Antibacterial effect and mechanism of theaflavin against Listeria monocytogenes and its application on apple skins.

Theaflavin 3,3'-digallate (TF3), a major polyphenolic component of black tea, exhibits antibacterial effects against many foodborne pathogens. However, the antibacterial mechanisms of TF3 against Listeria monocytogenes remain unclear. In this study, we investigated the effects of TF3 on viability, biofilm, and membrane function of L. monocytogenes by the conventional plating method, crystal violet staining, and microscopy using fluorescent dyes JC-1 and Laurdan, respectively. It was found that TF3 showed excellent antibacterial activity against L. monocytogenes with the minimum inhibitory concentration of 62.5 mg/L. The viable count determined on TSA decreased by 3 log after the treatment for 2 h with TF3 at 62.5 mg/L. The viable count determined on TSA containing 4% NaCl decreased by more than 4 log after the treatment for 30 min with TF3 at the same concentration, suggesting that TF3 gave damage on the cells, enhancing the antibacterial action of 4% NaCl, but the damage was recoverable in the absence of 4% NaCl. To explore the antibacterial mechanisms of TF3, the effects of TF3 on membrane potential and membrane fluidity were investigated. TF3 reduced both membrane potential and fluidity of L. monocytogenes at 62.5 mg/L, suggesting that TF3 damaged the structural integrity of the cell membrane. TF3 reduced biofilm mass of mature biofilm of L. monocytogenes. Moreover, THEAFLAVIN TF40, a commercially available Camellia sinensis leaf extract containing TF3, reduced viable count of L. monocytogenes by 2 log on apple skin. These results suggest the potential of theaflavins as a natural anti-Listeria disinfectant.

Genetic Characterization, Antibiotic Resistance, and Virulence Genes Profiling of Bacillus cereus Strains from Various Foods in Japan.

Bacillus cereus sensu stricto is a foodborne pathogen that causes food poisoning. Their spore and biofilm-forming abilities persist in various environments and foods. This study investigated the prevalence, virulence, antibiotic resistance, and genetic diversity of B. cereus s. s. strains isolated from various food samples. Of 179 samples, 22.34% were positive for B. cereus s. s., with significantly high detection rates in milk products and raw chicken meat. Forty strains were isolated from positive samples. Matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis revealed nine distinct clusters and multi-locus sequence typing revealed 34 sequence types including 23 novel sequences, demonstrating high genetic diversity among the isolates. PCR analysis revealed that all the strains contained at least one toxin gene, but none contained the cytK gene. Antibiotic resistance tests revealed that all isolates were classified as multidrug-resistant, with high resistance levels, particularly to ß-lactam antibiotics and vancomycin, but were susceptible to gentamicin. All isolates showed variations in biofilm formation. This study highlights the significant public health risk due to B. cereus s. s. and underscores the need for stringent monitoring and control measures in food production to manage antimicrobial resistance and ensure food safety.

Applications of bacteriophage in combination with nisin for controlling multidrug-resistant Bacillus cereus in broth and various food matrices.

This study focused on the isolation and characterization of bacteriophages with specific activity against toxin-producing and multidrug-resistant strains of Bacillus cereus sensu stricto (B. cereus s. s.). Ten different samples yielded six bacteriophages by utilizing the double-layer agar technique. The most promising phage, vB_BceS-M2, was selected based on its broad host range and robust lytic activity against various B. cereus s. s. strains. The phage vB_BceS-M2 had a circular double-stranded DNA genome of 56,482 bp. This phage exhibited stability over a wide range of temperatures and pH values, which is crucial for its potential application in food matrices. The combined effect of phage vB_BceS-M2 and nisin, a widely used antimicrobial peptide, was investigated to enhance antimicrobial efficacy against B. cereus in food. The results suggested that nisin showed synergy and combined effect with the phage, potentially overcoming the growth of phage-resistant bacteria in the broth. Furthermore, practical applications were conducted in various liquid and solid food matrices, including whole and skimmed milk, boiled rice, cheese, and frozen meatballs, both at 4 and 25 °C. Phage vB_BceS-M2, either alone or in combination with nisin, reduced the growth rate of B. cereus in foods other than whole milk. The combination of bacteriophage and nisin showed promise for the development of effective antimicrobial interventions to counteract toxigenic and antibiotic-resistant B. cereus in food.

Single and combined application of bacteriophage and cinnamon oils against pathogenic Listeria monocytogenes in milk and smoked salmon.

Nowadays, the discovery of alternative natural antimicrobial substances such as bacteriophages, essential oils, and other physical and chemical agents is developing in the food industry. In this study, nine bacteriophages were isolated from various parts of raw chickens and exhibited lytic activities against L. monocytogenes and various Listeria spp. The characterization of phage vB_LmoS-PLM9 was stable at 4 to 50 °C and pH range from 4 to 10. Phage vB_LmoS-PLM9 had a circular, double-stranded genomic DNA with 38,345 bp having endolysin but no antibiotic resistance or virulence genes. Among the eight essential oils tested at 10 %, cinnamon bark, and cassia oils showed the strongest antilisterial activities. The combined use of phage vB_LmoS-PLM9 and cinnamon oils indicated higher efficiency than single treatments. The combination of phage (MOI of 10) and both cinnamon oils (0.03 %) reduced the viable counts of L. monocytogenes and inhibited the regrowth of resistant cell populations in broth at 30 °C. Furthermore, treatment with the combination of phage (MOI of 100) and cinnamon oil (0.125 %) was effective in milk, especially at 4 °C by reducing the viable count to less than lower limit of detection. These results suggest combining phage and cinnamon oil is a potential approach for controlling L. monocytogenes in milk.

Identification of Enterococcus spp. from food sources by matrix-assisted laser desorption ionization-time of flight mass spectrometry and characterization of virulence factors, antibiotic resistance, and biofilm formation.

The continuous detection of multi-drug-resistant enterococci in food source environments has aroused widespread concern. In this study, 198 samples from chicken products, animal feces, raw milk, and vegetables were collected in Japan and Egypt to investigate the prevalence of enterococci and virulence characterization. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed for species identification and taxonomic analysis of the isolates. The results showed that the rates of most virulence genes (efaA, gelE, asa1, ace, and hyl) in the Japanese isolates were slightly higher than those in the Egyptian isolates. The rate of efaA was the highest (94.9 %) among seven virulence genes detected, but the cylA gene was not detected in all isolates, which was in accordance with γ-type hemolysis phenotype. In Enterococcus faecalis, the rate of kanamycin-resistant strains was the highest (84.75 %) among the antibiotics tested. Moreover, 78 % of E. faecalis strains exhibited multi-drug resistance. Four moderately vancomycin-resistant strains were found in Egyptian isolates, but none were found in Japanese isolates. MALDI-TOF MS analysis correctly identified 98.5 % (68/69) of the Enterococcus isolates. In the principal component analysis dendrogram, strains isolated from the same region with the same virulence characteristics and similar biofilm-forming abilities were characterized by clustered distribution in different clusters. This finding highlights the potential of MALDI-TOF MS for classifying E. faecalis strains from food sources.

Characterization of selected phages for biocontrol of food-spoilage pseudomonads.

Pseudomonas spp., such as P. fluorescens group, P. fragi, and P. putida, are the major psychrophilic spoilage bacteria in the food industry. Bacteriophages (phages) are a promising tool for controlling food-spoilage and food-poisoning bacteria; however, there are few reports on phages effective on food-spoilage bacteria such as Pseudomonas spp. In this study, 12 Pseudomonas phages were isolated from chicken and soil samples. Based on the host range and lytic activity at 30 °C and 4 °C and various combinations of phages, phages vB_PflP-PCS4 and vB_PflP-PCW2 were selected to prepare phage cocktails to control Pseudomonas spp. The phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 showed the strongest lytic activity and retarded regrowth of P. fluorescens and P. putida at 30 °C, 8 °C, and 4 °C at a multiplicity of infection of 100. Nucleotide sequence analysis of the genomic DNA indicated that vB_PflP-PCS4 and vB_PflP-PCW2 phages were lytic phages of the Podoviridae family and lacked tRNA, toxin, or virulence genes. A novel endolysin gene was found in the genomic DNA of phage vB_PflP-PCS4. The results of this study suggest that the phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 is a promising tool for the biocontrol of psychrophilic food-spoilage pseudomonads during cold storage and distribution.

Isolation, characterization of Enterococcus phages and their application in control of E. faecalis in milk.

AIMS: Isolation and characterization of Enterococcus phages and application of phage cocktail to control E. faecalis in milk. METHODS AND RESULTS: For phage isolations, double layer agar method was used. Host range of the phages were determined by the spot test. Twelve phages with varying host ranges were isolated. Phages PEF1, PEF7b, and PEF9 with different host ranges and lytic activities were selected for phage cocktails. Compared to two-phages cocktails tested, the cocktail containing all the three phages displayed stronger antibacterial and biofilm removal activities. The cocktail treatment reduced viable E. faecalis in biofilm by 6 log within 6 h at both 30°C and 4°C. In milk, the cocktail gradually reduced the viable count of E. faecalis and the count reached below the lower limit of detection at 48 h at 4°C. CONCLUSION: The strong bactericidal and biofilm removal activities of the phage cocktail suggest the potential of this cocktail as a natural biocontrol agent for combating E. faecalis in milk.

Potential application of phage vB_EfKS5 to control Enterococcus faecalis and its biofilm in food.

Contaminated food with antibiotic-resistant Enterococcus spp. could be the vehicle for transmitting Enterococcus to humans and accordingly cause a public health problem. The accumulation of biogenic amines produced by Enterococcus faecalis (E. faecalis) in food may have cytological effects. Bacteriophages (phage in short) are natural antimicrobial agents and can be used alone or in combination with other food preservatives to reduce food microbial contaminants. The aim of this study was to isolate a novel phage against E. faecalis and determine its host range to evaluate its potential application. Bacteriophage, vB_EfKS5, with a broad host range, was isolated to control the growth of E. faecalis. The vB_EfKS5 genome is 59,246 bp in length and has a GC content of 39.7%. The computational analysis of phage vB_EfKS5 genome confirmed that it does not contain any lysogenic, toxic, or virulent genes. Phage vB_EfKS5 exhibited lytic activity against most E. faecalis isolates with different multiplicities of infections and it infected 75.5% (22/29) of E. faecalis isolates and 42.3% (3/7) of E. faecium isolates. It was also able to destroy the biofilm formed by E. faecalis with different MOIs. Phage vB_EfKS5 alone or in combination with nisin could control the growth of E. faecalis in broth and milk. Based on its high productivity, stability, short latent period, and large burst size, phage vB_EfKS5 has a high potential for applications both in food and medical applications.

Comparison of prevalence, characterization, antimicrobial resistance and pathogenicity of foodborne Listeria monocytogenes in recent 5 years in Japan.

This study investigated the prevalence, serotype, antimicrobial resistance (AMR), virulence potential, and biofilm formation of Listeria monocytogenes isolated in 2022 in Japan and compared their profiles with those of isolates in 2012 and 2017. A total of 85 chicken samples were randomly collected from different supermarkets in Fukuoka in 2022. L. monocytogenes were isolated by conventional method and characterized by MALDI-TOF MS. Among 85 samples tested in 2022, 9 (10.6%) were positive for L. monocytogenes and 17 strains were isolated from the positive samples. The isolates were serotyped as 1/2b (41.2%), 3a (29.4%), 3b (23.5%) and 1/2a (5.9%). Antimicrobial susceptibility tests of the 2022 isolates showed susceptibility to majority of the antibiotics, except cefoxitin, oxacillin, and fosfomycin. Compared to the previous surveillance results, the prevalence of L. monocytogenes in 2022 (10.6%) was significantly lower (p < 0.05) than those of the isolates in 2017 (24%) and 2012 (52.9%). The distribution of serotypes 1/2a and 1/2b decreased over time, and serotype 4b was not detected in the 2022 isolates. The proportion of multidrug resistant strains in 2022 (16.7%) was significantly lower than those in 2012 (46.7%) and 2017 (82.6%). Moreover, a total of 36 isolates (12 isolates/ year) were used to detect the virulence genes (hlyA, plcA, clpC, and inlA) and biofilm-forming capacity. Most of the isolates from different years harboured four virulence genes. The biofilm formation of the 2022 isolates was significantly weaker (p < 0.05) than those of the 2012 and 2017 isolates. Thus, despite the low rates of contamination in chicken meat and AMR of the isolates, virulent L. monocytogenes contamination in food should still be acknowledged.

Biocontrol of Salmonella Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast by using a novel bacteriophage with broad host range.

Salmonella spp., one of the most frequently reported bacteria, causes foodborne illness and economic losses. Due to the threat of increasing antibiotic resistant foodborne pathogens, application of bacteriophages as novel antibacterial agents in food matrices has become an emerging strategy. In this study, a novel Salmonella phage PS3-1 with high lytic activity against Salmonella Typhimurium was identified from previously isolated phages. PS3-1 belonged to the class Caudoviricetes with a broad host range, and had relatively short latent period (15 min), large burst size (92 PFU/cell), high pH stability (pH 3.0-11.0) and thermal tolerance (4-60 °C). Genome sequencing analysis showed that PS3-1 genome consisted of 107,110 bp DNA, without antibiotic resistance and virulence related genes. The results of growth curve and time-kill assay showed that PS3-1 not only inhibited the growth of S. Typhimurium, but also effectively decreased the viable cell counts (0.30-4.72 log) after 24-h incubation at 7, 25 and 37 °C (P < 0.05). Moreover, >1.28 log of established biofilm cells were effectively removed after 24-h treatment with PS3-1. Besides, PS3-1 significantly reduced the viability of S. Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast at different temperatures (P < 0.05). These results demonstrated that PS3-1 may be an excellent antibacterial agent for controlling S. Typhimurium in food industry.

Antimicrobial resistance profiles of Listeria monocytogenes isolated from chicken meat in Fukuoka, Japan.

In this study, the antimicrobial resistance profiles of L. monocytogenes isolated from chicken meat in Fukuoka in 2017 were compared with the isolates of 2012. A total of 85 and 50 chicken meat samples, including different body parts, were collected from different supermarkets in Fukuoka in 2012 and 2017, respectively. Detection, isolation, identification, and characterization of L. monocytogenes were performed according to the conventional methods. Forty-five among 85 samples (53%) were positive for L. monocytogenes in 2012, while 12 among 50 samples in 2017 (24%) tested positive. One hundred fifty-three and 29 L. monocytogenes strains were isolated in 2012 and 2017, respectively. The serotypes of isolates in 2012 were 1/2a (21.5%), 1/2b (73.9%), 1/2c (1.5%), and 4b/4e (3.1%). In contrast, the 2017 isolates showed 1/2a (48.3%) and 1/2b (51.7%) serotypes. While all isolates in 2012 were positive for hlyA (listeriolysin O) in the PCR assay with hlyA primer set 7, only 17 hlyA positive isolates were seen in 2017. Moreover, 75 isolates with different ribotypes in 2012 and 29 isolates in 2017, respectively, were tested for antimicrobial susceptibility by broth microdilution for 18 different antimicrobial agents. Most of the 2012 and 2017 isolates displayed antimicrobial susceptibility. However, among the 2012 and 2017 isolates, 98.7% and 100% of the isolates were resistant to cefoxitin, 57.3% and 95.7% to fosfomycin, 72.0% and 82.6% to oxacillin, 8.0% and 17.4% to clindamycin, respectively. In addition, 2.7% of the isolates in 2012 were resistant to flomoxef and 4.3% of the isolates in 2017 to linezolid. Multidrug resistance (MDR) to 3 or more antimicrobials was observed in 35/75 (46.7%) isolates of 2012 and 19/23 (82.6%) in 2017. Detection of antimicrobial resistance (AMR) genes by PCR showed that the resistant isolates of 2012 were positive for mecA (96.3%) and ermC (83.3%), whereas the resistant isolates in 2017 screened positive for mecA (94.7%) and mefA (25.0%). Other cfxA, ermA, ermB, fosA, fosB, and fosC genes were absent in the PCR assay for any of the isolates. This study investigated for the first time the change in the L. monocytogenes contamination of chicken meat and antibiotic resistance of the isolated L. monocytogenes strains in Fukuoka, Japan, in the course of 5 years. Although the contamination rate of L. monocytogenes in 2017 was found to be lower than that in 2012, AMR of the isolates in 2017 was higher.