Direct metagenomics investigation of non-surgical hard-to-heal wounds: a review.

BACKGROUND: Non-surgical chronic wounds, including diabetes-related foot diseases (DRFD), pressure injuries (PIs) and venous leg ulcers (VLU), are common hard-to-heal wounds. Wound evolution partly depends on microbial colonisation or infection, which is often confused by clinicians, thereby hampering proper management. Current routine microbiology investigation of these wounds is based on in vitro culture, focusing only on a limited panel of the most frequently isolated bacteria, leaving a large part of the wound microbiome undocumented. METHODS: A literature search was conducted on original studies published through October 2022 reporting metagenomic next generation sequencing (mNGS) of chronic wound samples. Studies were eligible for inclusion if they applied 16 S rRNA metagenomics or shotgun metagenomics for microbiome analysis or diagnosis. Case reports, prospective, or retrospective studies were included. However, review articles, animal studies, in vitro model optimisation, benchmarking, treatment optimisation studies, and non-clinical studies were excluded. Articles were identified in PubMed, Google Scholar, Web of Science, Microsoft Academic, Crossref and Semantic Scholar databases. RESULTS: Of the 3,202 articles found in the initial search, 2,336 articles were removed after deduplication and 834 articles following title and abstract screening. A further 14 were removed after full text reading, with 18 articles finally included. Data were provided for 3,628 patients, including 1,535 DRFDs, 956 VLUs, and 791 PIs, with 164 microbial genera and 116 species identified using mNGS approaches. A high microbial diversity was observed depending on the geographical location and wound evolution. Clinically infected wounds were the most diverse, possibly due to a widespread colonisation by pathogenic bacteria from body and environmental microbiota. mNGS data identified the presence of virus (EBV) and fungi (Candida and Aspergillus species), as well as Staphylococcus and Pseudomonas bacteriophages. CONCLUSION: This study highlighted the benefit of mNGS for time-effective pathogen genome detection. Despite the majority of the included studies investigating only 16 S rDNA, ignoring a part of viral, fungal and parasite colonisation, mNGS detected a large number of bacteria through the included studies. Such technology could be implemented in routine microbiology for hard-to-heal wound microbiota investigation and post-treatment wound colonisation surveillance.

Bacteriological analysis and antibiotic resistance in patients with diabetic foot ulcers in Dhaka.

The primary objective of this study was to isolate bacteria from diabetic foot ulcers and subsequently assess their antibiotic resistance capabilities. Seventy-five patients diagnosed with diabetic foot ulcers were investigated. A number of these patients (97.33%) had type 2 diabetes, with a significant proportion of them having been diagnosed for 1-5 years (29.33%). Notably, a substantial number of these individuals were on insulin usage (78.66%). Among the patients under examination, 49.33% reported having no use of tobacco products, alcohol, or betel leaf. The ulcers analyzed in this study were classified into grades 1-5 according to the Wagner scale. Wagner grade 2 diabetic foot ulcers had the highest number of culture-positive patients, at 33.33%. Pus samples collected from patients were cultured on selective media, and bacterial identity was confirmed by biochemical tests and polymerase chain reaction. A total of 141 isolates were isolated. Among the isolates, 82.97% gram-negative bacteria and 17.02% gram-positive bacteria were detected. Klebsiella pneumoniae was the most common isolate. Proteus spp., Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were also detected. Approximately 61.33% of the ulcers exhibited were polybacterial. In this study, it was observed that all bacterial isolates, except for Proteus spp., were primarily detected in patients classified under Wagner's grade 2. Moreover, antibiotic susceptibility was also tested on these 141 isolates. Among them, Escherichia coli showed the highest multidrug resistance, 81.81%. Most of the gram-negative bacteria were resistant to ampicillin. All of the gram-negative isolates exhibited high levels of susceptibility to piperacillin-tazobactam, and these levels were Klebsiella pneumoniae (97.56%), Pseudomonas aeruginosa (95.24%), Escherichia coli (81.82%), and Proteus spp. (80%). On the other hand, gram-positive Staphylococcus aureus mostly showed sensitivity towards vancomycin and norfloxacin (79.17%).

Selective Delivery of Clindamycin Using a Combination of Bacterially Sensitive Microparticle and Separable Effervescent Microarray Patch on Bacteria Causing Diabetic Foot Infection.

INTRODUCTION: Diabetic foot infection (DFI) is one of the complications of diabetes mellitus. Clindamycin (CLY) is one of the antibiotics recommended to treat DFI, but CLY given orally and intravenously still causes many side effects. METHODS: In this study, we encapsulated CLY in a bacteria sensitive microparticle system (MP-CLY) using polycaprolactone (PCL) polymer. MP-CLY was then delivered in a separable effervescent microarray patch (MP-CLY-SEMAP), which has the ability to separate between the needle layer and separable layer due to the formation of air bubbles when interacting with interstitial fluid in the skin. RESULT: The characterization results of MP-CLY proved that CLY was encapsulated in large amounts as the amount of PCL polymer used increased, and there was no change in the chemical structure of CLY. In vitro release test results showed increased CLY release in media cultured with Staphylococcus aureus bacteria and showed controlled release. The characterization results of MPCLY-SEMAP showed that the developed formula has optimal mechanical and penetration capabilities and can separate in 56 ± 5.099 s. An ex vivo dermatokinetic test on a bacterially infected skin model showed an improvement of CLY dermatokinetic profile from MP-CLY SEMAP and a decrease in bacterial viability by 99.99%. CONCLUSION: This research offers proof of concept demonstrating the improved dermatokinetic profile of CLY encapsulated in a bacteria sensitive MP form and delivered via MP-CLY-SEMAP. The results of this research can be developed for future research by testing MP-CLY-SEMAP in vivo in appropriate animal models.

16S rRNA seq-identified Corynebacterium promotes pyroptosis to aggravate diabetic foot ulcer.

BACKGROUND: Diabetic foot ulcer (DFU) is one of the main chronic complications caused by diabetes, leading to amputation in severe cases. Bacterial infection affects the wound healing in DFU. METHODS: DFU patients who met the criteria were selected, and the clinical data were recorded in detail. The pus exudate from the patient's foot wound and venous blood were collected for biochemical analysis. The distribution of bacterial flora in pus exudates of patients was analyzed by 16S rRNA sequencing, and the correlation between DFU and pathogenic variables, pyroptosis and immunity was analyzed by statistical analysis. Then, the effects of key bacteria on the inflammation, proliferation, apoptosis, and pyroptosis of polymorphonuclear leukocytes were investigated by ELISA, CCK-8, flow cytometry, RT-qPCR and western blot. RESULTS: Clinical data analysis showed that Wagner score was positively correlated with the level of inflammatory factors, and there was high CD3+, CD4+, and low CD8+ levels in DFU patients with high Wagner score. Through alpha, beta diversity analysis and species composition analysis, Corynebacterium accounted for a large proportion in DFU. Logistics regression model and Person correlation analysis demonstrated that mixed bacterial infections could aggravate foot ulcer, and the number of bacteria was closely related to inflammatory factors PCT, PRT, immune cells CD8+, and pyroptosis-related proteins GSDMD and NLRP3. Through in vitro experiments, Corynebacterium inhibited cell proliferation, promoted inflammation (TNF-α, PCT, CRP), apoptosis and pyroptosis (IL-1ß, LDH, IL-18, GSDMD, NLRP3, and caspase-3). CONCLUSION: Mixed bacterial infections exacerbate DFU progression with a high predominance of Corynebacterium, and Corynebacterium promotes inflammation, apoptosis and pyroptosis to inhibit DFU healing.

Correlation between inflammatory factors, autophagy protein levels, and infection in granulation tissue of diabetic foot ulcer.

OBJECTIVE: To observe the expression of inflammatory factors and autophagy-related proteins in granulation tissue of diabetic foot ulcer (DFU) patients and analyze their relationship with infection. METHODS: This is a retrospective cohort study. One hundred and fifty-two patients with DFU in our hospital from July 2020 to March 2022 were selected as the DFU group, including 98 cases in infection stage group and 54 cases in infection control group. The patients were further graded as the mild (51 cases), the moderate (65 cases), and the severe infection group (36 cases) according to the Wagner grading criteria. Sixty-seven patients with foot burns during the same period were selected as the control group. The distribution of pathogenic bacteria on the ulcer surface was examined using fully automated bacterial analyzer. The expression of inflammatory factors (procalcitonin [PCT], tumor necrosis factor-α [TNF-α], and interleukin-6 [IL-6]) was valued by real-time fluorescence quantitative PCR (qRT-PCR). Protein expression was measured by immunohistochemistry (IHC). The correlation was analyzed by Pearson. RESULTS: The surface infection of DFU patients was mostly induced by gram-negative and gram-positive bacteria, with Pseudomonas aeruginosa predominating among the Gram-negative bacteria and Staphylococcus aureus among the gram-positive bacteria. The infection stage group had higher content of PCT, TNF-α, and IL-6 and lower content of Beclin-1 and LC3 than the infection control group (p < .001). The levels of PCT, TNF-α, and IL-6 in the DFU patients with cardiovascular events were higher than those in the nonoccurrence group (p < .001). Glycated hemoglobin in patients with DFU was positively correlated with PCT, TNF-α, and IL-6 levels (p < .05), and negatively correlated with Beclin-1 and LC3 levels (p < .001). CONCLUSION: P. aeruginosa and S. aureus were predominant bacterial in DFU infections. Inflammatory factor and autophagy protein expression were closely correlated with the degree of infection.

L-arginine supplementation abrogates hypoxia-induced virulence of Staphylococcus aureus in a murine diabetic pressure wound model.

Diabetic foot ulcers (DFUs) are the most common complications of diabetes resulting from hyperglycemia leading to ischemic hypoxic tissue and nerve damage. Staphylococcus aureus is the most frequently isolated bacteria from DFUs and causes severe necrotic infections leading to amputations with a poor 5-year survival rate. However, very little is known about the mechanisms by which S. aureus dominantly colonizes and causes severe disease in DFUs. Herein, we utilized a pressure wound model in diabetic TALLYHO/JngJ mice to reproduce ischemic hypoxic tissue damage seen in DFUs and demonstrated that anaerobic fermentative growth of S. aureus significantly increased the virulence and the severity of disease by activating two-component regulatory systems leading to expression of virulence factors. Our in vitro studies showed that supplementation of nitrate as a terminal electron acceptor promotes anaerobic respiration and suppresses the expression of S. aureus virulence factors through inactivation of two-component regulatory systems, suggesting potential therapeutic benefits by promoting anaerobic nitrate respiration. Our in vivo studies revealed that dietary supplementation of L-arginine (L-Arg) significantly attenuated the severity of disease caused by S. aureus in the pressure wound model by providing nitrate. Collectively, these findings highlight the importance of anaerobic fermentative growth in S. aureus pathogenesis and the potential of dietary L-Arg supplementation as a therapeutic to prevent severe S. aureus infection in DFUs.IMPORTANCES. aureus is the most common cause of infection in DFUs, often resulting in lower-extremity amputation with a distressingly poor 5-year survival rate. Treatment for S. aureus infections has largely remained unchanged for decades and involves tissue debridement with antibiotic therapy. With high levels of conservative treatment failure, recurrence of ulcers, and antibiotic resistance, a new approach is necessary to prevent lower-extremity amputations. Nutritional aspects of DFU treatment have largely been overlooked as there has been contradictory clinical trial evidence, but very few in vitro and in vivo modelings of nutritional treatment studies have been performed. Here we demonstrate that dietary supplementation of L-Arg in a diabetic mouse model significantly reduced duration and severity of disease caused by S. aureus. These findings suggest that L-Arg supplementation could be useful as a potential preventive measure against severe S. aureus infections in DFUs.

Commonly associated aerobic microbial pathogens and their antibiotic susceptibility profile in diabetic foot ulcers in tertiary care centre in Western Maharashtra.

PURPOSE: Diabetes mellitus (DM-II) is a metabolic disorder either due to reduced insulin production or reduced insulin sensitivity. Diabetic foot ulcer (DFU) is one of the most devastating complications of DM-II. This study was performed to assess commonly isolated micro-organisms and their anti-microbial sensitivity pattern in diabetic foot ulcers in a tertiary care centre in Western Maharashtra. METHODS: Adult patients with a known case of DM-II with foot lesions, suspected to be a Diabetic Foot Infections (DFIs) at the tertiary care hospital from Aug 2022 to Sept 2022 were included in the study. After obtaining informed written consent, pus sample was collected with sterile swab from lesion's base and submitted to Microbiology Laboratory for aerobic culture and sensitivity. RESULTS: Out of 56 enrolled patients, 47 (83.9%) patients tested positive for bacteriological growth and there was 'no growth' in 9 (16.07%) patients at the end of 48 h of aerobic incubation. There was male preponderance and patients were in age group of 35-85 years. The most commonly isolated micro-organisms were P. aeruginosa (17.8%), followed by S. aureus (14.2%), K. pneumonia and P. mirabilis (12.5% each). The resistance markers observed was ESBL producer, AmpC producer, MBL producer, Methicillin resistance and Inducible Clindamycin Resistance (ICR). CONCLUSION: Due to the injudicious use of antibiotics, antibiotic resistance has been increased in all types of soft tissue infections. The empirical formula for the treatment of DFIs should be decided for given geographical reasons according to antimicrobial susceptibility profile from particular geographical area or health care institute.

Factores de riesgo asociados a osteomielitis por Corynebacterium striatum en pacientes con pie diabético / Risk factors associated with osteomyelitis due to Corynebacterium striatum in patients with diabetic foot

Objetivo Corynebacterium striatum (C. striatum) es un microorganismo emergente en la infección de pie diabético poco estudiado. El objetivo es evaluar los factores de riesgo (FR) relacionados con osteomielitis por C. striatum en paciente con pie diabético. Métodos Estudio casos-controles en la Unidad de Pie Diabético entre 2015 y 2021. Un número de 44 pacientes con osteomielitis por C. striatum (casos) y 44 con osteomielitis por microorganismo distinto (controles) fueron incluidos. Resultados Enfermedad arterial periférica (odds ratio [OR]: 2,8, p = 0,037), fibrilación auricular (OR: 3,7, p = 0,034), pie diabético isquémico (OR: 3,3, p = 0,020) y antibioterapia previa prolongada más de 14 días (OR: 3,4, p = 0,012) fueron FR para la osteomielitis por C. striatum. En el análisis multivariado la antibioterapia más de 14 días fue el único FR independiente (OR: 3,46; p = 0,017) para osteomielitis por C. striatum. Conclusiones La antibioterapia previa recibida durante más de 14 días es un FR independiente y estadísticamente significativo de osteomielitis por C. striatum en pacientes con pie diabético (AU)

Objective Corynebacterium striatum (CS) is an emerging micro-organism in diabetic foot infection for which there are currently few studies. The objective was to analyze the risk factors (RF) related to CS osteomyelitis in patients with diabetic foot. Methods A case–control study was conducted in the Diabetic Foot Unit between 2015 and 2021. Forty-four patients with osteomyelitis due to CS (cases) and 44 patients with osteomyelitis due a different micro-organism (controls) were included. Results Peripheral artery disease (OR: 2.8, p = 0.037), atrial fibrillation (OR: 3.7, p = 0.034), ischemic diabetic foot (OR: 3.3, p = 0.020) and previous prolonged antibiotic therapy more than 14 days (OR: 3.4, p = 0.012) were identified as RF for osteomyelitis due to CS. When performing the multivariate analysis antibiotic therapy >14 days was independent RF (OR: 3.46; p = 0.017). Conclusions Previous antibiotic therapy received more than 14 days is an independent and statistically significant RF for CS osteomyelitis in patients with diabetic foot (AU)

Analysis of risk factors of infection in diabetic foot patients.

This cross-sectional study assessed the risk factors for infection in 150 diabetic foot patients admitted to the Xiamen University Hospital between October 2020 and October 2022. Patients were categorised as infected (n = 80) or uninfected (n = 70) cohorts. The diabetic foot was evaluated using the American Diabetic Foot Grading system, whereas ulcers were categorised using Wagner's method. Analysed were patient-specific information, clinical data and risk factors including neuropathy, arterial disease and foot deformities. Our findings revealed no statistically significant differences between infected and uninfected cohorts concerning age, body mass index, gender, duration of diabetes or ankle-brachial index values (p > 0.05). However, infected group had a higher proportion of smokers and reduced socio-economic status (p < 0.05). Wagner grades indicated a greater severity in the infected group, with grade 3, grade 4 and grade 5 differing significantly (p < 0.05). Comparative analysis of ulcer characteristics revealed no statistically significant differences in ulcer surface area and depth, but the infected group had a higher prevalence of osteomyelitis and a greater number of ulcers (p > 0.05). Blood vessel complications, retinopathy, the presence of three or more ulcers, osteomyelitis and diabetic nephropathy were substantially more prevalent in the infected group, as determined by univariate analysis (p < 0.05). Subsequent multivariate logistic analysis revealed that patients with blood vessel complications, retinopathy, osteomyelitis, diabetic nephropathy and three or more ulcers were at increased risk for infection (p < 0.05). In addition, lifestyle factors, such as smoking, sedentary behaviour, inadequate foot hygiene, obesity and poor glycaemic control, were also associated with higher infection rates. A multivariate analysis of foot wound factors revealed that deeper, longer and recurrent lesions increased the likelihood of infection. Escherichia coli was the most frequently isolated bacterium from the infected group's bacterial culture, followed by Pseudomonas aeruginosa and Staphylococcus aureus. The study enhanced our comprehension of the multifactorial risk factors associated with infections in diabetic foot patients, highlighting the need for thorough clinical evaluation, lifestyle modification and vigilant infection control.

Diagnosis of infection in the foot of patients with diabetes: A systematic review.

BACKGROUND: Securing an early accurate diagnosis of diabetic foot infections and assessment of their severity are of paramount importance since these infections can cause great morbidity and potential mortality and present formidable challenges in surgical and antimicrobial treatment. METHODS: In June 2022, we searched the literature using PubMed and EMBASE for published studies on the diagnosis of diabetic foot infection (DFI). On the basis of pre-determined criteria, we reviewed prospective controlled, as well as non-controlled, studies in English. We then developed evidence statements based on the included papers. RESULTS: We selected a total of 64 papers that met our inclusion criteria. The certainty of the majority of the evidence statements was low because of the weak methodology of nearly all of the studies. The available data suggest that diagnosing diabetic foot infections on the basis of clinical signs and symptoms and classified according to the International Working Group of the Diabetic Foot/Infectious Diseases Society of America scheme correlates with the patient's likelihood of the need for hospitalisation, lower extremity amputation, and risk of death. Elevated levels of selected serum inflammatory markers such as erythrocyte sedimentation rate (ESR), C-reactive protein and procalcitonin are supportive, but not diagnostic, of soft tissue infection. Culturing tissue samples of soft tissues or bone, when care is taken to avoid contamination, provides more accurate microbiological information than culturing superficial (swab) samples. Although non-culture techniques, especially next-generation sequencing, are likely to identify more bacteria from tissue samples including bone than standard cultures, no studies have established a significant impact on the management of patients with DFIs. In patients with suspected diabetic foot osteomyelitis, the combination of a positive probe-to-bone test and elevated ESR supports this diagnosis. Plain X-ray remains the first-line imaging examination when there is suspicion of diabetic foot osteomyelitis (DFO), but advanced imaging methods including magnetic resonance imaging (MRI) and nuclear imaging when MRI is not feasible help in cases when either the diagnosis or the localisation of infection is uncertain. Intra-operative or non-per-wound percutaneous biopsy is the best method to accurately identify bone pathogens in case of a suspicion of a DFO. Bedside percutaneous biopsies are effective and safe and are an option to obtain bone culture data when conventional (i.e. surgical or radiological) procedures are not feasible. CONCLUSIONS: The results of this systematic review of the diagnosis of diabetic foot infections provide some guidance for clinicians, but there is still a need for more prospective controlled studies of high quality.

A study on prevalence, profile, and risk factors of developing fungal infection in patients with diabetic foot ulcer.

BACKGROUND: Many chronic nonhealing diabetic foot ulcers (DFUs) with increased rates of amputation are frequently associated with fungal infections. PURPOSE: To evaluate the prevalence, profile, and risk factors of developing a fungal infection in patients with DFU. METHODS: This prospective observational study was carried out from October 2018 to July 2020. All adult patients with DFUs admitted to the surgery ward were recruited. Patients on antifungal therapy or who received such therapy within 6 weeks prior to admission were excluded. Three deep tissue samples were sent for bacterial culture, fungal culture, and histopathological examination of fungal elements. RESULTS: A total of 251 patients were enrolled in the study. Of the 23.3% of patients with positive fungal growth (n = 47/202), 2% (n = 4/202) had pure fungal growth and 21.3% (n = 43/202) had mixed growth with bacteria in their ulcers (ie, non-contaminated samples). A significant association was found between wound grade (P = .027), ulcer duration (P = .028), and positive fungal growth in DFUs. CONCLUSIONS: In this study, the prevalence of fungal infection in DFUs was 23.3%; Candida tropicalis (27.08%) was the most common isolate, followed by C. albicans (20.83%). The rate of fungal infections was high in patients with mild diabetic foot infection or DFU of 7 to 14 days' duration.

Isolation and Characterization of New Bacteriophages against Staphylococcal Clinical Isolates from Diabetic Foot Ulcers.

Staphylococcus sp. is the most common bacterial genus in infections related to diabetic foot ulcers (DFUs). The emergence of multidrug-resistant bacteria places a serious burden on public health systems. Phage therapy is an alternative treatment to antibiotics, overcoming the issue of antibiotic resistance. In this study, six phages (SAVM01 to SAVM06) were isolated from effluents and were used against a panel of staphylococcal clinical samples isolated from DFUs. A genomic analysis revealed that the phages belonged to the Herelleviridae family, with sequences similar to those of the Kayvirus genus. No lysogeny-associated genes, known virulence or drug resistance genes were identified in the phage genomes. The phages displayed a strong lytic and antibiofilm activity against DFU clinical isolates, as well as against opportunistic pathogenic coagulase-negative staphylococci. The results presented here suggest that these phages could be effective biocontrol agents against staphylococcal clinical isolates from DFUs.

Identification of contributing factors, microorganisms and antimicrobial resistance involved in the complication of diabetic foot ulcer treatment.

Diabetic foot ulcer (DFU) is a neurological and peripherical complication of diabetes with unknown etiology that is often associated with polymicrobial infections. The present study was conducted to investigate the contributing factors in 285 DFU patients, which included 200 patients with diabetic foot infections (DFI). Identification and characterization of infecting bacterial isolates were done followed by assessment of their pattern of susceptibility to commonly used antibiotics. Among the studied subjects, type 2 diabetes mellitus (T2DM), ulcer type, depth, grade, loss of sensation, infection type, affected foot, recurrence, smoking status, Body Mass Index (BMI), and obesity levels revealed significant disease risk association. Ulcer grades 1 and 2 were more common in males while grade 3 in females. Recurrent infections were significantly higher in females (P = 0.03). Diabetic duration, hyperglycemia, ulcer type, infection type and BMI were positively correlated with delayed wound healing. In DFI samples, 40.2% consisted of gram-negative bacteria, with Pseudomonas aeruginosa (37.5%) being the most common, while in the 60% gram-positive isolates Staphylococcus aureus (40.5%) was the predominant species. Staphylococcus epidermidis was found more frequently in females (P = 0.05). The isolated bacterial strains presented higher resistance against the tested antibiotics; however, ceftriaxone was effective against most of the pathogens. In the current study T2DM along with diabetes duration, obesity, ulcer severity with polymicrobial infection was found to play a strong role in DFI development, where gender predisposition was also observed in ulcer grade and infection. DFI was correlated with loss of sensation, infection type, affected foot, smoking status, BMI and obesity levels.

New insight into the virulence and inflammatory response of Staphylococcus aureus strains isolated from diabetic foot ulcers.

Staphylococcus aureus strains isolated from diabetic foot ulcers (DFUs) have less virulence, but still cause severe infections. Furthermore, hypovirulent S. aureus strains appear to be localized in the deep tissues of diabetic foot osteomyelitis, indicating that the unique environment within DFUs affects the pathogenicity of S. aureus. In this study, the cell-free culture medium (CFCM) of S. aureus strains isolated from DFUs exhibited higher cytotoxicity to human erythrocytes than those isolated from non-diabetic patients with sepsis or wounds. Among these S. aureus strains isolated from DFUs, ß-toxin negative strains have less virulence than ß-toxin positive strains, but induced a higher expression of inflammatory cytokines. Our study and previous studies have shown that the synergistic effect of phenol-soluble modulin α and ß-toxin contributes to the higher hemolytic activity of ß-toxin positive strains. However, lysis of human erythrocytes by the CFCM of ß-toxin negative strains was greatly inhibited by an autolysin inhibitor, sodium polyanethole sulfonate (SPS). A high level of glucose greatly reduced the hemolytic activity of S. aureus, but promoted the expression of interleukin-6 (IL-6) in human neutrophils. However, 5 mM glucose or glucose-6-phosphate (G6P) increased the hemolytic activity of SA118 (a ß-toxin negative strain) isolated from DFUs. Additionally, patients with DFUs with growth of S. aureus had lower level of serum IL-6 than those with other bacteria, and the CFCM of S. aureus strains significantly reduced lipopolysaccharide-induced IL-6 expression in human neutrophils. Therefore, the virulence and inflammatory response of S. aureus strains isolated from DFUs are determined by the levels of glucose and its metabolites, which may explain why it is the predominant bacteria isolated from DFUs.

Concordance of bone culture and deep tissue culture during the operation of diabetic foot osteomyelitis and clinical characteristics of patients.

PURPOSE: To retrospectively analyze the concordance of bacterial culture between bone tissue and deep soft tissue in diabetic foot osteomyelitis (DFO) patients and clinical characteristics of patients. METHODS: This study collected samples from 155 patients with suspected DFO (who required amputation after clinical evaluation). Bacterial culture and drug susceptibility tests were performed on the patients' deep soft tissue and bone tissue, and the consistency between the two was compared. In addition, the differences among DFO patients with different degrees of infection were compared classified by the PEDIS classifications. RESULTS: Among the 155 patients diagnosed with DFO, the positive rate of bone culture was 78.7% (122/155). This study cultured 162 strains, including 73 Gram-positive bacteria, 83 Gram-negative bacteria, and 6 fungi. Staphylococcus aureus (33 strains) was the most common bacteria. The overall agreement between bone culture and tissue culture was 42.8%, with Staphylococcus aureus and Enterobacteria having the best (64.3%) and least agreements (27.3%), respectively. The drug sensitivity results in bone culture showed that Staphylococcus aureus was the main Gram-positive bacteria. The bacteria were sensitive to linezolid and vancomycin. Proteus mirabilis was the main Gram-negative bacteria. These were more sensitive than biapenem and piperacillin/tazobactam. Fungi were more sensitive to voriconazole and itraconazole. CONCLUSION: The culture results of deep soft tissues near the bone cannot accurately represent the true pathogen of DFO. For DFO patients, bone culture should be taken as much as possible, and appropriate antibiotics should be selected according to the drug susceptibility results.

Toward non-invasive collection methods for sampling the microbiome of diabetic foot ulcers.

Identifying the microbiome within chronic diabetic foot ulcers is essential if effective antimicrobial therapies are to be administered. Using culture and 16S rRNA gene sequencing, the aim of this study was to compare the microbiome of paired tissue scraping samples with swab samples, collected from participants during attendance at a high-risk foot clinic. The mean richness of cultured swab and tissue scraping samples was consistent, with anaerobes infrequently isolated from both sample types. Comparing percentage frequencies of detection of selected genera of known and potential pathogens namely Staphylococcus, Streptococcus, Enterococcus, Corynebacterium, Enterobacteriaceae and Pseudomonas from cultured and sequenced swab and tissue scrapings indicated that both collection methods captured varying percentages of all the selected genera. The mean abundance of sequenced samples was not significantly different between swabs and tissue scrapings. The mean richness or number of distinct operational taxonomic units (OTUs) and Shannon's H diversity index were not significantly different between the two collection methods. The mean relative abundance of the selected genera of known and potential pathogens, including anaerobes Anaerococcus and Finegoldia, was higher in swabs compared with tissue scrapings and significantly so in Staphylococcus and Pseudomonas genera. Multivariate analyses confirmed no significant differences between the bacterial community compositions of the paired samples. These results suggest that tissue scrapings and swabs can effectively capture the microbiome of chronic DFUs using culture and 16S rRNA gene sequencing.

Concordance between culture, Molecular Culture and Illumina 16S rRNA gene amplicon sequencing of bone and ulcer bed biopsies in people with diabetic foot osteomyelitis.

BACKGROUND: In clinical practice the diagnosis of diabetic foot osteomyelitis (DFO) relies on cultures of bone or ulcer bed (UB) biopsies, of which bone biopsy is reference standard. The slow growth or fastidious nature of some bacteria, hamper expeditious detection and identification. Rapid molecular techniques may solve both issues, but their additional value for everyday practice is unknown. We investigated the concordance between conventional culture, the molecular techniques Molecular Culture (MC), and illumina 16S rRNA gene amplicon (16S) sequencing in people with DFO. METHODS: In the BeBoP trial, bone and UB biopsies were obtained from people with DFO who visited Amsterdam UMC. These biopsies were analysed using 1) conventional culture, 2)MC, a rapid broad range PCR analysing the 16S-23S ribosomal-interspace-region, and 3) 16S sequencing, and evaluated concordance among these techniques. RESULTS: We analysed 20 samples (11 bone and 9 UB) of 18 people. A total of 84 infectious agents were identified, 45 (54%) by all techniques, an additional 22 (26.5%, overall 80.5%) by both MC and 16S, and the remaining 16 species by culture and MC or 16S, or by a single method only. MC and 16S identified anaerobes not detected by culturing in 5 samples, and the presence of bacteria in 7 of 8 culture-negative (6 bone, 2 UB) samples. CONCLUSION: The high level of concordance between MC and 16S and the additional ability of molecular techniques to detect various bacteria not detected by culturing opens up prospects for routine use of fast molecular techniques, in clinical settings including DFO. TRIAL REGISTRATION: The BeBoP trial is retrospectively registered on 05-03-2019 in Netherlands Trial Register: NL 7582.

The ROSA-Like Prophage Colonizing Staphylococcus aureus Promotes Intracellular Survival, Biofilm Formation, and Virulence in a Chronic Wound Environment.

BACKGROUND: The transition from colonization to invasion is critical in diabetic foot ulcer (DFU). Staphylococcus aureus can colonize DFU, or invade the underlying tissues, causing serious infections. The ROSA-like prophage has previously been implicated in strain colonization characteristics of S aureus isolates in uninfected ulcers. METHODS: In this study, we investigated this prophage in the S aureus-colonizing strain using an in vitro chronic wound medium mimicking the chronic wound environment. RESULTS: Chronic wound medium reduced bacterial growth and increased biofilm formation and virulence in a zebrafish model. CONCLUSIONS: The ROSA-like prophage promoted intracellular survival of S aureus-colonizing strain in macrophages, keratinocytes, and osteoblasts.

Antibiotic-laden bone cement for diabetic foot infected wounds: A systematic review and meta-analysis.

Objective: A large body of literature has demonstrated the significant efficacy of antibiotic bone cement in treating infected diabetic foot wounds, but there is less corresponding evidence-based medical evidence. Therefore, this article provides a meta-analysis of the effectiveness of antibiotic bone cement in treating infected diabetic foot wounds to provide a reference basis for clinical treatment. Methods: PubMed, Embase, Cochrane library, Scoup, China Knowledge Network (CNKI), Wanfang database, and the ClinicalTrials.gov were searched, and the search time was from the establishment of the database to October 2022, and two investigators independently. Two investigators independently screened eligible studies, evaluated the quality of the literature using the Cochrane Evaluation Manual, and performed statistical analysis of the data using RevMan 5.3 software. Results: A total of nine randomized controlled studies (n=532) were included and, compared with the control group, antibiotic bone cement treatment reduced the time to wound healing (MD=-7.30 95% CI [-10.38, -4.23]), length of hospital stay (MD=-6.32, 95% CI [-10.15, -2.48]), time to bacterial conversion of the wound (MD=-5.15, 95% CI [-7.15,-2.19]), and the number of procedures (MD=-2.35, 95% CI [-3.68, -1.02]). Conclusion: Antibiotic bone cement has significant advantages over traditional treatment of diabetic foot wound infection and is worthy of clinical promotion and application. Systematic review registration: PROSPERO identifier, CDR 362293.

Point-of-care fluorescence imaging reveals extent of bacterial load in diabetic foot ulcers.

Elevated levels of bacteria, including biofilm, increase the risk of chronic wound infection and inhibit healing. Addressing asymptomatic high bacterial loads is challenged by a lack of clinical terminology and diagnostic tools. This post-hoc multicenter clinical trial analysis of 138 diabetic foot ulcers investigates fluorescence (FL)-imaging role in detecting biofilm-encased and planktonic bacteria in wounds at high loads. The sensitivity and specificity of clinical assessment and FL-imaging were compared across bacterial loads of concern (104 -109 CFU/g). Quantitative tissue culture confirmed the total loads. Bacterial presence was confirmed in 131/138 ulcers. Of these, 93.9% had loads >104 CFU/g. In those wounds, symptoms of infection were largely absent and did not correlate with, or increase proportionately with, bacterial loads at any threshold. FL-imaging increased sensitivity for the detection of bacteria across loads 104 -109 (P < .0001), peaking at 92.6% for >108 CFU/g. Imaging further showed that 84.2% of ulcers contained high loads in the periwound region. New terminology, chronic inhibitory bacterial load (CIBL), describes frequently asymptomatic, high bacterial loads in diabetic ulcers and periwound tissues, which require clinical intervention to prevent sequelae of infection. We anticipate this will spark a paradigm shift in assessment and management, enabling earlier intervention along the bacterial-infection continuum and supporting improved wound outcomes.