Characterisation of Australian MRSA Strains ST75- and ST883-MRSA-IV and Analysis of Their Accessory Gene Regulator Locus

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 autoinducing 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

A Field Guide to Pandemic, Epidemic and Sporadic Clones of Methicillin-Resistant Staphylococcus aureus

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

A Field Guide to Pandemic, Epidemic and Sporadic Clones of Methicillin-Resistant Staphylococcus aureus

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

Genotyping of Chlamydia trachomatis strains from culture and clinical samples using an ompA-based DNA microarray assay.

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 (TM) format for individual samples and the ArrayStrip (TM) 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. (C) 2010 Elsevier Ltd. All rights reserve

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