[Characterization of enterohemorrhagic Escherichia coli O26 and development of its isolation media].

We studied 101 strains of Enterohemorrhagic Escherichia coli (EHEC) O26 isolated from diarrhea patients in six prefectural institutes of public health in Japan during June 1996 and December 1997 and tried to establish an isolation medium for EHEC O26. None of the 101 EHEC O26 strains fermented rhamnose; Whereas all of the other EHEC including O157 and non-EHEC (166 strains) fermented rhamnose except 1 strain of non-EHEC. All of the randomly selected EHEC O26 (14 strains of O26:H11.2 strains of O26:H-) showed a very high resistance to potassium tellurite (Minimal Inhibitory Concentration (MIC) > or = 50 micrograms/ml), whereas all of the randomly selected non-EHEC (26 strains) but 1 showed a high sensitivity (MIC < or = 6.25 micrograms/ml) to this compound. On the basis of these results, we developed a Rhamnose MacConkey (RMAC) medium in which lactose in the MacConkey medium was replaced by rhamnose, and Cefixime-Potassium Tellurite-RMAC (CT-RMAC) medium in which Cefixime (0.05mg/l) and Potassium Tellurite (25mg/l) was added to RMAC for the isolation of EHEC O26 strains. We then evaluated the specifcity of these selective media by growing a selected number of O26 (24 strains) and 9 selected strains of bacteria. All of the EHEC O26 strains generated rhamnose non-fermented colonies (white color) on both media. In contrast to the EHEC O26, the vast majority of E. coli strains (166/167 = 99.4%) other than EHEC O26 were theoretically assumed to generate red colonies on the RMAC medium because of their rhamnose fermenting character and most of them were assumed not to grow on CT-RMAC medium because of their sensitivity to potassium tellurite. These findings and results indicate that EHEC O26 can be easily distinguished from other strains of E. coli including O157. Although EHEC O26 strains showed somewhat poor growth on CT-RMAC medium compared with that on RMAC medium, these O26 showed almost the same degree of growth on CT-RMAC as they showed on DHL media. The results of the present study demonstrated that the use of RMAC and CT-MRAC media for the isolation of EHEC O26 is very reliable and efficient with RMAC having good sensitivity and CT-RMAC having a better specificity for the isolation of this strain of EHEC.

[Epidemiological characteristics and virulence factor of verotoxin-producing Eschericia coli O121:H19 isolated in Akita Prefecture in July 1997].

Epidemiological characteristics and virulence factors of VTEC O121:H19 strains isolated in July 1997 from a 15 year old female and a 20 year old male patient suffering from bloody diarrhea and severe abdominal pain were examined. The 2 VTEC O121:H19 isolates showed identical antibiotic susceptibility patterns, biochemical characteristics and plasmid profile while slight differences were observed in their Xba I and Not I PFGE patterns, suggesting that closely related 2 VTEC O121:H19 strains evoked the sporadic infectious cases in July 1997. The 2 VTEC O121:H19 isolates, as well as VTEC O157:H7, possessed eaeA gene and a ca. 60 MDa plasmid which hybridised with CVD 419 probe and produced enterohemolysin. In addition, the VTEC O121:H19 isolates produced almost the same amount of VT-2 in vitro as VTEC O157:H7 did. These results suggested that VTEC O121:H19 possesed the virulence factor comparable to that of VTEC O157:H7. Incidence, molecular epidemiology and infectious source of VTEC O121:H19 in this country have not been sufficiently understood. Antiserum for E. coli serogroup O121 should be manufactured to clarify the epidemiology of the highly virulent VTEC strain.

[A familial outbreak of verotoxin-producing Escherichia coli O103:H2 infection in which a calf was the suspected infectious source].

A familial outbreak of Verotoxin-producing Escherichia coli (VTEC) infection occurred in July 1996 in AKITA prefecture. Four VTEC strains harboring VT-1 and eaeA genes were isolated from three patients and a calf, breeding farm for which was located as close as 4 meters from the house where the patients lived in. All of the 4 VTEC isolates were serotyped as O63:H2 using commercially available sera kits. However, a patient isolate, EC-281, was serotyped as 0103:H2 at the International Escherichia and Klebsiella Centre. Titration and absorption tests using rabbit antisera raised against EC-281 confirmed that the serogroup of the remaining 3-VTEC isolates was also O103. Epidemiological characteristics including plasmid profile, antibiotic susceptibility patterns and pulsed-field gel electrophoresis patterns of the 4 VTEC isolates were completely the same, indicating that these isolates originated from a common source. These findings in conjunction with the results of epidemiological survey conducted by the Health Center suggested that a possible infectious source for this outbreak is the calf. Our present results strengthen the significance of calf as an infectious source of VTEC infection.

[Characteristics of enterotoxigenic Escherichia coli and E. coli harboring enteroaggregative E. coli heat-stable enterotoxin-1 (EAST-1) gene isolated from a water-borne outbreak].

A water-borne outbreak occurred in A Town in Akita prefecture on March 1995. Enterotoxigenic Escherichia coli (ETEC) strains were isolated from 6 of 13 feces of patients with food poisoning disease and from 1 of 4 drinking water samples. In addition, E. coli strains harboring Enteroaggregative E. coli (EAggEC) heat-stable enterotoxin-1 (EAST-1) gene were isolated from 5 of 13 patient's feces and 1 feces sample obtained from the septic tank. Both of the E. coli strains were isolated from the 3 patient's feces, suggesting that this outbreak was a mixed infectious case. All of the ETEC strains possessed both heat-stable enterotoxin (ST) and EAST-1 genes and their serotype was O148:H28. The EAST-1 gene was detected on a ca. 80 kb plasmid by a southern blot analysis using EAST-1 DNA probe in the 5 of 7 ETEC strains. The southern blot analysis suggested that the location of the EAST-1 gene was genome in the rest of the 2 ETEC strains. A southern blot analysis using ST DNA probe also suggested that the location of the ST gene was genome in all of the ETEC strains. On the other hand, all of the 6 E. coli strains harboring EAST-1 gene could not be serotyped with commercially available OH sera. The location of the EAST-1 gene in all of the isolates was suggested to be genome by the southern blot analysis. All of the isolates lacked aggA gene which has been demonstrated to be involved in expression of aggregative adherence phenotype in EAggEC, suggesting that the EAST-1 gene-harboring strains isolated in this case were distinct from EAggEC. These results indicated that the EAST-1 gene was also harbored by E. coli strains distinct from EAggEC. In addition, a possibility was also suggested that the EAST-1 gene might be a transposon, as well as ST gene. Further study should be conducted in order to elucidate the significance of EAST-1 as a vilurence factor of diarrheagenic E. coli.

[Enteropathogenic Escherichia coli strains harboring enteroaggregative Escherichia coli (EAggEC) heat-stable enterotoxin-1 gene isolated from a food-borne like outbreak].

A food-poisoning outbreak occurred in G Town in Minami-Akita District, Akita Prefecture on 16 January 1995. As the causative agent of the outbreak, Enteropathogenic Escherichia coli (EPEC) O126:NM were isolated. The isolates showed the same plasmid profile and antibiotic susceptibility patterns suggesting that the EPEC strains originated from same infectious source. The isolates lacked eae and EAF genes which were considered to play a significant role in the diarrheagenic mechanism of EPEC. On the otherhand, the isolates possessed Enteroaggregative E. coli (EAggEC) heat-stable enterotoxin-1 (EAST-1) gene, though they did not possess the agg A gene in which the coded structural subunit of Aggregative adherence fimbriae 1 (AAF/1) has been demonstrated to be involved in the expression of ability for EAggEC to adhere to cultured cells in aggregative pattern, indicating that the EPEC strains apparently differed from EAggEC. These data suggested that EAST-1 showed its enterotoxic activity to human, and that EPEC represented multiple category of E. coli strains with different diarrheagenic mechanism, in which both eae and EAST-1 might be involved.

[Some characteristics of verotoxin-producing Escherichia coli strains isolated from sporadic diarrhea in Akita Prefecture].

Six sporadic cases of VTEC infection have been confirmed in Akita Prefecture from June. 1991 to Nov. 1994. Six VTEC strains isolated in these cases were examined for their serotype, Vero toxin type, existence of eae gene and 60 MDA plasmid, and CVD probe reactivity. Of the 6 isolates, 5 were O157:H7. Two isolates of which possessed TV-1 and VT-2 genes and the rest of 3 possessed VT-1 and VT-2vh genes, VT-2 gene, respectively. One isolate was O26:NM possessing VT-1 gene. A primer pair, designated as EA-1 and EA-2, was designed for detecting eae gene in VTEC. Examination of the 6 VTEC isolates by PCR using EA-1 and EA-2 primers indicated that all of the isolates were eae positive. This was further confirmed by dot blot hybridization using a eae probe. All of the 6 isolates also harbored approximately 60 MDa plasmid, which hybridized with the CVD419 probe in southern blot analysis. These results supported the hypothesis that eae gene and the 60 MDa plasmid might play a role in the mechanism for VTEC to cause diseases including diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans. Diversity observed in plasmid profile and VT type of the 6 isolates suggested that they were originated from independent sources, which could not be identified.

[Characterization by RFLP of DNAs from Campylobacter jejuni Lior serotype reference strains and clinical isolates and detection of C. jejuni by DNA-probe].

The Lior serotype reference strains and clinical isolates of Campylobacter jejuni were compared in the restriction fragment length polymorphism (RFLP) pattern to distinguish C. jejuni strains. These reference strains showed RFLP patterns different from one to another, while the patterns of some isolates were not coincident with those of the same serotype reference strains. Furthermore, we tried to hybridize HindIII-digested fragments from these strains with the DNA probe encoding the 46-kDa protein of C. jejuni by Southern and slot blottings. The 1.8-kbp fragments from all strains of C. jejuni hybridized with this probe, but those from other species of Campylobacter or enterobacteria did not. These results indicate that the Lior serotype is unrelated with the RFLP pattern of DNA of C. jejuni strains, but the DNA probe is useful to detect C. jejuni.

Specific detection of Campylobacter jejuni by means of polymerase chain reaction in chicken litter.

A routine detection assay of Campylobacter jejuni was developed with the polymerase chain reaction (PCR). A pair of oligonucleotide primers from the flaA of C. jejuni were used in detection by PCR. Although the primer pair specifically detected C. jejuni strains, they did not detect strains of C. coli, C. fetus, Salmonella spp. or Escherichia coli. In litter inoculated with C. jejuni, specific detection of C. jejuni was obtained by PCR with this primer pair.

Inhibition of superoxide generation and of increase in intracellular Ca2+ concentration by zinc in rat neutrophils stimulated with zymosan.

The effect of Zn2+ on the O2- generation and change in intracellular Ca2+ concentration ([Ca2+]i) of rat peritoneal neutrophils was studied. Zymosan (serum-treated zymosan (STZ))-induced O2- generation was inhibited by Zn2+ at concentrations as low as 10 microM. A large amount of the inhibition was observed in the absence of extracellular Ca2+ but the inhibition could not be restored by increasing the extracellular Ca2+ concentration, indicating that Zn2+ does not necessarily inhibit the O2- generation competitively with extracellular Ca2+. In the absence of extracellular Ca2+, Zn2+ inhibited STZ-induced transient increase in [Ca2+]i in the concentration range that evoked a marked inhibition in the O2- generation. On the other hand, Zn2+ did not inhibit significantly STZ-induced uptake of 45Ca2+ from extracellular medium by the cells. From these results, it is suggested that Zn2+ inhibits STZ-induced release of Ca2+ from intracellular storage sites, resulting in the suppression of the activation mechanism of neutrophils.

Enhancement of zymosan-induced respiratory burst of rat neutrophils by lead in vitro.

The effect of Pb2+ on serum-treated zymosan (STZ)-induced O2 consumption of rat peritoneal neutrophils was studied. Pb2+ was found to mimic effectively the enhancing action of Ca2+ on the O2 consumption depending on the concentration up to about 80 microM. However, at concentrations over 80 microM, Pb2+ inhibited the O2 consumption. The enhancing effect of Pb2+ on the O2 consumption was further examined using the intracellularly Ca2(+)-depleted neutrophils. Pb2+ also enhanced the O2 consumption of the Ca2(+)-depleted cells as effectively as Ca2+. The Pb2(+)-enhanced O2 consumption of the Ca2(+)-depleted cells was inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) based on its calmodulin antagonistic action. The effect of Pb2+ on the activity of activator-deficient 3',5'-cyclic nucleotide phosphodiesterase (PDE), a calmodulin-dependent enzyme, was examined. Pb2+ activated PDE as effectively as Ca2+ only in the presence of calmodulin. The Pb2(+)-activated PDE activity was also inhibited by W-7 only in the presence of calmodulin. These results indicated that Pb2+ could replace Ca2+ in the activation process(es) of the respiratory burst, suggesting a possible involvement of calmodulin in the enhancing mechanism of the O2 consumption by Pb2+.