Can molecular DNA-based techniques unravel the truth about diabetic foot infections?

Diabetes foot infections are a common condition and a major causal pathway to lower extremity amputation. Identification of causative pathogens is vital in directing antimicrobial therapy. Historically, clinicians have relied upon culture-dependent techniques that are now acknowledged as both being selective for microorganisms that thrive under the physiological and nutritional constraints of the microbiology laboratory and that grossly underestimate the microbial diversity of a sample. The amplification and sequence analysis of the 16S rRNA gene has revealed a diversity of microorganisms in diabetes foot infections, extending the view of the diabetic foot microbiome. The interpretation of these findings and their relevance to clinical care remains largely unexplored. The advent of molecular methods that are culture-independent and employ massively parallel DNA sequencing technology represents a potential 'game changer'. Metagenomics and its shotgun approach to surveying all DNA within a sample (whole genome sequencing) affords the possibility to characterize not only the microbial diversity within a diabetes foot infection (i.e. 'which microorganisms are present') but the biological functions of the community such as virulence and pathogenicity (i.e. 'what are the microorganisms capable of doing'), moving the focus from single species as pathogens to groups of species. This review will examine the new molecular techniques for exploration of the microbiome of infected and uninfected diabetic foot ulcers, exploring the potential of these new technologies and postulating how they could translate to improved clinical care. Copyright © 2016 John Wiley & Sons, Ltd.

Staphylococcus aureus dry-surface biofilms are not killed by sodium hypochlorite: implications for infection control.

BACKGROUND: Dry hospital environments are contaminated with pathogenic bacteria in biofilms, which suggests that current cleaning practices and disinfectants are failing. AIM: To test the efficacy of sodium hypochlorite solution against Staphylococcus aureus dry-surface biofilms. METHODS: The Centers for Disease Control and Prevention Biofilm Reactor was adapted to create a dry-surface biofilm, containing 1.36 × 10(7)S. aureus/coupon, by alternating cycles of growth and dehydration over 12 days. Biofilm was detected qualitatively using live/dead stain confocal laser scanning microscopy (CLSM), and quantitatively with sonicated viable plate counts and crystal violet assay. Sodium hypochlorite (1000-20,000parts per million) was applied to the dry-surface biofilm for 10min, coupons were rinsed three times, and residual biofilm viability was determined by CLSM, plate counts and prolonged culture up to 16 days. Isolates before and after exposure underwent minimum inhibitory concentration (MIC) and minimum eradication concentration (MEC) testing, and one pair underwent whole-genome sequencing. FINDINGS: Hypochlorite exposure reduced plate counts by a factor of 7 log10, and reduced biofilm biomass by a factor of 100; however, staining of residual biofilm showed that live S. aureus cells remained. On prolonged incubation, S. aureus regrew and formed biofilms. Post-exposure S. aureus isolates had MICs and MECs that were not significantly different from the parent strains. Whole-genome sequencing of one pre- and post-exposure pair found that they were virtually identical. CONCLUSIONS: Hypochlorite exposure led to a 7-log kill but the organisms regrew. No resistance mutations occurred, implying that hypochlorite resistance is an intrinsic property of S. aureus biofilms. The clinical significance of this warrants further study.

Carriage of an ACME II variant may have contributed to methicillin-resistant Staphylococcus aureus sequence type 239-like strain replacement in Liverpool Hospital, Sydney, Australia.

Approximately 39% of methicillin-resistant Staphylococcus aureus (MRSA) sequence type 239 (ST239)-like bloodstream isolates from Liverpool Hospital (obtained between 1997 and 2008) carry an arginine catabolic mobile element (ACME). Whole-genome sequencing revealed that an ACME II variant is located between orfX and SCCmec III, and based on pulsed-field gel electrophoresis patterns and temporal relationships of all ST239-like isolates (n = 360), ACME carriage may have contributed to subpulsotype strain replacement.