ST2249-MRSA-III: a second major recombinant methicillin-resistant Staphylococcus aureus clone causing healthcare infection in the 1970s.

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.

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

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

Molecular fingerprinting of Staphylococcus aureus from bone and joint infections.

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

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

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

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

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

The molecular epidemiology and evolution of the Panton-Valentine leukocidin-positive, methicillin-resistant Staphylococcus aureus strain USA300 in Western Australia.

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.

Molecular epidemiology of Staphylococcus aureus in asymptomatic carriers.

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

DNA microarray-based genotyping of methicillin-resistant Staphylococcus aureus strains from Eastern Saxony.

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.

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

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

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

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

The use of DNA microarray technology for detection and genetic characterisation of chlamydiae.

Due to its highly parallel approach, DNA microarray technology opens up new possibilities that may be particularly beneficial for laboratory diagnosis of infectious diseases. We developed a microarray assay for detection and differentiation of all currently defined chlamydial species belonging to the genera Chlamydia and Chlamydophila using the ArrayTube system, which we found to be particularly user-friendly and economical. The test includes PCR amplification of a 23S rDNA target region with concurrent biotinylation and subsequent hybridisation in the ArrayTube, a micro-reaction tube carrying the microarray chip on the bottom. In addition to high specificity, the assay was shown to allow detection and genetic characterisation of single PCR-amplifiable target DNA copies.

Microarray-based characterisation of a Panton-Valentine leukocidin-positive community-acquired strain of methicillin-resistant Staphylococcus aureus.

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.

An alternative method to amplify RNA without loss of signal conservation for expression analysis with a proteinase DNA microarray in the ArrayTube format.

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.

The IMB Jena Image Library of biological macromolecules.

The IMB Jena Image Library of Biological Macro-molecules (http://www. imb-jena.de/IMAGE.html ) is aimed at a better dissemination of information on three-dimensional biopolymer structures with an emphasis on visualization and analysis. It provides access to all structure entries deposited at the Protein Data Bank (PDB) and Nucleic Acid Database (NDB). By combining automatic and manual processing it is possible to keep pace with the rapidly growing number of known biopolymer structures and to provide, for selected entries, information not available from automatic procedures. Each entry page contains basic information on the structure, various visualization and analysis tools as well as links to other databases. The visualization techniques adopted include static mono/stereo raster or vector graphics representations, virtual reality modeling (VRML), RasMol/Chime scripts and Java applets. A helix and bending analysis tool provides consistent information on about 750 DNA and RNA duplex structures. Access to metal-containing PDB entries is possible via the Periodic Table of Elements. Finally, general information on amino acids, cis -peptide bonds, structural elements in proteins, base pairs, nucleic acid model conformations and experimental methods for biopolymer structure determination is provided.

Molecular modelling and footprinting studies of DNA minor groove binders: bisquaternary ammonium heterocyclic compounds.

We report new quantitative footprinting data which reveal differences in binding constants of bisquaternary ammonium heterocyclic compounds (BQA) with AT-rich DNA sites depending on the ligand structure and on the size and sequence of the DNA binding site. In an attempt to understand the dependence of binding affinity on the ligand structure we have performed quantum-chemical AM1 calculations on the BQA compounds and on subunits to explore the conformational space and to calculate the electronic and structural features of individual ligand conformations. Due to the properties of the rotatable backbone bonds, there is a large number of possible conformations with almost equal energy. We present a new method for the calculation of the radius of curvature of molecular structures. Assuming that strong binders should have a shape complementary to the DNA minor groove, this measure is used to select the optimum conformations for DNA-drug binding. The approach yields the correct ligand conformation for SN6999, for which an X-ray DNA-drug structure is known. The curvature of the optimum conformations of all ligands is compared with the experimental binding constants. A correlation is found between curvature and binding constant provided other structural factors do not vary. Therefore, we conclude that within structurally similar BQA compounds the extent of curvature is the relevant quantity which modulates the binding affinity.

Recognition of GC base pairs by triplex forming oligonucleotides containing nucleosides derived from 2-aminopyridine.

We have attempted to alleviate the pH dependency of triplex recognition of guanine by using intermolecular triplexes containing 2-amino-5-(2-deoxy-d-ribofuranosyl)pyridine (AP) as an analogue of 2'-deoxycytidine (dC). We find that for the beta-anomer of AP, the complex between (AP)6T6and the target site G6A6*T6C6is stable, generating a clear DNase I footprint at oligonucleotide concentrations as low as 0.25 microM at pH 5.0, in contrast to 50 microM C6T6which has no effect on the cleavage pattern. This complex is still stable at pH 6.5 producing a footprint with 1 microM oligonucleotide. Oligonucleotides containing the alpha-anomer of AP are much less effective than the beta-anomer, though in some instances they are more stable than the unmodified oligonucleotides. The results of molecular dynamics studies on a range of AP-containing triplexes has rationalized the observed stability behaviour in terms of hydrogen-bonding behaviour.