Inhibitory effects of Flavourzyme on biofilm formation, quorum sensing, and virulence genes of foodborne pathogens Salmonella Typhimurium and Escherichia coli.

Salmonella enterica and Shiga toxin-producing (or verotoxin-producing) Escherichia coli are major foodborne pathogens, posing substantial food safety risks. Due to the negative effects of chemical treatment against foodborne pathogens, the application of enzyme-based techniques is currently receiving great attention. Here, we evaluated the inhibitory properties of Flavourzyme, a commercial peptidase, against these two foodborne pathogens. We noticed 4.0 and 5.5 log inhibition of biofilm formation by S. Typhimurium and E. coli, respectively, while treated with sub-minimum inhibitory concentrations of Flavourzyme for 24 h. For both bacteria, the enzyme exhibited quorum-quenching activity, preventing autoinducer-2 production completely by E. coli. In addition, Flavourzyme significantly suppressed the relative expression levels of biofilm-forming, quorum sensing, and virulence regulatory genes as measured by qRT-PCR. Based on our results, we suggest the use of Flavourzyme as a preventive agent against foodborne pathogens that possibly acts by inhibiting bacterial self-defense mechanisms following disruption of cellular proteins. This finding may shed light on how enzymes can be applied as a novel weapon to control foodborne illnesses to ensure food safety and public health.

Research Note: Identification and characterization of Salmonella spp. in mechanically deboned chickens using pulsed-field gel electrophoresis.

Salmonella is one of the common foodborne bacteria, causing 80.3 million illnesses every year worldwide. This study was conducted to isolate and identify Salmonella enterica serovars from poultry samples responsible for causing foodborne poisoning in the Mississippi area, United States. A total of 55 S. enterica serovars-Enteritidis (6), Oranienburg (1), Schwarzengrund (8), Heidelberg (4), Kentucky (22), 4, [5], 12:i:- (1), Montevideo (2), Infantis (9), and multi serotypes (2)-were isolated from approximately 110 poultry samples. Through pulsed-field gel electrophoresis (PFGE) analysis, 8 to 13 bands were obtained. The profiles showed >90% similarity in strains within the same type. Consequently, PFGE could be a useful tool to determine chromosomal similarity (clonality of strains) that can be used to trace down epidemiologic sources and geographical origins of Salmonella.

Efficacy of flavourzyme against Salmonella Typhimurium, Escherichia coli, and Pseudomonas aeruginosa biofilms on food-contact surfaces.

Food contamination is a major public health concern, with Salmonella Typhimurium, Escherichia coli, and Pseudomonas aeruginosa being the prominent causal agents. They often produce resistant shields in food through biofilm formation and are difficult to remove from food-contact surfaces using conventional cleaning agents. In the current study, we investigated the efficacy of flavourzyme, an industrial peptidase, in biofilm removal from ultra-high molecular weight polyethylene (UHMWPE) and rubber surfaces and compared the corresponding efficacies with those of the commonly used DNase I. We noticed a significant reduction of young (24-h-old) and mature (72-h-old) biofilms on both surfaces after treatment with flavourzyme. The overall reduction potentiality of flavourzyme was higher than that of DNase I. The flavourzyme-mediated removal of biofilms appears to be caused by the gradual disruption of amide (NH) and polysaccharide (C-O-C) stretching bands of the extracellular polymeric substances (EPS) released by the microbes. EPS elimination and the cell-friendly behavior of flavourzyme were further confirmed by field emission scanning electron microscopy. Based on these findings, we suggest that flavourzyme can reduce microbial EPS formation, thus possibly controlling microbial food contamination. This finding reveals a new opportunity for the development of a novel method for controlling foodborne illness as well as food spoilage.

Antibacterial and antibiofilm mechanism of eugenol against antibiotic resistance Vibrio parahaemolyticus.

The objective of this study was to investigate the antibacterial and antibiofilm activity of eugenol against V. parahaemolyticus planktonic and biofilm cells and the involved mechanisms as well. Atime-kill assay, a biofilm formation assay on the surface of crab shells, an assay to determine the reduction of virulence using eugenol at different concentrations, energy-filtered transmission electron microscope (EF-TEM), field emission scanning electron microscopy (FE-SEM), confocal laser scanning microscope (CLSM) and high-performance liquid chromatography (HPLC) were performed to evaluate the antibacterial and antibiofilm activity of eugenol. The results indicated that different concentrations of eugenol (0.1-0.6%) significantly reduced biofilm formation, metabolic activities, and secretion of extracellular polysaccharide (EPS), with effective antibacterial effect. Eugenol at 0.4% effectively eradicated the biofilms formed by clinical and environmental V. parahaemolyticus on crab surface by more than 4.5 and 4 log CFU/cm2, respectively. At 0.6% concentration, the reduction rates of metabolic activities for ATCC27969 and NIFS29 were 79% and 68%, respectively. Whereas, the reduction rates of EPS for ATCC27969 and NIFS29 were 78% and 71%, respectively. On visual evaluation, significant results were observed for biofilm reduction, live/dead cell detection, and quorum sensing (QS). This study demonstrated that eugenol can be used to control V. parahaemolyticus biofilms and biofilm-related infections and can be employed for the protection of seafood.

Advances and Future Prospects of Enzyme-Based Biofilm Prevention Approaches in the Food Industry.

Food poisoning and foodborne diseases are a growing public health concern worldwide. Approximately 30 known and many unknown pathogens are the main culprits for these conditions. Biofilms are a heterogeneous living-form of pathogens and are considered a safe haven for their pathogenicity. In the field of food processing, the persistence of biofilms results in an increased likelihood of food contamination, which ultimately compromises overall food quality and safety. Because of the robust heterogeneity and resistant phenotypic nature of biofilms, the impairment of biofilms is very challenging when using conventional cleaning agents/antibiotics. Therefore, the development of alternative approaches is of great interest to the food industry. Recently, many researchers have found that use of enzymes can provide an exciting and effective therapeutic approach for solving biofilm-associated problems in the food industry, because enzymes are involved in almost every stage of biofilm detachment and degradation. Here, we describe biofilm-associated problems in the food industry and recent advances in enzyme-based biofilm impairment strategies. We also highlight major limitations, challenges, and possible prospects of enzyme-based biofilm-targeting technologies.