Jun Ogasawara

An outbreak of gastroenteritis in Osaka, Japan due to Escherichia coli serogroup O166:H15 that had a coding gene for enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1).

In an outbreak of gastroenteritis on 23 July 1996, in Osaka, Japan, 54 of 91 persons who had attended a meeting the previous day became ill. Escherichia coli O166:H15 was isolated from stool specimens of patients (29/33, 88%). Laboratory tests for other bacterial pathogens and viruses were negative. The E. coli 0166 organisms did not adhere to HEp-2 cells in a localized, diffuse, or enteroaggregative manner. The organisms did not express known enterotoxigenic E. coli (ETEC) colonization factors. In polymerase chain reaction tests, the bacteria did not have coding genes for shigatoxin of enterohemorrhagic E. coli (EHEC), heat-labile, or heat-stable enterotoxin of ETEC, attachment and effacement (eaeA) of EPEC, or invasion (invE) of enteroinvasive E. coli (EIEC). Consequently, they could not be assigned to any of the recognized diarrhoeagenic groups of E. coli: EPEC, ETEC, EHEC, EIEC, enteroaggregative E. coli (EAggEC), or diffusely adhering E. coli. However, the organisms possessed the EAggEC heat-stable enterotoxin (EAST1) gene. To our knowledge, this is the first report of an outbreak caused by E. coli that did not have well-characterized virulence genes other than EAST1. The isolates showed the same DNA banding pattern in pulsed-field gel electrophoresis after digestion with the restriction enzymes XbaI or NotI. Three O166:H15 strains isolated from two sporadic cases and another outbreak during 1997-8 were distinct, indicating that multiple clones have spread already. We propose that diarrhoeal specimens should be examined for E. coli possessing the EAST1 gene.

Multiplex real-time PCR for exhaustive detection of diarrhoeagenic Escherichia coli

Aims:  The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real‐time PCR assays for exhaustive detection of DEC.

Interleukin-8 Secretion by Epithelial Cells Infected with Diffusely Adherent Escherichia coli Possessing Afa Adhesin-Coding Genes

Escherichia coli that adhere sparsely to human epithelial (HEp‐2) cells are known as diffusely adherent E. coli (DAEC) and considered potentially diarrheagenic. The role of the afimbrial adhesive sheath (Afa)—identified originally as a uropathogenic factor—in diffuse adhesion is now understood. However, the role of DAEC in diarrheal disease remains controversial. Recently, ability to induce interleukin‐8 (IL‐8) secretion from intestinal epithelial cells has been suggested as one of the properties of enterovirulent bacteria. In this study, we examined whether DAEC strains possessing Afa genes induced IL‐8 in cultures of human carcinoma epithelial cells (e.g., HEp‐2, Caco‐2, and T84). Nineteen afa‐positive DAEC strains were examined for their ability to induce IL‐8 secretion, and only 7 strains (37%; 7/19) induced IL‐8 as much as enteroaggregative E. coli did. No marked differences in adhesion were observed between high and low inducers. Diffusive adhesiveness itself is unlikely to be sufficient to induce IL‐8. All high inducers were motile and others were nonmotile. Additional stimulation by flagella may be required to cause high levels of chemokine induction. Motility or presence of flagella can be an important criterion to predict DAEC diarrheagenicity at clinical laboratories.

Enhancement of Shiga Toxin Production in Enterohemorrhagic Escherichia coli Serotype O157:H7 by DNase Colicins

ABSTRACT Colicins are proteins produced by and active against several strains of Escherichia coli. Previously we reported that colicinogenic bacteria seemed beneficial in preventing the clinical manifestations of infectious disease caused by enterohemorrhagic E. coli O157 in humans. The inhibitory effects could be due to a decrease in O157 levels and/or pathogenicity. This study investigated the effects of colicinogenic E. coli on the production of Shiga toxin (Stx) by O157. Standard strains of colicinogenic bacteria carrying plasmids for each type of colicin (E3/5/8/9) were used for the study. The O157 strains were cultured in the presence of colicinogenic bacteria or extracted colicins. Compared with results for controls, DNase colicins (E8/9) facilitated an 8- to 64-fold increase in production of Stx2, while RNase colicins (E3/5) suppressed Stx production in only two strains. Stx prophages were induced in synchrony with Stx production. Semiquantitative real-time reverse transcription-PCR (RT-PCR) was then performed to examine SOS gene expression. The RT-PCR results clearly indicated a marked increase in mRNA levels of SOS reaction-associated genes after the addition of DNase colicins. We believe that Stx prophages are induced by the SOS response to DNA damage caused by DNase colicins, thus leading to higher Stx production. These findings suggest that while colicinogenic bacteria can be antagonistic to O157 infection, DNase colicins may enhance Stx production. Thus, colicinogenic flora is likely to be involved in the complex pathogenic pathways of O157 infection, and further investigation should be performed before the use of colicinogenic bacteria as an intervention method.

Epidemiology and properties of heat-stable enterotoxin-producing Escherichia coli serotype O169:H41.

Enterotoxigenic Escherichia coli (ETEC) serotype O169:H41 organisms have become the most prevalent ETEC in Japan since the first outbreak in 1991. It was assumed that the outbreaks were due to clonal spread of this new ETEC serotype. The relationship of 32 strains isolated from 6 outbreaks were examined for biotype, antibiotic susceptibility, enterotoxigenicity, protein banding pattern, lipopolysaccharide banding pattern, plasmid analysis, and ribotyping. Further, the strains were examined by haemagglutination, surface hydrophobicity, and the ability to adhere to HEp-2 cells. The present study suggests that the outbreaks were caused by multiple clones of STp-producing O169:H41 since they showed differences in ribotype and outer membrane protein banding patterns. The strains did not agglutinate human or bovine red blood cells in a mannose-resistant manner. They adhered to HEp-2 cells in a manner resembling enteroaggregative E. coli. Five strains were examined by dot-blot tests for the colonization factor antigens CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, PCFO159, PCFO166 and CFA/III. Although four strains expressed CS6, no structure for CS6 was identified. A strain that the anti-CS6 MAbs did not react with could adhere to HEp-2 cells in mannose resistant manner; thus, it is unlikely that CS6 play an important role in the adhesion to the cells. Electron microscopy studies of the O169:H41 strains suggested that curly fimbriae, a possible new colonization factor, may be playing an important role in the adhesion of the bacteria to HEp-2 cells. In conclusion, outbreaks due to ETEC O169:H41 were caused by multiple clones, and the strains should be examined in detail for a possible new colonization factor.

Epithelial Cells Secrete Interleukin-8 in Response to Adhesion and Invasion of Diffusely Adhering Escherichia coli Lacking Afa/Dr Genes

Escherichia coli that sparsely adhere to human epithelial cells are known as diffusely adherent E. coli (DAEC), and the role of the Afa/Dr family of adhesins is now understood. Strains that do not possess Afa/Dr, however, comprise another group of DAEC, of which the pathogenicity remains unknown. The ability to induce interleukin‐8 (IL‐8) secretion from intestinal epithelial cells might be a feature of enterovirulent bacteria. We previously found that some Afa/Dr DAEC strains induce IL‐8 by stimulating epithelial cells with flagella. The present study examines whether non‐Afa/Dr DAEC can induce IL‐8 in epithelial cells (HEp‐2, INT407, and T84). Among 21 strains, 11 (52%; 11/21) induced as much IL‐8 as high inducer strains of Afa/Dr DAEC. Adhesion did not significantly differ between high and low inducers; therefore diffuse adhesion alone is probably insufficient to induce IL‐8. It was shown that IL‐8 induction and the number of intracellular bacteria directly correlated. Wortmannin, an inhibitor of the phosphatidylinositol‐3‐phosphate kinase, reduced both intracellular bacteria and IL‐8 secretion. Motile strains were significantly more prevalent among high (10/11) than low (4/10) inducers. However, 4 low invasive strains hardly induced IL‐8 despite their motility. In conclusion, some non‐Afa/Dr DAEC invoke the induction of high levels of inflammatory cytokines. Unlike Afa/Dr DAEC, however, non‐Afa/Dr strains may require invasion to cause strong induction. These non‐Afa/Dr high inducers can be enteropathogenic for the cytokine‐inducing properties.

Solubilization and characterization of the acceptor for Clostridium botulinum type B neutroxin from rat brain synaptic membranes

The acceptor for Clostridium botulinum type B neurotoxin was solubilized from rat brain synaptic membrane with nonionic detergent, nonanoyl-N-methylglucamide (MEGA-9). The solubilized acceptor was assayed for the binding activity by precipitating the acceptor with acetone in the presence of phosphatidylcholine. 125Ilabeled neurotoxin specifically bound to the lipid vesicles having incorporated the acceptor together with gangliosides. The lipid vesicles having incorporated either the acceptor or gangliosides alone showed extremely low binding activity. The treatment of the solubilized acceptor with lysyl endopeptidase and glycopeptidase F but not with sialidase resulted in decreased toxin binding, indicating that the putative acceptor is a glycoprotein accompanying an N-linked carbohydrate moiety. The observations suggest also that a protein acceptor/ganglioside complex may be required to form the functional toxin receptor.

Association of IL-8-inducing strains of diffusely adherent Escherichia coli with sporadic diarrheal patients with less than 5 years of age

The role of diffusely adherent Escherichia coli (DAEC) in diarrheal disease has been controversial. However, DAEC strains were recently implicated in diarrheal disease in developing countries. To clarify whether DAEC are prevalent among sporadic cases of diarrheal illness in Osaka City, Japan, E. coli strains isolated between July 1997 and March 2000 during diarrheagenic E. coli (DEC) investigation were retrospectively examined. DAEC strains were recognized among 41 (4.4%) of 924 patients and formed the biggest subgroup of DEC. Previously, we reported that some DAEC strains caused epithelial cells to secrete as much IL-8 as enteroaggregative E. coli strains did. In this study, we attempted to evaluate epidemiologically whether the ability of DAEC to induce IL-8 was involved in the pathogenesis. Relationship among patient age, symptoms, Afa adhesins, season and IL-8 induction were examined. The subgroup of DAEC that possessed Afa genes and/or induced a high level of IL-8 was significantly prevalent among patients age 1 to 4 years; however total DAEC was not significantly high among the children compared to other age group. IL-8 inducing DAEC seems to play a role in causing sporadic diarrheal illnesses, particularly in pediatric fields. Investigations highlighting the relationship between IL-8 induction and enteropathogenicity are clearly necessary to confirm the role of DAEC in infectious enteritis.

Molecular typing to trace Listeria monocytogenes isolated from cold-smoked fish to a contamination source in a processing plant

In this study, Listeria monocytogenes contamination in a cold-smoked fish processing plant in Osaka, Japan, was examined from 2002 to 2004. A total of 430 samples were collected and divided into five categories: raw fish, materials during processing, processing equipment, environment, and finished products. A total of 59 finished products were examined throughout this study. L. monocytogenes was isolated from four of these samples during summer and autumn but was not found during winter or spring. During the warmer seasons, L. monocytogenes was more prevalent on processing equipment, especially slicing machines (8 of 54 samples in summer and autumn versus 1 of 50 samples in winter and spring). L. monocytogenes was not detected on whole skins removed from 23 frozen raw fish. L. monocytogenes strains isolated from 56 samples were characterized by serotyping, pulsed-field gel electrophoresis, and three PCR-based methods. Seventy-seven L. monocytogenes strains were recognized as contaminants of the samples: 2 distinguishable strains were identified in each of 13 samples, 3 strains were identified in 2 samples, 5 strains were identified in 1 sample, and the other 40 strains were identified in 40 samples. Combining the results from these techniques, 77 strains were classified into 13 different types. Three of these types prevailed throughout the plant, and two of the three were also isolated from final products. The DNA subtype found in the product was also found on the slicing machines. Our findings suggest that the slicing machines at this plant were the source of the product contamination. Implementing an appropriate cleaning regime for the slicing machines was effective in preventing contamination.

Heat and acid sensitivity of motile Aeromonas : a comparison with other food-poisoning bacteria

The present study was undertaken to compare the heat and acid sensitivity of aeromonads with those of other food-poisoning bacteria. It became obvious that aeromonads were more sensitive to heat than Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella typhimurium. Aeromonads were killed in peptone water within 2 min at 55 degrees C, while the other bacteria survived heating at 55 degrees C for more than 15 min. Aeromonas cells were also less resistant to heat in hamburger steaks. These findings suggest that Aeromonas infection can easily be prevented by heat treatment, although correct handling of food is required to avoid recontamination since aeromonads are very common in various kinds of food. E. coli, S. aureus and S. typhimurium cells survived in buffer at pH 3.2 and in foods seasoned with vinegar. By contrast, Aeromonas cells were found to be highly sensitive to acid. However, the resistance of Aeromonas to acid may be sufficient to allow it to infect the gastrointestinal tract since Vibrio parahaemolyticus, which causes numerous outbreaks of food-poisoning every year in Japan, was susceptible to acid to the same extent as Aeromonas.