La technologie des barrières utilisant des enzymes à pouvoir déstructurant et du carvacrol microencapsulés pour lutter contre les biofilms de bactéries pathogènes

L'environnement opératoire dans les domaines alimentaire et médical permet aux bactéries de se fixer et de se développer sur les surfaces, ce qui entraîne la formation de biofilms bactériens pathogènes et résistants. Ces structures pathogènes sont responsables de nombreuses maladies d'origine alimentaire et d'infections associées aux soins. Par conséquent, pour lutter contre ce fléau de santé publique, plusieurs stratégies ont récemment été proposées, notamment l'élimination chimique et mécanique. Ce travail présente les différents facteurs qui influencent l'adhésion bactérienne et la formation de biofilms sur des surfaces abiotiques, ainsi que la résistance des biofilms aux désinfectants. La microencapsulation par la méthode de séchage par pulvérisation pour la formulation de composants actifs anti-biofilm en vue d'assurer leur stabilité et améliorer leurs activités biologiques est également présentée. Dans ce contexte, une étude a été menée en utilisant le carvacrol, un agent antimicrobien naturel, pour contrôler les biofilms de Pseudomonas aeruginosa et Enterococcus faecalis. En effet, ces deux bactéries sont responsables de nombreuses infections dans le monde en raison de leur persistance sur des surfaces abiotiques dans les hôpitaux et les industries agroalimentaires. Par ailleurs, afin de renforcer l'activité antimicrobienne du carvacrol et de réduire sa volatilité et sa faible solubilité dans l'eau, des émulsions ont été préparées avec du caséinate de sodium et des maltodextrines, puis séchées par atomisation pour obtenir des microcapsules de carvacrol sèches. Les résultats ont montré que le carvacrol exerce une forte activité antimicrobienne contre les deux biofilms bactériens. De plus, nos résultats ont révélé que la microencapsulation par séchage par pulvérisation a augmenté de manière significative l'activité antimicrobienne du carvacrol tout en réduisant les quantités utilisées. En effet, le carvacrol microencapsulé a été capable de réduire le biofilm en dessous de la limite de détection pour Pseudomonas aeruginosa et de 5.5 log CFU mL-1 pour Enterococcus faecalis après 15 min de traitement. L'efficacité de la technologie hurdle pour éliminer les biofilms en utilisant différentes stratégies est discutée dans ce travail. Une des approches de la technologie hurdle est l'utilisation d'enzymes qui peuvent dégrader la matrice et disperser les bactéries intégrées dans les biofilms pour une désinfection plus efficace lorsqu'elles sont combinées avec des agents biocides. En effet, deux enzymes protéolytiques, la pepsine et la trypsine, ciblant les protéines de la matrice, ont été étudiées pour leur potentiel de dégradation des biofilms de Pseudomonas aeruginosa et Enterococcus faecalis et leur effet synergique lorsqu'elles sont combinées au carvacrol. L'analyse directe par microscopie à épifluorescence a permis de visualiser l'activité dispersive des protéases et l'activité létale du carvacrol contre les deux biofilms bactériens. En outre, le traitement combiné avec la pepsine ou la trypsine et le carvacrol a entraîné une réduction plus significative des deux biofilms par rapport au traitement avec le carvacrol seul. De plus, cette réduction était plus importante après un traitement séquentiel avec les deux enzymes suivies du carvacrol. Cependant, l'activité enzymatique est fortement influencée par les facteurs environnementaux et n'est optimale que dans des conditions restreintes. Un autre inconvénient de l'utilisation des enzymes est l'auto-dégradation, qui entraîne leur instabilité. En effet, des microcapsules de protéase contenant de la pepsine ou de la trypsine complexées avec de la pectine et de la maltodextrine ont été préparées.The ambient operating environments in the food and medical sectors allow bacteria to adhere and develop on the substrates, resulting in the growth of resistant pathogenic bacterial biofilms. These pathogenic structures are responsible for several foodborne diseases and health-care associated infections. Consequently, to combat this public health burden, several strategies have recently been proposed which include chemical and mechanical removal. This work presents the different factors that influence bacterial adhesion and biofilm formation on abiotic surfaces, as well as biofilm resistance to disinfectants. The different strategies for biofilm prevention and eradication are described. Microencapsulation using spray-drying method for the formulation of anti-biofilm active components as a tool to ensure their stability and improves their biological activities are also presented. In this context, a study was conducted using carvacrol, a natural antimicrobial agent, to control biofilms of Pseudomonas aeruginosa and Enterococcus faecalis. Indeed, these two bacteria are responsible for several infections worldwide due to their persistence on abiotic surfaces in hospitals and food processing industries. Furthermore, in order to enhance the antimicrobial activity of carvacrol and reduce its volatility and low solubility in water, feed emulsions were prepared with sodium caseinate and maltodextrins and then spray dried to obtain dry carvacrol microcapsules. The results showed that carvacrol had a strong antimicrobial activity against both bacterial biofilms. Furthermore, our findings revealed that microencapsulation by spray drying significantly increased the antimicrobial activity of carvacrol while reducing the amounts used. Indeed, microencapsulated carvacrol was able to reduce biofilm below the detection limit for Pseudomonas aeruginosa and 5.5 log CFU mL-1 for Enterococcus faecalis after 15 min of treatment. However, the complete removal of biofilms from abiotic surfaces in medical and food sectors has proven difficult with the single use of disinfection strategy due to the high protection of the biofilm cells by the extracellular polymeric matrix. This matrix provides an initial protective barrier for the biofilm cells, and makes biofilms highly resistant to antimicrobial agents. The effectiveness of hurdle technology in removing biofilms using different strategies is discussed in this work. One of the hurdle technology approaches is the use of matrix-degrading enzymes that can disperse bacteria embedded in biofilms for more efficient disinfection when combined with biocide agents. Indeed, two proteolytic enzymes, pepsin and trypsin, targeting matrix proteins, have been studied for their potential to degrade biofilms of Pseudomonas aeruginosa and Enterococcus faecalis and their synergistic effect when combined with carvacrol. The direct analysis using epifluorescence microscopy allowed visualization of the dispersive activity of proteases and the lethal activity of carvacrol against the two bacterial biofilms. In addition, the combined pepsin or trypsin treatment with carvacrol showed more significant reduction of both biofilms compared to carvacrol treatment alone. Moreover, this reduction was more substantial after sequential treatment of both enzymes followed by carvacrol. However, the enzyme activity is highly influenced by environmental factors and is only optimal under restricted conditions. Another disadvantage of using enzymes is self-degradation, leading to instability. Indeed, protease microcapsules containing pepsin or trypsin complexed with pectin and maltodextrin have been prepared

Enhanced antimicrobial, antibiofilm and ecotoxic activities of nanoencapsulated carvacrol and thymol as compared to their free counterparts

Essential oils active components emerged as captivating antimicrobials to control biofilms developed on food contact surfaces. Free and nanoencapsulated carvacrol (CAR) and thymol (THY) were assessed as antimicrobials against Salmonella Enteritidis biofilms adhered to stainless steel. The developed spherical nanocapsules using the spray-drying technique showed an average size ranged between 159.25 and 234.76 nm and zeta potential values ranged between 23.60 and 24.66 mV. The minimal inhibitory concentrations (MIC) of free THY and CAR were both 1.25 mg L−1. Nanoencapsulation reduced MIC values to 0.62 mg L−1 (THY) and 0.31 mg L−1 (CAR). Furthermore, the exposure to free and nanoencapsulated CAR and THY induced a destabilization of bacterial membranes with obvisous morphological deformations and a pronounced leakage of potassium ions and green fluorescent proteins. Eradication of S. Enteritidis biofilms developed on stainless steel was achieved following a 15 min treatment with nanoencapsulated CAR and THY at 2 MIC. Free antimicrobial exposures induced up to 4.27 log CFU cm−2 reductions. Additionally, the ecotoxicity tests against Daphnia magna crustaceans reported a non-toxicity of both free and nanoencapsulated CAR and THY after 48 h exposure. Thereby, both CAR and THY antimicrobials proved to be promising natural surface disinfectants that require further exploration and incorporation in food industries.This work was supported by the Partenariat Hubert Curien (PHC)- Cèdre program [42281SD]. The Chevreul Institute is also thanked for its help in the development of this work through the ARCHI-CM project supported by the “Ministère de l’Enseignement Supérieur de la Recherche et de l’Innovation”, the region “Hauts-de-France”, the ERDF program of the European Union and the “Métropole Européenne de Lille

Microencapsulation of carvacrol as an efficient tool to fight Pseudomonas aeruginosa and Enterococcus faecalis biofilms.

Biofilms are involved in serious problems in medical and food sectors due to their contribution to numerous severe chronic infections and foodborne diseases. The high resistance of biofilms to antimicrobial agents makes their removal as a big challenge. In this study, spray-drying was used to develop microcapsules containing carvacrol, a natural antimicrobial agent, to enhance its activity against P. aeruginosa and E. faecalis biofilms. The physicochemical properties and microscopic morphology of the realized capsules and cells were characterized. The minimum inhibitory concentration of encapsulated carvacrol (E-CARV) (1.25 mg mL-1) was 4-times lower than that of free carvacrol (F-CARV) (5 mg mL-1) against P. aeruginosa, while it remained the same against E. faecalis (0.625 mg mL-1). E-CARV was able to reduce biofilm below the detection limit for P. aeruginosa and by 5.5 log CFU ml-1 for E. faecalis after 15 min of treatment. Results also showed that F-CARV and E-CARV destabilize the bacterial cell membrane leading to cell death. These results indicate that carvacrol exhibited a strong antimicrobial effect against both bacterial biofilms. In addition, spray-drying could be used as an effective tool to enhance the antibiofilm activity of carvacrol, while reducing the concentrations required for disinfection of abiotic surfaces

Pepsin and Trypsin Treatment Combined with Carvacrol: An Efficient Strategy to Fight <i>Pseudomonas aeruginosa</i> and <i>Enterococcus faecalis</i> Biofilms

Biofilms consist of microbial communities enclosed in a self-produced extracellular matrix which is mainly responsible of biofilm virulence. Targeting this matrix could be an effective strategy to control biofilms. In this work, we examined the efficacy of two proteolytic enzymes, pepsin and trypsin, to degrade P. aeruginosa and E. faecalis biofilms and their synergistic effect when combined with carvacrol. The minimum dispersive concentrations (MDCs) and the contact times of enzymes, as well as the minimal inhibitory concentrations (MICs) and contact times of carvacrol, were determined against biofilms grown on polystyrene surfaces. For biofilms grown on stainless steel surfaces, the combined pepsin or trypsin with carvacrol treatment showed more significant reduction of both biofilms compared with carvacrol treatment alone. This reduction was more substantial after sequential treatment of both enzymes, followed by carvacrol with the greatest reduction of 4.7 log CFU mL−1 (p P. aeruginosa biofilm and 3.3 log CFU mL−1 (p E. faecalis biofilm. Such improved efficiency was also obvious in the epifluorescence microscopy analysis. These findings demonstrate that the combined effect of the protease-dispersing activity and the carvacrol antimicrobial activity could be a prospective approach for controlling P. aeruginosa and E. faecalis biofilms