Single nucleotide polymorphism (SNP) - genotyping of Community Acquired Methicillin-Resistant Staphylococcus aureus, including the subtyping of PVL toxin producers using Real-Time PCR

Staphylococcus aureus is a common pathogen that causes a variety of infections including soft tissue infections, impetigo, septicemia toxic shock and scalded skin syndrome. Traditionally, Methicillin-Resistant Staphylococcus aureus (MRSA) was considered a Hospital-Acquired (HA) infection. It is now recognised that the frequency of infections with MRSA is increasing in the community, and that these infections are not originating from hospital environments. A 2007 report by the Centers for Disease Control and Prevention (CDC) stated that Staphylococcus aureus is the most important cause of serious and fatal infections in the USA. Community-Acquired MRSA (CA-MRSA) are genetically diverse and distinct, meaning they are able to be identified and tracked by way of genotyping. Genotyping of MRSA using Single nucleotide polymorphisms (SNPs) is a rapid and robust method for monitoring MRSA, specifically ST93 (Queensland Clone) dissemination in the community. It has been shown that a large proportion of CA-MRSA infections in Queensland and New South Wales are caused by ST93. The rationale for this project was that SNP analysis of MLST genes is a rapid and cost-effective method for genotyping and monitoring MRSA dissemination in the community. In this study, 16 different sequence types (ST) were identified with 41% of isolates identified as ST93 making it the predominate clone. Males and Females were infected equally with an average patient age of 45yrs. Phenotypically, all of the ST93 had an identical antimicrobial resistance pattern. They were resistant to the β-lactams – Penicillin, Flu(di)cloxacillin and Cephalothin but sensitive to all other antibiotics tested. Virulence factors play an important role in allowing S. aureus to cause disease by way of colonising, replication and damage to the host. One virulence factor of particular interest is the toxin Panton-Valentine leukocidin (PVL), which is composed of two separate proteins encoded by two adjacent genes. PVL positive CA-MRSA are shown to cause recurrent, chronic or severe skin and soft tissue infections. As a result, it is important that PVL positive CA-MRSA is genotyped and tracked. Especially now that CA-MRSA infections are more prevalent than HA-MRSA infections and are now deemed endemic in Australia. 98% of all isolates in this study tested positive for the PVL toxin gene. This study showed that PVL is present in many different community based ST, not just ST93, which were all PVL positive. With this toxin becoming entrenched in CA-MRSA, genotyping would provide more accurate data and a way of tracking the dissemination. PVL gene can be sub-typed using an allele-specific Real-Time PCR (RT-PCR) followed by High resolution meltanalysis. This allows the identification of PVL subtypes within the CA-MRSA population and allow the tracking of these clones in the community.

Chi viaggia ha delle storie da raccontare : those who travel have stories to tell

This review article uses the work of Italian scholar Milly Buonanno to review the state and future of television scholarship, given that the ‘age of television’ has been overtaken by the age of computer-based media. In particular, it discusses the role of open-ended narrative through which we collectively explore the human condition.

Diversity of community acquired MRSA carrying the PVL gene in Queensland and New South Wales, Australia

Emergence and dissemination of community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strains are being reported with increasing frequency in Australia and worldwide. These strains of CA-MRSA are genetically diverse and distinct in Australia. Genotyping of CA-MRSA using eight highly-discriminatory single nucleotide polymorphisms (SNPs) is a rapid and robust method for monitoring the dissemination of these strains in the community. In this study, a SNP genotyping method was used to investigate the molecular epidemiology of 249 community acquired non-multiresistant MRSA (nm-MRSA) isolates over a 12-month period from routine diagnostic specimens. A real-time PCR for the presence of Panton-Valentine leukocidin (PVL) was also performed on these isolates. The CA-MRSA isolates were sourced from a large private laboratory in Brisbane, Australia that serves a wide geographic region encompassing Queensland and Northern New South Wales. This study identified 16 different STs and 98% of the CA-MRSA isolates were positive for the PVL gene. The most common ST was ST93 with 41% of isolates testing positive for this clone.

Comparing the cost-effectiveness of MRSA control strategies between ICU and non-ICU settings

Introduction / objectives Many strategies are used to control MRSA in hospitals. Only a few have been assessed in clinical trials and it is not obvious how findings should be generalised between settings. Uncertainty remains about which strategies represent the most appropriate use of scarce resources. We assess the cost-effectiveness of alternative MRSA screening and infection control strategies in England and Wales and discuss international relevance. Methods Models of MRSA transmission in ICUs and general medical (GM) wards were developed and used to evaluate different screening methods combined with decolonisation or isolation. Strategies were compared in terms of costs and health benefits (quality adjusted life years, QALYs). Different prevalences, proportions of high risk patients and ward sizes were investigated, and probabilistic sensitivity analyses (PSA) conducted. Results Decolonisation strategies were cost-saving in ICUs at a 5% admission prevalence, with admission and weekly PCR screening the most cost-effective (£3,929/QALY). In ICUs, screening and isolation reduced infection rates by ~10%. With admission prevalence ≤5%, targeting screening and isolation to high risk patients was optimal. In GM wards decolonisation and isolation strategies, though able to reduce MRSA infection rates up to ~50%, were not cost-effective. Conclusion The largest reductions in MRSA infection were achieved by screening and decolonisation strategies, and were cost-effective in ICU settings. In comparison, there is limited potential for screening and control strategies to be cost-effective in GM wards due to lower infection and mortality rates.

Replacement of healthcare-associated MRSA by community-associated MRSA in Queensland : confirmation by genotyping

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.

Typing early Australian healthcare-associated MRSA : confirmation of major clones and emergence of ST1-MRSA-IV and novel ST2249-MRSA-III

AIMS: To investigate the evolutionary origins of Australian healthcare-associated (HCA) methicillin-resistant Staphylococcus aureus (MRSA) strains from a panel of historical isolates typed using current genotyping techniques. METHODS: Nineteen MRSA isolates from 1965 to 1981 were examined and antibiotic susceptibility profiles determined. Genetic characterisation included real-time (RT) polymerase chain reaction (PCR) assays to identify single nucleotide polymorhpism (SNP) clonal complexes (SNP CC) and sequence type (SNP ST), multi locus sequence typing (MLST) and staphylococcal chromosomal cassette mec typing. RESULTS: All SNP CC30 isolates belonged to a novel sequence type, ST2249. All SNP CC239 isolates were confirmed as ST239-MRSA-III, except for a new single locus variant of ST239, ST2275. A further new type, ST2276, was identified. CONCLUSIONS: The earliest MRSA examined from 1965 was confirmed as ST250-MRSA-I, consistent with archaic European types. Identification of ST1-MRSA-IV in 1981 is the earliest appearance of this clinically important lineage which manifested in Australia and the United States in the 1990s. A previously unknown multi-resistant clone, ST2249-MRSA-III, was identified from 1973. Gentamicin resistance first appeared in this novel strain from 1976 and not ST239 as previously suspected. Thus, ST2249 was present in the earliest phase of the HCA MRSA epidemic in eastern Australia and was perhaps related to the emergence of the globally epidemic strain ST239.

Invasive phenotype of MCF10A cells overexpressing c-Ha-ras and c-erbB-2 oncogenes

Infection with erbB-2 (E) of Ha-ras (H) oncogene-transfected cells has been previously shown to cooperatively induce anchorage-independent growth of the MCF10A human mammary epithelial cell line in vitro, but not to induce nude mouse tumorigenicity. Here we show that oncogene-transformed MCF10A are able to halt in the lungs of nude mice, a sign of organ colonization potential. We have therefore studied the transformants for in vitro migratory and invasive properties known to correlate with the metastatic potential of human mammary carcinoma cells in nude mice. MCF10A transfected with Ha-ras, infected with a recombinant retroviral vector containing the human c-erB-2 proto-oncogene (MCF10A-HE cells), show a higher invasive index than either the single transfectant (MCF10A-H) or MCF10A-erB-2(MCF10A-E) cells in the Boyden chamber chemotaxis and chemoinvasion assays. The MCF10A-HE cells also adopted an invasive stellate growth pattern when plated or embedded in Matrigel, in contrast to the spherical colonies formed by the single transformants MCF10A-H, MCF10A-E, and the parental cells. Dot-blot analysis of gelatinase A and TIMP-2 mRNA levels revealed increasing gelatinase A mRNA levels (HE > E > H > MCF10A) and reduced TIMP-2 expression in both single and double transformants. Furthermore, MCF10A-HE cells show more MMP-2 activity than parental MCF10A cells or the single transformants. CD44 analysis revealed differential isoform banding for the MCF10A-HE cells compared to parental cells, MCF10A-H and MCF10A-E, accompanied by increased binding of hyaluronan by the double transformants. Our results indicate that erB-2 and Ha-ras co-expression can induce a more aggressive phenotype in vitro, representative of the malignancy of mammary carcinomas.

Evidence for a simple linear relationship between MRSA rates and hand-washing compliance [Comment]

Is there a threshold above which hand-rub solution consumption is efficient for decreasing MRSA incidence? [J Hosp Infect. 2009] Association between an index of consumption of hand-rub solution and the incidence of acquired meticillin-resistant Staphylococcus aureus in an intensive care unit.

An Australian native plant: A novel antimicrobial for the treatment of MRSA

Chronic wounds, often associated with venous and arterial ulcers, diabetes and pressure sores, is an area of great concern. In Australia, the cost of treating chronic wounds is conservatively estimated at $285 million/annum for the treatment of pressure ulcers and $654 million annually for the treatment and management of leg ulcers. Current figures indicate that more than seven million people suffer from chronic wounds worldwide with Australians accounting for approximately 600,000 of this number. Bacterial infection of the wound site is a major issue as contamination of a chronic wound with methicillin-resistant Staphylococcus aureus (MRSA) significantly delays wound healing. Further, once systemic, current antibiotic therapies capable of treating the infection are limited. Aboriginal bush medicine has been used for thousands of years for the treatment of wounds and sores. Hence, we selected a native Australian plant to evaluate its bactericidal activity against MRSA.