Acid injured bacteria are sensitive to selective agents. Enumeration of such bacteria in selective media therefore results in lower estimation of viable cell counts. In this study, several antibiotic resistant Escherichia coli O157: H7 strains were constructed and their recovery in acidic conditions on several agar media was validated. Introduction of nalidixic acid or rifampicin-resistant strains did not affect either their growth rate or sensitivity against acidic challenge. Recovery of such resistant strains on tryptic-soy agar (TSA) supplemented with corresponding antibiotic was higher than that of other selective media used, and the use of antibiotic-resistant strains or TSA medium supplemented with corresponding antibiotic did not affect the estimation of recovery. These results suggested that the antibiotic resistant strains could be useful for the challenge tests of acidic food.
The survival of Listeria monocytogenes IID 581 was determined in filtered sterilized artificial seawater (ASW), natural seawater (NSW) and ultra pure water (UPW) microsoms. L. monocytogenes IID581 maintained the culturable state longer at lower temperature in ASW and NSW. However, the pathogen lost culturability in UPW rapidly at all temperatures examined (5, 10 and 30°C). The culturability of L. monocytogenes IID 581 cells was determined by spread plate count (TSAYE), and the cell activity was observed under a fluorescence microscope, using the LIVE/DEAD BacLightTM bacteria viability kit. There were significant differences between the results obtained from those two methods at all temperatures (10, 20 and 30°C) in ASW. Greater differences were seen between the above two methods in ASW at higher temperature, while there was little difference between them in UPW. The results indicate that L. monocytogenes IID581 enters a viable but non-culturable (VBNC) state in ASW and both temperature and the concentration of inorganic salts influence the transition to the VBNC state.
To clarify the route of STEC (Shiga toxin-producing Escherichia coli) infection in humans, we examined the eae (intimin type) and saa genes of STEC strains isolated from humans and various animals. The eae gene was found in 79 of 92 (85 .9%), 44 of 87 (50.6%) and 3 of 9 (33.3%) strains from human, cattle and sheep respectively . Intimin γ was associated with E. coli serogroups O111 and O157, intimin β was found in O26, O119 was associated with α and O103, O121 and O165 showed ε. On the other hand, the saa gene was found in 31.0%, 11.1%, 4.3%and 4.0% of strains from cattle, sheep.human and swine, respectively. The saa gene was detected more frequently in STEC from cattle than in those from any other animals. This epidemiological surveys have revealed that cattle are regarded as the principal reservoir of STEC strains possessing eae and saa genes.
In order to evaluate the index organisms in fecal contamination of food, isolation and analysis of coliforms and Escherichia coli distributed in 191 samples of foods (meats: 53, vegetables: 138) and 47 samples of environmental material (river water: 25, soil: 22) were carried out. The results were as follows. 1) Isolation frequencies of coliforms, fecal coliforms and enteropathogenic E. coli from food were 92, 49 and 0%, respectively, in the case of meat, and 68, 28 and 0%, respectively, in the case of vegetables. Two hundred and six strains of isolated coliforms were identified in more than 16 species. The dominant species were Citrobacter freundii, E. coli, Klebsiella pneumoniae, Klebsiella oxytoca and Enterobacter cloacae amongthe coliforms, and E. coli among the fecal coliforms. Isolation frequencies of E. coli, C. freundii, K. pneumoniae, K. oxytoca and E. cloacae from food were 49, 36, 19, 13 and 17%, respectively, in the case of meat, and 29, 20, 17, 15 and 7%, respectively, in the case of vegetables. The majority of the coliform species, other than E. coli, failed to grow at 44.5°C, but did at 7°C. 2) Cold-adapted coliforms were detected at a frequency of 88% from river water and 73% from soil. The majority of cold-adapted coliforms isolated were identified as K. pneumoniae, K. oxytoca and C. freundii which failed to grow at 44.5°C. Isolation frequencies of cold-adapted C. freundii, K. pneumoniae and K. oxytoca were 12, 28 and 68%, respectively, in the case of river water, and 45, 0 and 32%, respectively, in the case of soil. These results suggest that most of the coliforms distributed in meats and vegetables are cold-adapted K. pneumoniae, K. oxytoca and C. feundii which are also found in the environment.
We investigated the characteristics of 15 gastroenteritis outbreaks due to viruses occurring in the 1999/2000, 2000/2001 and 2001/2002 cold seasons. Norovirus was identified in 14 of 15 outbreaks and one was caused by Sapovirus. The rates of vomiting and nausea were as high as that of diarrhea in Norovirus outbreaks. The rate of vomiting in Sapovirus was inclined to be lower than that due to Norovirus. The modes of transmission were identified as person-to-person contact in 3 cases, consumption of foods contaminated by food handlers in 4 cases and oysters in 3 cases, based on molecular investigations. The predominant probe type in Norovirus was G2F in genogroup II, followed by G2E and G1A, according to the classification of Fukuda et al. The Sapovirus was London type. Seasonal differences in the probe types of Norovirus were found. The involvement of multiple probe types was found in cases caused by the consumption of oysters.
In this investigation we typed Campylobacter isolates by RAPD PCR from the cecum contents of broiler chickens obtained at a slaughterhouse in Shizuoka prefecture. Campylobacter was isolated from 91 (56.9%) out of 160 birds from five out of eight flocks . Of the five Campylobacter positive flocks, three flocks possessed multiple RAPD types of Campylobacter, and two flocks possessed a single type. In the flocks where multiple types of Campylobacter were obtained, all the birds were positive and multiple RAPD types of Campylobacter were isolated from individual broiler samples. In these flocks, multiple episodes of Campylobacter contamination may have occurred . On the other hand, the three Campylobacter negative flocks were considered to have had no Campylobacter contaminations . The differences in Campylobacter colonization of the flocks in this study may, at least in part, reflect the differences in the hygiene measures of the broiler houses in which these birds are raised.
ES Colimark agar contains two chromagenic substrates, 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside (Magenta-GAL) and 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GLUC).Various bacteria were tested for growth and colony color with ES Colimark agar. Also, for enumerating coliforms from viable bacteria, ES Colimark agar and Desoxycholate agar were compared in 413 food samples. Magenta colonies and dark blue to violet colonies that grew on ES Colimark agar were identified. The results of examination are as follows. 1) All gram negative bacteria (45 strains) that included coliforms grew on ES Colimark agar and appeared clearly in colorful colonies. 2) Coliforms were detected from 198 (47.9%) of 413 food samples by ES Colimark agar, significantly more than by Desoxycholate agar 48 (11.6%). 3) Bacteria of 150 magenta colonies that grew on ES Colimark agar and did not grow on Desoxycolate agar were identified as 12 species which included P. agglomerans (43 strains) and E. cloacae (27 strains). 4) Eighty-eight dark blue to violet colonies on ES Colimark agar were identified as E. coli (87 strains, 98.9%) and only 1 strain was not identified. These results confirmed that ES Colimark agar could detect coliforms more broadly than the conventional method. Therefore, ES Colimark agar was considered useful for food safety science.