Modèles d'infection de la plaie du pied chez le diabétique : approche in vitro et in vivo de la formation de biofilms de bactéries pathogènes seules ou en association

Chronic wounds are a real public health problem. One of the main complications is the recurrent progress towards infection with an underlying risk of amputation. The difficulty of management lies in the presence of a polymicrobial biofilm at the wound bed. The objective of this thesis was to promote the development of the state-of-the-art of this important pathophysiology phenomenon of biofilm but also to promote new approaches for therapeutic treatment of chronic wounds to reduce risk of morbid infections. This doctoral work enabled: i) to perfect diagnostic tools by creating a new in vitro model mimicking the environment in which bacteria evolve at the level of chronic wounds; ii) to study behavior (virulence, fitness, genomics, morphology, early biofilm formation, expression of key genes for biofilm formation) of reference strains and clinical strains of Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) isolated on a patient wound, based on short or long-term exposure to a conventional or a chronic wound like environment. The results showed that exposure under stressful environmental conditions reduced the virulence and fitness of the strains in favor of a biofilm-oriented behavior; iii) demonstrate the impact of multiple bacteria in an environment. This work has particularly highlighted the decrease of the pathogenicity and the evolution of the expression of some genes involved in the biofilm of SA strain when in contact with PA strain. iv) the development of a dynamic biofilm formation and confocal imaging technique allowing the visualization of a three-dimensional polymicrobial biofilm and the evaluation of antimicrobial molecules on each bacterial species composing the biofilm polymicrobial after an automatized mechanical debridement. The combination of our new chronic wound-like medium and the microfluidic biofilm formation and 3D visualization system of the polymicrobial biofilm represents powerful tools to improve the understanding of the interactions governing biofilms but also to evaluate the effectiveness of candidate molecules in chronic wounds management.Key words: Chronic wounds, infections, biofilms, bacterial cooperationLes plaies chroniques sont un réel problème de santé publique. L’une des principales complications est l’évolution fréquente vers l’infection avec un risque sous-jacent d'amputation. La difficulté de prise en charge repose sur la présence d’un biofilm polymicrobien au niveau du lit de la plaie. L'objectif de cette thèse était de promouvoir l'amélioration de l'état de l'art relatif à la compréhension de ce phénomène physiopathologique important mais également de développer de nouvelles approches concernant le traitement thérapeutique des plaies chroniques pour réduire les risques d'infections morbides. Ce travail de doctorat a permis : i) de parfaire les outils diagnostic de par la création d’un nouveau modèle in vitro mimant l’environnement dans lequel les bactéries évoluent au niveau des plaies chroniques ; ii) d’étudier le comportement (virulence, fitness, génomique, morphologie, formation de biofilm précoce, expression de gènes clés pour la formation de biofilm) de souches de références et de souches cliniques de Staphylococcus aureus (SA) et Pseudomonas aeruginosa (PA). Ces souches, isolées au sein d’une même plaie, étaient exposées de façon courte ou prolongée, à un milieu classique ou mimant l’environnement chronique d’une plaie. Les résultats ont montré qu’une exposition dans des conditions environnementales stressantes réduisaient la virulence et le fitness des souches au profit d’un comportement plus tourné vers la formation de biofilm ; iii) de démontrer l’impact de la présence de plusieurs bactéries au sein d’un environnement. Ce travail a particulièrement mis en évidence la baisse de pathogénicité et l’évolution de l’expression de certains gènes impliqués dans le biofilm de souche de SA en présence de PA. Enfin iv) la mise au point d’une technique de formation de biofilm dynamique et d’imagerie confocale permettant la visualisation d’un biofilm polymicrobien en trois dimensions et l’évaluation de molécules antimicrobiennes sur chaque espèce bactérienne composant le biofilm polymicrobien après un débridement mécanique automatisé. La combinaison de notre nouveau milieu « plaie chronique » et du système de formation en flux et visualisation en 3D du biofilm polymicrobien représente des outils puissants pour améliorer la compréhension des interactions régissant les biofilms mais aussi pour évaluer l’efficacité de molécules candidates dans la prise en charge des plaies chroniques.Mots clés : Plaies chroniques, infections, biofilms, coopération bactérienn

Modèles d'infection de la plaie du pied chez le diabétique : approche in vitro et in vivo de la formation de biofilms de bactéries pathogènes seules ou en association

Chronic wounds are a real public health problem. One of the main complications is the recurrent progress towards infection with an underlying risk of amputation. The difficulty of management lies in the presence of a polymicrobial biofilm at the wound bed. The objective of this thesis was to promote the development of the state-of-the-art of this important pathophysiology phenomenon of biofilm but also to promote new approaches for therapeutic treatment of chronic wounds to reduce risk of morbid infections. This doctoral work enabled: i) to perfect diagnostic tools by creating a new in vitro model mimicking the environment in which bacteria evolve at the level of chronic wounds; ii) to study behavior (virulence, fitness, genomics, morphology, early biofilm formation, expression of key genes for biofilm formation) of reference strains and clinical strains of Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) isolated on a patient wound, based on short or long-term exposure to a conventional or a chronic wound like environment. The results showed that exposure under stressful environmental conditions reduced the virulence and fitness of the strains in favor of a biofilm-oriented behavior; iii) demonstrate the impact of multiple bacteria in an environment. This work has particularly highlighted the decrease of the pathogenicity and the evolution of the expression of some genes involved in the biofilm of SA strain when in contact with PA strain. iv) the development of a dynamic biofilm formation and confocal imaging technique allowing the visualization of a three-dimensional polymicrobial biofilm and the evaluation of antimicrobial molecules on each bacterial species composing the biofilm polymicrobial after an automatized mechanical debridement. The combination of our new chronic wound-like medium and the microfluidic biofilm formation and 3D visualization system of the polymicrobial biofilm represents powerful tools to improve the understanding of the interactions governing biofilms but also to evaluate the effectiveness of candidate molecules in chronic wounds management.Key words: Chronic wounds, infections, biofilms, bacterial cooperationLes plaies chroniques sont un réel problème de santé publique. L’une des principales complications est l’évolution fréquente vers l’infection avec un risque sous-jacent d'amputation. La difficulté de prise en charge repose sur la présence d’un biofilm polymicrobien au niveau du lit de la plaie. L'objectif de cette thèse était de promouvoir l'amélioration de l'état de l'art relatif à la compréhension de ce phénomène physiopathologique important mais également de développer de nouvelles approches concernant le traitement thérapeutique des plaies chroniques pour réduire les risques d'infections morbides. Ce travail de doctorat a permis : i) de parfaire les outils diagnostic de par la création d’un nouveau modèle in vitro mimant l’environnement dans lequel les bactéries évoluent au niveau des plaies chroniques ; ii) d’étudier le comportement (virulence, fitness, génomique, morphologie, formation de biofilm précoce, expression de gènes clés pour la formation de biofilm) de souches de références et de souches cliniques de Staphylococcus aureus (SA) et Pseudomonas aeruginosa (PA). Ces souches, isolées au sein d’une même plaie, étaient exposées de façon courte ou prolongée, à un milieu classique ou mimant l’environnement chronique d’une plaie. Les résultats ont montré qu’une exposition dans des conditions environnementales stressantes réduisaient la virulence et le fitness des souches au profit d’un comportement plus tourné vers la formation de biofilm ; iii) de démontrer l’impact de la présence de plusieurs bactéries au sein d’un environnement. Ce travail a particulièrement mis en évidence la baisse de pathogénicité et l’évolution de l’expression de certains gènes impliqués dans le biofilm de souche de SA en présence de PA. Enfin iv) la mise au point d’une technique de formation de biofilm dynamique et d’imagerie confocale permettant la visualisation d’un biofilm polymicrobien en trois dimensions et l’évaluation de molécules antimicrobiennes sur chaque espèce bactérienne composant le biofilm polymicrobien après un débridement mécanique automatisé. La combinaison de notre nouveau milieu « plaie chronique » et du système de formation en flux et visualisation en 3D du biofilm polymicrobien représente des outils puissants pour améliorer la compréhension des interactions régissant les biofilms mais aussi pour évaluer l’efficacité de molécules candidates dans la prise en charge des plaies chroniques.Mots clés : Plaies chroniques, infections, biofilms, coopération bactérienn

Models of diabetic foot ulcers : an in vitro and in vivo approach to uderstand the formation of biofilms of pathogenic bacteria alone or in combination

Les plaies chroniques sont un réel problème de santé publique. L’une des principales complications est l’évolution fréquente vers l’infection avec un risque sous-jacent d'amputation. La difficulté de prise en charge repose sur la présence d’un biofilm polymicrobien au niveau du lit de la plaie. L'objectif de cette thèse était de promouvoir l'amélioration de l'état de l'art relatif à la compréhension de ce phénomène physiopathologique important mais également de développer de nouvelles approches concernant le traitement thérapeutique des plaies chroniques pour réduire les risques d'infections morbides. Ce travail de doctorat a permis : i) de parfaire les outils diagnostic de par la création d’un nouveau modèle in vitro mimant l’environnement dans lequel les bactéries évoluent au niveau des plaies chroniques ; ii) d’étudier le comportement (virulence, fitness, génomique, morphologie, formation de biofilm précoce, expression de gènes clés pour la formation de biofilm) de souches de références et de souches cliniques de Staphylococcus aureus (SA) et Pseudomonas aeruginosa (PA). Ces souches, isolées au sein d’une même plaie, étaient exposées de façon courte ou prolongée, à un milieu classique ou mimant l’environnement chronique d’une plaie. Les résultats ont montré qu’une exposition dans des conditions environnementales stressantes réduisaient la virulence et le fitness des souches au profit d’un comportement plus tourné vers la formation de biofilm ; iii) de démontrer l’impact de la présence de plusieurs bactéries au sein d’un environnement. Ce travail a particulièrement mis en évidence la baisse de pathogénicité et l’évolution de l’expression de certains gènes impliqués dans le biofilm de souche de SA en présence de PA. Enfin iv) la mise au point d’une technique de formation de biofilm dynamique et d’imagerie confocale permettant la visualisation d’un biofilm polymicrobien en trois dimensions et l’évaluation de molécules antimicrobiennes sur chaque espèce bactérienne composant le biofilm polymicrobien après un débridement mécanique automatisé. La combinaison de notre nouveau milieu « plaie chronique » et du système de formation en flux et visualisation en 3D du biofilm polymicrobien représente des outils puissants pour améliorer la compréhension des interactions régissant les biofilms mais aussi pour évaluer l’efficacité de molécules candidates dans la prise en charge des plaies chroniques.Mots clés : Plaies chroniques, infections, biofilms, coopération bactérienneChronic wounds are a real public health problem. One of the main complications is the recurrent progress towards infection with an underlying risk of amputation. The difficulty of management lies in the presence of a polymicrobial biofilm at the wound bed. The objective of this thesis was to promote the development of the state-of-the-art of this important pathophysiology phenomenon of biofilm but also to promote new approaches for therapeutic treatment of chronic wounds to reduce risk of morbid infections. This doctoral work enabled: i) to perfect diagnostic tools by creating a new in vitro model mimicking the environment in which bacteria evolve at the level of chronic wounds; ii) to study behavior (virulence, fitness, genomics, morphology, early biofilm formation, expression of key genes for biofilm formation) of reference strains and clinical strains of Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) isolated on a patient wound, based on short or long-term exposure to a conventional or a chronic wound like environment. The results showed that exposure under stressful environmental conditions reduced the virulence and fitness of the strains in favor of a biofilm-oriented behavior; iii) demonstrate the impact of multiple bacteria in an environment. This work has particularly highlighted the decrease of the pathogenicity and the evolution of the expression of some genes involved in the biofilm of SA strain when in contact with PA strain. iv) the development of a dynamic biofilm formation and confocal imaging technique allowing the visualization of a three-dimensional polymicrobial biofilm and the evaluation of antimicrobial molecules on each bacterial species composing the biofilm polymicrobial after an automatized mechanical debridement. The combination of our new chronic wound-like medium and the microfluidic biofilm formation and 3D visualization system of the polymicrobial biofilm represents powerful tools to improve the understanding of the interactions governing biofilms but also to evaluate the effectiveness of candidate molecules in chronic wounds management.Key words: Chronic wounds, infections, biofilms, bacterial cooperatio

Polymicrobial Biofilm Organization of Staphylococcus aureus and Pseudomonas aeruginosa in a Chronic Wound Environment

Biofilm on the skin surface of chronic wounds is an important step that involves difficulties in wound healing. The polymicrobial nature inside this pathogenic biofilm is key to understanding the chronicity of the lesion. Few in vitro models have been developed to study bacterial interactions inside this chronic wound. We evaluated the biofilm formation and the evolution of bacteria released from this biofilm on the two main bacteria isolated in this condition, Staphylococcus aureus and Pseudomonas aeruginosa, using a dynamic system (BioFlux™ 200) and a chronic wound-like medium (CWM) that mimics the chronic wound environment. We observed that all species constituted a faster biofilm in the CWM compared to a traditional culture medium (p < 0.01). The percentages of biofilm formation were significantly higher in the mixed biofilm compared to those determined for the bacterial species alone (p < 0.01). Biofilm organization was a non-random structure where S. aureus aggregates were located close to the wound surface, whereas P. aeruginosa was located deeper in the wound bed. Planktonic biofilm-detached bacteria showed decreased growth, overexpression of genes encoding biofilm formation, and an increase in the mature biofilm biomass formed. Our data confirmed the impact of the chronic wound environment on biofilm formation and on bacterial lifecycle inside the biofilm

Effect of Antibiotic Exposure on <i>Staphylococcus epidermidis</i> Responsible for Catheter-Related Bacteremia

Coagulase-negative staphylococci (CoNS) and especially Staphylococcus epidermidis are responsible for health care infections, notably in the presence of foreign material (e.g., venous or central-line catheters). Catheter-related bacteremia (CRB) increases health care costs and mortality. The aim of our study was to evaluate the impact of 15 days of antibiotic exposure (ceftobiprole, daptomycin, linezolid and vancomycin) at sub-inhibitory concentration on the resistance, fitness and genome evolution of 36 clinical strains of S. epidermidis responsible for CRB. Resistance was evaluated by antibiogram, the ability to adapt metabolism by the Biofilm Ring test® and the in vivo nematode virulence model. The impact of antibiotic exposure was determined by whole-genome sequencing (WGS) and biofilm formation experiments. We observed that S. epidermidis strains presented a wide variety of virulence potential and biofilm formation. After antibiotic exposure, S. epidermidis strains adapted their fitness with an increase in biofilm formation. Antibiotic exposure also affected genes involved in resistance and was responsible for cross-resistance between vancomycin, daptomycin and ceftobiprole. Our data confirmed that antibiotic exposure modified bacterial pathogenicity and the emergence of resistant bacteria

Bacterial Interactions in the Context of Chronic Wound Biofilm: A Review

International audienceChronic wounds, defined by their resistance to care after four weeks, are a major concern, affecting millions of patients every year. They can be divided into three types of lesions: diabetic foot ulcers (DFU), pressure ulcers (PU), and venous/arterial ulcers. Once established, the classical treatment for chronic wounds includes tissue debridement at regular intervals to decrease biofilm mass constituted by microorganisms physiologically colonizing the wound. This particular niche hosts a dynamic bacterial population constituting the bed of interaction between the various microorganisms. The temporal reshuffle of biofilm relies on an organized architecture. Microbial community turnover is mainly associated with debridement (allowing transitioning from one major representant to another), but also with microbial competition and/or collaboration within wounds. This complex network of species and interactions has the potential, through diversity in antagonist and/or synergistic crosstalk, to accelerate, delay, or worsen wound healing. Understanding these interactions between microorganisms encountered in this clinical situation is essential to improve the management of chronic wounds

Biofilms in Diabetic Foot Ulcers: Significance and Clinical Relevance

International audienceFoot infections are the main disabling complication in patients with diabetes mellitus. These infections can lead to lower-limb amputation, increasing mortality and decreasing the quality of life. Biofilm formation is an important pathophysiology step in diabetic foot ulcers (DFU)—it plays a main role in the disease progression and chronicity of the lesion, the development of antibiotic resistance, and makes wound healing difficult to treat. The main problem is the difficulty in distinguishing between infection and colonization in DFU. The bacteria present in DFU are organized into functionally equivalent pathogroups that allow for close interactions between the bacteria within the biofilm. Consequently, some bacterial species that alone would be considered non-pathogenic, or incapable of maintaining a chronic infection, could co-aggregate symbiotically in a pathogenic biofilm and act synergistically to cause a chronic infection. In this review, we discuss current knowledge on biofilm formation, its presence in DFU, how the diabetic environment affects biofilm formation and its regulation, and the clinical implications

Antibiofilm Properties of Antiseptic Agents Used on Pseudomonas aeruginosa Isolated from Diabetic Foot Ulcers

In diabetic foot ulcers (DFUs), biofilm formation is a major challenge that promotes wound chronicity and delays healing. Antiseptics have been proposed to combat biofilms in the management of DFUs. However, there is limited evidence on the activity of these agents against biofilms, and there are questions as to which agents have the best efficiency. Here, we evaluated the antibiofilm activity of sodium hypochlorite, polyvinylpyrrolidoneIodine (PVPI), polyhexamethylenebiguanide (PHMB) and octenidine against Pseudomonas aeruginosa strains using static and dynamic systems in a chronic-wound-like medium (CWM) that mimics the chronic wound environment. Using Antibiofilmogram&reg;, a technology assessing the ability of antiseptics to reduce the initial phase of biofilm formation, we observed the significant activity of antiseptics against biofilm formation by P. aeruginosa (at 1:40 to 1:8 dilutions). Moreover, 1:100 to 1:3 dilutions of the different antiseptics reduced mature biofilms formed after 72 h by 10-log, although higher concentrations were needed in CWM (1:40 to 1:2). Finally, in the BioFlux200TM model, after biofilm debridement, sodium hypochlorite and PHMB were the most effective antiseptics. In conclusion, our study showed that among the four antiseptics tested, sodium hypochlorite demonstrated the best antibiofilm activity against P. aeruginosa biofilms and represents an alternative in the management of DFUs

Biofilm Formation in Methicillin-Resistant Staphylococcus aureus Isolated in Cystic Fibrosis Patients Is Strain-Dependent and Differentially Influenced by Antibiotics

International audienceCystic fibrosis (CF) is a genetic disease with lung abnormalities making patients particularly predisposed to pulmonary infections. Staphylococcus aureus is the most frequently identified pathogen, and multidrug-resistant strains (MRSA, methicillin-resistant S. aureus ) have been associated with more severe lung dysfunction leading to eradication recommendations. Diverse bacterial traits and adaptive skills, including biofilm formation, may, however, make antimicrobial therapy challenging. In this context, we compared the ability of a collection of genotyped MRSA isolates from CF patients to form biofilm with and without antibiotics (ceftaroline, ceftobiprole, linezolid, trimethoprim, and rifampicin). Our study used standardized approaches not previously applied to CF MRSA, the BioFilm Ring test® (BRT®), the Antibiofilmogram®, and the BioFlux™ 200 system which were adapted for use with the artificial sputum medium (ASM) mimicking conditions more relevant to the CF lung. We included 63 strains of 10 multilocus sequence types (STs) isolated from 35 CF patients, 16 of whom had chronic colonization. The BRT® showed that 27% of the strains isolated in 37% of the patients were strong biofilm producers. The Antibiofilmogram® performed on these strains showed that broad-spectrum cephalosporins had the lowest minimum biofilm inhibitory concentrations (bMIC) on a majority of strains. A focus on four chronically colonized patients with inclusion of successively isolated strains showed that ceftaroline, ceftobiprole, and/or linezolid bMICs may remain below the resistance thresholds over time. Studying the dynamics of biofilm formation by strains isolated 3years apart in one of these patients using BioFlux™ 200 showed that inhibition of biofilm formation was observed for up to 36h of exposure to bMIC and ceftaroline and ceftobiprole had a significantly greater effect than linezolid. This study has brought new insights into the behavior of CF MRSA which has been little studied for its ability to form biofilm. Biofilm formation is a common characteristic of prevalent MRSA clones in CF. Early biofilm formation was strain-dependent, even within a sample, and not only observed during chronic colonization. Ceftaroline and ceftobiprole showed a remarkable activity with a long-lasting inhibitory effect on biofilm formation and a conserved activity on certain strains adapted to the CF lung environment after years of colonization

Biofilm formation in MRSA isolated in Cystic Fibrosis patients is strain-dependent and differentially influenced by antibiotics

International audienc