Dr Angela Oates

In recent years the application of high throughput DNA sequencing for analyzing the composition of the skin microbiota has identified a highly diverse group of microorganisms. Although the factors determining the composition of the skin microbiota are not completely understood, it is clear that an array of bacteria including Staphylococcus epidermidis, coynebacteria, propionibacteria, micrococci, several types of fungi and also small mites known as Demodex, are able to reside on normal human skin, generally without causing disease. Other classes of microorganism that have been associated with skin include viruses and eukaryotic parasites, in addition to species of fungi or bacteria not normally considered part of the skin microbiota, and more often associated with disease. Despite these generalizations, resident microbes can also be associated with superficial and invasive disease when the integrity of skin is compromised. Thus, infections occur more frequently and with greater severity in individuals with a compromized skin barrier or immune status. A wide range of microbes therefore interact with the surface of the skin, with pathogenicity varying as a function of intrinsic characteristics including the expression of virulence factors, combined with host factors which may predispose the skin to infection.

Background: Venous leg ulceration is common in older adults in the United Kingdom. The gold-standard treatment is compression therapy. There are several compression bandage and hosiery systems that can be prescribed or purchased, but it was unclear what types of compression systems are currently being used to treat venous leg ulceration within the UK. This online scoping survey of registered nurses sought to (1) to identify what compression systems are available across the UK, (2) how frequently these are in use and (3) if there are any restrictions on their use. Results: The results showed that registered nurses who treat patients with venous leg ulceration use a wide range of compression systems. The most frequently used systems are the ‘less bulky’ two-layer elastic and inelastic compression bandaging systems whilst two-layer hosiery was used less frequently and four-layer bandaging used infrequently. Nurses report that certain compression systems are less accessible through the usual procurement routes but this appears to be related to concerns about competency in application techniques. Conclusions: The data in this survey provides some important insights into the issues around the use of compression therapy for venous leg ulceration in the UK. Limiting access to certain types of compression may promote patient safety but limit patient choice. There may be underuse of the types of compression that promote patient independence, such as hosiery, and over-use of potentially sub-therapeutic therapy such as ‘reduced compression’. Overall, this study suggests that further consideration is needed about the provision of compression therapy to UK patients with venous leg ulceration to optimise care and patient choice.

Abstract

In addition to clinical signs of infection (e.g. inflammation, purulence and pain), a microbial count of ≥105 colony‐forming units/g has historically been used to define wound infection. However, it is increasingly recognised that, rather than a high bioburden level alone being detrimental to wound healing, it is the virulence of the invading microorganism and the host's immune status that can affect clinical outcomes. Bacteria, such as Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis, have developed a range of virulence factors to help them overcome host defences and proliferate within the underlying soft tissue. More specifically, bacterial proteases are one such virulence factor that has been implicated in promoting the invasion and destruction of the host tissue. Because of the complexities of microorganisms, the proteases can negatively impact the wound environment, leading to delayed wound healing. The aim of the present paper is to describe various extracellular bacterial proteases; review the impact they have on the wound environment, the host immune response and biofilms; and discuss potential wound management strategies against them. The evidence discussed suggests that proteases may play a profound role in wound infections, contribute to the development of an inflammatory response and impede wound healing.

ABSTRACT

Wound debridement samples and contralateral (healthy) skin swabs acquired from 26 patients attending a specialist foot clinic were analyzed by differential isolation and eubacterium-specific PCR-denaturing gradient gel electrophoresis (DGGE) in conjunction with DNA sequencing. Thirteen of 26 wounds harbored pathogens according to culture analyses, with Staphylococcus aureus being the most common (13/13). Candida (1/13), pseudomonas (1/13), and streptococcus (7/13) were less prevalent. Contralateral skin was associated with comparatively low densities of bacteria, and overt pathogens were not detected. According to DGGE analyses, all wounds contained significantly greater eubacterial diversity than contralateral skin ( P < 0.05), although no significant difference in total eubacterial diversity was detected between wounds from which known pathogens had been isolated and those that were putatively uninfected. DGGE amplicons with homology to Staphylococcus sp. (8/13) and S. aureus (2/13) were detected in putatively infected wound samples, while Staphylococcus sp. amplicons were detected in 11/13 noninfected wounds; S. aureus was not detected in these samples. While a majority of skin-derived DGGE consortial fingerprints could be differentiated from wound profiles through principal component analysis (PCA), a large minority could not. Furthermore, wounds from which pathogens had been isolated could not be distinguished from putatively uninfected wounds on this basis. In conclusion, while chronic wounds generally harbored greater eubacterial diversity than healthy skin, the isolation of known pathogens was not associated with qualitatively distinct consortial profiles or otherwise altered diversity. The data generated support the utility of both culture and DGGE for the microbial characterization of chronic wounds.

Diabetic foot wounds are commonly colonised by taxonomically diverse microbial communities and may additionally be infected with specific pathogens. Since biofilms are demonstrably less susceptible to antimicrobial agents than are planktonic bacteria, and may be present in chronic wounds, there is increasing interest in their aetiological role. In the current investigation, the presence of structured microbial assemblages in chronic diabetic foot wounds is demonstrated using several visualization methods. Debridement samples, collected from the foot wounds of diabetic patients, were histologically sectioned and examined using bright-field, fluorescence, and environmental scanning electron microscopy and assessed by quantitative differential viable counting. All samples (n= 26) harboured bioburdens in excess of 5 log10CFU/g. Microcolonies were identified in 4/4 samples by all three microscopy methods, although bright-field and fluorescence microscopy were more effective at highlighting putative biofilm morphology than ESEM. Results in this pilot study indicate that bacterial microcolonies and putative biofilm matrix can be visualized in chronic wounds using florescence microscopy and ESEM, but also using the simple Gram stain.

An aerobic, Gram-stain-negative, non-motile coccus, designated strain GVCNT2T, was isolated from the tonsils of a healthy adult female. Cells were oxidase- and catalase-positive, positive for the production of esterase (C4), esterase lipase (C8) and leucine arylamidase, and weakly positive for naphthol-AS-BI-phosphohydrolase and alkaline phosphatase. Cells were also capable of hydrolysing DNA. Growth was observed at 20–37 °C and in the presence of up to 1.5 % NaCl. Phylogenetic analysis of near full-length 16S rRNA gene sequences indicated that the strain exhibited closest sequence similarity to Moraxella boevrei ATCC 700022T (94.68 %) and an uncultured, unspeciated bacterial clone (strain S12-08; 99 %). The major fatty acids were C18 : 1ω9c, C18 : 0, C16 : 0 and C16 : 1ω6c/C16 : 1ω7c. The DNA G+C content of strain GVCNT2T was 40.7 mol%. The major respiratory quinone identified was Q-8. Strain GVCNT2T exhibited a comparable phenotypic profile to other members of the genus Moraxella but could be distinguished based on its ability to produce acid (weakly) from d-glucose, melibiose, l-arabinose and rhamnose and on its ability to hydrolyse DNA. On the basis of phenotypic and phylogenetic differences from other members of the family Moraxellaceae , strain GVCNT2T is considered to represent a novel species of a new genus, for which the name Faucicola mancuniensis gen. nov., sp. nov. is proposed. The type strain of Faucicola mancuniensis is GVCNT2T ( = DSM 28411T = NCIMB 14946T).

Sessile cultures of the skin bacteria Staphylococcus saprophyticus and Corynebacterium xerosis were grown using novel fine-celled foam substrata to test the outcome of challenge by methicillin-resistant Staphylococcus aureus or Pseudomonas aeruginosa under three growth medium regimens (simulated sweat, simulated serum or simulated sweat substituted with simulated serum during the microbial challenge). S. saprophyticus and C. xerosis significantly limited MRSA and P. aeruginosa immigration respectively, under the simulated sweat and serum medium regimes. Under the substitution medium regime however, MRSA and P. aeruginosa integrated into pre-established biofilms to a significantly greater extent, attaining cell densities similar to the axenic controls. The outcome of challenge was influenced by the medium composition and test organism but could not be predicted based on planktonic competition assays or growth dynamics. Interactions between skin and wound isolates could be modelled using the fine-celled foam-based system. This model could be used to further investigate interactions and also in preclinical studies of antimicrobial wound care regimens.

Although many antibiotics are active against Gram-positive bacteria, fewer also show activity against Gram-negative bacteria. Here, we present a combination of in silico (electron ion-interaction potential, molecular docking, ADMET), NMR, and microbiological investigations of selected macrolides (14-membered, 15-membered, and 16-membered), aiming to discover the pattern of design for macrolides active against Gram-negative bacteria. Although the conformational studies of 14-membered and 15-membered macrolides are abundant in the literature, 16-membered macrolides, and their most prominent representative tylosin A, have received relatively little research attention. We therefore report the complete 1H and 13C NMR assignment of tylosin A in deuterated chloroform, as well as its 3D solution structure determined through molecular modelling (conformational search) and 2D ROESY NMR. Additionally, due to the degradation of tylosin A in deuterated chloroform, other species were also detected in 1D and 2D NMR spectra. We additionally studied the anti-bacterial activity of tylosin A and B against selected Gram-positive and Gram-negative bacteria.

Aim The management of infected diabetic foot ulcers (DFUs) requires balancing the need for timely interventions against the desire for targeted antibiotic therapy, which relies on laboratory results. This study aimed to evaluate concordance between molecular and conventional culture and sensitivity (C&S) methods in identifying bacteria from infected DFUs. Methods This study was conducted alongside CODIFI2, a Phase III randomised controlled trial comparing tissue sampling with wound swabbing. It assessed C&S and metagenomic 16S rRNA gene sequencing (M16s) in DFUs with suspected mild or moderate infections using both tissue and swab samples. Results In 145 participants, C&S identified 248 microorganisms across 25 genera including eight anaerobic genera. M16s identified a greater number and diversity of microorganisms, detecting 455 across 40 genera, including 173 anaerobes from 15 distinct genera. No bacterial growth was reported in 25.5% (95% CI:18.0%-32.3%) of C&S samples whereas M16s identified at least one organism in all samples. While observed agreement between methods was high (75%), Cohen’s Kappa revealed low concordance overall, except for Pseudomonas spp. and Streptococcus spp. (Kappa ≥0.5). Conclusions M16s detected a broader microbial spectrum, including fastidious anaerobes, but its low concordance with C&S and lack of antibiotic sensitivity data, challenge its suitability as a replacement for C&S in mild or moderate DFU infections. It may offer advantages in infections where increased sensitivity is beneficial, particularly where extended culture approaches are recommended to detect fastidious or low-abundance organisms. For mild to moderate diabetic foot ulcer infections, our findings support continued reliance on conventional C&S testing.

Aims/hypothesis CODIFI2 compared wound swabbing and tissue sampling in people with infected diabetic foot ulcers (DFU) to determine effects on clinical outcomes. Methods Multicentre, Phase III, prospective, non-blind, 2-arm parallel group, randomised controlled trial comparing time to ulcer healing (primary outcome), proportions healed, antimicrobial regimen, ulcer area reduction, hospitalisation duration, and time to death, for swab compared to tissue sampling. Allocation was via central and independent randomisation system, with minimisation by DFU site, number, type, size, location, and duration. Follow-up was 52-104 weeks, with healing confirmed by a blinded assessor. Sample-size target was 730 participants for 90% power to detect 12.5% difference in healing at 52-weeks. Results Between May 2019 and May 2022 149 participants were recruited (75 Swab, 74 Tissue) from 21 UK sites. The 52-week cumulative incidence of confirmed healing as first event was 45.3% (33.5% to 56.4%) and 44.6% (33.0 to 55.6%) for swab vs tissue. Hazard ratio (HR) for healing for tissue vs swab was 1.01 (95% CI 0.65 to 1.55). Median (IQR) days in hospital was 17 (12 to 39) for swab and 16 (10 to 32) for tissue. Seventeen swab and 7 tissue participants died during follow-up and 18.7% and 24.3% participants in the swab and tissue groups respectively had an amputation. Conclusions/interpretation This trial was underpowered to determine whether swab or tissue sampling impacted rate of healing or time to healing. Clinical prescribing and patient outcomes differed slightly between groups, hence the clinical benefit of tissue sampling is not established.

Hyaluronan (HA) is among the most important bioactive polymers in mammals, playing a key role in a number of biological functions. In the last decades, it has been increasingly studied as a biomaterial for drug delivery systems, thanks to its physico-chemical features and ability to target and enter certain cells. The most important receptor of HA is ‘Cluster of Differentiation 44’ (CD44), a cell surface glycoprotein over-expressed by a number of cancers and heavily involved in HA endocytosis. Moreover, CD44 is highly expressed by keratinocytes, activated macrophages and fibroblasts, all of which can act as ‘reservoirs’ for intracellular pathogens. Interestingly, both CD44 and HA appear to play a key role for the invasion and persistence of such microorganisms within the cells. As such, HA is increasingly recognised as a potential target for nano-carriers development, to pursuit and target intracellular pathogens, acting as a ‘Trojan Horse’. This review describes the biological relationship between HA, CD44 and the entry and survival of a number of pathogens within the cells and the subsequent development of HA-based nano-carriers for enhancing the intracellular activity of antimicrobials.

This multi-centre, Phase III, prospective, unblinded, two-arm parallel group, randomised controlled trial compared clinical (reported elsewhere) and economic outcomes of swab versus tissue sampling over a 52–104 week period. Resource use was logged using case record forms and patient questionnaire at weeks 4, 12, 26, 39, 52 and 104, costed using laboratory and published sources from the UK NHS perspective, at 2021/2022 price-year. EQ-5D-3L questionnaires issued at these time points were used to derive quality-adjusted life-years (QALYs). To account for imbalances such as age, a regression-based approach was used to estimate survival, expected costs and QALYs between the sampling arms. Available case analysis (ACA) and multiple imputation methods were applied for self reported missing data, and ACA for researcher-collected data (survival, hospitalisations and antibiotic use). Probabilistic sensitivity analysis was used to assess the uncertainty of economic results. Results We recruited 149 participants (75 swab, 74 tissue) from 21 UK sites, between 07 May 2019 and 28 April 2022 (last follow-up 28 April 2023). Planned sample size was 730 participants, for 90% power to detect 12.5% difference in healing at 52 weeks, but the trial stopped early due to low recruitment. Expected QALYs in the swab-sampling arm were greater than in the tissue-sampling arm at weeks 26, 52 and 104. The cost of tissue sampling was greater than of swabbing when including antibiotics and hospitalisation. Swab sampling participants had higher QALYs and lower costs across weeks 26–52, reducing slightly by week 104. Conclusions Because of higher costs, lower QALYs and lack of evidence of benefit, potentially due to the trial being underpowered, tissue sampling was dominated by wound swabbing in the cost-effectiveness analysis.

Abstract

Staphylococcus aureus is one of the most significant human pathogens that is frequently isolated in a wide range of superficial and systemic infections. The ability of S. aureus to invade and survive within host cells such as keratinocytes and host immune cells has been increasingly recognized as a potential factor in persistent infections and treatment failures. The incorporation of antibiotics into hyaluronan‐cholesterol nanohydrogels represents a novel paradigm in the delivery of therapeutic agents against intracellular bacteria. The work presented herein shows that NHs quickly enter human keratinocytes and accumulate into lysosomes. When used for targeting intracellular S. aureus the antimicrobial activity of loaded levofloxacin is enhanced, possibly changing the antibiotic intracellular fate from cytosol to lysosome. Indeed, gentamicin, an antibiotic that predominantly accumulates in lysosomes, shows significant and equal antibacterial activity when entrapped into NHs. These results strongly suggest that lysosomal formulations may display preferential activity toward intracellular S. aureus, opening new avenues for the use of HA‐based NHs for treatment of such skin infections.

Background Foot ulcers affecting people with diabetes (diabetic foot ulcers [DFUs]) often become infected, potentially leading to amputation. Suspected DFU infection is treated with immediate empiric antimicrobials, with wound samples for culture and sensitivity (C&S) collected to optimise antibiotic therapy. Collecting samples with swabs is easier than obtaining tissue , but report fewer pathogens and more contaminants. Compared with standard C&S laboratory methods, molecular microbiology identifies more organisms. How these differences affect clinical decisions or outcomes is currently unknown. Objectives Main study: To determine if taking tissue samples, versus swabs from suspected infected DFU affects ulcer healing, antibiotic prescribing, costs of care, and patient safety. Sub-study 1 (SS1). To determine the agreement between microbiology results from C&S versus molecular techniques, and assess whether intention of prescribers to change antimicrobials differs based on sampling methods. Sub-study 2 (SS2). A health-economic perspective of the expected application of empiric and/or targeted treatment regimens and the cost-consequences of treatment decisions based on SS1. Sub-study 3 (SS3): To compare questionnaire response rates for theoretically-informed versus standard participant letters. Sub-study 4 (SS4). To explore clinician perspectives on DFU sampling and processing techniques. Design Main study: Randomised controlled trial of results of performing tissue sampling versus swabbing of wounds in people with suspected mild or moderate infected DFU. Individually randomised (allocation concealed), 1:1, tissue or swab sampling for suspected DFU infection. Follow-up 12 - 24-months. A priori sample size estimate 730. SS1: Cross-sectional agreement study of microbiology results from molecular versus C&S techniques and virtual clinic. DFUs sampled for standard microbiology and central laboratory analysis (molecular). SS2: Exploratory cost-consequence analysis of molecular processing and the likelihood of empiric and targeted treatment based on treatment decisions from SS1. SS3: Randomised trial of theoretically-informed versus standard participant letters. SS4: Qualitative study explored clinicians’ perspectives regarding sampling and processing techniques. Setting and participants 21 UK DFU clinics. Participants with suspected mild or moderate infected DFUs. Main outcome measures Main study: Time to ulcer healing (primary outcome blinded assessment), proportion of ulcers healed, antibiotic usage, ulcer area reduction at 4-weeks, hospitalisation duration, time to death, quality of life, and cost-effectiveness. SS1: Extent of agreement regarding presence or absence of pathogens from standard versus molecular microbiology. Decision to revise antimicrobials based on sampling method. SS2: Modelled costs (from virtual clinic) of antimicrobial change for standard or molecular analysis. SS3: Response rate for questionnaires. Results The trial was stopped early, after enrolling only 149 participants, due to poor recruitment. Main study: The hazard ratio for wound healing for patients undergoing tissue versus swab sampling was 1.01 (95% CI 0.65 to 1.55). The swab group had both higher QALYs and lower costs across most time points. SS1: Agreement between C&S and molecular microbiology was low. A higher proportion of molecular versus C&S, vignettes led to recommendations to change antimicrobials (difference 20.5% (95% CI 8.9 to 31.1%)). SS2: The modelled costs of molecular processing versus C&S were £120 higher per wound. SS3: Response rates were 14.8% higher at 52 weeks (95% CI -3.2% to 31.2%) using a theoretically informed versus standard cover letter. SS4: Clinicians were not confident about replacing C&S with molecular microbiology, as the result reporting was unfamiliar to them. Limitations The trial was underpowered.. Conclusions Trial recruitment was challenging during the COVID pandemic and its aftermath. Whilst the results leave substantial uncertainty regarding differences in healing between the sampling methods, tissue sampling appeared costlier and was associated with lower QALYs than swabbing.

An in vitro model was developed to assess the effects of topical antimicrobials on taxonomically defined wound biofilms. Biofilms were exposed over seven days to povidone-iodine, silver acetate or polyhexamethylene biguanide (PHMB) at concentrations used in wound dressings. The rank order of tolerance in multi-species biofilms, based on an analysis of the average bacterial counts over time was P. aeruginosa > methicillin-resistant Staphylococcus aureus (MRSA) > B. fragilis > S. pyogenes. The rank order of effectiveness for the antimicrobials in the biofilm model was povidone-iodine > PHMB > silver acetate. None of the test compounds eradicated P. aeruginosa or MRSA from the biofilms although all compounds except silver acetate eliminated S. pyogenes. Antimicrobial effectiveness against bacteria grown in multi-species biofilms did not correlate with planktonic susceptibility. Defined biofilm populations of mixed-species wound pathogens could be maintained in the basal perfusion model, facilitating the efficacy testing of treatments regimens and potential dressings against multi-species biofilms composed of wound isolates.

The increasing prevalence of artificial intelligence in educational domains raises both opportunities and challenges in the context of academic integrity and pedagogical efficacy. This study outlines an innovative project that investigates the use of ChatGPT as a tool for enhancing the critical evaluation skills of master’s students in biomedical science. Using a dual approach combining AI-generated essay writing with subsequent student-led critical evaluation, this project sought to foster deeper critical evaluation skills in learners. By having participants critically assess AI-generated essays, supported critical evaluation based on peer-reviewed literature, the project aimed to deepen their evaluative skills. Outputs from the tasks were compared against academic benchmarks considering factors such as marks, writing, and overall quality. Participant perceptions were collected through a combination of a focus group session and an evaluation questionnaire. The key finding of this project was that while ChatGPT demonstrated proficiency in structural coherence and grammatical accuracy, it did not augment academic performance– participant marks for the AI-generated essays aligned closely with their overall module marks, showing no overall improvement. However, this study did see an increase in marks for participants’ critical evaluations. This suggests that ChatGPT was more effective as an assessment tool when used for critical evaluation tasks, aligning with pedagogical emphasis on nurturing critical evaluation skills. User interaction with AI emerged as a significant variable that influenced the tool’s efficacy, highlighting the need for a nuanced approach to its integration into educational settings. The study concludes that while ChatGPT offers promising avenues for both drafting and assessment, and demonstrated a high level of factual accuracy, it is not a substitute for human-led academic enquiry, and students preferred writing their own essays.