Chronic wounds: Treatment consensus.

The Wound Healing Foundation (WHF) recognised a need for an unbiased consensus on the best treatment of chronic wounds. A panel of 13 experts were invited to a virtual meeting which took place on 27 March 2021. The proceedings were organised in the sub-sections diagnosis, debridement, infection control, dressings, grafting, pain management, oxygen treatment, outcomes and future needs. Eighty percent or better concurrence among the panellists was considered a consensus. A large number of critical questions were discussed and agreed upon. Important takeaways included that wound care needs to be simplified to a point that it can be delivered by the patient or the patient's family. Another one was that telemonitoring, which has proved very useful during the COVID-19 pandemic, can help reduce the frequency of interventions by a visiting nurse or a wound care center. Defining patient expectations is critical to designing a successful treatment. Patient outcomes might include wound specific outcomes such as time to heal, wound size reduction, as well as improvement in quality of life. For those patients with expectations of healing, an aggressive approach to achieve that goal is recommended. When healing is not an expectation, such as in patients receiving palliative wound care, outcomes might include pain reduction, exudate management, odour management and/or other quality of life benefits to wound care.

Biofilm and catheter-related bloodstream infections.

Careful attention to detail and adherence to procedure guidelines when inserting and managing intravascular catheters has decreased the incidence of catheter-related bloodstream infections (CRBSIs). In order to limit these, health professionals must understand the underlying microbiology. Biofilms can explain the clinical findings most often seen with CRBSIs, yet they are poorly understood within medicine. Bacteria growing on solid surfaces such as a catheter are predominantly in biofilm phenotype, with a group of genes active that allow the bacteria to be tolerant to antiseptics and antibiotics by producing a self-secreted protective matrix. It is unclear whether it is planktonic seeding or small fragments of biofilm breaking off into the bloodstream that eventually results in the acute infection. The literature identifies four routes for microbes to adhere to a catheter and start biofilm formation: catheter contact, catheter insertion, catheter management and non-catheter-related sources. Routine clinical culture methods are inadequate to fully identify microbes producing catheter biofilm and/or bloodstream infection, therefore DNA methods may be required to diagnose CRBSIs. Treatment is removal and reinsertion of the catheter in a different site when possible. However, antibiofilm strategies can be employed to try to salvage the catheter. The use of high-dose antiseptics or antibiotics for long durations inside the catheter and hub (antibiotic/antiseptic lock) can suppress biofilm enough to reduce the seeding of the blood below a level where the patient's immune system can prevent bloodstream infection.

Pseudomonas aeruginosa transcriptome during human infection.

Laboratory experiments have uncovered many basic aspects of bacterial physiology and behavior. After the past century of mostly in vitro experiments, we now have detailed knowledge of bacterial behavior in standard laboratory conditions, but only a superficial understanding of bacterial functions and behaviors during human infection. It is well-known that the growth and behavior of bacteria are largely dictated by their environment, but how bacterial physiology differs in laboratory models compared with human infections is not known. To address this question, we compared the transcriptome of Pseudomonas aeruginosa during human infection to that of P. aeruginosa in a variety of laboratory conditions. Several pathways, including the bacterium's primary quorum sensing system, had significantly lower expression in human infections than in many laboratory conditions. On the other hand, multiple genes known to confer antibiotic resistance had substantially higher expression in human infection than in laboratory conditions, potentially explaining why antibiotic resistance assays in the clinical laboratory frequently underestimate resistance in patients. Using a standard machine learning technique known as support vector machines, we identified a set of genes whose expression reliably distinguished in vitro conditions from human infections. Finally, we used these support vector machines with binary classification to force P. aeruginosa mouse infection transcriptomes to be classified as human or in vitro. Determining what differentiates our current models from clinical infections is important to better understand bacterial infections and will be necessary to create model systems that more accurately capture the biology of infection.

Wound Biofilm: Current Perspectives and Strategies on Biofilm Disruption and Treatments

The presence of bio lm remains a challenging factor that contributes to the delayed healing of many chronic wounds. The major threat of chronic wound bio lms is their substantial protection from host immunities and extreme tolerance to antimicrobial agents. To help guide the development of wound treatment strategies, a panel of experts experienced in clinical and laboratory aspects of biofilm convened to discuss what is understood and not yet understood about biofilms and what is needed to better identify and treat chronic wounds in which biofilm is suspected. This article reviews evidence of the problem of biofilms in chronic wounds, summarizes literature-based and experience-based recommendations from the panel meeting, and identities future and emerging technologies needed to address the current gaps in knowledge. While currently there is insufficient evidence to provide an accurate comparison of the effectiveness of current therapies/products in reducing or removing biofilm, research has shown that in addition to debridement, appropriate topical antimicrobial application can suppress biofilm reformation. Because the majority of the resistance of bacteria in a biofilm population is expressed by its own secreted matrix of extracellular polymeric substance (EPS), panel members stressed the need for a paradigm shift toward biofilm treatment strategies that disrupt this shield. High-osmolarity surfactant solution technology is emerging as a potential multimodal treatment that has shown promise in EPS disruption and prevention of biofilm formation when used immediately post debridement. Panel members advocated incorporating an EPS-disrupting technology into an antibiofilm treatment approach for all chronic wounds. The activity of this panel is a step toward identifying technology and research needed to improve biofilm management of chronic wounds.

Clinically Addressing Biofilm in Chronic Wounds.

BACKGROUND: A chronic wound is a wound that is arrested in the inflammatory phase of wound healing and cannot progress further. Over 90% of chronic wounds contain bacteria and fungi living within a biofilm construct. THE PROBLEM: Each aggregation of microbes creates a distinct biofilm with differing characteristics so that a clinical approach has to be tailored to the specifics of a given biofilm. Defining the characteristics of that biofilm and then designing a therapeutic option particular to that biofilm is currently being defined. BASIC/CLINICAL SCIENCE ADVANCES: Biofilm becomes resistant to therapeutic maneuvers at 48-96 h after formation. By repeatedly attacking it on a regular schedule, one forces biofilm to reattach and reform during which time it is susceptible to antibiotics and host defenses. Identifying the multiple bacteria and fungi that make up a specific biofilm using polymerase chain reaction (PCR) allows directed therapeutic maneuvers such as application of specific topical antibiotics and biocides to increase the effectiveness of the debridement. CLINICAL CARE RELEVANCE: Most chronic wounds contain biofilm that perpetuate the inflammatory phase of wound healing. Combining debridement with using PCR to identify the bacteria and fungi within the biofilm allows for more targeted therapeutic maneuvers to eliminate a given biofilm. CONCLUSION: Therapeutic options in addition to debridement are currently being evaluated to address biofilm. Using PCR to direct adjunctive therapeutic maneuvers may increase the effectiveness of addressing biofilm in a chronic wound.

Colonic mucosal DNA methylation, immune response, and microbiome patterns in Toll-like receptor 2-knockout mice.

The connection between intestinal microbiota and host physiology is increasingly becoming recognized. The details of this dynamic interaction, however, remain to be explored. Toll-like receptor 2 (Tlr2) is important for its role in bacterial recognition, intestinal inflammation, and obesity-related metabolic changes. Therefore, we sought to determine the epigenomic and metagenomic consequences of Tlr2 deficiency in the colonic mucosa of mice to gain insights into biological pathways that shape the interface between the gut microbiota and the mammalian host. Colonic mucosa from wild type (WT) and Tlr2(-/-) C57BL/6 mice was interrogated by microarrays specific for DNA methylation and gene expression. The mucosal microbiome was studied by next-generation pyrosequencing of bacterial 16S rRNA. The expression of genes involved in immune processes was significantly modified by the absence of Tlr2, a number of which correlated with DNA methylation changes. The epigenomic and transcriptomic modifications associated with alteration in mucosal microbial composition. Several bacterial species, including members of the Firmicutes were significantly different in abundance between WT and Tlr2(-/-) animals. This manuscript highlights the intimate interrelationships between expression of immune-related genes and immunity pathways in the host with compositional and functional differences of the mammalian microbiome.

The effect of the macrolide antibiotic tylosin on microbial diversity in the canine small intestine as demonstrated by massive parallel 16S rRNA gene sequencing.

BACKGROUND: Recent studies have shown that the fecal microbiota is generally resilient to short-term antibiotic administration, but some bacterial taxa may remain depressed for several months. Limited information is available about the effect of antimicrobials on small intestinal microbiota, an important contributor to gastrointestinal health. The antibiotic tylosin is often successfully used for the treatment of chronic diarrhea in dogs, but its exact mode of action and its effect on the intestinal microbiota remain unknown. The aim of this study was to evaluate the effect of tylosin on canine jejunal microbiota. Tylosin was administered at 20 to 22 mg/kg q 24 hr for 14 days to five healthy dogs, each with a pre-existing jejunal fistula. Jejunal brush samples were collected through the fistula on days 0, 14, and 28 (14 days after withdrawal of tylosin). Bacterial diversity was characterized using massive parallel 16S rRNA gene pyrosequencing. RESULTS: Pyrosequencing revealed a previously unrecognized species richness in the canine small intestine. Ten bacterial phyla were identified. Microbial populations were phylogenetically more similar during tylosin treatment. However, a remarkable inter-individual response was observed for specific taxa. Fusobacteria, Bacteroidales, and Moraxella tended to decrease. The proportions of Enterococcus-like organisms, Pasteurella spp., and Dietzia spp. increased significantly during tylosin administration (p < 0.05). The proportion of Escherichia coli-like organisms increased by day 28 (p = 0.04). These changes were not accompanied by any obvious clinical effects. On day 28, the phylogenetic composition of the microbiota was similar to day 0 in only 2 of 5 dogs. Bacterial diversity resembled the pre-treatment state in 3 of 5 dogs. Several bacterial taxa such as Spirochaetes, Streptomycetaceae, and Prevotellaceae failed to recover at day 28 (p < 0.05). Several bacterial groups considered to be sensitive to tylosin increased in their proportions. CONCLUSION: Tylosin may lead to prolonged effects on the composition and diversity of jejunal microbiota. However, these changes were not associated with any short-term clinical signs of gastrointestinal disease in healthy dogs. Our results illustrate the complexity of the intestinal microbiota and the challenges associated with evaluating the effect of antibiotic administration on the various bacterial groups and their potential interactions.

Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) for microbiome studies: bacterial diversity in the ileum of newly weaned Salmonella-infected pigs.

The microbiota of an animal's intestinal tract plays a vital role in the animal's overall health. There is a surprising scarcity of information on the microbial diversity in the gut of livestock species such as cattle and swine. Here we describe a bacterial 16S-based tag-encoded FLX amplicon pyrosequencing (bTEFAP) method that we have developed as a high-throughput universal tool for bacterial diversity, epidemiology, and pathogen detection studies. This method will allow hundreds of samples to be run simultaneously but analyzed individually or as groups. To test this new methodology, we individually evaluated the bacterial diversity in the ileum of 21 pigs. Ubiquitous bacteria detected in the newly weaned pigs were Clostridium spp., Lactobacillus spp., and Helicobacter spp. Many of the pigs had surprisingly low concentrations of beneficial bacteria such as Bifidobacterium spp. Only four of the pigs were shown to be positive for Salmonella spp. using traditional culture methods. A total of eight pigs were bTEFAP positive for Salmonella spp., including all four of the pigs that had been culture positive. Two of the pigs sampled were also positive for Campylobacter spp. tentative identified as jejuni. Using rarefaction curves modeled with the Richards equation, we estimated the maximum number of unique species level (3% dissimilarity) operational taxonomic units in the ileum of these pigs. These predictions indicated that there may be as many as 821 different species associated with the ileum in pigs. Together these data indicate a powerful potential of this technology in food safety and epidemiological and bacterial diversity applications. Using bTEFAP, we can expect to gain a better understanding of how the microbiome of an animal contributes to its health and well-being.