Yoshiko Sugita-Konishi

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.

Multivariate Analyses Revealed Distinctive Features Differentiating Human and Cattle Isolates of Shiga Toxin-Producing Escherichia coli O157 in Japan

ABSTRACT Genotypes of Shiga toxin-producing Escherichia coli (STEC) O157 isolated from humans and cattle were analyzed by uni- and multivariable logistic regression, and population structure methods, to gain insight into transmission and the nature of human infection. Eleven genotyping assays, including PCR typing of five virulence factors (stx 1, stx 2, stx 2c, eae, and ehxA) and a lineage-specific polymorphism assay using six markers (LSPA6), were considered in the analyses. The prevalence of the stx 1, stx 2, and stx 2c virulence factors was significantly different between human and cattle isolates. However, multivariable regression revealed that the presence of only the stx 2 gene was significantly associated with human isolates after controlling for confounding effects. LSPA6 typing demonstrated an apparent difference in the distribution of LSPA6 lineages between human and cattle isolates and a strong association between stx genotypes and LSPA6 genotypes. Population genetics tools identified three genetically distinct clusters of STEC O157. Each cluster was characterized by stx genotypes and LSPA6 genotypes. The human isolates typically comprised LSPA6 lineage I with stx 1 stx 2 strains and LSPA6 lineage I/II with stx 2 or stx 2 stx 2c strains. In contrast, the cattle isolates comprised LSPA6 lineage II strains with stx 2c or stx 2 stx 2c strains in addition to the clusters identified for the human isolates. Our analyses provide new evidence that the stx 2 gene is the most distinctive feature in human isolates compared to cattle isolates in Japan, and only a subset of the genetically diverse population isolated from cattle is involved in human illnesses. Our results may contribute to international comparisons and risk assessments of STEC O157.

Virulence Gene Profiles and Population Genetic Analysis for Exploration of Pathogenic Serogroups of Shiga Toxin-Producing Escherichia coli

ABSTRACT Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) is a serious public health concern, causing severe diarrhea and hemolytic-uremic syndrome. Patient symptoms are varied among STEC strains, possibly implying the presence of markers for STEC virulence other than Stx. To reveal the genotypic traits responsible for STEC virulence, we investigated 282 strains of various serogroups for the presence of 17 major virulence genes, i.e., stx 1, stx 2a, stx 2c, stx 2d, stx 2e, stx 2f, eae, tir, espB, espD, iha, saa, subA, ehxA, espP, katP, and stcE. Next, we examined the prevalence of virulence genes according to the seropathotypes in which serotypes were classified (seropathotypes A through E) based on the reported frequencies in human illness, as well as known associations with outbreaks and with severe disease. Our results demonstrate that the presence of both katP and stcE in STEC, in addition to the genes located in the locus of enterocyte effacement (LEE), including eae, tir, espB, and espD, may indicate the most pathogenic genotype of STEC. A population structure analysis of the profiles of virulence genes statistically supported the pathogenic genotype and, furthermore, revealed that there are serogroups with potentially higher pathogenicity than previously thought. Some strains in serogroups O26, O145, and O165 may have high virulence equivalent to that of serogroup O157. Several serogroups, including O14, O16, O45, O63, O74, 119, O128, and O untypeable, also may be potentially pathogenic, although rarely in humans.

Variation in Stress Resistance Patterns among stx Genotypes and Genetic Lineages of Shiga Toxin-Producing Escherichia coli O157

ABSTRACT To evaluate the relationship between bacterial genotypes and stress resistance patterns, we exposed 57 strains of Shiga toxin-producing Escherichia coli (STEC) O157 to acid, freeze-thaw, heat, osmotic, oxidative, and starvation stresses. Inactivation rates were calculated in each assay and subjected to univariate and multivariate analyses, including principal component analysis (PCA) and cluster analysis. The stx genotype was determined for each strain as was the lineage-specific polymorphism assay (LSPA6) genotype. In univariate analyses, strains of the stx 1 stx 2 genotype showed greater resistance to heat than strains of the stx 1 stx 2c genotype; moreover, strains of the stx 1 stx 2 genotype showed greater resistance to starvation than strains of the stx 2 or stx 2c genotypes. LSPA6 lineage I (LI) strains showed greater resistance to heat and starvation than LSPA6 lineage II (LII) strains. PCA revealed a general trend that a strain with greater resistance to one type of stress tended to have greater resistance to other types of stresses. In cluster analysis, STEC O157 strains were grouped into stress-resistant, stress-sensitive, and intermediate clusters. In stx genotypes, all strains of the stx 1 stx 2 genotype were grouped with the stress-resistant cluster, whereas 72.7% (8/11) of strains of the stx 1 stx 2c genotype grouped with the stress-sensitive cluster. In LI strains, 77.8% (14/18) of the strains were grouped with the stress-resistant cluster, whereas 64.7% (11/17) of LII strains were grouped with the stress-sensitive cluster. These results indicate that the genotypes of STEC O157 that are frequently associated with human illness, i.e., LI or the stx 1 stx 2 genotype, have greater multiple stress resistance than do strains of other genotypes.

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.

Reactivity of Secretory IgA Antibodies in Breast Milk from 107 Japanese Mothers to 20 Environmental Antigens

The secretory IgA (sIgA) antibody response to 20 environmental antigens, including microorganisms, toxins, food, and inhaled allergens, was evaluated in the breast milk from 107 Japanese mothers 1–10 days after delivery. Specific sIgA antibody responses were detected in most milk samples against almost all of the antigens tested, although there was a wide variation in the specific sIgA antibody profiles of each individual’s milk. With regard to twelve bacterial antigens, highly specific sIgA antibody responses were detected against Escherichia coli, Yersiniaenterocolitica, and Pseudomonasaeruginosa. With regard to eight nonbacterial antigens, highly specific sIgA antibody responses were detected against rotavirus, cholera, and pertussis toxins. Similar sIgA antibody profiles were obtained when the 107 milk specimens were divided into colostrum (milk 1–5 days after delivery, n = 36) and transitional milk (milk 6–10 days after delivery, n = 71). This study provides information on the possible protective role of human milk sIgA antibodies and will serve as a baseline for future studies.

Penicillium camemberti and Penicillium roqueforti Enhance the Growth and Survival of Shiga Toxin-Producing Escherichia coli O157 under Mild Acidic Conditions

The effects of secondary starter molds of common mold-ripened cheeses on the Shiga toxin-producing Escherichia coli (STEC) O157 were assessed in 3 model systems. In the 1st model, 8 STEC O157 strains were incubated in the spent culture of Penicillium camemberti or Penicillium roqueforti under mild acidic conditions at 25 °C. In the spent cultures of the mold at pH 4.8 to 5.0, the lag times of STEC O157 growth were significantly shorter than those observed in fresh medium. Analyses of the spent culture of P. camemberti showed that the causative agents of the growth enhancement were produced by the mold in response to an acidic environment and were not fully inactivated in heat treatment. In the 2nd model, P. camemberti and STEC O157 were cocultured in acidified milk at 25 °C. The population of STEC O157 reached 10(8) CFU/mL in the presence of the mold, whereas the population steadily declined in the absence of the mold. Although this growth enhancement was partially attributable to alkalization by the mold, it was observed even when the pH of this model was stabilized. In the 3rd model, 2 STEC O157 strains were incubated in the spent cultures of molds at pH 4.5 at 10 °C. In the spent culture, proportions of injured cells were significantly lower and D values were significantly higher than those in control, except one STEC O157 strain in the spent culture of P. camemberti. These results showed that the molds could enhance the growth and survival of STEC O157 by changing the environment. Practical Application:u2003 This study demonstrated that molds in foods can improve the growth and survival of the Shiga toxin-producing Escherichia coli O157. Because microbial interactions are ubiquitous in food, our results provide an important insight for understanding the behavior of microorganisms in food.

Effects of Carrageenans on the Binding, Phagocytotic, and Killing Abilities of Macrophages to Salmonella

The effects of carrageenans (CGNs) on the host defense mechanisms of macrophages against Salmonella infection were examined in vitro by using macrophage-like J774.1 cells. Iota-CGN reduced the Salmonella-binding and phagocytotic activities of J774.1 cells, but it increased the killing activity of the cells. Kappa-CGN increased the binding activity, but reduced the killing ability. CGNs would affect the host defense mechanisms by modulating the macrophage functions.

Spread and change in stress resistance of Shiga toxin‐producing Escherichia coli O157 on fungal colonies

To elucidate the effect of fungal hyphae on the behaviour of Shiga toxin‐producing Escherichia coli (STEC) O157, the spread and change in stress resistance of the bacterium were evaluated after coculture with 11 species of food‐related fungi including fermentation starters. Spread distances of STEC O157 varied depending on the co‐cultured fungal species, and the motile bacterial strain spread for longer distances than the non‐motile strain. The population of STEC O157 increased when co‐cultured on colonies of nine fungal species but decreased on colonies of Emericella nidulans and Aspergillus ochraceus. Confocal scanning microscopy visualization of green fluorescent protein‐tagged STEC O157 on fungal hyphae revealed that the bacterium colonized in the water film that existed on and between hyphae. To investigate the physiological changes in STEC O157 caused by co‐culturing with fungi, the bacterium was harvested after 7 days of co‐culturing and tested for acid resistance. After co‐culture with eight fungal species, STEC O157 showed greater acid resistance compared to those cultured without fungi. Our results indicate that fungal hyphae can spread the contamination of STEC O157 and can also enhance the stress resistance of the bacteria.