Radish, cress and alfalfa seeds contaminated with Escherichia coli O157 and Salmonella Enteritidis were stored at 4°C for 8 months. As a result, at contamination levels of 104 colony forming units (cfu) /10g, populations of S. Enteritidis in all seeds and those of.E. coli O157 in cress seeds were maintained for up to 8 months. When the seeds were grown, the edible parts of sprouts were heavily contaminated with the microorganisms. Although the population of E.coli O157 in radish and alfalfa seeds had greatly decreased, the microorganism was detected in the edible parts of sprouts from the seeds. At contamination levels of 102 cfu/10g, S. Enteritidis survived in alfalfa seeds at low population levels for up to 8 months and were detected at high population levels in sprouts from the seeds. These results suggested that E. coli O157 and S. Enteritidis could survive in seeds refrigerated for a long time and contaminate the sprouts from the seeds. Furthermore, E. coli O157 and S. Enteritidis were able to grow in the soaking water of seeds. Under a scanning electron microscope, we observed that the surface substrates of seeds were removed after soaking for a short time. Infrared spectroscopy revealed that the substrates were mainly composed of saccharides. These findings suggested that the growth of bacterial pathogens during sprouting of seeds depends on the surface saccharides of the seeds.
In Japan, there have been an increasing number of food poisoning cases caused by pathogenic-bacteria-contaminated foods containing vegetables. Enterohemorrhagic Escherichia coli O157:H7 (O157) and Salmonella are the main causative agents of such poisoning cases. The cases have been caused by not only contaminated food materials but also food contaminatedduring cooking and arrangement. For prevention of such food poisoning, the proliferation of O157, Salmonella Infantis (SI) and Salmonella Enteritidis (SE) were studied in the following conditions. 1. Proliferations of these bacteria in distilled water (DW), physiological saline (PS) and tryptic soy broth (TSB) for 2, 4, 6 and 8 hr at 25, 30 and 35°C. The bacteria could not proliferate in DW or PS but could survive at any temperature tested and proliferated 101-104 times in 8 hr at 30 and 35°C in soy broth. 2. Spinach, lettuce, cucumber, Japanese radish, cabbage, celery, turnips and leeks were cut and soaked, and the water used for soaking (instead of washing) was tested for proliferation. O157 did not proliferate in the water leeks had soaked in. SI could not proliferate in the water that celery, turnips and leeks had soaked in, but proliferated fully in the soak water from other vegetables. 3. Water samples from boiled spinach, qinggeng-cai and cabbage were tested for proliferation of SE and SI. Water samples from boiled qinggeng-cai and cabbage were found to be good media for proliferation of SI, but not for SE.
We studied the incidence of Vibrio parahaemolyticus sporadic diarrhea, the distribution of thermostable direct hemolysin (TDH) gene (tdh) - and TDH-related hemolysin (TRH) gene (trh) -positive V. parahaemolyticus, and the molecular epidemiological relationship between V. parahaemolyticus isolated from patients and the sea environment in the Tohoku district in northeastern Japan. V. parahaemolyticus sporadic diarrhea was prevalent in August 1999 in the Tohoku district. A marked rise in the temperature of sea water was observed in mid July in Aomori, Akita, and Miyagi prefectures, followed by an increase in the number of sporadic V. parahaemolyticus patients, suggesting that the rise in the temperature of sea water could serve as an indication for V. parahaemolyticus sporadic diarrhea cases. Serotyping of 1, 265 V. parahaemolyticus strains isolated from patients across the Tohoku district revealed that O3:K6 was the predominant serotype, representing 86.8% of the total isolates. O3: Kut trh + was isolated from sea mud in Miyagi prefecture, and O3:K6 tdh + and O3:K7 tdh + were isolated from sea mud in Fukushima prefecture, confirming that tdh- and trh-positive V. parahaemolyticus strains are disseminated in the sea environment of the Tohoku district. V. parahaemolyticus O3:K6 tdh + strains isolated from sea mud in Fukushima prefecture showed Type A Notl PFGE pattern, which was the predominant type among strains isolated from patients in Fukushima prefecture, suggesting an etiologic role of environmental V. parahaemolyticus O3:K6 tdh + in human disease. Dynamics of tdh- and trh-positive V. parahaemolyticus strains in the environment should be further elucidated to better understand the epidemiology of V. parahaemolyticus O3:K6 tdh + infection, including the mechanism of food contamination with tdh- and trh-positive V. parahaemolyticus strains from environmental sources.
Rapid and simple methods are required to detect viable pathogenic microbes in foods and drinks. In this study, fluorescent vital staining was used for the rapid enumeration of Escherichia coli O157 with physiologic activity in drinking water. FITC-labeled antibody (FA) was used for the specific detection of target microbes. 5-Cyano-2, 3-ditolyl tetrazolium chloride (CTC) was used to enumerate respiring bacteria. Natural mineral water was inoculated with E. coli O157:H7 cells, and analyzed by both fluorescent microscopy and flow cytometry following double staining with FA and CTC. Respiring E. coli O157:H7 cells inoculated into natural mineral water showed strong fluorescence of both FA (green) and CTC (red), thus they could be clearly and specifically distinguished from respiring indigenous bacteria or inactive cells in natural mineral water. A good correlation was achieved in flow cytometric analysis between the numbers of inoculated viable E. coli O157:H7 and those determined by flow cytometry. Respiring E. coli O157 cells in natural mineral water can be detected specifically by this method within a few hours. This double staining method with flow cytometry may be useful for microbial monitoring of drinking water.
A total of 358 shiga toxin producing Escherichia coli (STEC) isolates recovered from humans between 1987 and 2002 in Shizuoka prefecture were examined to determine the prevalence of serovar, shiga toxin (Stx) type, drug-resistance and phage type (O157). On serological and Stx typing test, the majority (213; 60.7%) of STEC isolates were identified as O157:H7 (130 producing Stx1/2, 80 producing Stx2, and 3 producing Stx1). On antibiotic susceptibility test using 12 drugs (ABPC, TC, SM, CPFX, KM, NA, FOM, CP, CTX, ST, TMP and GM), 116 STEC isolates (36.6%) were resistant to some of the drugs, and they showed 12 drug-resistance patterns. The most prevalent drug-resistance pattern was SM/TC (35;30.2%). 7 STEC 0111 isolates (53.8%) were resistant to 3 to 7 drugs. On phage typing scheme (0157), 222 STEC O157 isolates were classified into 27 phage pes (PT 1, 2, 4, 8, 14, 21, 23, 24, 28, 31, 32, 33, 34, 37, 41, 42, 43, 45, 46, 48, 49, 54, 56, 61, 67, 72 and 74). The most prevalent phage type was PT2 (32;14.4%). Phage types of STEC O157 isolates from the same outbreak (group or familial) were the same. These results suggest that phage typing scheme (O157) is useful in studying causative factors in epidemiological investigations.
An arabinogalactan 3-β-D-galactanohydrolase (EC 3. 2. 1. 90) from a culture broth of Aspergillus fumigatus No. 232 was isolated using ion-exchange chromatography on SP-Sephadex G-50 and gel filtration on Sephadex G-100. The galactanase apparent specific activity increased 19.7-fold after purification. This enzyme migrated as a single band in SDS-PAGE and had a molecular mass of 86, 000. The optimal activity was measured at pH 4.6 and 45°C and the optimal stability was observed at pH 4.0 and below 50°C. All enzymatic activity was inhibited completely by the addition Hg2+ ions. Lineweaver-Burk plots showed a Michaelis constant (Km) of 0.89 and 4.74 mg/m/ and a maximum reaction velocity (Vmax) of 3.16 and 6.54 unit/mg/min for hydrolysis of arabinogalactan from coffee bean and arabinogalactan from larch wood, respectively. This enzyme hydrolysed coffee bean arabinogalactan, larch wood arabinogalactan, gum arabic, and curdlan, all of which contains β- (1→3) and α- (1→3) -linkages in its structure, The hydrolysis products were arabinose, galactose, and galactobiose. It is likely that the substrate selectivity of the enzyme purified from Aspergillus fumigatus No. 232 was less specific to that of other galactanase.