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.

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.

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.

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.

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.

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.

The association between bacteria and outcome and the influence of sampling method, in people with a diabetic foot infection.

PURPOSE: Different bacteria lead to divers diabetic foot infections (DFIs), and some bacteria probably lead to higher amputation and mortality risks. We assessed mortality and amputation risk in relation to bacterial profiles in people DFI and investigated the role of sampling method. METHODS: We included people (> 18 years) with DFI in this retrospective study (2011-2020) at a Dutch tertiary care hospital. We retrieved cultures according to best sampling method: (1) bone biopsy; (2) ulcer bed biopsy; and (3) swab. We aggregated data into a composite determinant, consisting of unrepeated bacteria of one episode of infection, clustered into 5 profiles: (1) Streptococcus and Staphylococcus aureus; (2) coagulase-negative Staphylococcus, Cutibacterium, Corynebacterium and Enterococcus; (3) gram-negative; (4) Anaerobic; and (5) less common gram-positive bacteria. We calculated Hazard Ratio's (HR's) using time-dependent-Cox regression for the analyses and investigated effect modification by sampling method. RESULTS: We included 139 people, with 447 person-years follow-up and 459 episodes of infection. Sampling method modified the association between bacterial profiles and amputation for profile 2. HR's (95% CI's) for amputation for bacterial profiles 1-5: 0.7 (0.39-1.1); stratified analysis for profile 2: bone biopsy 0.84 (0.26-2.7), ulcer bed biopsy 0.89 (0.34-2.3), swab 5.9*(2.9-11.8); 1.3 (0.78-2.1); 1.6 (0.91-2.6); 1.6 (0.58-4.5). HR's (95% CI's) for mortality for bacterial profiles 1-5: 0.89 (0.49-1.6); 0.73 (0.38-1.4); 2.6*(1.4-4.8); 1.1(0.58-2.2); 0.80(0.19-3.3). CONCLUSIONS: In people with DFI, there was no association between bacterial profiles in ulcer bed and bone biopsies and amputation. Only in swab cultures, low-pathogenic bacteria (profile 2), were associated with a higher amputation risk. Infection with gram-negative bacteria was associated with a higher mortality risk. This study underlined the possible negative outcome of DFI treatment based on swabs cultures.

Microbiological Pattern, Antimicrobial Resistance and Prevalence of MDR/XDR Organisms in Patients With Diabetic Foot Infection in an Indian Tertiary Care Hospital.

Foot infections are the most prevalent problem in persons with diabetes. The burden of multidrug resistant (MDR) microorganisms in diabetic foot infections (DFIs) is rising day by day. Given that, the present study aims to determine the variety of microorganisms isolated from the diabetic foot ulcers (DFUs), and their antibiotic sensitivity pattern. This prospective observational study was conducted for 1 year at Bharati Hospital and Research Centre, Pune, India. Clinically infected patients with DFU admitted to the surgery ward were included in this study. The specimen for microbiological studies is obtained from the wound swabs, soft tissue, and bone tissue as a part of routine clinical care. All demographic, clinical data, microbial culture results were collected, and evaluated for each case. Antimicrobial susceptibility testing to different agents was carried out using the VITEK-2® machine. A total of 110 microorganisms were isolated from 76 specimens, with an average of 1.4 organisms per lesion. Staphylococcus aureus (n = 27, 24.5%) and Escherichia coli (n = 17, 15.4%) were the most prevalent Gram-positive and Gram-negative organisms isolated, respectively. MDR organisms constituted up to 52 (47.2%), while 6 (5.4%) of the samples were extensively drug resistant (XDR). Methicillin-resistant S aureus (MRSA) accounted for up to 19 (70.3%) of the S aureus isolates, likewise extended-spectrum beta-lactamase producing microorganisms constituted 16 (14.5%) of total isolates in this study. Oxacillin and benzyl penicillin exhibited least susceptibility against Gram-positive bacteria, among Gram-negative organisms; cefuroxime, ceftriaxone, and ciprofloxacin were least sensitive. As most of the S aureus isolate in our study was MRSA, empirical antimicrobial therapy may include coverage for MRSA in a patient with risk factors associated with this pathogen. A crucial observation is the presence of XDR strains of Proteus mirabilis in DFIs, which is resistant to almost all the antimicrobials, tested. Appropriate antimicrobial selection may reduce the morbidity and the emergence of MDR organisms in DFIs.

The Effects of Antimicrobial Resistance and the Compatibility of Initial Antibiotic Treatment on Clinical Outcomes in Patients With Diabetic Foot Infection.

We aimed to determine pathogen microorganisms, their antimicrobial resistance patterns, and the effect of initial treatment on clinical outcomes in patients with diabetic foot infection (DFI). Patients with DFI from 5 centers were included in this multicenter observational prospective study between June 2018 and June 2019. Multivariate analysis was performed for the predictors of reinfection/death and major amputation. A total of 284 patients were recorded. Of whom, 193 (68%) were male and the median age was 59.9 ± 11.3 years. One hundred nineteen (41.9%) patients had amputations, as the minor (n = 83, 29.2%) or major (n = 36, 12.7%). The mortality rate was 1.7% with 4 deaths. A total of 247 microorganisms were isolated from 200 patients. The most common microorganisms were Staphylococcus aureus (n = 36, 14.6%) and Escherichia coli (n = 32, 13.0%). Methicillin resistance rates were 19.4% and 69.6% in S aureus and coagulase-negative Staphylococcus spp., respectively. Multidrug-resistant Pseudomonas aeruginosa was detected in 4 of 22 (18.2%) isolates. Extended-spectrum beta-lactamase-producing Gram-negative bacteria were detected in 20 (38.5%) isolates of E coli (14 of 32) and Klebsiella spp. (6 of 20). When the initial treatment was inappropriate, Klebsiella spp. related reinfection within 1 to 3 months was observed more frequently. Polymicrobial infection (p = .043) and vancomycin treatment (p = .007) were independent predictors of reinfection/death. Multivariate analysis revealed vascular insufficiency (p = .004), hospital readmission (p = .009), C-reactive protein > 130 mg/dL (p = .007), and receiving carbapenems (p = .005) as independent predictors of major amputation. Our results justify the importance of using appropriate narrow-spectrum empirical antimicrobials because higher rates of reinfection and major amputation were found even in the use of broad-spectrum antimicrobials.

Culture Concordance in Different Sections of the Metatarsal Head: Interpretations of Microbiological Results.

Microbiological cultures of per-wound bone biopsies have shown a lack of correlation and a high rate of false-negative results when compared with bone biopsy cultures in diabetic foot osteomyelitis. The selection of samples from the area of active osteomyelitis, which contains a complete census of the microorganisms responsible for the infection, is essential to properly guide antimicrobial treatment. We aimed to comparatively evaluate the quantitative and qualitative cultures taken from different areas, in metatarsal heads resected for osteomyelitis. For this purpose, we consecutively selected 13 metatarsal heads from 12 outpatients with plantar ulcers admitted to our diabetic foot unit. Metatarsal heads were divided transversally into 3 portions: plantar (A), central (B), and dorsal (C), and the 39 resulting samples were cultured. Qualitative and quantitative microbiological analysis was performed, and the isolated species and bacterial load, total and species specific, were compared between the 3 metatarsal bone segments. The primary outcome of the study was the bacterial diversity detected in the different bone sections. Cultures were positive in 12 of the 13 included metatarsal heads (92%). A total of 34 organisms were isolated from all specimens. Ten of the 12 cultures (83%) were polymicrobial. Ten of the 13 metatarsal heads (77%) had identical microbiological results in each of the 3 bone sections. The largest number of microorganisms was found in the central section. The overall concordance between sections was 91%. The predominant microorganisms were coagulase-negative staphylococci (41%). Statistical differences were not found in the bioburden between sections (range 3.25-3.41 log10 colony-forming unit/g for all sections; P = .511). The results of our study suggest that microorganisms exhibit a high tendency to spread along the metatarsal bone and that the degree of progression along the bone is species dependent. The central portions of metatarsal bones tend to accumulate a higher diversity of species. Thus, we recommend this area of bone for targeted biopsy in patients with suspected osteomyelitis.

Waterjet in Bacterial Clearance of Diabetic Lower Extremity Contaminated Wounds: A Retrospective Cohort Study.

The waterjet debridement is now a standard practice in contaminated or infected diabetic lower extremity wounds. The bacterial clearance of the waterjet debridement remains an important parameter that should be predicted in this application. This study aimed to investigate the waterjet in reducing the diabetic lower extremity wound contaminants. A retrospective cohort study was conducted. Patients' etiology and pathogen diagnosis were established as diabetic lower extremity contaminated wound. The high-power waterjet (Versajet™, Smith-Nephew) was used in the treatment group and conventional surgical methods were used in the control group. The bacteriological swab samples were collected before and after the debridement. The results of bacterial culture were analyzed. A total of 74 patients were included in our study, 40 patients in the treatment group and 34 in the control group. Patient characteristics were well matched. The preoperative bacteriological swab samples of the 2 groups showed no significant difference between each other with a P value of .1022. The culture result of postoperative bacteriological swab samples in the treatment group was significantly lower than control with a P value of .0099. The odds of bacterial clearance were greater in the treatment group than in the control group (odds ratio, 5.139; 95% confidence interval, 1.386-18.41). As demonstrated by this retrospective research, waterjet debridement reduced the bacterial load in the diabetic lower extremity contaminated wounds.