RESUMEN
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.
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Antiinfecciosos/uso terapéutico , Pie Diabético/tratamiento farmacológico , Yodo/uso terapéutico , Antiinfecciosos/administración & dosificación , Antiinfecciosos/farmacología , Biopelículas/efectos de los fármacos , Pie Diabético/microbiología , Humanos , Yodo/administración & dosificación , Yodo/farmacologíaRESUMEN
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.
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Microbiota , Polimorfismo de Nucleótido Simple , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Infecciones Estafilocócicas , Staphylococcus epidermidis , Cicatrización de Heridas/genética , Infección de Heridas , Animales , Enfermedad Crónica , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Ratones , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patología , Talina/genética , Talina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Infección de Heridas/genética , Infección de Heridas/metabolismo , Infección de Heridas/microbiología , Infección de Heridas/patologíaRESUMEN
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.
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Criopreservación , Microbiota , Heridas y Lesiones/microbiología , Animales , Carga Bacteriana , Supervivencia Celular , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Congelación , Humanos , RatonesRESUMEN
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.
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Proliferación Celular/fisiología , Pseudomonas aeruginosa/crecimiento & desarrollo , Cicatrización de Heridas/fisiología , Adulto , Animales , Bacterias/crecimiento & desarrollo , Infecciones Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Modelos Animales de Enfermedad , Femenino , Aptitud Genética , Interacciones Microbiota-Huesped/fisiología , Humanos , Masculino , Ratones , Infecciones por Pseudomonas , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , Virulencia/fisiología , Infección de Heridas/metabolismo , Infección de Heridas/microbiologíaRESUMEN
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.
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Antiinfecciosos Locales/administración & dosificación , Biopelículas , Consenso , Cicatrización de Heridas/fisiología , Infección de Heridas/terapia , Administración Tópica , Animales , Enfermedad Crónica , HumanosRESUMEN
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.
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Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Biopelículas/efectos de los fármacos , Enfermedad Crónica/epidemiología , Cicatrización de Heridas/efectos de los fármacos , Infección de Heridas/microbiología , Biopelículas/crecimiento & desarrollo , ADN Bacteriano/genética , Humanos , Microbiota/efectos de los fármacos , Microbiota/genética , Técnicas de Diagnóstico Molecular , Análisis de Secuencia de ADN , Análisis Espacio-Temporal , Texas , Cicatrización de Heridas/genética , Infección de Heridas/tratamiento farmacológico , Infección de Heridas/genéticaRESUMEN
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.
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Antibacterianos/uso terapéutico , Biopelículas/crecimiento & desarrollo , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/crecimiento & desarrollo , Infección de Heridas/microbiología , Heridas y Lesiones/microbiología , Enfermedades Transmisibles/tratamiento farmacológico , Enfermedades Transmisibles/microbiología , Farmacorresistencia Bacteriana , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/patogenicidad , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/patogenicidad , Humanos , Pruebas de Sensibilidad Microbiana , Infección de Heridas/tratamiento farmacológico , Infección de Heridas/patologíaRESUMEN
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.
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Infecciones por Corynebacterium/epidemiología , Pie Diabético/microbiología , Úlcera por Presión/microbiología , Infecciones por Pseudomonas/epidemiología , Infecciones Estafilocócicas/epidemiología , Infecciones Estreptocócicas/epidemiología , Herida Quirúrgica/microbiología , Úlcera Varicosa/microbiología , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad Crónica , Corynebacterium/genética , Corynebacterium/aislamiento & purificación , Infecciones por Corynebacterium/microbiología , Femenino , Humanos , Masculino , Microbiota , Persona de Mediana Edad , Pseudomonas/genética , Pseudomonas/aislamiento & purificación , Infecciones por Pseudomonas/microbiología , ARN Ribosómico 16S/genética , Estudios Retrospectivos , Infecciones Estafilocócicas/microbiología , Staphylococcus/genética , Staphylococcus/aislamiento & purificación , Infecciones Estreptocócicas/microbiología , Streptococcus/genética , Streptococcus/aislamiento & purificación , Estados Unidos/epidemiología , Heridas y Lesiones/microbiologíaRESUMEN
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.
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Bacterias/aislamiento & purificación , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/microbiología , Técnicas Bacteriológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Infección de Heridas/microbiología , Bacterias/genética , Bacterias/crecimiento & desarrollo , Enfermedad Crónica , ADN Ribosómico/genética , Humanos , ARN Ribosómico 16S/genética , Estados UnidosRESUMEN
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.
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Fibrosis Quística/genética , Pulmón/microbiología , Metagenoma/fisiología , Esputo/microbiología , Tráquea/microbiología , Antibacterianos/farmacología , Bacterias/genética , Humanos , Pulmón/metabolismo , Neumología/métodos , ARN Ribosómico 16S/metabolismo , Análisis de Secuencia de ADN , Especificidad de la Especie , Manejo de EspecímenesRESUMEN
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.
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Bacterias/aislamiento & purificación , Técnicas de Tipificación Bacteriana/métodos , Tipificación Molecular/métodos , Heridas y Lesiones/microbiología , Bacterias/genética , Enfermedad Crónica , Humanos , ARN Ribosómico 16S/genéticaRESUMEN
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. .
RESUMEN
Chronic wound infections are typically polymicrobial; however, most in vivo studies have focused on monospecies infections. This project was designed to develop an in vivo, polymicrobial, biofilm-related, infected wound model in order to study multispecies biofilm dynamics and in relation to wound chronicity. Multispecies biofilms consisting of both Gram negative and Gram positive strains, as well as aerobes and anaerobes, were grown in vitro and then transplanted onto the wounds of mice. These in vitro-to-in vivo multi-species biofilm transplants generated polymicrobial wound infections, which remained heterogeneous with four bacterial species throughout the experiment. We observed that wounded mice given multispecies biofilm infections displayed a wound healing impairment over mice infected with a single-species of bacteria. In addition, the bacteria in the polymicrobial wound infections displayed increased antimicrobial tolerance in comparison to those in single species infections. These data suggest that synergistic interactions between different bacterial species in wounds may contribute to healing delays and/or antibiotic tolerance.
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Bacterias/clasificación , Bacterias/crecimiento & desarrollo , Infecciones Bacterianas/microbiología , Biopelículas/crecimiento & desarrollo , Interacciones Microbianas , Infección de Heridas/microbiología , Animales , Antibacterianos/farmacología , Bacterias/patogenicidad , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/genética , Biodiversidad , Enfermedad Crónica , ADN Bacteriano/genética , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Especificidad de la Especie , Cicatrización de Heridas/efectos de los fármacos , Infección de Heridas/tratamiento farmacológicoRESUMEN
Comprehensive evaluation of microbial diversity in almost any environment is now possible. Questions such as "Does the addition of fiber to the diet of humans change the gastrointestinal microbiota?" can now be answered easily and inexpensively. Tag-encoded FLX-amplicon pyrosequencing (TEFAP) has been utilized to evaluate bacterial, archaeal, fungal, algal, as well as functional genes. Using the new tag-encoded FLX amplicon pyrosequencing (bTEFAP) approach, we have evaluated the microbial diversity using a more cost-effective and largely reproducible method that would allow us to sequence the ribosomal RNA genes of microorganisms (hereafter focused on bacteria), without the need for the inherent bias of culture methods. These developments have ushered in a new age of microbial ecology studies, and we have utilized this technology to evaluate the microbiome in a wide range of systems in almost any conceivable environment.
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Bacterias/genética , ADN Bacteriano/genética , Variación Genética , Análisis de Secuencia de ADN/métodos , Animales , Bacterias/aislamiento & purificación , Biodiversidad , Cartilla de ADN/genética , ADN Bacteriano/aislamiento & purificación , ADN Ribosómico/genética , ADN Ribosómico/aislamiento & purificación , Metagenoma/genética , Microesferas , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Decubitus ulcers, also known as bedsores or pressure ulcers, affect millions of hospitalized patients each year. The microflora of chronic wounds such as ulcers most commonly exist in the biofilm phenotype and have been known to significantly impair normal healing trajectories. METHODS: Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), a universal bacterial identification method, was used to identify bacterial populations in 49 decubitus ulcers. Diversity estimators were utilized and wound community compositions analyzed in relation to metadata such as Age, race, gender, and comorbidities. RESULTS: Decubitus ulcers are shown to be polymicrobial in nature with no single bacterium exclusively colonizing the wounds. The microbial community among such ulcers is highly variable. While there are between 3 and 10 primary populations in each wound there can be hundreds of different species present many of which are in trace amounts. There is no clearly significant differences in the microbial ecology of decubitus ulcer in relation to metadata except when considering diabetes. The microbial populations and composition in the decubitus ulcers of diabetics may be significantly different from the communities in non-diabetics. CONCLUSIONS: Based upon the continued elucidation of chronic wound bioburdens as polymicrobial infections, it is recommended that, in addition to traditional biofilm-based wound care strategies, an antimicrobial/antibiofilm treatment program can be tailored to each patient's respective wound microflora.
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Bacterias/genética , Bacterias/aislamiento & purificación , Biodiversidad , Úlcera por Presión/microbiología , Análisis de Secuencia de ADN/métodos , Fenómenos Fisiológicos Bacterianos , Biopelículas , Enfermedad Crónica , ADN Bacteriano/genética , Femenino , Humanos , Masculino , Fenotipo , Análisis de Componente PrincipalRESUMEN
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.
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Trastorno Autístico , Heces/microbiología , Metagenoma , Adolescente , Niño , Preescolar , Femenino , Humanos , Masculino , Análisis de Secuencia de ADN/métodosRESUMEN
An extensive portion of the healthcare budget is allocated to chronic human infection. Chronic wounds in particular are a major contributor to this financial burden. Little is known about the types of bacteria which may contribute to the chronicity, biofilm and overall bioburden of the wound itself. In this study we compare the bacteriology of wounds and associated intact skin. Wound and paired intact skin swabs (from a contralateral location) were collected. The bacterial diversity was determined using bacterial Tag-encoded FLX amplicon pyrosequencing (bTEFAP). Diversity analysis showed intact skin to be significantly more diverse than wounds on both the species and genus levels (3% and 5% divergence). Furthermore, wounds show heightened levels of anaerobic bacteria, like Peptoniphilus, Finegoldia, and Anaerococcus, and other detrimental genera such as Corynebacterium and Staphylococcus. Although some of these and other bacterial genera were found to be common between intact skin and wounds, notable opportunistic wound pathogens were found at lower levels in intact skin. Principal Component Analysis demonstrated a clear separability of the two groups. The findings of the study not only greatly support the hypothesis of differing bacterial composition of intact skin and wounds, but also contribute additional insight into the ecology of skin and wound microflora. The increased diversity and lowered levels of opportunistic pathogens found in skin make the system highly distinguishable from wounds.
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INTRODUCTION: Conventional cultures have implicated Staphylococcus aureus (SA) and coagulase-negative Staphylococcus (CNS) as principal pathogens in chronic rhinosinusitis (CRS). These results are questioned by recent studies in which molecular probes implicate Haemophilus influenzae instead. OBJECTIVES: To identify all bacterial species present on sinonasal mucosa using molecular culture (bacterial tag-encoded FLX amplicon pyrosequencing [bTEFAP]) and to compare them with those identified with conventional methods. METHODS: A prospective study of 18 patients undergoing endoscopic sinus surgery for CRS and 9 control patients with pituitary adenomas was conducted. Per-operative mucosal biopsies were assessed with bTEFAP by sequencing the species-specific 16S ribosomal deoxyribonucleic acid (DNA) fragment for genetic identification of bacteria and then compared with simultaneous swab culture. RESULTS: Standard cultures showed mainly SA and CNS. Molecular cultures identified up to 20 organisms per sample. Surprisingly, anaerobic species predominated (Diaphorobacter and Peptoniphilus). SA was nevertheless detected in 50%. CONCLUSION: Molecular cultures such as bTEFAP are sensitive tools for bacterial identification in CRS and suggest that anaerobe involvement may be more frequent than presumed.
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Bacterias/clasificación , Rinitis/microbiología , Sinusitis/microbiología , Técnicas de Tipificación Bacteriana , Biopelículas , Biopsia , Enfermedad Crónica , Endoscopía , Femenino , Genes Bacterianos , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
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.