Infected Diabetic Foot Ulcers Research Articles

The Influence of Multidrug-Resistant Bacteria on Clinical Outcomes of Diabetic Foot Ulcers: A Systematic Review.

Multidrug-resistant organism infections have become important in recent years due to the increased prevalence of diabetic foot ulcers and their possible consequences. This study aimed to systematically review and evaluate ulcer duration, healing time, hospital stay, amputation, and mortality rates in patients with diabetic foot ulcers caused by infection with multidrug-resistant organisms. PubMed, the Cochrane Library, and Web of Science were searched in May 2020 to find observational studies in English about the clinical outcomes of multidrug-resistant organism infection in diabetic foot ulcers. Eight studies met the inclusion criteria, and these studies included 923 patients. The overall methodological quality of the study was moderate. Ulcer duration was described in six studies, and there was no practical association with multidrug-resistant organisms. Two out of three studies reported a longer healing time in multidrug-resistant organism infections than in non-multidrug-resistant organism infections. Clinical outcomes included the duration of hospitalisation, surgeries, amputations, and deaths. Lower limb amputation was the most reported clinical outcome in the included studies, and was more prevalent in the multidrug-resistant organism infections. We concluded that there was not enough evidence that multidrug-resistant organisms hindered the healing of diabetic foot ulcers. In contrast to the clinical outcomes, multidrug-resistant organisms affect both amputation rates and mortality rates.

The efficacy of negative pressure wound therapy compared with conventional dressing in treating infected diabetic foot ulcers: a randomized controlled trial

Despite advances made in diagnosing and controlling diabetes mellitus (DM), treatment of diabetic foot ulcers (DFUs) is still among challenges faced by physicians. Negative pressure wound therapy (NPWT) is one of newer modalities proposed in the treatment of DFUs. However, there is lack of evidence to support its mileage in this regard. This study was conducted with the aim of assessing the efficacy of NPWT in healing process of DFUs. Sixty patients with DM were randomly allocated into two groups consisting of 30 patients each: the intervention group received sub-atmospheric pressure of − 75 to − 100 mmHg (5 min on, 2 min off) with dressings changed every 48 h, and the control group was treated with silver sulfadiazine dressings, changed twice daily. Patients were followed up until complete closure of ulcers, with a mean duration of 3 months. Of the total 60 patients, 27 patients (45%) were females. Most of the patients in both groups had DFUs of grade 2 according to Wegner’s classification. Rate of healing of the ulcers was significantly higher by using NPWT (p-value 0.01). NPWT also caused a significant reduction in ulcer surface area, depth, size, major and minor amputations, and disability duration (p-values 0.008, 0.002, 0.02, 0.03, and 0.01, respectively). No significant decrease in occurrence of complications was seen with NPWT. NPWT seems to be more efficacious than conventional dressing in treating infected DFUs.

Associating Insulin Instillation with Negative Pressure Wound Therapy Reduces Insulin Resistance in Diabetic Patients with Infected Foot Wounds

In this work we investigated whether a joint treatment with insulin instillation and negative pressure wound therapy (NPWT) would reduce insulin resistance (IR) in diabetic patients with infected diabetic foot ulcers (DFUs) and improve their healing. We also studied the effects of insulin+NPWT treatment on the inflammatory response coupled with wound healing. Seventy-five diabetic patients with infected DFUs were recruited and randomly divided into equal (n=25) groups treated respectively with NPWT alone, NPWT+insulin, and conventional dressings (controls). Thereafter, the ulcers’ healing progress was assessed. The serum levels of Tumor Necrosis Factor-α (TNF-α), Interleukin-2 (IL-2), and Interleukin-6 (IL-6) were measured at 1 day before and at 7, 14, and 21 days after each treatment using specific double-antibody sandwich enzyme-linked immunosorbent assays (ELISA). Serum glucose was measured via a glucose-oxidase method, and serum insulin via radioimmunoassay. In each patient, IR was appraised via the Homeostasis Model Assessment (HOMA-IR) and the quantitative insulin sensitivity check index (QUICKI). Our results showed that at 7, 14 and 21 days post-treatment, with respect to the control group the two NPWT-treated groups had (i) significantly (p < 0.05) lower serum levels of TNF-α, IL-2 and IL-6; (ii) a significantly (p < 0.01) greater reduction in IR; and (iii) significantly (p < 0.05) higher wound healing rates. Moreover, at 14- and 21-days post-treatment, the NPWT+insulin-treated group had a significantly (p < 0.05) higher wound healing rate than the NPWT alone-treated group. In conclusion, the combined NPWT+insulin instillation treatment gave superior results by improving wound healing, decreasing inflammation at the wounds surface, and reducing IR in diabetic patients with infected DFUs.

Incidence and Prevalence of Candidiasis in Diabetic Foot Ulcer Patients

The aim of our present study was to estimate the prevalence of Candida infection in foot ulcer patients and spectrum of Candida species and their drug resistant pattern. A total of 100 Swabs was taken from diabetic foot ulcer patients from January 2016 to June 2016. Samples were cultured on SDA agar medium. Candida spp. were differentiated by culture on Hi CHROM agar, Sugar assimilation test, fermentation test and antifungal sensitivity test. Out of 100 samples obtained from diabetic patients with a foot ulcer, 32 (32%) were positive for Candida sp by culture. It was more significant in males 22 (68.75%) than females 10 (31.25%) Candida albicans was found to be the predominant isolate followed by C.tropicalis. Resistance to fluconazole was observed 17 (17%) in our study. C.albicans was more resistant to azoles than non albicans. Our results will help physicians to treat fungal infections of diabetic foot ulcers, as well as their drug resistant pattern. Fluconazole resistance is a public health concern and the rational use of this drug is important in community.

Patient perceptions of phage therapy for diabetic foot infection.

Infections of diabetic foot ulcers are common, generally recalcitrant and often complicated by antibiotic resistance. Alternative antimicrobial strategies are needed. Phage therapy is a promising alternative that is being rediscovered. Despite phage therapy’s 100-year history, there have been no investigations into patient thoughts and concerns. This study aimed to explore patient awareness of and concern about antibiotic resistance and gain insight into the perceptions of phage therapy among a patient group that could potentially benefit from phage therapy. Patients with an active or resolved (healed or amputated) diabetic foot ulcer were eligible to participate. A survey was distributed digitally to eligible patients across Scotland via the NHS Research Scotland Diabetes Network and hard copies were available in diabetic foot clinics at the Royal Infirmary of Edinburgh and Queen Elizabeth University Hospital, Glasgow. A focus group of five survey respondents was held in Glasgow. Fifty-five survey responses were obtained. There was a high level of awareness (76.4%; N = 55) and concern (83.3%; N = 54) about antibiotic resistance. While largely aware of viruses, most patients had not heard of phage or phage therapy. Patients were no more concerned about phage than antibiotic therapy, with most suggesting more information could alleviate any concerns. Patient acceptability of phage therapy was high, a finding confirmed by the focus group. Patients are concerned about antibiotic resistance and supportive of ‘new’ antimicrobials. We have demonstrated that patients are supportive, enthusiastic and accepting of phage therapy. Although ‘Western’ phage therapy remains in its infancy, an understanding of patient ideas, concerns and expectations will be important in eventually shaping and successfully reintroducing phage therapy.

Wound care using metronidazole in a patient with diabetic foot ulcer: a case report

The control of infection in diabetic foot ulcers is essential to prevent injuries from getting worse. Infection in diabetic foot ulcers is generally caused by bacteria existence in the wound. Antibiotics may not be orally able to exterminate bacteria rapidly in the wound area, so antibiotics are needed directly and topically to the wound to reduce the infection process. This case describes wound care intervention using gauze added by antibiotic metronidazole as a wound compress in a 46-year-old male patient with diabetic foot ulcers treated at Cempaka Ward Banyumas Regional Public Hospital. This case is interesting because the medical intervention given to patients is rarely implemented in clinical practice. The conclusion is that a metronidazole antibiotic used to compress wounds in patients with diabetic foot ulcers can reduce the wound infection process.

Bacteriological Profile and Antibiotic Sensitivity Pattern in Infected Diabetic Foot Ulcers

Objectives: To identify the common bacterial pathogens responsible for infection in diabetic foot ulcer and their sensitivity pattern to different antibiotics. Methods: This prospective observational study was conducted from 24th June 2019 to 27th December 2019 at Services Institute of Medical Sciences Lahore. Specimens of discharge from diabetic foot ulcers (DFU) were received from Diabetes Management Centre (DMC). Cultures were put up and bacteria isolated were identified by standard methods. Antibiotic sensitivity was determined by Kirby-Bauer disc diffusion method. Results: Samples from DFU of 50 patients were processed. Thirty-six (72%) samples were from males and 14(28%) were from females; mean age of patients was 53±9.5 years. Forty-nine (98%) patients had unsatisfactory glycemic control. Forty three (86%) samples were growth positive while 07(14%) were bacteriologically sterile. Monomicrobial infection was observed in 38 (76%) cases while polymicrobial infection was seen in 12 cases (24%). The most common isolates were Staphylococcus aureus 14(28%), Pseudomonas species 11 (22%) and Proteus species 10 (20%). Forty three percent of Staphylococcus aureus, were methicillin resistant (MRSA). All MRSA remained sensitive to vancomycin and linezolid. In Pseudomonas species, resistance to third generation cephalosporins, ceftazidime , was 27%, while resistance to imipenem was seen in 3 (9%) of isolates. In Proteus species, resistance to third generation cephalosporins was 90% while to imipenem resistance was 60%. Resistance to commonly prescribed quinolones was more than 70% among all the bacterial isolates. Conclusions: Common Gram positive and Gram negative organisms responsible for infection in DFU were Staphylococcus aureus, Pseudomonas species and Proteus species. The isolates were multi-drug resistant (MDR). Resistance to antibiotics used as empiric therapy was high. Keywords: Diabetic foot ulcer, antibiotic resistance, empiric therapy

Silver Sulfadiazine Eradicates Antibiotic-Tolerant Staphylococcus aureus and Pseudomonas aeruginosa Biofilms in Patients with Infected Diabetic Foot Ulcers.

Infections are among the most frequent and challenging events in diabetic foot ulcers (DFUs). Pathogenic bacteria growing in biofilms within host tissue are highly tolerant to environmental and chemical agents, including antibiotics. The present study was aimed at assessing the use of silver sulfadiazine (SSD) for wound healing and infection control in 16 patients with DFUs harboring biofilm-growing Staphylococcus aureus and Pseudomonas aeruginosa. All patients received a treatment based on a dressing protocol including disinfection, cleansing, application of SSD, and application of nonadherent gauze, followed by sterile gauze and tibio-breech bandage, in preparation for toilet surgery after 30 days of treatment. Clinical parameters were analyzed by the T.I.M.E. classification system. In addition, the activity of SSD against biofilm-growing S. aureus and P. aeruginosa isolates was assessed in vitro. A total of 16 patients with S. aureus and P. aeruginosa infected DFUs were included in the study. Clinical data showed a statistically significant (p < 0.002) improvement of patients’ DFUs after 30 days of treatment with SSD with significant amelioration of all the parameters analyzed. Notably, after 30 days of treatment, resolution of infection was observed in all DFUs. In vitro analysis showed that both S. aureus and P. aeruginosa isolates developed complex and highly structured biofilms. Antibiotic susceptibility profiles indicated that biofilm cultures were significantly (p ≤ 0.002) more tolerant to all tested antimicrobials than their planktonic counterparts. However, SSD was found to be effective against fully developed biofilms of both S. aureus and P. aeruginosa at concentrations below those normally used in clinical preparations (10 mg/mL). These results strongly suggest that the topical administration of SSD may represent an effective alternative to conventional antibiotics for the successful treatment of DFUs infected by biofilm-growing S. aureus and P. aeruginosa.

Gene Expression Profiling of Multiple Histone Deacetylases (HDAC) and Its Correlation with NRF2-Mediated Redox Regulation in the Pathogenesis of Diabetic Foot Ulcers.

Nuclear factor erythroid-2-related factor 2 (Nrf2) is a protein of the leucine zipper family, which mitigates inflammation and employs cytoprotective effects. Attempting to unravel the epigenetic regulation of type 2 diabetes mellitus (T2DM) and diabetic foot ulcer (DFU), we profiled the expression of eleven isoform-specific histone deacetylases (HDACs) and correlated them with NRF2 and cytokines. This study recruited a total of 60 subjects and categorized into DFU patients (n = 20), T2DM patients (n = 20), and healthy controls (n = 20). The DFU patients were subcategorized into uninfected and infected DFU (n = 10 each). We observed a progressive decline in the expression of NRF2 and its downstream targets among T2DM and DFU subjects. The inflammatory markers IL-6 and TNF-α were significantly upregulated, whereas anti-inflammatory marker IL-10 was significantly downregulated in DFU. Of note, a significant upregulation of HDAC1, 3, 4, 11, SIRT3 and downregulation of HDAC2,8, SIRT1, SIRT2, SIRT3, SIRT7 among DFU patients were observed. The significant positive correlation between NRF2 and SIRT1 in DFU patients suggested the vital role of NRF2/SIRT1 in redox homeostasis and angiogenesis. In contrast, the significant negative correlation between NRF2 and HDAC1, 3 and 4, implied an imbalance in NRF2-HDAC1, 3, 4 circuit. Furthermore, a significant positive correlation was observed between HDAC4 and IL-6, and the negative correlation between SIRT1 and IL-6 suggested the pro-inflammatory role of HDAC4 and the anti-inflammatory role of SIRT1 in NRF2 signaling. In conclusion, the epigenetic changes such as upregulation of HDAC1, 3, 4, 11, SIRT3 and downregulation of HDAC2, 8, SIRT1, SIRT2, SIRT6, SIRT7 and their association with NRF2 as well as inflammatory markers are suggestive of their roles in pathophysiology of T2DM and DFU.

Next-Generation Sequencing for Pathogen Identification in Infected Foot Ulcers

Category:OtherIntroduction/Purpose:Foot infections are often polymicrobial with diverse microbiomes. Accurate identification of the main pathogen in diabetic foot ulcer (DFU) remain challenging due to contamination or negative cultures often leading to ineffective post-surgical antibiotic treatment. Application of molecular diagnostics, such as next generation sequencing (NGS) has been explored as an alternative to standard culture in orthopaedic infections. NGS is highly sensitive and detects an entire bacterial genome along with pharmacologic resistant genes in a given sample. We sought to investigate the potential use of NGS for accurate diagnosis and quantification of various species in infected DFU. We hypothesize that NGS will provide a more accurate means of diagnosing and profiling microorganisms in infected DFU compared to the standard culture method.Methods:We investigated 30 infected DFU patients who underwent surgical treatment by a single academic orthopaedic surgeon from October 2018 to September 2019. The average age of the patient was 60.4 (range 33-82) years-old. Surgical procedures performed were irrigation and debridement (12), toe or ray amputation (13), calcanectomies (4), and below-knee amputation (1). Infected bone specimens were obtained intraoperatively and processed for standard culture and NGS. Quantitative PCR was performed to determine the bacterial burden present in the sample. DNA was amplified by PCR from a highly conserved region of the rRNA gene in the bacteria (16S rRNA). Once a high level of DNA was generated and determined, it was compared against NIH GenBank database. Concordance between the standard culture and NGS was assessed.Results:In 28 of 29 patients, pathogens were identified by both NGS and culture, with complete consistency of organisms in 13 cases (concordance rate: 43.3%). NGS provided relative quantitative measures and the presence of antibiotic resistant genes for each pathogen. In NGS, Anaerococcus species (79.3%) was the most common organism, followed by Streptococcus species (44.8%), Prevotella species (44.8%), Finegoldia magna (44.8%). In culture, S. aureus (58.6%) was the most common, followed by Streptococcus species (34.5%), coagulase-negative Staphylococci (24.1%), Corynebacterium species (20.7%). On average, NGS revealed 5.1 (1-11) number of pathogens, whereas standard culture revealed 2.6 (1-6) pathogens in a given sample. NGS identified 2 cases with false positive standard culture and detected antibiotic resistant organisms in 15 specimens.Conclusion:NGS is an emerging method of microbial identification in orthopedic infection. It is particularly helpful in profiling diverse microbes in polymicrobial infected DFU. It can identify major pathogens and may correct false positive or false negative culture. NGS may allow a faster invitation of postoperative targeted antibiotic therapy.

Group B Streptococcus Infected Tenosynovitis in Diabetic Foot Ulcers

Category:Diabetes; OtherIntroduction/Purpose:The incidence of Streptococcus agalactiae (Group B Streptococcus, GBS) infection in diabetic foot ulcers (DFU) has been on the rise. Severe soft tissue damage, which often leads to septicemia and amputation, has been reported in many cases. With the paucity of literature, we aimed to investigate the clinical outcome of GBS infected DFU patients. We hypothesize that GBS patients have a greater severity of infection as indicated by elevated inflammatory markers, more frequent wound complication, and a higher rate of unplanned readmission and reoperation.Methods:Data was retrospectively collected in a single academic orthopedic surgeon’s practice from February 2015 to October 2019. Seventy-eight patients with infected DFUs who underwent surgical treatment formed the basis of this study. Infected bone samples were obtained intraoperatively and sent for standard culture. The microbe data, demographic data (age, gender, race, ethnicity, and BMI), comorbidities, and initial lab values (HgA1C, CRP, ESR, WBC, and glucose) were recorded for all patients. Sixteen GBS infected DFU patients (20.5%) were identified. Among them, GBS infection occurred in 9 acute (<2 wks), 2 subacute (4-6 wks), and 5 chronic (>6 wks) DFUs. Clinical outcome was assessed by surgical outcome, wound healing status, post-operative complications, unplanned readmission, and unplanned reoperation within 3 months following initial surgery. Mean, standard deviation, percentage and range were calculated for patient demographics and inflammatory markers. Statistical significance of inflammatory markers between patients with and without GBS was also calculated.Results:The initial procedures were irrigation and debridement (n=11), toe amputation (n=1), ray amputation (n=2), transmetatarsal amputation (n=1), and a partial calcanectomy (n=1). Five GBS patients (31.3%), as compared to eighteen (29%) DFU patients without GBS, developed post-surgical complications (wound dehiscence, recurrent infection, septicemia) which required unplanned readmission and reoperation. Repeat operations were irrigation and debridement (n=1), metatarsal ray amputation (n=1), ray amputation (n=1), and below knee amputation (n=2) with average number of 2 repeat operations (range: 1 - 5). Hemoglobin A1C (p=.0067) was statistically higher in GBS patients. When comparing acute GBS ulcers (n=9) and acute ulcers without GBS (n=18), CRP (p=.037), HgA1C (p=.026), and blood glucose (p=.046) were all found to be significantly higher in patients with GBS DFUs.Conclusion:GBS infected DFU patients generally showed more extensive and severe soft tissue inflammation, as indicated by higher inflammatory markers at initial presentation. Compared to other patients with DFUs, GBS patients had significantly higher HgA1C values, and in those experiencing acute ulcers, had higher CRP, HgA1C, and blood glucose values. They have higher rates of post-operative complications that required unplanned readmission and reoperation at more proximal level. Surgeons should consider time sensitive and aggressive surgical treatment for GBS infected DFUs and counsel patients on the high risk of post- operative complications and repeat surgery.

Species-Specific Immunoassay for Diagnosis and Tracking Treatment Response in Foot and Ankle Infection

Category:Basic Sciences/Biologics; DiabetesIntroduction/Purpose:Staphylococcus aureus is the major pathogen foot and ankle infections and osteomyelitis (>50%). There are currently no sensitive and specific diagnostic tools for monitoring a pathogen’s ongoing infection or providing prognostic measures. We have developed a novel immunoassay for S. aureus, and have applied this to diagnose and monitor its infectivity. We hypothesize that: 1) the species-specific immunoassay can serve as a reliable diagnostic tool for S. aureus foot and ankle infections and 2) the immunoassay provides a measure of treatment response and prognosis of clinical outcome to antibiotics therapy for S. aureus foot and ankle infections.Methods:From July 2015 to July 2019, 83 infected diabetic foot ulcer (DFU) patients undertaking surgical treatment were recruited. Blood were drawn from subjects at initial, 4-week, 8-week, and 12-week after surgery. Clinical wound healing status was determined by a fellowship-trained orthopaedic foot and ankle surgeon. Serum antibodies and plasmablast cultured antibodies (newly synthesized antibodies: NSA) were harvested. Eight unique S. aureusantigens from distinct functional classes were used for the immunoassay. All serum and NSA samples were run on a flow cytometer (Bio-Plex 200; Bio-Rad, Life Sciences Research) in duplicates and assessed for predictive ability in discriminating infection status and healing status using receiver operating characteristic (ROC) curve analysis, with accuracy summarized by the area under the ROC curve (AUC). Nonparametric estimates and 95% confidence intervals for the AUC were computed for each predictor along with p-values for testing the significance of each AUC.Results:Analysis of serum immunoassay showed significant difference in three anti-S. aureus antigens titers (IsdH (p = 0.037; AUC = 0.638), ClfB (p = 0.025; AUC = 0.644), and SCIN (p = 0.005; AUC = 0.677)) between S. aureus infected versus non- S. aureus infected DFU patients at initial presentation. NSA immunoassay showed elevation of two different S. aureus specific antigens, IsdH and LukS-PV, for S. aureus infected versus non- S. aureus infected DFU patients in ratios of approximately five and four, respectively. Changes of NSA based anti-S. aureus antibody titers over 12 weeks period, as single antigen or in combination, significantly correlated with clinical resolution of infection and wound healing status. Four anti-S. aureus antigen combinations achieved the highest AUC (Figure 1).Conclusion:Our results demonstrate that both the serum and NSA immunoassay can diagnose S. aureus infected DFUs. Furthermore, changes in NSA titers over a period of time against various S. aureus specific antigens significantly correlated with clinical representation of infection and wound healing status. The novel species-specific immunoassay can serve as a promising diagnostic system, tracking tool and prognostic potential in management of S. aureus associated foot and ankle infection.

Bacterial diversity and inflammatory response at first-time visit in younger and older individuals with diabetic foot infection (DFI).

Infection of a diabetic foot ulcer (DFU) is common. More than the half of DFUs become infected and 15-20% of them necessitate amputation in course of treatment. Diabetic foot infection (DFI) is therefore the major cause for non-traumatic lower limb amputation in Germany. Prompt and effective treatment of DFI is mandatory to safe limbs and lives. We investigated if there are relevant differences in evoked inflammatory response between different species and age-separated groups. We further investigated if there is an impact of ulcer localization on bacterial diversity. For a 12-month period, we investigated 353 individuals with infected DFU, their laboratory results and bacterial diversity at first-time visit in a Diabetic Foot Care Center in Southern Germany. The ulcer microbiota was dominated by gram-positive species, primary Staphylococcus aureus. The gram-negative sector was mainly formed by Pseudomonas aeruginosa and Enterobacteriaceae (Proteus spp., Enterobacter spp., Escherichia coli and Klebsiella spp.). With increase in age, P. aeruginosa and S. aureus became more frequent, while Streptococci decreased. Ischemic and/or deep wounds were more likely to bear gram-negative species. Inflammatory response did not differ between gram-positive and gram-negative species, while Streptococci and Proteus spp. induced the highest serum inflammation reaction in their category. Streptococci, Enterobacter spp. and E. coli were more frequent in summer, while Enterococci spp., coagulase-negative Staphylococci and P.aeruginosa were more prevalent in winter half-year. DFIs of the forefoot and plantar side are mostly caused by gram-positive species, while Enterobacteriaceae were most frequent in plantar ulcerations. Gram-positive species dominate bacterial spectrum in DFI. With increase in age, S. aureus, Streptococci and Pseudomonas aeruginosa became more frequent. The inflammatory response did not differ significantly between different species, but gram-negative species were slightly but not significant more frequent in ischemic wounds. Climatic distinction like summer or winter half-year as well as foot ulcer localization seems to influence bacterial diversity in DFUs.

Selective MMP-9 Inhibitor (R)-ND-336 Alone or in Combination with Linezolid Accelerates Wound Healing in Infected Diabetic Mice.

Diabetic foot ulcers (DFUs) are a common complication of diabetes that are recalcitrant to healing due to persistent inflammation. The majority of DFUs have bacterial biofilms, with Staphylococcus epidermidis as a predominant bacterium, requiring infection control with antibiotics before treatment of the wound. Matrix metalloproteinases (MMPs) play roles in the pathology and repair of DFUs. However, defining the roles of the 24 human MMPs has been challenging due to the presence of three forms for each MMP, of which only one is catalytically competent, and the lack of convenient methods to distinguish among the three forms of MMPs. Using an affinity resin that binds only to the active forms of MMPs, with identification and quantification by mass spectrometry, we found that infected wounds in mice had increased levels of active MMP-9 compared to uninfected ones, paralleling infected human DFUs. MMP-9 activity prevents diabetic wounds from healing. We evaluated the efficacy of the selective small-molecule MMP-9 inhibitor, (R)-ND-336, in the infected diabetic mouse model of wound healing and showed that (R)-ND-336 alone or in combination with the antibiotic linezolid improves wound healing by inhibiting the detrimental MMP-9, mitigating macrophage infiltration to diminish inflammation, and increasing angiogenesis to restore the normal wound healing process. An advantage of this strategy is the ability to administer (R)-ND-336 concurrently with an antibiotic.

Pathogenesis of Staphylococcus haemolyticus on primary human skin fibroblast cells

ABSTRACT Staphylococcus haemolyticus (S. haemolyticus) is one of the Coagulase-negative staphylococci (CoNS) that inhabits the skin as a commensal. It is increasingly implicated in opportunistic infections, including diabetic foot ulcer (DFU) infections. In contrast to the abundance of information available for S. aureus and S. epidermidis, little is known about the pathogenicity of S. haemolyticus, despite the increased prevalence of this pathogen in hospitalized patients. We described, for the first time, the pathogenesis of different clinical isolates of S. haemolyticus isolated from DFU on primary human skin fibroblast (PHSF) cells. Virulence-related genes were investigated, adhesion and invasion assays were carried out using Giemsa stain, transmission electron microscopy (TEM), MTT and flowcytometry assays. Our results showed that most S. haemolyticus carried different sets of virulence-related genes. S. haemolyticus adhered to the PHSF cells to variable degrees. TEM showed that the bacteria were engulfed in a zipper-like mechanism into a vacuole inside the cell. Bacterial internalization was confirmed using flowcytometry and achieved high intracellular levels. PHSF cells infected with S.haemolyticus suffered from amarked decrease in viability and increased apoptosis when treated with whole bacterial suspensions or cell-free supernatants but not with heat-treated cells. After co-culture with PBMCs, S. haemolyticus induced high levels of pro-inflammatory cytokines. This study highlights the significant development of S. haemolyticus, which was previously considered a contaminant when detected in cultures of clinical samples. Their high ability to adhere, invade and kill the PHSF cells illustrate the severe damage associated with DFU infections. Abbreviations CoNS, coagulase-negative staphylococci; DFU, diabetic foot ulcer; DM, diabetes mellitus; DMEM, Dulbecco’s Modified Eagle Medium; MTT, 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide; PBMCs,peripheral blood mononuclear cells; PHSF, primary human skin fibroblast; CFU, colony-forming unit.

Advanced Glycation End Products Enhance Biofilm Formation by Promoting Extracellular DNA Release Through sigB Upregulation in Staphylococcus aureus.

Bacterial biofilms do serious harm to the diabetic foot ulcer (DFU) because they play a crucial role in infection invasion and spread. Staphylococcus aureus, the predominant Gram-positive bacteria in diabetic foot infection (DFI), is often associated with colonization and biofilm formation. Through biofilm formation tests in vitro, we observed that S. aureus bacteria isolated from DFU wounds were more prone to form biofilms than those from non-diabetic patients, while there was no difference in blood sugar between the biofilm (+) diabetics (DB+) and biofilm (-) diabetics (DB-). Furthermore, we found that advanced glycation end products (AGEs) promoted the biofilm formation of S. aureus in clinical isolates and laboratory strains in vitro, including a methicillin-resistant strain. Analysis of biofilm components demonstrated that the biofilms formed mainly by increasing extracellular DNA (eDNA) release; remarkably, the S. aureus global regulator sigB was upregulated, and its downstream factor lrgA was downregulated after AGE treatments. Mechanism studies using a sigB-deleted mutant (Newman-ΔsigB) confirmed that AGEs decreased expression of lrgA via induction of sigB, which is responsible for eDNA release and is a required component for S. aureus biofilm development. In conclusion, the present study suggests that AGEs promote S. aureus biofilm formation via an eDNA-dependent pathway by regulating sigB. The data generated by this study will provide experimental proof and theoretical support to improve DFU infection healing.

Role of Nrf2 in MALAT1/ HIF-1α loop on the regulation of angiogenesis in diabetic foot ulcer

Diabetic non healing wounds often result in significant morbidity and mortality. The number of effective targets to detect these wounds are meagre. Slow lymphangiogenesis is one of the complex processes involved in impaired healing of wounds. Long non coding RNAs (lncRNAs) have been importantly recognized for their role in pathological conditions. Multiple studies highlighting the role of lncRNAs in the regulation of several biological processes and complex diseases. Herein, we investigated the role of lncRNA Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in the progression of diabetic foot ulcer (DFU). We report a significant reduction in the expression of lncRNA MALAT1 in the infected DFU subjects which was positively correlated with the expression of angiogenic factors such as Nrf2, HIF-1α and VEGF. Further, expression of pro-inflammatory markers TNF-α and IL-6 were found to be increased while, the expression of anti-inflammatory marker IL-10 was decreased in infected DFU tissues. Involvement of lncRNA MALAT1 in angiogenesis in EA.hy926 cells was demonstrated by silencing the expression of Nrf2, HIF-1α, and VEGF through interference mediated by MALAT1. In addition, its inflammatory role was demonstrated by decreased expression of TNF-α, IL-6 and not affecting the expression of IL-10. Further, CRISPR-Cas9 knock out of Nrf2 decreased the expression of lncRNA MALAT1, HIF-1α and VEGF which revealed the association of Nrf2 in regulating MALAT1/HIF-1α loop through positive feedback mechanism. Collectively, our results suggested the role of Nrf2 on MALAT1/HIF-1α loop in the regulation of angiogenesis, which could act as a novel target in the treatment of diabetic wounds.

MON-626 Frequency and Associated Factors with Multidrug-Resistant Organism Infection in Diabetic Foot Ulcers in a Peruvian Public Hospital

Objective: To determine the frequency and associated factors with multidrug-resistant organism (MDRO) infection among patients with diabetic foot ulcers in a Peruvian Public Hospital. Materials and methods. Cross-sectional survey was conducted from January 2017 -December 2018 at National Hospital in Lima Perú. Ulcers with clinical signs of infection (erythema, edema, pain, purulent exudate) according Infectious Diseases Society of America clinical practice guideline were included1. Wounds with only skin involvement were excluded. On admission, specimens for culture were obtained after cleansing and debriding of the wound. Samples were promptly sent to the microbiology laboratory for culture using appropriate transport media. Bacterial identification and antibiotic susceptibility testing were performed using the VITEK® 2 automated system (BioMérieux Laboratory, Argentina). Multidrug-resistant organisms were identified according to the recommendations of International Expert Proposal2. Prevalence ratios derived from bivariate analysis are given with their 95% CI, which was performed to study factors associated with the presence of multidrug-resistant bacteria; and a multivariate analysis with a lineal model to associated variables found in the bivariate analysis. This study has the approval of the Research Ethics Committee of the María Auxiliadora Hospital. Results Among 153 selected subjects, 75% were male, with an average age of 59 yo, 70% had ≥10 years of diabetes duration and only 16% had HbA1C <7%. A frequency of 85% of patients with MDRO infection was found and was associated with minor amputation RP 1.18 (95% CI 1.01-1.44) and with hospitalization time of ≥ 28 days RP 1.21 (95% CI 1.03-1.30). Conclusion. 6 of 7 patients have MDRO infection among patients with diabetic foot ulcers and are associated with the occurrence of minor amputation and hospitalization time ≥ 28 days.

Comparative Microbiological and Whole-Genome Analysis of Staphylococcus aureus Populations in the Oro-Nasal Cavities, Skin and Diabetic Foot Ulcers of Patients With Type 2 Diabetes Reveals a Possible Oro-Nasal Reservoir for Ulcer Infection.

Patients with type 2 diabetes are at higher risk for periodontal disease and diabetic foot ulcer infections (DFUIs), the latter of which are predominantly caused by staphylococcal bacteria. Staphylococci have also been detected in the mouth, nose and gums (the oro-nasal cavity) of patients with periodontal disease and can move between the mouth and nose. The present study investigated if the oro-nasal cavity and/or periodontal pockets (PPs) in diseased gum tissue can provide a microbial reservoir for DFUIs. Eighteen patients with type 2 diabetes and at least three natural teeth (13 patients with ulcers and 5 patients without ulcers) underwent non-invasive microbiological sampling of PP, oro-nasal, skin and ulcer sites. Staphylococci were recovered using selective chromogenic agar, definitively identified and subjected to DNA microarray profiling, whole-genome sequencing and core-genome multilocus sequence typing (cgMLST). Staphylococcus aureus and Staphylococcus epidermidis were recovered from both the oro-nasal and ulcer sites of 6/13 and 5/13 patients with ulcers, respectively. Molecular typing based on the staphylococcal protein A (spa) gene and DNA microarray profiling indicated that for each patient investigated, S. aureus strains from oro-nasal and ulcer sites were identical. Comparative cgMLST confirmed that isolates from multiple anatomical sites of each individual investigated grouped into closely related, patient-distinct clusters (Clusters 1–7). Isolates belonging to the same cluster exhibited an average of 2.9 allelic differences (range 0–11). In contrast, reference genomes downloaded from GenBank selected as representatives of each sequence type identified in the present study exhibited an average of 227 allelic differences from the most closely related isolate within each cluster.

Pexiganan in Combination with Nisin to Control Polymicrobial Diabetic Foot Infections.

Diabetic foot ulcers (DFUs) are major complications of Diabetes mellitus being responsible for significant morbidity and mortality. DFUs frequently become chronically infected by a complex community of bacteria, including multidrug-resistant and biofilm-producing strains of Staphylococcus aureus and Pseudomonas aeruginosa. Diabetic foot infections (DFI) are often recalcitrant to conventional antibiotics and alternative treatment strategies are urgently needed. Antimicrobial Peptides (AMPs), such as pexiganan and nisin, have been increasingly investigated and reported as effective antimicrobial agents. Here, we evaluated the antibacterial potential of pexiganan and nisin used in combination (dual-AMP) to control the growth of planktonic and biofilm co-cultures of S. aureus and P. aeruginosa clinical strains, co-isolated from a DFU. A DFU collagen three-dimensional (3D) model was used to evaluate the distribution and efficacy of AMPs locally delivered into the model. The concentration of pexiganan required to inhibit and eradicate both planktonic and biofilm-based bacterial cells was substantially reduced when used in combination with nisin. Moreover, incorporation of both AMPs in a guar gum delivery system (dual-AMP biogel) did not affect the dual-AMP antimicrobial activity. Importantly, the application of the dual-AMP biogel resulted in the eradication of the S. aureus strain from the model. In conclusion, data suggest that the local application of the dual-AMPs biogel constitutes a potential complementary therapy for the treatment of infected DFU.