Search

•We studied the presence of mastitis-associated bovine Staphylococcus aureusin Rwanda.•Two clonal lineages, CC3666 and CC3591, have an important role in cattle health and dairy production in Rwanda.•The dominance of CC97 underlines the widespread global distribution of this cattle-adapted clonal lineage.•The occurrence of human-associated CC152 suggests interspecies transmission.

Methicillin-resistant Staphylococcus aureus(MRSA) are a major concern for public health, and broiler farms are a potential source of MRSA isolates. In this study, a total of 56 MRSA isolates from 15 broiler farms from 4 different counties in Germany were characterised phenotypically and genotypically. Spatypes, drutypes, SCCmectypes, and virulence genes as well as resistance genes were determined by using a DNA microarray or specific PCR assays. In addition, PFGE profiles of isolates were used for analysis of their epidemiological relatedness. While half of the isolates belonged to spatype t011, the other half was of spatypes t1430 and t034. On 3 farms, more than 1 spatype was found. The most common drutype was dt10a (n = 19), followed by dt11a (n = 17). Susceptibility testing of all isolates by broth microdilution revealed 21 different resistance phenotypes and a wide range of resistance genes was present among the isolates. Up to 10 different resistance phenotypes were found on individual farms. Resistance to tetracyclines (n = 53), MLSBantibiotics (n = 49), trimethoprim (n = 38), and elevated MICs of tiamulin (n = 29) were most commonly observed. Microarray analysis detected genes for leucocidin (lukF/S), haemolysin gamma (hlgA), and other haemolysines in all isolates. In all t1430 isolates, the egccluster comprising of genes encoding enterotoxin G, I, M, N, O, U, and/or Y was found. The splitstree analysis based on microarray and PCR gene profiles revealed that all CC9/SCCmecIV/t1430/dt10a isolates clustered apart from the other isolates. These findings confirm that genotypic patterns were specific for clonal lineages rather than for the origin of isolates from individual farms.

Aim:A newly designed multiplex real-time PCR (rt-PCR) was validated to detect four clinically relevant Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniaeand Pseudomonas aeruginosa). Materials & methods:Serial dilutions of genomic DNA were used to determine the limit of detection. Colony PCR was performed with isolates of the four selected species and other species as negative controls. Isolates were characterized genotypically and phenotypically to evaluate the assay. Results:Specific signals of all target genes were detected with diluted templates comprising ten genomic equivalents. Using colony rt-PCR, all isolates of the target species were identified correctly. All negative control isolates were negative. Conclusion:The genes gad, basC, kheand ecfXcan reliably identify these four species via multiplex colony rt-PCR.

Aim:A DNA microarray-based assay for the detection of antimicrobial resistance (AMR) genes was used to study carbapenemase-producing organisms at the Kidney Center of Rawalpindi, Pakistan. Methods:The evaluation of this assay was performed using 97 reference strains with confirmed AMR genes. Testing of 7857 clinical samples identified 425 Gram-negative bacteria out of which 82 appeared carbapenem resistant. These isolates were analyzed using VITEK-2 for phenotyping and the described AMR assay for genotyping. Results:The most prevalent carbapenemase gene was blaNDM and in 12 isolates we detected two carbapenemase genes (e.g., blaNDM/blaOXA-48). Conclusion:Our prevalence data from Pakistan show that – as in other parts of the world – carbapenemase-producing organisms with different underlying resistance mechanisms are emerging, and this warrants intensified and constant surveillance.

•We determined the MICs of the novel cephalosporin compound ceftaroline against a collection of MRSA harbouring mecC/SCCmecXI.•Isolates were found to be susceptible to ceftaroline.

•We characterised 30 methicillin-resistant Staphylococcus aureus(MRSA) isolates collected in Ghana between 2010 and 2013 from patients and healthy carriers.•DNA microarray analysis revealed that tet(M), tet(K), aphA3and aacA–aphDwere the common resistant genes associated with these MRSA isolates.•Whereas no resistance to glycopeptides, linezolid, daptomycin and tigecycline was detected, resistance to tetracycline, norfloxacin, moxifloxacin, erythromycin, clindamycin, gentamicin, kanamycin and ceftaroline was frequent among the isolates.•USA300 [containing arginine catabolic mobile element (ACME) and Panton–Valentine leukocidin (PVL)] was among the multidrug-resistant MRSA detected in Ghana.

ABSTRACTStreptococcus agalactiaefrequently colonizes the urogenital tract, and it is a major cause of bacterial septicemia, meningitis, and pneumonia in newborns. For typing purposes, a microarray targeting group B streptococcus (GBS) virulence-associated markers and resistance genes was designed and validated with reference strains, as well as clinical and veterinary isolates. Selected isolates were also subjected to multilocus sequence typing. It was observed that putative typing markers, such as alleles of the alpha-like protein or capsule types, vary independently of each other, and they also vary independently from the affiliation to their multilocus sequence typing (MLST)-defined sequence types. Thus, it is not possible to assign isolates to sequence types based on the identification of a single distinct marker, such as a capsule type or alpallele. This suggests the occurrence of frequent genomic recombination. For array-based typing, a set of 11 markers (bac, alp, pil1locus, pepS8, fbsB, capsule locus, hylB, abiG-I/-II plus Q8DZ34, pil2locus, nssplus srrplus rogB2, and rgfC/A/D/B) was defined that provides a framework for splitting the tested 448 S. agalactiaeisolates into 76 strains that clustered mainly according to MLST-defined clonal complexes. There was evidence for region- and host-specific differences in the population structure of S. agalactiae, as well as an overrepresentation of strains related to sequence type 17 among the invasive isolates. The arrays and typing scheme described here proved to be a convenient tool for genotyping large numbers of clinical/veterinary isolates and thus might help obtain insight into the epidemiology of S. agalactiae.

ABSTRACTIn this study, we developed a new rapid, economic, and automated microarray-based genotyping test for the standardized subtyping of Shiga toxins 1 and 2 of Escherichia coli. The microarrays from Alere Technologies can be used in two different formats, the ArrayTube and the ArrayStrip (which enables high-throughput testing in a 96-well format). One microarray chip harbors all the gene sequences necessary to distinguish between all Stx subtypes, facilitating the identification of single and multiple subtypes within a single isolate in one experiment. Specific software was developed to automatically analyze all data obtained from the microarray. The assay was validated with 21 Shiga toxin-producing E. coli(STEC) reference strains that were previously tested by the complete set of conventional subtyping PCRs. The microarray results showed 100% concordance with the PCR results. Essentially identical results were detected when the standard DNA extraction method was replaced by a time-saving heat lysis protocol. For further validation of the microarray, we identified the Stx subtypes or combinations of the subtypes in 446 STEC field isolates of human and animal origin. In summary, this oligonucleotide array represents an excellent diagnostic tool that provides some advantages over standard PCR-based subtyping. The number of the spotted probes on the microarrays can be increased by additional probes, such as for novel alleles, species markers, or resistance genes, should the need arise.

ABSTRACTThere has been a worldwide increase in community-associated (CA) methicillin-resistant Staphylococcus aureus(MRSA) infections. CA-MRSA isolates commonly produce the Panton-Valentine leukocidin toxin encoded by the pvlgenes lukF-PVand lukS-PV. This study investigated the clinical and molecular epidemiologies of pvl-positive MRSA and methicillin-susceptible S. aureus(MSSA) isolates identified by the Irish National MRSA Reference Laboratory (NMRSARL) between 2002 and 2011. All pvl-positive MRSA (n= 190) and MSSA (n= 39) isolates underwent antibiogram-resistogram typing, spatyping, and DNA microarray profiling for multilocus sequence type, clonal complex (CC) and/or sequence type (ST), staphylococcal cassette chromosome mectype assignment, and virulence and resistance gene detection. Where available, patient demographics and clinical data were analyzed. The prevalence of pvl-positive MRSA increased from 0.2% to 8.8%, and that of pvl-positive MSSA decreased from 20% to 2.5% during the study period. The pvl-positive MRSA and MSSA isolates belonged to 16 and 5 genotypes, respectively, with CC/ST8-MRSA-IV, CC/ST30-MRSA-IV, CC/ST80-MRSA-IV, CC1/ST772-MRSA-V, CC30-MSSA, CC22-MSSA, and CC121-MSSA predominating. Temporal shifts in the predominant pvl-positive MRSA genotypes and a 6-fold increase in multiresistant pvl-positive MRSA genotypes occurred during the study period. An analysis of patient data indicated that pvl-positive S. aureusstrains, especially MRSA strains, had been imported into Ireland several times. Two hospital and six family clusters of pvl-positive MRSA were identified, and 70% of the patient isolates for which information was available were from patients in the community. This study highlights the increased burden and changing molecular epidemiology of pvl-positive S. aureusin Ireland over the last decade and the contribution of international travel to the influx of genetically diverse pvl-positive S. aureusisolates into Ireland.

ABSTRACTClonal replacement of predominant nosocomial methicillin-resistant Staphylococcus aureus(MRSA) strains has occurred several times in Ireland during the last 4 decades. However, little is known about sporadically occurring MRSA in Irish hospitals or in other countries. Eighty-eight representative pvl-negative sporadic MRSA isolates recovered in Irish hospitals between 2000 and 2012 were investigated. These yielded unusual pulsed-field gel electrophoresis and antibiogram-resistogram typing patterns distinct from those of the predominant nosocomial MRSA clone, ST22-MRSA-IV, during the study period. Isolates were characterized by spatyping and DNA microarray profiling for multilocus sequence type (MLST) clonal complex (CC) and/or sequence type (ST) and SCCmectype assignment, as well as for detection of virulence and antimicrobial resistance genes. Conventional PCR-based SCCmecsubtyping was undertaken when necessary. Extensive diversity was detected, including 38 spatypes, 13 MLST-CCs (including 18 STs among 62 isolates assigned to STs), and 25 SCCmectypes (including 2 possible novel SCCmecelements and 7 possible novel SCCmecsubtypes). Fifty-four MLST-spa-SCCmectype combinations were identified. Overall, 68.5% of isolates were assigned to nosocomial lineages, with ST8-t190-MRSA-IID/IIE ± SCCM1predominating (17.4%), followed by CC779/ST779-t878-MRSA-ψSCCmec-SCC-SCCCRISPR(7.6%) and CC22/ST22-t032-MRSA-IVh (5.4%). Community-associated clones, including CC1-t127/t386/t2279-MRSA-IV, CC59-t216-MRSA-V, CC8-t008-MRSA-IVa, and CC5-t002/t242-MRSA-IV/V, and putative animal-associated clones, including CC130-t12399-MRSA-XI, ST8-t064-MRSA-IVa, ST398-t011-MRSA-IVa, and CC6-t701-MRSA-V, were also identified. In total, 53.3% and 47.8% of isolates harbored genes for resistance to two or more classes of antimicrobial agents and two or more mobile genetic element-encoded virulence-associated factors, respectively. Effective ongoing surveillance of sporadic nosocomial MRSA is warranted for early detection of emerging clones and reservoirs of virulence, resistance, and SCCmecgenes.

Methicillin-resistant Staphylococcus aureus(MRSA) is a major nosocomial pathogen worldwide. Malta is one of the countries with the highest MRSA prevalence in Europe, as identified from hospital blood cultures [1]. However, community prevalence of MRSA has never previously been investigated. This study aimed at establishing the prevalence of community MRSA nasal colonization in Maltese individuals and identifying the clonal characteristics of the detected isolates. Nasal swabs were collected from 329 healthy individuals who were also asked to complete a brief questionnaire about risk factors commonly associated with MRSA carriage and infection. The swabs were transported and enriched in a nutrient broth supplemented with NaCl. The presence of MRSA was then determined by culturing on MRSA Selectchromogenic agar and then confirming by several assays, including catalase, coagulase and PBP2a agglutination tests. The isolates were assayed for antibiotic susceptibilities and typed by microarray analysis to determine the clonal characteristics of each strain. The prevalence of MRSA nasal colonization in the healthy Maltese population was found to be 8.81% (95% confidence interval [CI], 5.75–11.87%), much higher than that found in other studies carried out in several countries. No statistical association was found between MRSA carriage and demographics or risk factors; however, this was hindered by the small sample size. Almost all the isolates were fusidic-acid resistant. The majority were found to belong to a local endemic clone (CC5) which seems to be replacing the previously prevalent European clone UK-EMRSA-15 in the country. A new clone (CC50-MRSA-V) was also characterized. The presence of such a significant community reservoir of MRSA increases the burdens already faced by the local healthcare system to control the MRSA epidemic. Colonization of MRSA in otherwise healthy individuals may represent a risk for endogenous infection and transmission to hospitalized patients after admission to a healthcare facility, leading to longer hospital stays and, consequently, increased healthcare costs.

ABSTRACTPanton-Valentine leukocidin (PVL) is a virulence factor of Staphylococcus aureus, which is associated with skin and soft-tissue infections and necrotizing pneumonia. To develop a rapid phenotypic assay, recombinant PVL F component was used to generate monoclonal antibodies by phage display. These antibodies were spotted on protein microarrays and screened using different lukF-PVpreparations and detection antibodies. This led to the identification of the optimal antibody combination that was then used to establish a lateral flow assay. This test was used to detect PVL in S. aureuscultures. The detection limit of the assay with purified native and recombinant antigens was determined to be around 1 ng/ml. Overnight cultures from various solid and liquid media proved suitable for PVL detection. Six hundred strains and clinical isolates from patients from America, Europe, Australia, Africa, and the Middle East were tested. Isolates were genotyped in parallel by DNA microarray hybridization for confirmation of PVL status and assignment to clonal complexes. The sensitivity, specificity, and positive and negative predictive values of the assay in this trial were 99.7, 98.3, 98.4, and 99.7%, respectively. A total of 302 clinical isolates and reference strains were PVL positive and were assigned to 21 different clonal complexes. In summary, the lateral flow test allows rapid and economical detection of PVL in a routine bacteriology laboratory. As the test utilizes cultures from standard media and does not require sophisticated equipment, it can be easily integrated into a laboratory's workflow and might contribute to timely therapy of PVL-associated infections.

Methicillin-resistant Staphylococcus aureus(MRSA) is a major nosocomial pathogen worldwide. Malta is one of the countries with the highest MRSA prevalence in Europe, as identified from hospital blood cultures [1]. However, community prevalence of MRSA has never previously been investigated. This study aimed at establishing the prevalence of community MRSA nasal colonization in Maltese individuals and identifying the clonal characteristics of the detected isolates. Nasal swabs were collected from 329 healthy individuals who were also asked to complete a brief questionnaire about risk factors commonly associated with MRSA carriage and infection. The swabs were transported and enriched in a nutrient broth supplemented with NaCl. The presence of MRSA was then determined by culturing on MRSA Select chromogenic agar and then confirming by several assays, including catalase, coagulase and PBP2a agglutination tests. The isolates were assayed for antibiotic susceptibilities and typed by microarray analysis to determine the clonal characteristics of each strain. The prevalence of MRSA nasal colonization in the healthy Maltese population was found to be 8.81% (95% confidence interval [CI], 5.75–11.87%), much higher than that found in other studies carried out in several countries. No statistical association was found between MRSA carriage and demographics or risk factors; however, this was hindered by the small sample size. Almost all the isolates were fusidic-acid resistant. The majority were found to belong to a local endemic clone (CC5) which seems to be replacing the previously prevalent European clone UK-EMRSA-15 in the country. A new clone (CC50-MRSA-V) was also characterized. The presence of such a significant community reservoir of MRSA increases the burdens already faced by the local healthcare system to control the MRSA epidemic. Colonization of MRSA in otherwise healthy individuals may represent a risk for endogenous infection and transmission to hospitalized patients after admission to a healthcare facility, leading to longer hospital stays and, consequently, increased healthcare costs.

ABSTRACTIn this study, 18 methicillin-resistant Staphylococcus aureus(MRSA) isolates harboring staphylococcal cassette chromosome mec(SCCmec) type XI, recovered in the Dutch-German Euregio, were characterized by DNA microarrays. In contrast to previous data, we found two MRSA strains of different clonal lineages possessing SCCmecXI that carried important virulence determinants. The worrisome emergence of such toxigenic MRSA strains raises concerns that MRSA strains with enhanced virulence potential and impaired detectability by standard molecular assays may spread in Europe.

ABSTRACTMethicillin-resistant Staphylococcus aureus(MRSA) has been a major cause of nosocomial infection in Irish hospitals for 4 decades, and replacement of predominant MRSA clones has occurred several times. An MRSA isolate recovered in 2006 as part of a larger study of sporadic MRSA exhibited a rare spa(t878) and multilocus sequence (ST779) type and was nontypeable by PCR- and DNA microarray-based staphylococcal cassette chromosome mec(SCCmec) element typing. Whole-genome sequencing revealed the presence of a novel 51-kb composite island (CI) element with three distinct domains, each flanked by direct repeat and inverted repeat sequences, including (i) a pseudo SCCmecelement (16.3 kb) carrying mecAwith a novel mecclass region, a fusidic acid resistance gene (fusC), and two copper resistance genes (copBand copC) but lacking ccrgenes; (ii) an SCC element (17.5 kb) carrying a novel ccrAB4allele; and (iii) an SCC element (17.4 kb) carrying a novel ccrCallele and a clustered regularly interspaced short palindromic repeat (CRISPR) region. The novel CI was subsequently identified by PCR in an additional 13 t878/ST779 MRSA isolates, six from bloodstream infections, recovered between 2006 and 2011 in 11 hospitals. Analysis of open reading frames (ORFs) carried by the CI showed amino acid sequence similarity of 44 to 100% to ORFs from S. aureusand coagulase-negative staphylococci (CoNS). These findings provide further evidence of genetic transfer between S. aureusand CoNS and show how this contributes to the emergence of novel SCCmecelements and MRSA strains. Ongoing surveillance of this MRSA strain is warranted and will require updating of currently used SCCmectyping methods.

ABSTRACTTo screen isolates and to identify mecAalleles, published mecAsequences were analyzed, and a microarray for the rapid discrimination of mecAalleles was designed. A GenBank analysis yielded 135 full-length gene sequences annotated as mecA. These sequences clustered into 32 different alleles corresponding to 28 unique amino acid sequences and to 15 distinct hybridization patterns on this microarray. A collection of 78 clinical and veterinary isolates of Staphylococcusspp. was characterized using this assay. Nine of the 15 expected patterns, as well as one as-yet-unknown pattern, were identified. These patterns were detected in various epidemic methicillin-resistant Staphylococcus aureusstrains, in S. pseudintermedius, and in coagulase-negative species such as S. epidermidis, S. fleurettii, or S. haemolyticus. There was no correlation between the different mecAhybridization patterns and the SCCmectype. Determination of MICs showed that mecAalleles corresponding to only four of these nine patterns were associated with β-lactam resistance. The mecAalleles that did not confer β-lactam resistance were largely restricted to coagulase-negative staphylococci of animal origin, such as S. sciuriand S. vitulinus. Because of the diversity of sequences and the different impact on β-lactam susceptibility, the existence of different mecAalleles needs to be taken into account when designing diagnostic assays for the detection of mecA.

ABSTRACTOne hundred seventy-five isolates representative of methicillin-resistant Staphylococcus aureus(MRSA) clones that predominated in Irish hospitals between 1971 and 2004 and that previously underwent multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec(SCCmec) typing were characterized by spatyping (175 isolates) and DNA microarray profiling (107 isolates). The isolates belonged to 26 sequence type (ST)-SCCmectypes and subtypes and 35 spatypes. The array assigned all isolates to the correct MLST clonal complex (CC), and 94% (100/107) were assigned an ST, with 98% (98/100) correlating with MLST. The array assigned all isolates to the correct SCCmectype, but subtyping of only some SCCmecelements was possible. Additional SCCmec/SCC genes or DNA sequence variation not detected by SCCmectyping was detected by array profiling, including the SCC-fusidic acid resistance determinant Q6GD50/fusC. Novel SCCmec/SCC composite islands (CIs) were detected among CC8 isolates and comprised SCCmecIIA-IIE, IVE, IVF, or IVg and a ccrAB4-SCC element with 99% DNA sequence identity to SCCM1from ST8/t024-MRSA, SCCmecVIII, and SCC-CI in Staphylococcus epidermidis. The array showed that the majority of isolates harbored one or more superantigen (94%; 100/107) and immune evasion cluster (91%; 97/107) genes. Apart from fusidic acid and trimethoprim resistance, the correlation between isolate antimicrobial resistance phenotype and the presence of specific resistance genes was ≥97%. Array profiling allowed high-throughput, accurate assignment of MRSA to CCs/STs and SCCmectypes and provided further evidence of the diversity of SCCmec/SCC. In most cases, array profiling can accurately predict the resistance phenotype of an isolate.

ABSTRACTMembrane-based spoligotyping has been converted to DNA microarray format to qualify it for high-throughput testing. We have shown the assay's validity and suitability for direct typing from tissue and detecting new spoligotypes. Advantages of the microarray methodology include rapidity, ease of operation, automatic data processing, and affordability.

ABSTRACTMethicillin resistance in staphylococci is mediated by penicillin binding protein 2a (PBP 2a), encoded by mecAon mobile staphylococcal cassette chromosome mec(SCCmec) elements. In this study, two clonal complex 130 (CC130) methicillin-resistant Staphylococcus aureus(MRSA) isolates from patients in Irish hospitals were identified that were phenotypically PBP 2a positive but lacked mecAby conventional PCR and by DNA microarray screening. The isolates were identified as methicillin-susceptible S. aureususing the GeneXpert real-time PCR assay. Whole-genome sequencing of one isolate (M10/0061) revealed a 30-kb SCCmecelement encoding a class E meccomplex with highly divergent blaZ-mecA-mecR1-mecI, a type 8 cassette chromosome recombinase (ccr) complex consisting of ccrA1-ccrB3, an arsenic resistance operon, and flanking direct repeats (DRs). The SCCmecelement was almost identical to that of SCCmectype XI (SCCmecXI) identified by the Sanger Institute in sequence type 425 bovine MRSA strain LGA251 listed on the website of the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements. The open reading frames (ORFs) identified within SCCmecXI of M10/0061 exhibited 21 to 93% amino acid identity to ORFs in GenBank. A third DR was identified ca. 3 kb downstream of SCCmecXI, indicating the presence of a possible SCC remnant. SCCmecXI was also identified in the second CC130 MRSA isolate by PCR and sequencing. The CC130 MRSA isolates may be of animal origin as previously reported CC130 S. aureusstrains were predominantly from bovine sources. The highly divergent nature of SCCmecXI relative to other SCCmecelements indicates that it may have originated in another taxon.

ABSTRACTThe arginine catabolic mobile element (ACME) is prevalent among methicillin-resistant Staphylococcus aureus(MRSA) isolates of sequence type 8 (ST8) and staphylococcal chromosomal cassette mec(SCCmec) type IVa (USA300) (ST8-MRSA-IVa isolates), and evidence suggests that ACME enhances the ability of ST8-MRSA-IVa to grow and survive on its host. ACME has been identified in a small number of isolates belonging to other MRSA clones but is widespread among coagulase-negative staphylococci (CoNS). This study reports the first description of ACME in two distinct strains of the pandemic ST22-MRSA-IV clone. A total of 238 MRSA isolates recovered in Ireland between 1971 and 2008 were investigated for ACME using a DNA microarray. Twenty-three isolates (9.7%) were ACME positive, and all were either MRSA genotype ST8-MRSA-IVa (7/23, 30%) or MRSA genotype ST22-MRSA-IV (16/23, 70%). Whole-genome sequencing and comprehensive molecular characterization revealed the presence of a novel 46-kb ACME and staphylococcal chromosomal cassette mec(SCCmec) composite island (ACME/SCCmec-CI) in ST22-MRSA-IVh isolates (n= 15). This ACME/SCCmec-CI consists of a 12-kb DNA region previously identified in ACME type II in S. epidermidisATCC 12228, a truncated copy of the J1 region of SCCmectype I, and a complete SCCmectype IVh element. The composite island has a novel genetic organization, with ACME located within orfXand SCCmeclocated downstream of ACME. One PVL locus-positive ST22-MRSA-IVa isolate carried ACME located downstream of SCCmectype IVa, as previously described in ST8-MRSA-IVa. These results suggest that ACME has been acquired by ST22-MRSA-IV on two independent occasions. At least one of these instances may have involved horizontal transfer and recombination events between MRSA and CoNS. The presence of ACME may enhance dissemination of ST22-MRSA-IV, an already successful MRSA clone.

ABSTRACTThe staphylococcal cfrgene mediates resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A, a phenotype that has been termed PhLOPSA. The cfrgene has mainly been associated with coagulase-negative staphylococcal isolates from animals, and only a few cfr-positive methicillin-resistant Staphylococcus aureus(MRSA) isolates have been described so far. This study reports the first description of a cfr-positive MRSA isolate (M05/0060) belonging to the pandemic Panton-Valentine leukocidin (PVL)-positive sequence type 8 MRSA IVa/USA300 (ST8-MRSA-IVa/USA300) clone. The cfrgene was detected in M05/0060 using a DNA microarray which was used to screen PVL-positive MRSA isolates for the presence of virulence genes, typing markers, and antimicrobial resistance genes. Antimicrobial susceptibility testing revealed that M05/0060 exhibited the cfr-associated resistance phenotype. Molecular analysis identified the presence of cfrand a second phenicol resistance gene, fexA, on a novel 45-kb conjugative plasmid, which was designated pSCFS7. Within pSCFS7, a DNA segment consisting of cfr, a truncated copy of insertion sequence IS21-558, and a region with homology to the DNA invertase gene bin3of transposon Tn552from Bacillus mycoideswas integrated into the transposase gene tnpBof the fexA-carrying transposon Tn558. The emergence of a multidrug-resistant cfr-positive variant of ST8-MRSA-IVa/USA300 is alarming and requires ongoing surveillance. Moreover, the identification of a novel conjugative plasmid carrying the cfrgene indicates the ability of cfrto spread to other MRSA strains.

ABSTRACTClonal complex 59 (CC59) community-associated methicillin-resistant Staphylococcus aureus(CA-MRSA) strains were characterized using pulsed-field gel electrophoresis, spatyping, multilocus sequence typing, diagnostic DNA microarrays, and PCRs targeting staphylococcal cassette chromosome mec(SCCmec) elements and Panton-Valentine leukocidin (PVL). Six distinct groups within CC59 were characterized. At least seven different variants of SCCmecelements were identified (IVa [2B], IVb [2B], IVd [2B], IV variant [2B], IVa [2B&5], V variant [5C2], and V [5C2&5]). (The structural type is indicated by a Roman numeral, with a lowercase letter indicating the subtype, and the ccrcomplex and the meccomplex are indicated by an Arabic numeral and an uppercase letter, respectively. Where there is an extra ccrelement, this is indicated by “&” and an Arabic numeral designating the ccrtype.) The first group is similar to the American sequence type 59 (ST59) MRSA-IV CA-MRSA strain USA1000. The second group includes a PVL-negative ST87 strain with an SCCmecelement of subtype IVb (2B). The third group comprises PVL-variable ST59 MRSA-IV strains harboring multiple SCCmecIV subtypes. PVL-negative ST59 MRSA strains with multiple or composite SCCmecelements (IVa [2B&5]) form the fourth group. Group 5 corresponds to the internationally known “Taiwan clone,” a PVL-positive strain with a variant SCCmecelement (V [5C2&5]). This strain proved to be the most common CC59 MRSA strain isolated in Western Australia. Finally, group 6 encompasses the ST59 MRSA-V variant (5C2). The differentiation of CC59 into groups and strains indicates a rapid evolution and spread of SCCmecelements. Observed differences between groups of strains as well as intrastrain variability within a group facilitate the tracing of their spread.

Despite the increasing importance of methicillin-resistant Staphylococcus aureus(MRSA) in veterinary medicine, knowledge about the epidemiology of the pathogen in horses is still poor. The phylogenetic relationship of strains of human and equine origins has been addressed before, usually by analyzing results of common standard classification methods for MRSA. This work intends to go beyond the baseline of typing procedures in order to comparatively characterize equine and human MRSA strains with similar phylogenetic backgrounds. In addition to multilocus sequence typing, pulsed-field gel electrophoresis, spatyping, staphylococcal cassette chromosome mectyping, and a PCR for Panton-Valentine leukocidin gene detection, a microarray analysis of a total of 185 structural, virulence-associated, and resistance loci was applied. The results showed that clonal complex 8 (CC8) was absolutely predominant (16 strains) in 19 investigated equine MSRA strains. Of the CC8 strains, 13 belonged to sequence type 254 (ST254) and the other 3 to ST8. This genotype has been isolated from different equine patients in various regions over several years, substantiating the apparent predominance of CC8 STs in MRSA strains of horses worldwide. Furthermore, comparatively investigated human strains of ST254 displayed molecular-typing results indistinguishable from those for strains of equine origin. Two further equine strains (ST22 and ST1117) showed similarity to ST22 human strains (CC22). One equine strain belonged to ST398, a genotype recently described as being frequently isolated from specimens from pigs and pig farmers. These data provide evidence for the adaptation of certain MRSA genotypes to more than one mammalian species, reflecting their extended host spectra.

ABSTRACTA West Australian methicillin-resistant Staphylococcus aureusstrain (WA MRSA-59) was characterized by microarray and sequencing. Its pseudo-staphylococcal cassette chromosome mec(SCCmec) element comprised dcs, Q9XB68-dcs, mvaS-SCC, Q5HJW6, dru, ugpQ, ydeM, mecA-mecR-mecI, txbi mecI, tnpIS431, copA2-mco(copper resistance), ydhK, arsC-arsB-arsR(arsenic resistance), open reading frame PT43, and per-2. Recombinase genes, xylR(mecR2), and PSM-mec(phenol-soluble modulin) were absent. We suggest that meccomplex A should be split into two subtypes. One harbors PSM-mecand xylR(mecR2). It is found in SCCmectypes II, III, and VIII. The second subtype, described herein, is present in WA MRSA-59 and some coagulase-negative staphylococci.

ABSTRACTStaphylococcus aureusbacteremia is common in both nosocomial and community settings, and the pathogenicity of the microbe depends upon a large repertoire of virulence factors. S. aureusbacteremia isolates (n= 126) were characterized using DNA microarrays. Clonal complexes 5, 8, 15, 30, and 45 accounted for 74.6% of the isolates. We identified geographical differences in dominating clones and toxin gene profiles. One isolate was methicillin resistant. Potential associations between age and genotype were detected.

ABSTRACTOxacillin-susceptible, mecA-positive Staphylococcus aureusisolates create a treatment challenge for the clinician. In this article, we describe two cases of bacteremia from isolates that carried the mecAgene but were susceptible to oxacillin (oxacillin-susceptible methicillin-resistant S. aureus[OS-MRSA]). DNA microarray analysis was used to characterize these isolates as a mecA-positive, clonal complex 5, pediatric strain and a mecA-positive, clonal complex 8, USA300 strain.

The Bengal Bay clone (ST772) is a community-associated and multidrug-resistant Staphylococcus aureuslineage first isolated from Bangladesh and India in 2004. In this study, we showed that the Bengal Bay clone emerged from a virulent progenitor circulating on the Indian subcontinent. Its subsequent global transmission was associated with travel or family contact in the region. ST772 progressively acquired specific resistance elements at limited cost to its fitness and continues to be exported globally, resulting in small-scale community and health care outbreaks. The Bengal Bay clone therefore combines the virulence potential and epidemiology of community-associated clones with the multidrug resistance of health care-associated S. aureuslineages. This study demonstrates the importance of whole-genome sequencing for the surveillance of highly antibiotic-resistant pathogens, which may emerge in the community setting of regions with poor antibiotic stewardship and rapidly spread into hospitals and communities across the world.