Phages and engineered lysins as an effective tool to combat Gram-negative foodborne pathogens.

One of the biggest challenges faced by food producers is ensuring microbiological safety. Despite strict criteria for food products, foodborne diseases are a global problem and pose a real risk to consumers. Therefore, it is necessary to identify new and more effective methods for eliminating pathogens from food and the food processing environment. According to the European Food Safety Authority (EFSA), the most common foodborne diseases are caused by Campylobacter, Salmonella, Yersinia, Escherichia coli, and Listeria. Out of the five listed, four are Gram-negative bacteria. Our review focuses on the use of bacteriophages, which are ubiquitous bacterial viruses, and bacteriophage endolysins to eliminate Gram-negative pathogens. Endolysins cleave specific bonds within the peptidoglycan (PG) of the bacterial cell, causing the cell to burst. Single phages or phage cocktails, which are, in some instances, commercially available products, eliminate pathogenic bacteria in livestock and various food matrices. Endolysins have matured as the most advanced class of antibacterial agents in the clinical sector, but their use in food protection is highly unexplored. Advanced molecular engineering techniques, different formulations, protein encapsulation, and the addition of outer membrane (OM) permeabilization agents enhance the activity of lysins against Gram-negative pathogens. This creates space for groundbreaking research on the use of lysins in the food sector.

Molecular characterization of the PhiKo endolysin from Thermus thermophilus HB27 bacteriophage phiKo and its cryptic lytic peptide RAP-29.

Introduction: In the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here. Methods: Enzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3'-Dipropylthiacarbocyanine iodide (DiSC3(5)). Results and discussion: The PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7-7.1 log units and reaching the minimum inhibitory concentration values in the range of 2-31 µM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously.

Overview of the Importance of Biotics in Gut Barrier Integrity.

Increased gut permeability is suggested to be involved in the pathogenesis of a growing number of disorders. The altered intestinal barrier and the subsequent translocation of bacteria or bacterial products into the internal milieu of the human body induce the inflammatory state. Gut microbiota maintains intestinal epithelium integrity. Since dysbiosis contributes to increased gut permeability, the interventions that change the gut microbiota and correct dysbiosis are suggested to also restore intestinal barrier function. In this review, the current knowledge on the role of biotics (probiotics, prebiotics, synbiotics and postbiotics) in maintaining the intestinal barrier function is summarized. The potential outcome of the results from in vitro and animal studies is presented, and the need for further well-designed randomized clinical trials is highlighted. Moreover, we indicate the need to understand the mechanisms by which biotics regulate the function of the intestinal barrier. This review is concluded with the future direction and requirement of studies involving biotics and gut barrier.

Operational culture conditions determinate benzalkonium chloride resistance in L. monocytogenes-E. coli dual species biofilms.

Biofilms pose a serious challenge to the food industry. Higher resistance of biofilms to any external stimuli is a major hindrance for their eradication. In this study, we compared the growth dynamics and benzalkonium chloride (BAC) resistance of dual species Listeria monocytogenes-Escherichia coli 48 h biofilms formed on stainless steel (SS) coupons surfaces under batch and fed-batch cultures. Differences between both operational culture conditions were evaluated in terms of total viable adhered cells (TVAC) in the coupons during 48 h of the mixed-culture and of reduction of viable adhered cells (RVAC) obtained after BAC-treatment of a 48 h biofilm of L. monocytogenes-E. coli formed under both culture conditions. Additionally, epifluorescence microscopy (EFM) and confocal scanning microscopy (CLSM) permitted to visualize the 2D and 3D biofilms structure, respectively. Observed results showed an increase in the TVAC of both strains during biofilm development, being the number of E. coli adhered cells higher than L. monocytogenes in both experimental systems (p < 0.05). Additionally, the number of both strains were higher approximately 2.0 log CFU/coupon in batch conditions compared to fed-batch system (p < 0.05). On the contrary, significantly higher resistance to BAC was observed in biofilms formed under fed-batch conditions. Furthermore, in batch system both strains had a similar reduction level of approximately 2.0 log CFU/coupon, while significantly higher resistance of E. coli compared to L. monocytogenes (reduction level of 0.69 and 1.72 log CFU/coupon, respectively) (p < 0.05) was observed in fed-batch system. Microscopic image visualization corroborated these results and showed higher complexity of 2D and 3D structures in dual species biofilms formed in batch cultures. Overall, we can conclude that the complexity of the biofilm structure does not always imply higher resistance to external stimuli, and highlights the need to mimic industrial operational conditions in the experimental systems in order to better assess the risk associated to the presence of pathogenic bacterial biofilms.