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From 2009 to 2011 [transmission period (TP) 1] and 2014 to 2017 (TP2), two outbreaks involving community-associated clonal complex (CC) 88-MRSA spa types t186 and t786, respectively, occurred in the Neonatal Intensive Care Unit (NICU) of an Irish hospital (H1). This study investigated the relatedness of these isolates, their relationship to other CC88 MRSA from Ireland and their likely geographic origin, using whole-genome sequencing (WGS). All 28 CC88-MRSA isolates identified at the Irish National MRSA Reference Laboratory between 2009 and 2017 were investigated including 20 H1 patient isolates, two H1 isolates recovered from a single healthcare worker (HCW) 2 years apart, three patient isolates from a second hospital (H2) and one patient isolate from each of three different hospitals (H3, H4, and H5). All isolates underwent DNA microarray profiling. Thirteen international isolates with similar microarray profiles to at least one Irish isolate were selected from an extensive global database. All isolates underwent Illumina MiSeq WGS. The majority of Irish isolates (25/28; all H1 isolates, two H2 isolates and the H3 isolate) were identified as ST78-MRSA-IVa and formed a large cluster, exhibiting 1-71 pairwise allelic differences, in a whole-genome MLST-based minimum spanning tree (MST) involving all Irish isolates. A H1/H2, H1/H3, and H1 HCW/patient isolate pair each exhibited one allelic difference. The TP2 isolates were characterised by a different spa type and the loss of hsdS . The three remaining Irish isolates (from H2, H4, and H5) were identified as ST88-MRSA-IVa and dispersed at the opposite end of the MST, exhibiting 81-211 pairwise allelic differences. Core-genome MLST and sequence-based plasmid analysis revealed the recent shared ancestry of Irish and Australian ST78-MRSA-IVa, and of Irish and French/Egyptian ST88-MRSA-IVa. This study revealed the homogeneity of isolates recovered during two NICU outbreaks (despite spa type and hsdS carriage variances), HCW involvement in the outbreak transmission chain and the strain's spread to two other Irish hospitals. The outbreak strain, CC88/ST78-MRSA-IVa, was likely imported from Australia, where it is prevalent. CC88/ST88-MRSA-IVa was also identified in Irish hospitals and was likely imported from Africa, where it is predominant, and/or a country with a large population of African descent.

Introduction: Healthcare workers (HCWs) colonized with Staphylococcus aureus may serve as a reservoir of infection. This study was carried to determine the genetic make-up of S. aureus nasal colonizers in HCWs., Methodology: Nasal swabs were obtained from 93 HCWs and molecular characterization of identified S. aureus isolates was carried out using the StaphyType DNA microarray (Alere Technologies GmbH, Jena, Germany)., Results: Twenty-nine HCWs (31%) were colonized with S. aureus (MSSA = 23; MRSA = 6). Thus the overall MRSA carriage rate was 6.5% (n/N = 6/93) and 20.7% (n/N = 6/29) of those colonized with S. aureus harboured MRSA. The S. aureus isolates belonged to 16 clonal complexes (CC). MSSA isolates included three each for CC15, CC188, ST2867; two each for CC5, CC97, CC367 as well as one each for CC1, CC8, CC30, CC45, CC101, CC121, ST291/813 and CC1153. The staphylococcal cassette chromosome recombinase genes ccrA-1; ccrB-1 and the fusidic acid resistance gene (fusC) were present in two MSSA isolates (CC1 and CC8). The six MRSA isolates included CC5-MRSA-[VI+fusC] (n = 2); one each of CC5-MRSA-V; CC22-MRSA-IV (tst1+); CC80-MRSA-IV [pvl+] ("European CA-MRSA Clone") and CC97-MRSA-[V+fusC]., Conclusion: There is wide clonal diversity of S. aureus colonizers with associated high MRSA carriage among the HCWs. The presence of genetically stable MSSA isolates with the capability to transform into MRSA isolates is of concern., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2018 Abiola Catherine Senok, Ali Somily, Muhabat Raji, Ghada Garaween, Maha Kabil, Atef Shibl, Stefan Monecke, Ralf Ehricht.)

Shiga toxin-producing Escherichia coli (STEC) cause bloody diarrhea (BD), hemorrhagic colitis (HC), and even hemolytic uremic syndrome (HUS). In Nordic countries, STEC are widely spread and usually associated with gastrointestinal symptoms and HUS. The objective of this study was to investigate the occurrence of STEC in Swedish patients over 10 years of age from 2003 through 2015, and to analyze the correlation of critical STEC virulence factors with clinical symptoms and duration of stx shedding. Diarrheal stool samples were screened for presence of stx by real-time PCR. All STEC isolates were characterized by DNA microarray assay and PCR to determine serogenotypes, stx subtypes, and presence of intimin gene eae and enterohaemolysin gene ehxA . Multilocus sequencing typing (MLST) was used to assess phylogenetic relationships. Clinical features were collected and analyzed using data from the routine infection control measures in the county. A total of 14,550 samples were enrolled in this 12-years period study, and 175 (1.2%) stools were stx positive by real-time PCR. The overall incidence of STEC infection was 4.9 cases per 100,000 person-years during the project period. Seventy-five isolates, with one isolate per sample were recovered, among which 43 were from non-bloody stools, 32 from BD, and 3 out of the 75 STEC positive patients developed HUS. The presence of stx2 in both stools and isolates were associated with BD ( p = 0.008, p = 0.05), and the presence of eae in isolates was related to BD ( p = 0.008). The predominant serogenotypes associated with BD were O157:H7, O26:H11, O121:H19, and O103:H2. Isolates from HUS were O104:H4 and O98: H21 serotypes. Phylogenetic analysis revealed our strains were highly diverse, and showed close relatedness to HUS-associated STEC collection strains. In conclusion, the presence of stx2 in stool was related to BD already at the initial diagnostic procedure, thus could be used as risk predictor at an early stage. STEC isolates with stx2 and eae were significantly associated with BD. The predominant serotypes associated with BD were O157:H7, O26:H11, O121:H19, and O103:H2. Nevertheless, the pathogenic potential of other serotypes and genotypes should not be neglected.

Antimicrobial resistance of Staphylococcus aureus is a major problem in human and veterinary medicine. The aim of this study was to characterise S. aureus isolates from wild and zoo animals mainly associated with bacterial infections. In total, 23 S. aureus isolates, including nine from wild animals and 14 from zoo animals, were obtained during routine diagnostics. All isolates were subjected to multilocus sequence typing (MLST), spa typing, macrorestriction analysis with subsequent SmaI pulsed-field gelelectrophoresis (PFGE), antimicrobial susceptibility testing and S. aureus-specific DNA-microarray analysis. Resistant isolates were also tested for their respective resistance genes by PCR. Isolates from zoo animals and wildlife showed a high diversity of MLST types, spa types and PFGE patterns. Nineteen different spa types were identified, including three novel types and 16 main macrorestriction patterns. Only few isolates were resistant to members of four classes of antimicrobial agents and harboured the respective resistance genes (β-lactams [blaZ, mecA, mecC], tetracyclines [tet(K), tet(L)] and chloramphenicol [cat pC221 ]) or mutations (fluoroquinolones). The DNA microarray analysis identified one isolate from a zoo animal harbouring the toxic shock syndrome toxin gene tst1. Moreover, several enterotoxin genes were detected in five S. aureus isolates. All isolates were negative for Panton-Valentine leukocidin (PVL) genes, but the animal-associated leukocidin genes lukM/lukF-P83 were found in three isolates from two animals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Pakistan is known to be high, but very few studies have described the molecular epidemiology of the different MRSA clones circulating in the country. Forty-four MRSA isolates were collected from two tertiary care hospitals of the Rawalpindi district of Pakistan. All strains were identified by a conventional phenotypic method and then subjected to genotyping by microarray hybridisation. Six clonal complexes (CCs) and 19 strains were identified. The most commonly identified strains were: (i) Panton-Valentine leucocidin (PVL)-positive CC772-MRSA-V, "Bengal Bay Clone" (ten isolates; 22.3%), (ii) ST239-MRSA [III + ccrC] (five isolates) and (iii) a CC8-MRSA-IV strain, as well as CC6-MRSA-IV (both with four isolates; 9.1% each). Several of the strains detected indicated epidemiological links to the Middle Eastern/Arabian Gulf region. Further studies are needed to type MRSA from countries with less known epidemiology and to monitor the distribution and spread of strains, as well as possible links to global travel, migration and commerce.

The most common livestock-associated lineage of methicillin-resistant Staphylococcus aureus (MRSA) in Western Europe is currently clonal complex (CC) 398. CC398-MRSA spread extensively across livestock populations in several Western European countries, and livestock-derived CC398-MRSA strains can also be detected in humans. Based on their SCCmec elements, different CC398 strains can be distinguished. SCCmec elements of 100 veterinary and human CC398-MRSA isolates from Germany and Austria were examined using DNA microarray-based assays. In addition, 589 published SCC and/or genome sequences of CC398-MRSA (including both, fully finished and partially assembled sequences) were analysed by mapping them to the probe sequences of the microarrays. Several isolates and sequences showed an insertion of a large fragment of CC9 genomic DNA into the CC398 chromosome. Fifteen subtypes of SCCmec elements were detected among the 100 CC398 isolates and 41 subtypes could be discerned among the published CC398 sequences. Eleven of these were also experimentally detected within our strain collection, while four subtypes identified in the isolates where not found among the sequences. A high prevalence of heavy metal resistance genes, especially of czrC, was observed among CC398-MRSA. A possible co-selection of resistances to antibiotics and zinc/copper supplements in animal feed as well as a spill-over of SCCmec elements that have evolved in CC398-MRSA to other, possibly more virulent and/or medically relevant S. aureus lineages might pose public health problems in future., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Serological analysis of Chlamydia (C.) spp. infections is still mainly based on micro-immunofluorescence and ELISA. To overcome the limitations of conventional serology, we have designed a novel microarray carrying 52 synthetic peptides representing B-cell epitopes from immunodominant proteins of all 11 chlamydial species. The new assay has been validated using monospecific mouse hyperimmune sera. Subsequently, serum samples from cattle, sheep and humans with a known history of chlamydial infection were examined. For instance, the specific humoral response of sheep to treatment with a C. abortus vaccine has been visualized against a background of C. pecorum carriership. In samples from humans, dual infection with C. trachomatis and C. pneumoniae could be demonstrated. The experiments revealed that the peptide microarray assay was capable of simultaneously identifying specific antibodies to each Chlamydia spp. The actual assay represents an open platform test that can be complemented through future advances in Chlamydia proteome research. The concept of the highly parallel multi-antigen microarray proven in this study has the potential to enhance our understanding of antibody responses by defining not only a single quantitative response, but also the pattern of this response. The added value of using peptide antigens will consist in unprecedented serodiagnostic specificity.

Mupirocin is used for eradicating methicillin-resistant S. aureus (MRSA) in nasal colonization. A plasmid-borne gene, mupA , is associated with high-level mupirocin resistance. Despite the fact that, among all MRSA from a tertiary care center in the German state of Saxony, the prevalence of mupA , encoding high-level mupirocin resistance, was approximately 1% over a 15-year period from 2000-2015, a sharp increase to nearly 20% was observed in 2016/2017. DNA microarray profiling revealed that this was due to the dissemination of a variant of CC22-MRSA-IV ("Barnim Epidemic Strain" or "UK-EMRSA-15"), which, in addition to mecA , harbors mupA , aacA-aphD , qacA , and - in most isolates - erm (C). In order to prevent therapy failures and a further spread of this strain, the use of mupirocin should be more stringently controlled as well as guided by susceptibility testing. In addition, MRSA decolonization regimens that rely on other substances, such as betaisodona, polyhexanide or octenidine, should be considered., Competing Interests: The authors declare that they have no competing interests.

Objective: ATCC25923 is a Staphylococcus aureus strain that is positive for the Panton Valentin leukocidin. It has been used for decades as reference strain. We observed that two separately maintained clones of ATCC25923 ("G477 and G478") differed grossly in the expression of this toxin. For that reason, both clones were sequenced using an Illumina MiSeq instrument. After assembling, the final sequences were analyzed and mapped to a previously published ATCC25923 sequence (GenBank CP009361) using bl2seq from the NCBI Blast2 package., Results: The genomes of G477 and G478 size 2778,859 and 2792,213 nucleotides, respectively. Both genomes include a circular plasmid of 27,490 nucleotides. The sequence of the G477 chromosome maps nearly exactly to CP009361. G478 has a slightly larger size because of the presence of an additional transposable element tnp13k. The second copy of that tnp13k element is located in an intergenic region between the genes mazF and rsbU. The sequences of the ATCC25923 clones G477 and G478 differ mainly in the insertion of a second tnp13k element between the genes mazF and rsbU. That insertion may lead to a different transcription of that genome region resulting in upregulation of the expression of the Panton-Valentine leukocidin in the ATCC25923 clone G478.

A novel multiplex real-time PCR was designed to detect the clinically most important carbapenemase genes, blaKPC, blaVIM, blaNDM and blaOXA-48. The multiplex assay was verified testing genomic DNA of 24 carbapenemase-producing strains. It was validated using a blinded panel of 82 carbapenemase-producing and 50 non-producing isolates by direct colony PCR., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Staphylococcus aureus ( S. aureus ) is a major agent of dairy cow intramammary infections: the different prevalences of mastitis reported might be related to a combination of S. aureus virulence factors beyond host factors. The present study considered 169 isolates from different Italian dairy herds that were classified into four groups based on the prevalence of S. aureus infection at the first testing: low prevalence (LP), medium-low (MLP), medium-high (MHP) and high (HP). We aimed to correlate the presence of virulence genes with the prevalence of intramammary infections in order to develop new strategies for the control of S. aureus mastitis. Microarray data were statistically evaluated using binary logistic regression and correspondence analysis to screen the risk factors and the relationship between prevalence group and gene. The analysis showed: (1) 24 genes at significant risk of being detected in all the herds with infection prevalence >5%, including genes belonging to microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), immune evasion and serine proteases; and (2) a significant correlation coefficient between the genes interacting with the host immune response and HP isolates against LP ones. These results support the hypothesis that virulence factors, in addition to cow management, could be related to strain contagiousness, offering new insights into vaccine development., Competing Interests: The authors declare no conflict of interest.

Between 1 August and 6 September 2013, an outbreak of Legionnaires' disease (LD) with 78 cases confirmed by positive urinary antigen tests occurred in Warstein, North Rhine-Westphalia, Germany. Legionella (L.) pneumophila, serogroup (Sg) 1, monoclonal antibody (mAb) subgroup Knoxville, sequence type (ST) 345, was identified as the epidemic strain. This strain was isolated from seven patients. To detect the source of the infection, epidemiological typing of clinical and environmental strains was performed in two consecutive steps. First, strains were typed by monoclonal antibodies. Indistinguishable strains were further subtyped by sequence-based typing (SBT) which is the internationally recognized standard method for epidemiological genotyping of L. pneumophila. In an early stage of the outbreak investigation, many environmental isolates were found to belong to the mAb subgroup Knoxville, but to two different STs, namely to ST 345, the epidemic strain, and to ST 600. A majority of environmental isolates belonged to ST 600 whereas the epidemic ST 345 strain was less common in environmental samples. To rapidly distinguish both Knoxville strains, we applied a novel typing method based on DNA-hybridization on glass chips. The new assay can easily and rapidly discriminate L. pneumophila Sg 1 strains. Thus, we were able to quickly identify the sources harboring the epidemic strain, i.e., two cooling towers of different companies, the waste water treatment plants (WWTP) of the city and one company as well as water samples of the river Wester and its branches., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Background: Shiga toxin (Stx) producing Escherichia coli ( E. coli ) (STEC) is the most frequent cause of diarrhoea-positive haemolytic uraemic syndrome (D + HUS) in humans. In 2011, a huge outbreak with an STEC O104:H4 strain in Germany highlighted the limited possibilities for causative treatment of this syndrome. The responsible STEC strain was found to combine Stx production with adherence mechanisms normally found in enteroaggregative E. coli (EAEC). Pathotypes of E. coli evolve and can exhibit different adhesion mechanisms. It has been shown previously that neonatal gnotobiotic piglets are susceptible for infection with STEC, such as STEC O157:H7 as well as for EAEC, which are considered to be the phylogenetic origin of E. coli O104:H4. This study was designed to characterise the host response to infection with the STEC O104:H4 outbreak strain in comparison to an STEC O157:H7 isolate by evaluating clinical parameters (scoring) and markers of organ dysfunction (biochemistry), as well as immunological (flow cytometry, assessment of cytokines/chemokines and acute phase proteins) and histological alterations (light- and electron microscopy) in a gnotobiotic piglet model of haemolytic uraemic syndrome., Results: We observed severe clinical symptoms, such as diarrhoea, dehydration and neurological disorders as well as attaching-and-effacing lesions (A/E) in the colon in STEC O157:H7 infected piglets. In contrast, STEC O104:H4 challenged animals exhibited only mild clinical symptoms including diarrhoea and dehydration and HUS-specific/severe histopathological, haematological and biochemical alterations were only inconsistently presented by individual piglets. A specific adherence phenotype of STEC O104:H4 could not be observed. Flow cytometric analyses of lymphocytes derived from infected animals revealed an increase of natural killer cells (NK cells) during the course of infection revealing a potential role of this subset in the anti-bacterial activity in STEC disease., Conclusions: Unexpectedly, E. coli O104:H4 infection caused only mild symptoms and minor changes in histology and blood parameters in piglets. Outcome of the infection trial does not reflect E.   coli O104:H4 associated human disease as observed during the outbreak in 2011. The potential role of cells of the innate immune system for STEC related disease pathogenesis should be further elucidated.

The secretion of antimicrobial compounds is an ancient mechanism with clear survival benefits for microbes competing with other microorganisms. Consequently, mechanisms that confer resistance are also ancient and may represent an underestimated reservoir in environmental bacteria. In this context, β-lactamases (BLs) are of great interest due to their long-term presence and diversification in the hospital environment, leading to the emergence of Gram-negative pathogens that are resistant to cephalosporins (extended spectrum BLs = ESBLs) and carbapenems (carbapenemases). In the current study, protein sequence databases were used to analyze BLs, and the results revealed a substantial number of unknown and functionally uncharacterized BLs in a multitude of environmental and pathogenic species. Together, these BLs represent an uncharacterized reservoir of potentially transferable resistance genes. Considering all available data, in silico approaches appear to more adequately reflect a given resistome than analyses of limited datasets. This approach leads to a more precise definition of BL clades and conserved motifs. Moreover, it may support the prediction of new resistance determinants and improve the tailored development of robust molecular diagnostics.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human health problem and recently, domestic animals are described as carriers and possible reservoirs. Twenty seven S. aureus isolates from five turkey farms ( n = 18) and two broiler farms ( n = 9) were obtained by culturing of choana and skin swabs from apparently healthy birds, identified by Taqman-based real-time duplex nuc - mec A-PCR and characterized by spa typing as well as by a DNA microarray based assay which covered, amongst others, a considerable number of antibiotic resistance genes, species controls, and virulence markers. The antimicrobial susceptibility profiles were tested by agar diffusion assays and genotypically confirmed by the microarray. Five different spa types (3 in turkeys and 2 in broilers) were detected. The majority of MRSA isolates (24/27) belonged to clonal complex 398-MRSA-V. The most frequently occurring spa types were accordingly t011, t034, and t899. A single CC5-MRSA-III isolated from turkey and CC398-MRSA with an unidentified/truncated SCC mec element in turkey and broiler were additionally detected. The phenotypic antimicrobial resistance profiles of S. aureus isolated from both turkeys and broilers against 14 different antimicrobials showed that all isolates were resistant to ampicillin, cefoxitin, oxacillin, doxycycline, and tetracycline. Moreover, all S. aureus isolated from broilers were resistant to erythromycin and azithromycin. All isolates were susceptible to gentamicin, chloramphenicol, sulphonamides, and fusidic acid. The resistance rate against ciprofloxacin was 55.6% in broiler isolates and 42.1% in turkey isolates. All tetracycline resistant isolates possessed genes tet K/M. All erythromycin-resistant broiler isolates carried ermA . Only one broiler isolate (11.1%) carried genes ermA, ermB , and ermC , while 55.6% of turkey isolates possessed erm A and erm B genes. Neither PVL genes ( luk F/S-PV), animal-associated leukocidin ( luk M and luk -P83) nor the gene encoding the toxic shock syndrome toxin ( tst 1) were found in turkey and broiler isolates. In conclusion, the detection of MRSA in healthy turkeys and broilers with even additional antibiotic resistance markers is of major public health concern. The difference in antibiotic resistance and virulence markers between MRSA isolates from turkeys and broilers was addressed.

Staphylococcus aureus is a well-known colonizer and cause of infection among animals and it has been described from numerous domestic and wild animal species. The aim of the present study was to investigate the molecular epidemiology of S. aureus in a convenience sample of European wildlife and to review what previously has been observed in the subject field. 124 S. aureus isolates were collected from wildlife in Germany, Austria and Sweden; they were characterized by DNA microarray hybridization and, for isolates with novel hybridization patterns, by multilocus sequence typing (MLST). The isolates were assigned to 29 clonal complexes and singleton sequence types (CC1, CC5, CC6, CC7, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC49, CC59, CC88, CC97, CC130, CC133, CC398, ST425, CC599, CC692, CC707, ST890, CC1956, ST2425, CC2671, ST2691, CC2767 and ST2963), some of which (ST2425, ST2691, ST2963) were not described previously. Resistance rates in wildlife strains were rather low and mecA-MRSA isolates were rare (n = 6). mecC-MRSA (n = 8) were identified from a fox, a fallow deer, hares and hedgehogs. The common cattle-associated lineages CC479 and CC705 were not detected in wildlife in the present study while, in contrast, a third common cattle lineage, CC97, was found to be common among cervids. No Staphylococcus argenteus or Staphylococcus schweitzeri-like isolates were found. Systematic studies are required to monitor the possible transmission of human- and livestock-associated S. aureus/MRSA to wildlife and vice versa as well as the possible transmission, by unprotected contact to animals. The prevalence of S. aureus/MRSA in wildlife as well as its population structures in different wildlife host species warrants further investigation., Competing Interests: Competing Interests: R. Ehricht, E. Müller, A. Reissig and S. Monecke are employees of Alere Technologies, the company that manufactures the microarrays used for this study. The arrays used herein are (or will be developed to be) a marketed product. This does not alter our adherence to PLOS ONE policies on sharing data and materials

Food products of animal origin can serve as a vehicle for Staphylococcus (S.) aureus, a facultative pathogen involved in a variety of diseases. As a result, international trade and illegal transportation of foodstuffs can facilitate the distribution of S. aureus over long distances. In this study, we investigated S. aureus isolates recovered from meat products confiscated from passengers returning from non-EU countries at two German airports and from samples of legally imported meats from non-EU countries. The aim was to characterize isolates in regard to their genetic relatedness as well as their antimicrobial resistance profiles and major virulence factors in order to assess potential risks associated with these products. The isolates were characterized by spa typing, MLST, macrorestriction analysis, microarray analysis and antimicrobial susceptibility testing. MRSA isolates were further characterized by dru typing. The characteristics of the majority of the isolates indicated a human origin, rather than an association with livestock. The results further revealed a considerable heterogeneity among the MRSA isolates, despite their common origin. Overall, a plenitude of major virulence factors and antimicrobial resistances was detected among the isolates, highlighting the potential risks associated with contaminated meat products and the transportation of such products among different countries., Competing Interests: The authors of this manuscript declare the following competing interests: R. Ehricht and S. Monecke are employees of Alere Technologies GmbH, the company that manufactures the microarray used in this study. This funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Objectives: CC22-MRSA-IV, UK-EMRSA-15/Barnim EMRSA, is a common and pandemic strain of methicillin-resistant Staphylococcus aureus (MRSA) that has been found mainly in Western Europe, but also in other parts of the world including some Gulf countries. One suspected case of an infection with this strain in a patient who was admitted to the surgical unit in Riyadh, Kingdom of Saudi Arabia (KSA) was investigated in order to check whether this strain has reached KSA., Methods: Besides the index isolate, 46 additional isolates of CC22-MRSA-IV from patients from KSA, Abu Dhabi, Kuwait, and Germany (patients with a history of travel in the Middle East), were characterized by microarray hybridization., Results: The study revealed a regional presence of as many as six distinct 'strains' of CC22-MRSA-IV that could be distinguished based on carriage of SCCmec IV subtypes and virulence factors. No true UK-EMRSA-15/Barnim EMRSA was identified in Riyadh; all suspected isolates from Riyadh were assigned to other, albeit related strains. However, this strain was identified in Abu Dhabi and Kuwait., Conclusions: CC22-MRSA-IV from KSA could be linked to other epidemic strains from the Middle East and possibly India, rather than to the Western European UK-EMRSA-15/Barnim EMRSA. High-resolution typing methods, including SCCmec subtyping, might help to differentiate related epidemic strains and to monitor routes of transmission., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

SCCmec elements are very important mobile genetic elements in Staphylococci that carry beta-lactam resistance genes mecA/mecC, recombinase genes and a variety of accessory genes. Twelve main types and a couple of variants have yet been described. In addition, there are also other SCC elements harbouring other markers. In order to subtype strains of methicillin-resistant S. aureus (MRSA) based on variations within their SCCmec elements, 86 markers were selected from published SCC sequences for an assay based on multiplexed primer extension reactions followed by hybridisation to the specific probes. These included mecA/mecC, fusC, regulatory genes, recombinase genes, genes from ACME and heavy metal resistance loci as well as several genes of unknown function. Hybridisation patterns for published genome or SCC sequences were theoretically predicted. For validation of the microarray based assay and for stringent hybridisation protocol optimization, real hybridization experiments with fully sequenced reference strains were performed modifying protocols until yielded the results were in concordance to the theoretical predictions. Subsequently, 226 clinical isolates from two hospitals in the city of Dresden, Germany, were characterised in detail. Beside previously described types and subtypes, a wide variety of additional SCC types or subtypes and pseudoSCC elements were observed as well as numerous composite elements. Within the study collection, 61 different such elements have been identified. Since hybridisation cannot recognise the localisation of target genes, gene duplications or inversions, this is a rather conservative estimate. Interestingly, some widespread epidemic strains engulf distinct variants with different SCCmec subtypes. Notable examples are ST239-MRSA-III, CC5-, CC22-, CC30-, and CC45-MRSA-IV or CC398-MRSA-V. Conversely, identical SCC elements were observed in different strains with SCCmec IVa being spread among the highest number of Clonal Complexes. The proposed microarray can help to distinguish isolates that appear similar or identical by other typing methods and it can be used as high-throughput screening tool for the detection of putative new SCC types or variants that warrant further investigation and sequencing. The high degree of diversity of SCC elements even within so-called strains could be helpful for epidemiological typing. It also raises the question on scale and speed of the evolution of SCC elements., Competing Interests: EM, AR, PS, RE, and SM are employees of Alere Technologies, the company that manufactures the microarrays used for this study. This does not affect the authors' adherence to all the PLOS policies on sharing data and materials. The arrays used herein are (or potentially will be developed to be) a marketed product. The other authors do not have competing interests to declare.

Microarray-based serogenotyping, antimicrobial susceptibility tests and the detection of relevant resistance genes were performed on isolates of Salmonella spp. from retail meat samples obtained in Lagos, Nigeria. Out of 151 meat samples, 33 Salmonella isolates were obtained. Nine different Salmonella serovars (S. Amoutive, S. Bargny, S. Drac, S. Ealing, S. Urbana, S. Hadar, S. Nyborg, S. Anatum and S. Havana) were identified by microarray-based serogenotyping and confirmed afterwards using classical serotyping. Antibiotic susceptibility tests with 17 antibiotics showed that almost all isolates were fully susceptible to this panel. The results of this study indicated a high prevalence of Salmonella in retail meat, the presence of some previously rather rarely described Serovars in retail meat samples from Lagos, and a need to monitor for Salmonella and their antibiotic resistance determinants. The microarray-based system used herein proved to be perfectly suited as epidemiological tool to replace classical serotyping., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: The environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking., Methods and Principal Findings: In this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response., Conclusions: Our protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our high-throughput assay might be useful for the detection of anti-B. pseudomallei antibodies in epidemiological studies. Further studies are needed to elucidate the clinical and diagnostic significance of the different antibody kinetics observed during melioidosis.

Introduction: Industrial livestock farming is a possible source of multi-resistant Gram-negative bacteria, including producers of extended spectrum beta-lactamases (ESBLs) conferring resistance to 3rd generation cephalosporins. Limited information is currently available on the situation of ESBL producers in livestock farming outside of Western Europe. A surveillance study was conducted from January to May in 2014 in four dairy cattle farms in different areas of the Nile delta, Egypt., Materials and Methods: In total, 266 samples were collected from 4 dairy farms including rectal swabs from clinically healthy cattle (n = 210), and environmental samples from the stalls (n = 56). After 24 h pre-enrichment in buffered peptone water, all samples were screened for 3rd generation cephalosporin-resistant Escherichia coli using Brilliance™ ESBL agar. Suspected colonies of putatively ESBL-producing E. coli were sub-cultured and subsequently genotypically and phenotypically characterized. Susceptibility testing using the VITEK-2 system was performed. All suspect isolates were genotypically analyzed using two DNA-microarray based assays: CarbDetect AS-1 and E. coli PanType AS-2 kit (ALERE). These tests allow detection of a multitude of genes and their alleles associated with resistance toward carbapenems, cephalosporins, and other frequently used antibiotics. Serotypes were determined using the E. coli SeroGenotyping AS-1 kit (ALERE)., Results: Out of 266 samples tested, 114 (42.8%) ESBL-producing E. coli were geno- and phenotypically identified. 113 of 114 phenotypically 3rd generation cephalosporin-resistant isolates harbored at least one of the ESBL resistance genes covered by the applied assays [blaCTX-M15 (n = 105), blaCTX-M9 (n = 1), blaTEM (n = 90), blaSHV (n = 1)]. Alarmingly, the carbapenemase genes blaOXA-48 (n = 5) and blaOXA-181 (n = 1) were found in isolates that also were phenotypically resistant to imipenem and meropenem. Using the array-based serogenotyping method, 66 of the 118 isolates (55%) could be genotypically assigned to O-types., Conclusion: This study is considered to be a first report of the high prevalence of ESBL-producing E. coli in dairy farms in Egypt. ESBL-producing E. coli isolates with different underlying resistance mechanisms are common in investigated dairy cattle farms in Egypt. The global rise of ESBL- and carbapenemase-producing Gram-negative bacteria is a big concern, and demands intensified surveillance.

Limited data exist from the Gulf Cooperation Council states on the prevalence and population dynamics of Staphylococcus aureus colonizing livestock or contaminating retail meat. This study was designed to determine the presence and genetic characteristics of Staphylococcus aureus isolated from raw retail meat sold in Riyadh, Saudi Arabia. Over a period of 9 months, different raw retail meat types were aseptically processed using the double broth enrichment technique, characteristic colonies from chromogenic and mannitol salt agar were further identified using conventional methods. Susceptibility to 9 antibiotics was determined using the disc diffusion technique. Interpretation of inhibition zone was done according to Clinical and Laboratory Standards Institute guidelines. Molecular characterization was carried out using the StaphyType DNA microarray technology. Twenty-five meat samples yielded Staphylococcus aureus isolates. Camel meat had the highest contamination rate with Methicillin resistant Staphylococcus aureus (MRSA) (20%) and Methicillin susceptible Staphylococcus aureus (28%), while poultry meat had the least contamination rate with MRSA (4%). The MRSA isolates were grouped into 4 clonal complexes (CCs) namely CC1-MRSA-IV/SCCfus (n = 2), CC15-MRSA-V/SCCfus (n = 4), CC80-MRSA-IV/PVL+ (n = 5), and CC88-MRSA-IV/PVL+ (n = 2). All CC15-MRSA-V/SCCfus isolates were obtained from camel meat. This is the first study to demonstrate the novel CC15-MRSA-V/SCCfus in retail camel meat. We recommend that surveillance studies should be incorporated in public health and food hygiene programs.

Background: Meticillin-resistant staphylococci (MRS) are pathogens of increasing importance to human and animal health worldwide. Transmission of meticillin-resistant Staphylococcus aureus (MRSA) between animals and humans has been well documented. By contrast, information about transmission of other Staphylococcus spp. is limited., Hypothesis/objectives: The aim of this study was to screen animals and humans on a small farm for nasal carriage of MRS and to assess interspecies exchange., Methods: After detection of MRSA in a lung sample of a deceased cat, which lived on a small mixed farm, nasal swabs were taken within two weeks, four and 16 months from other animals of various species and humans living on the farm. Swabs were cultured for MRS which were then characterized molecularly., Results: MRSA and meticillin-resistant coagulase negative staphylococci (MRCoNS), including Staphylococcus haemolyticus, S. epidermidis and S. fleurettii, were isolated from humans and different animal species. Typing of the MRS revealed isolates with the same characteristics in different human and animal hosts., Conclusions and Clinical Importance: To the best of the authors' knowledge, this is the first report of carriage of both MRSA and MRCoNS among humans and various animals within a shared environment. The detection of strains with indistinguishable molecular characteristics strongly suggested transmission of these MRS between the various animal species and humans., (© 2016 ESVD and ACVD.)

Ceftaroline is a new cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA). A collection of 17 clinical and veterinary mecC-positive MRSA isolates was tested to evaluate the in vitro efficacy of ceftaroline against recently emerged mecC-MRSA isolates. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of ceftaroline for the 17 isolates were determined by broth microdilution using the methodology and interpretive criteria of the Clinical and Laboratory Standards Institute (CLSI). Additional susceptibility tests were performed using ceftaroline M.I.C.Evaluator (M.I.C.E.™) strips. All isolates showed susceptibility according to CLSI breakpoints, with MICs of ceftaroline ranging from 0.125mg/L to 0.25mg/L. MBCs were identical or up to a twofold dilution step higher. In conclusion, all tested isolates, from various sources and belonging to several clonal complexes (CCs), but predominantly to CC130, were found to be susceptible to ceftaroline. Ceftaroline could thus be an option for the treatment of mecC-MRSA infections., (Copyright © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

The aim of this study was to investigate the presence of β-lactamase producing or fluoroquinolone-resistant members of the family Enterobacteriaceae in European mouflons (Ovis orientalis musimon). The mouflon samples originated from nasal and perineal swabs and/or organ samples in cases of a suspected infection. Only one of the 32 mouflons was tested positive for the presence of Enterobacteriaceae that displayed either an ESBL/AmpC phenotype or were resistant to ciprofloxacin. The positively tested swab originated from a sample of the jejunal mucosa of a four-year old female mouflon. Two different colony morphotypes were identified as Escherichia coli and Klebsiella pneumoniae. These isolates were phenotypically and genotypically characterized in detail by a polyphasic approach. Both isolates were multi-drug resistant. The E. coli isolate belonged to the phylogenetic group B1 and sequence type (ST) 744 and harboured the β-lactamase genes blaCTX-M-15 and blaOXA-1. The K. pneumoniae, identified as ST11, harboured the β-lactamase genes blaSHV-11, blaOXA-1, and blaDHA-1 as well as the plasmid-mediated quinolone resistance (PMQR) gene qnrB55. The present study demonstrates that wild animals can acquire human-derived resistance determinants and such findings may indicate environmental pollution with resistance determinants from other sources.

Aim: To countermeasure the global spread of β-lactamases, we developed a rapid molecular test for the highly variable OXA-β-lactamases that allows minimizing the time to effective treatment., Methods: OXA-mRNA was specifically enriched from total RNA using group-specific biotinylated DNA probes and streptavidin-coated magnetic beads. Phylogenetic OXA groups were distinguished by PCR product size., Results: This mRNA fishing method is highly sensitive, yielding specific results from as little as 1 ng total RNA. It enables discrimination of OXA-extended substrate spectrum β-lactamases and carbapenemases and the semi-quantitative detection of highly expressed ISAba1-controlled variants., Conclusion: Targeting mRNA with specific probes on magnetic beads will allow for adaptation to automated systems, such as point-of-care diagnostics.

The pathogen Staphylococcus aureus causes a broad range of severe diseases and is feared for its ability to rapidly develop resistance to antibiotic substances. The increasing number of highly resistant S. aureus infections has accelerated the search for alternative treatment options to close the widening gap in anti-S. aureus therapy. This study analyses the humoral immune response to vaccination of Balb/c mice with sublethal doses of live S. aureus. The elicited antibody pattern in the sera of intravenously and intramuscularly vaccinated mice was determined using of a recently developed protein array. We observed a specific antibody response against a broad set of S. aureus antigens which was stronger following i.v. than i.m. vaccination. Intravenous but not intramuscular vaccination protected mice against an intramuscular challenge infection with a high bacterial dose. Vaccine protection was correlated with the strength of the anti-S. aureus antibody response. This study identified novel vaccine candidates by using protein microarrays as an effective tool and showed that successful vaccination against S. aureus relies on the optimal route of administration.

Aim: Limited information is currently available about the prevalence of carbapenemase-producing Enterobacteriaceae (CPE) in Romania., Materials & Methods: Routine tests of 1,993 clinical isolates at a hospital in Iaşi yielded 46 isolates that were resistant to carbapenems. All 46 isolates were phenotypically and genotypically analyzed using VITEK-2 and DNA microarray-based assays., Results: Isolates were assigned to Klebsiella pneumoniae and Enterobacter cloacae. For 39 isolates, carbapenem resistance was confirmed and 37 harbored at least one carbapenem resistance gene. Two isolates were probably resistant due to AmpC β-lactamases in combination with a porin loss. The overall concordance between detected phenotype and genotype was 95%., Conclusion: Our data show that carbapenemase-producing isolates with different underlying resistance mechanisms are still rare in Iaşi, but the global rise of CPE warrants intensified surveillance.

Sugar beet root rot diseases that occur during the cropping season or in storage are accompanied by high yield losses and a severe reduction of processing quality. The vast diversity of microorganism species involved in rot development requires molecular tools allowing simultaneous identification of many different targets. Therefore, a new microarray technology (ArrayTube) was applied in this study to improve diagnosis of sugar beet root rot diseases. Based on three marker genes (internal transcribed spacer, translation elongation factor 1 alpha, and 16S ribosomal DNA), 42 well-performing probes enabled the identification of prevalent field pathogens (e.g., Aphanomyces cochlioides), storage pathogens (e.g., Botrytis cinerea), and ubiquitous spoilage fungi (e.g., Penicillium expansum). All probes were proven for specificity with pure cultures from 73 microorganism species as well as for in planta detection of their target species using inoculated sugar beet tissue. Microarray-based identification of root rot pathogens in diseased field beets was successfully confirmed by classical detection methods. The high discriminatory potential was proven by Fusarium species differentiation based on a single nucleotide polymorphism. The results demonstrate that the ArrayTube constitute an innovative tool allowing a rapid and reliable detection of plant pathogens particularly when multiple microorganism species are present.

Purpose: The mortality rate of patients with Staphylococcus aureus infections is alarming and urgently demands new strategies to attenuate the course of these infections or to detect them at earlier stages., Experimental Design: To study the adaptive immune response to S. aureus antigens in healthy human volunteers, a protein microarray containing 44 S. aureus proteins was developed using the ArrayStrip platform technology., Results: Testing plasma samples from 15 S. aureus carriers and 15 noncarriers 21 immunogenic S. aureus antigens have been identified. Seven antigens were recognized by antibodies present in at least 60% of the samples, representing the core S. aureus immunome of healthy individuals. S. aureus-specific serum immunoglobulin G (IgG) levels were significantly lower in noncarriers than in carriers specifically anti-IsaA, anti-SACOL0479, and anti-SACOL0480 IgGs were found at lower frequencies and quantities. Twenty-two antigens present on the microarray were encoded by all S. aureus carrier isolates. Nevertheless, the immune system of the carriers was responsive to only eight of them and with different intensities., Conclusion and Clinical Relevance: The established protein microarray allows a broad profiling of the S. aureus-specific antibody response and can be used to identify S. aureus antigens that might serve as vaccines or diagnostic markers., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins., Methods: In this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays., Results: 110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate., Conclusions: The multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers.

A West Australian methicillin-resistant Staphylococcus aureus strain (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, tnp IS431, 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 mec complex A should be split into two subtypes. One harbors PSM-mec and xylR (mecR2). It is found in SCCmec types II, III, and VIII. The second subtype, described herein, is present in WA MRSA-59 and some coagulase-negative staphylococci., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The aim of this study was to provide the first detailed insight into the population structure of Staphylococcus aureus in one modern dairy farm (Gamasa) and several household cows and buffaloes in Dakahlia Governorate, Egypt. Eight hundred seventy-two quarter milk samples of 218 dairy cattle and buffaloes with clinical and subclinical mastitis were investigated. Bacteria were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and staphylococci were further characterized by DNA sequencing of 16S rRNA genes and microarray analysis. Staphylococcus aureus was present in 5.6% of all collected samples, whereas methicillin-resistant S. aureus (MRSA) represented 24.5% of all identified S. aureus (12/49). Six clonal complexes (CC) of S. aureus were detected. Staphylococcus aureus CC398 (ST291/813)-MSSA (methicillin-susceptible S. aureus) was identified frequently in the Gamasa farm in addition to a few CC5-MRSA-V isolates. However, a small number of different isolates of S. aureus were found in household cattle and buffaloes harboring different CC. The presence of these genotypes of S. aureus in milk might indicate a public health hazard, because all of these CC have previously been isolated from human patients. Thus, a recommendation was given to the owner of the dairy farm to review the hygiene regimen on the farm. In perspective, further investigation regarding S. aureus screening of all lactating cows and personnel on the farm is warranted., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

This study compares the characteristics of Staphylococcus epidermidis (SE) and Staphylococcus haemolyticus (SH) isolates from epidemiologically unrelated infections in humans (Hu) (28 SE-Hu; 8 SH-Hu) and companion animals (CpA) (12 SE-CpA; 13 SH-CpA). All isolates underwent antimicrobial susceptibility testing, multilocus sequence typing and DNA microarray profiling to detect antimicrobial resistance and SCCmec-associated genes. All methicillin-resistant (MR) isolates (33/40 SE, 20/21 SH) underwent dru and mecA allele typing. Isolates were predominantly assigned to sequence types (STs) within a single clonal complex (CC2, SE, 84.8%; CC1, SH, 95.2%). SCCmec IV predominated among MRSE with ST2-MRSE-IVc common to both Hu (40.9%) and CpA (54.5%). Identical mecA alleles and nontypeable dru types (dts) were identified in one ST2-MRSE-IVc Hu and CpA isolate, however, all mecA alleles and 2/4 dts detected among 18 ST2-MRSE-IVc isolates were closely related, sharing >96.5% DNA sequence homology. Although only one ST-SCCmec type combination (ST1 with a non-typeable [NT] SCCmec NT9 [class C mec and ccrB4]) was common to four MRSH-Hu and one MRSH-CpA, all MRSH isolates were closely related based on similar STs, SCCmec genes (V/VT or components thereof), mecA alleles and dts. Overall, 39.6% of MR isolates harbored NT SCCmec elements, and ACME was more common amongst MRSE and CpA isolates. Multidrug resistance (MDR) was detected among 96.7% of isolates but they differed in the prevalence of specific macrolide, aminoglycoside and trimethoprim resistance genes amongst SE and SH isolates. Ciprofloxacin, rifampicin, chloramphenicol [fexA, cat-pC221], tetracycline [tet(K)], aminoglycosides [aadD, aphA3] and fusidic acid [fusB] resistance was significantly more common amongst CpA isolates. SE and SH isolates causing infections in Hu and CpA hosts belong predominantly to STs within a single lineage, harboring similar but variable SCCmec genes, mecA alleles and dts. Host and staphylococcal species-specific characteristics were identified in relation to antimicrobial resistance genes and phenotypes, SCCmec and ACME.

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.

The biofilm-associated protein (Bap) of Staphylococcus aureus is a high molecular weight cell-wall-anchored protein involved in biofilm formation, first described in bovine mastitis strains from Spain. So far, studies regarding Bap were mainly based on the Spanish strain V329 and its mutants, but no information on the genetic variability of bap-positive Staph. aureus strains is yet available in the literature. The present study investigated the molecular characteristics of 8 bap-positive Staph. aureus strains from subclinical bovine mastitis, isolated in 5 herds; somatic cell counts (SCC) of milk samples were also registered. Strains were characterised using MLST, SPA typing and microarray and the results were compared with V329. All isolates from this study and V329 were assigned to ST126, t605, but some molecular differences were observed. Only herd A and B strains harboured the genes for β-lactams resistance; the leukocidin D/E gene, a type I site-specific deoxyribonuclease subunit, 3rd locus gene and serin-protease A and B were carried by all strains, but not by V329, while serin-protease E was absent in V329 and in another isolate. Four isolates and V329 harboured the fibronectin-binding protein B gene. SCC showed the highest value in the milk sample affected by the only strain carrying all the virulence factors considered. Potential large variability of virulence was evidenced among V329 and all bap-positive Staph. aureus strains considered: the carriage of fnb could enhance the accumulation of biofilm, but the lack of lukD/E and splA, B or E might decrease the invasiveness of strain.

Typing of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) from Australia in the 1970s revealed a novel clone, ST2249-MRSA-III (CC45), present from 1973 to 1979. This clone was present before the Australian epidemic caused by the recombinant clone, ST239-MRSA-III. This study aimed to characterize the genome of ST2249-MRSA-III to establish its relationship to other MRSA clones. DNA microarray analysis was conducted and a draft genome sequence of ST2249 was obtained. The recombinant structure of the ST2249 genome was revealed by comparisons to publicly available ST239 and ST45 genomes. Microarray analysis of genomic DNA of 13 ST2249 isolates showed gross similarities with the ST239 chromosome in a segment around the origin of replication and with ST45 for the remainder of the chromosome. Recombination breakpoints were precisely determined by the changing pattern of nucleotide polymorphisms in the genome sequence of ST2249 isolate SK1585 compared with ST239 and ST45. One breakpoint was identified to the right of oriC, between sites 1014 and 1065 of the gene D484_00045. Another was identified to the left of oriC, between sites 1185 and 1248 of D484_01632. These results indicate that ST2249 inherited approximately 35.3% of its chromosome from an ST239-like parent and 64.7% from an ST45-like parent. ST2249-MRSA-III resulted from a major recombination between parents that resemble ST239 and ST45. Although only limited Australian archival material is available, the oldest extant isolate of ST2249 predates the oldest Australian isolate of ST239 by 3 years. It is therefore plausible that these two recombinant clones were introduced into Australia separately., (Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Spoligotyping is a widely used typing method for the Mycobacterium tuberculosis complex. Protocols and platforms can be adapted for direct use on patient samples. Serial dilutions of genomic DNA from Mycobacterium bovis BCG strain DSM45071 were spoligotyped by array hybridization using 32 different commercial PCR polymerase preparations. In samples with very low concentrations of mycobacterial DNA, commercially available PCR polymerases differed in their performance, and some yielded no, or false, identification. Direct spoligotyping from samples with very low concentrations of mycobacterial DNA thus requires careful selection of polymerase and strict standardization., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Staphylococcus aureus is one of the most relevant pathogens causing clinical and subclinical, chronic mastitis in dairy animals. Routinely, mastitis pathogens are isolated and classified to genus or species level, and regarded as single entities. However, S. aureus includes a broad range of genotypes with distinct pathogenic and epidemiologic characteristics. The objective of the present study was to assess the host-specificity of S. aureus causing mastitis in dairy animals, based on phylogenetic and genotypic characterization as well as the presence of virulence and antimicrobial resistance genes in the pathogen genome. S. aureus isolates from mastitis in cows, sheep and goats in Israel, and from cows in Germany, the USA and Italy, were compared by the following methods: a. Bayesian phylogenetic comparison of sequences of genes nuc, coa, lukF and clfA, b. genotyping by spa and agr typing, and assignment to MLST Clonal Complexes (MLST CC), and c. the presence of a broad array of virulence and antimicrobial resistance genes. Overall, phylogenetic, virulence and genotyping approaches agreed with each other. Cow isolates could be differentiated from sheep and goat isolates with all three methods, with different resolution. In two phylogenetic clusters, segregation was found also between cow isolates from Israel and abroad. Sheep and goats' isolates showed less variability than isolates from cows in all methods used. In conclusion, different S. aureus lineages are associated to cows in contrast to goats and sheep, suggesting co-evolution between pathogen and host species. Modern diagnostics approaches should aim to explore molecular data for a better understanding and cost-effective management of mastitis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The anterior nares have been regarded as the major carriage site of Staphylococcus aureus. From here, the organism can spread to other parts of the body where it might act as harmless commensal or cause mild to severe infections. Nasal sinuses are normally sterile, but in patients with chronic rhinosinusitis (CRS), the finding of S. aureus in maxillary sinus cultures is common. Isolates were obtained from the nares and maxillary sinus of patients with CRS and the nares of healthy controls. A significantly higher frequency of S. aureus was found in nares samples from patients (24/42) compared to controls (16/57) (p = 0.004). There is no consensus regarding whether S. aureus is a relevant pathogen in CRS. A DNA microarray was used to investigate the prevalence of S. aureus virulence genes with focus on staphylococcal enterotoxins, toxic shock syndrome toxin-1, agr types, and cell wall-associated proteins. The genotyping of S. aureus isolates revealed only small and non-significant differences in gene prevalence between isolates collected from patients with CRS and those collected from healthy nasal carriers. This study provides an increased knowledge of the genetic pattern of virulence genes among S. aureus collected in CRS., (© 2014 APMIS. Published by John Wiley & Sons Ltd.)

Staphylococcus aureus is one of the major pathogens that causes bacteremia; therefore, it is important to understand the long-term molecular epidemiology of S. aureus bacteremia infections. In particular, little is known about the population structure of methicillin-sensitive S. aureus (MSSA) compared to that of methicillin-resistant S. aureus. We investigated potential changes in the MSSA molecular epidemiology in Örebro County, Sweden, from 1980 through 2010. 400 MSSA bacteremia isolates, the first 100 isolated each decade from 1980 through 2010, were retrospectively identified and analyzed regarding assignment to clonal complexes (CCs), presence of virulence genes and antibiotic resistant determinants with DNA microarray-based genotyping. 24 different CCs were identified. Most isolates (80%) belonged to 6 predominant lineages. Of those, the number of isolates assigned to CC5 and CC15 increased, and those assigned to CC8, CC25, and CC30 decreased. The most prevalent clone, CC45, did not show a significant change in prevalence during the study period. A change in prevalence was observed for some of the virulence genes, mainly attributed with their association to certain CCs. With the exception of the common blaZ gene (encoding penicillinase), antibiotic resistance genes were only sporadically detected. In conclusion, the MSSA population structure was genetically diverse. We observed decadal changes in assignments to five predominant clones, and corresponding changes in the prevalence of some virulence genes linked to CC affiliation. In light of the restrictive antibiotics prescriptions and extensive infection control procedures in Sweden, antibiotic resistance genes were rarely detected and their prevalence unaffected during the study period.

Streptococcus agalactiae frequently 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 alp allele. This suggests the occurrence of frequent genomic recombination. For array-based typing, a set of 11 markers (bac, alp, pil1 locus, pepS8, fbsB, capsule locus, hylB, abiG-I/-II plus Q8DZ34, pil2 locus, nss plus srr plus rogB2, and rgfC/A/D/B) was defined that provides a framework for splitting the tested 448 S. agalactiae isolates 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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Staphylococcus aureus is a human pathogen that can harbour several genes encoding exotoxins including leukocidins. A clinically most relevant factor is Panton-Valentine leukocidin (PVL) because of its association with chronic, recurrent or severe skin and soft tissue infections. In this study an antibody array was designed and used to obtain an overview about the in vitro PVL expression levels of 266 clinical isolates of MRSA as well as of MSSA belonging to a wide variety of clonal complexes. For that purpose, a novel precipitation based method was used. Unknown PVL concentrations were determined by mapping the signal intensities for spotted monoclonal antibodies to calibration curves that resulted from experiments with known concentrations of recombinant LukF-PV. In most cases, isolates belonging to one clonal complex (CC) showed similar PVL expressions. However, there were also CCs with widely varying PVL concentrations. First analyses, based on in vitro PVL measurements, showed low PVL concentrations in isolates from severe and fatal conditions that are not associated with PVL, such as sepsis, while isolates from skin and soft tissue infections yielded higher concentrations. Agr-group I and IV isolates generally produced more PVL than isolates from agr-groups II and III. The few isolates harbouring the gene encoding toxic shock syndrome toxin (tst1) were particularly low level PVL producers. However, these issues warrant further studies. The method described herein allows rapid quantification of expressed proteins such as PVL in collections of clinical isolates in order to correlate with clinical or genotypic data with a potential for further parallelisation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In 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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)