Efficacy of a topical wound agent methanesulfonic acid and dimethylsulfoxide on in vitro biofilms

A topical desiccating wound agent containing methanesulfonic acid, dimethylsulfoxide and amorphous silica was evaluated in three in vitro models for its efficacy against biofilms produced by Pseudomonas aeruginosa (ATCC-15442) and Staphylococcus aureus (ATCC-6538). The in vitro biofilm models used were; the MBEC Assay®, Centre for Disease Control (CDC) Biofilm Reactor® and a Semi-solid biofilm model. A 30-s exposure of a topical wound desiccating agent was used in each model. A complete eradication of viable cells was demonstrated in all models for both strains (p < 0.0001). Imaging with scanning electron microscopy (SEM) was performed where possible. All three models demonstrated complete eradication of viable cells with a 30 s application of a topical wound desiccating agent

Sonication versus the conventional method for evaluation of the dental microbiome: a prospective pilot study

Objectives: To investigate sonication as a new tool in microbiological probing of dental infections. Methods: Comparison of a standard probing method: intraoperative swab, with sonication, and vortex of the removed tooth, was performed on 20 carious destructed teeth. Illumina high throughput sequencing of the 16S-rRNA-gene was used for assessing the microbial composition. Antibiotic susceptibility has been assigned based on known resistances of each detected species. Probing procedures were compared using Bland-Altmann-Test, and antibiotic susceptibility using the Friedmann-Test and alpha-adjusted post-hoc-analysis. Results: In total, 60 samples were analysed: 20 intraoperative swabs, 20 vortex fluids, and 20 sonication fluids. Sonication fluid yielded the highest number of bacterial sequencing reads in all three procedures. Comparing the operational taxonomic units (OTUs) of the identified bacteria, significantly more OTUs were found in sonication fluid samples. Phylum and order abundances varied between the three procedures. Significantly more Actinomycetales have been found in sonication fluid samples compared to swab samples. The assigned resistance rates for the identified bacteria (1.79-31.23%) showed no differences between the tested probing procedures. The lowest resistance rates were found for amoxicillin + clavulanate (3.95%) and levofloxacin (3.40%), with the highest in amoxicillin (30.21%) and clindamycin (21.88%). Conclusions: By using sonication on extracted teeth, it is possible to get a more comprehensive image of the residing microbial flora compared to the standard procedure. If sonication is not available, vortexing is a potential alternative. In immunocompromised patients, especially when actinomycosis is suspected, sonication should be considered for a more detailed microbiological evaluation of the potential disease-causing microbiome. Due to the high rates of antibiotic resistance, a more targeted antibiotic therapy is favourable. Levofloxacin should be considered as a first-line alternative to amoxicillin + clavulanate in patients with an allergy to penicillin

A metatranscriptomic approach to explore longitudinal tissue specimens from non-healing diabetes related foot ulcers

Cellular mechanisms and/or microbiological interactions which contribute to chronic diabetes related foot ulcers (DRFUs) were explored using serially collected tissue specimens from chronic DRFUs and control healthy foot skin. Total RNA was isolated for next-generation sequencing. We found differentially expressed genes (DEGs) and enriched hallmark gene ontology biological processes upregulated in chronic DRFUs which primarily functioned in the host immune response including: (i) Inflammatory response; (ii) TNF signalling via NFKB; (iii) IL6 JAK-STAT3 signalling; (iv) IL2 STAT5 signalling and (v) Reactive oxygen species. A temporal analysis identified RN7SL1 signal recognition protein and IGHG4 immunoglobulin protein coding genes as being the most upregulated genes after the onset of treatment. Testing relative temporal changes between healing and non-healing DRFUs identified progressive upregulation in healed wounds of CXCR5 and MS4A1 (CD20), both canonical markers of lymphocytes (follicular B cells/follicular T helper cells and B cells, respectively). Collectively, our RNA-seq data provides insights into chronic DRFU pathogenesis

Efficacy of a topical concentrated surfactant gel on microbial communities in non-healing diabetic foot ulcers with chronic biofilm infections : a proof-of-concept study

This proof-of-concept study sought to determine the effects of standard of care (SOC) and a topically applied concentrated surfactant gel (SG) on the total microbial load, community composition, and community diversity in non-healing diabetic foot ulcers (DFUs) with chronic biofilm infections. SOC was provided in addition to a topical concentrated SG, applied every 2 days for 6 weeks. Wound swabs were obtained from the base of ulcers at baseline (week 0), week 1, mid-point (week 3), and end of treatment (week 6). DNA sequencing and real-time quantitative polymerase chain reaction (qPCR) were employed to determine the total microbial load, community composition, and diversity of patient samples. Tissue specimens were obtained at baseline and scanning electron microscopy and peptide nucleic acid fluorescent in situ hybridisation with confocal laser scanning microscopy were used to confirm the presence of biofilm in all 10 DFUs with suspected chronic biofilm infections. The application of SG resulted in 7 of 10 samples achieving a reduction in mean log10 total microbial load from baseline to end of treatment (0.8 Log10 16S copies, ±0.6), and 3 of 10 samples demonstrated an increase in mean Log10 total microbial load (0.6 log10 16S copies, ±0.8) from baseline to end of treatment. Composition changes in microbial communities were driven by changes to the most dominant bacteria. Corynebacterium sp. and Streptococcus sp. frequently reduced in relative abundance in patient samples from week 0 to week 6 but did not disappear. In contrast, Staphylococcus sp., Finegoldia sp., and Fusobacterium sp., relative abundances frequently increased in patient samples from week 0 to week 6. The application of a concentrated SG resulted in varying shifts to diversity (increase or decrease) between week 0 and week 6 samples at the individual patient level. Any shifts in community diversity were independent to changes in the total microbial loads. SOC and a topical concentrated SG directly affect the microbial loads and community composition of DFUs with chronic biofilm infections

Monitoring wound progression to healing in diabetic foot ulcers using three-dimensional wound imaging

Aim: 3D wound imaging has provided clinicians with even greater wound measurement options. No data is available to guide clinicians as to which 3D measurements may yield the most reflective marker of wound progression to healing. Method: A prospective pilot study was undertaken to assess the accuracy of five 3D wound measurements that best reflect metrics of interest to clinicians. Twenty-one diabetic foot ulcers were enrolled from initial ulcer presentation, through to healing. The relationship between mean wound healing measurement variables was examined using linear regression and Pearsons correlation coefficient, in addition to assessing clinician inter-rater reliability of measurements using Intra-class correlation coefficients (ICC). Results: Statistical analysis demonstrated a linear healing slope for each wound measurement as having a value greater than R 0.70 and a statistical significance of p = 0.0001. This suggests that all five wound measurements are useful prognostic markers of wound progression to healing. Low variability of measurements between users indicates good inter-observer reliability. Conclusion: 3D wound measurements demonstrate a linear correlation between the measurement and time to healing. This suggests they could be effective prognostic markers of a wounds progression to healing and closure. It may also provide important early identification of wounds not responding to standard care. Larger studies are required to validate our results

[In Press] Host-microbe metatranscriptome reveals differences between acute and chronic infections in diabetes-related foot ulcers

Virtually all diabetes-related foot ulcers (DRFUs) will become colonized by microorganisms that may increase the risk of developing an infection. The reasons why some ulcerations develop acute clinical infections (AI-DRFUs) whilst others develop chronic infection (CI-DRFUs) and the preceding host-microbe interactions in vivo remain largely unknown. Establishing that acute and chronic infections are distinct processes requires demonstrating that these are two different strategies employed by microbes when interacting with a host. In this study, dual-RNA seq was employed to differentiate the host-microbe metatranscriptome between DRFUs that had localized chronic infection or acute clinical infection. Comparison of the host metatranscriptome in AI-DRFUs relative to CI-DRFUs identified upregulated differentially expressed genes (DEGs) that functioned as regulators of vascular lymphatic inflammatory responses, Tcell signalling and olfactory receptors. Conversely, CI-DRFUs upregulated DEGs responsible for cellular homeostasis. Gene set enrichment analysis using Hallmark annotations revealed enrichment of immune and inflammatory profiles in CI-DRFUs relative to AI-DRFUs. Analysis of the microbial metatranscriptome identified the DEGs being enriched within AI-DRFUs relative to CI-DRFUs included several toxins, two-component systems, bacterial motility, secretion systems and genes encoding for energy metabolism. Functions relevant to DRFU pathology were further explored, including biofilm and bacterial pathogenesis. This identified that the expression of biofilm-associated genes was higher within CI-DRFUs compared to that of AI-DRFUs, with mucR being the most highly expressed gene. Collectively, these data provide insights into the host-microbe function in two clinically-distinct infective phenotypes that affect DRFUs. The data reveal that bacteria in acutely infected DRFUs prioritize motility over biofilm and demonstrate greater pathogenicity and mechanisms, which likely subvert host cellular and immune pathways to establish infection. Upregulation of genes for key vascular inflammatory mediators in acutely infected ulcers may contribute, in part, to the clinical picture of a red, hot, swollen foot, which differentiates an acutely infected ulcer from that of a chronic infection

Effect on total microbial load and community composition with two vs six-week topical Cadexomer Iodine for treating chronic biofilm infections in diabetic foot ulcers

This study compares two vs six weeks of topical antimicrobial therapy with Cadexomer Iodine in patients with diabetic foot ulcers (DFUs) complicated by chronic biofilm infections. Patients with non‐healing DFUs with suspected chronic biofilm infections were eligible for enrolment. Patients were randomised to receive either two or six weeks of treatment with topical Cadexomer Iodine. Tissue biopsies from the ulcers were obtained pre‐and‐post treatment and underwent DNA sequencing and real‐time quantitative polymerase chain reaction (PCR) to determine the total microbial load, community composition, and diversity of bacteria. Scanning electron microscopy confirmed biofilm in all 18 ulcers with suspected chronic biofilm infections. Cadexomer Iodine resulted in 14 of 18 (78%) samples achieving a mean 0.5 log10 reduction in microbial load. Regardless of treatment duration, there was no statistical difference in the reduction of total microbial loads. No difference in the rate of wound healing in the two groups was seen at 6 weeks. Cadexomer Iodine reduces the total microbial load in DFUs with chronic biofilm infections and affects microbial community composition and diversity. All ulcers in both groups showed an initial reduction in wound size with application of Cadexomer Iodine, which might reflect its effect on biofilms

Analysis of proximal bone margins in diabetic foot osteomyelitis by conventional culture, DNA sequencing and microscopy

Multiple approaches were employed to detect pathogens from bone margins associated with Diabetic Foot Osteomyelitis (DFO). Intra‐operative bone specimens of 14 consecutive subjects with suspected DFO were collected over a six‐month study period from Liverpool Hospital. Infected bone and a proximal bone margins presumed to be ‘clean/non‐infected’ were collected. Bone material was subjected to conventional culture, DNA sequencing and microscopy. In total, eight of 14 (57%) proximal bone margins had no growth by conventional culture but were identified in all proximal bone specimens by DNA sequencing. Proximal margins had lower median total microbial counts than infected specimens, but these differences were not statistically significant. Pathogens identified by sequencing in infected specimens were identified in proximal margins and the microbiomes were similar (ANOSIM = 0.02, p = 0.59). Using a combination of SEM and/or PNA‐FISH, we visualized the presence of microorganisms in infected bone specimens and their corresponding proximal margins of seven patients (50%) with DFO. We identify that bacteria can still reside in what seems to be proximal ‘clean’ margins. The significance and implications of clinical outcomes requires further analysis from a larger sample size that incorporates differences in surgical and post‐operative approaches, correlating any outcomes back to culture‐sequence findings

Analysis of proximal bone margins in diabetic foot osteomyelitis by conventional culture, DNA sequencing and microscopy

Multiple approaches were employed to detect pathogens from bone margins associated with Diabetic Foot Osteomyelitis (DFO). Intra‐operative bone specimens of 14 consecutive subjects with suspected DFO were collected over a six‐month study period from Liverpool Hospital. Infected bone and a proximal bone margins presumed to be ‘clean/non‐infected’ were collected. Bone material was subjected to conventional culture, DNA sequencing and microscopy. In total, eight of 14 (57%) proximal bone margins had no growth by conventional culture but were identified in all proximal bone specimens by DNA sequencing. Proximal margins had lower median total microbial counts than infected specimens, but these differences were not statistically significant. Pathogens identified by sequencing in infected specimens were identified in proximal margins and the microbiomes were similar (ANOSIM = 0.02, p = 0.59). Using a combination of SEM and/or PNA‐FISH, we visualized the presence of microorganisms in infected bone specimens and their corresponding proximal margins of seven patients (50%) with DFO. We identify that bacteria can still reside in what seems to be proximal ‘clean’ margins. The significance and implications of clinical outcomes requires further analysis from a larger sample size that incorporates differences in surgical and post‐operative approaches, correlating any outcomes back to culture‐sequence findings