staphylococcus aureus and Escherichia coli, among residents in a nursing home and people in unassisted living situations. METHODS: A cross-sectional study of 73 elderly residents from a village in south-central Sweden was conducted. Of these, 35 were residents of a nursing home, and 34 lived in an own place of residence in the same geographical area. Samples of two representative bacterial species were collected from multiple body sites and analysed for molecular diversity. FINDINGS: Combining all body sites, 47% of the participants were colonized with S. aureus and 93% with E. coli. The nursing home group, the group in unassisted living situations, and both units combined, held 16, 17, and 29 different S. aureus spa types, respectively. The corresponding numbers of different E. coli serogenotypes were 34, 28, and 48. Diabetes mellitus was associated with more frequent colonization with S. aureus. CONCLUSION: The molecular diversity of bacteria found within different forms of accommodation was within the same range. Hospital quality hygiene might have contributed to the absence of homogenization of the molecular diversity within the nursing home group. Diabetes mellitus might have played a role in a patient selection characterized by advanced age.

Disseminated cutaneous and pulmonary abscesses in an injecting drug user caused by a Panton-Valentine leucocidin-positive, methicillin-susceptible Staphylococcus aureus strain.

Staphylococcus aureus is an important pathogen in injecting drug users. We illustrate this with a case of a young female patient who was admitted with multiple cutaneous and pulmonary abscesses. The causative strain was characterised using diagnostic microarrays. Genes encoding Panton-Valentine leucocidin (PVL) were detected, mecA was absent. The overall hybridisation profile allowed us to assign this isolate to ST152, being related to an epidemic community-acquired methicillin-resistant S. aureus (caMRSA) strain from south-eastern Europe. While PVL-positive MRSA has frequently been observed in injecting drug users, methicillin-susceptible isolates are usually not screened for the presence of PVL genes. The use of diagnostic microarrays contributes to patient care by the simultaneous detection of resistance and virulence markers. It also facilitates the study of phylogenetic relationships of epidemic strains.

Diversity of methicillin-resistant Staphylococcus aureus (MRSA) isolated from Austrian ruminants and New World camelids.

The aim of this study was to determine the prevalence, the antimicrobial resistance patterns and the genetic diversity of methicillin-resistant Staphylococcus aureus (MRSA) from Austrian ruminants and New World camelids that were treated at the University of Veterinary Medicine, Vienna. Between April 2014 and January 2017, 723 nasal swabs originating from ruminants and New World camelids were examined. MRSA isolates were characterized by mecA/mecA1/mecC PCRs and by DNA microarray analysis. They were genotyped by spa typing, dru typing, MLST and MLVA. Glycopolymer fingerprinting by FTIR spectroscopy was also performed. Antimicrobial susceptibility testing was conducted by agar disk diffusion. Twelve MRSA isolates were mecA-positive, whereas three were mecC-positive. The MRSA isolates carried five different SCCmec elements, and belonged to three sequence types (ST45, ST130, ST398). The MRSA isolates displayed seven different resistance phenotypes. The present study describes for the first time mecC-carrying MRSA isolates originating from domesticated animals in Austria. More systematic studies are needed to unravel the role of ruminants and New World camelids as reservoirs for MRSA as a potential risk for zooanthropogenic transmission.

High diversity of Panton-Valentine leukocidin-positive, methicillin-susceptible isolates of Staphylococcus aureus and implications for the evolution of community-associated methicillin-resistant S. aureus.

In total, 100 Staphylococcus aureus isolates from diverse cases of skin and soft-tissue infection at a university hospital in Saxony, Germany, were characterised using diagnostic microarrays. Virulence factors, including Panton-Valentine leukocidin (PVL), were detected and the isolates were assigned to clonal groups. Thirty isolates were positive for the genes encoding PVL. Only three PVL-positive methicillin-resistant S. aureus (MRSA) isolates were found, two of which belonged to European clone ST80-MRSA IV and one to USA300 strain ST8-MRSA IV. The remaining methicillin-susceptible PVL-positive isolates belonged to a variety of different multilocus sequence types. The predominant strains were agrI/ST22, agrII/CC5, agrIII/CC30 and agrIV/ST121. In order to check for possible bias caused by regional or local outbreak strains, an additional 18 methicillin-susceptible, PVL-positive isolates from the UK were tested. Approximately two-thirds of the UK isolates belonged to types that also comprised approximately two-thirds of the isolates from Saxony. Some methicillin-susceptible PVL-positive isolates (agrI/ST152, agrIII/ST80 and agrIII/ST96) closely resembled known epidemic community-acquired MRSA (CaMRSA) strains. These findings indicate that the current rise in PVL-positive CaMRSA could be caused by the dissemination of novel SCCmec elements among pre-existing PVL-positive strains, rather than by the spread of PVL phages among MRSA strains.

Comparative genomics and DNA array-based genotyping of pandemic Staphylococcus aureus strains encoding Panton-Valentine leukocidin.

Within the last few years, methicillin-resistant Staphylococcus aureus (MRSA) strains encoding Panton-Valentine leukocidin (PVL) have emerged and spread worldwide. This epidemic can be attributed to a small number of distinct clones. The present study used a novel assay, based on multiplex linear DNA amplification and subsequent microarray hybridisation, to simultaneously detect all relevant exotoxins, antimicrobial resistance determinants and the allelic variants of agr. The genes of the staphylococcal exotoxin-like (set) locus were also included for typing purposes. This assay, together with multilocus sequence typing (MLST) and spa typing, was applied to 56 clinical isolates and reference strains representing all major pandemic PVL-MRSA lineages, as well as to phylogenetically-related strains and putative ancestors. Array hybridisation results allowed the assignment of isolates to clonal groups, which were in accordance with MLST and spa typing data. Ten distinct clonal groups of PVL-MRSA (ST1, ST5, ST8, ST22, ST30, ST59/359, ST80/583, ST88, ST93 and ST152), including 12 MLST types, were identified and analysed with regard to resistance determinants and genes coding for exotoxins. The array hybridisation data confirmed that pandemic PVL-positive strains originate from very diverse genetic backgrounds, and provided insights into the evolution of some lineages. The DNA microarray technique provides a valuable epidemiological tool for the detailed characterisation of clinical isolates and comparison of strains at a global level.