Metagenomics of Toenail Onychomycosis in Three Victorian Regions of Australia.

Onychomycosis is a fungal disease of the nail that is found worldwide and is difficult to diagnose accurately. This study used metagenomics to investigate the microbiology of 18 clinically diagnosed mycotic nails and two normal nails for fungi and bacteria using the ITS2 and 16S loci. Four mycotic nails were from Bass Coast, six from Melbourne Metropolitan and eight from Shepparton, Victoria, Australia. The mycotic nails were photographed and metagenomically analysed. The ITS2 sequences for T. rubrum and T. interdigitale/mentagrophytes averaged over 90% of hits in 14/18 nails. The high abundance of sequences of a single dermatophyte, compared to all other fungi in a single nail, made it the most likely infecting agents (MLIA). Trichophyton rubrum and T. interdigitale/mentagrophytes were found in Bass Coast and Shepparton while only T. interdigitale/mentagrophytes was found in Melbourne. Two nails with T. interdigitale/mentagrophytes mixed with high abundance non-dermatophyte moulds (NDMs) (Aspergillus versicolor, Acremonium sclerotigenum) were also observed. The two control nails contained chiefly Fusarium oxysporum and Malassezia slooffiae. For bacteria, Staphylococcus epidermidis was in every nail and was the most abundant, including the control nails, with an overall mean rate of 66.01%. Rothia koreensis, Corynebacterium tuberculostearicum, and Brevibacterium sediminis also featured.

Garlic alters the expression of putative virulence factor genes SIR2 and ECE1 in vulvovaginal C. albicans isolates.

Vulvovaginal candidiasis causes sufferers much discomfort. Phytotherapy with garlic has been reported to be a possible alternative form of treatment; however, it is unknown why patients report varying success with this strategy. Fresh garlic extract has been shown to down-regulate the putative virulence gene, SIR2 in C. albicans. Our study aimed to see if previous observations were reproducible for the gene responsible for Candidalysin (ECE1). Two clinical strains from patients with reported variable efficacy of using garlic for the treatment of vulvovaginal candidiasis were compared through biofilm assays and antimicrobial susceptibility. Real-time PCR was used to assess changes in gene expression when exposed to garlic. Treatment with fresh garlic extract and pure allicin (an active compound produced in cut garlic) resulted in a decrease in SIR2 expression in all strains. In contrast, ECE1 expression was up-regulated in a reference strain and an isolate from a patient unresponsive to garlic therapy, while in an isolate from a patient responsive to garlic therapy, down-regulation of ECE1 occurred. Future studies that investigate the effectiveness of phytotherapies should take into account possible varying responses of individual strains and that gene expression may be amplified in the presence of serum.

Predominance of Trichophyton interdigitale Revealed in Podiatric Nail Dust Collections in Eastern Australia.

Toenail onychomycosis caused by dermatophytes is a significant medical and financial worldwide burden. Relatively scant research has been undertaken as to the predominant species and strains causing this condition in Australia, which is a unique isolated continent with diverse geographical, climatic and population regions. Four regions were selected in Eastern Australia: Far North Queensland, Rural Victoria, Melbourne Metropolitan and Tasmania. From each of these areas, communal nail dust bags from podiatric physicians' work were collected and analysed. A total of 32 dust bags were collected: 10 from Far North Queensland, 8 from Melbourne Metropolitan, 8 from Rural Victoria and 6 from Tasmania. Dermatophyte test medium was used to isolate dermatophytes from the dust, and the colonies were subcultured to Potato Dextrose Agar. Of the bags collected, in total 69% were positive for dermatophytes: 40% from Far North Queensland, 75% from Melbourne Metropolitan, 88% from Rural Victoria and 83% from Tasmania. The internal transcribed spacer (ITS) region of ribosomal DNA was used to identify and compare isolates. A total of 148 dermatophyte strains were identified. The predominant species isolated was Trichophyton interdigitale (125 isolates), which was found in all four regions. This species was further subdivided into four ITS genotypes: the first two were present in all regions, but the third was found only in the Melbourne Metropolitan area and the fourth only in Tasmania. Only one strain of Trichophyton rubrum was found and only in Rural Victoria. Eighteen isolates of Arthroderma quadrifidum were cultured from Rural Victoria and Tasmania and were further classified into three ITS genotypes. Some isolates rarely reported in clinical material were identified as Paraphyton cookei, Arthroderma tuberculatum and Arthroderma crocatum. A potentially new species of Arthroderma was also found in Melbourne Metropolitan. These findings reveal a unique dermatophyte fingerprint in toenails for Eastern Australia.

Visibility and transmission: complexities around promoting hand hygiene in young children - a qualitative study.

BACKGROUND: Effective hand hygiene practice can reduce transmission of diseases such as respiratory tract infections (RTIs) and gastrointestinal infections, especially in young children. While hand hygiene has been widely promoted within Australia, primary care providers' (PCPs) and parents' understanding of hand hygiene importance, and their views on hand hygiene in reducing transmission of diseases in the community are unclear. Therefore, the aim of this study was to explore the views of PCPs and parents of young children on their knowledge and practice of hand hygiene in disease transmission. METHODS: Using a cross-sectional qualitative research design, we conducted 30 in-depth interviews with PCPs and five focus groups with parents (n = 50) between June 2014 and July 2015 in Melbourne, Australia. Data were thematically analysed. RESULTS: Participants agreed that hand hygiene practice was important in reducing disease transmissions. However, barriers such as variations of hand hygiene habits, relating visibility to transmission; concerns around young children being obsessed with washing hands; children already being 'too clean' and the need to build their immunity through exposure to dirt; and scepticism that hand hygiene practice was achievable in young children, all hindered participants' motivation to develop good hand hygiene behaviour in young children. CONCLUSION: Despite the established benefits of hand hygiene, sustained efforts are needed to ensure its uptake in routine care. To overcome the barriers identified in this study a multifaceted intervention is needed that includes teaching young children good hand hygiene habits, PCPs prompting parents and young children to practice hand hygiene when coming for an RTI consultation, reassuring parents that effective hand hygiene practice will not lead to abnormal psychological behaviour in their children, and community health promotion education campaigns.

Dissonant views - GPs' and parents' perspectives on antibiotic prescribing for young children with respiratory tract infections.

BACKGROUND: Antibiotics are not recommended for treating uncomplicated respiratory tract infections (RTIs), despite this, antibiotic prescribing for this is widespread. General practitioners (GPs) report parental pressure and fear of losing patients if they do not prescribe antibiotics, however, parental views on antibiotics for RTIs are unclear. Therefore, this study examined GPs' and parents' perceptions regarding antibiotic prescribing for RTIs in young children. METHODS: We conducted semi-structured interviews with 20 GPs, and a survey and focus groups with 50 parents and carers of children under the age of five between June 2014 and July 2015 in Melbourne, Australia. Qualitative data were thematically analysed using NVivo and quantitative data were analysed using SPSS. RESULTS: GPs believed that parents expect antibiotics for RTIs and were more likely to prescribe them if parents were insistent. They believed parents would go elsewhere if they did not prescribe antibiotics. GPs suggested that there would be less conflict if parents were better educated on appropriate antibiotics use. In contrast, parents demonstrated good knowledge of RTIs and appropriate antibiotic use. Their main expectation from GPs was to obtain a diagnosis, discuss management, and receive reassurance that the illness was not serious. Parental satisfaction with GPs was not dependent on receiving antibiotics (r = 0.658, p < 0.001), and they would not seek another GP if antibiotics were not prescribed (r = 0.655, p < 0.001). CONCLUSION: GPs and parents have dissonant views on antibiotic prescribing for RTI in young children. GPs perceived parents wanting a diagnosis and reassurance that their child is not severely ill as pressure to prescribe antibiotic. To overcome these barriers, targeted training for both GPs and parents to improve communication and reassurance that satisfaction is not related to receiving antibiotics may reduce unnecessary antibiotic prescribing for RTI in young children.

The safety and sensitivity of a telemetric capsule to monitor gastrointestinal hydrogen production in vivo in healthy subjects: a pilot trial comparison to concurrent breath analysis.

BACKGROUND: Intestinal gases are currently used for the diagnosis of disorders including small intestinal bacterial overgrowth and carbohydrate malabsorption. AIM: To compare the performance of measuring hydrogen production within the gut directly with the telemetric gas-sensing capsule with that of indirect measurement through breath testing. METHODS: Using standard breath testing protocols, the capsules and breath tests were simultaneously evaluated in a single-blinded trial in 12 healthy subjects. Eight received a single dose of 1.25-40 g inulin and four 20 or 40 g glucose. Safety and reliability of the capsules were also assessed. RESULTS: There were no reported adverse events. All capsules were retrieved and operated without failure. Capsule measurements were in agreement with breath test measurements in magnitude but not in timing; minimal hydrogen production was observed after glucose ingestion and capsule measurements correlated with breath hydrogen after ingestion of 40 g inulin. A dose-dependent increase in concentration of hydrogen was observed from the capsule following ingestion of inulin as low as 1.25 g compared with >10 g for breath measurements. Specifically, the capsule measured >3000 times higher concentrations of hydrogen compared to breath tests, resulting in a signal-to-noise ratio of 23.4 for the capsule compared to 4.2 for the breath test. CONCLUSIONS: The capsule showed high sensitivity and signal-to-noise ratio in measuring luminal hydrogen concentrations, provided information on the site of intestinal gas production, and demonstrated safety and reliability. The capsule has potential for improving diagnostic precision for disorders such as small intestinal bacterial overgrowth.

Why do we not want to recommend influenza vaccination to young children? A qualitative study of Australian parents and primary care providers.

INTRODUCTION: Influenza vaccination has been shown to be safe and effective against influenza and in the prevention of complicating secondary respiratory illnesses. However, its uptake in young children remains low. This study explored the views, attitudes and practices of parents and primary care providers (PCPs) on their knowledge and acceptance of influenza vaccination in children under 5. METHODS: Using a cross-sectional qualitative research design, we conducted 30 in-depth interviews with PCPs (i.e., general practitioners, practice nurses, maternal and child health nurses, and pharmacists) and five focus groups with parents (n = 50) between June 2014 and July 2015 in Melbourne, Australia. Data were thematically analysed. RESULTS: Parents thought the vaccine could cause influenza, and influenza vaccination was not necessary for their children as they needed to build their own 'immunity'. Parents said that they would consider vaccinating their children if recommended by their GP and if the influenza vaccine was part of the immunisation schedule. PCPs also expressed concerns regarding the efficacy of the vaccine as well as out-of-pocket costs incurred by families, and uncertainty regarding the mortality and morbidity of influenza in otherwise healthy children. However, they said they would recommend the vaccine to high-risk groups (e.g. children with chronic disease(s), and asthma). CONCLUSION: Despite the established safety of influenza vaccines, barriers to uptake include concerns regarding the iatrogenic effects of vaccination, its administration schedule, and knowledge of influenza severity. Updated information on influenza and the efficacy of the vaccine, and incorporating influenza vaccination into the immunisation schedule may overcome some of these barriers to increase influenza vaccination in this vulnerable cohort.

Management of respiratory tract infections in young children-A qualitative study of primary care providers' perspectives.

Respiratory tract infections in young children are the most common cause of general practice visits in Australia. Despite the availability of clinical practice guidelines, the treatment and management of respiratory tract infections in young children is inconsistent. The aim of the study was to explore the management of respiratory tract infections in young children from a multi-disciplinary perspective using across-sectional qualitative research design based on the theoretical domains framework and the Capability, Opportunity and Motivation-B model. In-depth interviews were conducted with 30 primary care providers to explore their knowledge, views and management of respiratory tract infections in young children. Interviews focused on symptomatic management, over-the-counter medications and antibiotic use, and data were thematically analysed. Our findings showed that factors such as primary care providers' time constraints, parental anxiety, general practitioners' perception of what parents want, perceived parental pressure, and fear of losing patients were some of the reasons why primary care providers did not always adhere to guideline recommendations. Primary care providers also provided conflicting advice to parents concerning over-the-counter medications and when children should resume normal activities. Overall, this study showed that complex interactions involving emotional and psychological factors influenced the decision making process of primary care providers' management of respiratory tract infections in young children. A team care approach with consistent advice, and improved communication between primary care providers and parents is vital to overcome some of these barriers and improve guideline adherence. The findings of this research will inform the development of interventions to better manage respiratory tract infections in young children. RESPIRATORY TRACT INFECTIONS: CLINICIANS SWAYED BY PARENTAL ANXIETY AND PRESSURE: The emotions and psychology of both parents and clinicians influence how respiratory tract infections (RTIs) are managed in young children. Researchers in Australia, led by Ruby Biezen from Monash University, interviewed 30 primary care clinicians about their views on how to care for children with RTIs, such as the common cold. The interviews focused on symptomatic management, over-the-counter medications and antibiotic use. Despite the availability of best-practice guidelines, clinicians did not always follow the recommendations owing to factors such as time constraints, parental anxiety, perceived parental pressure, and fear of losing patients. These are some of the reasons why clinicians sometimes advise or prescribe unnecessary medications. The authors suggest that a team approach involving multiple healthcare professionals who deliver consistent advice could improve guideline adherence.

Potential of in vivo real-time gastric gas profiling: a pilot evaluation of heat-stress and modulating dietary cinnamon effect in an animal model.

Gastroenterologists are still unable to differentiate between some of the most ordinary disorders of the gut and consequently patients are misdiagnosed. We have developed a swallowable gas sensor capsule for addressing this. The gases of the gut are the by-product of the fermentation processes during digestion, affected by the gut state and can consequently provide the needed information regarding the health of the gut. Here we present the first study on gas sensor capsules for revealing the effect of a medical supplement in an animal (pig) model. We characterise the real-time alterations of gastric-gas in response to environmental heat-stress and dietary cinnamon and use the gas profiles for understanding the bio-physiological changes. Under no heat-stress, feeding increases gastric CO2 concentration, while dietary cinnamon reduces it due to decrease in gastric acid and pepsin secretion. Alternatively, heat-stress leads to hyperventilation in pigs, which reduces CO2 concentration and with the cinnamon treatment, CO2 diminishes even more, resulting in health improvement outcomes. Overall, a good repeatability in gas profiles is also observed. The model demonstrates the strong potential of real-time gas profiler in providing new physiological information that will impact understanding of therapeutics, presenting a highly reliable device for monitoring/diagnostics of gastrointestinal disorders.

Anti-amyloidogenic properties of some phenolic compounds.

A family of 21 polyphenolic compounds consisting of those found naturally in danshen and their analogues were synthesized and subsequently screened for their anti-amyloidogenic activity against the amyloid beta peptide (Aß42) of Alzheimer's disease. After 24 h incubation with Aß42, five compounds reduced thioflavin T (ThT) fluorescence, indicative of their anti-amyloidogenic propensity (p < 0.001). TEM and immunoblotting analysis also showed that selected compounds were capable of hindering fibril formation even after prolonged incubations. These compounds were also capable of rescuing the yeast cells from toxic changes induced by the chemically synthesized Aß42. In a second assay, a Saccharomyces cerevisiae AHP1 deletant strain transformed with GFP fused to Aß42 was treated with these compounds and analyzed by flow cytometry. There was a significant reduction in the green fluorescence intensity associated with 14 compounds. We interpret this result to mean that the compounds had an anti-amyloid-aggregation propensity in the yeast and GFP-Aß42 was removed by proteolysis. The position and not the number of hydroxyl groups on the aromatic ring was found to be the most important determinant for the anti-amyloidogenic properties.

Respiratory tract infections among children younger than 5 years: current management in Australian general practice.

OBJECTIVE: To explore the current management in Australian general practice of common respiratory tract infections (RTIs) in children younger than 5 years. DESIGN, SETTING AND PARTICIPANTS: Analysis of data from a sample of 4522 general practitioners who participated in the Bettering the Evaluation and Care of Health (BEACH) cross-sectional survey, April 2007 to March 2012. Consultations with children younger than 5 years were analysed. MAIN OUTCOME MEASURES: GPs' management of four common RTIs (acute upper RTI [URTI], acute bronchitis/bronchiolitis, acute tonsillitis, and pneumonia) in association with six management options: antibiotic medications; prescribed or supplied non-antibiotic medications; medications advised for over-the-counter purchase; referrals; pathology testing; and counselling. RESULTS: Of 31 295 encounters recorded, at least one of the four selected paediatric RTIs was managed at 8157 encounters. URTI was managed 18.6 times per 100 GP patient encounters, bronchitis/bronchiolitis 4.2 times, acute tonsillitis 2.7 times, and pneumonia 0.6 times per 100 encounters. Antibiotics were prescribed most frequently for tonsillitis and least frequently for URTI. Male GPs prescribed antibiotics for URTI significantly more often than female GPs, while older GPs prescribed antibiotics for URTI more often than younger GPs. CONCLUSION: GP management of paediatric RTIs in Australia varied according to the clinical problem and with age and sex of the GP. Further research into parents' and health professionals' attitudes and practices regarding the role of antibiotics, over-the-counter medications, and hygiene will help maintain favourable management practices.

A simple and inexpensive device for biofilm analysis.

The Calgary Biofilm Device (CBD) has been described as a technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. In this study a simple and inexpensive alternative to the CBD was developed from polypropylene (PP) microcentrifuge tubes and pipette tip boxes. The utility of the device was demonstrated using Candida glabrata, a yeast that can develop antimicrobial-resistant biofilm communities. Biofilms of C. glabrata were formed on the outside surface of microcentrifuge tubes and examined by quantitative analysis and scanning electron microscopy. Growth of three C. glabrata strains, including a clinical isolate, demonstrated that biofilms could be formed on the microcentrifuge tubes. After 24 h incubation the three C. glabrata strains produced biofilms that were recovered into cell suspension and quantified. The method was found to produce uniform and reproducible results with no significant differences between biofilms formed on PP tubes incubated in various compartments of the device. In addition, the difference between maximum and minimum counts for each strain was comparable to those which have been reported for the CBD device.

Reprint of New opportunities for improved ribotyping of C. difficile clinical isolates by exploring their genomes.

Clostridium difficile causes outbreaks of infectious diarrhoea, most commonly occurring in healthcare institutions. Recently, concern has been raised with reports of C. difficile disease in those traditionally thought to be at low risk i.e. community acquired rather than healthcare acquired. This has increased awareness for the need to track outbreaks and PCR-ribotyping has found widespread use to elucidate epidemiologically linked isolates. PCR-ribotyping uses conserved regions of the 16S rRNA gene and 23S rRNA gene as primer binding sites to produce varying PCR products due to the intergenic spacer (ITS1) regions of the multiple operons. With the explosion of whole genome sequence data it became possible to analyse the start of the 23S rRNA gene for a more accurate selection of regions closer to the end of the ITS1. However the following questions must still be asked: (i) Does the chromosomal organisation of the rrn operon vary between C. difficile strains? and (ii) just how conserved are the primer binding regions? Eight published C. difficile genomes have been aligned to produce a detailed database of indels of the ITS1's from the rrn operon sets. An iPad Filemaker Go App has been constructed and named RiboTyping (RT). It contains detail such as sequences, ribotypes, strain numbers, GenBank numbers and genome position numbers. Access to various levels of the database is provided so that details can be printed. There are three main regions of the rrn operon that have been analysed by the database and related to each other by strain, ribotype and operon: (1) 16S gene (2) ITS1 indels (3) 23S gene. This has enabled direct intra- and inter-genomic comparisons at the strain, ribotype and operon (allele) levels in each of the three genomic regions. This is the first time that such an analysis has been done. By using the RT App with search criteria it will be possible to select probe combinations for specific strains/ribotypes/rrn operons for experiments to do with diagnostics, typing and recombination of operons. Many more incomplete C. difficile whole genome sequencing projects are recorded in GenBank as underway and the rrn operon information from these can also be added to the RT App when available. The RT App will help simplify probe selection because of the complexity of the ITS1 in C. difficile even in a single genome and because other allele-specific regions (16S and 23S genes) of variability can be relationally compared to design extra probes to increase sensitivity.

Associations with asymptomatic colonization with Candida in women reporting past vaginal candidiasis: an observational study.

OBJECTIVE: Asymptomatic vaginal colonization with Candida species is a known risk factor for vulvovaginal candidiasis (VVC). Taking known risk factors for symptomatic VVC, the authors sought to identify factors associated with asymptomatic colonization. STUDY DESIGN: As part of a randomized controlled trial which compared vaginal candidal colony counts in women taking garlic tablets or placebo, 192 asymptomatic women collected a baseline screening swab for Candida species. Eligibility for this study included at least one self-reported episode of VVC in the previous 12 months and age 18-50 years. Known risk factors for VVC were compared in women colonized with candida and those without colonization. RESULTS: 37% of asymptomatic women who self-reported VVC in the previous 12 months were colonized with vaginal Candida species. Using multivariate analysis, two factors were associated with asymptomatic colonization: a current sexual partner (P=0.02) and being born outside of Australia (P=0.05). Use of oral contraceptives was not statistically significant (P=0.27). CONCLUSIONS: Clinical relevance of asymptomatic colonization with vaginal yeast and its link to episodes of VVC warrants further investigation.

New opportunities for improved ribotyping of C. difficile clinical isolates by exploring their genomes.

Clostridium difficile causes outbreaks of infectious diarrhoea, most commonly occurring in healthcare institutions. Recently, concern has been raised with reports of C. difficile disease in those traditionally thought to be at low risk i.e. community acquired rather than healthcare acquired. This has increased awareness for the need to track outbreaks and PCR-ribotyping has found widespread use to elucidate epidemiologically linked isolates. PCR-ribotyping uses conserved regions of the 16S rRNA gene and 23S rRNA gene as primer binding sites to produce varying PCR products due to the intergenic spacer (ITS1) regions of the multiple operons. With the explosion of whole genome sequence data it became possible to analyse the start of the 23S rRNA gene for a more accurate selection of regions closer to the end of the ITS1. However the following questions must still be asked: (i) Does the chromosomal organisation of the rrn operon vary between C. difficile strains? and (ii) just how conserved are the primer binding regions? Eight published C. difficile genomes have been aligned to produce a detailed database of indels of the ITS1's from the rrn operon sets. An iPad Filemaker Go App has been constructed and named RiboTyping (RT). It contains detail such as sequences, ribotypes, strain numbers, GenBank numbers and genome position numbers. Access to various levels of the database is provided so that details can be printed. There are three main regions of the rrn operon that have been analysed by the database and related to each other by strain, ribotype and operon: (1) 16S gene (2) ITS1 indels (3) 23S gene. This has enabled direct intra- and inter-genomic comparisons at the strain, ribotype and operon (allele) levels in each of the three genomic regions. This is the first time that such an analysis has been done. By using the RT App with search criteria it will be possible to select probe combinations for specific strains/ribotypes/rrn operons for experiments to do with diagnostics, typing and recombination of operons. Many more incomplete C. difficile whole genome sequencing projects are recorded in GenBank as underway and the rrn operon information from these can also be added to the RT App when available. The RT App will help simplify probe selection because of the complexity of the ITS1 in C. difficile even in a single genome and because other allele-specific regions (16S and 23S genes) of variability can be relationally compared to design extra probes to increase sensitivity.

Premenstrual vaginal colonization of Candida and symptoms of vaginitis.

Although premenstrual exacerbation of vulvovaginal symptoms attributed to Candida spp. is well documented, the causation of these symptoms is not well understood. This study describes the daily vaginal colonization of Candida in three women. A single pilot study was designed to test the methodology of the proposed randomized controlled trial, Garlic and Candida. This study reports the colonization of Candida spp. in three women. Ten women aged 18-50 who reported at least one episode of vulvovaginal candidiasis were recruited by the University of Melbourne. Each participant took daily vaginal swabs for 2 weeks during the luteal phase of their menstrual cycle, which were analysed for quantitative colony counts of Candida spp. Of these, three women were colonized with Candida spp. For the first time, to our knowledge, daily colonization of Candida during the luteal phase of the menstrual cycle is described in three women, demonstrating an increase in the colony count preceding symptom development. This small study demonstrated the colonization of Candida spp. during the luteal phase of the menstrual cycle in three women. Candida colonization is poorly understood, yet investigating the relevance of the link between symptom exacerbation and the menstrual cycle in those women who experience recurrent episodes of vulvovaginal candidiasis may influence the management of this condition.

A novel method for simultaneous Enterococcus species identification/typing and van genotyping by high resolution melt analysis.

In order to develop a typing and identification method for van gene containing Enterococcus faecium, two multiplex PCR reactions were developed for use in HRM-PCR (High Resolution Melt-PCR): (i) vanA, vanB, vanC, vanC23 to detect van genes from different Enterococcus species; (ii) ISR (intergenic spacer region between the 16S and 23S rRNA genes) to detect all Enterococcus species and obtain species and isolate specific HRM curves. To test and validate the method three groups of isolates were tested: (i) 1672 Enterococcus species isolates from January 2009 to December 2009; (ii) 71 isolates previously identified and typed by PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing); and (iii) 18 of the isolates from (i) for which ISR sequencing was done. As well as successfully identifying 2 common genotypes by HRM from the Austin Hospital clinical isolates, this study analysed the sequences of all the vanB genes deposited in GenBank and developed a numerical classification scheme for the standardised naming of these vanB genotypes. The identification of Enterococcus faecalis from E. faecium was reliable and stable using ISR PCR. The typing of E. faecium by ISR PCR: (i) detected two variable peaks corresponding to different copy numbers of insertion sequences I and II corresponding to peak I and II respectively; (ii) produced 7 melt profiles for E. faecium with variable copy numbers of sequences I and II; (iii) demonstrated stability and instability of peak heights with equal frequency within the patient sample (36.4±4.5 days and 38.6±5.8 days respectively for 192 patients); (iv) detected ISR-HRM types with as much discrimination as PFGE and more than MLST; and (v) detected ISR-HRM types that differentiated some isolates that were identical by PFGE and MLST. In conjunction with the rapid and accurate van genotyping method described here, this ISR-HRM typing and identification method can be used as a stable identification and typing method with predictable instability based on recombination and concerted evolution of the rrn operon that will complement existing typing methods.

High resolution melt analysis to track infections due to ribotype 027 Clostridium difficile.

The increased prevalence of hypervirulent ribotype 027 Clostridium difficile requires rapid identification of isolates in order to implement timely infection control strategies. High resolution melt (HRM) analysis of PCR products can identify strain variation amongst genera of bacteria. The intergenic (16S-23S rDNA) spacer region contains sequence regions conserved within genera and other sequence region variables between species within genera. We wished to investigate whether HRM analysis of PCR ribotyping products could identify ribotype 027 C. difficile. Ribotyping was performed on 93 clinical isolates and five control strains and band patterns were analysed using GelCompar II (Applied Maths, USA). Real-time PCR using ribotyping primers was performed and normalised melt curves were generated. The HRM data was then imported into ScreenClust software (QIAGEN) to generate principal component analysis graphs depicting clustered relationships of strains. Ribotyping produced clear PCR bands for 88/98 isolates tested. Dendrograms generated by GelCompar showed a diversity of ribotype patterns amongst these 88 isolates with 18 groups identified with 70% homology. One clinical isolate showed 100% homology with the control 027 strains. ScreenClust analysis of the same 88 HRM results showed clustering of isolates, with 027 strains identifiable as a unique cluster. HRM analysis correctly identified the control 027 stains and the clinical isolate shown to be 027. HRM combined with ScreenClust analysis of real-time PCR products of the 16S-23S rDNA spacer region successfully identified ribotype 027 strains. For infection control purposes this was achieved within 2-3 h of colony isolation.