Metatranscriptomic analysis reveals active bacterial communities in diabetic foot infections

Despite the extended view of the composition of diabetic foot infections (DFIs), little is known about which transcriptionally active bacterial communities are pertinent to infection, and if any differences are associated with increased infection severity. We applied a RNA sequencing approach to analyze the composition, function, and pathogenicity of the active bacterial communities in DFIs. Taxonomic profiling of bacterial transcripts revealed the presence of 14 bacterial phyla in DFIs. The abundance of the Spiroplasma, Vibrio, and Mycoplasma were significantly different in different infection severities (P < 0.05). Mild and severe stages of infections were dominated by Staphylococcus aureus and Porphyromonas asaccharolytica, respectively. A total of 132 metabolic pathways were identified of which ribosome and thiamin being among the most highly transcribed pathways. Moreover, a total of 131 antibiotic resistance genes, primarily involved in the multidrug efflux pumps/exporters, were identified. Furthermore, iron acquisition systems (synthesize and regulation of siderophores) and pathways involved in the synthesis and regulation of cell-surface components associated with adhesion, colonization, and movement of bacterial cells were the most common virulence factors. These virulence factors may help bacteria compete for scares resources and survive the host wound proteases. Characterization of transcriptionally active bacterial communities can help to provide an understanding of the role of key pathogens in the development of DFIs. Such information can be clinically useful allowing replacement of DFIs empirical therapy with targeted treatment

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

Utilisation of the 2019 IWGDF diabetic foot infection guidelines to benchmark practice and improve the delivery of care in persons with diabetic foot infections

Aims: To utilise the 2019 International Working Group on the Diabetic Foot (IWGDF) - diabetic foot infection (DFI) guidelines as an audit tool for clinical practice in patients with diabetes attending a High-Risk Foot Service. Methods: Data from 93 consecutive patients were collected over a 19-month period in patients attending a High-Risk Foot Service. The diagnosis and management of each patient in the sample were compared against the 2019 IWGDF DFI guidelines, grouped into four categories: Diagnosis, Microbiology, Treatment of soft tissue infection, and Surgical treatment and osteomyelitis. Deficits in performance were recorded using the recommendations as a benchmark standard. Results: There were 109 DFI events. Nineteen (63%) of the recommendations were met, 7 (24%) were partially met, and four (13%) recommendations were not met. Fourteen of the sample had no documented requests for full blood counts. Tissue was obtained for culture in 32 (29%) of the sample. No percutaneous bone biopsies were performed. Only 13 (28%) patients had intraoperative bone specimens sent for culture and sensitivities, with no bone specimens sent for histopathology. Modification of antibiotic therapy following available culture results was low, occurring in 12 out of 63 possible occasions (19%). The duration of antibiotic regimens in PEDIS 2 infections and osteomyelitis was greater than that recommended. Conclusions: Utilising the IWGDF DFI guidelines to benchmark clinical practice is a useful tool to identify gaps in clinical performance or service delivery and may help to improve patient care

Elastomeric pump infusion failures caused by inadequate Luer lock connector engagement to needleless connectors

Needleless connectors are used widely across all types of vascular access devices and provide safe, needleless administration of intravenous fluids and medications. An analysis of patients from an outpatient parenteral antimicrobial therapy program is presented in which elastomeric pumps had failed to flow due to incomplete tightening of Luer lock needleless connections. An alert was issued to community nursing staff responsible for daily elastomeric pump changes to ensure that needleless connectors were properly checked for full tightening. The frequency of failure of flow events before and after the alert was reviewed. Force and torque profiles required to activate the internal mechanism of connectors were measured in the 4 most frequently used needleless connectors in the outpatient parenteral antimicrobial therapy program. The degree of torque and force required to activate the different needleless connectors varied and was identified as a factor contributing to inadequate connection with the elastomeric pump and consequent failure of flow. Repeated feedback to nursing staff over the study period about the force and torque required for needleless connector flow activation resulted in a highly significant decrease in the rate of failure of flow events per elastomeric pump from a rate of 0.0147 events per elastomeric pump per year in the last 3 months of 2018 to 0.0003 in the first 6 months of 2020 (difference = 0.0144 [CI, 0.0097-0.02]; P < .0001)

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 X = 32,014) compared to tissue (X = 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. SOME OF THE SCIENTIC SYMBOLS CAN NOT BE REPRESENTED CORRECTLY IN THE ABSTRACT. PLEASE READ WITH CAUTION AND REFER TO THE ORIGINAL PUBLICATION

The effect of continuous diffusion of oxygen treatment on cytokines, perfusion, bacterial load, and healing in patients with diabetic foot ulcers

To evaluate continuous diffusion of oxygen therapy (CDO) on cytokines, perfusion, and bacterial load in diabetic foot ulcers we evaluated 23 patients for 3 weeks. Tissues biopsies were obtained at each visit to evaluate cytokines and quantitative bacterial cultures. Perfusion was measured with hyperspectral imaging and transcutaneous oxygen. We used paired T tests to compare continuous variables and independent T tests to compare healers and nonhealers. There was an increase from baseline to week 1 in TGF-β (P =.008), TNF-α (P =.014), VEGF (P =.008), PDGF (P =.087), and IGF-1 (P =.058); baseline to week 2 in TGF-β (P =.010), VEGF (P =.051), and IL-6 (P =.031); and baseline to week 3 with TGF-β (P =.055) and IL-6 (P =.054). There was a significant increase in transcutaneous oxygen after 1 week of treatment on both medial and lateral foot (P =.086 and.025). Fifty-three percent of the patients had at least a 50% wound area reduction (healers). At baseline, there were no differences in cytokines between healers and nonhealers. However, there was an increase in CXCL8 after 1 week of treatment (P =.080) and IL-6 after 3 weeks of treatment in nonhealers (P =.099). There were no differences in quantitative cultures in healers and nonhealers

Lyopreserved amniotic membrane is cellularly and clinically similar to cryopreserved construct for treating foot ulcers

We compared cellular viability between cryopreserved and lyopreserved amniotic membranes and clinical outcomes of the lyopreserved construct in a prospective cohort study of 40 patients with neuropathic foot ulcers. Patients received weekly application of lyopreserved membrane for 12 weeks with standard weekly debridement and offloading. We evaluated the proportion of foot ulcers that closed, time to closure, closure trajectories, and infection during therapy. We used chi-square tests for dichotomous variables and independent t-tests for continuous variables with an alpha of α =.10. Cellular viability was equivalent between cryo- and lyopreserved amniotic tissues. Clinically, 48% of subjects' wounds closed in an average of 40.0 days. Those that did not close were older (63 vs 59 years, P =.011) and larger ulcers at baseline (7.8 vs 1.6 cm2, P =.012). Significantly more patients who achieved closure reached a 50% wound area reduction in 4 weeks compared with non-closed wounds (73.7% vs 47.6%, P =.093). There was no difference in the slope of the wound closure trajectories between closed and non-closed wounds (0.124 and 0.159, P =.85), indicating the rate of closure was similar. The rate of closure was 0.60 mm/day (SD = 0.47) for wounds that closed and 0.50 mm/day (SD = 0.58) for wounds that did not close (P =.89)

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

Consensus guidelines for the identification and treatment of biofilms in chronic nonhealing wounds

Background: Despite a growing consensus that biofilms contribute to a delay in the healing of chronic wounds, conflicting evidence pertaining to their identification and management can lead to uncertainty regarding treatment. This, in part, has been driven by reliance on in vitro data or animal models, which may not directly correlate to clinical evidence on the importance of biofilms. Limited data presented in human studies have further contributed to the uncertainty. Guidelines for care of chronic wounds with a focus on biofilms are needed to help aid the identification and management of biofilms, providing a clinical focus to support clinicians in improving patient care through evidence-based medicine. Methods: A Global Wound Biofilm Expert Panel, comprising 10 clinicians and researchers with expertise in laboratory and clinical aspects of biofilms, was identified and convened. A modified Delphi process, based on published scientific data and expert opinion, was used to develop consensus statements that could help identify and treat biofilms as part of the management of chronic nonhealing wounds. Using an electronic survey, panel members rated their agreement with statements about biofilm identification and treatment, and the management of chronic nonhealing wounds. Final consensus statements were agreed on in a face-to-face meeting. Results: Participants reached consensus on 61 statements in the following topic areas: understanding biofilms and the problems they cause clinicians; current diagnostic options; clinical indicators of biofilms; future options for diagnostic tests; treatment strategies; mechanical debridement; topical antiseptics; screening antibiofilm agents; and levels of evidence when choosing antibiofilm treatments. Conclusion: This consensus document attempts to clarify misunderstandings about the role of biofilms in clinical practice, and provides a basis for clinicians to recognize biofilms in chronic nonhealing wounds and manage patients optimally. A new paradigm for wound care, based on a stepped-down treatment approach, was derived from the consensus statements

Biofilm-based wound care : the importance of debridement in biofilm treatment strategies

Emerging evidence from the last century on the behaviour of microorganisms in their natural environments (i.e. exploration of aquatic/marine environments) identified that microorganisms have a natural tendency to associate with surfaces, with each other, and prefer a sessile (stationary, slow growth) lifestyle (Geesey et al, 1978). A significant proportion of this work was conducted on environmental samples and has provided a platform for the contemporary medical models that we have come to understand as microbial biofilms. The concept of biofilms in human health and disease is now universally accepted in periodontal disease and dental caries (Marsh and Bradshaw, 1995), cystic fibrosis (Costerton, 2001; Bjarnsholt et al, 2009) and indwelling medical device infections (Donlan, 2001), Otitis media and other upper respiratory infections (Hall-Stoodley et al, 2006) and chronic wounds (James et al, 2008; Bjarnsholt et al, 2008; Malone et al, 2017a). Unlike planktonic counterparts, microorganisms in biofilm phenotypes demonstrate a remarkable tolerance to many forms of treatments. This presents a challenge to wound care clinicians dealing with chronic wounds complicated by biofilm. This article will focus on the treatment of wound biofilm that is supported by evidence-based practice