Immersion of debrided diabetic foot ulcer tissue in electrochemically generated pH neutral hypochlorous acid significantly reduces the microbial bioburden: whole-genome sequencing of Staphylococcus aureus, the most prevalent species recovered.

BACKGROUND: Diabetic foot ulcer infections (DFUIs) are the leading cause of lower-limb amputations, mediated predominantly by Staphylococcus aureus. pH-neutral electrochemically generated hypochlorous acid (anolyte) is a non-toxic, microbiocidal agent with significant potential for wound disinfection. AIMS: To investigate both the effectiveness of anolyte for microbial bioburden reduction in debrided ulcer tissues and the population of resident S. aureus. METHODS: Fifty-one debrided tissues from 30 people with type II diabetes were aliquoted by wet weight and immersed in 1- or 10-mL volumes of anolyte (200 parts per million) or saline for 3 min. Microbial loads recovered were determined in colony forming units/g (cfu/g) of tissue following aerobic, anaerobic and staphylococcal-selective culture. Bacterial species were identified and 50 S. aureus isolates from 30 tissues underwent whole-genome sequencing (WGS). FINDINGS: The ulcers were predominantly superficial, lacking signs of infection (39/51, 76.5%). Of the 42/51 saline-treated tissues yielding ≥105 cfu/g, a microbial threshold reported to impede wound-healing, only 4/42 (9.5%) were clinically diagnosed DFUIs. Microbial loads from anolyte-treated tissues were significantly lower than saline-treated tissues using 1 mL (1065-fold, 2.0 log) and 10 mL (8216-fold, 2.1 log) immersion volumes (P<0.0005). S. aureus was the predominant species recovered (44/51, 86.3%) and 50 isolates underwent WGS. All were meticillin susceptible and comprised 12 sequence types (STs), predominantly ST1, ST5 and ST15. Whole-genome multi-locus sequence typing identified three clusters of closely related isolates from 10 patients indicating inter-patient transmission. CONCLUSIONS: Short immersions of debrided ulcer tissue in anolyte significantly reduced microbial bioburden: a potential novel DFUI treatment.

An emerging Panton-Valentine leukocidin-positive CC5-meticillin-resistant Staphylococcus aureus-IVc clone recovered from hospital and community settings over a 17-year period from 12 countries investigated by whole-genome sequencing.

BACKGROUND: A novel Panton-Valentine leukocidin (PVL)-positive meticillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC)5-MRSA-IVc ('Sri Lankan' clone) was recently described from Sri Lanka. Similar isolates caused a recent Irish hospital outbreak. AIM: To investigate the international dissemination and diversity of PVL-positive CC5-MRSA-IVc isolates from hospital and community settings using whole-genome sequencing (WGS). METHODS: Core-genome single nucleotide polymorphism (cgSNP) analysis, core-genome multi-locus sequence typing (cgMLST) and microarray-based detection of antimicrobial-resistance and virulence genes were used to investigate PVL-positive CC5-MRSA-IVc (N = 214 including 46 'Sri Lankan' clone) from hospital and community settings in 12 countries over 17 years. Comparators included 29 PVL-positive and 23 PVL-negative CC5/ST5-MRSA-I/II/IVa/IVc/IVg/V. RESULTS: Maximum-likelihood cgSNP analysis grouped 209/214 (97.7%) CC5-MRSA-IVc into Clade I; average of 110 cgSNPs between isolates. Clade III contained the five remaining CC5-MRSA-IVc; average of 92 cgSNPs between isolates. Clade II contained seven PVL-positive CC5-MRSA-IVa comparators, whereas the remaining 45 comparators formed an outlier group. Minimum-spanning cgMLST analysis revealed a comparably low average of 57 allelic differences between all CC5/ST5-MRSA-IVc. All 214 CC5/ST5-MRSA-IVc were identified as 'Sri Lankan' clone, predominantly spa type t002 (186/214) with low population diversity and harboured a similar range of virulence genes and variable antimicrobial-resistance genes. All 214 Sri Lankan clone isolates and Clade II comparators harboured a 9616-bp chromosomal PVL-encoding phage remnant, suggesting both arose from a PVL-positive meticillin-susceptible ancestor. Over half of Sri Lankan clone isolates were from infections (142/214), and where detailed metadata were available (168/214), most were community associated (85/168). CONCLUSIONS: Stable chromosomal retention of pvl may facilitate Sri-Lankan clone dissemination.

Multiple distinct outbreaks of Panton-Valentine leucocidin-positive community-associated meticillin-resistant Staphylococcus aureus in Ireland investigated by whole-genome sequencing.

BACKGROUND: Panton-Valentine leucocidin (PVL)-positive community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA) is increasingly associated with infection outbreaks. AIM: To investigate multiple suspected PVL-positive CA-MRSA outbreaks using whole-genome sequencing (WGS). METHODS: Forty-six suspected outbreak-associated isolates from 36 individuals at three separate Irish hospitals (H1-H3) and from separate incidents involving separate families associated with H2 were investigated by whole-genome multi-locus sequence typing (wgMLST). FINDINGS: Two clusters (CH1 and CH2) consisting of 8/10 and 6/6 PVL-positive t008 ST8-MRSA-IVa isolates from H1 and H2, respectively, were identified. Within each cluster, neighbouring isolates were separated by ≤5 allelic differences; however, ≥73 allelic differences were identified between the clusters, indicating two independent outbreaks. Isolates from the H3 maternity unit formed two clusters (CH3-SCI and CH3-SCII) composed of four PVL-negative t4667 ST5-MRSA-V and 14 PVL-positive t002 ST5-MRSA-IVc isolates, respectively. Within clusters, neighbouring isolates were separated by ≤24 allelic differences, whereas both clusters were separated by 1822 allelic differences, indicating two distinct H3 outbreaks. Eight PVL-positive t127 ST1-MRSA-V+fus and three PVL-negative t267 ST97-MRSA-V+fus isolates from two distinct H2-associated families FC1 (N = 4) and FC2 (N = 7) formed three separate clusters (FC1 (t127), FC2 (t127) and FC2 (t267)). Neighbouring isolates within clusters were closely related and exhibited ≤7 allelic differences. Intrafamilial transmission was apparent, but the detection of ≥48 allelic differences between clusters indicated no interfamilial transmission. CONCLUSION: The frequent importation of PVL-positive CA-MRSA into healthcare settings, transmission and association with outbreaks is a serious ongoing concern. WGS is a highly discriminatory, informative method for deciphering such outbreaks conclusively.