Thesis

Développement d'une thérapie combinatoire à base d’agents anti-biofilms et de particules d’acide poly-lactique pour la délivrance d'antibiotiques contre des biofilms bactériens

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Abstract

Après l’âge d’or de la découverte des antibiotiques, les infections bactériennes représentent toujours un challenge considérable pour la santé publique mondiale. Le mode de croissance en biofilms est le principal responsable des infections chroniques pour lesquelles les thérapies antibiotiques sont en échec clinique, pointant la nécessité d’élaborer de nouvelles stratégies thérapeutiques. L’objectif de cette thèse a porté sur le développement d’une thérapie combinatoire à base d’agents anti-biofilms et de particules biodégradables d’acide poly-lactique pour la délivrance d’antibiotiques au cœur des biofilms bactériens. Une stratégie de formulation d’antibiotiques a été développée, aboutissant à des particules stables et fortement chargées en rifampicine. La charge de surface des particules a été inversée vers des valeurs positives par adsorption d’un peptide cationique, la poly-lysine. L’intérêt d’une telle formulation a été évalué in vitro sur des biofilms de Staphylococcus aureus. Capables d’interagir via des liaisons électrostatiques avec les biofilms et les bactéries, les particules cationiques sont retenues en plus grande quantité dans les biofilms que les particules anioniques qui peuvent être éliminées par lavage. Les particules permettant une délivrance progressive de l’antibiotique, l’inversion de leur charge de surface qui renforce ces interactions permet de réduire les quantités d’antibiotique nécessaires pour maintenir une efficacité anti-biofilm. La combinaison de ce traitement avec la DNase, une enzyme capable de dégrader la matrice de biofilms, permet de potentialiser la dégradation des biofilms sans toutefois augmenter l’activité bactéricide de l’antibiotique. Des évaluations in vivo de l’efficacité de cette stratégie thérapeutique permettraient de confirmer son intérêt pour le traitement des biofilms bactériens.

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Methicillin-resistant Staphylococcus aureus (MRSA) presents one of the most serious health concerns worldwide. The WHO labeled it as a “high-priority” pathogen in 2017, also citing the more recently emerged vancomycin-intermediate and -resistant strains. With the spread of antibiotic resistance due in large part to the selective pressure exerted by conventional antibiotics, the use of antivirulence strategies has been recurrently proposed as a promising therapeutic approach. In MRSA, virulence is chiefly controlled by quorum sensing (QS); inhibitors of QS are called quorum quenchers (QQ). In S. aureus , the majority of QS components are coded for by the accessory gene regulator (Agr) system. Although much work has been done to develop QQs against MRSA, only a few studies have progressed to in vivo models. Those studies include both prophylactic and curative models of infection as well as combination treatments with antibiotic. For most, high efficacy is seen at attenuating MRSA virulence and pathogenicity, with some studies showing effects such as synergy with antibiotics and antibiotic resensitization. This minireview aims to summarize and derive conclusions from the literature on the in vivo efficacy of QQ agents in MRSA infection models. In vitro data are also summarized to provide sufficient background on the hits discussed. On the whole, the reported in vivo effects of the reviewed QQs against MRSA represent positive progress at this early stage in drug development. Follow-up studies that thoroughly examine in vitro and in vivo activity are needed to propel the field forward and set the stage for lead optimization.
Thesis
L'objectif de cette thèse était de mettre en oeuvre le développement d'une thérapie des plaies cutanées profondes basée sur l'utilisation de nanoparticules (NP) biodégradables de poly(acide lactique) (NP-PLA) vectrices de médiateurs de la cicatrisation. Le but était d'accélérer la cicatrisation cutanée et de favoriser la reconstruction d'un derme fonctionnel. La méthode a été de (i) réduire la réaction inflammatoire pour en contenir les effets délétères et (ii) stimuler la réépithélialisation pour accélérer la cicatrisation et réduire le risque infectieux. Les moyens ont été l'utilisation d'un antioxydant, la vitamine E (VE) et d'un facteur de croissance des fibroblastes (le FGF2) vectorisés par des nanoparticules biocompatibles et biodégradables de poly(acide lactique) (PLA). Nos NP-PLA contiennent l'antioxydant (VE) dans leur coeur hydrophobe, et portent le facteur de croissance fibroblastique (FGF2) à leur surface. Ces formulations ont été (i) caractérisées par des méthodes physico-chimiques et (ii) testées par des méthodes in vitro pour évaluer leurs effets potentiels, en tant que système de délivrance de VE et de FGF2, sur la cicatrisation des plaies. Des modèles expérimentaux in vivo ont été développés et caractérisés pour mettre en évidence l'efficacité des NP-PLA fonctionnalisées pour la cicatrisation cutanée et la reconstruction dermique fonctionnelle. Nos résultats montrent que l'activité antioxydante de la VE n'est pas perturbée par l'encapsulation dans des NP-PLA et qu'elle est légèrement supérieure à celle de la VE libre dans un système in vitro. De même, l'activité biologique du FGF2 sur la prolifération et la migration des fibroblastes dans un système in vitro n'est pas altérée par son adsorption sur des NP-PLA. Aucune de ces deux NP-PLA fonctionnalisées n'a de cytotoxicité avérée in vitro. Deux modèles expérimentaux de plaies cutanées profondes ont été développés sur souris sans poils SKH1 saines : (i) Un modèle robuste de brûlure cutanée thermique de 3ème degré qui se caractérise par une inflammation massive de la plaie et par un stade de granulation tardif après 16 jours de cicatrisation. (ii) Un modèle de plaie d'excision cutanée a également été utilisé. Un modèle de cicatrisation retardée a été obtenu par induction chimique d'un diabète de type I stable avant réalisation des plaies d'excision ou de brûlure. Ces modèles de plaies cutanées ont été caractérisés tout au long du processus de cicatrisation par des études (i) macroscopiques de cinétique de fermeture des plaies, (ii) histologiques d'inflammation, de nécrose et de réépithélialisation, (iii) physiologiques de perfusion sanguine cutanée. L'expression de 84 gènes impliqués dans le processus de cicatrisation a été étudiée sur le tissu cicatriciel 14 jours après formation de la plaie. Pour conclure, nos résultats mettent en évidence le potentiel de vectorisation de molécules thérapeutiques des NP de PLA pour le développement de futures stratégies de délivrance ciblée de VE et de FGF2 dans les plaies cutanées profondes. Les modèles expérimentaux in vivo développés et caractérisés, ouvrent la voie aux études précliniques d'efficacité des NP-PLA fonctionnalisées dans le processus de cicatrisation des plaies profondes
Article
An urgent demand exists for the development of effective carrier systems that systematically enhance the cellular uptake and localization of antibiotic drugs for the treatment of intracellular pathogens. Commercially available antibiotics suffer from poor cellular penetration, restricting their efficacy against pathogens hosted and protected within phagocytic cells. In this study, the potency of the antibiotic, rifampicin, against intracellular small colony variants (SCV) of Staphylococcus aureus was improved through encapsulation within a strategically engineered cell-penetrant delivery system, composed of lipid nanoparticles encapsulated within a poly(lactic-co¬-glycolic) acid (PLGA) nanoparticle matrix. PLGA-lipid hybrid (PLH) microparticles were synthesized through the process of spray drying, whereby rifampicin was loaded within both the polymer and lipid phase, to create a nanoparticle-in-microparticle system capable of efficient redispersion in aqueous biorelevant media and with programmable release kinetics. The ability for PLH particles to disintegrate into nanoscale agglomerates of the precursor nanoparticles was shown to be instrumental in optimizing rifampicin uptake in RAW264.7 macrophages, with a 7.2- and 1.6- fold increase in cellular uptake, when compared to the pure drug and PLGA microparticles (of an equivalent initial particle size), respectively. The enhanced phagocytosis and extended drug release mechanism (under the acidic macrophage environment) associated with PLH particles induced a 2.5-log reduction in CFU, compared to initial colonies at 2.50 µg/mL rifampicin dose. Thus, the ability for PLH particles to reduce the intracellular viability of S. aureus, without demonstrating significant cellular toxicity, satisfies the requirements necessary for the safe and efficacious delivery of antibiotics to macrophages for the treatment of intracellular infections.
Article
Deoxyribose nuclease I recombinant (DNase I) was successfully loaded and delivered by silver-doped mesoporous silica nanoparticles (MSN-Ag) for biofilm treatment. This nanofomulation showed enhanced antibacterial effects to both gram-negative and...
Article
Background: Shortcomings of inhaled antibiotic treatments for Pseudomonas aeruginosa infection in patients with cystic fibrosis (CF) include poor drug penetration, inactivation by sputum, poor efficiency due to protective biofilm, and short residence in the lung. Methods: Eligible patients with forced expiratory volume in 1 s (FEV1) ≥25% of predicted value at screening and CF with chronic P. aeruginosa infection were randomly assigned to receive 3 treatment cycles (28 days on, 28 days off) of amikacin liposome inhalation suspension (ALIS, 590 mg QD) or tobramycin inhalation solution (TIS, 300 mg BID). The primary endpoint was noninferiority of ALIS vs TIS in change from baseline to day 168 in FEV1 (per-protocol population). Secondary endpoints included change in respiratory symptoms by Cystic Fibrosis Questionnaire-Revised (CFQ-R). Results: The study was conducted February 2012 to September 2013. ALIS was noninferior to TIS (95% CI, -4.95 to 2.34) for relative change in FEV1 (L) from baseline. The mean increases in CFQ-R score from baseline on the Respiratory Symptoms scale suggested clinically meaningful improvement in both arms at the end of treatment in cycle 1 and in the ALIS arm at the end of treatment in cycles 2 and 3; however, the changes were not statistically significant between the 2 treatment arms. Treatment-emergent adverse events (TEAEs) were reported in most patients (ALIS, 84.5%; TIS, 78.8%). Serious TEAEs occurred in 17.6% and 19.9% of patients, respectively; most were hospitalisations for infective pulmonary exacerbation of CF. Conclusions: Cyclical dosing of once-daily ALIS was noninferior to cyclical twice-daily TIS in improving lung function. ClinicalTrials.gov Identifier: NCT01315678.
Article
The negative consequences of biofilms are widely reported. A defining feature of biofilms is the extracellular matrix, a complex mixture of biomacromolecules, termed EPS, which contributes to reduced antimicrobial susceptibility. EPS targeting is a promising, but underexploited, approach to biofilm control allowing disruption of the matrix and thereby increasing the susceptibility to antimicrobials. Nanoparticles (NPs) can play a very important role as 'carriers' of EPS matrix disruptors, and several approaches have recently been proposed. In this review, we discuss the application of nanoparticles as antibiofilm technologies with a special emphasis on the role of the EPS matrix in the physicochemical regulation of the nanoparticle–biofilm interaction. We highlight the use of nanoparticles as a platform for a new generation of antibiofilm approaches.
Article
Nowadays, the bacterial drug resistance leads to serious healthy problem worldwide due to the long-term use and the abuse of traditional antibiotics result in drug resistance of bacteria. Finding a new antibiotic is becoming more and more difficult. Antimicrobial peptides (AMPs) are the host defense peptides with most of them being the cationic (positively charged) and amphiphilic (hydrophilic and hydrophobic) α-helical peptide molecules. The membrane permeability is mostly recognized as the well-accepted mechanism to describe the action of cationic AMPs. These cationic AMPs can bind and interact with the negatively charged bacterial cell membranes, leading to the change of the electrochemical potential on bacterial cell membranes, inducing cell membrane damage and the permeation of larger molecules such as proteins, destroying cell morphology and membranes and eventually resulting in cell death. These AMPs have been demonstrated to have their own advantages over the traditional antibiotics with a broad-spectrum of antimicrobial activities including anti-bacteria, anti-fungi, anti-viruses, and anti-cancers, and even overcome bacterial drug-resistance. The natural AMPs exist in a variety of organisms and are not stable with a short half-life, more or less toxic side effects, and particularly may have severe hemolytic activity. To open the clinical applications, it is necessary and important to develop the synthetic and long-lasting AMP analogs that overcome the disadvantages of their natural peptides and the potential problems for the drug candidates.
Article
Designing potent and safe-of-use therapies against cancers and infections remains challenging despite the emergence of novel molecule classes like checkpoint inhibitors or Toll-Like-Receptor ligands. The latest therapeutic perspectives under development for immune modulator administration exploits vectorization, and biodegradable delivery systems are one of the most promising vehicles. Nanoparticles based on Poly (D,L) Lactic Acid (PLA) as polymer for formulation are widely investigated due to its bioresorbable, biocompatible and low immunogen properties. We propose a PLA-based nanoparticle delivery system to vectorize Pam3CSK4, a lipopeptide TLR1/2 ligand and a potent activator of the proinflammatory transcription factor NF-κB that shows a self-assembling behavior from 30 µg/mL onwards. We demonstrate successful encapsulation of Pam3CSK4 in PLA nanoparticles by nanoprecipitation in a 40-180 µg/mL concentration range, with 99% of entrapment efficiency. By molecular modelling, we characterize drug/carrier interactions and conclude that Pam3CSK4 forms clusters onto the nanoparticle and is not encapsulated into the hydrophobic core. In silico predictions provide nanoprecipitation optimization and the mechanistic understanding of the particle dynamics. The loaded-Pam3CSK4 maintains bioactivity on TLR2, confirmed by in vitro experiments using reporter cell line HEK-Blue hTLR2. Our presented data and results are convincing evidence that Pam3CSK4-loaded in PLA nanoparticles represent a promising immune modulating system.
Article
Biofilm resistance is one of the severe complication associated with chronic wound infections which impose the extreme microbial tolerance against antibiotic therapy. Interestingly, DNase-I has empirically proved efficacy to improve the antibiotics susceptibility against biofilm-associated infections. DNase-I hydrolyzes the extracellular DNA, a key component of the biofilm responsible for the cell adhesion and strength. Moreover, Silver sulfadiazine, a frontline therapy in burn wound infections, exhibit delayed wound healing due to fibroblast toxicity. In this study, solid lipid nanoparticles of silver sulfadiazine (SSD-SLNs) laden chitosan gel supplemented with DNase-I has been developed to reduce the fibroblast cytotoxicity and overcome the biofilm imposed resistance. The extensive optimization by using Box-Behnken Design (BBD) resulted in the formation of SSD-SLNs with smooth surface as confirmed by scanning electron microscopy and controlled release (83%) for up to 24h. The compatibility between the SSD and other formulation excipients was confirmed by FTIR, differential scanning calorimetry and powder X-ray diffraction studies. Developed SSD-SLNs demonstrated improved cell viability (90.3±3.8%) as compared to SSD alone (76.9±4.2%) and combination of SSD-SLNs with DNase-I, inhibited around 96.8% of biofilm of Pseudomonas aeruginosa as compared to SSD with DNase-I (82.9%). In line with our hypothesis, SSD-SLNs were found less toxic (cell viability 90.3±3.8% at 100 µg/mL) in comparison with SSD (Cell viability 76.9±4.2 %) against human dermal fibroblasts cell line. Eventually, the results of in-vivo wound healing study showed complete wound healing after 21 days’ treatment with SSD-SLNs with DNase-I, whereas, marketed formulation, SSD and SSD-LSNs showed incomplete healing after 21 days. Data in hand suggest, SSD-SLNs with DNase-I as an effective treatment strategy against the biofilm-associated wound infections and to accelerate the wound healing.
Article
Bacterial biofilms pose a major threat to public health because they are resistant to most current therapeutics. Conventional antibiotics exhibit limited penetration and weakened activity in the acidic microenvironment of a biofilm. Here, the development of biofilm-responsive nanoantibiotics (rAgNAs) composed of self-assembled silver nanoclusters and pH-sensitive charge reversal ligands, whose bactericidal activity can be selectively boosted in the biofilm microenvironment, is reported. Under neutral physiological conditions, the bactericidal activity of rAgNAs is self-quenched because the toxic silver ions’ release is largely inhibited; however, upon entry into the acidic biofilm microenvironment, the rAgNAs not only exhibit charge reversal to facilitate local accumulation and retention but also disassemble into small silver nanoclusters, thus enabling deep penetration and accelerated silver ions release for dramatically amplified bactericidal activity. The superior antibiofilm activity of rAgNAs is demonstrated both in vitro and in vivo, and the mortality rate of mice with multi-drug-resistant biofilm-induced severe pyomyositis can be significantly reduced by rAgNAs treatment, indicating the immense potential of rAgNAs as highly efficient nanoscale antibacterial agents to combat resistant bacterial biofilm-associated infections.
Article
Most bacteria use a communication system known as quorum sensing which relies on the secretion and perception of small molecules called autoinducers enabling bacteria to adapt their behavior according to the population size and synchronize the expression of genes involved in virulence, antimicrobial resistance and biofilm formation. Methods have emerged to inhibit bacterial communication and limit their noxious traits. Chemical inhibitors, sequestering antibodies and degrading enzymes have been developed and proved efficient to decrease bacterial virulence both in vitro and in vivo. This strategy, named quorum quenching, also showed synergistic effects with traditional antibacterial treatments by increasing bacterial susceptibility to antibiotics. Thereby quorum quenching constitutes an interesting therapeutic strategy to fight against bacterial infections and limit the consequences of antibiotic resistance. © 2019 médecine/sciences – Inserm.
Article
Background In patients with non-cystic fibrosis bronchiectasis, lung infection with Pseudomonas aeruginosa is associated with frequent pulmonary exacerbations and admission to hospital for treatment, reduced quality of life, and increased mortality. Although inhaled antibiotics are conditionally recommended for long-term management of non-cystic fibrosis bronchiectasis with frequent exacerbations, there is no approved therapy. We investigated the safety and efficacy of inhaled liposomal ciprofloxacin (ARD-3150) in two phase 3 trials. Methods ORBIT-3 and ORBIT-4 were international, randomised, double-blind, placebo-controlled, phase 3 trials run concurrently in similar geographical regions. Eligible patients had non-cystic fibrosis bronchiectasis, had had at least two pulmonary exacerbations treated with antibiotics in the previous 12 months, and had a history of chronic P aeruginosa lung infection. Patients were randomly assigned (2:1) to receive either ARD-3150 or placebo. ARD-3150 (3 mL liposome encapsulated ciprofloxacin 135 mg and 3 mL free ciprofloxacin 54 mg) or 6 mL placebo (3 mL dilute empty liposomes mixed with 3 mL of saline) was self-administered once daily for six 56-day treatment cycles, for 48 weeks. The primary endpoint was time to first pulmonary exacerbation from the date of randomisation to week 48. We did primary and secondary efficacy, safety, and microbiology analyses on the full analysis population, which comprised all randomised patients who received at least one dose of study drug. ORBIT-3 and ORBIT-4 are registered with ClinicalTrials.gov, numbers NCT01515007 and NCT02104245, respectively. Findings Between March 31, 2014, and Aug 19, 2015, we screened 514 patients in ORBIT-3 and 533 patients in ORBIT-4. The full analysis populations consisted of 278 patients in ORBIT-3 (183 patients received at least one dose of ARD-3150 and 95 received placebo) and 304 patients in ORBIT-4 (206 patients received at least one dose of ARD-3150 and 98 received placebo). In ORBIT-4, the median time to first pulmonary exacerbation was 230 days in the ARD-3150 group compared with 158 days in the placebo group, a statistically significant difference of 72 days (hazard ratio [HR] 0·72 [95% CI 0·53–0·97], p=0·032). In ORBIT-3, the median time to first pulmonary exacerbation was 214 days in the ARD-3150 group and 136 days in the placebo group, a non-statistically significant difference of 78 days (HR 0·99 [95% CI 0·71–1·38], p=0·97). In a pooled analysis of data from both ORBIT-3 and ORBIT-4, the median time to first pulmonary exacerbation was 222 days in the ARD-3150 group and 157 days in the placebo group, a non-statistically significant difference of 65 days (0·82 [0·65–1·02], p=0·074). The numbers of adverse events and serious adverse events were similar in both groups in ORBIT-3 and ORBIT-4. Interpretation In patients with non-cystic fibrosis bronchiectasis and chronic P aeruginosa lung infection requiring antibiotic therapy in the preceding year, ARD-3150 led to a significantly longer median time to first pulmonary exacerbation compared with placebo in ORBIT-4, but not in ORBIT-3 or the pooled analysis. Inconsistency between the trials suggests further research is needed into the heterogeneity of non-cystic fibrosis bronchiectasis and optimal outcome measures for inhaled antibiotics. Funding Aradigm Corporation.
Article
Bacterial-infections are mostly due to bacteria in an adhering, biofilm-mode of growth and not due to planktonically growing, suspended-bacteria. Biofilm-bacteria are much more recalcitrant to conventional antimicrobials than planktonic-bacteria due to (1) emergence of new properties of biofilm-bacteria that cannot be predicted on the basis of planktonic properties, (2) low penetration and accumulation of antimicrobials in a biofilm, (3) disabling of antimicrobials due to acidic and anaerobic conditions prevailing in a biofilm, and (4) enzymatic modification or inactivation of antimicrobials by biofilm inhabitants. In recent years, new nanotechnology-based antimicrobials have been designed to kill planktonic, antibiotic-resistant bacteria, but additional requirements rather than the mere killing of suspended bacteria must be met to combat biofilm-infections. The requirements and merits of nanotechnology-based antimicrobials for the control of biofilm-infection form the focus of this Tutorial Review.
Article
Biofilms formed by pathogenic bacteria have been one of the most important reasons for multidrug resistance. One of the major limitations in the biofilm treatment is the existence of intensive matrices which greatly block the diffusion of antimicrobial agents. In the current study, we design poly(aspartamide) derived micelles self-assembled from cationic copolymers with azithromycin conjugated and pH-sensitive copolymers, followed by loading cis-aconityl-D-tyrosine (CA-Tyr) via electrostatic interactions. In response to the acidic microenvironment of biofilm matrix, the hydrophilic transition of pH-sensitive copolymers and the removal of CA-Tyr lead to a sharp decrease in the micelle size from 107 to 54 nm and a rapid shifting of zeta potentials from -11.7 to +26.4 mV, which facilitates the penetration of micelles into biofilms. The acid-labile release of D-tyrosine disintegrates the biofilm matrix, and the lipase-triggered release of azithromycin eradicates bacteria in biofilms. The in vitro test is performed on pre-established P. aeruginosa biofilms in microwells, while biofilms grown on catheters are surgically implanted in rats for in vivo evaluation. It demonstrates the capabilities of size/surface charge-adaptive micelles in the intensive infiltration into biofilm matrix and spatiotemporal release of biofilm dispersion and antibacterial agents for the comprehensive treatment of biofilm-relevant infections.
Article
Introduction: Quorum sensing (QS) is a cell density-dependent phenomenon in which specific pathways are activated after autoinducers (AIs) outside the microorganism reach a threshold concentration. QS creates a positive feedback loop that induces a cascade of gene expression and causes biofilm formation, virulence and sporulation. QS signals are diverse, acyl-homoserine lactone (AHL), AI peptide (AIP) and AI-2 are three major categories of QS signals. QS inhibitors (QSIs) can disrupt or prevent the formation of biofilm and reduce virulence while exerting less selective pressure on the bacteria, suggesting that QSIs are potential alternatives for antibiotics. Areas covered: This review summarized the pertinent patents on quorum sensing inhibition available from 2014 to 2018. The authors analyze these patents and provided an overview of them and their potential applications. Expert opinion: The main strategy for QS inhibition is to use the analogues of various QS signals to block downstream signal transducers. The inactivation of signal molecules or the stimulation of the immune response are also attractive strategies to inhibit QS. However, additional clinical trials are needed to assess their efficacy in mammals. In sum, QS inhibition can reduce the virulence of bacteria without affecting their growth or killing them and the reduced pressure may minimize the increasingly resistance.
Article
Background: Wound infections are the main cause of sepsis in patients with burns and increase burn-related morbidity and mortality. Bacteriophages, natural bacterial viruses, are being considered as an alternative therapy to treat infections caused by multidrug-resistant bacteria. We aimed to compare the efficacy and tolerability of a cocktail of lytic anti-Pseudomonas aeruginosa bacteriophages with standard of care for patients with burns. Methods: In this randomised phase 1/2 trial, patients with a confirmed burn wound infection were recruited from nine burn centres in hospitals in France and Belgium. Patients were eligible if they were aged 18 years or older and had a burn wound clinically infected with P aeruginosa. Eligible participants were randomly assigned (1:1) by use of an interactive web response system to a cocktail of 12 natural lytic anti-P aeruginosa bacteriophages (PP1131; 1 × 106 plaque-forming units [PFU] per mL) or standard of care (1% sulfadiazine silver emulsion cream), both given as a daily topical treatment for 7 days, with 14 days of follow-up. Masking of treatment from clinicians was not possible because of the appearance of the two treatments (standard of care a thick cream, PP1131 a clear liquid applied via a dressing), but assignments were masked from microbiologists who analysed the samples and patients (treatment applied while patients were under general anaesthetic). The primary endpoint was median time to sustained reduction in bacterial burden by at least two quadrants via a four-quadrant method, assessed by use of daily swabs in all participants with a microbiologically documented infection at day 0 who were given at least one sulfadiazine silver or phage dressing (modified intention-to-treat population). Safety was assessed in all participants who received at least one dressing according to protocol. Ancillary studies were done in the per-protocol population (all PP1131 participants who completed 7 days of treatment) to assess the reasons for success or failure of phage therapy. This trial is registered with the European Clinical Trials database, number 2014-000714-65, and ClinicalTrials.gov, number NCT02116010, and is now closed. Findings: Between July 22, 2015, and Jan 2, 2017, across two recruitment periods spanning 13 months, 27 patients were recruited and randomly assigned to receive phage therapy (n=13) or standard of care (n=14). One patient in the standard of care group was not exposed to treatment, giving a safety population of 26 patients (PP1131 n=13, standard of care n=13), and one patient in the PP1131 group did not have an infection at day 0, giving an efficacy population of 25 patients (PP1131 n=12, standard of care n=13). The trial was stopped on Jan 2, 2017, because of the insufficient efficacy of PP1131. The primary endpoint was reached in a median of 144 h (95% CI 48-not reached) in the PP1131 group versus a median of 47 h (23-122) in the standard of care group (hazard ratio 0·29, 95% CI 0·10-0·79; p=0·018). In the PP1131 group, six (50%) of 12 analysable participants had a maximal bacterial burden versus two (15%) of 13 in the standard of care group. PP1131 titre decreased after manufacturing and participants were given a lower concentration of phages than expected (1 × 102 PFU/mL per daily dose). In the PP1131 group, three (23%) of 13 analysable participants had adverse events versus seven (54%) of 13 in the standard of care group. One participant in each group died after follow-up and the deaths were determined to not be related to treatment. The ancillary study showed that the bacteria isolated from patients with failed PP1131 treatment were resistant to low phage doses. Interpretation: At very low concentrations, PP1131 decreased bacterial burden in burn wounds at a slower pace than standard of care. Further studies using increased phage concentrations and phagograms in a larger sample of participants are warranted. Funding: European Commission: Framework Programme 7.
Article
Bacterial biofilms play a key role during infections, which are associated with an increased morbidity and mortality. The classical antibiotic therapy cannot eradicate biofilm-related infections because biofilm bacteria display high drug resistance due to biofilm matrix. Thus, novel drug delivery to overcome biofilm resistance and eliminate biofilm-protected bacteria is needed to be developed. In this study, positively charged chitosan nanoparticles (CSNP) loaded with oxacillin (Oxa) and Deoxyribonuclease I (CSNP-DNase-Oxa) were fabricated. The antibiofilm activity was evaluated against Staphylococcus aureus biofilms. Biofilm architecture on silicone surfaces was investigated by scanning electron microscopy (SEM). Confocal laser scanning microscopy (CLSM) was used to examine live/dead organisms within biofilm. CSNP-DNase-Oxa exhibited higher antibiofilm activity than Oxa-loaded nanoparticles without DNase (CSNP-Oxa) and free Oxa (Oxa and Oxa + DNase) at each concentration in all in-vitro tests. CSNP-DNase-Oxa inhibited biofilm formation in-vitro and eradicated mature biofilm effectively. CSNP-DNase-Oxa could disrupt the biofilm formation through degradation of eDNA, reduced biofilm thickness and the amount of viable cells on silicone. Repeated treatment with CSNP-DNase-Oxa for two days resulted in 98.4% biofilm reduction. Moreover, CSNP-DNase-Oxa was not only able to affect the biofilm of a standard S. aureus strain, but also showed the highest eradication of biofilms of clinical isolates compared with control groups. These results suggest the potential applicability of NPs for the treatment of biofilm-related infections and provide a platform for designing novel drug delivery with more functions.
Thesis
Les biofilms constituent un mode de vie microbien extrêmement répandu, aussi bien en milieu naturel que dans les environnements anthropisés. Dans ce dernier cas, les structures morphologique et microbiologique du biofilm vont conditionner son impact sur le système, que cet impact lui soit bénéfique ou préjudiciable. L'objectif de cette thèse est d'approfondir notre connaissance des phénomènes de structuration du biofilm afin, à terme, d'optimiser les performances de procédé. Afin de représenter au mieux les conditions industrielles, des régimes d'écoulement turbulents et des consortia microbiens complexes ont été utilisés. Une première partie se focalise sur l'impact des forces de cisaillement sur l'adhésion microbienne. Les résultats démontrent un changement progressif de la flore bactérienne fixée et de sa distribution spatiale. Dans un second temps, le projet s'est intéressé aux étapes de développement du biofilm et ont permis d'identifier un effet mémoire du biofilm mature. Il s'agit d'une conservation des structures morphologique et microbiologique au cours du temps en dépit d'un changement de régime hydrodynamique. Enfin la dernière partie a consisté en la mise au point d'une méthode de quantification des prédateurs mobiles dans les biofilms. Ces prédateurs participent à la structuration du biofilm et leur quantification peut s'avérer utile dans le contexte de l'épuration des eaux.
Chapter
Pathogenic Pseudomonas species produce virulence elements and form biofilm through quorum sensing (QS) system. Clinical infections caused by Pseudomonas aeruginosa are becoming increasingly tough to treat on account of wide spread drug resistance. The drugs have lost their efficacy due to the virulence elements and biofilms, which are responsible for increased severity of the infection. The search for novel drugs has increased and novel modes of action against Gram-negative pathogenic bacteria are been much more of important. Plants secondary metabolites are widely used for treating the many bacterial diseases. Plant-derived anti-QS and anti-biofilm compounds that do not negatively affect the growth of the bacterial cells, but rather attenuates the QS controlled virulence factors, that might allow the host defense to act more effectively to washout the P. aeruginosa infection. In this review mainly focus on overview of pathogenicity, QS controlled virulence factors and biofilm formation of P. aeruginosa infection. This review describes a brief account of QS inhibitors and anti-biofilm compounds, which exhibit alternative medicine possible for treating drug resistant P. aeruginosa.
Article
The use of medical devices in modern medicine is constantly increasing. Despite the multiple precautionary strategies that are being employed in hospitals, which include increased hygiene and sterilization measures, bacterial infections on these devices still happen frequently. Staphylococci are among the major causes of medical device infection. This is mostly due to the strong capacity of those bacteria to form device‐associated biofilms, which provide resistance to chemical and physical treatments as well as attacks by the host's immune system. Biofilm development is a multi‐step process with specific factors participating in each step. It is tightly regulated to provide a balance between biofilm expansion and detachment. Detachment from a biofilm on a medical device can lead to severe systemic infection, such as bacteremia and sepsis. While our understanding of staphylococcal biofilm formation has increased significantly and staphylococcal biofilm formation on medical devices is among the best understood biofilm‐associated infections, the extensive effort put in pre‐clinical studies with the goal to find novel therapies against staphylococcal device‐associated infections has not yet resulted in efficient, applicable therapeutic options for that difficult‐to‐treat type of disease. This article is protected by copyright. All rights reserved.
Article
Pseudomonas aeruginosa exhibits the biofilm mode of growth and causes chronic as well as acute infections in humans. Several reports have shown that the treatments with sub-minimum inhibitory concentrations (sub-MICs) of antimicrobial agents influence biofilm formation by P. aeruginosa. The antibiotic ceftazidime (CAZ) is used to treat P. aeruginosa infections, but few studies have examined the effects of β-lactams on biofilm formation by P. aeruginosa. In this study, we investigated the role of sub-MICs of CAZ in the formation of P. aeruginosa biofilms. 1/4 × MIC CAZ reduced the biofilm volume of P. aeruginosa PAO1, as quantified by crystal violet staining. The formation of P. aeruginosa PAO1 biofilms treated with 1/4 × MIC CAZ were observed by confocal laser scanning microscopy. They were more heterogeneous than the PAO1 biofilms without CAZ treatment. Furthermore, sub-MICs of CAZ inhibited the twitching motility, which played an important role in mature biofilm formation. 1/4 × MIC CAZ also reduced the gene expressions of lecA, lecB, pel and psl, which mediate the adhesion and polysaccharide matrix synthesis of P. aeruginosa. These effects suggest that sub-MICs of CAZ may affect a number of stages of biofilm formation. Investigating the effects of sub-MIC antibiotics on targeted bacterial biofilm may lead to the development of future antibiotic treatment modalities.
Article
Introduction: Biofilm formation represents a protected mode of growth that renders bacterial cells less susceptible to antimicrobials and to killing by host immune effector mechanisms and so enables the pathogens to survive in hostile environments and also to disperse and colonize new niches. Biofilm disease includes device-related infections, chronic infections in the absence of a foreign body, and even malfunction of medical devices. Areas covered: This review puts forward a new medical entity that represents a major public health issue, which we have named “biofilm-related disease”. We highlight the characteristics of biofilm disease including its pathogenesis, microbiological features, clinical presentation, and treatment challenges. Expert commentary: The diversity of biofilm-associated infections is increasing over time and its impact may be underestimated. This peculiar form of development endows associated bacteria with a high tolerance to conventional antimicrobial agents. A small percentage of persister cells developing within the biofilm is known to be highly tolerant to antibiotics and has typically been involved in causing relapse of infections. Knowledge of the pivotal role played by biofilm-growing microorganisms in related infections will provide new treatment dynamics for this biofilm-related disease.