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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 β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bacterial food poisoning cases due to Salmonella and E. coli O157:H7 have been linked with the consumption of a variety of food products, threatening public health around the world. This study describes the combined effects of a phage cocktail (STG2, SEG5, and PS5), EDTA, nisin, and polylysine against the bacterial cocktail consisting of S. typhimurium, S. enteritidis, and E. coli O157:H7. Overall, phage cocktail (alone or in combination with nisin or/and polylysine) not only showed great antibacterial effects against bacterial cocktail at different temperatures (4 °C, 24 °C, and 37 °C), but also totally inhibited the emergence of phage resistance during the incubation period. These results suggest that the combination of phages with nisin or/and polylysine has great potential to simultaneously control S. typhimurium, S. enteritidis, and E. coli O157:H7. [ABSTRACT FROM AUTHOR]

To develop a method for the identification of shiikuwasha (Citrus depressa Hayata) and calamondin (Citrus madurensis Lour.), trnL-trnF and trnT-trnL intergenic spacer regions of their chloroplast DNA were amplified using PCR and the nucleotide sequences were determined. In each region, a single nucleotide polymorphism (SNP) site specific to the respective citrus species (shiikuwasha and calamondin) was found. For species discrimination using PCR, two forward primers containing the allele-specific SNP site at the 3'- end and a mismatched nucleotide at the 3rd base from the 3'-end were designed. The allele-specific forward primers specific to shiikuwasha and calamondin were respectively designated CiDeLF-F and CiMaTL-F. To confirm the specificity of the designed primers, PCR was carried out with DNA prepared from citrus peel or hand-squeezed juice as the template. Results showed that shiikuwasha and calamondin fruits and juices were identifiable by PCR using the allele-specific primers. Furthermore, this allele-specific PCR method can be applied to industrially processed and concentrated juice by amplifying DNA in advance. [ABSTRACT FROM AUTHOR]

Detection of foodborne pathogens at very low levels is still a challenge. A custom-built multichannel surface plasmon resonance (SPR) biosensor and simultaneous enrichment broth (SEB) were used to develop a simultaneous detection method for 3 important foodborne pathogens, Escherichia coli O157:H7 (O157:H7), Salmonella enteritidis, and Listeria monocytogenes, at a very low level. These 3 foodborne pathogens at a very low level (14, 6, and 28 CFU/25 g (mL) for O157:H7, S. enteritidis, and L. monocytogenes, respectively) were inoculated in SEB and incubated at 37 ˚C for 24 h. Sample prepared from the simultaneous enrichment culture was analyzed using the multichannel SPR biosensor and sensor chip immobilized with polyclonal antibodies specific to each of the target pathogens. O157:H7, S. enteritidis, and L. monocytogenes in chicken were detected simultaneously at an inoculum dose of 14, 6, and 28 CFU/25 g, respectively. Our method using a custom-built multichannel SPR biosensor and enrichment in SEB is expected as a rapid and simultaneous detection method for low levels of O157:H7, S. enteritidis, and L. monocytogenes in food. Practical Application Our method is expected as a rapid and simultaneous detection method for pathogens at very low levels. It has great potential for safety control of food and microbiological detection applications. [ABSTRACT FROM AUTHOR]

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Tomatoes have recently been implicated as an important vehicle in outbreaks of produce-associated salmonellosis. Traceback reports suggested that pre-harvest contamination of Salmonella enterica might be the main reason for these outbreaks; however, the site of pathogen attachment remains unclear. Therefore, it is important to investigate the mechanisms of Salmonella contamination of fresh produce. To trace the presence of Salmonella in soil and plants, Salmonella Enteritidis transformed with a pEGFP plasmid vector (S. Enteritidis-EGFP) was used. Soil was artificially contaminated with S. Enteritidis-EGFP at 104, s 106 or l0 CFU/g, followed by cultivation of tomato plants in the contaminated soil. Samples of the soil and each organ of the tomato (fruit, stems/leaves, and root) were assayed for Salmonella by plating onto Tryptic Soy Agar and using the MPN method. Salmonella levels in the soil gradually decreased over time, and soil persistence was dependent on the initial inoculation level. Salmonella levels were below the detection limit (< 100 CFU/g) in fruits and stems/leaves, regardless of the level of soil contamination. Moreover, S. Enteritidis-EGFP was not detected in the tomato fruits after root injury. These results indicate that the internalization of Salmonella in tomato fruits might not occur after cultivation in contaminated soil. [ABSTRACT FROM AUTHOR]

In this study, primer sets of L. monocytogenes virulence gene hlyA were designed for the L. monocytogenes-specific PCR assay. To evaluate the performance of these primer sets, the detection sensitivity and specificity were examined and the most suitable primer set was selected. Subsequently, it was subjected to compare the performance for detection of L. monocytogenes with commercially available kits (TaKaRa detection kit and ABI detection kit) on DNA level. Results of real-time PCR showed that the efficiency of this new primer set was 101.6% while TaKaRa and ABI detection kit were 101.1% and 107.4%, respectively. The detection sensitivity of all three methods was equivalent to less than 1 copy per reaction using purified genomic DNA as template. Detection specificity were 100% when testing L. monocytogenes isolates, and for other Listeria isolates were 15.4%, 76.9% and 100% by the method using hlyA L. monocytogenes detection primer set 7, TaKaRa and ABI detection kit, respectively, in summary, this new PCR detection method is relatively sensitive and more specific to L. monocytogenes under certain conditions, could serve as a rapid detection method and has the potential for detection of L. monocytogenes from contaminated food. [ABSTRACT FROM AUTHOR]

The article focuses on a study on the role of a chloroplastic NADPH-dependent thioredoxin (Trx) reductase (NTRC) in the freezing tolerance of Chlorella vulgaris. NTR and Trx-like dithiol-disulfide oxidoreductase activities were determined in Escherichia coli after a mature CvNTRC protein was expressed. Results show that CvNTRC functions as an antioxidant with 2-Cys peroxiredoxin (Prx) in the acquisition of freezing tolerance of Chlorella and has maintained at a level during hardening.

Proteins and genes involved in the recovery of heat-injured Salmonella Enteritidis were investigated. Salmonella Enteritidis cells cultured overnight in tryptic soy broth (TSB; nonselective medium) were suspended in citric acid-disodium hydrogen phosphate buffer (pH 6). After heat treatment at 55°C for 15 min, the culturable counts measured by tryptic soy agar (TSA; nonselective medium) decreased from 108 to 107 CFU/ml. On the other hand, culturable counts measured by desoxycholate-hydrogen sulfite-lactose (DHL) agar (selective medium) were decreased from 108 to 104 CFU/ml by the same treatment. The results suggest that 99.9% of Salmonella Enteritidis detected on TSA were injured but recoverable. When injured Salmonella Enteritidis was incubated in TSB, the culturable count measured by TSA did not increase for 2 h, whereas that by DHL agar increased after incubation for 30 min. After incubation for 2 h, the culturable count measured by DHL agar reached a similar level with that by TSA, indicating that Salmonella Enteritidis had recovered. The two-dimensional polyacrylamide gel electrophoresis analysis revealed that elongation factor G (FusA) and pyruvate kinase (PykF) specifically increased in the cells just after heat treatment and in the recovery cells. The levels of transcription of 86 stress-inducible genes were also investigated by reverse transcription PCR. Nineteen heat-inducible (clpB, clpX, degP, dnaJ, fkpA, frsJ, gapA, hflB, hslJ, hslU, hslV, htpG, htrA, lon, mopA, mopB, mreB, rpoE, and ppiD), and 12 oxidative-stress and DNA damage-inducible (ahpC, ahpF, fldB, fur, grxA, dinF, katG, mutM, recA, soxR, trxC, and zwf) genes were transcribed extensively during recovery in TSB. The results obtained in this study will be used to develop the media or culture conditions that will promote recovery for the detection of food poisoning bacteria, including injured cells from food products. [ABSTRACT FROM AUTHOR]

Describes the purification and characterization of two isoforms of glucose-6-phosphate dehydrogenase (G6PDH) from Chlorella vulgaris C-27. Properties of the purified G6PDH isoforms; Effects of DDT and metal ions on G6PDH; Amino acid sequences of peptide fragments of the two isoforms.

A rapid and sensitive method for detection of staphylococcal enterotoxin B (SEB) in raw and dry milk samples with the use of antibody-based immunomagnetic separation (IMS) in conjunction with flow cytometry (FCM) was developed. Sheep anti-SEB immunoglobulin G (IgG) was immobilized on Dynabeads M-280. The SEB initially binds to the capturing antibody, which is bound on the magnetic beads. The rabbit anti-SEB IgG binds to the captured toxin and is further labeled with a Cy5-labeled goat anti-rabbit IgG antibody. The percentage of the beads that were fluorescent was measured by FCM. FCM was carried out for 1 min, and the data obtained were expressed as histograms for particle size (forward light scatter) and histograms for fluorescence intensity. A peak corresponding to the magnetic beads was clearly distinguished from a peak derived from contaminating particles in the sample solution. In the absence of SEB, about 10% of the beads emitted fluorescence. The percentage of fluorescent beads and the fluorescence intensity increased with increasing SEB concentrations. For this IMSFCM assay, the lower limits of detection for SEB were estimated to be 0.01 and 0.25 ng/ml for buffer and milk samples, respectively. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen. Baicalein (5,6,7-trihydroxylflavone), a flavone isolated from the roots of Scutellaria baicalensis, is considered as a potential antibacterial agent to control foodborne pathogens. Among seven compounds selected by in silico screening of the natural compound database, baicalein inhibited the cytotoxicity of both Shiga toxins 1 and 2 (Stx1 and Stx2) against Vero cells after pretreatment at 0.13 mmol/L. In addition, baicalein reduced the susceptibility of Vero cells to both Stx1 and Stx2. Real-time qPCR showed that baicalein increased transcription of stx1 but not of stx2. However, baicalein had no effects on production or secretion of Stx1 or Stx2. Docking models suggested that baicalein formed a stable structure with StxB pentamer with low intramolecular energy. The results demonstrate that inhibitory activity of baicalein against the cytotoxicity of both Stx1 and Stx2 might be due to of the formation of a binding structure inside the pocket of the Stx1B and Stx2B pentamers. [ABSTRACT FROM AUTHOR]

Chicken meats are considered as main sources associated with Salmonella infections in humans. In this study, lytic phages against Salmonella were isolated and examined for their efficacy to control Salmonella . Eighteen lytic phages were isolated from raw chicken skin and gizzard. Five phages belonging to Myoviridae and Siphoviridae families were characterized and selected for bacterial challenge tests. The treatment of raw chicken breast samples contaminated with S. Enteritidis and S. Typhimurium at 8 °C by the cocktail of five phages significantly reduced ( P  < 0.05) viable counts by 1.41 and 1.86 log CFU/piece, respectively. When incubated at 25 °C, the highest reductions of viable counts of S. Enteritidis and S. Typhimurium in the phage-treated samples were 3.06 and 2.21 log CFU/piece, respectively ( P  < 0.05). These data suggested that the phages isolated from raw chicken meats are potential agents for controlling Salmonella in raw meats. [ABSTRACT FROM AUTHOR]

Extended Spectrum Beta-Lactamase-producing Escherichia coli (ESBLEC) were isolated from 12/13 (92.3%) raw chicken meat samples by using selective culture and PCR. Of the 27 ESBLEC analyzed, 33.3% (9/27) of isolates were positive for ESBL of CTX-M group 1 followed by TEM (22.2%), SHV (22.2%), CTX-M group 2 (11.1%), and CTX-M group 9 (11.1%). None of ESBLEC tested were positive for stx 1 , stx 2 , eae , ehxA , saa , and subAB genes of Shiga toxin-producing E. coli . Among 22 isolated phages, phages PBL66-CL1 and PBL116-CS6 infected 21 (77.7%) and 20 (74%) of 27 ESBLEC isolates examined, respectively. The remaining phages lysed less than 50% of the hosts tested. Compared to non-treatment, the treatment of isolate EBL116 in the broth medium with phage cocktail of PBL66-CL1 and PBL116-CS6 at 25 °C and 5 °C after 6 h significantly reduced bacterial viable counts by 5.06 and 1.33 log CFU/mL, respectively. When the treatment was performed on raw chicken meat samples, viable counts of EBL116 were also decreased by 2.02 and 1.67 log CFU/4 cm 2 meat piece after 6 h at 25 °C and 5 °C, respectively. This study demonstrates a high prevalence of ESBLEC in raw chicken meat and possible use of lytic phages as bactericide for controlling ESBLEC. [ABSTRACT FROM AUTHOR]

Abstract: Shiga toxin-producing Escherichia coli (STEC) can cause severe illnesses in humans such as hemorrhagic colitis and hemolytic-uremic syndrome. In this study, we carried out genotypic analysis of the Shiga toxin (stx) gene in 120 clinical isolates of STEC and enterohemorrhagic E. coli (EHEC) from patients in a southern district of Japan. We identified 88 stx1 + and 103 stx2 + strains. We further identified 12 stx1 + and stx2 + isolates expressing little or no Shiga toxin 1 (Stx1) and/or 2 (Stx2) by reversed passive latex agglutination (RPLA) and Vero cell toxicity assays. Among them, 1 strain could not produce Stx1, 8 could not produce Stx2, and 3 strains could produce neither. Two of the latter three strains were of the non-O157 serotype. Most of the Stx RPLA-negative strains belonged to the stx1 /stx2 subtype (11/12, [91.7%]). Our quantitative reverse transcription PCR analysis indicated that the stx genes were not effectively transcribed in the RPLA-negative strains. This is the first report of the isolation of stx-positive strains showing Stx-negative phenotype from stx1 -bearing strains and non-O157 strains. [ABSTRACT FROM AUTHOR]

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. • L. monocytogenes was positive in chicken meat at 53% and 24% in 2012 and 2017. • Some L. monocytogenes isolates were resistant against oxacillin, cefoxitin, fosfomycin, clindamycin and linezolid. • 46.7% (2012) and 82.6% (2017) of L. monocytogenes isolates showed multidrug resistance. • mecA gene was positive by PCR in most of resistant isolates. [ABSTRACT FROM AUTHOR]

Abstract Sodium chloride maintains osmotic pressure of living cells including bacteria. Heat treatment is common for decontamination of bacteria in food. In this study, effects of NaCl on heat resistance of Salmonella Typhimurium were investigated. After cultivation in TSB containing 0.5% (TSB), 4% (4SC) and 8% (8SC) NaCl, S. Typhimurium cells were heated at 60 °C for 20 min. Total viable counts including intact cells and injured but recoverable cells determined by the plating method using TSA of S. Typhimurium cultured in 4SC and 8SC were significantly higher than those of the cells cultured in TSB. Meanwhile, changes of gene transcription were analyzed by DNA microarray. Transcription of genes involved in the colanic acid synthesis largely increased after cultivation in 4SC and 8SC. The amount of colanic acid significantly increased in the cells cultured in 4SC compared to that in M9-glucose medium. After recovery culture for 3 h, the genes involved in the phage shock response strongly up-regulated, suggesting contribution of these gene products in recovery of heat injured cells. The outcome of this study contributes to understand the mechanism of cross protection in Salmonella. Highlights • Salmonella cultured in the presence of 4% and 8% NaCl increased heat resistance. • Transcription of 366 and 463 genes changed under 4% and 8% NaCl, respectively. • Colanic acid production was induced with high concentration of NaCl. • Phage shock proteins seem important for recovery from heat injury under 4% NaCl. [ABSTRACT FROM AUTHOR]

Catechins are major polyphenolic compounds of green tea. To investigate mechanism for antibacterial action of catechins, 11 monoclonal antibodies (MAbs) were raised against a 3-succinyl-epicatechin (EC)-keyhole limpet hemocyanin (KLH) conjugate. Amino acid sequences of variable regions determined for MAbs b-1058, b-1565, and b-2106 confirmed their innovative character. MAb b-1058 strongly interacted with its target substances in the following order of magnitude: theaflavin-3,3′-di- O -gallate (TFDG) > theaflavin-3- O -gallate (TF3G) ≥ theaflavin-3′- O -gallate (TF3’G) > gallocatechin gallate (GCg) > penta- O -galloyl-β- d -glucose (PGG) > epigallocatechin gallate (EGCg), as determined using surface plasmon resonance (SPR) on MAb-immobilized sensor chips. The affinity profiles of MAbs b-1058 and b-2106 to the various polyphenols tested suggested that flavan skeletons with both carbonyl oxygen and hydroxyl groups are important for this interaction to take place. S. aureus cells treated with EGCg showed green fluorescence around the cells after incubation with FITC-labeled MAb b-1058. The fluorescence intensity increased with increasing concentrations of EGCg. These MAbs are effective to investigate antibacterial mechanism of catechins and theaflavins. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic or Shiga toxin-producing Escherichia coli is a food-poisoning bacterium that grows in the intestine to produce Shiga toxin (Stx). In this study, the effects of 20 polyphenols on the cytotoxicity of Stx1 and Stx2 in Vero cells were investigated. Among these, epigallocatechin gallate, butein, isorhapontigenin, hesperetin, morin, luteolin, resveratrol, and rhapontigenin showed inhibitory effects on the cytotoxicity of Stxs at 0.4 mmol/L. Furthermore, Vero cells pre-treated with these polyphenols were resistant to Stx at 0.4 mmol/L. However, luteolin showed the most potent inhibitory and cytoprotective effect against Stxs at 0.08 mmol/L or more. This inhibitory mechanism of luteolin was determined using a cell-free protein synthesis system and quantitative reverse transcription PCR assay to detect depurination of 28S rRNA in Vero cells. Luteolin did not inhibit the cell-free protein synthesis by Stxs, suggesting that the enzymatic activity of the Stx A subunit was not inhibited by luteolin. The depurination of 28S rRNA by Stxs was also investigated in Vero cells. The 28S rRNA depurination by Stxs was suppressed in Vero cells treated with Stxs which had been pretreated with luteolin. These results suggest that luteolin inhibits the incorporation of Stxs into Vero cells. This is the first report to show that luteolin inhibits the cytotoxicity of both Stx1 and Stx2 by inhibiting the incorporation of Stxs into Vero cells. • Luteolin inhibited cytotoxicity of Stxs and protected Vero cells from the toxins. • Luteolin did not suppress the inhibition of cell-free protein synthesis by Stxs. • Stxs pretreated with luteolin suppressed the depurination of 28S rRNA by Stxs in Vero cells. • Luteolin seems to inhibit incorporation of Stxs into Vero cells. [ABSTRACT FROM AUTHOR]

As one major foodborne pathogen, Salmonella can cause serious food poisoning outbreaks worldwide. Bacteriophage therapy is increasingly considered as one of the promising antibacterial agents for the biocontrol of foodborne pathogens. In the current study, a lytic phage STG2 capable of infecting S. enteritidis and S. typhimurium was characterized, and its efficacy in reducing these foodborne pathogens in both planktonic and biofilm forms was evaluated on cabbage and various surfaces. Genomic characterization revealed that phage STG2 was Siphoviridae phage (Epseptimavirus genus) with a dsDNA genome comprising of 114,275 bp and its genome does not contain any genes associated to antibiotic resistance, toxins, lysogeny, or virulence factors. Additionally, phage STG2 exhibited great efficacy in reducing (>2 Log) planktonic cells on cabbage as well as the biofilms formed on cabbage, polystyrene, and stainless steel, suggesting that phage STG2 is capable of simultaneously controlling both S. enteritidis and S. typhimurium contaminations on food and food-related surfaces. • Lytic phage STG2 has high stress tolerance, and a genome free of virulence genes. • STG2 genome encodes many lytic proteins and tail fiber proteins containing chaperone of endosialidase at C-terminal ends. • Phage STG2 showed great antimicrobial and antibiofilm efficacy against S. enteritidis and S. typhimurium. • Phage STG2 has the potential to reduce the risk of Salmonella infection in food. [ABSTRACT FROM AUTHOR]

Clostridium perfringens is a major cause of foodborne disease in developed countries. The aim of this study was to isolate and characterize phages specific to C. perfringens to evaluate the most efficient phage cocktail for the biocontrol of C. perfringens , both in vitro and in curry roux. In this study, four phages were isolated from chicken meat and were morphologically and genetically characterized along with two phages previously isolated in our laboratory that display different host lysis spectra. Phage cocktail CP11, consisting of phages CPQ3, 7, 8, and 10, showed the broadest host range. Electron micrograph images suggested that all four phages belong to the Podoviridae family, and none of them carry any antibiotic resistance or toxin genes. Notably, the phages were stable at various pH values and in curry roux. Cocktails consisting of six, five, and four phages at the same concentrations were examined to determine the most effective phage cocktail. Phage cocktail PC11 significantly decreased the viable count of C. perfringens to a value less than the lower detection limit up to 48 h at both 8 and 37 °C in broth and at 24 °C in the curry roux. These results suggest that phage cocktail PC11 is a promising natural biocontrol agent against C. perfringens in vitro and in curry roux. • Phage cocktail PC11, including CPQ 3, 7, 8, and 10, displayed the broadest lytic spectrum. • Some phage cocktails suppressed the regrowth of resistant bacterial cells. • Phage cocktail PC11 displayed strong lytic activity in broth and curry roux. [ABSTRACT FROM AUTHOR]

Salmonella enterica subsp. enterica serotype Typhimurium (S. Typhimurium) is one of the most prevalent foodborne pathogens responsible for food poisoning and is spread through the consumption of contaminated poultry products. In this study, four antimicrobial peptides (AMPs) with varying hydrophobicity and helical structure-forming tendencies were designed and synthesized based on the amino acid sequences of peptides from egg white hydrolysate. Two of these AMPs, P1R3 (KSWKKHVVSGFFLR) and P1C (KSWKKHVVSGFFLRLWVHKK), exhibited inhibitory activity against S. Typhimurium and compromised its biofilm-forming ability. Investigation of their modes of action revealed that P1R3 and P1C interact with and permeabilize the cytoplasmic membrane of bacteria, leading to membrane potential dissipation, damage to membrane integrity, and consequent bacterial death. P1R3 also bound to S. Typhimurium DNA, resulting in DNA aggregation or precipitation. Moreover, both peptides showed negligible cytotoxicity to Vero cells, and P1C displayed significant antimicrobial activity in chicken meat. Peptides P1R3 and P1C, therefore, have the potential to be developed as promising food preservatives, especially against pathogenic S. Typhimurium. • P1R3 and P1C with higher helicity and hydrophobicity exhibited improved antimicrobial activities. • P1R3 and P1C damaged membrane functions. • P1R3 and P1C exhibited low cytotoxicity to mammalian cells. • P1C reduced viability of S. Typhimurium in chicken meat at 4 °C. [ABSTRACT FROM AUTHOR]

The study was conducted to examine the adhesion inhibition and antibacterial activities by combined use of some selected food additives such as Sucrose Fatty Acid Ester (SE) C18, Gardenia Yellow (GY), Monascus Pigment (MP), Protamine (PT), ε-polylysine (PL) and Milk Serum Protein (MSP), against Salmonella Enteritidis, Salmonella Typhimurium, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus. The adhesion of those pathogenic bacteria was reduced by several combination of food additives compared to that of each of the single use. The combinations decreased the relative adhesion more than 10% compared to that of each of the single use were taken into consideration. The following combinations such as SE18 & GY, PT & MSP, and PL & MSP were effective in inhibiting the attachment of S. Enteritidis onto microtiter plate compared to that of each of the single use. In case of S. Typhimurium, the combination of MSP & MP, SE18 & GY, SE18 & PT, SE18 & PL, SE18 & MP, SE18 & MSP, MP & PT, MSP & PL, GY & PL, and MSP & PT were effective in adhesion inhibition. The combination of SE18 & GY, GY & MSP, and PL & MSP were effective in inhibiting the attachment of P. aeruginosa onto microtiter plate compared to that of each of the single use. The combination of GY & MSP, MSP & PT or PL and MP & PL were effective to inhibit the attachment of L. monocytogenes. The adhesion of S. aureus was reduced by combined use of SE18 & PT, GY & PT, GY & PL, MSP & GY, MSP & PL, SE18 & GY, SE18 & MP, and MSP & PT compared to that of each of the single use. On the other hand, there were no such significant changes in viable counts of all pathogenic bacteria tested by using combination of food additives compared to that of each of the single use. However, the viable counts of S. Enteritidis and P. aeruginosa decreased drastically in the presence of PL (0.01%) and PT (1%), respectively and reached to the lower detection limit. In addition, the viable counts of L. monocytogenes decreased around 4 Log in the presence of PL. These results clearly showed that the combination of MSP and PT or PL effectively reduced adhesion of all the Gram-positive and negative pathogens tested on the microtiter plate. The pretreatment of cabbage leaves with combination of PL & MSP was also effective to reduce the viable counts of secondary-contaminated S. Enteritidis on the cabbage leaves by washing with water compared to that of untreated cabbage leaves. [ABSTRACT FROM AUTHOR]

Abstract: Mechanism of inhibitory action of 8 catechins and teaflavin was investigated at low concentration against Shiga-like toxin (Stx). Viability of Vero cells largely decreased in the presence of Stx1 and Stx2 preparations. Cytotoxicity of Stx1 decreased after preincubation with gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) at 100 mg/L. However, the cytotoxicity of Stx2 was not inhibited by the preincubation with catechins and teaflavin tested. The inhibitory activity of GCg and EGCg at 15 mg/L (0.0327 mM) was investigated against Stx preparations with various concentrations. The cytotoxicity of Stx1 at the concentration ranging from 1.6 to 50 ng/mL significantly reduced (p < 0.01) by the preincubation of Stx1 with GCg at 15 mg/L. Similarly, the cytotoxicity of Stx1 at the concentration ranging from 3.1 to 25 ng/mL was significantly reduced (p < 0.01) by the preincubation with EGCg at 15 mg/L. In contrast, GCg and EGCg did not inhibit cytotoxicity of Stx2 at any concentrations tested. On the other hand, EGC showed no significant effects on cytotoxicity of both Stx1 and Stx2 at the same concentrations tested. The pocket sizes formed at the center of the Stx1B and Stx2B pentamers were calculated to be 778Å3 and 475 Å3, respectively. Docking simulations were conducted with EGCg positioned in the center of the pore of StxB pentamers. The docking models showed that EGCg formed 7 hydrogen bonds with side chains of amino acids faced inside the pocket of the Stx1B pentarmer with the lowest intramolecular energy (strain energy + electrostatic energy) of −0.1 kcal/mol. In contrast, in the case of Stx2B pentamer, EGCg formed 6 hydrogen bonds with the lowest intramolecular energy of 5.2 kcal/mol. In silico study suggested that EGCg forms more stable structure with Stx1B pentamer than Stx2B pentamer. These results indicated that both GCg and EGCg specifically inhibited cytotoxicity of Stx1 but not of Stx2. [Copyright &y& Elsevier]

Clostridium perfringens is one of the most important foodborne pathogens in developed countries. It causes severe food poisoning outbreaks worldwide, along with mortality and economic losses. Recently, bacteriophages have been investigated as an alternative tool to control pathogenic bacteria in the food industry. In this study, 19 Clostridium perfringens and 6 Clostridium perfringens bacteriophages were isolated from chicken meat. According to host range and stability tests, bacteriophage CPQ1 showed high thermostability and the broadest host range. The electron micrograph image of this bacteriophage suggested that it belongs to the Picovirinae subfamily of the Podoviridae family. Nucleotide sequence analysis of the genomic DNA indicated the absence of any antibiotic resistance, toxin, or virulence genes. In broth, CPQ1 showed strong lytic activity with a low MOI of 1, decreasing the OD 600 of Clostridium perfringens cell suspension from 0.2 to 0.02 at 37 °C in 2 h. In pasteurized milk and chicken meat, CPQ1 with an MOI of 10 also caused a significant decrease in viable counts of Clostridium perfringens compared to the bacteriophageless control at both 24 °C and 37 °C. This is the first report on the application of bacteriophage to control Clostridium perfringens in foods. • All the 19 C. perfringens isolated from meat were identified as C. perfringens type A. • Among 6 phages isolated against C. perfringens , phage CPQ1 showed the broadest host range. • Phage CPQ1 had a double-stranded DNA composed of 18,535 bp, huge burst size and thermostability. • Phage CPQ1 showed strong lytic activity in broth, milk, and chicken meat. [ABSTRACT FROM AUTHOR]

The gene encoding LysSTG2, a novel endolysin from Salmonella- lytic bacteriophage STG2, has been cloned, overexpressed, and characterized previously. In this study, LysSTG2 was used to control Pseudomonas aeruginosa and P. putida , which showed high sensitivity to LysSTG2, in bottled water, milk, chicken breast, salmon, and the biofilms formed on the surface of polystyrene resin and stainless steel. In bottled water, LysSTG2 combined with EDTA reduced the viable counts of P. aeruginosa and P. putida by 2.2 log and to lower than the limit of detection, respectively. However, there was no significant decrease in viable counts in pasteurized milk artificially contaminated with these bacteria. Meanwhile, the addition of 1 mg/mL LysSTG2 alone reduced the viable counts of P. aeruginosa and P. putida by 0.6 log in chicken and 1.1 log in salmon samples contaminated with these bacteria, respectively. Further reduction in viable counts in combination with EDTA was not observed. Additionally, a strong effect of LysSTG2 on the removal of P. aeruginosa and P. putida biofilms was observed on the polystyrene resin and stainless steel surfaces, displaying more than 99.9% decrease in viable biofilm cells after 2 h of treatment at 37 °C. The results of this study suggest that LysSTG2 has the potential to control both planktonic and biofilm cells of Pseudomonas. Further investigations are required to optimize and improve the use of the endolysin for application in food and food processing facilities. [Display omitted] • LysSTG2 was highly effective to planktonic and biofilm cells of Pseudomonas. • LysSTG2 effectively killed the P. aeruginosa and P. putida in bottled water, chicken and salmon. • LysSTG2 showed strong antibiofilm effects against Pseudomonas on polystyrene and stainless steel. [ABSTRACT FROM AUTHOR]

Abstract: We have developed a method for quantifying gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) using a catechin-binding peptide (part of the 67-kDa laminin receptor). Using micro titer plates, we investigated various conditions, including the quantifiable range of EGCg concentrations, the optimal concentration of the catechin-binding peptide, and the optimal reaction conditions. In this microplate assay, after each well was coated with bovine serum albumin, sample containing GCg and EGCg was added at pH 8.0, and allowed to stand at 37 °C for 2 h. After washing, biotinylated-peptide solution was added at 1 μg mL− 1 and allowed to react for 1 h at 37 °C. Each well was added with streptavidin–horseradish peroxidase conjugate, followed by chromogenic reaction for 25 min at room temperature. After the reaction, absorbance was measured at 405 nm. Our method is capable of quantifying EGCg in the range of approximately 0.1–2.0 mg L− 1 with a high degree of sensitivity and a high correlation (R 2 = 0.98) between EGCg concentration and absorbance. The method was specific to GCg and EGCg and seems capable of estimating GCg and EGCg contents in the presence of other catechin compounds. The method is simple and highly sensitive for quantitative GCg and EGCg measurement that requires no special equipment or operation and can measure multiple samples simultaneously. [Copyright &y& Elsevier]