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.     

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.     

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.     

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.     

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.     

Verotoxigenic Escherichia coli (VTEC): A major public health threat in Canada

BACKGROUND: Verotoxigenic Escherichia coli (VTEC) was first described in Canada during the 1980s as an emerging foodborne disease in association with morbidity and mortality in outbreaks of hemorrhagic colitis caused by E coli O157:H7. OBJECTIVE: To describe the surveillance activities and epidemiological laboratory markers of VTEC that are used at the National Laboratory for Enteric Pathogens (NLEP) to investigate sporadic cases and outbreaks of E coli O157:H7 and non-O157 VTEC in Canada. METHODS: Passive surveillance was conducted by obtaining data on laboratory confirmed cases of VTEC from the Provincial Laboratories of Public Health across Canada. The laboratory epidemiological markers generated for isolates of VTEC included biotyping, serotyping, phage typing, toxin detection and characterization, and molecular typing using pulsed-field gel electrophoresis. RESULTS: Major outbreaks of VTEC O157:H7 disease have been associated with ground beef, unpasteurized apple juice, salami and untreated water. In 1999 and 2000, a total of 46 outbreaks of E coli O157:H7 disease were investigated. Among those, one outbreak was associated with contact at a petting zoo and a second with the consumption of salami. An outbreak in 2000 in Ontario was associated with water and resulted in more than 1000 cases of human illness, with six deaths. The NLEP has also identified more than 100 non-O157 VTEC serotypes from cattle and meat products. At least 23 VTEC serotypes found in humans were also identical to those found in cattle and meat products. CONCLUSIONS: The laboratory-based information that is generated is used to define the incidence, sources of infection, risk factors, trends, distribution and transmission of VTEC to humans from food, water and animal sources. Prevention and control of outbreaks are high-priority health concerns.     

Drug sensitivity of vero toxin-producing Escherichia coli O157:H7 (VTEC O157:H7) isolated from human diarrhea and healthy cows

As a part of studies on the source of infection of Vero toxin-producing Escherichia coli (VTEC), O157:H7 strains isolated from human infectious enteritis between 1986 and 1995 and O157:H7 strains isolated from feces of milk cows between 2001 and 2003 were subjected to drug sensitivity test with drugs widely used as therapeutic drugs for various infectious diseases in humans and animals, and the following results were obtained. 1) Drug sensitivity tests with 20 drugs were performed in 52 strains derived human from diarrhea and 100 strains derived from milk cows, and resistance was noted in 115 strains (75.7%): 36 of the 52 human diarrhea-derived strains (69.2%) and 79 of the 100 milk cow-derived strains (79.0%). 2) The human diarrhea-derived strains and milk cow-derived strains were compared with regard to MIC90 of each drug. The antibacterial activity of the drugs was generally higher against the human diarrhea-derived strains than against the milk cow-derived strains. 3) In the 115 strains exhibiting resistance, the most frequent pattern of drug resistance was single drug resistance noted in 80 strains (68.4%), and multidrug resistance was noted in 35 strains (30.4%) consisting of 17 strains with resistance to 3 drugs, 14 strains with resistance to 2 drugs, and 2 strains each with resistance to 4 drugs and 5 drugs. More strains were multidrug-resistant in the milk cow-derived strains.     

浙江省O157大肠杆菌监测及其分子生物学特性

目的了解肠出血性大肠杆菌O157∶H7和其他O157大肠杆菌在浙江省动物、人群中的分布、流行以及PFGE分型、毒力基因携带状况。方法按全国O157∶H7监测方案在5-10月份肠道传染病高发季节,采集全省各地(市)肠道门诊腹泻病人粪便,进行O157大肠杆菌分离培养,并用免疫磁珠分离法对浙江省5个监测点的宿主动物进行O157∶H7分离培养、鉴定,可疑菌株以PCR法检测O157∶H7抗原、志贺样毒素(stx1和stx2)、粘附抹平因子(eaeA)及溶血素(hly)4种毒力基因。用脉冲场凝胶电泳(pulse field gel electrophoresis,PFGE)方法进行同源性分析,同时选择14种抗生素进行药敏试验,并将分析结果与本省首株患者粪便中分离的产志贺样毒素的O157∶H7菌株进行比较。结果全省5个监测点2006年共监测动物粪便标本2377份,分离到4株O157∶H7菌株,阳性率为0.17%;同时在绍兴、舟山肠道门诊腹泻病人粪便中分离到2株O157:H?菌株。4株O157∶H7菌株,stx2、Hly、eaeA均阳性,stx1均阴性;2株O157∶H?菌株仅1株携带eaeA毒力基因。脉冲场凝胶电泳分型显示,4株O157∶H7菌株分3个型,除金华地区2006年分离所得2株完全相似外,其它相同地区不同年代分离的O157∶H7菌株及相同年代不同地区分离的O157∶H7菌株则完全不相似。2株O157∶H?菌株1株PF-GE电泳条带降解,另1株与其它O157∶H7菌株电泳条带差异明显。结论浙江省大肠杆菌0157菌株在动物中以携带stx2毒力基因的O157∶H7菌株为主,但在腹泻患者中则以不带志贺样毒素的O157∶H?菌株为主。不同地区分离的O157∶H7菌株PFGE分型差异明显。羊、奶牛是携带stx2毒力基因的O157∶H7大肠杆菌的主要宿主。各级疾控应加强对宿主动物和腹泻病人大肠杆菌O157的分离监测和流行病学调查。     

Escherichia coli 'O' group serology of a haemolytic uraemic syndrome (HUS) epidemic

This is the first comprehensive serological analysis of a haemolytic uraemic syndrome (HUS) outbreak. A wide range of 'O' group Escherichia coli antibody responses in patients and controls was examined. The study provides a unique insight into the epidemiology of such epidemics, points a way to the most appropriate investigation of these and indicates possible answers to a number of issues related to severity of disease. In order to be able to test for a wide variety of E. coli 'O' antigens, a microagglutination assay was used to examine E. coli 'O' group serological responses of 22 children admitted to hospital with HUS and 14 contemporaneous age-matched controls. A total of 51 'O' serogroup strains were used. These included 'O' groups reported to be associated with cases of HUS, with 6 isolates from patients associated with the Adelaide outbreak (O26, O111, O123 and O157), environmental Verocytotoxigenic/Shiga-toxin producing Escherichia coli (VTEC/STEC) strains and common human commensal strains. Sixteen clinically confirmed HUS cases (72.7%) of 22 seroconverted to 1 or more serogroups of which 11 (50%) seroconverted to O111 (the serogroup isolated from 16 patients). In addition, 11 (50%) and 10 (45.5%) developed antibody to O137 and O145, respectively, although no stool isolates of these serogroups were made. Seventeen (77.3%) of 22 HUS patients had antibody to serogroup O157, with 11 (50%) seroconversions, however, O157:H- was isolated from only 2 of these. Overall, titres ranged from 100 to 6400, some of the highest in 3 patients were against O157, whose faeces yielded only Enterohaemorrhagic E. coli (EHEC) O111, and only 1 developed O111 antibody. Mixed infection was demonstrated serologically by microagglutination (confirmed by Western blot) and was consistent with the findings of multiple serogroups of VTEC found in the mettwurst incriminated as the source, and suggests further strains (not found in the source or in patients' faeces) were probably also involved. In HUS associated with EHEC infection, multiple strain infection may be the rule rather than the exception. A relationship with clinical severity deserves further investigation. Non-O157 EHEC (in addition to O157) should be sought in all future outbreaks of EHEC disease.     

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.     

Epidemiological and bacteriological investigation of enterohemorrhagic Escherichia coli infection in the Chugoku-Shikoku area

We investigated the occurring tendency of enterohemorrhagic Escherichia coli (EHEC) infection in the prefectural and municipal public health institutes in the Chugoku-Shikoku area from 1996 to 1999, and the bacteriological characteristics of EHEC isolated from these cases. Consequently, epidemiological analysis of the EHEC infection in this district was performed. 22 outbreaks in the various facilities showed the tendency occurred in infants and aged groups, and the serotypes of EHEC isolated from these outbreaks were O26, O111 etc. as well as O157. In 4 cases, EHEC were isolated from specimens of buckwheat noodles, salad, sand box, and goat feces, and these were determined as the source of infection. In 898 sporadic cases, including familial infection, the EHEC isolates were classified into 24 serotypes, and the genotypes of EHEC O157:H7 isolates by pulsed-field gel electrophoresis (PFGE) also varied. Moreover, since many asymptomatic carriers were detected in the adult group with familial infection, the existence of healthy carriers is as important as the source of infection. The drug-resistance test of EHEC isolates showed that 24% of the 924 isolates were resistant to drugs.     

Hemolytic-uremic syndrome and Vero cytotoxin-producing Escherichia coli infection in Italy. The HUS Italian Study Group.

In a 3-year prospective study, 49 Italian children with the hemolytic-uremic syndrome (HUS) were examined for evidence of infection with Vero cytotoxin-producing Escherichia coli (VTEC). Diagnosis of infection was established in 37 patients (75.5%) by the combined use of stool examination for VTEC and for free fecal neutralizable Vero cytotoxin and serum analysis for antibodies to the Vero toxins and the lipopolysaccharides (LPS) of three major VTEC serogroups (O157, O26, O111). Anti-LPS antibodies were detected in sera from 30 patients: 25 had antibody to O157 LPS, 4 to O26, and 1 to O111. In as many as 27 patients (55.1%), diagnosis of infection relied only on serologic findings, and the presence of antibody to LPS was the sole evidence of VTEC infection in 20 patients (40.8%). The use of LPS from different E. coli serogroups provided evidence that in Italy O157 strains are the most prevalent VTEC involved in HUS.     

Hemolytic-uremic syndrome associated with enterohemorrhagic Escherichia coli O26:H infection and consumption of unpasteurized cow's milk.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O26 has emerged as a significant cause of hemolytic-uremic syndrome (HUS). The source and the vehicle of contamination with EHEC O26 are not often identified. We report two Austrian cases of HUS due to E. coli O26:H- affecting an 11-month-old boy and a 28-month-old girl in which transmission through unpasteurized cow's milk was positively identified. METHODS AND RESULTS: Using automated ribotyping and pulsed-field gel electrophoresis (PFGE), the isolates (which yielded the virulence genes stx2, eae, and hly) were indistinguishable from each other. An epidemiologic investigation revealed that the children had stayed in the same hotel. Both patients had consumed unpasteurized cow's milk from the breakfast buffet. Fecal samples were taken from the cows of the farm producing the incriminating milk, and one of three cattle EHEC O26:H- isolates had a PFGE pattern indistinguishable from that of the patients' strains. CONCLUSIONS: These two cases of E. coli O26 infection illustrate the hazards associated with the consumption of raw milk, and underline the importance of microbiological diagnostic approaches able to detect sorbitol-fermenting, non-O157 EHEC.     

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.     

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.     

Serotypes, virulence genes, and intimin types of shiga toxin-producing Escherichia coli and enteropathogenic Escherichia coli isolated from mastitic milk relevant to human health in Egypt

Some foodborne pathogens can cause mastitis, in which the organism is directly excreted into milk. Therefore, we undertook the steps to determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) isolates from bovine mastitic milk in Egypt. Forty milk samples from dairy cattle showing mastitis were collected and examined for the presence of E. coli. Following enrichment and plating on selective agar, confirmation of the isolates was based on biochemical tests and the isolates were determined at the species level using cytochrome oxidase, triple sugar iron agar, urea, and indole tests as putatively E. coli. About 77.4% of the isolates belonged to four different O serogroups (O26, O86, O111, and O127). The multiplex polymerase chain reaction (PCR) found that the seven isolates revealed positive amplification of the Eagg gene from the extracted DNA of the E. coli isolates in an incidence of 100%. Also, the selected isolates were subjected to a simple PCR for the detection of 12 of the most important E. coli genes associated with virulence. Those genes detected were stx1, stx2, hylA, Flic(h7), stb, F41, K99, sta, F17, LT-I, LT-II, and eaeA. A total of seven E. coli isolates that were non-O157 isolates were investigated. Among the seven isolates, none was stx positive, and all seven lacked F41, K99, LT-I, LT-II, and Flic(h7). Of these seven isolates, three (42.85%) were enterohemorrhagic E. coli hlyA positive and two (28.57%) were eaeA positive. STEC isolates were not found in bovine mastitic milk in Egypt. Isolates from mastitic milk were potentially pathogenic for human in that they belonged to serogroups associated with diarrhea and hemolytic-uremic syndrome, and some of them were hylA, stb, sta, F17, and eaeA positive.     

Molecular characterization of enterohemorrhagic Escherichia coli O157:H7 isolates dispersed across Japan by pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis

We identified seven distinct subtypes of enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were derived from sporadic cases and outbreaks from multiple prefectures in Japan in 2005. A surveillance system utilizing pulsed-field gel electrophoresis (PFGE), PulseNet Japan, was used. Some strains showed indistinguishable PFGE patterns using another restriction enzyme (BlnI or SpeI) in each subtype of EHEC O157:H7 isolates that were routinely subtyped by the XbaI PFGE pattern. In order to examine the genotypic relatedness of these strains, we carried out a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA). By using the MLVA system, we found that three of seven subtypes of EHEC O157:H7 strains that were isolated from sporadic cases dispersed across multiple prefectures within a few months showed indistinguishable PFGE patterns and identical MLVA types. Strains belonging to the other four subtypes of EHEC O157:H7 in the PFGE analysis were further classified into different clusters of EHEC O157:H7. Therefore, compared to PFGE, MLVA showed greater discriminatory power with respect to analysis of the isolates in this study.     

Escherichia coli O157 infections and unpasteurised milk

We report on two children with Escherichia coli O157 infection, one of whom developed haemolytic uraemic syndrome (HUS). Both had drunk raw cows or goats milk in the week before their illness. Molecular subtyping identified a sorbitol fermenting Escherichia coli O157:H isolate from a dairy cow. This isolate differed from Shiga toxin producing O157:H strains isolated from the 6 year old boy with HUS. This result underlines the need to search for other causes of infection, despite documented consumption of unpasteurised milk. In the second patient, human sorbitol non-fermenting O157:H isolates and animal isolates from goats were indistinguishable. The isolation of indistinguishable sorbitol non-fermenting Escherichia coli O157:H from contact animals supports the association between HUS and consumption of raw goats milk, and re-emphasises the importance of pasteurising milk.     

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 of Shiga toxin-producing Escherichia coli O157:H7 from processed salmon roe associated with the outbreaks in Japan, 1998, and a molecular typing of the isolates by pulsed-field gel electrophoresis

Shiga toxin-producing Escherichia coil (STEC) O157 were isolated from processed salmon roe which had been a suspected food item in sporadic infections which occurred in Japan in 1998. A total of 45 samples of the processed salmon roe were pre-enriched in trypticase soy broth (TSB) at 36 degrees C for 6 h and novobiocin-supplemented modified EC broth (mEC-NB) at 42 degrees C for 18 h. After the pre-enrichments, the cultures were examined for possible occurrence of STEC O157, using an immunomagnetic separation (IMS) method. From the examination, a total of 84 strains of STEC O157:H7 that were positive for both stx 1 and stx 2 genes were isolated. By applying the most-probable-number technique, it was estimated that the number of STEC O157 was in the range of 0.73-1.5 per 10 g of the processed salmon roe. Subsequent analysis of the isolates by a pulsed-field gel electrophoresis (PFGE) revealed a pattern commonly seen in 82 isolates and another pattern in two isolates. Clinical isolates from 7 patients also showed an identical pattern to those of the 82 isolates and one isolate from a patient showed the other pattern identical to those of the two isolates. The isolates were found to belong to the phage type 14.