Next Generation DNA Sequencing of Tissues from Infected Diabetic Foot Ulcers.
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ABSTRACT:
SUBMITTER: Malone M
PROVIDER: S-EPMC5514496 | biostudies-literature | 2017 Jul
REPOSITORIES: biostudies-literature
Publications
Next Generation DNA Sequencing of Tissues from Infected Diabetic Foot Ulcers.
Malone M M Johani K K Jensen S O SO Gosbell I B IB Dickson H G HG Hu H H Vickery K K
EBioMedicine 20170627
We used next generation DNA sequencing to profile the microbiome of infected Diabetic Foot Ulcers (DFUs). The microbiota was correlated to clinical parameters and treatment outcomes to determine if directed antimicrobial therapy based on conventional microbiological cultures are relevant based on genomic analysis. Patients≥18years presenting with a new Diabetic Foot Infection (DFI) who had not received topical or oral antimicrobials in the two weeks prior to presentation, were eligible for enrol ...[more]
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Project description:BackgroundAccurate identification of primary pathogens in foot infections remains challenging due to the diverse microbiome. Conventional culture may show false-positive or false-negative growth, leading to ineffective postoperative antibiotic treatment. Next-generation sequencing (NGS) has been explored as an alternative to standard culture in orthopedic infections. NGS is highly sensitive and can detect an entire bacterial genome along with genes conferring antibiotic resistance in a given sample. We investigated the potential use of NGS for accurate identification and quantification of microbes in infected diabetic foot ulcer (DFU). We hypothesize that NGS will aid identification of dominant pathogen and provide a more complete profile of microorganisms in infected DFUs compared to the standard culture method.MethodsData were prospectively collected from 30 infected DFU patients who underwent operative treatment by a fellowship-trained orthopedic foot and ankle surgeon from October 2018 to September 2019. The average age of the patient was 60.4 years. Operative procedures performed were irrigation and debridement (12), toe or ray amputation (13), calcanectomies (4), and below-the-knee amputation (1). Infected bone specimens were obtained intraoperatively and processed for standard culture and NGS. Concordance between the standard culture and NGS was assessed.ResultsIn 29 of 30 patients, pathogens were identified by both NGS and culture, with a concordance rate of 70%. In standard culture, Staphylococcus aureus (58.6%) was the most common pathogen, followed by coagulase-negative Staphylococcus (24.1%), Corynebacterium striatum (17.2%), and Enterococcus faecalis (17.2%). In NGS, Finegoldia magna (44.8%) was the most common microorganism followed by S. aureus (41.4%), and Anaerococcus vaginalis (24.1%). On average, NGS revealed 5.1 (range, 1-11) pathogens in a given sample, whereas culture revealed 2.6 (range, 1-6) pathogens.ConclusionNGS is an emerging molecular diagnostic method of microbial identification in orthopedic infection. It frequently provides different profiles of microorganisms along with antibiotic-resistant gene information compared to conventional culture in polymicrobial foot infection. Clinical use of NGS for management of foot and ankle infections warrants further investigation.Level of evidenceLevel II, diagnostic study.
| S-EPMC8702686 | biostudies-literature
Project description:Diabetic foot ulcer (DFU) is a devastating complication, affecting around 15% of diabetic patients and representing a leading cause of non-traumatic amputations. Notably, the risk of mixed bacterial-fungal infection is elevated and highly associated with wound necrosis and poor clinical outcomes. However, it is often underestimated in the literature. Therefore, polymicrobial infection control must be considered for effective management of DFU. It is noteworthy that antimicrobial resistance is constantly rising overtime, therefore increasing the need for new alternatives to antibiotics and antifungals. Antimicrobial peptides (AMPs) are endogenous peptides that are naturally abundant in several organisms, such as bacteria, amphibians and mammals, particularly in the skin. These molecules have shown broad-spectrum antimicrobial activity and some of them even have wound-healing activity, establishing themselves as ideal candidates for treating multi-kingdom infected wounds. Furthermore, the role of AMPs with antifungal activity in wound management is poorly described and deserves further investigation in association with antibacterial agents, such as antibiotics and AMPs with antibacterial activity, or alternatively the application of broad-spectrum antimicrobial agents that target both aerobic and anaerobic bacteria, as well as fungi. Accordingly, the aim of this review is to unravel the molecular mechanisms by which AMPs achieve their dual antimicrobial and wound-healing properties, and to discuss how these are currently being applied as promising therapies against polymicrobial-infected chronic wounds such as DFUs.
| S-EPMC8699205 | biostudies-literature
Project description:BackgroundCosts related to diabetic foot ulcer (DFU) care are greater than $1 billion annually and rising. We sought to describe the impact of diabetes mellitus (DM) on foot ulcer admissions in the United States, and to investigate potential explanations for rising hospital costs.MethodsThe Nationwide Inpatient Sample (2005-2010) was queried using International Classification of Diseases, 9th Revision (ICD-9) codes for a primary diagnosis of foot ulceration. Multivariable analyses were used to compare outcomes and per-admission costs among patients with foot ulceration and DM versus non-DM.ResultsIn total, 962,496 foot ulcer patients were admitted over the study period. The overall rate of admissions was relatively stable over time, but the ratio of DM versus non-DM admissions increased significantly (2005: 10.2 vs. 2010: 12.7; P < 0.001). Neuropathy and infection accounted for 90% of DFU admissions, while peripheral vascular disease accounted for most non-DM admissions. Admissions related to infection rose significantly among DM patients (2005: 39,682 vs. 2010: 51,660; P < 0.001), but remained stable among non-DM patients. Overall, DM accounted for 83% and 96% of all major and minor amputations related to foot ulcers, respectively, and significantly increased cost of care (DM: $1.38 vs. non-DM: $0.13 billion/year; P < 0.001). Hospital costs per DFU admission were significantly higher for patients with infection compared with all other causes ($11,290 vs. $8,145; P < 0.001).ConclusionsDiabetes increases the incidence of foot ulcer admissions by 11-fold, accounting for more than 80% of all amputations and increasing hospital costs more than 10-fold over the 5 years. The majority of these costs are related to the treatment of infected foot ulcers. Education initiatives and early prevention strategies through outpatient multidisciplinary care targeted at high-risk populations are essential to preventing further increases in what is already a substantial economic burden.
| S-EPMC6048950 | biostudies-literature
Project description:BackgroundPoor wound healing is a significant complication of diabetes, which is commonly caused by neuropathy, trauma, deformities, plantar hypertension and peripheral arterial disease. Diabetic foot ulcers (DFU) are difficult to heal, which makes patients susceptible to infections and can ultimately conduce to limb amputation or even death in severe cases. An increasing number of studies have found that epigenetic alterations are strongly associated with poor wound healing in diabetes.Aim of reviewThis work provides significant insights into the development of therapeutics for improving chronic diabetic wound healing, particularly by targeting and regulating DNA methylation and demethylation in DFU. Key scientific concepts of review: DNA methylation and demethylation play an important part in diabetic wound healing, via regulating corresponding signaling pathways in different breeds of cells, including macrophages, vascular endothelial cells and keratinocytes. In this review, we describe the four main phases of wound healing and their abnormality in diabetic patients. Furthermore, we provided an in-depth summary and discussion on how DNA methylation and demethylation regulate diabetic wound healing in different types of cells; and gave a brief summary on recent advances in applying cellular reprogramming techniques for improving diabetic wound healing.
| S-EPMC10703625 | biostudies-literature
Project description:We examined the feasibility of single cell RNA sequencing (scRNA-seq) analysis to evaluate human chronic wound samples acquired in the clinic, subjected to prolonged cold ischemia time, and processed without FACS sorting. Wound tissue from human diabetic and non-diabetic plantar foot ulcers were evaluated using an optimized 10X Genomics scRNA-seq platform and analyzed using a modified data pipeline designed for low-yield specimens. Cell subtypes were identified informatically and their distributions and transcriptional programs were compared between diabetic and non-diabetic tissue. 139,000 diabetic and non-diabetic wound cells were delivered for 10X capture after either 90 or 180 min of cold ischemia time. cDNA library concentrations were 858.7 and 364.7 pg/µL, respectively, prior to sequencing. Among all barcoded fragments, we found that 83.5% successfully aligned to the human transcriptome and 68% met the minimum cell viability threshold. The average mitochondrial mRNA fraction was 8.5% for diabetic cells and 6.6% for non-diabetic cells, correlating with differences in cold ischemia time. A total of 384 individual cells were of sufficient quality for subsequent analyses; from this cell pool, we identified transcriptionally-distinct cell clusters whose gene expression profiles corresponded to fibroblasts, keratinocytes, neutrophils, monocytes, and endothelial cells. Fibroblast subpopulations with differing fibrotic potentials were identified, and their distributions were found to be altered in diabetic vs. non-diabetic cells.
2023-11-25 | GSE248247 | GEO
Project description:Background: Recent advances in high-throughput single-cell sequencing technologies have led to their increasingly widespread adoption for clinical applications. However, challenges associated with tissue viability, cell yield, and delayed time-to-capture have created unique obstacles for data processing. Chronic wounds, in particular, represent some of the most difficult target specimens, due to the significant amount of fibrinous debris, extracellular matrix components, and non-viable cells inherent in tissue routinely obtained from debridement. Methods: Here, we examined the feasibility of single cell RNA sequencing (scRNA-seq) analysis to evaluate human chronic wound samples acquired in the clinic, subjected to prolonged cold ischemia time, and processed without FACS sorting. Wound tissue from human diabetic and non-diabetic plantar foot ulcers were evaluated using an optimized 10X Genomics scRNA-seq platform and analyzed using a modified data pipeline designed for low-yield specimens. Cell subtypes were identified informatically and their distributions and transcriptional programs were compared between diabetic and non-diabetic tissue. Results: 139,000 diabetic and non-diabetic wound cells were delivered for 10X capture after either 90 or 180 min of cold ischemia time. cDNA library concentrations were 858.7 and 364.7 pg/µL, respectively, prior to sequencing. Among all barcoded fragments, we found that 83.5% successfully aligned to the human transcriptome and 68% met the minimum cell viability threshold. The average mitochondrial mRNA fraction was 8.5% for diabetic cells and 6.6% for non-diabetic cells, correlating with differences in cold ischemia time. A total of 384 individual cells were of sufficient quality for subsequent analyses; from this cell pool, we identified transcriptionally-distinct cell clusters whose gene expression profiles corresponded to fibroblasts, keratinocytes, neutrophils, monocytes, and endothelial cells. Fibroblast subpopulations with differing fibrotic potentials were identified, and their distributions were found to be altered in diabetic vs. non-diabetic cells. Conclusions: scRNA-seq of clinical wound samples can be achieved using minor modifications to standard processing protocols and data analysis methods. This simple approach can capture widespread transcriptional differences between diabetic and non-diabetic tissue obtained from matched wound locations.
| S-EPMC7570277 | biostudies-literature
Project description:BackgroundInfected diabetic foot ulcers (IDFU) are a major complication of diabetes mellitus. These potentially limb-threatening ulcers are challenging to treat due to impaired wound healing characterizing diabetic patients and the complex microbial environment of these ulcers.AimTo analyze the microbiome of IDFU in association with clinical outcomes.MethodsWound biopsies from IDFU were obtained from hospitalized patients and were analyzed using traditional microbiology cultures, 16S rRNA sequencing and metagenomic sequencing. Patients' characteristics, culture-based results and sequencing data were analyzed in association with clinical outcomes.ResultsA total of 31 patients were enrolled. Gram-negative bacteria dominated the IDFU samples (79%, 59% and 54% of metagenomics, 16S rRNA and cultures results, respectively, p<0.001). 16S rRNA and metagenomic sequencing detected significantly more anaerobic bacteria, as compared to conventional cultures (59% and 76%, respectively vs. 26% in cultures, p=0.001). Culture-based results showed that Staphylococcus aureus was more prevalent among patients who were treated conservatively (p=0.048). In metagenomic analysis, the Bacteroides genus was more prevalent among patients who underwent amputation (p<0.001). Analysis of metagenomic-based functional data showed that antibiotic resistance genes and genes related to biofilm production and to bacterial virulent factors were more prevalent in IDFU that resulted in amputation (p<0.001).ConclusionSequencing tools uncover the complex biodiversity of IDFU and emphasize the high prevalence of anaerobes and Gram-negative bacteria in these ulcers. Furthermore, sequencing results highlight possible associations among certain genera, species, and bacterial functional genes to clinical outcomes.
| S-EPMC8987016 | biostudies-literature
Project description:Diabetic foot ulcers (DFUs) are a devastating complication of diabetes. In order to identify systemic and local factors associated with DFU healing, we examined the cellular landscape of DFUs by single-cell RNA-seq analysis of foot and forearm skin specimens, as well as PBMC samples, from 10 non-diabetic subjects, and 17 diabetic patients, 11 with, and 6 without DFU. Our analysis shows enrichment of a unique inflammatory fibroblast population in DFU patients with healing wounds. The patients with healing DFUs also depicted enrichment of macrophages with M1 polarization, as opposed to more M2 macrophages in non-healing wounds. These findings were verified using Immunohistochemistry and Spatial Transcriptomics.
2021-11-01 | GSE165816 | GEO
Project description:BackgroundDiabetes foot ulcer (DFU) is a complication of diabetes mellitus. Accurate diagnosis of DFU severity through inflammatory markers will assist in reducing impact on quality of life. We aimed to ascertain the diagnostic test accuracy of commonly used inflammatory markers such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), procalcitonin (PCT), and white cell count (WCC) for the diagnosis and differentiation between DFU grades based on the International Working Group on the Diabetic Foot classification system.MethodsThis systematic review explored studies that investigated one or more of the above-listed index tests aiding in diagnosing infected DFU. This review was registered on PROSPERO database (ID = CRD42021255618) and searched 5 databases including an assessment of the references of included studies. Records were manually screened as per Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. A total of 16 studies were included which were assessed for quality using QUADAS-2 tool and meta-analysed using Meta-Disc v1.4.ResultsCRP had the greatest area under the curve (AUC) of 0.893 for diagnosing grade 2 DFU. This returned a pooled sensitivity and specificity of 77.4% (95% CI: 72% to 82%) and 84.3% (95% CI: 79% to 89%) respectively. In terms of diagnosing grade 3 DFU, procalcitonin had the highest AUC value of 0.844 when compared with other markers. The pooled sensitivity of PCT was calculated as 85.5% (95% CI: 79% to 90%) and specificity as 68.9% (95% CI: 63% to 75%).ConclusionCRP and PCT are the best markers for diagnosing grade 2 and grade 3 DFU respectively. Other markers are also valuable when used in conjunction with clinical judgement. The findings accentuate the necessity of further research to establish standardised cut-off values for these inflammatory markers in diagnosing diabetic foot ulcers.
| S-EPMC9045669 | biostudies-literature
Project description:BackgroundThe study aimed at (i) characterizing the mode of transmission of bla(CTX-M) and bla(TEM-1) among extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli strains isolated from infected diabetic foot ulcers, and (ii) identifying the risk factors for "sex-associated multidrug resistant Gram-negative bacterial (MDRGNB)-infection status" of the ulcers.MethodsSeventy-seven diabetic patients having clinically infected foot ulcers were studied in a consecutive series. The E. coli strains isolated from the ulcers were screened for bla(CTX-M), bla(TEM-1), armA, rmtA and rmtB during the 2-year study-period. PCR amplified bla(CTX-M) genes were cloned and sequenced. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used for the analysis of genetic relatedness of the ESBL-producers. Risk factors for "sex-associated MDRGNB-infection status" of ulcers were assessed. Modeling was performed using Swiss-Model-Server and verified by Procheck and verify3D programmes. Discovery Studio2.0 (Accelrys) was used to prepare Ramachandran plots. Z-scores were calculated using 'WHAT IF'-package. Docking of cefotaxime with modeled CTX-M-15 enzyme was performed using Hex5.1.ResultsAmong 51 E. coli isolates, 14 (27.5%) ESBL-producers were identified. Only 7 Class1 integrons, 2 bla(CTX-M-15), and 1 bla(TEM-1) were detected. Ceftazidime and cefotaxime resistance markers were present on the plasmidic DNA of both the bla(CTX-M-15) positive strains and were transmissible through conjugation. The residues Asn132, Glu166, Pro167, Val172, Lys234 and Thr235 of CTX-M-15 were found to make important contacts with cefotaxime in the docked-complex. Multivariate analysis proved 'Glycemic control at discharge' as the single independent risk factor.ConclusionsMale diabetic patients with MDRGNB-infected foot ulcers have poor glycemic control and hence they might have higher mortality rates compared to their female counterparts. Plasmid-mediated conjugal transfer, albeit at a low frequency might be the possible mechanism of transfer of bla(CTX-M-15) resistance marker in the present setting. Since the docking results proved that the amino acid residues Asn132, Glu166, Pro167, Val172, Lys234 and Thr235 of CTX-M-15 (enzyme) make important contacts with cefotaxime (drug) in the 'enzyme-drug complex', researchers are expected to duly utilize this information for designing more potent and versatile CTX-M-inhibitors.
| S-EPMC2821376 | biostudies-literature