The chronic wound characterisation study and biobank: a study protocol for a prospective observational cohort investigation of bacterial community composition, inflammatory responses and wound-healing trajectories in non-healing wounds.

Introduction: Chronic wounds affect 1%-2% of the global population, with rising incidence due to ageing and lifestyle-related diseases. Bacterial biofilms, found in 80% of chronic wounds, and scattered single-cell bacteria may hinder healing. Microbes are believed to negatively impact healing by exacerbating inflammation and host immune response.

Novel sampling technique maintaining the two-dimensional organization of microbes during cultivation from chronic wounds: The Imprint method.

This study aimed to develop and validate "the Imprint method,", a technique for sampling microbes from chronic wounds while preserving their two-dimensional spatial organization. We used nylon filters to sample bacteria and compared with sampling using Eswabs in 12 patients. The Imprint method identified a mean of 0.

Inhibition of quorum sensing by chemical induction of the MexEF-oprN efflux pump.

The cell-to-cell communication system quorum sensing (QS), used by various pathogenic bacteria to synchronize gene expression and increase host invasion potentials, is studied as a potential target for persistent infection control. To search for novel molecules targeting the QS system in the Gram-negative opportunistic pathogen , a chemical library consisting of 3,280 small compounds from LifeArc was screened. A series of 10 conjugated phenones that have not previously been reported to target bacteria were identified as inhibitors of QS in .

Single cells and bacterial biofilm populations in chronic wound infections.

Chronic wounds and chronic ulcers are an increasing problem associated with high health care burden and patient burden. The arrested healing of chronic wounds has, in part, been attributed to the presence of biofilms. Substantial research has documented the presence of biofilms in chronic wounds, and many mechanisms of host-pathogen interactions have been uncovered to explain the arrested healing.

What's in a name? Characteristics of clinical biofilms.

In vitro biofilms are communities of microbes with unique features compared to individual cells. Biofilms are commonly characterized by physical traits like size, adhesion, and a matrix made of extracellular substances. They display distinct phenotypic features, such as metabolic activity and antibiotic tolerance.

The structure-function relationship of Pseudomonas aeruginosa in infections and its influence on the microenvironment.

Pseudomonas aeruginosa is a human pathogen associated with both acute and chronic infections. While intensively studied, the basic mechanisms enabling the long-term survival of P. aeruginosa in the host, despite massive immune system attack and heavy antimicrobial treatment, remain to be identified.

Biofilm Survival Strategies in Chronic Wounds.

Bacterial biofilms residing in chronic wounds are thought to have numerous survival strategies, making them extremely difficult to eradicate and resulting in long-term infections. However, much of our knowledge regarding biofilm persistence stems from in vitro models and experiments performed in vivo in animal models. While the knowledge obtained from such experiments is highly valuable, its direct translation to the human clinical setting should be undertaken with caution.

Nitric-oxide-driven oxygen release in anoxic .

Denitrification supports anoxic growth of in infections. Moreover, denitrification may provide oxygen (O) resulting from dismutation of the denitrification intermediate nitric oxide (NO) as seen in . To examine the prevalence of NO dismutation we studied O release by in airtight vials.

Identification of small molecules that interfere with c-di-GMP signaling and induce dispersal of Pseudomonas aeruginosa biofilms.

Microbial biofilms are involved in a number of infections that cannot be cured, as microbes in biofilms resist host immune defenses and antibiotic therapies. With no strict biofilm-antibiotic in the current pipelines, there is an unmet need for drug candidates that enable the current antibiotics to eradicate bacteria in biofilms. We used high-throughput screening to identify chemical compounds that reduce the intracellular c-di-GMP content in Pseudomonas aeruginosa.

Induction of Native c-di-GMP Phosphodiesterases Leads to Dispersal of Pseudomonas aeruginosa Biofilms.

A decade of research has shown that the molecule c-di-GMP functions as a central second messenger in many bacteria. A high level of c-di-GMP is associated with biofilm formation, whereas a low level of c-di-GMP is associated with a planktonic single-cell bacterial lifestyle. c-di-GMP is formed by diguanylate cyclases and is degraded by specific phosphodiesterases.

Sampling challenges in diagnosis of chronic bacterial infections.

In recent decades there has been an increase in knowledge of the distribution, species diversity and growth patterns of bacteria in human chronic infections. This has challenged standard diagnostic methods, which have undergone a development to both increase the accuracy of testing as well as to decrease the occurrence of contamination. In particular, the introduction of new technologies based on molecular techniques into the clinical diagnostic process has increased detection and identification of infectious pathogens.

Small Molecule Anti-biofilm Agents Developed on the Basis of Mechanistic Understanding of Biofilm Formation.

Microbial biofilms are the cause of persistent infections associated with various medical implants and distinct body sites such as the urinary tract, lungs, and wounds. Compared with their free living counterparts, bacteria in biofilms display a highly increased resistance to immune system activities and antibiotic treatment. Therefore, biofilm infections are difficult or impossible to treat with our current armory of antibiotics.

Oxidative stress response plays a role in antibiotic tolerance of Streptococcus mutans biofilms.

Knowledge about biofilm-associated antibiotic tolerance mechanisms is warranted in order to develop effective treatments against biofilm infections. We performed a screen of a Streptococcus mutans transposon mutant library for mutants with reduced biofilm-associated antimicrobial tolerance, and found that the spxA1 gene plays a role in tolerance towards gentamicin and other antibiotics such as vancomycin and linezolid. SpxA1 is a regulator of genes involved in the oxidative stress response in S.

Qualitative and Quantitative Determination of Quorum Sensing Inhibition In Vitro.

The formation of biofilms in conjunction with quorum sensing (QS) regulated expression of virulence by opportunistic pathogens contributes significantly to immune evasion and tolerance to a variety of antimicrobial treatments. The present protocol describes methods to determine the in vitro efficacy of potential QS inhibitors (QSIs). Work on Pseudomonas aeruginosa has shown that chemical blockage of QS is a promising new antimicrobial strategy.

Imaging N-Acyl Homoserine Lactone Quorum Sensing In Vivo.

In order to study N-acyl homoserine lactone (AHL)-based quorum sensing in vivo, we present a protocol using an Escherichia coli strain equipped with a luxR-based monitor system, which in the presence of exogenous AHL molecules expresses a green fluorescent protein (GFP). Lungs from mice challenged intratracheally with alginate beads containing both a Pseudomonas aeruginosa strain together with the E. coli monitor strain can be investigated at different time points postinfection.

Metagenomic and metatranscriptomic analysis of saliva reveals disease-associated microbiota in patients with periodontitis and dental caries.

The taxonomic composition of the salivary microbiota has been reported to differentiate between oral health and disease. However, information on bacterial activity and gene expression of the salivary microbiota is limited. The purpose of this study was to perform metagenomic and metatranscriptomic characterization of the salivary microbiota and test the hypothesis that salivary microbial presence and activity could be an indicator of the oral health status.

Bacterial Biofilm Control by Perturbation of Bacterial Signaling Processes.

The development of effective strategies to combat biofilm infections by means of either mechanical or chemical approaches could dramatically change today's treatment procedures for the benefit of thousands of patients. Remarkably, considering the increased focus on biofilms in general, there has still not been invented and/or developed any simple, efficient and reliable methods with which to "chemically" eradicate biofilm infections. This underlines the resilience of infective agents present as biofilms and it further emphasizes the insufficiency of today's approaches used to combat chronic infections.

Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits -Like Characteristics.

Alginate beads represent a simple and highly reproducible model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with and followed for up to 72 h. Confocal microscopy revealed that formed dense clusters similar in size to aggregates observed in cystic fibrosis lungs and chronic wounds.

Fusaric acid and analogues as Gram-negative bacterial quorum sensing inhibitors.

Taking advantage of microwave-assisted synthesis, efficient and expedite procedures for preparation of a library of fusaric acid and 39 analogues are reported. The fusaric acid analogues were tested in cell-based screening assays for inhibition of the las and rhl quorum sensing system in Pseudomonas aeruginosa and the lux quorum sensing system in Vibrio fischeri. Eight of the 40 compounds in the library including fusaric acid inhibited lux quorum sensing and one compound inhibited activity of the las quorum sensing system.

Disulfide Bond-Containing Ajoene Analogues As Novel Quorum Sensing Inhibitors of Pseudomonas aeruginosa.

Since its discovery 22 years ago, the bacterial cell-to-cell communication system, termed quorum sensing (QS), has shown potential as antipathogenic target. Previous studies reported that ajoene from garlic inhibits QS in opportunistic human pathogen Pseudomonas aeruginosa. In this study, screening of an in-house compound library revealed two sulfur-containing compounds which possess structural resemblance with ajoene and inhibit QS in bioreporter assay.

Comparative systems biology analysis to study the mode of action of the isothiocyanate compound Iberin on Pseudomonas aeruginosa.

Food is now recognized as a natural resource of novel antimicrobial agents, including those that target the virulence mechanisms of bacterial pathogens. Iberin, an isothiocyanate compound from horseradish, was recently identified as a quorum-sensing inhibitor (QSI) of the bacterial pathogen Pseudomonas aeruginosa. In this study, we used a comparative systems biology approach to unravel the molecular mechanisms of the effects of iberin on QS and virulence factor expression of P.

Complete genome sequence of the cystic fibrosis pathogen Achromobacter xylosoxidans NH44784-1996 complies with important pathogenic phenotypes.

Achromobacter xylosoxidans is an environmental opportunistic pathogen, which infects an increasing number of immunocompromised patients. In this study we combined genomic analysis of a clinical isolated A. xylosoxidans strain with phenotypic investigations of its important pathogenic features.