Patient genetics is linked to chronic wound microbiome composition and healing.

The clinical importance of microbiomes to the chronicity of wounds is widely appreciated, yet little is understood about patient-specific processes shaping wound microbiome composition. Here, a two-cohort microbiome-genome wide association study is presented through which patient genomic loci associated with chronic wound microbiome diversity were identified. Further investigation revealed that alternative TLN2 and ZNF521 genotypes explained significant inter-patient variation in relative abundance of two key pathogens, Pseudomonas aeruginosa and Staphylococcus epidermidis. Wound diversity was lowest in Pseudomonas aeruginosa infected wounds, and decreasing wound diversity had a significant negative linear relationship with healing rate. In addition to microbiome characteristics, age, diabetic status, and genetic ancestry all significantly influenced healing. Using structural equation modeling to identify common variance among SNPs, six loci were sufficient to explain 53% of variation in wound microbiome diversity, which was a 10% increase over traditional multiple regression. Focusing on TLN2, genotype at rs8031916 explained expression differences of alternative transcripts that differ in inclusion of important focal adhesion binding domains. Such differences are hypothesized to relate to wound microbiomes and healing through effects on bacterial exploitation of focal adhesions and/or cellular migration. Related, other associated loci were functionally enriched, often with roles in cytoskeletal dynamics. This study, being the first to identify patient genetic determinants for wound microbiomes and healing, implicates genetic variation determining cellular adhesion phenotypes as important drivers of infection type. The identification of predictive biomarkers for chronic wound microbiomes may serve as risk factors and guide treatment by informing patient-specific tendencies of infection.

Bacterial fitness in chronic wounds appears to be mediated by the capacity for high-density growth, not virulence or biofilm functions.

While much is known about acute infection pathogenesis, the understanding of chronic infections has lagged. Here we sought to identify the genes and functions that mediate fitness of the pathogen Pseudomonas aeruginosa in chronic wound infections, and to better understand the selective environment in wounds. We found that clinical isolates from chronic human wounds were frequently defective in virulence functions and biofilm formation, and that many virulence and biofilm formation genes were not required for bacterial fitness in experimental mouse wounds. In contrast, genes involved in anaerobic growth, some metabolic and energy pathways, and membrane integrity were critical. Consistent with these findings, the fitness characteristics of some wound impaired-mutants could be represented by anaerobic, oxidative, and membrane-stress conditions ex vivo, and more comprehensively by high-density bacterial growth conditions, in the absence of a host. These data shed light on the bacterial functions needed in chronic wound infections, the nature of stresses applied to bacteria at chronic infection sites, and suggest therapeutic targets that might compromise wound infection pathogenesis.

A review of iodine-based compounds, with a focus on biofilms: results of an expert panel.

Biofilms play a central role in the chronicity of non-healing lesions such as venous leg ulcers and diabetic foot ulcers. Therefore, biofilm management and treatment is now considered an essential part of wound care. Many antimicrobial treatments, whether topical or systemic, have been shown to have limited efficacy in the treatment of biofilm phenotypes. The antimicrobial properties of iodine compounds rely on multiple and diverse interactions to exert their effects on microorganisms. An expert panel, held in Las Vegas during the autumn Symposium on Advanced Wound Care meeting in 2018, discussed these properties, with the focus on iodine and iodophors and their effects on biofilm prevention and treatment.

A post-planktonic era of in vitro infectious models: issues and changes addressed by a clinically relevant wound like media.

Medical science is pitted against an ever-increasing rise in antibiotic tolerant microorganisms. Concurrently, during the past decade, biofilms have garnered much attention within research and clinical practice. Although the significance of clinical biofilms is becoming very apparent, current methods for diagnostics and direction of therapy plans in many hospitals do not reflect this knowledge; with many of the present tools proving to be inadequate for accurately mimicking the biofilm phenomenon. Based on current findings, we address some of the fundamental issues overlooked by clinical labs: the paradigm shifts that need to occur in assessing chronic wounds; better simulation of physiological conditions in vitro; and the importance of incorporating polymicrobial populations into biofilm models. In addition, this review considers using a biofilm relevant in vitro model for cultivating and determining the antibiotic tolerance and susceptibility of microorganisms associated with chronic wounds. This model presents itself as a highly rapid and functional tool that can be utilized by hospitals in an aim to improve bedside treatments.

Temporal dynamics of relative abundances and bacterial succession in chronic wound communities.

Polymicrobial bacterial infection is an important factor contributing to wound chronicity. Consequently, clinicians frequently adopt a biofilm-based wound care approach, in which wounds are treated utilizing DNA sequencing information about microbial communities. While more successful than treatment not using community information, there is little information about temporal dynamics of wound communities and optimal approaches over the course of treatment. To characterize these dynamics, temporal analysis over three sampling points was conducted for 167 chronic wounds. Across sampling intervals, wound communities from the same patients changed in composition, and most commonly shared less than 50% of observed species. There was a significant relationship between community similarity and time between sampling. Classifying wounds into state types, we found that communities frequently transitioned from Pseudomonas or Staphylococcus dominated, into a highly variable state type. Although low abundance microbial species are typically disregarded due to uncertainty of biological importance, we found that 80% of wound microbiomes included common or dominant species at subsequent time points that were in low abundance in earlier samples. Moreover, these species were often those known to frequently infect wounds. Results document compositional shifts through the course of treatment and suggest that routine consideration of low abundance species may improve biofilm-based wound care. Moreover, findings indicate that integrating ecological modeling to understand wound microbiome succession may lead to more informed therapy.

Consensus guidelines for the identification and treatment of biofilms in chronic nonhealing wounds.

BACKGROUND: Despite a growing consensus that biofilms contribute to a delay in the healing of chronic wounds, conflicting evidence pertaining to their identification and management can lead to uncertainty regarding treatment. This, in part, has been driven by reliance on in vitro data or animal models, which may not directly correlate to clinical evidence on the importance of biofilms. Limited data presented in human studies have further contributed to the uncertainty. Guidelines for care of chronic wounds with a focus on biofilms are needed to help aid the identification and management of biofilms, providing a clinical focus to support clinicians in improving patient care through evidence-based medicine. METHODS: A Global Wound Biofilm Expert Panel, comprising 10 clinicians and researchers with expertise in laboratory and clinical aspects of biofilms, was identified and convened. A modified Delphi process, based on published scientific data and expert opinion, was used to develop consensus statements that could help identify and treat biofilms as part of the management of chronic nonhealing wounds. Using an electronic survey, panel members rated their agreement with statements about biofilm identification and treatment, and the management of chronic nonhealing wounds. Final consensus statements were agreed on in a face-to-face meeting. RESULTS: Participants reached consensus on 61 statements in the following topic areas: understanding biofilms and the problems they cause clinicians; current diagnostic options; clinical indicators of biofilms; future options for diagnostic tests; treatment strategies; mechanical debridement; topical antiseptics; screening antibiofilm agents; and levels of evidence when choosing antibiofilm treatments. CONCLUSION: This consensus document attempts to clarify misunderstandings about the role of biofilms in clinical practice, and provides a basis for clinicians to recognize biofilms in chronic nonhealing wounds and manage patients optimally. A new paradigm for wound care, based on a stepped-down treatment approach, was derived from the consensus statements.

Analysis of the chronic wound microbiota of 2,963 patients by 16S rDNA pyrosequencing.

The extent to which microorganisms impair wound healing is an ongoing controversy in the management of chronic wounds. Because the high diversity and extreme variability of the microbiota between individual chronic wounds lead to inconsistent findings in small cohort studies, evaluation of a large number of chronic wounds using identical sequencing and bioinformatics methods is necessary for clinicians to be able to select appropriate empiric therapies. In this study, we utilized 16S rDNA pyrosequencing to analyze the composition of the bacterial communities present in samples obtained from patients with chronic diabetic foot ulcers (N = 910), venous leg ulcers (N = 916), decubitus ulcers (N = 767), and nonhealing surgical wounds (N = 370). The wound samples contained a high proportion of Staphylococcus and Pseudomonas species in 63 and 25% of all wounds, respectively; however, a high prevalence of anaerobic bacteria and bacteria traditionally considered commensalistic was also observed. Our results suggest that neither patient demographics nor wound type influenced the bacterial composition of the chronic wound microbiome. Collectively, these findings indicate that empiric antibiotic selection need not be based on nor altered for wound type. Furthermore, the results provide a much clearer understanding of chronic wound microbiota in general; clinical application of this new knowledge over time may help in its translation to improved wound healing outcomes.

Direct sampling of cystic fibrosis lungs indicates that DNA-based analyses of upper-airway specimens can misrepresent lung microbiota.

Recent work using culture-independent methods suggests that the lungs of cystic fibrosis (CF) patients harbor a vast array of bacteria not conventionally implicated in CF lung disease. However, sampling lung secretions in living subjects requires that expectorated specimens or collection devices pass through the oropharynx. Thus, contamination could confound results. Here, we compared culture-independent analyses of throat and sputum specimens to samples directly obtained from the lungs at the time of transplantation. We found that CF lungs with advanced disease contained relatively homogenous populations of typical CF pathogens. In contrast, upper-airway specimens from the same subjects contained higher levels of microbial diversity and organisms not typically considered CF pathogens. Furthermore, sputum exhibited day-to-day variation in the abundance of nontypical organisms, even in the absence of clinical changes. These findings suggest that oropharyngeal contamination could limit the accuracy of DNA-based measurements on upper-airway specimens. This work highlights the importance of sampling procedures for microbiome studies and suggests that methods that account for contamination are needed when DNA-based methods are used on clinical specimens.

Clinical identification of bacteria in human chronic wound infections: culturing vs. 16S ribosomal DNA sequencing.

BACKGROUND: Chronic wounds affect millions of people and cost billions of dollars in the United States each year. These wounds harbor polymicrobial biofilm communities, which can be difficult to elucidate using culturing methods. Clinical molecular microbiological methods are increasingly being employed to investigate the microbiota of chronic infections, including wounds, as part of standard patient care. However, molecular testing is more sensitive than culturing, which results in markedly different results being reported to clinicians. This study compares the results of aerobic culturing and molecular testing (culture-free 16S ribosomal DNA sequencing), and it examines the relative abundance score that is generated by the molecular test and the usefulness of the relative abundance score in predicting the likelihood that the same organism would be detected by culture. METHODS: Parallel samples from 51 chronic wounds were studied using aerobic culturing and 16S DNA sequencing for the identification of bacteria. RESULTS: One hundred forty-five (145) unique genera were identified using molecular methods, and 68 of these genera were aerotolerant. Fourteen (14) unique genera were identified using aerobic culture methods. One-third (31/92) of the cultures were determined to be < 1% of the relative abundance of the wound microbiota using molecular testing. At the genus level, molecular testing identified 85% (78/92) of the bacteria that were identified by culture. Conversely, culturing detected 15.7% (78/497) of the aerotolerant bacteria and detected 54.9% of the collective aerotolerant relative abundance of the samples. Aerotolerant bacterial genera (and individual species including Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis) with higher relative abundance scores were more likely to be detected by culture as demonstrated with regression modeling. CONCLUSION: Discordance between molecular and culture testing is often observed. However, culture-free 16S ribosomal DNA sequencing and its relative abundance score can provide clinicians with insight into which bacteria are most abundant in a sample and which are most likely to be detected by culture.

Comparison of culture and molecular identification of bacteria in chronic wounds.

Clinical diagnostics of chronic polymicrobial infections, such as those found in chronic wounds, represent a diagnostic challenge for both culture and molecular methods. In the current retrospective study, the results of aerobic bacterial cultures and culture-free bacterial identification using DNA analyses were compared. A total of 168 chronic wounds were studied. The majority of bacteria identified with culture testing were also identified with molecular testing, but the majority of bacteria identified with the molecular testing were not identified with culture testing. Seventeen (17) different bacterial taxa were identified with culture, and 338 different bacterial taxa were identified with molecular testing. This study demonstrates the increased sensitivity that molecular microbial identification can have over culture methodologies, and previous studies suggest that molecular bacterial identification can improve the clinical outcomes of patients with chronic wounds.

The effect of a hydroconductive dressing on the suppression of wound biofilm.

UNLABELLED: Although excessive exudate has been associated with poor wound healing outcomes, exudate is still not well understood in the pathophysiology of chronic wounds. Wound exudate is believed to be the result of wounds that are trapped in a persistent, hyper inflammatory state. Biofilm, bacteria of multiple species living in community, has multiple well-defined molecular pathways that produce hyper inflammation. The exudate that is produced in wounds is a potentially important nutrient source for biofilm; therefore, rapidly removing exudate may rob the biofilm of important nutrients and suppress its negative effects. METHODS: A hydroconductive fiber dressing that possesses excellent capillary action properties was utilized to rapidly remove wound exudate in 10 patients. RESULTS: The data demonstrate an average 62% reduction in wound volume for these 10 wounds over a 4-week period. Two wounds completely closed during the 2 weeks, and all but 1 wound significantly improved. Only 6 of the 10 wounds showed fewer bacteria at the end of the 4-week study period, suggesting there is not a 1:1 correlation with reduction in the number of bacteria in the wound and wound healing. CONCLUSION: Rapid removal of the nutrient source from wound biofilm, while not diminishing the number of bacteria, may suppress a biofilm's negative effects on wound healing. .

Microarray analysis and draft genomes of two Escherichia coli O157:H7 lineage II cattle isolates FRIK966 and FRIK2000 investigating lack of Shiga toxin expression.

The existence of two separate genetic lineages of Escherichia coli O157:H7 has previously been reported, and research indicates that lineage I could be more pathogenic toward human hosts than lineage II. We have previously shown that lineage I as a group expresses higher levels of Shiga toxin 2 (Stx2) than lineage II. To help evaluate why lineage II strains do not express appreciable levels of this toxin, whole-genome microarrays were performed using Agilent custom microarrays. Gene expression of the two representative bovine lineage II strains (FRIK966 and FRIK2000) were compared with gene expression of E. coli O157:H7 EDL933 (lineage I clinical type strain). Missing or differentially expressed genes and pathways were identified. Quantitative reverse transcription-polymerase chain reaction was performed to validate the microarray data. Draft genomes of FRIK966 and FRIK2000 were sequenced using Roche Applied Science/454 GS-FLX technology shotgun and paired-end approaches followed by de novo assembly. These assemblies were compared with the lineage I genome sequences from E. coli O157:H7 EDL933. The bacteriophage 933W, which encodes the Stx2 genes, showed a notable repression in gene expression. Polymerase chain reaction primers, based upon EDL933 genomic information, were also designed against all of the potentially missing genes of this bacteriophage. Most of the structural genes associated with the bacteriophage were found to be absent from the genome of the two bovine strains. These analyses, combined with evaluation of the genomic information, suggest that transposon (IS629) rearrangements may be associated with disruption of the bacteriophage genome in the FRIK strains. The results support the hypothesis that lineage II strains may be less of a risk as human foodborne pathogens. The microarray and genome data have been made available to the scientific community to allow continuing analysis of these cattle-isolated lineage II genomes and their gene expression.

Pyrosequencing study of fecal microflora of autistic and control children.

There is evidence of genetic predisposition to autism, but the percent of autistic subjects with this background is unknown. It is clear that other factors, such as environmental influences, may play a role in this disease. In the present study, we have examined the fecal microbial flora of 33 subjects with various severities of autism with gastrointestinal symptoms, 7 siblings not showing autistic symptoms (sibling controls) and eight non-sibling control subjects, using the bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) procedure. The results provide us with information on the microflora of stools of young children and a compelling picture of unique fecal microflora of children with autism with gastrointestinal symptomatology. Differences based upon maximum observed and maximum predicted operational taxonomic units were statistically significant when comparing autistic and control subjects with p-values ranging from <0.001 to 0.009 using both parametric and non-parametric estimators. At the phylum level, Bacteroidetes and Firmicutes showed the most difference between groups of varying severities of autism. Bacteroidetes was found at high levels in the severely autistic group, while Firmicutes were more predominant in the control group. Smaller, but significant, differences also occurred in the Actinobacterium and Proteobacterium phyla. Desulfovibrio species and Bacteroides vulgatus are present in significantly higher numbers in stools of severely autistic children than in controls. If the unique microbial flora is found to be a causative or consequent factor in this type of autism, it may have implications with regard to a specific diagnostic test, its epidemiology, and for treatment and prevention.

Evaluation of the bacterial diversity among and within individual venous leg ulcers using bacterial tag-encoded FLX and titanium amplicon pyrosequencing and metagenomic approaches.

BACKGROUND: Approximately 1 out of every 100 individuals has some form of venous insufficiency, which can lead to chronic venous disease and Venous Leg Ulcer (VLU). There are known underlying pathologies which contribute to the chronic nature of VLU including biofilm phenotype infections. RESULTS: Using pyrosequencing based approaches we evaluated VLU to characterize their microbial ecology. Results show that VLU infections are polymicrobial with no single bacterium colonizing the wounds. The most ubiquitous and predominant organisms include a previously uncharacterized bacteroidales, various anaerobes, Staphylococcus, Corynebacterium, and Serratia. Topological analysis of VLU show some notable differences in bacterial populations across the surface of the wounds highlighting the importance of sampling techniques during diagnostics. Metagenomics provide a preliminary indication that there may be protozoa, fungi and possibly an undescribed virus associated with these wounds. CONCLUSION: The polymicrobial nature of VLU and previous research on diabetic foot ulcers and surgical site infections suggest that the future of therapy for such wounds lies in the core of the logical and proven multiple concurrent strategy approach, which has been termed "biofilm-based wound care" and the use of individualized therapeutics rather than in a single treatment modality.

Chronic wound microbiome colonization on mouse model following cryogenic preservation.

Chronic wound infections are increasingly recognized to be dynamic and polymicrobial in nature, necessitating the development of wound models which reflect the complexities of infection in a non-healing wound. Wound slough isolated from human chronic wounds and transferred to mice was recently shown to create polymicrobial infection in mice, and there is potential this tool may be improved by cryogenic preservation. The purpose of this study was to investigate the application of cryogenic preservation to transferring polymicrobial communities, specifically by quantifying the effects of cryopreservation and wound microbiome transplantation. Slough from an established murine polymicrobial surgical excision model and five patients were subjected to three preservation strategies: refrigeration until infection, freezing in liquid nitrogen, or freezing in liquid nitrogen with glycerol solution prior to infection in individual mice. Four days following inoculation onto mice, wound microbiota were quantified using either culture isolation or by 16s rRNA gene community profiling and quantitative PCR. Cryogenic preservation did not significantly reduce bacterial viability. Reestablished microbial communities were significantly associated with patient of origin as well as host context (i.e., originally preserved from a patient versus mouse infection). Whereas preservation treatment did not significantly shape community composition, the transfers of microbiomes from human to mouse were characterized by reduced diversity and compositional changes. These findings indicated that changes should be expected to occur to community structure after colonization, and that compositional change is likely due to the rapid change in infection context as opposed to preservation strategy. Furthermore, species that were present in higher relative abundance in wound inoculate were more likely to colonize subsequent wounds, and wound inoculate with higher bacterial load established wound communities that were more compositionally similar. Results inform expectations for the complementation of chronic wound in vivo modeling with cryogenic preservation archives.

Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP).

BACKGROUND: The microbiota of an animal's intestinal tract plays important roles in the animal's overall health, productivity and well-being. There is still a scarcity of information on the microbial diversity in the gut of livestock species such as cattle. The primary reason for this lack of data relates to the expense of methods needed to generate such data. Here we have utilized a bacterial tag-encoded FLX 16s rDNA amplicon pyrosequencing (bTEFAP) approach that is able to perform diversity analyses of gastrointestinal populations. bTEFAP is relatively inexpensive in terms of both time and labor due to the implementation of a novel tag priming method and an efficient bioinformatics pipeline. We have evaluated the microbiome from the feces of 20 commercial, lactating dairy cows. RESULTS: Ubiquitous bacteria detected from the cattle feces included Clostridium, Bacteroides, Porpyhyromonas, Ruminococcus, Alistipes, Lachnospiraceae, Prevotella, Lachnospira, Enterococcus, Oscillospira, Cytophage, Anaerotruncus, and Acidaminococcus spp. Foodborne pathogenic bacteria were detected in several of the cattle, a total of 4 cows were found to be positive for Salmonella spp (tentative enterica) and 6 cows were positive for Campylobacter spp. (tentative lanienae). CONCLUSION: Using bTEFAP we have examined the microbiota in the feces of cattle. As these methods continue to mature we will better understand the ecology of the major populations of bacteria the lower intestinal tract. This in turn will allow for a better understanding of ways in which the intestinal microbiome contributes to animal health, productivity and wellbeing.

Survey of bacterial diversity in chronic wounds using pyrosequencing, DGGE, and full ribosome shotgun sequencing.

BACKGROUND: Chronic wound pathogenic biofilms are host-pathogen environments that colonize and exist as a cohabitation of many bacterial species. These bacterial populations cooperate to promote their own survival and the chronic nature of the infection. Few studies have performed extensive surveys of the bacterial populations that occur within different types of chronic wound biofilms. The use of 3 separate16S-based molecular amplifications followed by pyrosequencing, shotgun Sanger sequencing, and denaturing gradient gel electrophoresis were utilized to survey the major populations of bacteria that occur in the pathogenic biofilms of three types of chronic wound types: diabetic foot ulcers (D), venous leg ulcers (V), and pressure ulcers (P). RESULTS: There are specific major populations of bacteria that were evident in the biofilms of all chronic wound types, including Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp. Each of the wound types reveals marked differences in bacterial populations, such as pressure ulcers in which 62% of the populations were identified as obligate anaerobes. There were also populations of bacteria that were identified but not recognized as wound pathogens, such as Abiotrophia para-adiacens and Rhodopseudomonas spp. Results of molecular analyses were also compared to those obtained using traditional culture-based diagnostics. Only in one wound type did culture methods correctly identify the primary bacterial population indicating the need for improved diagnostic methods. CONCLUSION: If clinicians can gain a better understanding of the wound's microbiota, it will give them a greater understanding of the wound's ecology and will allow them to better manage healing of the wound improving the prognosis of patients. This research highlights the necessity to begin evaluating, studying, and treating chronic wound pathogenic biofilms as multi-species entities in order to improve the outcomes of patients. This survey will also foster the pioneering and development of new molecular diagnostic tools, which can be used to identify the community compositions of chronic wound pathogenic biofilms and other medical biofilm infections.

In vitro multispecies Lubbock chronic wound biofilm model.

Multispecies biofilms are becoming increasingly recognized as the naturally occurring state in which bacteria reside. One of the primary health issues that is now recognized to be exacerbated by biofilms are chronic, nonhealing wounds such as venous leg ulcers, diabetic foot ulcers, and pressure ulcers. Arguably three of the most important species associated with multispecies biofilms that our group sees clinically are Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus. This study was conducted to address the need for a chronic pathogenic biofilm laboratory model that allows for cooperative growth of these three organisms. We have developed a novel media formulation, simple laboratory system, quantitative polymerase chain reaction for monitoring population dynamics, and methods for objectively and subjectively measuring biofilm formation. The Lubbock chronic wound pathogenic biofilm withstood treatment with a 50-fold higher concentration of bleach than that which was completely bacteriocidal for fully turbid planktonic cultures. The Lubbock chronic wound pathogenic biofilm when treated with biofilm effectors such as gallium nitrate and triclosan responded with selective inhibition of Pseudomonas aeruginosa or Staphylococcus aureus, respectively, as has been reported in the literature. The ability of this 24-hour model to react as predicted using known biofilm effectors suggests that it will lend itself to future work in the development and testing of first-generation chronic wounds pathogenic biofilm therapeutics. We have defined a realistic in vitro multispecies biofilm model simulating the functional characteristics of chronic pathogenic biofilms and developed effective tools for its characterization and analyses.