Prevalence and serotypes of verocytotoxin-producing Escherichia coli (VTEC) isolates from dairy cattle]

To clarify the source of infection and route of transmission of Verocytotoxin-producing Escherichia coli (VTEC) in humans, we collected fresh feces from healthy dairy cattle reared in Hokkaido, Fukushima, Kanagawa and Okinawa prefectures between June 1996 and March 1997, and attempted to isolate VTEC. The results are described below. 1) VTEC was isolated from 68 (27.1%) of 251 fecal samples tested. VTEC was isolated from 14 (28.0%) of 50 in Hokkaido, 13 (26.0%) of 50 in Fukushima, 20 (39.2%) of 51 in Kanagawa and 21 (21.0%) of 100 in Okinawa. There were no difference in the prevalence among the prefectures. 2) Toxin type and serotype of 85 isolates were determined. Thirty-three isolaties (38.8%) were classified into VT1 toxin and VT2 toxin, respectively, and 19 isolates (22.4%) were classified as the strain that produces both VT1 and VT2 toxins. The toxin types of these isolates were divided by serotypes. The VT1-producing isolates were the most frequent among O111:H-. The VT2-producing isolates included O2:H12, O2:H29, O2:H-, O82:H8, O82:HUT, O153:H19, O153:H42 and O153:H-. Among the isolates producing both VT1 and VT2 toxins, O153:H19 was relatively frequent. Based on findings that many bacterial strains coinciding with toxin types and serotypes of human-derived VTEC isolated from dairy cattle, it was suggested that dairy cattle are closely related to VTEC infection in human as a source of infection.     

Biological properties and drug susceptibility of verotoxin-producing Escherichia coli (VTEC) isolated from healthy goats in Okinawa Prefecture

Fecal samples from 116 healthy goats out of 25 randomly selected farms were examined for verotoxin-producing Escherichia coli (VTEC) during 1996 and 1998 in Okinawa Prefecture. VTECs were detected 204 (15.0%) from 1,361 E. coli strains, 36 (31.0%) goats out of 13 (52.0%) farms. Randomly selected 88 strains were further characterized according to VT types, serotypes, virulence markers, biochemical properties and drug susceptibility. VT types were classified as VT1 (46.6%), VT2 (6.8%), and VT1/VT2 (46.6%) by means of reversed latex agglutination test. The VTEC belonged to 18 different O serogroups: O1, O6, O22, O27, O48, O75, O76, O77, O78, O82, O91, O103, O111, O123, O125, O128, O146, and O158. Serotypes O91:H- (13 strains), O27:H- (10 strains), O22:H19 (6 strains) are considered to be predominant, whereas O serotypes O157 and O26 were not isolated. eaeA gene was detected only in 5 strains (5.7%):O103:H2 and O111:H-, in contrast, hlyA gene was found frequent in 45 strains (51.1%) belong to various O serogroups, except for O146 (8 strains). On the basis of 20 biochemical features in all isolates, characteristic patterns were divided into 14 distinct types:47 strains (57.3%) were classified as one type. The VTECs examined were resistant to streptomycin (26.7%), ampicillin (12.2%), kanamycin (8.9%), oxytetracyline (8.9%), and oxolinic acid (3.3%), respectively. The current results indicate that goats harbored VTEC at high frequencies and may be a potential reservoir of human VTEC infection.     

Isolation and serotypes of Vero toxin-producing Escherichia coli (VTEC) from pigeons and crows

To clarify the source and route of infection with Vero toxin-producing Escherichia coli (VTEC) in humans, we sampled gastrointestinal contents and isolated VTEC from wild birds captured to exterminate harmful birds between August 1997 and January 1998. Pigeons were caught in Sagamihara-shi and crows were caught in Sagamihara-shi, Kawasaki-shi, Yokohama-shi, and the Tokyo metropolitan area. The following results were obtained. 1) VTEC was isolated from 32 of 521 birds (6.1%) examined. Among pigeons, VTEC was isolated from 25 of 262 birds (9.5%) captured in Sagamihara-shi. Among crows, VTEC was isolated from 7 of 184 birds (3.8%) captured in Sagamihara-shi, but not isolated from any bird of 11.4, and 60 birds captured in Yokohama-shi, Kawasaki-shi, and the Tokyo metropolitan area, respectively. 2) Toxin was typed in 33 isolates. There were four VT1-producing isolates (6.5%), 27 VT2-producing isolates (88.7%), and two VT1, VT2-producing isolates (4.8%). 3) The serotypes of the isolates were: O78: H-, 10; O152: H-, 7; O153: H19.2; O164: H-, 1; O128: H-, 1; O164/143: H-, and O1: HUT, 1. The serotype was unknown in 10 isolates. Among 10 isolates for which the serotype could not be determined, auto-aggregation was observed in one isolate. 4) EaeA was investigated in the 33 isolates, and 31 isolates (93.9%) possessed eaeA. The above findings showed that strains with same toxin types and serotypes of human diarrhea-derived VTEC were isolated from pigeons and crows, and the isolates frequently possessed eaeA, which is considered to have an important association with its pathology, suggesting that birds are involved in VTEC infection in humans as a source of infection.     

Isolation of vero-cytotoxin-producing Escherichia coli from cattle and serotyping and toxin-typing of the isolated strains]

Two hundred and sixty-six piglets with diarrhea (from 4 farms), 73 healthy pregnant pig (from 2 farms), 27 calves with diarrhea (from 9 farms) and 47 healthy milk cows (from 1 farm) were examined for Vero-cytotoxin-producing Escherichia coli (VTEC), and 52, 11, 15 and 67 strains of VTEC were isolated from 17 piglets, 11 pregnant pigs, 6 calves and 23 milk cows, respectively. All VTEC strains from the piglets produced only VT2vp, while the strains from the healthy pigs did not produce VT2vp, but did VT1 and/or VT2. Most VTEC strains from calves and cows produced VT2vhb and some produced VT2 and VT1. Serotyping of the isolated strains showed that many strains from the piglets belonged either O139:H1, O141:H4 or O141:HUT, but the strains from the pigs were either R-form or O-untypable. Many strains from the calves and cows were serotyped into O116 or O113, but there were several R-form and O-untypable. From these results, it is suggested that VTEC strains, especially from the pregnant pigs, calves with diarrhea and healthy milk cows, which produced the same type of Verotoxins to that produced by human isolates, may become sources of human infections.     

Isolation of verocytotoxin-producing Escherichia coli from cattle and pets]

During a three-year period (1987 & 1990), a comprehensive attempt was made to isolate verocytotoxin-producing Escherichia coli (VTEC) from 96 bovine, 89 porcine, 67 canine and 18 feline cases suffering from gastroenteritis. VTEC were isolated from 11 cows and 1 cat. Bead-ELISA and oligonucteotide probes were used to type the verotoxins (VT) and it was found that the VTEC strains from cows produced not only the currently recognized VT1, VT2 and VT2vh but also produced two new VT's tentatively designated as VTx and VTy. The strain from the cat produced VTy. Serotyping of the strains revealed that some animal strains belonged to similar serotypes as those isolated from human patients with hemorrhagic colitis and hemolytic uremic syndrome. Cattle, especially cows, and domestic pets apparently are reservoirs of VTEC and probable sources of infection in our country as has been previously documented in Canada and USA.     

Vero cytotoxin-producing strains of Escherichia coli in children with haemolytic uraemic syndrome and diarrhoea in Czechoslovakia

Summary The presence of verotoxin-producing strains ofEscherichia coli (VTEC) was examined in six children with haemolytic uraemic syndrome and one child with haemorrhagic colitis. Stools were screened for strains of serogroup O157 on sorbitol-MacConkey agar for VTEC of other serogroups by serotyping. Verotoxin (VT) was tested on Vero cell monolayers: the antigenic variant of VT was assessed by neutralization experiments. Strains producing verotoxin 1 or verotoxin 2 or both were detected in the stools of all seven children. Three strains belonged to serogroup O157 (two of them to serotype O157:H7, one was non-motile) and another five belonged to serogroups O26 (two strains), O1, O5 and O18. The faeces of five children available for testing contained free VT. Production of VT was also examined retrospectively in 32E. coli strains of serotype O26:H11 isolated from children with diarrhoea; eight (25%) of them produced moderate to high levels of verotoxin 1 despite several years storagein vitro. In conclusion, VTEC including strains of serogroup O157 seem to be an important cause of haemolytic uraemic syndrome, haemorrhagic colitis and diarrhoea in children in Czechoslovakia.

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Vero Cytotoxin bildende Stämme von Escherichia coli bei Kindern mit hämolytisch-urämischem Syndrom und Diarrhoe in der Tschechoslowakei

Zusammenfassung Bei sechs Kindern mit hämolytisch-urämischem Syndrom und einem Kind mit hämorrhagischer Kolitis wurde nach Verotoxin bildenden Stämmen vonEscherichia coli (VTEC) gesucht. Das Stuhl-Screening auf Stämme derSerogruppe O157 erfolgte auf Sorbitol-MacConkey Agar; zum Nachweis von VTEC und anderen Serogruppen wurde die Serotypisierung eingesetzt. Verotoxin (VT) wurde auf Monolayer-Verozellkulturen nachgewiesen; die Bestimmung der Antigenvariante von VT erfolgte durch Neutralisationsversuche. Bei allen sieben Kindern konnten im Stuhl Stämme nachgewiesen werden, die Verotoxin 1 oder Verotoxin 2 bildeten. Drei Stämme gehörten der Serogruppe O157 an (zwei davon Serotyp O157:H7, einer war ohne Motilität) und die übrigen fünf gehörten zu den Serogruppen O26 (zwei Stämme), O1, O5 und O18. Freies VT konnte in fünf Stühlen nachgewiesen werden; diese Untersuchung war nur bei fünf Kindern durchführbar. 32E. coli-Stämme vom Serotyp O26:H11, Isolate von Kindern mit Diarrhoe, wurden retrospektiv ebenfalls auf Bildung von VT untersucht. Davon bildeten achtin vitro (25%) noch mittel- bis hohe Spiegel von Verotoxin 1 obwohl sie schon mehrere Jahre lang gelagert waren. VTEC einschließlich der Stämme der Serogruppe O157 stellen folglich wichtige Erreger des hämolytischurämischen Syndroms, der hämorrhagischen Kolitis und anderer Formen der Diarrhoe bei Kindern in der Tschechoslowakei dar.

     

Haemolytic-uraemic syndrome caused by vero toxin-producing Escherichia coli serotype Rough: K-: H49.

The first case of haemolytic-uraemic syndrome (HUS) caused by Vero toxin-producing Escherichia coli (VTEC) which belonged to a novel serotype, Rough: K-: H49, is reported. The case was initially diagnosed as nephropathia epidemica caused by Puumala virus, but the subsequent diagnosis of HUS caused by VTEC was made after bacteriological investigation. The strain isolated fermented sorbitol produced VT2 toxin but not enterohaemolysin, nor did it carry the eaeA gene. In VTEC strains, the O antigen, the eaeA gene and enterohaemolysin production have been characterized as virulence-associated factors and believed to have an effect on pathogenesis of these strains to cause haemorrhagic colitis or HUS. The findings of this study demonstrate that there is a need for further studies to evaluate the pathogenetic mechanism of VTEC and need for easy diagnostic methods exploiting other properties than O157 antigen and non-fermentation of sorbitol to find all VTEC in human infections.     

Isolation and serotyping of Vero toxin-producing Escherichia coli (VTEC) from pig

As a part of basic studies to elucidate the source of infection of Verotoxin-producing Escherichia coli (VTEC) infectious disease, fresh feces were collected from pigs raised in Kanto District (A and B Prefectures) and Kyushu District (C and D Prefectures) between April and October in 2000, and isolation, serotyping, toxin typing, and drug sensitivity test of VTEC were performed. 1) Of 411 fecal samples tested, VTEC was isolated from 44 samples (10.7%), consisting of 12 of 112 samples (10.7%) from A Prefecture, nine of 100 samples (9.0%) from B Prefecture, 18 of 99 samples (18.2%) from C Prefecture, and five of 100 samples (5.0%) from D Prefecture. 2) Forty-five isolates were serotyped. Four isolates (8.9%) were typed as type 3, but the remaining 41 isolates (91.1%) could not be typed. The four typed isolates consisted of two O112ac:H- isolates and one each of O126:H- and O157:H7. 3) Toxin was typed in 45 isolates. Twenty-seven (60.0%) and 17 isolates (37.8%) produced VT 2 and VT1, respectively, and one isolate (2.2%) produced both VT1 and VT2. 4) Drug sensitivity tests of 45 isolates were performed. All 45 isolates (100%) were multidrug-resistant that were resistant to multiple drugs. Nineteen, nine, four, four, seven, one, and one isolates were resistant to five, six, two, three, four eight, and nine drugs, respectively. The above findings confirmed contamination in all districts, although the VTEC isolation rate varied among the sampling districts. Serotyping clarified the presence of O157:H7 and O112ac:H- that are detected in human VTEC infectious disease. The drug sensitivity tests clarified the presence of many multidrug-resistant strains.     

Adhesion to cells in culture and plasmid profiles of enteropathogenic Escherichia coli isolated from outbreaks and sporadic cases of infant diarrhoea

Enteropathogenic strains of Escherichia coli (EPEC) that caused 10 outbreaks of infant diarrhoea in the U.K. between 1968 and 1986 were studied. All gave localised adherence (LA) to HEp-2 cells, HeLa cells and Intestine 407 cells in culture. All hybridised with the EPEC adherence factor (EAF) probe. The hybridising sequences were carried on plasmids ranging in size from 26 to 76 MDa. EPEC from sporadic cases of infant diarrhoea occurring between 1979 and 1986 that belonged to the same serotypes as the outbreak strains were also studied. All strains of serotypes O111ab.H2, O114.H2, O119.H6, O127.H6 and O142.H6 gave LA and were EAF-positive. In other serotypes, non-adhering strains or strains giving diffuse adherence were found also. In addition, strains of serotype O128.H2 which gave LA but did not hybridise with the EAF probe were identified. The strains isolated from sporadic cases of diarrhoea in the U.K. were similar, with respect to adhesion and hybridisation, to those isolated from sporadic cases of diarrhoea in developing countries.     

Verotoxigenic (enterohaemorrhagic) Escherichia coli in infants and toddlers in Czechoslovakia

Summary The results of the investigation indicate that verotoxigenicEscherichia coli (VTEC) belonging to enteropathogenic and other serogroups includingEscherichia coli O157 : H7 or H- are important enteropathogens in infants and toddlers in Czechoslovakia. As to enteropathogenic serotypes, verotoxin (VT) production was proved most frequently in strains of serogroup O26, and also O111 and O128. Diseases caused by them were as a rule manifested by febrile watery diarrhoea with mucus in the stool. In two of five infants withEscherichia coli O26 : H11 with VT1 production in titres of 1 : 512 (blood was present) in the stool and one suffered from marked abdominal pain. In one infant haemorrhagic colitis due toEscherichia coli O157 : H- was found. Haemolytic uraemic syndrome associated with VTEC of serogroups O157, O26, O18, O5 and O1 with VT1 and/or VT2 was observed in six children including five who contracted the disease during an outbreak in a small town, and the source of infection was probably contaminated water. Five children recovered and one died; the postmortem examination revealed haemorrhagic colitis and necrosis of the renal cortex. Haemorrhagic colitis caused byEscherichia coli O157 in infants and toddlers differed from the course hitherto described in older subjects by fever and the presence of mucus in the stools.

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Enterohämorrhagische verotoxigene Escherichia coli bei Säuglingen und Kleinkindern in der Tschechoslowakei

Zusammenfassung Die Ergebnisse der Arbeit zeigen, daß verotoxigeneEscherichia coli (VTEC), die zu enteropathogenen und anderen Serogruppen gehören, einschließlichEscherichia coli O157 : H7 evtl. H-, bedeutende Enteropathogene bei Säuglingen und Kleinkindern in der CSFR darstellen. Unter den enteropathogenen Serotypen wurde die Produktion von Verotoxin (VT) am häufigsten bei Stämmen der Serogruppe O26 und weiter bei O111 und O128 nachgewiesen. Diese Erkrankungen verliefen am häufigsten wie ein fieberhafter wäßriger Durchfall mit Schleimbeimengung. Von fünf Säuglingen mitEscherichia coli O 26 : H11 mit VT1-Produktion im Titer 1 : 512 hatten zwei Blutbeimengungen in Stühlen und in einem Fall wurden starke Bauchschmerzen beobachtet. Ein hämolytisch-urämisches Syndrom im Zusammenhang mit VTEC der Serogruppen O157, O26, O18, O5 und O1 mit VT1 und/oder VT2 wurde bei sechs Kindern beobachtet. Von diesen erkrankten fünf Kinder während einer Epidemie in einer kleinen Stadt und die Ansteckungsquelle war wahrscheinlich kontaminiertes Wasser. Fünf Kinder genasen und ein Kind starb; der Sektionsbefund bewies eine hämorrhagische Kolitis und Nierenrindennekrose. Der Verlauf der vonEscherichia coli O157 hervorgerufenen Kolitiden bei Säuglingen und Kleinkindern unterschied sich vom bisher bei älteren Personen beschriebenen Verlauf durch Fieber und Schleimbeimengungen in den Stühlen.

     

Evaluation of automated ribotyping system for characterization and identification of verocytotoxin-producing Escherichia coli isolated in Japan

The usefulness of an automated ribotyping system (RiboPrinter) was evaluated for characterizing and identifying clinical isolates of 37 verocytotoxin-producing Escherichia coli (VTEC) strains and 16 non-VTEC strains. All strains were successfully ribotyped with satisfactory reproducibility and stability and characterized into 10 different ribogroups. All VTEC O157 strains were characterized into a specific ribogroup and correctly typed into the specific DuPont ID for VTEC O157:H7, while all of the non-VTEC O157 strains were clearly distinguished from VTEC O157. VTEC O26 and O111 strains, the most prevalent VTEC serotypes after O157, were also well characterized into specific ribogroups and identified. These results suggest that the RiboPrinter may have an advantage over other typing systems in that it can rapidly and easily discriminate VTEC from non-VTEC strains of the most prevalent VTEC serotypes in Japan, even though it provides a lesser degree of discrimination than pulsed-field gel electrophoresis (PFGE). With a hierarchical or sequential typing combining the RiboPrinter and PFGE, rapid and accurate typing can be achieved during an outbreak of VTEC, which may be useful in clinical and public health settings.     

Verocytotoxin-producing Escherichia coli infection: The Hong Kong experience

Abstract Infection by verocytotoxin-producing Escherichia coli (VTEC) is prevalent in many parts of the world but relatively uncommon in Asia, except Japan. A territory wide screening for VTEC (April to August 1996) in diarrhoeal stool samples sent to six hospital microbiology laboratories in Hong Kong revealed only four isolates of VTEC and one isolate of E. coli O157:NM in 1003 specimens (incidence 0.5%). Two isolates carrying the verocytotoxin (VT) genes belonged to the O157:H7 serotype while the other two were non-O157. One non-toxigenic E. coli O157:NM was also isolated. All isolates positive for VT genes by polymerase chain reaction (PCR) were also positive for the Vero toxin assayed by the Vero cell culture. The 97 kDa eaeA outer membrane protein gene and 60 MDa fimbrial plasmid pcVD419 were present only in the two O157:H7 isolates. All patients presented with uncomplicated watery diarrhoea; no one suffered from haemorrhagic colitis or the haemolytic uraemic syndrome. All patients recovered uneventfully without antibiotic treatment. Although VTEC infection is still uncommon in Hong Kong, continued surveillance is essential to prevent future outbreaks.     

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.     

The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli

Forty pediatric patients with idiopathic hemolytic uremic syndrome (HUS) were investigated for evidence of infection by Verotoxin-producing Escherichia coli (VTEC). Fecal VTEC (belonging to at least six different O serogroups including O26, O111, O113, O121, O145, and O157) or specifically neutralizable free-fecal Verotoxin (VT) or both were detected in 24 (60%) patients but were not detected in 40 matched controls. Ten of 15 of the former developed fourfold or greater rises in VT-neutralizing antibody titers, as did six other patients who were negative for both fecal VTEC and VT. A total of 30 (75%) patients had evidence of VTEC infection by one or more criteria. We concluded that a significant association exists between idiopathic HUS and infection by VTEC. The detection of free-fecal VT was the most important procedure for the early diagnosis of this infection because, in our study, VTEC were never isolated in the absence of fecal VT, whereas fecal VT was often present even when VTEC were undetectable.     

Detection of verotoxin-producing Escherichia coli using polymerase chain reaction from dairy cattle]

The vero cytotoxin (VT) is responsible for hemorrhagic colitis and hemolytic uremic syndrome. Polymerase chain reaction (PCR) was used to detect VT-producing coliform bacteria from dairy cattle. It was found that 39 (33.3%) of the 117 fecal samples examined were recognized with VT genes in BGLB enrichment broth by the PCR method (named BGLB-PCR). Of the VT-positive samples, 31 samples (26.5%) were found to have VT-producing Escherichia coli. Frequencies of isolation in younger cattle (under 5 months) were 31.3-32.9%. On the other hand, the PCR method using the bacterial suspension of some colonies from DHL selective isolation medium (named DHL-PCR), was used for 105 samples. The DHL-PCR was validated according to the number of colonies tested for detecting VTEC. When using E. coli strains which have been stored after isolation by the conventional culture method, the VT-producing strains found were 7 (10.3%) of the 68 isolates tested. The 101 out of the 108 VTEC strains from cattle were classified into 14 O groups. 4 O serogroups (O26, O111, O145, O157) from 60% of VTEC positive cattle, were also the most common in humans with diarrhea. All E. coli O157:H7 isolates failed to ferment after 48 hrs and to hydrolyze 4-methyl-umbelliferyl-beta-D-glucuronide (MUG). These results suggests that cattle may play an important role in human VTEC infections. The BGL B-PCR technique is usefull in ecological studies for VT-producing pathogens.     

The serotype and Shiga toxin type of Shiga toxin-producing Escherichia coli from humans and various animals

To clear the route of STEC (Shiga toxin-producing Escherichia coli) infection to humans, we examined the serotype. Shiga toxin genotype and eae gene of STEC strains from humans and various animals. The most predominant serotype originated from humans was O157:H7, followed by O26:H11, and other serotypes were O91:H21, O103:H2, O111:NM, O121:H19, etc. The eae gene was found in 79 of 93 strains from human origin. The serotypes of STEC from cattle were significant by similar to that of STEC from humans. The eae gene was found in 44 of 87 strains from cattle. Shiga toxin genotypes possessed O157 strains from humans and cattle, were divided into six groups, stx1, stx2, stx2c, stx1 + stx2, stx1 + stx2c and stx2 + stx2c. Moreover, frequency rates of Shiga toxin genotypes of O157 were also similar to both human and cattle origins. The serotypes of STEC from sheep were also a little similar to that of STEC from humans. Seven of 8 strains from deer possessed stx2d gene that the strains from humans seldom possessed, and none of the strains possessed eae gene. In STEC originated from swine, 15 of 25 strains were O139:H1 that Shiga toxin genotype was stx2e. It was thought that the sources of STEC infection to human are cattle and sheep, and deer and swine had little possibility to human STEC infection.     

Hazard evaluation of livestock-derived, verotoxin-producing Escherichia coli by enterohemolysin production assay and eaeA gene detection

We examined enterohemolysin (Ehly) production, and detected the hlyA gene and the eaeA gene for the intestinal mucosal adherence factor intimin in 131 strains of human-derived verotoxin-producing Escherichia coli (VTEC) and 140 strains of livestock (cattle and swine) -derived VTEC to evaluate their hazards to humans. The hlyA gene was confirmed in 98.5% of human-derived, in 50.5% of cattle-derived, and in 10.3% of swine-derived VTEC strains. Ehly-positive rates were 96.2-97.7%, 45.9-55.0%, and 10.3-20.7% in human-, cattle-, and swine-derived VTEC strains, respectively. Thus, the positive rates differed among strains of different species origins. However, all 12 cattle-derived O157VTEC strains had hlyA, and were Ehly-positive. Although 97.7% of human-derived strains and all cattle-derived O157VTEC strains had eaeA, only 8.1% of cattle derived strains of serotypes other than O157 and 3.4% of swine-derived strains had eaeA. In human- and cattle-derived strains, the presence of eaeA was associated with Ehly: all eaeA-carrying strains had hlyA, and almost all of them were Ehly-positive. Cattle-derived eaeA-carrying strains accounted for 29.5-35.3% of Ehly-positive strains, compared to 100% in human-derived strains. Only 3-4% of Ehly-negative strains had eaeA, and none of the non-hlyA-carrying strains had eaeA. These findings suggest that 2 factors, eaeA and Ehly, serve as useful indicators for the evaluation of hazard to humans, and that Ehly is a useful indicator because cattle-derived Ehly-positive strains may have eaeA.     

Comparison of O-serotype distribution of Escherichia coli isolated from faecal specimens of patients with sporadic diarrhea and healthy persons and regional difference in Japan

To investigate the isolation frequency of O-serotype of Escherichia coli, a total of 1,563 faecal specimens obtained from patients with sporadic diarrhea in Ishikawa between July 1997 and June 1998, were examined. As a result of O-serotyping of isolated strains using commercially E. coli antisera (43 different types), 247 strains of 29 different O-serotypes were isolated. Isolation rate was 15.8%. Most predominant O-serotype was O1 (128 strains, 52%), followed by O18 (26 strains, 11%), O6 (17 strains, 7%), O111 (16 strains, 6%), and these 4 different O-serotypes took up three quarters of the isolated E. coli. Between August 1996 and May 1997, E. coli isolation from faecal samples of 51,893 healthy persons and O-serotyping of isolated strains using commercial antisera to 6 predominant O-serotypes (O-26, 111, 114, 128, 157 and O1) of VTEC/EHEC were carried out. Among 6 O-serotypes, the most predominant O-serotype was O1 (93% of isolates), followed by O26, 111, 128 (6%) and O114, 157 (1%). These isolation frequencies in patients were 80%, 18%, 2%, respectively, have resembled each other in healthy persons in many points. In a similar way, of these distributions of O-serotype of strains hemolysed on Beutin's blood agar plates, we compared patients with healthy persons. Fifty-six strains (3.6% of the total) of E. coli of different O-serotypes were isolated from 1,563 patients and 57 strains (2.8% of the total) belonging to 11 serotypes from 2,036 healthy persons. As a result of O-serotype frequency, both groups resembled each other. O18 and O6, the most predominant O-serotypes, occupied 64% of the isolated strains in patients and 74% in healthy persons. Next in patients, O1, 26 were 7%--level, O28 ac, 152, 157 were 4%--level, respectively, and in healthy persons. O1 was 5%--level, O28 ac, 55, 146, 152 were 4%--level respectively. In the comparison of O-serotype frequency of E. coli isolated from sporadic diarrhea in other 5 areas (Kanto district, Tokyo, Oita, Aichi and Ishikawa), O1, 6, 8, 18, 25, 26, 55, 86a, 111, 125, 126, 127a, 128, 146, 148, 157 and 166 (17 types) have covered a wide area. On the other hand, O29, 44, 78, 112ac, 115, 136, 143, 152, 168 and 169 (10 types) have a tendency to distribute in local areas, we believe that there are regional differences even in the same Japanese territory.     

Vero cytotoxin binding to polymorphonuclear leukocytes among households with children with hemolytic uremic syndrome

Hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in childhood, can be caused by different serotypes of vero cytotoxin (VT; i.e., Shiga toxin)-producing Escherichia coli (VTEC). Recently, VT was shown to bind to polymorphonuclear leukocytes (PMNL) in the systemic circulation of patients with HUS. This study investigated whether VT bound to PMNL could be detected in persons in households with patients with HUS. Serum antibodies against E. coli O157 and, when available, fecal samples from patients with HUS and household members were studied for the presence of VTEC infection. The circulating PMNL of 82% of the household members were positive for VT, whereas stool and/or serum examination showed only 21% positivity. Thus, current methods underestimate the number of infected persons in households with patients with HUS.     

Clonal diversity and virulence factors in strains of Escherichia coli of the classic enteropathogenic serogroup O114

Eighty-eight Escherichia coli strains of the enteropathogenic (EPEC) group O114 that were isolated from humans and animals in geographically different places and over more than 30 years were examined for virulence markers, O:H serotypes, and for electrophoretic types by multilocus enzyme electrophoresis. Four major genetically tightly related clusters of strains showed close correlation between electrophoretic types and other phenotypic characters. Cluster I contained 35 EPEC class II strains of serotypes O114:H9 and O114:H- and 5 enterotoxigenic E. coli belonging to O114:H21 and O114:H49. Clusters II and III comprised 36 O114:H4, O114:H32, and O114:H- strains; most were of doubtful pathogenicity except one Verotoxin-positive O114:H4 strain isolated from a human with diarrhea. Cluster IV contained 9 classic EPEC strains of serogroup O114:H2 that were characterized by localized adherence to HEp-2 cells and by the EPEC adherence factor.