Isolation, characterization, and application of bacteriophage on Vibrio parahaemolyticus biofilm to control seafood contamination.

OBJECTIVE: This study intended to isolate a Vibrio-particular phage from the natural environment, analyse its characteristics and genome sequence, and investigate its reduction effect on V. parahaemolyticus biofilm as a biocontrol agent in squid and mackerel. METHODS: Among 21 phages, phage CAU_VPP01, isolated from beach mud, was chosen for further experiments based on host range and EOP tests. When examining the reduction effect of phage CAU_VPP01 against Vibrio parahaemolyticus biofilms on surfaces (stainless steel [SS] and polyethylene terephthalate [PET]) and food surfaces (squid and mackerel). RESULTS: The phage showed the most excellent reduction effect at a multiplicity-of-infection (MOI) 10. Three-dimensional images acquired with confocal laser scanning microscopy (CLSM) analysis were quantified using COMSTAT, which showed that biomass, average thickness, and roughness coefficient decreased when treated with the phage. Colour and texture analysis confirmed that the quality of squid and mackerel was maintained after the phage treatment. Finally, a comparison of gene expression levels determined by qRT-PCR analysis showed that the phage treatment induced a decrease in the gene expression of flaA, vp0962, andluxS, as examples. CONCLUSION: This study indicated that Vibrio-specific phage CAU_VPP01 effectively controlled V. parahaemolyticus biofilms under various conditions and confirmed that the isolated phage could possibly be used as an effective biocontrol weapon in the seafood manufacturing industry.

Efficacy of disinfectant and bacteriophage mixture against planktonic and biofilm state of Listeria monocytogenes to control in the food industry.

Fresh produce and animal-based products contaminated with Listeria monocytogenes have been the main cause of listeriosis outbreaks for many years. The present investigation explored the potential of combination treatment of disinfectants with a bacteriophage cocktail to control L. monocytogenes contamination in the food industry. A mixture of 1 minimal inhibitory concentration (MIC) of disinfectants (sodium hypochlorite [NaOCl], hydrogen peroxide [H2O2], and lactic acid [LA]) and multiplicity of infection (MOI) 100 of phage cocktail was applied to both planktonic cells in vitro and already-formed biofilm cells on food contact materials (FCMs; polyethylene, polypropylene, and stainless steel) and foods (celery and chicken meat). All the combinations significantly lowered the population, biofilm-forming ability, and the expression of flaA, motB, hlyA, prfA, actA, and sigB genes of L. monocytogenes. Additionally, in the antibiofilm test, approximately 4 log CFU/cm2 was eradicated by 6 h treatment on FCMs, and 3 log CFU/g was eradicated within 3 days on celery. However, <2 log CFU/g was eradicated in chicken meat, and regrowth of L. monocytogenes was observed on foods after 5 days. The biofilm eradication efficacy of the combination treatment was proven through visualization using scanning electron microscopy (SEM) and confocal microscopy. In the SEM images, the unusual behavior of L. monocytogenes invading from the surface to the inside was observed after treating celery with NaOCl+P or H2O2 + P. These results suggested that combination of disinfectants (NaOCl, H2O2, and LA) with Listeria-specific phage cocktail can be employed in the food industry as a novel antimicrobial and antibiofilm approach, and further research of L. monocytogenes behavior after disinfection is needed.

Control of Listeria monocytogenes in food industry by a combination treatment of natural aromatic compound with Listeria-specific bacteriophage cocktail.

Most Listeria monocytogenes found in the food industry are listeriosis-causing pathogens and possess the ability to form biofilms on food and food contact materials (FCMs). This study aims to evaluate the efficacy of the combination treatment of natural aromatic compounds (thymol, eugenol, carvacrol, and citral) with a Listeria-specific phage cocktail in mitigating the threat posed by L. monocytogenes in the food industry. In vitro combination treatment of 1 minimal inhibitory concentration (MIC) of natural aromatic compound with phage cocktail at multiplicity of infection (MOI) 100 reduced more than 4 log CFU/mL of L. monocytogenes planktonic cells and inhibited biofilm formation. In addition, the expression of virulence-related genes (flaA, motB, hlyA, prfA, and actA) and the stress response (sigB) gene were significantly downregulated. The combination of natural aromatic compound with phage cocktail reduced the biofilm cell population on contaminated celery by more than 2 log CFU/g and by more than 2 log CFU/cm2 on already-formed biofilm on FCMs, but it was less effective on chicken meat, with an approximate reduction of only 1 log CFU/g. The antibiofilm activity toward preformed L. monocytogenes biofilms was also observed using field-emission scanning electron microscopy (FESEM) and confocal laser scanning microscopy (CLSM). COMSTAT analysis of the structural change of biofilms revealed that major biofilm structure parameters (biovolume, thickness, diffusion distance, and microcolonies at substratum) were reduced after treatment. Our findings suggest that the combination of natural aromatic compounds with a phage cocktail has enormous potential as an antimicrobial and antibiofilm agent for controlling L. monocytogenes in the food industry.

Biofilm eradication ability of phage cocktail against Listeria monocytogenes biofilms formed on food contact materials and effect on virulence-related genes and biofilm structure.

Listeria monocytogenes is a foodborne pathogen that can form biofilms in food processing facilities even under unfavorable growth environment. This study aimed to evaluate the biofilm eradication ability of Listeria-specific bacteriophage (phage) cocktail (LMPC01+02+03) against L. monocytogenes young (1 day) and mature (3 days) biofilms formed on food contact materials (FCMs: polyethylene, polypropylene, and stainless steel) at 4, 15, and 30 °C. In addition, virulence-related genes and biofilm structure parameters of the phage-treated biofilms were investigated. The biofilm eradication ability of the phage cocktail was evaluated on 96 well and MBEC plate, and the results revealed that a multiplicity-of-infection (MOI) 100 of the phage cocktail exhibited the ability of eradicate biofilms. Using MOI 100, the phage cocktail treatment on the biofilms formed on FCMs for 8 h reduced over 2 log CFU/cm2 of the young biofilms, and approximately 1 log CFU/cm2 of the mature biofilms. In addition, the phage treatment against the biofilms resulted in a significant up-regulation of two genes (flaA and motB), and up/down-regulation or no changes in three genes (hlyA, prfA, and actA). Confocal and scanning electron microscopy images revealed the loss of the biofilm matrix after the phage treatment, and quantitative analysis revealed a reduction in the structural parameters of the biofilm, except the microcolonies at the substratum level, which increased. These results suggested that MOI 100 of the phage cocktail (LMPC01+02+03) was an effective tool for eradicating L. monocytogenes biofilms formed on FCMs, and it is essential to develop a countermeasure to eradicate the biofilm remaining after phage treatment.