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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 SCC mec 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. [ABSTRACT FROM AUTHOR]

A diagnostic microarray was used to characterise a collection of methicillin-resistant Staphylococcus aureus (MRSA) isolates from hospitals in the German region of Eastern Saxony. The most abundant epidemic MRSA (EMRSA) strains were ST5-MRSA  II (Rhine–Hesse EMRSA, EMRSA-3), CC5/ST228-MRSA  I (South German EMRSA), ST22-MRSA  IV (Barnim EMRSA, EMRSA-15) and ST45-MRSA  IV (Berlin EMRSA). Other strains were found only as sporadic isolates or in minor outbreaks. These strains included ST1-MRSA  IV, ST8-MRSA  IV (Hannover EMRSA and others), clonal group 5 strains carrying SCC mec type IV elements (Paediatric clone), ST45-MRSA  V, CC8/ST239-MRSA  III and ST398-MRSA  V. Panton–Valentine leukocidin-positive MRSA isolates were still very rare. The predominant strain was ST80-MRSA  IV, although increasing numbers of different strains have recently been detected (ST8-MRSA  IV, ST30-MRSA  IV and ST59-MRSA  V). For more common MRSA strains, it was possible to detect variants that differed mainly in the carriage of additional resistance determinants and certain virulence-associated genes. Detection of such variants can sometimes allow epidemic strains to be resolved beyond spa types to a hospital-specific level, which is of significant value for epidemiological purposes. [ABSTRACT FROM AUTHOR]

We investigated why a clinical meticillin-resistant Staphylococcus aureus (MRSA) isolate yielded false-negative results with some commercial PCR tests for MRSA detection. We found that an epidemic European CC1-MRSA-IV clone generally exhibits this behaviour. The failure of the assays was attributable to a large insertion in the orfX /SCC mec integration site. To ensure the reliability of molecular MRSA tests, it is vital to monitor emergence of new SCC mec types and junction sites.

Background: Serological screening of pig herds at the abattoir is considered a potential tool to improve meat inspection procedures and herd health management. Therefore, we previously reported the feasibility of a miniaturised protein microarray as a new serological IgG screening test for zoonotic agents and production diseases in pigs. The present study investigates whether the protein microarray-based assay is applicable for high sample throughput using either blood serum or meat juice., Material and Methods: Microarrays with 12 different antigens were produced by Abbott (formerly Alere Technologies GmbH) Jena, Germany in a previously offered 'ArrayTube' platform and in an 'ArrayStrip' platform for large-scale use. A test protocol for the use of meat juice on both microarray platforms was developed. Agreement between serum and meat juice was analysed with 88 paired samples from three German abattoirs. Serum was diluted 1:50 and meat juice 1:2. ELISA results for all tested antigens from a preceding study were used as reference test to perform Receiver Operating Characteristic analysis for both test specimens on both microarray platforms., Results: High area under curve values (AUC > 0.7) were calculated for the analysis of T. gondii (0.87), Y. enterocolitica (0.97), Mycoplasma hyopneumoniae (0.84) and Actinobacillus pleuropneumoniae (0.71) with serum as the test specimen and for T. gondii (0.99), Y. enterocolitica (0.94), PRRSV (0.88), A. pleuropneumoniae (0.78) and Salmonella spp. (0.72) with meat juice as the test specimen on the ArrayStrip platform. Cohens kappa values of 0.92 for T. gondii and 0.82 for Y. enterocolitica were obtained for the comparison between serum and meat juice. When applying the new method in two further laboratories, kappa values between 0.63 and 0.94 were achieved between the laboratories for these two pathogens., Conclusion: Further development of a miniaturised pig-specific IgG protein microarray assay showed that meat juice can be used on microarray platforms. Two out of twelve tested antigens (T. gondii, Y. enterocolitica) showed high test accuracy on the ArrayTube and the ArrayStrip platform with both sample materials.

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. Spa types, dru types, SCCmec types, 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 spa type t011, the other half was of spa types t1430 and t034. On 3 farms, more than 1 spa type was found. The most common dru type 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), MLSB antibiotics (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 egc cluster 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/SCCmec IV/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., (© 2019 Poultry Science Association Inc.)

This study investigated the recent emergence of multidrug-resistant Panton-Valentine leukocidin (PVL)-negative CC1-MRSA-IV in Ireland and Germany. Ten CC1-MSSA and 139 CC1-MRSA isolates recovered in Ireland between 2004 and 2017 were investigated. These were compared to 21 German CC1-MRSA, 10 Romanian CC1-MSSA, five Romanian CC1-MRSA and two UAE CC1-MRSA, which were selected from an extensive global database, based on similar DNA microarray profiles to the Irish isolates. All isolates subsequently underwent whole-genome sequencing, core-genome single nucleotide polymorphism (cgSNP) analysis and enhanced SCCmec subtyping. Two PVL-negative clades (A and B1) were identified among four main clades. Clade A included 20 German isolates, 119 Irish isolates, and all Romanian MRSA and MSSA isolates, the latter of which differed from clade A MRSA by 47-130 cgSNPs. Eighty-six Irish clade A isolates formed a tight subclade (A1) exhibiting 0-49 pairwise cgSNPs, 80 of which harboured a 46 kb conjugative plasmid carrying both ileS2, encoding high-level mupirocin resistance, and qacA, encoding chlorhexidine resistance. The resistance genes aadE, aphA3 and sat were detected in all clade A MRSA and the majority (8/10) of clade A MSSA isolates. None of the clade A isolates harboured any enterotoxin genes other than seh, which is universally present in CC1. Clade B1 included the remaining German isolate, 17 Irish isolates and the two UAE isolates, all of which corresponded to the Western Australia MRSA-1 (WA MRSA-1) clone based on genotypic characteristics. MRSA within clades A and B1 differed by 188 cgSNPs and clade-specific SCCmec characteristics were identified, indicating independent acquisition of the SCCmec element. This study demonstrated the existence of a European PVL-negative CC1-MRSA-IV clone that is distinctly different from the well-defined PVL-negative CC1-MRSA-IV clone, WA MRSA-1. Furthermore, cgSNP analysis revealed that this newly defined clone may have originated in South-Eastern Europe, before spreading to both Ireland and Germany., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Clostridium difficile can cause antibiotic-associated diarrhea and a possibility of outbreaks in hospital settings warrants molecular typing. A microarray was designed that included toxin genes (tcdA/B, cdtA/B), genes related to antimicrobial resistance, the slpA gene and additional variable genes., Results: DNA of six reference strains and 234 clinical isolates from South-Western and Eastern Germany was subjected to linear amplification and labeling with dUTP-linked biotin. Amplicons were hybridized to microarrays providing information on the presence of target genes and on their alleles. Tested isolates were assigned to 37 distinct profiles that clustered mainly according to MLST-defined clades. Three additional profiles were predicted from published genome sequences, although they were not found experimentally., Conclusions: The microarray based assay allows rapid and high-throughput genotyping of clinical C. difficile isolates including toxin gene detection and strain assignment. Overall hybridization profiles correlated with MLST-derived clades.

In 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 SCCmec XI 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.

Staphylococcus aureus is one of the most important causal agents of bovine mastitis. Population studies on bovine Staphylococcus aureus isolates have identified considerable genetic heterogeneity among strains causing mastitis. The aim of this study was to investigate the contribution of different clonal complexes and the occurrence of virulence factors and resistance determinants within bovine Staphylococcus aureus isolates.A total of 189 Staphylococcus aureus isolates obtained from milk samples of 34 dairy herds in the German Federal State of Thuringia were characterised by microarray technology.The isolates were assigned to eleven different clonal complexes with CC151, CC479 and CC133 being dominant (together 80.5%). The methicillin resistance gene mecA was found in four isolates (2.1%), which belonged to CC398. Enterotoxin genes could be detected in 79.3% of analysed Staphylococcus aureus and 19 isolates (10.1%) harboured a distinct allele of the toxic shock syndrome toxin gene, tst-RF122. The most striking finding of the present study was that almost all except one isolate (151/152) belonging to CC151, CC479 and CC133 harboured the leukocidin genes lukF-P83 and lukM, whereas no further isolates from other lineages possessed these genes.The consistent occurrence of lukF-P83/lukM in the dominating clonal complexes suggests an essential role of this leukocidin in the etiology of bovine mastitis.

Worldwide, methicillin-resistant Staphylococcus aureus (MRSA) pose an increased risk for healthcare- and community-associated infections. Since the first report of MRSA in England in 1961, several distinct clones or strains have emerged. Changes within the MRSA population of whole countries, small regions or of single hospitals have been observed with some clones replacing others. In this study, the clonal replacement of MRSA isolates in a South-eastern German tertiary care hospital between 2000 and 2010 is described based on microarray analyses of 778 isolates and at least 50 MRSA per year. Within these eleven years, four common epidemic strains, CC22-MRSA-IV, CC45-MRSA-IV, CC5/ST228-MRSA-I (including a variant with a truncated SCCmec element) and CC5-MRSA-II were identified. The PVL-negative CC22-MRSA-IV strain (Barnim Epidemic Strain, UK-EMRSA-15) was detected for the first time in 2001 and its abundance increased since then to 58.6% in 2010. CC5-MRSA-II increased from 2% (2000) to about 30% (2003), and since then it fluctuates between 23 and 37% of isolates. CC5/ST228-MRSA-I decreased from about the half of tested isolates (2000) to 2.3% (2010). A similar trend was observed for CC45-MRSA-IV, which decreased drastically down to 3.4% in 2010 after reaching a maximum of 62.0% in 2002. Seventeen other PVL-negative MRSA strains were identified sporadically with no significant trend being observed. Seven PVL-positive MRSA strains were found, but they remained rare during the study period accounting together for 2.7% of isolates.

Objectives: Twenty-five MRSA ST398 isolates from cases of bovine clinical mastitis and two isolates from farm personnel collected from 17 dairy farms in Germany were investigated for genetic relatedness, antimicrobial resistance and virulence properties., Methods: Genomic relationships were determined by ApaI PFGE, spa typing, SCCmec typing and dru typing. Antimicrobial resistance phenotypes were determined by broth microdilution. Resistance and virulence genes were detected via a diagnostic DNA microarray and specific PCRs., Results: Nine major ApaI PFGE patterns were detected. Three spa types (t011, t034 and t2576) and two SCCmec types (IV and V) were identified. Five different dru types were seen with dt11a being predominant. All isolates were negative for Panton-Valentine leucocidin, enterotoxin and exfoliative toxin genes. Ten resistance patterns were observed with 11 (40.7%) isolates being resistant to only beta-lactam antibiotics and tetracyclines. Several resistance genes were detected: blaZ (penicillin resistance); tet(M), tet(K) and tet(L) (tetracycline resistance); erm(A), erm(B), erm(C) and erm(T) (macrolide/lincosamide/streptogramin B resistance); aacA-aphD, aphA3, aadD and spc (aminoglycoside or aminocyclitol resistance); fexA (phenicol resistance); dfrK (trimethoprim resistance); and vga(A) and vga(C) (pleuromutilin/lincosamide/streptogramin A resistance). The two human isolates were indistinguishable in their genotypic and phenotypic characteristics from the mastitis isolates of the same farm., Conclusions: As previously described for ST398 from swine, isolates of this sequence type from cases of bovine mastitis also demonstrated a high degree of variability when ApaI PFGE profiles and other genotypic and phenotypic characteristics were compared. A uniform virulence gene pattern appeared to be conserved between ST398 isolates from both animal species.

Objectives: Fifty-four methicillin-resistant Staphylococcus aureus (MRSA) ST398 isolates from unrelated diseased swine collected all over Germany were comparatively investigated for their antimicrobial resistance and virulence properties, and for their genomic relatedness., Methods: MICs of 30 antimicrobial agents were determined by broth microdilution. Resistance and virulence genes were detected via a diagnostic DNA microarray and specific PCRs. The genomic relationships were determined by ApaI-PFGE, spa typing and SCCmec typing., Results: Twenty-two distinct resistance patterns were observed. All 54 isolates were tetracycline resistant, mediated by tet(M), tet(K) and/or tet(L), with 14 isolates being only resistant to beta-lactam antibiotics and tetracyclines. Trimethoprim resistance, seen in 28 isolates, was mostly due to the gene dfrK or dfrG. Among the 24 macrolide/lincosamide-resistant isolates, the genes erm(A), erm(B) and/or erm(C) were detected. The two chloramphenicol/florfenicol-resistant isolates harboured the gene fexA. The eight gentamicin-resistant isolates carried the gene aacA/aphD. Fifty-three isolates harboured SCCmec type V elements while the remaining one carried mecA and ugpQ, but no recombinase genes. All isolates were PVL negative, but one and three isolates, respectively, were positive for the enterotoxin B and enterotoxin K and Q genes. Eight different spa types were identified with t011 being the most predominant. Six ApaI-PFGE clusters with up to nine individual patterns were detected., Conclusions: MRSA ST398 isolates varied slightly in their virulence properties and spa types but differed distinctly in their antimicrobial resistance pheno- and genotypes as well as their ApaI-PFGE patterns. These data underline the ability of ST398 to acquire genetic material that might increase antimicrobial resistance and virulence.

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.

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.

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.