Genetic diversity among community methicillin-resistant Staphylococcus aureus strains causing outpatient infections in Australia

Increasing reports of the appearance of novel nonmultiresistant methicillin-resistant Staphylococcus aureus MRSA (MRSA) strains in the community and of the spread of hospital MRSA strains into the community are cause for public health concern. We conducted two national surveys of unique isolates of S. aureus from clinical specimens collected from nonhospitalized patients commencing in 2000 and 2002, respectively. A total of 11.7% of 2,498 isolates from 2000 and 15.4% of 2,486 isolates from 2002 were MRSA. Approximately 54% of the MRSA isolates were nonmultiresistant (resistant to less than three of nine antibiotics) in both surveys. The majority of multiresistant MRSA isolates in both surveys belonged to two strains (strains AUS-2 and AUS-3), as determined by pulsed-field gel electrophoresis (PFGE) and resistogram typing. The 3 AUS-2 isolates and 10 of the 11 AUS-3 isolates selected for multilocus sequence typing (MLST) and staphylococcal chromosomal cassette mec (SCCmec) analysis were ST239-MRSA-III (where ST is the sequence type) and thus belonged to the same clone as the eastern Australian MRSA strain of the 1980s, which spread internationally. Four predominant clones of novel nonmultiresistant MRSA were identified by PFGE, MLST, and SCCmec analysis: ST22-MRSA-IV (strain EMRSA-15), ST1-MRSA-IV (strain WA-1), ST30-MRSA-IV (strain SWP), and ST93-MRSA-IV (strain Queensland). The last three clones are associated with community acquisition. A total of 14 STs were identified in the surveys, including six unique clones of novel nonmultiresistant MRSA, namely, STs 73, 93, 129, 75, and 80sIv and a new ST. SCCmec types IV and V were present in diverse genetic backgrounds. These findings provide support for the acquisition of SCCmec by multiple lineages of S. aureus. They also confirm that both hospital and community strains of MRSA are now common in nonhospitalized patients throughout Australia.

mecA locus diversity in methicillin-resistant Staphylococcus aureus isolates in Brisbane, Australia, and the development of a novel diagnostic procedure for the Western Samoan phage pattern clone

An emerging public health phenomenon is the increasing incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections that are acquired outside of health care facilities. One lineage of community-acquired MRSA (CA-MRSA) is known as the Western Samoan phage pattern (WSPP) clone. The central aim of this study was to develop an efficient genotyping procedure for the identification of WSPP isolates. The approach taken was to make use of the highly variable region downstream of mecA in combination with a single nucleotide polymorphism (SNP) defined by the S. aureus multilocus sequence typing (MLST) database. The premise was that a combinatorial genotyping method that interrogated both a highly variable region and the genomic backbone would deliver a high degree of informative power relative to the number of genetic polymorphisms-interrogated. Thirty-five MRSA isolates were used for this study, and their gene contents and order downstream of mecA were determined. The CA-MRSA isolates were found to contain a truncated mecA downstream region consisting of mecA-HVR-IS431 mec-dcs-Ins117, and a PCR-based method for identifying this structure was developed. The hospital-acquired isolates were found to contain eight different mecA downstream regions, three of which were novel. The Minimum SNPs computer software program was used to mine the S. aureus MLST database, and the arcC 2726 polymorph was identified as 82% discriminatory for ST-30. A real-time PCR assay was developed to interrogate this SNP. We found that the assay for the truncated mecA downstream region in combination with the interrogation of arcC position 272 provided an unambiguous identification of WSPP isolates.

Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms

The aim of this study was to identify a set of genetic polymorphisms that efficiently divides methicillin-resistant Staphylococcus aureus (MRSA) strains into groups consistent with the population structure. The rationale was that such polymorphisms could underpin rapid real-time PCR or low-density array-based methods for monitoring MRSA dissemination in a cost-effective manner. Previously, the authors devised a computerized method for identifying sets of single nucleoticle polymorphisms (SNPs) with high resolving power that are defined by multilocus sequence typing (MLST) databases, and also developed a real-time PCR method for interrogating a seven-member SNP set for genotyping S. aureus. Here, it is shown that these seven SNPs efficiently resolve the major MRSA lineages and define 27 genotypes. The SNP-based genotypes are consistent with the MRSA population structure as defined by eBURST analysis. The capacity of binary markers to improve resolution was tested using 107 diverse MRSA isolates of Australian origin that encompass nine SNP-based genotypes. The addition of the virulence-associated genes cna, pvl and bbplsdrE, and the integrated plasmids pT181, p1258 and pUB110, resolved the nine SNP-based genotypes into 21 combinatorial genotypes. Subtyping of the SCCmec locus revealed new SCCmec types and increased the number of combinatorial genotypes to 24. It was concluded that these polymorphisms provide a facile means of assigning MRSA isolates into well-recognized lineages.

Staphylococcus aureus genotyping using novel real-time PCR formats

One approach to microbial genotyping is to make use of sets of single-nucleotide polymorphisms (SNPs) in combination with binary markers. Here we report the modification and automation of a SNP-plus-binary-marker-based approach to the genotyping of Staphylococcus aureus and its application to 391 S. aureus isolates from southeast Queensland, Australia. The SNPs used were arcC210, tpi243, arcC162, gmk318, pta294, tpi36, tpi241, and pta383. These provide a Simpson's index of diversity (D) of 0.95 with respect to the S. aureus multilocus sequence typing database and define 61 genotypes and the major clonal complexes. The binary markers used were pvl, cna, sdrE, pT181, and pUB110. Two novel real-time PCR formats for interrogating these markers were compared. One of these makes use of light upon extension (LUX) primers and biplexed reactions, while the other is a streamlined modification of kinetic PCR using SYBR green. The latter format proved to be more robust. In addition, automated methods for DNA template preparation, reaction setup, and data analysis were developed. A single SNP-based method for ST-93 (Queensland clone) identification was also devised. The genotyping revealed the numerical importance of the South West Pacific and Queensland community-acquired methicillin-resistant S. aureus (MRSA) clones and the clonal complex 239 Aus-1/Aus-2 hospital-associated MRSA. There was a strong association between the community-acquired clones and pvl.

Methicillin-resistant Staphylococcus aureus in the Australian community: an evolving epidemic

Objective: To describe antimicrobial resistance and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) isolated in community settings in Australia. Design and setting: Survey of S. aureus isolates collected prospectively Australia-wide between July 2004 and February 2005; results were compared with those of similar surveys conducted in 2000 and 2002. Main outcome measures: Up to 100 consecutive, unique clinical isolates of S. aureus from outpatient settings were collected at each of 22 teaching hospital and five private laboratories from cities in all Australian states and territories. They were characterised by antimicrobial susceptibilities (by agar dilution methods), coagulase gene typing, pulsed-field gel electrophoresis, multilocus sequence typing, SCCmec typing and polymerase chain reaction tests for Panton-Valentine leukocidin (PVL) gene. Results: 2652 S. aureus isolates were collected, of which 395 (14.9%) were MRSA. The number of community-associated MRSA (CA-MRSA) isolates rose from 4.7% (118/2498) of S. aureus isolates in 2000 to 7.3% (194/2652) in 2004 (P=0.001). Of the three major CA-MRSA strains, WA-1 constituted 45/257 (18%) of MRSA in 2000 and 64/395 (16%) in 2004 (P=0.89), while the Queensland (OLD) strain increased from 13/257 (5%) to 58/395 (15%) (P=0.0004), and the south-west Pacific (SWP) strain decreased from 33/257 (13%) to 26/395 (7%) (P=0.01). PVL genes were detected in 90/195 (46%) of CA-MRSA strains, including 5/64 (8%) of WA-1, 56/58 (97%) of OLD, and 25/26 (96%) of SWP strains. Among health care-associated MRSA strains, all AUS-2 and AUS-3 isolates were multidrug-resistant, and UK EMRSA-15 isolates were resistant to ciprofloxacin and erythromycin (50%) or to ciprofloxacin alone (44%). Almost all (98%) of CA-MRSA strains were non-multiresistant. Conclusions: Community-onset MRSA continues to spread throughout Australia. The hypervirulence determinant PVL is often found in two of the most common CA-MRSA strains. The rapid changes in prevalence emphasise the importance of ongoing surveillance.

Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin genes: Worldwide emergence

Infections caused by community-acquired (CA)-methicillin-resistant Staphylococcus aureus (MRSA) have been reported worldwide. We assessed whether any common genetic markers existed among 117 CA-MRSA isolates from the United States, France, Switzerland, Australia, New Zealand, and Western Samoa by performing polymerase chain reaction for 24 virulence factors and the methicillin-resistance determinant. The genetic background of the strain was analyzed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The CA-MRSA strains shared a type IV SCCmec cassette and the Panton-Valentine leukocidin locus, whereas the distribution of the other toxin genes was quite specific to the strains from each continent. PFGE and MLST analysis indicated distinct genetic backgrounds associated with each geographic origin, although predominantly restricted to the agr3 background. Within each continent, the genetic background of CA-MRSA strains did not correspond to that of the hospital-acquired MRSA.

Application of inter simple sequence repeat (ISSR) markers to plant genetics

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single-locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5'-anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)(n), can be made with a degenerate 3'-anchor, such as (CA)(8)RG or (AGC)(6)TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with P-32 or P-33 via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.

Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system

Current serotyping methods classify Pasteurella multocida into five capsular serogroups (serogroups A, B, D, E, and F) and 16 somatic serotypes (serotypes 1 to 16). In the present study, we have developed a multiplex PCR assay as a rapid alternative to the conventional capsular serotyping system. The serogroup-specific primers used in this assay were designed following identification, sequence determination, and analysis of the capsular biosynthetic loci of each capsular serogroup. The entire capsular biosynthetic loci of P. multocida A:1 (X-73) and B:2 (M1404) have been cloned and sequenced previously (J. Y. Chung, Y. M. Zhang, and B. Adler, FEMS Microbiol. Lett. 166:289-296, 1998; J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121-134, 2000). Nucleotide sequence analysis of the biosynthetic region (region 2) from each of the remaining three serogroups, serogroups D, E, and F, identified serogroup-specific regions and gave an indication of the capsular polysaccharide composition. The multiplex capsular PCR assay was highly specific, and its results, with the exception of those for some serogroup F strains, correlated well with conventional serotyping results. Sequence analysis of the strains that gave conflicting results confirmed the validity of the multiplex PCR and indicated that these strains were in fact capsular serogroup A. The multiplex PCR will clarify the distinction between closely related serogroups A and F and constitutes a rapid assay for the definitive classification of P. multocida capsular types

Functional recycling of C2 domains throughout evolution: A comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches

The C2 domain is one of the most frequent and widely distributed calcium-binding motifs. Its structure comprises an eight-stranded beta-sandwich with two structural types as if the result of a circular permutation. Combining sequence, structural and modelling information, we have explored, at different levels of granularity, the functional characteristics of several families of C2 domains. At the coarsest level,the similarity correlates with key structural determinants of the C2 domain fold and, at the finest level, with the domain architecture of the proteins containing them, highlighting the functional diversity between the various subfamilies. The functional diversity appears as different conserved surface patches throughout this common fold. In some cases, these patches are related to substrate-binding sites whereas in others they correspond to interfaces of presumably permanent interaction between other domains within the same polypeptide chain. For those related to substrate-binding sites, the predictions overlap with biochemical data in addition to providing some novel observations. For those acting as protein-protein interfaces' our modelling analysis suggests that slight variations between families are a result of not only complementary adaptations in the interfaces involved but also different domain architecture. In the light of the sequence and structural genomic projects, the work presented here shows that modelling approaches along with careful sub-typing of protein families will be a powerful combination for a broader coverage in proteomics. (C) 2003 Elsevier Ltd. All rights reserved.

Antigenic and genetic typing of Whataroa viruses in Australia

We recently characterized three novel alphaviruses isolated from mosquitoes captured in New South Wales, Australia. Initial cross-neutralization studies revealed antigenic similarity to the Sindbis virus (SINV)-like Whataroa virus (WHAV), heretofore found only in New Zealand. Nucleotide sequence analysis showed that the WHAV-Iike viruses shared >99% nucleotide sequence similarity with each other, and 96-97% similarity with prototype WHAV. Enzyme-linked immunosorbent assay reactions of a panel of monoclonal antibodies to SINV showed that the novel WHAV-Iike viruses displayed identical binding patterns and were antigenically distinct from all SINV isolates examined. Although these viruses displayed a similar binding pattern to prototype WHAV, three monoclonal antibodies discriminated them from the New Zealand virus. Our results suggest that these novel alphaviruses are antigenic variants of WHAV and represent the first reported isolations of this virus from outside New Zealand. The monoclonal antibodies used in this study will be useful for typing new SINV and SINV-like isolates.

Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.

Algorithms for sequence analysis via mutagenesis

Despite many successes of conventional DNA sequencing methods, some DNAs remain difficult or impossible to sequence. Unsequenceable regions occur in the genomes of many biologically important organisms, including the human genome. Such regions range in length from tens to millions of bases, and may contain valuable information such as the sequences of important genes. The authors have recently developed a technique that renders a wide range of problematic DNAs amenable to sequencing. The technique is known as sequence analysis via mutagenesis (SAM). This paper presents a number of algorithms for analysing and interpreting data generated by this technique.

Unlocking hidden genomic sequence

Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and has been used to read previously intractable poly(A), AT-rich and GC-rich motifs.

Bellerophon: a program to detect chimeric sequences in multiple sequence alignments

Bellerophon is a program for detecting chimeric sequences in multiple sequence datasets by an adaption of partial treeing analysis. Bellerophon was specifically developed to detect 16S rRNA gene chimeras in PCR-clone libraries of environmental samples but can be applied to other nucleotide sequence alignments.