The Visualization of Biofilms in Chronic Diabetic Foot Wounds Using Routine Diagnostic Microscopy Methods

Diabetic foot wounds are commonly colonised by taxonomically diverse microbial communities and may additionally be infected with specific pathogens. Since biofilms are demonstrably less susceptible to antimicrobial agents than are planktonic bacteria, and may be present in chronic wounds, there is increasing interest in their aetiological role. In the current investigation, the presence of structured microbial assemblages in chronic diabetic foot wounds is demonstrated using several visualization methods. Debridement samples, collected from the foot wounds of diabetic patients, were histologically sectioned and examined using bright-field, fluorescence, and environmental scanning electron microscopy and assessed by quantitative differential viable counting. All samples (n = 26) harboured bioburdens in excess of 5 log10 CFU/g. Microcolonies were identified in 4/4 samples by all three microscopy methods, although bright-field and fluorescence microscopy were more effective at highlighting putative biofilm morphology than ESEM. Results in this pilot study indicate that bacterial microcolonies and putative biofilm matrix can be visualized in chronic wounds using florescence microscopy and ESEM, but also using the simple Gram stain

Panbacterial real-time PCR to evaluate bacterial burden in chronic wounds treated with Cutimed™ Sorbact™

The impact of polymicrobial bacterial infection on chronic wounds has been studied extensively, but standard bacteriological analysis is not always sensitive enough. Molecular approaches represent a promising alternative to the standard bacteriological analysis. This work aimed to assess the usefulness of a panbacterial quantitative real-time PCR reaction to quantitate the total bacterial load in chronic wounds treated with Cutimed™ Sorbact™, a novel therapeutic approach based on hydrophobic binding of bacteria to a membrane. The results obtained by panbacterial real-time PCR on conserved sequences of the bacterial 16S gene show that the bacterial burden significantly decreased in 10 out of 15 healing chronic wounds, and did not change in 5 out of 5 non-healing chronic wounds. On the contrary, classical culture for S. aureus and P. aeruginosa, and real-time PCR for Bacteroides and Fusobacterium did not show any correlation with the clinical outcome. Our study also shows that quantification of chronic wounds by panbacterial real-time PCR is to be performed on biopsies and not on swabs. These results show that panbacterial real-time PCR is a promising and quick method of determining the total bacterial load in chronic wounds, and suggest that it might be an important biomarker for the prognosis of chronic wounds under treatment

Polymicrobial Nature of Chronic Diabetic Foot Ulcer Biofilm Infections Determined Using Bacterial Tag Encoded FLX Amplicon Pyrosequencing (bTEFAP)

Diabetic extremity ulcers are associated with chronic infections. Such ulcer infections are too often followed by amputation because there is little or no understanding of the ecology of such infections or how to control or eliminate this type of chronic infection. A primary impediment to the healing of chronic wounds is biofilm phenotype infections. Diabetic foot ulcers are the most common, disabling, and costly complications of diabetes. Here we seek to derive a better understanding of the polymicrobial nature of chronic diabetic extremity ulcer infections. spp. and against difficult to culture bacteria such as anaerobes. While PCR methods also have bias, further work is now needed in comparing traditional culture results to high-resolution molecular diagnostic methods such as bTEFAP

Secondary bacterial infections of buruli ulcer lesions before and after chemotherapy with streptomycin and rifampicin

Buruli ulcer (BU), caused by Mycobacterium ulcerans is a chronic necrotizing skin disease. It usually starts with a subcutaneous nodule or plaque containing large clusters of extracellular acid-fast bacilli. Surrounding tissue is destroyed by the cytotoxic macrolide toxin mycolactone produced by microcolonies of M. ulcerans. Skin covering the destroyed subcutaneous fat and soft tissue may eventually break down leading to the formation of large ulcers that progress, if untreated, over months and years. Here we have analyzed the bacterial flora of BU lesions of three different groups of patients before, during and after daily treatment with streptomycin and rifampicin for eight weeks (SR8) and determined drug resistance of the bacteria isolated from the lesions. Before SR8 treatment, more than 60% of the examined BU lesions were infected with other bacteria, with Staphylococcus aureus and Pseudomonas aeruginosa being the most prominent ones. During treatment, 65% of all lesions were still infected, mainly with P. aeruginosa. After completion of SR8 treatment, still more than 75% of lesions clinically suspected to be infected were microbiologically confirmed as infected, mainly with P. aeruginosa or Proteus miriabilis. Drug susceptibility tests revealed especially for S. aureus a high frequency of resistance to the first line drugs used in Ghana. Our results show that secondary infection of BU lesions is common. This could lead to delayed healing and should therefore be further investigated

Growth factor-enriched autologous plasma improves wound healing after surgical debridement in odontogenic necrotizing fasciitis: a case report

<p>Abstract</p> <p>Background</p> <p>Odontogenic necrotizing fasciitis of the neck is a fulminant infection of odontogenic origin that quickly spreads along the fascial planes and results in necrosis of the affected tissues. It is usually polymicrobial, occurs frequently in immunocompromised patients, and has a high mortality rate.</p> <p>Case presentation</p> <p>A 69-year old Mexican male had a pain in the maxillar right-canine region and a swelling of the submental and submandibular regions. Our examination revealed local pain, tachycardia, hyperthermia (39°C), and the swelling of bilateral submental and submandibular regions, which also were erythematous, hyperthermic, crepitant, and with a positive Godet sign. Mobility and third-degree caries were seen in the right mandibular canine. Bacteriological cultures isolated <it>streptococcus pyogenes </it>and <it>staphylococcus aureus</it>. The histopathological diagnosis was odontogenic necrotizing fasciitis of the submental and submandibular regions. The initial treatment was surgical debridement and the administration of antibiotics. After cultures were negative, the surgical wound was treated with a growth factor-enriched autologous plasma eight times every third day until complete healing occurred.</p> <p>Conclusions</p> <p>The treatment with a growth factor-enriched autologous plasma caused a rapid healing of an extensive surgical wound in a patient with odontogenic necrotizing fasciitis. The benefits were rapid tissue regeneration, an aesthetic and a functional scar, and the avoidance of further surgery and possible complications.</p

Community Analysis of Chronic Wound Bacteria Using 16S rRNA Gene-Based Pyrosequencing: Impact of Diabetes and Antibiotics on Chronic Wound Microbiota

Background: Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. Methodology/Principal Findings: We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds-approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. Conclusions/Significance: The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure-reducing some bacteria while selecting for others

An airway epithelial IL-17A response signature identifies a steroid-unresponsive COPD patient subgroup

© 2019 American Society for Clinical Investigation. All rights reserved. BACKGROUND. Chronic obstructive pulmonary disease (COPD) is a heterogeneous smoking-related disease characterized by airway obstruction and inflammation. This inflammation may persist even after smoking cessation and responds variably to corticosteroids. Personalizing treatment to biologically similar "molecular phenotypes" may improve therapeutic efficacy in COPD. IL-17A is involved in neutrophilic inflammation and corticosteroid resistance, and thus may be particularly important in a COPD molecular phenotype. METHODS. We generated a gene expression signature of IL-17A response in bronchial airway epithelial brushings from smokers with and without COPD (n = 238), and validated it using data from 2 randomized trials of IL-17 blockade in psoriasis. This IL-17 signature was related to clinical and pathologic characteristics in 2 additional human studies of COPD: (a) SPIROMICS (n = 47), which included former and current smokers with COPD, and (b) GLUCOLD (n = 79), in which COPD participants were randomized to placebo or corticosteroids. RESULTS. The IL-17 signature was associated with an inflammatory profile characteristic of an IL-17 response, including increased airway neutrophils and macrophages. In SPIROMICS the signature was associated with increased airway obstruction and functional small airways disease on quantitative chest CT. In GLUCOLD the signature was associated with decreased response to corticosteroids, irrespective of airway eosinophilic or type 2 inflammation. CONCLUSION. These data suggest that a gene signature of IL-17 airway epithelial response distinguishes a biologically, radiographically, and clinically distinct COPD subgroup that may benefit from personalized therapy

A Gene in the Process of Endosymbiotic Transfer

BACKGROUND: The endosymbiotic birth of organelles is accompanied by massive transfer of endosymbiont genes to the eukaryotic host nucleus. In the centric diatom Thalassiosira pseudonana the Psb28 protein is encoded in the plastid genome while a second version is nuclear-encoded and possesses a bipartite N-terminal presequence necessary to target the protein into the diatom complex plastid. Thus it can represent a gene captured during endosymbiotic gene transfer. METHODOLOGY/PRINCIPAL FINDINGS: To specify the origin of nuclear- and plastid-encoded Psb28 in T. pseudonana we have performed extensive phylogenetic analyses of both mentioned genes. We have also experimentally tested the intracellular location of the nuclear-encoded Psb28 protein (nuPsb28) through transformation of the diatom Phaeodactylum tricornutum with the gene in question fused to EYFP. CONCLUSIONS/SIGNIFICANCE: We show here that both versions of the psb28 gene in T. pseudonana are transcribed. We also provide experimental evidence for successful targeting of the nuPsb28 fused with EYFP to the diatom complex plastid. Extensive phylogenetic analyses demonstrate that nucleotide composition of the analyzed genes deeply influences the tree topology and that appropriate methods designed to deal with a compositional bias of the sequences and the long branch attraction artefact (LBA) need to be used to overcome this obstacle. We propose that nuclear psb28 in T. pseudonana is a duplicate of a plastid localized version, and that it has been transferred from its endosymbiont

Aviation turbulence: dynamics, forecasting, and response to climate change

Atmospheric turbulence is a major hazard in the aviation industry and can cause injuries to passengers and crew. Understanding the physical and dynamical generation mechanisms of turbulence aids with the development of new forecasting algorithms and, therefore, reduces the impact that it has on the aviation industry. The scope of this paper is to review the dynamics of aviation turbulence, its response to climate change, and current forecasting methods at the cruising altitude of aircraft. Aviation-affecting turbulence comes from three main sources: vertical wind shear instabilities, convection, and mountain waves. Understanding these features helps researchers to develop better turbulence diagnostics. Recent research suggests that turbulence will increase in frequency and strength with climate change, and therefore, turbulence forecasting may become more important in the future. The current methods of forecasting are unable to predict every turbulence event, and research is ongoing to find the best solution to this problem by combining turbulence predictors and using ensemble forecasts to increase skill. The skill of operational turbulence forecasts has increased steadily over recent decades, mirroring improvements in our understanding. However, more work is needed—ideally in collaboration with the aviation industry—to improve observations and increase forecast skill, to help maintain and enhance aviation safety standards in the future

Histopathological Changes and Clinical Responses of Buruli Ulcer Plaque Lesions during Chemotherapy: A Role for Surgical Removal of Necrotic Tissue?

The tropical necrotizing skin disease Buruli ulcer (BU) caused by Mycobacterium ulcerans is associated with extensive tissue destruction and local immunosuppression caused by the macrolide exotoxin mycolactone. Chemotherapy with a combination of rifampicin and streptomycin for 8 weeks is the currently recommended treatment for all types of BU lesions, including both ulcerative and non-ulcerative stages (plaques, nodules and edema). Our histopathological analysis of twelve BU plaque lesions revealed extensive destruction of sub-cutaneous tissue. This frequently led to ulceration during antibiotic treatment. This should not be mistaken as a failure of the antimycobacterial chemotherapy, since we found no evidence for the persistence of active infection foci. Large necrotic areas were found to persist even after completion of antibiotic treatment. These may disturb wound healing and the role of wound debridement should therefore be formally tested in a clinical trial setting