Susumu Kumagai

Prevalence and contamination levels of Listeria monocytogenes in retail foods in Japan.

Retail foods in Japan were surveyed for the presence and contamination levels of L. monocytogenes. It was isolated from 12.2, 20.6, 37.0 and 25.0% of 41 minced beef, 34 minced pork, 46 minced chicken and 16 minced pork-beef mixture samples, respectively. MPN values were higher than 100/g in five (10.9%) minced chicken samples, but lower than 100/g in all minced beef, pork and pork-beef mixture samples. The organism was also isolated from 5.4% of the 92 smoked salmon samples at MPN values lower than 10/g, and from 3.3% of 213 ready-to-eat raw seafood samples at MPN values from lower than 0.3 to higher than 100/g. None of the 285 vegetable samples were contaminated with L. monocytogenes. These findings indicate that ready-to-eat raw seafoods are relatively high risk among the foods surveyed in this study.

Epigallocatechin gallate and gallocatechin gallate in green tea catechins inhibit extracellular release of Vero toxin from enterohemorrhagic Escherichia coli O157:H7.

We studied the effects of six catechin derivatives (catechin, epigallocatechin, epicatechin, epicatechin gallate, epigallocatechin gallate (EGCg) and gallocatechin gallate (GCg)) in green tea on the production and extracellular release of Vero toxins (VTs) from enterohemorrhagic Escherichia coli (EHEC) cultured at 37 degrees C for 24 h. EGCg and GCg in the culture medium markedly inhibited extracellular VTs release from EHEC cells into the culture supernatant fluid at concentrations of 0.05 mg/ml or higher, as estimated by both the reversed passive latex agglutination assay and cytotoxic assay using Vero cells. Production and extracellular release of maltose binding protein, a periplasmic protein, into the culture supernatant were also inhibited by EGCg and GCg, indicating that their inhibitory effect on release from periplasm into the outer milieu is not specific to VTs, but general to the proteins accumulated in EHEC periplasm.

In vitro effect of deoxynivalenol on the differentiation of human colonic cell lines Caco-2 and T84.

The effects of low concentrations of deoxynivalenol (DON) on structural and functional characteristics of human colonic adenocarcinoma cell lines Caco-2 and T84 were examined. Scanning electron microscopic (SEM) analysis of the apical surfaces of Caco-2 cells revealed reduction or abnormal formation of brush borders in the presence of 50, 100 and 200 ng/ml of DON. Monolayer integrity of Caco-2 and T84 cells was studied using cells which were cultured on permeable membranes. The transepithelial electrical resistance (TEER) of Caco-2 cells was significantly reduced at 50, 100 and 200 ng/ml of DON, significant increase in lucifer yellow (LY) permeability was also observed in these cells at 100 ng/ml of DON. The TEER of T84 cells was significantly reduced at 100 and 200 ng/ml of DON. LY permeability significantly increased at 200 ng/ml of DON in T84 cells. Enzyme activities in Caco-2 cells were also examined. Alkaline phosphatase activity was reduced from the 6th to 15th day of culture in the presense of 100 or 200 ng/ml of DON, whereas sucrase- isomaltase activity was significantly decreased by adding 50 or 100 ng/ml of DON for 15 or 20 days. Protein content was attenuated only by treatment with 200 ng/ml of DON thoughout the experimental period. The results indicate that DON interferes with structural and functional characteristics of differentiation in enterocytes at low doses.

Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef

For the evaluation of plating and immunological methods applicable to the detection of Escherichia coli O157:H7 from ground beef and radish sprouts, a collaborative study was conducted. It focused on a comparison of the efficiency of the plating and immunological methods using various plating agars and immuno-kits in combination with enrichment in modified E. coli broth supplemented with novobiocin (mEC + n), and using immunomagnetic separation. The plating media tested were sorbitol MacConkey agar (SMAC), SMAC supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (CT-SMAC), and agars containing beta-glucuronidase substrates such as BCM O157 and CHROMagar O157. The immuno-kits used were Now E. coli, Path-Stick O157, VIP, EHEC-Tek ELISA System and Rapiblot E. coli O157. The 20 participating laboratories attempted to detect E. coli O157:H7 in 25 g chilled and frozen samples of ground beef uninoculated and inoculated with E. coli O157:H7 at levels of 138.9 and 23.9 cfu/25 g, and in 25 g chilled and frozen samples of radish sprouts uninoculated and inoculated at levels of 20.4 and 1.7 cfu/25 g. E. coli O157:H7 was recovered well from ground beef by all of the methods except direct plating with SMAC. For radish sprouts, the IMS-plating methods with CT-SMAC, BCM O157 and CHROMagar O157 were most efficient at detecting E. coli O157:H7 in more than 90% of the chilled samples inoculated at the level of 20.4 cfu/25 g. All the methods were less sensitive when applied to similar levels of E. coli O157:H7 in radish sprouts (20.4 cfu/25 g) compared with ground beef (23.9 cfu/25 g) especially if the sprouts were frozen. The sensitivity of the immuno-kits appeared to be similar to the IMS-plating methods, but the specificity was lower. Based on the results, we recommend the IMS-plating method using CT-SMAC and agars containing beta-glucuronidase substrate in combination with static enrichment incubation in mEC + n at 42 degrees C.

Escherichia coli O26 detection from foods using an enrichment procedure and an immunomagnetic separation method

H. HARA‐KUDO, H. KONUMA, H. NAKAGAWA and S. KUMAGAI.2000.We found effective enrichment procedures for detecting Escherichia coli O26 in foods using methods that are used for E. coli O157. Ground beef or radish sprouts inoculated with ≈6 colony‐forming units of E. coli O26 were homogenized in 225 ml of various broths. After static incubation at 37 °C or 42 °C for 6 h or 18 h, we isolated the inoculated bacterium by plating onto Rainbow Agar O157 with novobiocin. In combination with the immunomagnetic separation method, E. coli O26 was isolated from all samples by using enrichment in tryptone soy broth at 37 °C for 6 h and in modified E. coli broth with novobiocin (mEC + n) at 42 °C for 18 h in ground beef and radish sprouts, respectively. Enrichment in mEC + n at 42 °C for 18 h was effective for isolating both E. coli O26 and E. coli O157 from both ground beef and radish sprouts.

Detection of Salmonella enteritidis in shell and liquid eggs using enrichment and plating.

Detection methods using various enrichment and plating media and immunoconcentration for Salmonella enteritidis in shell and liquid eggs were evaluated. For liquid egg samples naturally contaminated with S. enteritidis, pre-enrichment in 225 ml of buffered peptone water with cysteine followed by selective enrichment in 10 ml of tetrathionate broth was the superior, resulting in the detection of S. enteritidis in all samples on six of the seven types of selective agar substrate investigated. This enrichment procedure also enabled detection of S. enteritidis in most of artificially inoculated shell egg and pasteurized liquid egg samples.

Comparison of enrichment procedures for isolation of Escherichia coli O157:H7 from ground beef and radish sprouts

Abstract Enrichment procedures using Tryptic soy broth (TSB), modified TSB (mTSB), modified E. coli broth with novobiocin (mEC+n), mTSB (without novobiocin) with vancomycin, cefixime and cefsulodin (mTSB-VCC), or TSB with cefixime, tellurite and vancomycin (TSB-CTV) were evaluated by determining the rate of successful isolation of fifteen Escherichia coli O157:H7 strains from inoculated broth containing ground beef or radish sprout extract. E. coli O157:H7 tended to be isolated more efficiently after enrichment with TSB, mTSB and mEC+n than with the other broths. In order to identify the most efficient enrichmemt condition using these broths, E. coli O157:H7 were inoculated into 25 g ground beef or radish sprouts, which were then homogenized in 225 ml broth and incubated static at 37°C or 42°C for 6 h or 18 h. Attempts were made to isolate the inoculated bacteria by plating method in combination with the immunomagnetic separation method. The most effective enrichment condition was incubation in mTSB or mEC+n at 42°C for 18 h for ground beef, and in mEC+n at 42°C for 18 h for radish sprouts.

The fate and acute toxicity of aflatoxin B1 in the mastomys and rat

The fate and acute toxicity of aflatoxin B1 (AFB1) were studied in the mastomys (Praomys coucha) and compared with Fischer rats. The experiment regarding the fate of [3H]AFB1 showed that the radioactivity was excreted mainly through the feces, more rapidly in the mastomys than in the rat, regardless of whether [3H]AFB1 was given orally or intravenously. The levels of radioactivity bound to the liver DNA were lower in the mastomys than in the rat, indicating that the levels of AFB1 binding to the macromolecules in the liver were lower in the mastomys. Consistent with such differences in the fate of AFB1 between the two species, the mastomys were far more resistent to the acute effects of AFB1 than were the rats. Oral administration of AFB1 at a dose of 1.0 mg/kg to rats caused marked microscopic changes in the liver, involving hepatic necrosis and proliferation of bile ducts, but at a dose of 4.0 mg/kg to mastomys caused no pathological changes in the liver or kidneys, and at a dose of 10.0 mg/kg caused only glycogen deposition in hepatic cells in a limited area. The observed differences in susceptibility to the toxic effects of AFB1 and in the fate of AFB1 between the two species are in accord with our previous finding that liver cytosol in the mastomys inhibits microsome-mediated AFB1-DNA binding in vitro more strongly than in rat liver.

Detection of freeze-injured Escherichia coli O157:H7 cells from foods by resuscitation prior to selective enrichment

We tried to detect Escherichia coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 using an enrichment method with modified EC broth supplemented with novobiocin (mEC + n). When the samples were cultured for enrichment immediately after inoculation of freeze-injured cells, E. coli O157:H7 was not detected in 13 out of 18 samples. However, allowing the food samples to stand for 3 h at room temperature prior to enrichment in mEC + n remarkably improved recovery of E. coli O157:H7 except for some acidic foods. E. coli O157:H7 was detected in the acidic foods by introducing a resuscitation step of 3-h of incubation in a non-selective broth at room temperature prior to enrichment with mEC + n.

Aflatoxin B1-binding proteins in primary cultured hepatocytes of chicken embryo: studies in vivo and in vitro

Among several aflatoxin B1 (AFB1)-binding proteins in primary cultured hepatocytes of chicken embryo, a cytosolic 25 kDa protein proved to be an AFB1-specific binding protein. Binding of AFB1 to a cytosolic protein of the same molecular mass as that seen in vivo was also detectable in a cell-free system and was dependent on the microsomal components. The binding was observed in the in vitro systems derived from AFB1-sensitive animals such as chicken, rabbit, and rat. These findings suggest that the 25 kDa AFB1-binding protein(s) is involved in the intracellular process of AFB1-mediated toxicity.