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Mycoplasmas comprise a conglomerate of pathogens and commensals occurring in humans and animals. The genus Mycoplasma alone contains more than 120 species at present, and new members are continuously being discovered. Therefore, it seems promising to use a single highly parallel detection assay rather than develop separate tests for each individual species. In this study, we have designed a DNA microarray carrying 70 oligonucleotide probes derived from the 23S rRNA gene and 86 probes from the tuf gene target regions. Following a PCR amplification and biotinylation step, hybridization on the array was shown to specifically identify 31 Mycoplasma spp., as well as 3 Acholeplasma spp. and 3 Ureaplasma spp. Members of the Mycoplasma mycoides cluster can be recognized at subgroup level. This procedure enables parallel detection of Mollicutes spp. occurring in humans, animals or cell culture, from mono- and multiple infections, in a single run. The main advantages of the microarray assay include ease of operation, rapidity, high information content, and affordability. The new test's analytical sensitivity is equivalent to that of real-time PCR and allows examination of field samples without the need for culture. When 60 field samples from ruminants and birds previously analyzed by denaturing-gradient gel electrophoresis (DGGE) were tested by the microarray assay both tests identified the same agent in 98.3% of the cases. Notably, microarray testing revealed an unexpectedly high proportion (35%) of multiple mycoplasma infections, i.e., substantially more than DGGE (15%). Two of the samples were found to contain four different Mycoplasma spp. This phenomenon deserves more attention, particularly its implications for epidemiology and treatment.

In Australia the PVL-positive ST93-IV [2B], colloquially known as "Queensland CA-MRSA" has become the dominant CA-MRSA clone. First described in the early 2000s, ST93-IV [2B] is associated with skin and severe invasive infections including necrotizing pneumonia. A singleton by multilocus sequence typing (MLST) eBURST analysis ST93 is distinct from other S. aureus clones. To determine if the increased prevalence of ST93-IV [2B] is due to the widespread transmission of a single strain of ST93-IV [2B] the genetic relatedness of 58 S. aureus ST93 isolated throughout Australia over an extended period were studied in detail using a variety of molecular methods including pulsed-field gel electrophoresis, spa typing, MLST, microarray DNA, SCCmec typing and dru typing. Identification of the phage harbouring the lukS-PV/lukF-PV Panton Valentine leucocidin genes, detection of allelic variations in lukS-PV/lukF-PV, and quantification of LukF-PV expression was also performed. Although ST93-IV [2B] is known to have an apparent enhanced clinical virulence, the isolates harboured few known virulence determinants. All PVL-positive isolates carried the PVL-encoding phage ΦSa2USA and the lukS-PV/lukF-PV genes had the same R variant SNP profile. The isolates produced similar expression levels of LukF-PV. Although multiple rearrangements of the spa sequence have occurred, the core genome in ST93 is very stable. The emergence of ST93-MRSA is due to independent acquisitions of different dru-defined type IV and type V SCCmec elements in several spa-defined ST93-MSSA backgrounds. Rearrangement of the spa sequence in ST93-MRSA has subsequently occurred in some of these strains. Although multiple ST93-MRSA strains were characterised, little genetic diversity was identified for most isolates, with PVL-positive ST93-IVa [2B]-t202-dt10 predominant across Australia. Whether ST93-IVa [2B] t202-dt10 arose from one PVL-positive ST93-MSSA-t202, or by independent acquisitions of SCCmec-IVa [2B]-dt10 into multiple PVL-positive ST93-MSSA-t202 strains is not known.

Two isolates of enterohemorrhagic Escherichia coli (EHEC) O104:H4 were isolated in France in 2004 and 2009. Both were characterized and compared to the strain which caused the German outbreak in 2011 and to other O104:H4 strains. This suggests that different O104:H4 EHEC strains were present several years prior to the 2011 outbreak.

Brucellosis is a worldwide zoonosis leading to tremendous economic losses and severe human illness. Fast and reliable laboratory tests are needed to detect disease in both humans and animals and to monitor the production of safe food products and feed. For rapid identification of the genus Brucella and differentiation of its species, a multiplex polymerase chain reaction microarray assay based on 11 signature sequences and redundant oligonucleotide probes was developed. The gene targets included genus-specific sequences in bcsp31, perA, cgs, and omp2b, as well as chromosomal regions displaying species-specific hybridization patterns. Brucella reference strains and a representative panel of 102 field isolates were unambiguously identified by their hybridization patterns. The differentiation of species, however, was limited in members of the groups B. suis bv 3/4/B. canis and B. neotomae/B. microti. In summary, the newly developed Brucella ArrayTube® assay is an easy-to-handle molecular test for high-throughput and parallel analysis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

During a survey of fresh chicken and turkey meat as well as chicken and turkey meat products for the presence of methicillin-resistant Staphylococcus aureus (MRSA) isolates in Germany, 32 (37.2%) of 86 samples were MRSA positive. Twenty-eight of these MRSA isolates belonged to clonal complex 398 (CC398), which is widespread among food-producing animals. These CC398 isolates carried SCCmec elements of type IV or V and exhibited spa type t011, t034, t899, t2346 or t6574 and either the known dru types dt2b, dt6j, dt10a, dt10q, dt11a, dt11v, and dt11ab or the novel dru types dt6m, dt10as, and dt10at. In addition, two MRSA sequence type 9 (ST9) isolates with a type IV SCCmec cassette, spa type t1430, and dru type dt10a as well as single MRSA ST5 and ST1791 isolates with a type III SCCmec cassette, spa type t002, and dru type dt9v were identified. All but two isolates were classified as multiresistant. A wide variety of resistance phenotypes and genotypes were detected. All isolates were negative for the major virulence factors, such as Panton-Valentine leukocidin, toxic shock syndrome toxin 1, or exfoliative toxins. In contrast to the MRSA CC398 isolates, the four ST9, ST5, or ST1791 isolates harbored the egc gene cluster for enterotoxin G, I, M, N, O, and U genes. Although the relevance of contamination of fresh poultry meat or poultry products with MRSA is currently unclear, the presence of multiresistant and, in part, enterotoxigenic MRSA emphasizes the need for further studies to elucidate possible health hazards for consumers.

Background: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was first reported in remote regions of Western Australia and is now the predominant MRSA isolated in the state. The objective of this study is to determine the genetic relatedness of Western Australian CA-MRSA clones within different multilocus sequence type (MLST) clonal clusters providing an insight into the frequency of S. aureus SCCmec acquisition within a region., Results: The CA-MRSA population in Western Australia is genetically diverse consisting of 83 unique pulsed-field gel electrophoresis strains from which 46 MLSTs have been characterised. Forty five of these sequence types are from 18 MLST clonal clusters and two singletons. While SCCmec IV and V are the predominant SCCmec elements, SCCmec VIII and several novel and composite SCCmec elements are present. The emergence of MRSA in diverse S. aureus clonal clusters suggests horizontal transmission of the SCCmec element has occurred on multiple occasions. Furthermore DNA microarray and spa typing suggests horizontal transfer of SCCmec elements has also occurred within the same CC. For many single and double locus variant CA-MRSA clones only a few isolates have been detected., Conclusions: Although multiple CA-MRSA clones have evolved in the Western Australian community only three clones have successfully adapted to the Western Australian community environment. These data suggest the successful evolution of a CA-MRSA clone may not only depend on the mobility of the SCCmec element but also on other genetic determinants.

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

Typing of Chlamydia trachomatis is important to understanding its epidemiology. Currently used methods such as DNA sequencing of the ompA gene and multilocus sequence typing (MLST) either offer limited epidemiological resolution or are laborious and expensive, or both. DNA microarray technology using the ArrayStrip format is an affordable alternative for genotyping. In this study, we developed a new multilocus typing (MLT) DNA microarray, based on the target regions of a high-resolution MLST system as well as software for easy analysis. Validation of the array was done by typing 80 previously MLST-typed clinical specimens from unselected adolescents in school. The MLT array showed 100% specificity and provided 2.4-times-higher resolution than ompA sequencing, separating the commonly predominating ompA E/Bour genotype into 7 MLT array genotypes. The MLT array reproduced epidemiological findings revealed by the MLST system and showed sufficient sensitivity to work with clinical specimens. Compared to MLST analysis, the expenses needed for testing a sample with the MLT array are considerably lower. Moreover, testing can be completed within 1 working day rather than 3 or 4 days, with data analysis not requiring highly specialized personnel. The present MLT array represents a powerful alternative in C. trachomatis genotyping.

Staphylococcus aureus is a common cause of mastitis and other diseases in camels. In order to obtain data on population structure as well as on the carriage of toxin genes and resistance markers, a collection of 45 isolates from dromedaries of Dubai, United Arab Emirates, were genotyped. These isolates belonged to clonal complexes CC6 (twenty isolates; 44.44%), CC30 (sixteen isolates; 35.56%), CC188 (five isolates; 11.11%), CC152 (1 isolate, 2.2%) and to a previously un-described sequence type (ST1755: arcc-18, aroe-115, glpf-6, gmk-2 pta-109, tpi-50 and yqil-2; three isolates; 6.67%). Resistance genes proved to be rare. Only three out of 45 isolates (6.67%) carried the beta-lactamase operon. The tetracycline resistance gene tetK was also detected in three isolates (6.67%). Neither the mecA gene, defining MRSA, nor other resistance genes were found. Common virulence markers included leukocidin genes lukD+lukE (in twenty-five isolates; 55.56%), the staphylokinase gene sak (twenty-two isolates; 48.89%), the enterotoxin gene cluster egc (fifteen isolates; 33.33%), and a distinct variant of the enterotoxin A gene (sea-320E, GenBank AY196686.1; thirteen isolates; 28.89%). One CC152 isolate was positive for genes encoding the Panton-Valentine leukocidin (lukF-PV+lukS-PV). This study provides first genotyping data on the population structure and the presence of toxin genes and resistance markers of S. aureus strains in Middle Eastern camels., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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

In recent years, methicillin-resistant Staphylococcus aureus (MRSA) have become a truly global challenge. In addition to the long-known healthcare-associated clones, novel strains have also emerged outside of the hospital settings, in the community as well as in livestock. The emergence and spread of virulent clones expressing Panton-Valentine leukocidin (PVL) is an additional cause for concern. In order to provide an overview of pandemic, epidemic and sporadic strains, more than 3,000 clinical and veterinary isolates of MRSA mainly from Germany, the United Kingdom, Ireland, France, Malta, Abu Dhabi, Hong Kong, Australia, Trinidad & Tobago as well as some reference strains from the United States have been genotyped by DNA microarray analysis. This technique allowed the assignment of the MRSA isolates to 34 distinct lineages which can be clearly defined based on non-mobile genes. The results were in accordance with data from multilocus sequence typing. More than 100 different strains were distinguished based on affiliation to these lineages, SCCmec type and the presence or absence of PVL. These strains are described here mainly with regard to clinically relevant antimicrobial resistance- and virulence-associated markers, but also in relation to epidemiology and geographic distribution. The findings of the study show a high level of biodiversity among MRSA, especially among strains harbouring SCCmec IV and V elements. The data also indicate a high rate of genetic recombination in MRSA involving SCC elements, bacteriophages or other mobile genetic elements and large-scale chromosomal replacements.

Avian influenza viruses (AIVs) of the H5 and H7 subtypes can cause substantial economic losses in the poultry industry and are a potential threat to public health. Serosurveillance of poultry populations is an important monitoring tool and can also be used for control of vaccination campaigns. The purpose of this study was to develop broadly reactive, yet subtype-specific competitive ELISAs (cELISAs) for the specific detection of antibodies to the notifiable AIV subtypes H5 and H7 as an alternative to the gold standard haemagglutination inhibition assay (HI). Broadly reacting monoclonal competitor antibodies (mAbs) and genetically engineered subtype H5 or H7 haemagglutinin antigen, expressed and in vivo biotinylated in insect cells, were used to develop the cELISAs. Sera from galliform species and water fowl (n=793) were used to evaluate the performance characteristics of the cELISAs. For the H5 specific cELISA, 98.1% test sensitivity and 91.5% test specificity (97.7% and 90.2% for galliforms; 98.9% and 92.6% for waterfowl), and for the H7 cELISA 97.3% sensitivity and 91.8% specificity (95.3% and 98.9% for galliforms; 100% and 82.7% for waterfowl) were reached when compared to HI. The use of competitor mAbs with broad spectrum reactivity within an AIV haemagglutinin subtype allowed for homogenous detection with high sensitivity of subtype-specific antibodies induced by antigenically widely distinct isolates including antigenic drift variants. However, a trade-off regarding sensitivity versus nonspecific detection of interfering antibodies induced by phylo- and antigenically closely related subtypes, e.g., H5 versus H2 and H7 versus H15, must be considered. The observed intersubtype antibody cross-reactivity remains a disturbance variable in AIV subtype-specific serodiagnosis which negatively affects specificity., (Copyright © 2011 Elsevier B.V. All rights reserved.)

There is an urgent need for robust subtype-specific serological tests to diagnose influenza A virus infections in poultry and mammals, including humans. Such assays require reliable subtype-specific sources of soluble and authentically folded seroreactive hemagglutinin (HA), one of the integral membrane proteins that determine the serological subtype of influenza viruses. To this purpose, a bigenic pFastBacDual baculovirus transfer vector allowing efficient invivo biotinylation of soluble HA homo-oligomers expressed via the secretory pathway was developed. An Avi-Tag allowed site-specific biotinylation by a coexpressed genetically modified BirA biotin ligase retained in the endoplasmic reticulum (ER). Highly seroreactive mono-biotinylated HA of recent H5 and H7 influenza A subtypes was secreted from recombinant baculovirus infected High-Five insect cells at levels sufficient to directly load streptavidin-coated enzyme-linked immunosorbent assay (ELISA) matrices, thereby avoiding any purification steps. The recombinant antigens retained authentic antigenicity, including conformation-dependent epitopes involved in hemagglutination inhibition as detected by monoclonal antibodies. This is the first bigenic invivo biotinylation system established for use in insect cells with secretable recombinant membrane proteins biotinylated by an ER-retained variant of BirA biotin ligase. The proposed technique is expected to significantly increase flexibility in the design of subtype-specific assays, thereby expanding the power of influenzaA virus serodiagnosis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objectives: To investigate the occurrence of antimicrobial resistance genes of human clinical relevance in Salmonella isolated from livestock in Great Britain., Methods: Two hundred and twenty-five Salmonella enterica isolates were characterized using an antimicrobial resistance gene chip and disc diffusion assays. Plasmid profiling, conjugation experiments and identification of Salmonella genomic island 1 (SGI1) were performed for selected isolates., Results: Approximately 43% of Salmonella harboured single or multiple antimicrobial resistance genes with pig isolates showing the highest numbers where 96% of Salmonella Typhimurium harboured one or more resistance genes. Isolates harbouring multiple resistances divided into three groups. Group 1 isolates harboured ampicillin/streptomycin/sulphonamide/tetracycline resistance and similar phenotypes. This group contained isolates from pigs, cattle and poultry that were from several serovars including Typhimurium, 4,[5],12:i:-, Derby, Ohio and Indiana. All Group 2 isolates were from pigs and were Salmonella Typhimurium. They contained a non-sul-type class 1 integron and up to 13 transferrable resistances. All Group 3 isolates harboured a class 1 integron and were isolated from all animal species included in the study. Most isolates were Salmonella Typhimurium and harboured SGI1., Conclusions: Salmonella isolated from livestock was shown to harbour antimicrobial resistance genes although no or little resistance to third-generation cephalosporins or ciprofloxacin, respectively, was detected. The preponderance in pigs of multidrug-resistant Salmonella Typhimurium makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food chain and limit human therapeutic options.

Current typing methods of Chlamydia (C.) trachomatis are mainly based on the diversity of the ompA gene, which is coding for the major outer membrane protein A. The present study aimed at facilitating genotyping of strains of this obligate intracellular human pathogen by developing a DNA microarray assay using the ArrayTube™ format for individual samples and the ArrayStrip™ format for higher throughput. The new test is exploiting multiple discriminatory sites by involving a total of 61 oligonucleotide probes representing genotype-specific polymorphisms in variable domains 1, 2 and 4 of the ompA gene. After multiplex amplification of these domains using biotinylated primers, the sample is hybridized in the microarray vessel under highly stringent conditions. The resulting binding pattern is genotype specific, thus allowing direct identification. We were able to show that DNA from each of the currently accepted genotypes (serovars) yielded a unique, theoretically expected and distinct hybridization pattern. The assay was also shown to be highly sensitive as a dilution containing the equivalent of 1 inclusion-forming unit was still correctly genotyped. In addition, when 62 clinical samples were examined and compared to PCR-RFLP typing results, the genotype was correctly identified by the DNA microarray in all cases. The present test is easy to handle and economically affordable, and it allows genotyping of C. trachomatis to be accomplished within a working day, thus lending itself for epidemiological studies and routine diagnosis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

The staphylococcal cfr gene mediates resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A, a phenotype that has been termed PhLOPS(A). The cfr gene has mainly been associated with coagulase-negative staphylococcal isolates from animals, and only a few cfr-positive methicillin-resistant Staphylococcus aureus (MRSA) isolates have been described so far. This study reports the first description of a cfr-positive MRSA isolate (M05/0060) belonging to the pandemic Panton-Valentine leukocidin (PVL)-positive sequence type 8 MRSA IVa/USA300 (ST8-MRSA-IVa/USA300) clone. The cfr gene was detected in M05/0060 using a DNA microarray which was used to screen PVL-positive MRSA isolates for the presence of virulence genes, typing markers, and antimicrobial resistance genes. Antimicrobial susceptibility testing revealed that M05/0060 exhibited the cfr-associated resistance phenotype. Molecular analysis identified the presence of cfr and a second phenicol resistance gene, fexA, on a novel 45-kb conjugative plasmid, which was designated pSCFS7. Within pSCFS7, a DNA segment consisting of cfr, a truncated copy of insertion sequence IS21-558, and a region with homology to the DNA invertase gene bin3 of transposon Tn552 from Bacillus mycoides was integrated into the transposase gene tnpB of the fexA-carrying transposon Tn558. The emergence of a multidrug-resistant cfr-positive variant of ST8-MRSA-IVa/USA300 is alarming and requires ongoing surveillance. Moreover, the identification of a novel conjugative plasmid carrying the cfr gene indicates the ability of cfr to spread to other MRSA strains.

Background: Community-acquired methicillin-resistant Staphylococcus aureus have become a major problem in Australia. These strains have now been isolated throughout Australia including remote Indigenous communities that have had minimal exposure to healthcare facilities. Some of these strains, belonging to sequence types ST75 and ST883, have previously been reported to harbour highly divergent alleles of the housekeeping genes used in multilocus sequence typing., Methodology/principal Findings: ST75-MRSA-IV and ST883-MRSA-IV isolates were characterised in detail. Morphological features as well as 16S sequences were identical to other S. aureus strains. Although a partial rnpB gene sequence was not identical to previously known S. aureus sequences, it was found to be more closely related to S. aureus than to other staphylococci. Isolates also were screened using diagnostic DNA microarrays. These isolates yielded hybridisation results atypical for S. aureus. Primer directed amplification assays failed to detect species markers (femA, katA, sbi, spa). However, arbitrarily primed amplification indicated the presence of unknown alleles of these genes. Isolates could not be assigned to capsule types 1, 5 or 8. The allelic group of the accessory gene regulator (agr) locus was not determinable. Sequencing of a region of agrB, agrC and agrD (approximately 2,100 bp) revealed a divergent sequence. However, this sequence is more related to S. aureus agr alleles I and IV than to agr sequences from other Staphylococcus species. The predicted auto-inducing peptide (AIP) sequence of ST75 was identical to that of agr group I, while the predicted AIP sequence of ST883 was identical to agr group IV., Conclusions/significance: The genetic properties of ST75/ST883-MRSA may be due to a series of evolutionary events in ancient insulated S. aureus strains including a convergent evolution leading to agr group I- or IV-like AIP sequences and a recent acquisition of SCCmec IV elements.

Since enterohemorrhagic Escherichia coli (EHEC) isolates of serogroup O156 have been obtained from human diarrhea patients and asymptomatic carriers, we studied cattle as a potential reservoir for these bacteria. E. coli isolates serotyped by agglutination as O156:H25/H-/Hnt strains (n = 32) were isolated from three cattle farms during a period of 21 months and characterized by rapid microarray-based genotyping. The serotyping by agglutination of the O156 isolates was not confirmed in some cases by the results of DNA-based serotyping as only 25 of the 32 isolates were conclusively identified as O156:H25. In the multilocus sequence typing (MLST) analysis, all EHEC O156:H25 isolates were characterized as sequence type 300 (ST300) and ST688, which differ by a single-nucleotide exchange in the purA gene. Oligonucleotide microarrays allow simultaneous detection of a wider range of EHEC-associated and other E. coli virulence markers than other methods. All O156:H25 isolates showed a wide spectrum of virulence factors typical for EHEC. The stx(1) genes combined with the EHEC hlyA (hlyA(EHEC)) gene, the eae gene of the zeta subtype, as well as numerous other virulence markers were present in all EHEC O156:H25 strains. The behavior of eight different cluster groups, including four that were EHEC O156:H25, was monitored in space and time. Variations in the O156 cluster groups were detected. The results of the cluster analysis suggest that some O156:H25 strains had the genetic potential for a long persistence in the host and on the farm, while other strains did not. As judged by their pattern of virulence markers, E. coli O156:H25 isolates of bovine origin may represent a considerable risk for human infection. Our results showed that the miniaturized E. coli oligonucleotide arrays are an excellent tool for the rapid detection of a large number of virulence markers.

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

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.

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

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

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.

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

Rapid and reliable methods are fundamental for the comprehensive characterization of emerging and evolving avian influenza viruses. Although microarrays provide new possibilities with their parallel approach, their use in diagnostic laboratories is still limited due to economical and practical factors. An easy-to-use, low-cost microarray-based assay for haemagglutinin subtyping and pathotyping of avian influenza viruses and specific detection of highly pathogenic H5N1/Asia clade 2.2 is described as a novel diagnostic tool. The ArrayTube platform is user-friendly, inexpensive and allows processing of many samples. The sensitivity of the assay developed was comparable to real-time RT-PCR, and the simultaneous detection of different subtypes was possible. Validation with 90 influenza A virus isolates representing all 16 haemagglutinin subtypes and 44 field samples (cloacal swabs from wild and domestic birds) demonstrated the feasibility of the system for sensitive and specific characterization of AIV. Facilitating haemagglutinin subtyping and pathotyping for the majority of influenza A-positive cloacal swabs within 24h, the new assay enables detailed AIV diagnosis even in less well-equipped laboratories.

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

Rapid neuraminidase subtyping of avian influenza viruses from diagnostic samples is crucial considering the existence of permanently emerging and evolving strains. Here we report an easy-to-use, low-cost microarray for neuraminidase subtyping following fragment amplification by a generic, neuraminidase-specific reverse transcription-PCR (RT-PCR). This method enables highly specific characterization with a sensitivity equal to that of matrix gene-specific real-time RT-PCR.

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.

Between 2003 and 2008, 76 clinical isolates of the Panton-Valentine leukocidin-positive Staphylococcus aureus strain 'West Australian methicillin-resistant Staphylococcus aureus (MRSA)-12' (WA MRSA-12) were recovered from 72 patients living in the Perth area in Western Australia. These isolates were found to belong to multilocus sequence type 8, and had a USA300-like pulsed-field gel electrophoresis pulsotype. All isolates were genotyped using diagnostic DNA arrays covering species markers, resistance factors, virulence-associated, as well as MSCRAMM (microbial surface components recognizing adhesive matrix molecules) genes to prove the identity between WA MRSA-12 and the pandemic strain USA300, as well as to detect possible genetic variability. In general, WA MRSA-12 isolates were similar to USA300, and the most common variant was identical to USA300-TC1516. From this clone, most of the other variants may have evolved by a limited number of gene losses or acquisitions. Variations in carriage of virulence and resistance-associated genes allow distinction of variants or sub-clones. Altogether, 16 variants could be distinguished. They differed in the carriage of resistance genes (blaZ/I/R, ermC, msrA + mpbBM, aadD + mupR, aphA3 + sat, tetK, qacC, merA/B/R/T) of beta-haemolysin-converting phages and of enterotoxins (sek + seq, which were deleted in four isolates). Notably, the arginine catabolic mobile element (ACME) was absent in 12 isolates (15.8%). The mercury resistance (mer) operon, which is usually associated with SCCmec type III elements, was found in several ACME-negative isolates. The present study emphasises the importance of genotyping in detecting the introduction and evolution of significant MRSA strains within a community.

We developed a rapid oligonucleotide microarray assay based on genetic markers for the accurate identification and differentiation of Burkholderia (B.) mallei and Burkholderia pseudomallei, the agents of glanders and melioidosis, respectively. These two agents were clearly identified using at least 4 independent genetic markers including 16S rRNA gene, fliC, motB and also by novel species-specific target genes, identified by in silico sequence analysis. Specific hybridization signal profiles allowed the detection and differentiation of up to 10 further Burkholderia spp., including the closely related species Burkholderia thailandensis and Burkholderia-like agents, such as Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia vietnamiensis, Burkholderia ambifaria, and Burkholderia gladioli, which are often associated with cystic fibrosis (CF) lung disease. The assay was developed using the easy-to-handle and economical ArrayTube (AT) platform. A representative strain panel comprising 44 B. mallei, 32 B. pseudomallei isolates, and various Burkholderia type strains were examined to validate the test. Assay specificity was determined by examination of 40 non-Burkholderia strains.

The avian and human pathogen Chlamydophila (C.) psittaci represents a genetically heterogeneous species. To facilitate epidemiological surveys, more rapid yet highly specific molecular tests are needed. Currently used typing methods, i.e. serotyping and PCR-RFLP, have only limited sensitivity and are incapable of covering the wide spectrum of naturally occurring types of C. psittaci strains. In the present study, a new DNA microarray assay based on the ArrayTube (AT) technology was used to genotype C. psittaci in 98 isolates and 23 clinical tissue samples. The present array carries 35 oligonucleotide probes derived from variable domains 2 and 4 of the ompA gene. The assay proved highly sensitive, allowing correct genotyping of DNA from 2 inclusion-forming units. The results of DNA microarray genotyping of cultured strains proved highly concordant with the data from PCR-RFLP typing and serotyping. Sequencing of the ompA gene served as the reference test to verify the accuracy of AT genotyping results. In 15 instances (15.3%), strains were successfully typed by the AT assay, while serotyping and/or PCR-RFLP genotyping failed to produce unambiguous results. Eleven of these samples were ompA sequenced to confirm the AT findings. In addition to the currently accepted nine ompA genotypes, the microarray test was shown to recognise new provisional genotypes, such as Mat116 and YP84. In conclusion, the new AT assay proved to be suitable for rapid, sensitive and reproducible genotyping of C. psittaci strains and can be recommended for routine diagnosis.

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

In immunocompromised patients, the diagnosis of infections with herpesviruses and adenoviruses relies mainly on PCR amplification of viral genomic DNA from clinical samples. In the case of co-infections with two or more viruses, single amplification of viral DNA from clinical samples has proven to be time-consuming and expensive, hampering the efficient diagnosis and therapy of viral co-infections. In this study, a diagnostic DNA-microarray allowing simultaneous detection of herpes simplex virus types 1 and 2 (HSV 1/2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus-6 types A and B (HHV-6 A/B), and adenovirus in clinical samples was developed and validated. The assay displays a high analytical sensitivity (10genomeequivalents(GE)/reaction) and specificity, being cost-effective and time-saving. Because the DNA-microarray uses the same analytical conditions as real-time quantitative PCR, it can be used as a screening device for multiple viral infections, followed by selective viral load measurement depending on the clinical context. Those features make the DNA-microarray an attractive device for the management of viral infections in immunocompromised patients.

Microarray technology enables the fast and parallel analysis of a multitude of biologically relevant parameters. Not only nucleic acid-based tests, but also peptide, antigen, and antibody assays using different formats of microarrays evolved within the last decade. They offer the possibility to measure interactions in a miniaturised, economic, automated, and qualitative or quantitative way providing insights into the cellular machinery of diverse organisms. Examples of applications in research and diagnostics are, e.g., O-typing of pathogenic Escherichia coli, detection of bacterial toxins and other biological warfare agents (BW agents) from a variety of different samples, screening of complex antibody libraries, and epitope mapping. Conventional O- and H-serotyping methods can now be substituted by procedures applying DNA oligonucleotide and antibody-based microarrays. For simultaneous and sensitive detection of BW agents microarray-based tests are available, which include not only relevant viruses and bacteria, but also toxins. This application is not only restricted to the security and military sector but it can also be used in the fields of medical diagnostics or public health to detect, e.g., staphylococcal enterotoxins in food or clinical samples. Furthermore, the same technology could be used to detect antibodies against enterotoxins in human sera using a competitive assay. Protein and peptide microarrays can also be used for characterisation of antibodies. On one hand, peptide microarrays allow detailed epitope mapping. On the other hand, a set of different antibodies recognising the same antigen can be spotted as a microarray and labelled as detection antibodies. This approach makes it possible to test every combination, allowing to find the optimal pair of detection/capture antibody.

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.

A DNA microarray was designed for the rapid genotyping of Staphylococcus aureus. It covers 185 distinct genes and about 300 alleles thereof, including species-specific controls, accessory gene regulator (agr) alleles, genes encoding virulence factors, and microbial surface components recognizing adhesive matrix molecules, capsule type-specific genes, as well as resistance determinants. It was used to examine 100 clinical isolates and reference strains. Relationships of leukocidin and ssl/set (staphylococcal superantigen-like or exotoxin-like) genes were reviewed considering these experimental results as well as published sequences. A good correlation of overall hybridization pattern and multilocus sequence typing was found. Analysis of hybridization profiles thus allowed not only to assess virulence and drug resistance, but also to assign isolates to strains and to clonal complexes. Hybridization data were used to construct a split network tree and to analyse relationships between strains. Allelic variations of a number of genes indicate a division of S. aureus into three major branches that are not in accordance to agr group or capsule-type affiliations. Additionally, there are some isolated lineages, such as ST75, ST93, or ST152. These strains produce aberrant hybridization profiles, indicating that only a part of the gene pool of S. aureus has been investigated yet.

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 SCCmec 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.

We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum beta-lactamases. Validation of the array with control strains demonstrated a 99% correlation between polymerase chain reaction and array results. There was also good correlation between phenotypic and genotypic results for a large panel of Escherichia coli and Salmonella isolates. Some differences were also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the epidemiology of isolates and to detect gene linkage in bacterial populations.

Background: The currently used genotyping system for the avian zoonotic pathogen Chlamydophila (C.) psittaci has evolved from serology and is based on ompA sequence variations. It includes seven avian and two non-avian genotypes. Restriction enzyme cleavage of the amplified ompA gene and, less frequently, ompA sequencing are being used for examination, but, beside methodological limitations, an increasing number of recently tested strains could not be assigned to any established genotype., Results: Comprehensive analysis of all available ompA gene sequences has revealed a remarkable genetic diversity within the species C. psittaci, which is only partially covered by the present genotyping scheme. We suggest adjustments and extensions to the present scheme, which include the introduction of subgroups to the more heterogeneous genotypes A, E/B and D, as well as six provisional genotypes representing so far untypable strains. The findings of sequence analysis have been incorporated in the design of a new DNA microarray. The ArrayTubetrade mark microarray-based ompA genotyping assay has been shown to discriminate among established genotypes and identify so far untyped strains. Its high specificity, which allows detection of single-nucleotide polymorphisms, is due to the parallel approach consisting in the use of 35 hybridization probes derived from variable domains 2 and 4 of the ompA gene., Conclusion: The traditional genotyping system does not adequately reflect the extent of intra-species heterogeneity in ompA sequences of C. psittaci. The newly developed DNA microarray-based assay represents a promising diagnostic tool for tracing epidemiological chains, exploring the dissemination of genotypes and identifying non-typical representatives of C. psittaci.

While DNA microarrays have become a widely accepted tool for mRNA expression monitoring, their use in rapid diagnosis of bacterial and viral pathogens is only emerging. So far, insufficient sensitivity and high costs have been the major limiting factors preventing more widespread use of microarray platforms in direct testing of clinical samples. In the present study, a total of 339 samples, among them 293 clinical specimens from animals and humans, were examined by the ArrayTube (AT) DNA microarray assay to detect chlamydial DNA and identify the species of Chlamydia and Chlamydophila involved. Samples included nasal and conjunctival swabs, formalin-fixed, paraffin-embedded and fresh organ tissue, milk, feces and cell culture. Notably, the AT test was shown to detect mixed infections in clinical samples. The calculated median sensitivity of 0.81 over the entire panel of clinical samples was comparable to conventional 16S PCR, but slightly lower than real-time PCR and other PCR assays. However, when a panel of long-time stored swab samples was excluded from the calculation, the sensitivity was clearly higher (0.87) and equivalent to that of real-time PCR. Altogether, the data demonstrate the suitability of this DNA microarray assay for routine diagnosis.

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.

Staphylococcus aureus is a common pathogen which can colonise and infect not only man, but also domestic animals. Especially, infection of cattle is of high economic relevance as S. aureus is an important causal agent of bovine mastitis. In the present contribution, a DNA microarray was applied for the study of 144 different gene targets, including resistance genes and genes encoding exotoxins, in S. aureus isolated from cows. One hundred and twenty-eight isolates from Germany and Switzerland were tested. These isolates were assigned to 20 different strains and nine clonal complexes. The majority of isolates belonged either to apparently closely related clonal complexes 8, 25, and 97 (together 34.4%) or were related to the sequenced bovine strain RF122 (48.4%). Notable characteristics of S. aureus of bovine origin are the carriage of intact haemolysin beta (in 82% of isolates tested), the absence of staphylokinase (in 89.1%), the presence of allelic variants of several exotoxins such as toxic shock syndrome toxin and enterotoxin N, and the occurrence of the leukocidin lukF-P83/lukM (in 53.1%). Two isolates were methicillin-resistant S. aureus (MRSA). One of them was a clonal complex 8 MRSA related to the epidemic MRSA strain Irish 01. The other one belonged to ST398/spa-type 34 resembling a newly emerging MRSA strain which has been described to occur in humans as well as in domestic animals. The presence of these two strains highlights the possibility of transfers of S. aureus strains between different host species.

The detection of virulence determinants harbored by pathogenic Escherichia coli is important for establishing the pathotype responsible for infection. A sensitive and specific miniaturized virulence microarray containing 60 oligonucleotide probes was developed. It detected six E. coli pathotypes and will be suitable in the future for high-throughput use.

In a validation trial of a DNA microarray test for chlamydiae we repeatedly observed false-positive PCR amplicons from truly negative samples and non-template controls. Various PCR tests, microarray hybridization and DNA sequencing, revealed that residual Escherichia coli DNA from thermostable DNA polymerases was the cause of this cross-reaction. A subsequent survey showed that only five out of 23 commercial polymerases were free of E. coli DNA. When designing generic oligonucleotide sequences for PCR and PCR microarray-based assays one should be aware of such possible internal contamination, particularly when the target organism is E. coli.

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

Classical antibody-based serotyping of Escherichia coli is an important method in diagnostic microbiology for epidemiological purposes, as well as for a rough virulence assessment. However, serotyping is so tedious that its use is restricted to a few reference laboratories. To improve this situation we developed and validated a genetic approach for serotyping based on the microarray technology. The genes encoding the O-antigen flippase (wzx) and the O-antigen polymerase (wzy) were selected as target sequences for the O antigen, whereas fliC and related genes, which code for the flagellar monomer, were chosen as representatives for the H phenotype. Starting with a detailed bioinformatic analysis and oligonucleotide design, an ArrayTube-based assay was established: a fast and robust DNA extraction method was coupled with a site-specific, linear multiplex labeling procedure and hybridization analysis of the biotinylated amplicons. The microarray contained oligonucleotide DNA probes, each in duplicate, representing 24 of the epidemiologically most relevant of the over 180 known O antigens (O antigens 4, 6 to 9, 15, 26, 52, 53, 55, 79, 86, 91, 101, 103, 104, 111, 113, 114, 121, 128, 145, 157, and 172) as well as 47 of the 53 different H antigens (H antigens 1 to 12, 14 to 16, 18 to 21, 23 to 34, 37 to 43, 45, 46, 48, 49, 51 to 54, and 56). Evaluation of the microarray with a set of defined strains representing all O and H serotypes covered revealed that it has a high sensitivity and a high specificity. All of the conventionally typed 24 O groups and all of the 47 H serotypes were correctly identified. Moreover, strains which were nonmotile or nontypeable by previous serotyping assays yielded unequivocal results with the novel ArrayTube assay, which proved to be a valuable alternative to classical serotyping, allowing processing of single colonies within a single working day.

Recent years have witnessed the emergence of novel methicillin-resistant Staphylococcus aureus (MRSA) strains that produce the potent toxin Panton-Valentine leukocidin (PVL). PVL-positive strains can cause complicated skin infections or necrotising pneumonia with high mortality, and these strains have the potential for epidemic spread in the community. In 2004-2005, two case clusters and two isolated cases were observed in eastern Saxony and southern Brandenburg. These were the first known infections with PVL-positive community-acquired MRSA (caMRSA) in this part of Germany. The isolates belonged to agr type III, spa type 44 or spa type 131, and showed a SmaI macrorestriction pattern that corresponded to caMRSA of clonal group ST80. The isolates were susceptible to levofloxacin, macrolides, clindamycin, gentamicin and vancomycin. Most isolates showed resistance to tetracycline and fusidic acid because of the presence of the tetK and far1 genes. A novel plasmid (designated pUB102) harbouring far1, tetK and blaZ was characterised and partially sequenced. Microarray analysis revealed that the caMRSA isolates harboured genes encoding several bi-component toxins (lukF/S-PVL, lukD/E, lukS/F plus hlgA, and another putative leukocidin homologue). Neither tst1 nor genes for enterotoxins A-Y were detected, but the isolates harboured several staphylococcal enterotoxin-like toxin genes (set genes), as well as genes encoding an epidermal cell differentiation inhibitor (edinB) and exfoliative toxin D (etD). Comparative analysis of other isolates from Australia, Germany, Switzerland and the UK showed that these isolates were representative of a widespread clone of caMRSA.

Background: Recent developments in DNA microarray technology led to a variety of open and closed devices and systems including high and low density microarrays for high-throughput screening applications as well as microarrays of lower density for specific diagnostic purposes. Beside predefined microarrays for specific applications manufacturers offer the production of custom-designed microarrays adapted to customers' wishes. Array based assays demand complex procedures including several steps for sample preparation (RNA extraction, amplification and sample labelling), hybridization and detection, thus leading to a high variability between several approaches and resulting in the necessity of extensive standardization and normalization procedures., Results: In the present work a custom designed human proteinase DNA microarray of lower density in ArrayTube format was established. This highly economic open platform only requires standard laboratory equipment and allows the study of the molecular regulation of cell behaviour by proteinases. We established a procedure for sample preparation and hybridization and verified the array based gene expression profile by quantitative real-time PCR (QRT-PCR). Moreover, we compared the results with the well established Affymetrix microarray. By application of standard labelling procedures with e.g. Klenow fragment exo-, single primer amplification (SPA) or In Vitro Transcription (IVT) we noticed a loss of signal conservation for some genes. To overcome this problem we developed a protocol in accordance with the SPA protocol, in which we included target specific primers designed individually for each spotted oligomer. Here we present a complete array based assay in which only the specific transcripts of interest are amplified in parallel and in a linear manner. The array represents a proof of principle which can be adapted to other species as well., Conclusion: As the designed protocol for amplifying mRNA starts from as little as 100 ng total RNA, it presents an alternative method for detecting even low expressed genes by microarray experiments in a highly reproducible and sensitive manner. Preservation of signal integrity is demonstrated out by QRT-PCR measurements. The little amounts of total RNA necessary for the analyses make this method applicable for investigations with limited material as in clinical samples from, for example, organ or tumour biopsies. Those are arguments in favour of the high potential of our assay compared to established procedures for amplification within the field of diagnostic expression profiling. Nevertheless, the screening character of microarray data must be mentioned, and independent methods should verify the results.

A protein chip has been developed that allows the simultaneous detection of a multitude of different biowarfare agents. The chip was developed for the ArrayTube platform providing a cheap and easy to handle technology solution that combines a microtube-integrated protein chip with the classical procedure of a sandwich-enzyme-linked immunosorbent assay and signal amplification by streptavidin-poly-horseradish peroxidase. Specific immunoassays for Staphylococcus enterotoxin B, ricin, Venezuelan equine encephalitis virus, St. Louis encephalitis virus, West Nile virus, Yellow fever virus, Orthopox virus species, Francisella tularensis, Yersinia pestis, Brucella melitensis, Burkholderia mallei and Escherichia coli EHEC O157:H7 were developed and optimized. All assays could be completed within 1 to 1 1/2 h and detection levels were demonstrated to be as low as in well established ELISAs. Most interesting, as a result of careful antibody screening and testing, it is currently possible to analyse at least five of the "dirty dozen" agents on one single protein chip in parallel.