The microbiome of diabetic foot ulcers: a comparison of swab and tissue biopsy wound sampling techniques using 16S rRNA gene sequencing.

BACKGROUND: Health-care professionals need to collect wound samples to identify potential pathogens that contribute to wound infection. Obtaining appropriate samples from diabetic foot ulcers (DFUs) where there is a suspicion of infection is of high importance. Paired swabs and tissue biopsies were collected from DFUs and both sampling techniques were compared using 16S rRNA gene sequencing. RESULTS: Mean bacterial abundance determined using quantitative polymerase chain reaction (qPCR) was significantly lower in tissue biopsies (p = 0.03). The mean number of reads across all samples was significantly higher in wound swabs [Formula: see text] = 32,014) compared to tissue ([Formula: see text] = 15,256, p = 0.001). Tissue biopsies exhibited greater overall diversity of bacteria relative to swabs (Shannon's H diversity p = 0.009). However, based on a presence/absence analysis of all paired samples, the frequency of occurrence of bacteria from genera of known and potential pathogens was generally higher in wound swabs than tissue biopsies. Multivariate analysis identified significantly different bacterial communities in swabs compared to tissue (p = 0.001). There was minimal correlation between paired wound swabs and tissue biopsies in the number and types of microorganisms. RELATE analysis revealed low concordance between paired DFU swab and tissue biopsy samples (Rho = 0.043, p = 0.34). CONCLUSIONS: Using 16S rRNA gene sequencing this study identifies the potential for using less invasive swabs to recover high relative abundances of known and potential pathogen genera from DFUs when compared to the gold standard collection method of tissue biopsy.

Choose wisely: Network, ontology and annotation resources for the analysis of Staphylococcus aureus omics data.

Staphylococcus aureus (S. aureus) is a prominent human and livestock pathogen investigated widely using omic technologies. Critically, due to availability, low visibility or scattered resources, robust network and statistical contextualisation of the resulting data is generally under-represented. Here, we present novel meta-analyses of freely-accessible molecular network and gene ontology annotation information resources for S. aureus omics data interpretation. Furthermore, through the application of the gene ontology annotation resources we demonstrate their value and ability (or lack-there-of) to summarise and statistically interpret the emergent properties of gene expression and protein abundance changes using publically available data. This analysis provides simple metrics for network selection and demonstrates the availability and impact that gene ontology annotation selection can have on the contextualisation of bacterial omics data.

Heterogeneity of clinical and environmental isolates of Mycobacterium fortuitum using repetitive element sequence-based PCR: municipal water an unlikely source of community-acquired infections.

M. fortuitum is a rapidly growing mycobacterium associated with community-acquired and nosocomial wound, soft tissue, and pulmonary infections. It has been postulated that water has been the source of infection especially in the hospital setting. The aim of this study was to determine if municipal water may be the source of community-acquired or nosocomial infections in the Brisbane area. Between 2007 and 2009, 20 strains of M. fortuitum were recovered from municipal water and 53 patients' isolates were submitted to the reference laboratory. A wide variation in strain types was identified using repetitive element sequence-based PCR, with 13 clusters of ⩾2 indistinguishable isolates, and 28 patterns consisting of individual isolates. The clusters could be grouped into seven similar groups (>95% similarity). Municipal water and clinical isolates collected during the same time period and from the same geographical area consisted of different strain types, making municipal water an unlikely source of sporadic human infection.

Genotyping of Enterococcus faecalis and Enterococcus faecium isolates by use of a set of eight single nucleotide polymorphisms.

A single nucleotide polymorphism (SNP) genotyping method for Enterococcus faecalis and Enterococcus faecium was developed using the "Minimum SNPs" program. SNP sets were interrogated using allele-specific real-time PCR. SNP typing subdivided clonal complexes 2 and 9 of E. faecalis and 17 of E. faecium, members of which cause the majority of nosocomial infections globally.

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.

Prevalence of Staphylococcus aureus strains in an Australian cohort, 1989-2003: evidence for the low prevalence of the toxic shock toxin and Panton-Valentine leukocidin genes.

The purpose of this paper is to determine the prevalence of the toxic shock toxin gene (tst) and to enumerate the circulating strains of methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in Australian isolates collected over two decades. The aim was to subtype these strains using the binary genes pvl, cna, sdrE, pUB110 and pT181. Isolates were assayed using real-time polymerase chain reaction (PCR) for mecA, nuc, 16 S rRNA, eight single-nucleotide polymorphisms (SNPs) and for five binary genes. Two real-time PCR assays were developed for tst. The 90 MRSA isolates belonged to CC239 (39 in 1989, 38 in 1996 and ten in 2003), CC1 (two in 2003) and CC22 (one in 2003). The majority of the 210 MSSA isolates belonged to CC1 (26), CC5 (24) and CC78 (23). Only 18 isolates were tst-positive and only 15 were pvl-positive. Nine MSSA isolates belonged to five binary types of ST93, including two pvl-positive types. The proportion of tst-positive and pvl-positive isolates was low and no significant increase was demonstrated. Dominant MSSA clonal complexes were similar to those seen elsewhere, with the exception of CC78. CC239 MRSA (AUS-2/3) was the predominant MRSA but decreased significantly in prevalence, while CC22 (EMRSA-15) and CC1 (WA-1) emerged. Genetically diverse ST93 MSSA predated the emergence of ST93-MRSA (the Queensland clone).

Real-time PCR detection of pathogenic microorganisms in roof-harvested rainwater in Southeast Queensland, Australia.

In this study, the microbiological quality of roof-harvested rainwater was assessed by monitoring the concentrations of Escherichia coli, enterococci, Clostridium perfringens, and Bacteroides spp. in rainwater obtained from tanks in Southeast Queensland, Australia. Samples were also tested using real-time PCR (with SYBR Green I dye) for the presence of potential pathogenic microorganisms. Of the 27 rainwater samples tested, 17 (63%), 21 (78%), 13 (48%), and 24 (89%) were positive for E. coli, enterococci, C. perfringens, and Bacteroides spp., respectively. Of the 27 samples, 11 (41%), 7 (26%), 4 (15%), 3 (11%), and 1 (4%) were PCR positive for the Campylobacter coli ceuE gene, the Legionella pneumophila mip gene, the Aeromonas hydrophila lip gene, the Salmonella invA gene, and the Campylobacter jejuni mapA gene. Of the 21 samples tested, 4 (19%) were positive for the Giardia lamblia beta-giardin gene. The binary logistic regression model indicated a positive correlation (P < 0.02) between the presence/absence of enterococci and A. hydrophila. In contrast, the presence/absence of the remaining potential pathogens did not correlate with traditional fecal indicators. The poor correlation between fecal indicators and potential pathogens suggested that fecal indicators may not be adequate to assess the microbiological quality of rainwater and consequent health risk.

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates.

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.

Preliminary validation of a novel high-resolution melt-based typing method based on the multilocus sequence typing scheme of Streptococcus pyogenes.

The major limitation of current typing methods for Streptococcus pyogenes, such as emm sequence typing and T typing, is that these are based on regions subject to considerable selective pressure. Multilocus sequence typing (MLST) is a better indicator of the genetic backbone of a strain but is not widely used due to high costs. The objective of this study was to develop a robust and cost-effective alternative to S. pyogenes MLST. A 10-member single nucleotide polymorphism (SNP) set that provides a Simpson's Index of Diversity (D) of 0.99 with respect to the S. pyogenes MLST database was derived. A typing format involving high-resolution melting (HRM) analysis of small fragments nucleated by each of the resolution-optimized SNPs was developed. The fragments were 59-119 bp in size and, based on differences in G+C content, were predicted to generate three to six resolvable HRM curves. The combination of curves across each of the 10 fragments can be used to generate a melt type (MelT) for each sequence type (ST). The 525 STs currently in the S. pyogenes MLST database are predicted to resolve into 298 distinct MelTs and the method is calculated to provide a D of 0.996 against the MLST database. The MelTs are concordant with the S. pyogenes population structure. To validate the method we examined clinical isolates of S. pyogenes of 70 STs. Curves were generated as predicted by G+C content discriminating the 70 STs into 65 distinct MelTs.

Prevalence and occurrence of zoonotic bacterial pathogens in surface waters determined by quantitative PCR.

The prevalence and concentrations of Campylobacter jejuni, Salmonella spp. and enterohaemorrhagic Escherichia coli (EHEC) were investigated in surface waters in Brisbane, Australia using quantitative PCR (qPCR) based methodologies. Water samples were collected from Brisbane City Botanic Gardens (CBG) Pond, and two urban tidal creeks (i.e., Oxley Creek and Blunder Creek). Of the 32 water samples collected, 8 (25%), 1 (3%), 9 (28%), 14 (44%), and 15 (47%) were positive for C. jejuni mapA, Salmonella invA, EHEC O157 LPS, EHEC VT1, and EHEC VT2 genes, respectively. The presence/absence of the potential pathogens did not correlate with either E. coli or enterococci concentrations as determined by binary logistic regression. In conclusion, the high prevalence, and concentrations of potential zoonotic pathogens along with the concentrations of one or more fecal indicators in surface water samples indicate a poor level of microbial quality of surface water, and could represent a significant health risk to users. The results from the current study would provide valuable information to the water quality managers in terms of minimizing the risk from pathogens in surface waters.

Nasal carriage of Staphylococcus aureus, including community-associated methicillin-resistant strains, in Queensland adults.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are emerging in southeast Queensland, Australia, but the incidence of carriage of CA-MRSA strains is unknown. The aim of this study was to assess the nasal carriage rate of S. aureus, including CA-MRSA strains, in the general adult population of southeast Queensland. 396 patients presenting to general practices in two Brisbane suburbs and 303 volunteers randomly selected from the electoral rolls in the same suburbs completed a medical questionnaire and had nasal swabs performed for S. aureus. All isolates of S. aureus underwent antibiotic susceptibility testing and single-nucleotide polymorphism (SNP) and binary typing, including determination of Panton-Valentine leukocidin (PVL). The nasal carriage rate of methicillin-susceptible S. aureus (MSSA) was 202/699 (28%), a rate similar to that found in other community-based nasal carriage studies. According to multivariate analysis, nasal carriage of S. aureus was associated with male sex, young adult age group and Caucasian ethnicity. Only two study isolates (one MSSA and one CA-MRSA) carried PVL. The nasal carriage rate of MRSA was low, at 5/699 (0.7%), and only two study participants (0.3%) had CA-MRSA strains. CA-MRSA is an emerging cause of infection in southeast Queensland, but as yet the incidence of carriage of CA-MRSA in the general community is low.

Methicillin-susceptible, non-multiresistant methicillin-resistant and multiresistant methicillin-resistant Staphylococcus aureus infections: a clinical, epidemiological and microbiological comparative study.

Non-multiresistant methicillin-resistant Staphylococcus aureus (nmMRSA) infections are emerging worldwide and are often community-associated. This prospective case-cohort study compares features of 96 nmMRSA clinical isolates with 96 matched multiresistant MRSA (mMRSA) and 192 matched methicillin-susceptible S. aureus (MSSA) clinical isolates. Seventy-four percent of nmMRSA infections were healthcare-associated. nmMRSA infections were much more likely to involve skin and soft tissue (skin and soft tissue infections; SSTIs) and were much less likely to be treated appropriately with antibiotics than MSSA or mMRSA infections. Panton-Valentine leukocidin (PVL) genes were detected in 55% of nmMRSA, 16% of MSSA and 2% of mMRSA isolates. Independent of the methicillin-resistance phenotype, 59% of PVL-positive SSTIs presented as furunculosis compared to only 10% of PVL-negative SSTIs. Patients with PVL-positive infections were much younger than patients with PVL-negative infections. The proportion of PVL-positive infections peaked in the 10-29 years old age group, followed by a linear decline.

Life-threatening community-acquired methicillin-resistant Staphylococcus aureus infection in Australia.

Eight patients with invasive bacteremic community-acquired methicillin-resistant Staphylococcus aureus infection in southeast Queensland, Australia, are reported. One patient died of septic shock. Haematogenous seeding to lungs, bone, and other sites was common. All isolates carried the virulence factor Panton-Valentine leukocidin and were either the southwest Pacific clone or the newly described Queensland clone. Clinicians should consider community-acquired methicillin-resistant Staphylococcus aureus infection in any patient presenting to hospital with severe staphylococcal sepsis or pneumonia.

Vancomycin binding to cell walls of non-streptococcal vancomycin-resistant bacteria.

The availability of peptidoglycan for binding to vancomycin was investigated in intrinsically vancomycin-resistant clinical isolates of Leuconostoc mesenteroides, Lactobacillus viridescens and Pediococcus pentosaceus. Intrinsic vancomycin resistance expressed by L. mesenteroides and P. pentosaceus was the result of poor binding of vancomycin to native or SDS-treated cell walls. Pre-exposure of L. viridescens to vancomycin decreased the subsequent binding of vancomycin to the cell walls, suggesting an alternative mechanism to that found in high-level vancomycin-resistant enterococci and intrinsically-resistant L. mesenteroides and P. pentosaceus. Vancomycin binding was sensitive to substitution of larger side chains at the C-terminus. Variations of peptidoglycan peptide types found in L. mesenteroides, L. viridescens and P. pentosaceus could account for their intrinsic vancomycin resistance.

GABA(A) receptor alpha-subunit proteins in human chronic alcoholics.

Antibodies were raised against specific peptides from N-terminal regions of the alpha1 and alpha3 isoforms of the GABA(A) receptor, and used to assess the relative expression of these proteins in the superior frontal and primary motor cortices of 10 control, nine uncomplicated alcoholic and six cirrhotic alcoholic cases were matched for age and post-mortem delay. The regression of expression on post-mortem delay was not statistically significant for either isoform in either region. In both cortical areas, the regression of alpha1 expression on age differed significantly between alcoholic cases, which showed a decrease, and normal controls, which did not. Age had no effect on alpha3 expression. The alpha1 and alpha3 isoforms were found to be expressed differentially across cortical regions and showed a tendency to be expressed differentially across case groups. In cirrhotic alcoholics, alpha1 expression was greater in superior frontal than in motor cortex, whereas this regional difference was not significant in controls or uncomplicated alcoholics. In uncomplicated alcoholics, alpha3 expression was significantly lower in superior frontal than in motor cortex. Expression of alpha1 was significantly different from that of alpha3 in the superior frontal cortex of alcoholics, but not in controls. In motor cortex, there were no significant differences in expression between the isoforms in any case group.

Methicillin resistance and beta-lactamase production in Staphylococcus aureus.

Staphylococcus aureus isolates were either sensitive, resistant or partially resistant (minimum inhibitory concentration 90 of 4 micrograms/ml) to methicillin. Low-level methicillin resistance was shown to be due to beta-lactamase production. The clinical significance of this beta-lactamase-mediated resistance is still unclear, so it is recommended that these strains should, at present, be regarded as methicillin-resistant.