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.

Screening of genes involved in phage-resistance of Escherichia coli and effects of substances interacting with primosomal protein A on the resistant bacteria.

AIMS: The study was to identify the genes involved in phage resistance and to develop an effective biocontrol method to improve the lytic activity of phages against foodborne pathogens. METHODS AND RESULTS: A total of 3,909 single gene-deletion mutants of Escherichia coli BW25113 from the Keio collection were individually screened for genes involved in phage resistance. Phage S127BCL3 isolated from chicken liver, infecting both E. coli BW25113 and O157: H7, was characterized and used for screening. The 10 gene-deletion mutants showed increased susceptibility to phage S127BCL3. Among them, priA gene-deletion mutant strain showed significant susceptibility to the phages S127BCL3 and T7. Furthermore, we investigated the substances that have been reported to inhibit the function of primosomal protein A (PriA) and were used to confirm increased phage susceptibility in E. coli BW25113 (Parent strain) and O157: H7. CONCLUSION: PriA inhibitors at a low concentration showed combined effects with phage against E. coli O157: H7 and delayed the regrowth rate of phage-resistant cells.

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.

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.

Inactivation of mixed Escherichia coli O157:H7 biofilms on lettuce by bacteriophage in combination with slightly acidic hypochlorous water (SAHW) and mild heat treatment.

Escherichia coli O157:H7 is one of the most important foodborne pathogens that can persist in leafy green vegetables and subsequently produce biofilms. Biofilm formation is an ongoing concern in the food industry as biofilms are relatively resistant to a variety of antimicrobial treatments. In the present study, we evaluated the combined effects of phage FP43 and mild-heated slightly acidic hypochlorous water (SAHW) in reducing established biofilms on lettuce. Prior to the sequential treatments involving phage-SAHW and SAHW-phage for long-term storage, equal inoculum densities of E. coli O157:H7 and E. coli O91:H- were added on iceberg lettuce surfaces and the lettuce samples were stored at 10 °C for 48 h to allow biofilm formation. The sequential treatment with phage FP43 and SAHW significantly decreased the number of adhered cells, especially the combination of phage FP43 at 25 °C for 2 h and mild-heated SAHW, which considerably eliminated E. coli viable biofilm cells to undetectable levels (>3 log CFU/piece). However, the biofilms were not completely removed, as evidenced via SEM observation. Additionally, sequential treatment with SAHW and phage caused continuous reductions in viable counts, decreasing the viability of E. coli O157:H7 and total E. coli to the lower limit of detection after incubation for 5 d. Meanwhile, bacterial regrowth was observed after treatment with SAHW alone. These results indicated that the combination of phage and SAHW could be considered as a promising strategy to control the formation of E. coli O157:H7 biofilms on lettuce.

Endolysin LysSTG2: Characterization and application to control Salmonella Typhimurium biofilm alone and in combination with slightly acidic hypochlorous water.

The gene encoding LysSTG2, an endolysin from Salmonella-lytic bacteriophage STG2, was cloned, overexpressed, and characterized. LysSTG2 consists of a single domain belonging to the Peptidase_M15 superfamily. LysSTG2 showed strong lytic activity against chloroform-treated S. Typhimurium cells after incubation at 4-50 °C for 30 min, at pH ranging from 7.0 to 11.0, and in the presence of NaCl from 0 to 300 mmol/L. It also showed lytic activity against all the 14 tested Gram-negative strains treated with chloroform, including Salmonella, E. coli, and Pseudomonas aeruginosa, but not against the Gram-positive bacteria tested. In addition, LysSTG2 (100 µg/mL) reduced the viability of S. Typhimurium NBRC 12529 planktonic cells by 1.2 log and that of the biofilm cells after 1-h treatment. Sequential treatment of slightly acidic hypochlorous water (SAHW) containing 40 mg/L available chlorine and LysSTG2 (100 µg/mL) was effective on S. Typhimurium NBRC 12529 biofilm cells, removing more than 99% of biofilm cells. These results demonstrate that LysSTG2 alone can effectively kill S. Typhimurium cells after permeabilization treatment and successfully control S. Typhimurium in biofilms in combination with SAHW, suggesting that the combined use of LysSTG2 and SAHW might be a novel and promising method for combating S. Typhimurium in food industries.

Inhibition of phage-resistant bacterial pathogen re-growth with the combined use of bacteriophages and EDTA.

The combined effects of ethylenediaminetetraacetic acid (EDTA) and bacteriophage (phage) treatment of foodborne pathogens were investigated. Although viable counts for Campylobacter jejuni decreased by 1.5 log after incubation for 8 h in the presence of phage PC10, re-growth was observed thereafter. The combination of phage PC10 and 1 mM EDTA significantly inhibited the re-growth of C. jejuni. The viable counts for C. jejuni decreased by 2.6 log (P < 0.05) compared with that of the initial count after 24 h. Moreover, EDTA at 0.67 or 1.3 mM, combined with the specific lytic phages, also effectively inhibited the re-growth of phage-resistant cells of Campylobacter coli, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Typhimurium. In addition, the combined effects of lytic phages and EDTA were investigated on the viability of Campylobacter in BHI broth at low temperatures followed by the optimum growth temperature. The re-growth of C. coli was significantly inhibited by the coexistence of 1.3 mM EDTA, and the viable counts of surviving bacteria was about the same as the initial viable count after the incubation. This is the first study demonstrating the combined use of lytic phages and EDTA is effective in inhibiting the re-growth of phage-resistant bacteria in Gram-negative bacteria.

Role of Toxin-Antitoxin-Regulated Persister Population and Indole in Bacterial Heat Tolerance.

YafQ is an endoribonuclease toxin that degrades target gene transcripts such as that of tnaA, a gene encoding tryptophanase to synthesize indole from tryptophan. DinJ is the cognate antitoxin of YafQ, and the YafQ-DinJ system was reported to regulate persister formation by controlling indole production in Escherichia coli In this study, we investigated the role of YafQ-DinJ, indole production, and persister population in bacterial heat tolerance. yafQ (ΔyafQ), dinJ (ΔdinJ), and tnaA (ΔtnaA) single-gene knockout mutants showed approximately 10-fold higher heat tolerance than wild-type (WT) E. coli BW25113. Persister fractions of all mutants were slightly larger than that of the WT. Interestingly, these persister cells showed an approximately 100-fold higher heat tolerance than normal cells, but there was no difference among the persister cells of all mutants and the WT in terms of heat tolerance. Indole and its derivatives promoted a drastic reduction of bacterial heat tolerance by just 10 min of pretreatment, which is not sufficient to affect persister formation before heat treatment. Surprisingly, indole and its derivatives also reduced the heat tolerance of persister cells. Among the tested derivatives, 5-iodoindole exhibited the strongest effect on both normal and persister cells.IMPORTANCE Our study demonstrated that a small persister population exhibits significantly higher heat tolerance than normal cells and that this small fraction contributes to the heat tolerance of the total bacterial population. This study also demonstrated that indole, known to inhibit persister formation, and its derivatives are very promising candidates to reduce the heat tolerance of not only normal bacterial cells but also persister cells.

Transcriptional changes involved in inhibition of biofilm formation by ε-polylysine in Salmonella Typhimurium.

The pathogenicity of Salmonella Typhimurium, a foodborne pathogen, is mainly attributed to its ability to form biofilm on food contact surfaces. ε-polylysine, a polymer of positively charged lysine, is reported to inhibit biofilm formation of both gram-positive and gram-negative bacteria. To elucidate the mechanism underlying ε-polylysine-mediated inhibition of biofilm formation, the transcriptional profiles of ε-polylysine-treated and untreated Salmonella Typhimurium cells were comparatively analysed. The genome-wide DNA microarray analysis was performed using Salmonella Typhimurium incubated with 0.001% ε-polylysine in 0.1% Bacto Soytone at 30 °C for 2 h. The expression levels of genes involved in curli amyloid fibres and cellulose production, quorum sensing, and flagellar motility were downregulated, whereas those of genes associated with colanic acid synthesis were upregulated after treatment with ε-polylysine. The microarray results were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, treatment with ε-polylysine decreased the production of colanic acid in Salmonella Typhimurium. The findings of this study improved our understanding of the mechanisms underlying ε-polylysine-mediated biofilm inhibition and may contribute to the development of new disinfectants to control biofilm during food manufacturing and storage.

Isolation and application of bacteriophages alone or in combination with nisin against planktonic and biofilm cells of Staphylococcus aureus.

Staphylococcus aureus is a notorious foodborne pathogen since it has ability to produce variety of toxins including heat-stable enterotoxin, form biofilm, and acquire resistance to antibiotics. Biocontrol of foodborne pathogens by lytic bacteriophages garners increasing interest from both researchers and food industry. In the present study, 29 phages against S. aureus were successfully isolated from chicken, pork, and fish. Characterization of the isolates revealed that phage SA46-CTH2 belonging to Podoviridae family had a number of features suitable for food industry applications such as wide host range, short latent period, large burst size, high stress tolerance, and a genome free of virulence genes. Furthermore, phage SA46-CTH2 alone or in combination with nisin exhibited great efficacy in reducing planktonic and biofilm cells of S. aureus at various conditions tested. The combination of phage SA46-CTH2 and nisin was also found to be able to inhibit the regrowth of S. aureus at both 37 and 24 °C.Key points• A total of 29 S. aureus phages were successfully isolated from fish, pork, and chicken products. • Phage SA46-CTH2 was characterized by host range, morphology, and genome sequencing. • SA46-CTH2 significantly reduced both planktonic and biofilm cells of S. aureus. • Combination of SA46-CTH2 and nisin inhibited the regrowth of S. aureus.

Application of bacteriophages in simultaneously controlling Escherichia coli O157:H7 and extended-spectrum beta-lactamase producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 and extended-spectrum beta-lactamase (ESBL) producing E. coli (ESBLEC) are important bacteria of public health concern and frequently isolated from raw beef products. Bacteriophage-based methods have been increasingly exploited to control bacterial contamination in meats. Here, we describe the isolation, characterization, and application of a lytic phage PE37 for the simultaneous bio-control of STEC O157:H7 and ESBLEC. Phage PE37, isolated from the bovine intestine, was morphologically characterized as a member of the Myoviridae family, with a broad host range and great stability under various stress conditions. Sequencing analysis revealed that the genomic DNA of phage PE37 contains genes that contribute to virion structure, replication, assembly, and host lysis. PE37 significantly reduced the viable counts of STEC O157:H7 by 4.9 and 2.6 log CFU/mL in broth after 6 h of incubation at 25 and 8 °C, respectively. Application of phage PE37 to raw beef artificially contaminated with STEC O157:H7 resulted in significant reductions in the viable counts by 2.3 and 0.9 log CFU/piece after 24 h of storage at 25 and 8 °C, respectively. Treatment of raw beef contaminated with a bacterial cocktail of STEC O157:H7 and ESBLEC with PE37 also significantly decreased the viable counts of the bacterial mixture by 1.4 and 1.0 log CFU/piece after 24 h of incubation at 25 and 8 °C, respectively. These findings suggest that bacteriophage PE37 may be a potential bio-agent for controlling STEC O157:H7 and ESBLEC contamination in raw beef.

Functional Analysis of Genes Involved in the Biosynthesis of Enterocin NKR-5-3B, a Novel Circular Bacteriocin.

UNLABELLED: A putative biosynthetic gene cluster of the enterocin NKR-5-3B (Ent53B), a novel circular bacteriocin, was analyzed by sequencing the flanking regions around enkB, the Ent53B structural gene, using a fosmid library. A region approximately 9 kb in length was obtained, and the enkB1, enkB2, enkB3, and enkB4 genes, encoding putative biosynthetic proteins involved in the production, maturation, and secretion of Ent53B, were identified. We also determined the identity of proteins mediating self-immunity against the effects of Ent53B. Heterologous expression systems in various heterologous hosts, such as Enterococcus faecalis and Lactococcus lactis strains, were successfully established. The production and secretion of the mature Ent53B required the cooperative functions of five genes. Ent53B was produced only by those heterologous hosts that expressed protein products of the enkB, enkB1, enkB2, enkB3, and enkB4 genes. Moreover, self-immunity against the antimicrobial action of Ent53B was conferred by at least two independent mechanisms. Heterologous hosts harboring the intact enkB4 gene and/or a combination of intact enkB1 and enkB3 genes were immune to the inhibitory action of Ent53B. IMPORTANCE: In addition to their potential application as food preservatives, circular bacteriocins are now considered possible alternatives to therapeutic antibiotics due to the exceptional stability conferred by their circular structure. The successful practical application of circular bacteriocins will become possible only if the molecular details of their biosynthesis are fully understood. The results of the present study offer a new perspective on the possible mechanism of circular bacteriocin biosynthesis. In addition, since some enterococcal strains are associated with pathogenicity, virulence, and drug resistance, the establishment of the first multigenus host heterologous production of Ent53B has very high practical significance, as it widens the scope of possible Ent53B applications.

Gene cluster responsible for secretion of and immunity to multiple bacteriocins, the NKR-5-3 enterocins.

Enterococcus faecium NKR-5-3, isolated from Thai fermented fish, is characterized by the unique ability to produce five bacteriocins, namely, enterocins NKR-5-3A, -B, -C, -D, and -Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). Genetic analysis with a genome library revealed that the bacteriocin structural genes (enkA [ent53A], enkC [ent53C], enkD [ent53D], and enkZ [ent53Z]) that encode these peptides (except for Ent53B) are located in close proximity to each other. This NKR-5-3ACDZ (Ent53ACDZ) enterocin gene cluster (approximately 13 kb long) includes certain bacteriocin biosynthetic genes such as an ABC transporter gene (enkT), two immunity genes (enkIaz and enkIc), a response regulator (enkR), and a histidine protein kinase (enkK). Heterologous-expression studies of enkT and ΔenkT mutant strains showed that enkT is responsible for the secretion of Ent53A, Ent53C, Ent53D, and Ent53Z, suggesting that EnkT is a wide-range ABC transporter that contributes to the effective production of these bacteriocins. In addition, EnkIaz and EnkIc were found to confer self-immunity to the respective bacteriocins. Furthermore, bacteriocin induction assays performed with the ΔenkRK mutant strain showed that EnkR and EnkK are regulatory proteins responsible for bacteriocin production and that, together with Ent53D, they constitute a three-component regulatory system. Thus, the Ent53ACDZ gene cluster is essential for the biosynthesis and regulation of NKR-5-3 enterocins, and this is, to our knowledge, the first report that demonstrates the secretion of multiple bacteriocins by an ABC transporter.

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.

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.

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.

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.

Zirex: a novel zinc-regulated expression system for Lactococcus lactis.

Here, we report a new zinc-inducible expression system for Lactococcus lactis, called Zirex, consisting of the pneumococcal repressor SczA and PczcD. PczcD tightly regulates the expression of green fluorescent protein in L. lactis. We show the applicability of Zirex together with the nisin-controlled expression system, enabling simultaneous but independent regulation of different genes.

Antimicrobial resistance and biofilm formation of Escherichia coli in a Vietnamese Pangasius fish processing facility.

This study aimed to investigate the occurrence, antibiotic resistance, and biofilm formation of Escherichia coli in the Vietnamese Pangasius fish processing facility. Among 144 samples including Pangasius fish, wash water, food contact surfaces, and personnel gloves, 18 E. coli isolates was detected and characterized. The E. coli was detected most frequently in wash water samples (22%, 8/36), followed by Pangasius fish (18%, 8/45). According to the antibiotic susceptibility test by the disc diffusion method, isolates showed the highest resistance against sulfamethoxazole/trimethoprim (45%), followed by tetracycline (39%), whereas all the E. coli isolates were susceptible to meropenem and fosfomycin. Notably, 39% of the isolates (7/18) were found to be multidrug resistant while no E. coli isolates were confirmed as extended-spectrum ß-lactamase producers by the double-disk synergy test. The potency to form biofilm on the polystyrene surface of E. coli isolates indicated that 44% of the isolates (8/18) were classified as weak, 39% (7/18) as moderate, and 17% (3/18) as strong biofilm formers. Interestingly, multidrug resistant E. coli isolates were observed in moderate and strong biofilm producers. Additionally, either slightly acidic hypochlorous water with 40 mg/L of available chlorine or sodium hypochlorite with 100 mg/L of available chlorine exhibited a significant reduction in biofilm mass and biofilm cells of E. coli isolates. This study may provide helpful information about the actual state of E. coli isolates for effective control in the fish processing plant.