Isolation and Characterization of Bacillus cereus Bacteriophages from Foods and Soil.

The aim of this study was to isolate and characterize Bacillus cereus bacteriophages of various origins. Twenty-seven bacteriophages against B. cereus were isolated from various Korean traditional fermented foods and soils. Plaque size, transmission electron microscopy, virulence profile, and in vitro lytic activity of bacteriophage isolates were examined. Transmission electron microscopy confirmed B. cereus bacteriophages belonging to the family Siphoviridae. Among B. cereus bacteriophages with broad host range, 18 isolates (66.7%) did not harbor any B. cereus virulence factors. Among them, bacteriophage strain CAU150036, CAU150038, CAU150058, CAU150064, CAU150065, and CAU150066 effectively inhibited B. cereus in vitro within 1 h. Therefore, they are considered potential candidates for controlling the contamination of B. cereus in food or other applications.

Comparative sequence analysis of enteroaggregative Escherichia coli heat-stable enterotoxin 1 identified in Korean and Japanese Escherichia coli strains.

The aim of this study was to compare the sequence of the astA gene found in 8 Korean and 11 Japanese Escherichia coli isolates. Conventional PCR was used to amplify the astA gene from the chromosomal and plasmid DNA preparation samples of each isolate using commercial DNA extraction kits. Cloning of the PCR products, sequence analysis, and pulse field gel electrophoresis (PFGE) were sequentially performed. An identical copy of astA in each isolate were found for 8 Korean and 8 Japanese E. coli strains isolated from bovine, porcine, and healthy human carriers. Among these, 1 Korean and 4 Japanese isolates carried a stop mutation at residue 16. Three Japanese outbreak strains (V199, V638, and 96-127-23) carried multiple clones of astA gene with multiple amino acids changes at residues 11, 16, 20, 23, 30, 33, and 34. Compared with the non-diarrheal isolates, clonal diversity and sequence variations of the astA gene in outbreak isolates may be associated with virulence potential of EAST1.

Detection of Foodborne Pathogens and Mycotoxins in Eggs and Chicken Feeds from Farms to Retail Markets.

Contamination by foodborne pathogens and mycotoxins was examined in 475 eggs and 20 feed samples collected from three egg layer farms, three egg-processing units, and five retail markets in Korea. Microbial contamination with Salmonella species, Escherichia coli, and Arcobacter species was examined by bacterial culture and multiplex polymerase chain reaction (PCR). The contamination levels of aflatoxins, ochratoxins, and zearalenone in eggs and chicken feeds were simultaneously analyzed with high-performance liquid chromatography coupled with fluorescence detection after the post-derivatization. While E. coli was isolated from 9.1% of eggs, Salmonella species were not isolated. Arcobacter species were detected in 0.8% of eggs collected from egg layers by PCR only. While aflatoxins, ochratoxins, and zearalenone were found in 100%, 100%, and 85% of chicken feeds, their contamination levels were below the maximum acceptable levels (1.86, 2.24, and 147.53 µg/kg, respectively). However, no eggs were contaminated with aflatoxins, ochratoxins, or zearalenone. Therefore, the risk of contamination by mycotoxins and microbes in eggs and chicken feeds is considered negligible and unlikely to pose a threat to human health.

Simultaneous Detection and Prevalence of Allergens in Anisakis Species Isolated from Marine Fishes.

This study was conducted to develop a multiplex reverse transcription (RT) PCR for the detection of Anisakis allergens and to investigate the relationship between allergen profiles and anisakid larvae isolated from Scomber japonicus, Trichiurus lepturus, and Conger myriaster in Korea. The species of Anisakis was determined using Anisakis pegreffii-specific PCR and restriction fragment length polymorphism analysis. The prevalence and profiles of five Ani s allergens were examined by multiplex RTPCR. A. pegreffii and Anisakis typica accounted for 97.1 and 2.9%, respectively, of the 140 larvae examined. In A. pegreffii, allergen prevalence was 41.2% for Ani s 1, 72.1% for Ani s 2, 69.9% for Ani s 3, 86.7% for Ani s 4, and 93.4% for Ani s 5. Most A. pegreffii larvae had multiple allergen profiles, and 80.7% of A. pegreffii carried both Ani s 4 and Ani s 5, which are heat-resistant allergens. Fifty-two to 65% of A. pegreffii isolated from S. japonicus and C. myriaster carried all five Ani s allergens.

Inhibiting the Growth of Escherichia coli O157:H7 in Beef, Pork, and Chicken Meat using a Bacteriophage.

This study aimed to inhibit Escherichia coli (E. coli) O157:H7 artificially contaminated in fresh meat using bacteriophage. Among 14 bacteriophages, the highly lytic bacteriophage BPECO19 strain was selected to inhibit E. coli O157:H7 in artificially contaminated meat samples. Bacteriophage BPECO19 significantly reduced E. coli O157:H7 bacterial load in vitro in a multiplicity of infection (MOI)-dependent manner. E. coli O157:H7 was completely inhibited only in 10 min in vitro by the treatment of 10,000 MOI BPECO19. The treatment of BPECO19 at 100,000 MOI completely reduced 5 Log CFU/cm(2) E. coli O157:H7 bacterial load in beef and pork at 4 and 8h, respectively. In chicken meat, a 4.65 log reduction of E. coli O157:H7 was observed at 4 h by 100,000 MOI. The treatment of single bacteriophage BPECO19 was an effective method to control E. coli O157:H7 in meat samples.

Detection of viable murine norovirus using the plaque assay and propidium-monoazide-combined real-time reverse transcription-polymerase chain reaction.

Human norovirus (HuNoV) is the most common cause of gastroenteritis worldwide. The lack of a virus culture system makes it difficult to determine the viability of norovirus by only reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative RT-PCR (qRT-PCR). The aim of this study was to investigate the detection of viable murine norovirus (MNV) by combining propidium monoazide (PMA) or ethidium monoazide (EMA) with qRT-PCR. MNV (5.21log10PFU/mL) was subjected to heat treatment at room temperature, 65, 70, 75, 80, 85, or 90°C in a water bath for 1min. The plaque assay, qRT-PCR, PMA-combined qRT-PCR, and EMA-combined qRT-PCR were then performed with heat exposed MNV samples. The MNV titer was reduced by 0.38, 1.34, and 3.71log10PFU/mL at temperatures of 65, 70, and 75°C, respectively. MNV was reduced >4.21log10PFU/mL at 80, 85, and 90°C heat inactivation. PMA (EMA) value equation for the interpretation of the viability of MNV was derived as follows: PMA (EMA) value=-logRN-logRP (RN: the relative quantity value of the not-treated sample, and RP: the relative quantity value of the PMA- or EMA-treated sample as determined by qRT-PCR). By PMA-combined qRT-PCR, the viable PMA value was 0.32, 0.83, and 2.62 for the 65, 70, and 75°C preheated MNVs, respectively. The viable PMA values for the viruses heated at 80, 85, and 90°C were all greater than 3.0, which was the cutoff value for discriminating between live and dead MNVs. The results of EMA-combined qRT-PCR were similar to those of qRT-PCR. Thus, PMA-combined qRT-PCR correlated well with the plaque assay in detecting viable MNVs.