Two rare moulds, Helicostylum pulchrum and Scopulariopsis flava, were isolated from a brown stain found on the cut surface of commercially available Brie cheese. Spoilage yeast Debaryomyces hansenii was also present in the same portion. H. pulchrum is psychrophilic but not commonly found as spoilage mycobiota on natural cheese. It grew rapidly on malt extract agar or Brie cheese at 10°C, while D. hansenii and S. flava developed at a moderate and quite slow rate, respectively, under the same condition. To examine the relationship between the contaminants and a discoloration, fungal spike trials were carried out. A reddish or yellowish discoloration was found on the spiked cheese slices by the inoculum of D. hansenii alone or D. hansenii combined with H. pulchrum or/and S. flava. It was not pigments produced by spiked species, but caused by lactic acid bacteria. Concerning H. pulchrum- or D. hansenii-damaged cheeses, cytotoxic assay was done using human neuroblastoma cells. The extracts of these damaged cheeses exhibited no toxicity to the human cells.
A rapid live bacteria counter (Cassette Lab.), with a disposable cassette in which the number of live bacteria is counted rapidly, simply, and accurately, for ensuring food safety, was developed. Cassette Lab. has the following features: (1) Cassette Lab. automatically performs the following processes in the cassette: removing food debris, staining live and dead bacteria, and counting live bacteria by flow cytometry. (2) It uses multistaining (fluorescent-dye combination) optimized to make it easy to distinguish the fluorescence of live bacteria from the different fluorescence of food debris and dye particles. (3) When counting the number of live bacteria in meat and fish, bleaching fluorescent-dye after staining bacteria reduces false positive rates. The live bacteria count in test samples, obtained with Cassette Lab. and that obtained by conventional plating methods, are highly correlated (R (bacteria & yeasts): 0.90; R (vegetables): 0.96; and R (chicken leg): 0.97).
A case of the suspected food poisoning related to deer meat occurred in December, 2011 in Shiga prefecture in Japan. Four of 18 people showed transient diarrhea, abdominal pain, nausea, and vomiting within 5 to 16 hr after eating. No typical food poisonous bacteria and viruses were detected in the food samples. Parasitological tests were performed on the deer meat, and the Ministry of Health, Labour and Welfare of Japan for horsemeat food poisoning were officially notified. A 1,100-bp DNA fragment was amplified by PCR from three slices of the deer meat, suggesting the presence of Sarcocystis sp. Cysts and bradyzoites were detected in the specimens of the deer meat. Immunohistochemical staining of the cysts detected in the deer meat with an antibody against the toxic 15 kDa protein of S. fayeri showed a positive reaction. This indicated that a similar toxic protein originating from Sarcocystis cysts was present in the deer meat. This suggested that the deer meat containing Sarcocystis cysts was the causative food in these cases of food poisoning.
Some species of black Aspergilli have been recently found to produce fumonisin B2 (FB2). Ten strains belonging to three Aspergillus species isolated from moldy dried fruits, which were found to be contaminated by FB2 and ochratoxin A, were assessed for FB2 production on 5% NaCl added Czapek yeast extract agar (5% NaCl CYA) plates and sterilized grape berries. Among them, 4 strains identified as A. niger/A. awamori were found to be able to produce FB2 on 5% NaCl CYA in the range of 12–22 μg/g (average positive: 17 μg/g) and on grape berries in the range of 0.72–13 μg/g (average positive: 5.1 μg/g), respectively. None of 5 strains identified as A. tubingensis produced detectable amounts of the toxin. These findings indicate that A. niger/A. awamori are responsible for the natural occurrence of FB2 in dried fruits. This is the first record of freshly isolated A. niger/A. awamori strains producing FB2 in Japan.
To examine the relation of Campylobacter jejuni isolates to the development of Guillain–Barré syndrome, a total of 120 strains isolated from 56 gastroenteritis patients and 64 samples of chicken meat and giblet were characterized with serological test, PCR-detection of cst-II, cgtA, and cgtB genes associated with ganglioside-like mimicry of lipooligosaccharide (LOS), and antimicrobial susceptibility test. The 24 human-derived strains and the 19 chicken-derived strains were found to represent 12 and 10 different Penner's serogroups, respectively. Those three LOS genes were simultaneously detected from 9 human-derived strains and 9 chicken-derived strains. Among those 18 strains, only 1 chicken-derived strain carried cst-II (Asn51), and the other 17 strains carried cst-II (Thr51). The serogroups of the strains which harbored the three LOS genes were as follows: serogroup C (O : 3), O (O : 19) and R (O : 23, 36, 53), respectively in 3 human-derived strains; serogroup B (O : 2) in a chicken-derived strain; serogroup D (O : 4, 13, 16, 43, 50) in 4 chicken-derived strains. The antimicrobial sensitivity test showed a high frequency of resistance to 4 quinolones (nalidixic acid, norfloxacin, ofloxacin and ciprofloxacin) in 22 of 56 human-derived strains (39.3%) and 22 of 64 chicken-derived strains (34.4%). The frequency of tetracycline resistance was high: 24 of 56 human-derived strains (42.9%) and 16 of 64 chicken-derived strains (25.0%), however, that of fosfomycin resistance was low: 6 of 56 human-derived strains (10.7%) and 4 of 64 chicken-derived strains (6.3%). All 120 strains were sensitive to erythromycin.
We investigated the effectiveness of a loop-mediated isothermal amplification (LAMP) assay to screen meat samples to detect enterohemorrhagic Escherichia coli serovar O157 and O26. Ninety beef rumen samples were cultured in mEC broth at 37°C and noboviocin-containing mEC broth (N-mEC) at 42°C, and then assayed by LAMP reactions. Twenty-eight mEC culture samples were positive by LAMP assay but no samples were positive for EHEC O157 or O26 by the immuno-magnetic separation (IMS) method. Among N-mEC culture samples, 17 samples were positive by LAMP assay and EHEC O157 was isolated from one sample by IMS method, but no sample was positive for EHEC O26 by IMS method. The rate of concordance for negative results between LAMP assay and IMS method (true negative fraction) was 0.8202 for EHEC O157 and 0.8111 for EHEC O26 by N-mEC culturing. By mEC culturing, true negative fraction was 0.6889 for both EHEC O157 and O26. The effectiveness of LAMP assay for screening was also confirmed using retailed meat samples. Four out of 100 retailed meat samples had positive LAMP reactions, and no EHEC O157 or O26 was isolated from the positive samples. For retailed meat samples, true negative fraction was 0.98 for EHEC O157 or O26. These results suggested that LAMP assay was effective for screening meat samples for EHEC O157 and O26.