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Staphylococcus aureus infections of the central nervous system are serious and can be fatal. S. aureus is commonly present in the nasal cavity, and after injury to the nasal epithelium it can rapidly invade the brain via the olfactory nerve. The trigeminal nerve constitutes another potential route of brain infection. The glia of these nerves, olfactory ensheathing cells (OECs) and trigeminal nerve Schwann cells (TgSCs), as well as astrocytes populating the glia limitans layer, can phagocytose bacteria. Whilst some glial responses to S. aureus have been studied, the specific responses of different glial types are unknown. Here, we compared how primary mouse OECs, TgSCs, astrocytes and microglia responded to S. aureus. All glial types internalized the bacteria within phagolysosomes, and S. aureus-conjugated BioParticles could be tracked with subtle but significant differences in time-course of phagocytosis between glial types. Live bacteria could be isolated from all glia after 24 h in culture, and microglia, OECs and TgSCs exhibited better protection against intracellular S. aureus survival than astrocytes. All glial types responded to the bacteria by cytokine secretion. Overall, OECs secreted the lowest level of cytokines, suggesting that these cells, despite showing strong capacity for phagocytosis, have immunomodulatory functions that can be relevant for neural repair.

Purpose: Dressings containing chlorhexidine gluconate (CHG) are increasingly used in clinical environments for prevention of infection at central venous catheter insertion sites. Increased tolerance to this biocide in staphylococci is primarily associated with the presence of qacA/B and smr genes., Methodology: We used a culture-independent method to assess the prevalence of these genes in 78 DNA specimens recovered from the skin of 43 patients at catheter insertion sites in the arm that were covered with CHG dressings., Results: Of the 78 DNA specimens analysed, 52 (67 %) possessed qacA/B and 14 (18 %) possessed smr; all samples positive for smr were also positive for qacA/B. These prevalence rates were not statistically greater than those observed in a subsample of specimens taken from non-CHG treated contralateral arms and non-CHG-dressing exposed arms. A statistically greater proportion of specimens with greater than 72 h exposure to CHG dressings were qac-positive (P=0.04), suggesting that the patients were contaminated with bacteria or DNA containing qacA/B during their hospital stay. The presence of qac genes was not positively associated with the presence of DNA specific for Staphylococcusepidermidis and Staphylococcusaureus in these specimens., Conclusion: Our results show that CHG genes are highly prevalent on hospital patients' skin, even in the absence of viable bacteria.

The aim was to determine the evolutionary position of the Staphylococcus aureus clonal complex 75 (CC75) that is prevalent in tropical northern Australia. Sequencing of gap, rpoB, sodA, tuf, and hsp60 and the multilocus sequence typing loci revealed a clear separation between conventional S. aureus and CC75 and significant diversity within CC75.

Background: The staphylococcal protein A (spa) locus of Staphylococcus aureus contains a complex repeat structure and is commonly used for single-locus sequence-based genotyping. The real-time PCR platform supports genotyping methods that are single step and closed tube and potentially can be carried out simultaneously with diagnosis. We describe here a method for genotyping S. aureus using high-resolution melting (HRM) analysis of the spa polymorphic region X., Methods: The conventional PCR spa assay was modified and optimized for the Rotor-Gene 6000 instrument (Corbett Life Science). HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 22 known spa sequences obtained from 44 diverse methicillin-resistant S. aureus (MRSA) isolates. Criteria for calling pairs of melting curves "same" or "different" were developed empirically by converting the data to difference graph format with one curve defined as the control. HRM curve comparison between runs was done to determine the portability of the method. The assay performance was assessed by genotyping uncharacterized isolates, carrying out blind trials, and comparing HRM profiles from different runs., Results: HRM analysis of 44 diverse MRSA isolates generated 20 profiles from 22 spa sequence types. The 2 unresolved HRM spa types differed by only 1 bp. Two blind trials demonstrated complete reproducibility with respect to calling the different spa types. Interrun comparisons of HRM curves were successfully developed, indicating the robustness of the method., Conclusion: Analysis of the spa locus by HRM resolves spa sequence variants. This single- and closed-tube single-step method for S. aureus genotyping can be easily combined with the interrogation of other genetic markers.

The aim of this study was to identify optimized sets of genotyping targets for the staphylococcal cassette chromosome mec (SCCmec). We analyzed the gene contents of 46 SCCmec variants in order to identify minimal subsets of targets that provide useful resolution. This was achieved by firstly identifying and characterizing each available SCCmec element based on the presence or absence of 34 binary targets. This information was used as input for the software "Minimum SNPs," which identifies the minimum number of targets required to differentiate a set of genotypes up to a predefined Simpson's index of diversity (D) value. It was determined that 22 of the 34 targets were required to genotype the 46 SCCmec variants to a D of 1. The first 6, 9, 12, and 15 targets were found to define 21, 29, 35, and 39 SCCmec variants, respectively. The genotypes defined by these marker subsets were largely consistent with the relationships between SCCmec variants and the accepted nomenclature. Consistency was made virtually complete by forcing the computer program to include ccr1 and ccr5 in the target set. An alternative target set biased towards discriminating abundant SCCmec variants was derived by analyzing an input file in which common SCCmec variants were repeated, thus ensuring that markers that discriminate abundant variants had a large effect on D. Finally, it was determined that mecA single nucleotide polymorphisms (SNPs) can increase the overall genotyping resolution, as different mecA alleles were found in otherwise identical SCCmec variants.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged as a major public health problem in Australia, as in many other parts of the world. High rates of CA-MRSA skin and soft tissue infection have been reported from Aboriginal communities. We used a single-nucleotide polymorphism (SNP) genotyping typing system based on the multilocus sequence type (MLST) database to investigate the epidemiology of CA-MRSA and methicillin-sensitive S. aureus (MSSA) over a 12-month period in three remote Aboriginal communities of Northern Australia. This was supplemented by real-time PCR for Panton-Valentine leukocidin (PVL) genes, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial susceptibility testing. S. aureus was recovered from pyoderma lesions on 221 occasions and throat swabs on 44 occasions. The median monthly recovery rate of S. aureus from skin sores was 58% (interquartile range, 62 to 78%), and there was no seasonal variation. Twenty-three percent of isolates were CA-MRSA; the proportion was similar across the communities and did not vary over the study period. Erythromycin resistance was found in 47% of CA-MRSA and 21% of MSSA. SNP-based typing identified 14 different clonal complexes (cc); however, cc75 was predominant, accounting for 71% of CA-MRSA isolates. These were confirmed as ST75-like by using an additional SNP and MLST of selected isolates. All but one of the cc75 isolates had SSCmec type IV (one had type V), and all were PVL negative. Monthly tracking of SNP-based cc types showed a highly dynamic process. ST75-MRSA-IV appears to be unique to the region and probably evolved de novo in remote Aboriginal communities.

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.

Objective: To determine the prevalence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) carriage and infection among children living in an Indigenous community in Queensland., Design, Setting and Participants: Swabs for culture of S. aureus were collected from the nose, throat and skin wounds of primary school children., Main Outcome Measures: MRSA carriage, antibiotic sensitivity, genotype, and presence of the virulence factor Panton-Valentine leukocidin (PVL); and epidemiological risk factors for MRSA carriage., Results: 92 (59%) of 157 eligible children were included in the study. Twenty-seven (29%) carried S. aureus; 14 of these (15% of total) carried MRSA. MRSA was isolated from 29% of wound swabs, 8% of nose swabs, and 1% of throat swabs. Fourteen of 15 MRSA isolates were sensitive to all non-beta-lactam antibiotics tested. Eight children (9%) carried CA-MRSA clonal types: six carried the Queensland clone (ST93), and two carried the South West Pacific clone (ST30). All these isolates carried the virulence factor PVL. The remaining six children carried a hospital-associated MRSA strain (ST5), negative for PVL., Conclusions: We have identified a high prevalence of CA-MRSA carriage in school children from a Queensland Indigenous community. In this setting, antibiotics with activity against CA-MRSA should be considered for empiric therapy of suspected staphylococcal infection. Larger community-based studies are needed to improve our understanding of the epidemiology of CA-MRSA, and to assist in the development of therapeutic guidelines for this important infection.

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 nucleotide 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 bbp/sdrE, and the integrated plasmids pT181, pI258 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.

Background: A dramatic increase in multiple-drug-resistant (MDR) Gram-positive pathogens has occurred in recent times, leading to increased rates or morbidity and mortality and also associated with high costs for the treatment of these infections. It is clear that there is an urgent need for the development of effective antimicrobial agents. The anti-bacterial activity of seven 2,2,6,6-tetramethylpiperidine (TMP)-substituted phenazines, compared to clofazimine (B663), were tested against 70 clinical isolates of methicillin-resistant Staphylococcus aureus, MDR Streptococcus pneumoniae and resistant Enterococcus sp., Methods: Standard minimum inhibitory concentration agar dilution susceptibility tests were done on all isolates, including ATCC control strains., Results: All the TMP-substituted phenazines were more active than clofazimine against all isolates tested. Compound B4125 was the most active by inhibiting all growth of the organisms tested, including vancomycin-resistant Enterococcus faecium., Conclusion: Clofazimine has been shown to have anti-staphylococcal activity. We demonstrate enhanced anti-bacterial activity of TMP-substituted phenazines against drug-resistant Gram-positive organisms compared to clofazimine.

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 80slv 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.

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 272G 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.

A unified, bioinformatics-driven, single nucleotide polymorphism (SNP)-based approach to microbial genotyping has been developed. Multilocus sequence typing (MLST) databases consist of known variants of standardized housekeeping genes. Normally, seven fragments are defined; a sequence type (ST) consists of the variants of these fragments that are found in a particular isolate. A computer program that can identify highly informative sets of SNPs in entire MLST databases has been constructed. The SNPs either define a particular user-specified ST or provide a high value for Simpson's index of diversity (D), and may thus be generally applicable to that species. SNP sets that are diagnostic for Neisseria meningitidis ST-11 and ST-42, and high-D SNP sets for N. meningitidis and Staphylococcus aureus, were identified and real-time PCR methods to interrogate these SNPs were demonstrated. High-D SNP sets were also identified in other MLST databases. This widely applicable approach allows rapid genetic fingerprinting of infectious agents.

The region surrounding mecA in methicillin-resistant Staphylococcus aureus (MRSA) is highly variable. We describe an approach for the rapid genotyping of MRSA by assaying for the presence or absence of variable or mobile elements previously shown to be associated with the mecA region.

Background: Sequencing highly-variable 16S regions is a common and often effective approach to the study of microbial communities, and next-generation sequencing (NGS) technologies provide abundant quantities of data for analysis. However, the speed of existing analysis pipelines may limit our ability to work with these quantities of data. Furthermore, the limited coverage of existing 16S databases may hamper our ability to characterise these communities, particularly in the context of complex or poorly studied environments. Results: In this article we present the SigClust algorithm, a novel clustering method involving the transformation of sequence reads into binary signatures. When compared to other published methods, SigClust yields superior cluster coherence and separation of metagenomic read data, while operating within substantially reduced timeframes. We demonstrate its utility on published Illumina datasets and on a large collection of labelled wound reads sourced from patients in a wound clinic. The temporal analysis is based on tracking the dominant clusters of wound samples over time. The analysis can identify markers of both healing and non-healing wounds in response to treatment. Prominent clusters are found, corresponding to bacterial species known to be associated with unfavourable healing outcomes, including a number of strains of Staphylococcus aureus. Conclusions: SigClust identifies clusters rapidly and supports an improved understanding of the wound microbiome without reliance on a reference database. The results indicate a promising use for a SigClust-based pipeline in wound analysis and prediction, and a possible novel method for wound management and treatment. [ABSTRACT FROM AUTHOR]

Summary: Objective: To describe the changing prevalence of healthcare- and community-associated MRSA. Methods: Susceptibility phenotypes of MRSA were observed from 2000 to 2012 using routine susceptibility data. Phenotypic definitions of major clones were validated by genotyping isolates from a nested period prevalence survey in 2011. Results: The predominant healthcare-associated (AUS-2/3 like) MRSA phenotype decreased from 42 to 14 isolates per million occasions of service in outpatients (P < 0.0001) and from 650 to 75 isolates per million accrued patient days in inpatients (P 0.0005), while the respective rates of the healthcare-related EMRSA-15 like phenotype increased from 1 to 19 in outpatients (P < 0.0001) and from 11 to 83 in inpatients (P < 0.0001) and those of the community-associated MRSA phenotype increased from 17 to 296 in outpatients (P < 0.0001) and from 71 to 486 in inpatients (P < 0.0001). When compared with single nucleotide polymorphism genotyping the AUS-2/3 like phenotype had a sensitivity and positive predictive value (PPV) for CC239 of 1 and 0.791 respectively, while the EMRSA-15 like phenotype had a sensitivity and PPV for CC22 of 0.903 and 0.774. PVL-positive CA-MRSA, predominantly ST93 and CC30, accounted for 60.8% of MRSA, while PVL-negative CA-MRSA, mainly CC5 and CC1, accounted for 21.4%. Conclusions: The initially dominant healthcare-associated MRSA clone has been progressively replaced, mainly by four community-associated lineages. [Copyright &y& Elsevier]