Phage-typing of Vero-cytotoxin (VT) producing Escherichia coli O157 isolated in the United Kingdom

Vero-cytotoxin (VT) producing Escherichia coli serogroup O157 have been isolated from patients with diarrhoea, haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). A phage-typing scheme developed in Canada has been used to type 155 VT+ E. coli O157 serogroup isolated from sporadic infections in the UK since 1983, and 48 strains from HC or HUS outbreaks. Twelve phage types were identified of which three, types 49, 51 and 52, have not been found in North America. All strains carried a 60 x 10(6) plasmid and most VT1+VT2+ strains also had a 5 x 10(6) plasmid coding for colicin D production. The majority of strains producing both VT1 and VT2 belonged to phage type 1, or the related types 4, 8 and 14. Most strains producing only VT2 belonged to types 2 or 49. Four outbreaks were included in the survey. Three had strains of a single phage type while strains from the fourth outbreak were more variable. The distribution of phage types throughout the UK showed no marked geographical variations.     

Characterization of verocytotoxin-producing Escherichia coli O157 isolates from patients with haemolytic uraemic syndrome in Western Europe.

Fifty verocytotoxin (VT)-producing Escherichia coli (VTEC) strains of serogroup O157 were characterized by phage typing, polymerase chain reaction (PCR) for VT genes and the E. coli attaching and effacing (eae) gene, and random amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting. The collection represented isolates obtained from patients with diarrhoea-associated haemolytic-uraemic syndrome (D+ HUS) and their family contacts, isolated in the Netherlands, Belgium and Germany between 1989 and 1993. Based on isolates from separate families (n = 27) seven different phage types were identified, types 2 (44%) and 4 (33%) were predominant. Eighty-five percent of the strains contained only VT2 gene sequences and 15% both VT1 and VT2. All strains of the dominant phage types 2 and 4 carried the VT2 gene. Strains that belonged to the minor phage types 8, 14, 32 carried both VT1 and VT2 genes, with the exception of two isolates identified as phage types 49 and 54 which contained only VT2 genes. All O157 VTEC strains possessed the chromosomally-located eae gene, which indicates its usefulness as virulence marker. RAPD-PCR fingerprinting identified four distinct banding patterns, with one profile found among 79% of the strains. Based on the combined results of all typing methods used in this study, the collection of 50 O157 VTEC strains could be divided into nine distinct groups. Strains isolated from different persons within one family could not be distinguished by any of these methods. The data suggest that O157 VTEC strains are members of one clone that has become widely distributed.     

Cattle as a possible source of verocytotoxin-producing Escherichia coli O157 infections in man.

In May-June 1992 cases of infection with verocytotoxin-producing (VT+) Escherichia coli O157 in South Yorkshire could have been associated with prior consumption of beef from a local abattoir. During investigation of the abattoir, bovine rectal swabs and samples of meat and surface swabs from beef carcasses were examined for E. coli O157, isolates of which were tested for toxigenicity, plasmid content and phage type. E. coli O157 was isolated from 84 (4%) of 2103 bovine rectal swabs; of these 84, 78 (93%) were VT+, the most common phage types being 2 and 8, the types implicated in the cluster of human cases. Positive cattle were from diverse sources within England. E. coli O157 was isolated from 7 (30%) of 23 carcasses of rectal swab-positive cattle and from 2 (8%) of 25 carcasses of rectal swab-negative cattle. The study has shown that cattle may be a reservoir of VT+ E. coli O157, and that contamination of carcasses during slaughter and processing may be how beef and beef products become contaminated and thereby transmit the organism to man.     

Phage typing of Vero cytotoxin-producing Escherichia coli O 157 isolated in the United Kingdom: 1989-91.

Between 1989 and 1991 a total of 1092 Vero cytotoxin-producing Escherichia coli O 157 isolated in the United Kingdom were phage typed in the Laboratory of Enteric Pathogens (LEP). Twenty-three phage types was identified, the most frequent being types 2 (36.1%), 49 (29.6%), 1 (10.3%) and 4 (8.9%). Although isolations of O 157 VTEC have increased each year from 1 in 1982 to 532 in 1991, the predominant phage types have remained unchanged although the proportion of strains belonging to types 2 and 49 has increased. O 157 VTEC from 17 outbreaks were phage typed during this period with phage type 49 predominating (7 of 17 outbreaks).     

Molecular typing methods to investigate transmission of Escherichia coli O157:H7 from cattle to humans.

The utility of phage typing, pulsed-field gel electrophoresis (PFGE), and plasmid profile analysis was compared, to differentiate between Canadian Escherichia coli O157:H7 strains of human (n = 27) and cattle (n = 24) origin. The diversity indices for phage typing, plasmid analysis and PFGE were 0.85, 0.69 and 0.93, respectively. PFGE and phage typing were also applied to study the role of direct transmission of E. coli O157:H7 from cattle to humans on isolates collected from two separate farm outbreaks. PFGE showed that more than one E. coli O157:H7 strain with varying PFGE DNA subtype profiles, may be responsible for an outbreak, and that more than one E. coli O157:H7 subtype may be circulating on a particular farm at any one time. To our knowledge, this is one of the first reports where PFGE typing was used to verify the direct transmission of E. coli O157:H7 from cattle to humans.     

Characterization and epidemiologic subtyping of Shiga toxin-producing Escherichia coli strains isolated from hemolytic uremic syndrome and diarrhea cases in Argentina

Argentina has a high incidence of hemolytic uremic syndrome (HUS); 12.2 cases per 100,000 children younger than 5 years old were reported in 2002. Shiga toxin (Stx)-producing Escherichia coli (STEC) is the primary etiologic agent of HUS, and STEC O157 is the predominant serogroup isolated. The main objective of the present work was to establish the phenotypic and genotypic characteristics of the STEC strains in general isolated from Argentine children during a prospective study and the clonal relatedness of STEC O157:H7 strains using subtyping techniques. One hundred and three STEC strains isolated from 99 children were included. The phenotypic and genotypic features were established, and a polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) was performed to determine stx2 variants. The clonal relatedness of E. coli O157 isolates was established by phage typing and pulsed-field gel electrophoresis (PFGE). The 103 STEC strains belonged to 18 different serotypes, and 59% were of serotype O157:H7. Stx2 was identified in 90.3%, and stx1 in 9.7%. Among the 61 STEC O157 strains, 93.4% harbored the stx2/stx2vh-a genes; PT4 (39.3%) and PT2 (29.5%) were the predominant phage types. Using PFGE with the enzyme XbaI, a total of 41 patterns with at least 80% similarity were identified, and seven clusters with identical profiles were established. Some of the clusters were further split by PFGE using BlnI as the second enzyme. Isolates with indistinguishable PFGE patterns were with one exception also indistinguishable by phage typing and stx genotyping. These findings confirmed that some isolates were genetically related. However, no epidemiological linkages were identified. STEC strains with different genotypes and belonging to diverse serotypes were isolated in Argentina. Some STEC O157 strains could not be distinguished by applying subtyping techniques such as PFGE and phage typing.     

Vero cytotoxin-producing Escherichia coli, particularly serogroup O157, associated with human infections in England and Wales: 1992-4.

Investigations were performed by the Laboratory of Enteric Pathogens on Vero cytotoxin-producing Escherichia coli (VTEC) in England and Wales from 1992-4. Bacterial isolates, faeces and sera obtained from patients with diarrhoea, bloody diarrhoea and haemolytic uraemic syndrome were examined. Using serotyping, Vero cytotoxin gene probing and serodiagnostic tests for E. coli O157, evidence of infection was detected in 543, 434 and 491 individuals in 1992, 1993 and 1994 respectively; VTEC of serogroup O157 were isolated from 470, 385 and 411 cases. The O157 VTEC strains belonged to at least 19 different phage types (PT) although 84% belonged to PT2, PT49, PT8, PT1 or PT4. Antibodies to E. coli O157 lipopolysaccharide were detected in 13% of the cases. The average annual rate of infection with O157 VTEC was 0.83/100000 and 12% of the 1458 individuals with evidence of infection with VTEC or E. coli O157 developed haemolytic uraemic syndrome. There were at least 18 general outbreaks and many family outbreaks.     

E. coli O157 phage type 21/28 outbreak in North Cumbria associated with pasteurized milk

In March 1999, a large community outbreak of Escherichia coli O157 infection occurred in North Cumbria. A total of 114 individuals were reported to the Outbreak Control Team (OCT); 88 had laboratory confirmed E. coli O157. Twenty-eight (32%) of the confirmed cases were admitted to hospital, including three children (3.4%) with haemolytic uraemic syndrome. There were no deaths. A case-control study found that illness was strongly associated with drinking pasteurized milk from a local farm (P = <0.0001) on single variable analysis. Microbiological investigations at the farm revealed E. coli O157 phage type (PT) 21/28 VT 2 which was indistinguishable from the human isolates by pulsed field gel electrophoresis. At the time of occurrence this was the largest E. coli O157 outbreak in England and Wales and the first E. coli O157 PT 21/28 VT 2 outbreak associated with pasteurized milk. This outbreak highlights lessons to be learnt regarding on-farm pasteurization.     

Extended phage-typing scheme for Campylobacter jejuni and Campylobacter coli.

The extended phage-typing scheme described for Campylobacter jejuni and Campylobacter coli has established 46 different phage types using 19 typing phages. Altogether 754 campylobacter isolates, 672 C. jejuni and 82 C. coli, isolated from human and non-human sources received from 17 different countries were phage-typed. Overall, 80.6% of the total isolates were typable. Among typable strains, 9 phage types (3, 5, 10, 11, 18, 19, 23, 26 and 44) represented 57.0% of the strains, 21.3% of the strains belonged to another 37 phage types and the remaining 2.3% of isolates were designated atypical. The most common phage type 11 (140/754) was frequently observed among C. jejuni isolates from human (113/561) and non-human sources (18/111), whereas type 44 was frequent among C. coli isolates from human (22/59) and from non-human sources (8/23). A study of the animal host-associations of common phage types showed that contaminated cattle and poultry appear to be the most common sources of human infection. The greatest variety of phage types was observed in Canada (24 phage types), followed by Portugal (17 types) and the UK (14 types), reflecting the larger sample sizes from these countries. Phage type 11 was encountered in 12 different countries and prevalence of other phage types varied from one country to another. The number of isolates typable with the scheme varied from 93.2% (261/280) in Canada to 61% (47/77) in Thailand. However, the number and diversity of phage types makes phage typing the method of choice in epidemiological studies of campylobacter infections.     

Persistence of Escherichia coli O157:H7 in dairy cattle and the dairy farm environment.

The persistence of Escherichia coli O157:H7 in cattle and the farm environment was investigated on eight Ontario dairy farms positive for E. coli O157:H7 in a longitudinal study commenced one year previously. Faecal samples from cows, calves, humans, cats, rodents, wild birds, a composite fly sample and numerous composite and individual environmental samples were cultured and tested for verotoxin-producing E. coli (VTEC). VTEC isolates were serotyped and E. coli O157:H7 isolates were phage typed. E. coli O157:H7 phage type 34 was isolated from one calf on each of two farms. The same phage type had been isolated on one of these farms 12 months earlier. Most E. coli O157:H7-positive animals and farms became culture-negative within 2 and 3 months, respectively. E. coli O157:H7 was not isolated from any environmental samples, although evidence of VTEC was found in composite samples from calf feeders (19.1%), calf barn surfaces (18%), cow feeders (14.9%), flies (12.5%), cow barn surfaces (11.3%), and individual milk filters (12.5%). VTEC belonging to 21 non-O157 serotypes were isolated from 24 cows (8.2%), 21 calves (18.3%), 2 cow feeder samples (3.0%), and 1 calf feeder sample (4.8%). Shedding of E. coli O157:H7 by infected dairy cattle appears to be transient and persistence of E. coli O157:H7 was not demonstrated from the farm environment sites tested.     

2001年中国食源性致病菌及其耐药性主动监测研究

目的　旨在监测中国食源性致病菌及其耐药性。方法　中国疾病预防控制中心营养与食品安全所建立的全国食品污染物监测网的食源性致病菌监测部分 ,2 0 0 1年在全国 11个省市设点采样并检测了七大类 (生肉、熟肉、生奶、冰激淋、酸奶、水产品和蔬菜 )共 4 0 34份样品。结果　沙门氏菌、大肠杆菌O15 7∶H7和单核细胞增生李斯特氏菌 ,总阳性率 5 5 0 %。其中沙门氏菌 3 32 % ,单核细胞增生李斯特氏菌 1 2 9% ,大肠杆菌O15 7∶H70 82 %。生肉的污染情况最为严重 ,阳性率 12 96 %。共分离出沙门氏菌 137株 ,德比沙门氏菌、阿贡纳沙门氏菌、肠炎沙门氏菌、里定沙门氏菌、鸭沙门氏菌、明斯特沙门氏菌及鼠伤寒沙门氏菌 7种血清型占 80 %。结论　沙门氏菌的血清型分布和耐药性各个省不同。自生、熟肉中分离到O15 7∶H7的强毒株。对分离的沙门氏菌、大肠杆菌检测了 14种抗生素的耐药性 ,结果表明沙门氏菌和大肠杆菌中都有多重耐药株。     

A longitudinal study of Vero cytotoxin producing Escherichia coli in cattle calves in Sri Lanka.

Two cohorts of 10 and 16 calves were followed at weekly or fortnightly intervals from 4-28 and 1-9 weeks respectively to determine whether natural infection by Vero cytotoxin (VT) producing Escherichia coli (VTEC) occurred. Ninety-one of 171 (53%) faecal specimens were VTEC positive and 20-80% of animals at any given time excreted VTEC. Of 104 VTEC strains studied further, 6 different serogroups (O 22.H16; O 25.H5; O 49.H-; O 86.H26; O 88.H25; O 153.H12) and an untypable strain (O? .H21) were identified. All strains belonging to the same serotype had identical profiles of reactivity with DNA probes to toxins VT1 or 2, LTI or II and a probe (CVD419) derived from a plasmid carried by enterohaemorrhagic Escherichia coli O 157.H7. Four of these serotypes were found in the faecal flora of the calves, taken as a group, throughout the 4-month study period. Sixty percent of the strains hybridized with the probe for VT1, 4% with the probe for VT2, and 36% with both probes. Faecal VTEC were significantly associated with overt diarrhoeal illness in animals < 10 weeks of age, but no characteristic profile of markers (serotype or hybridization pattern) in E. coli isolates was associated with diarrhoea. A serological response to VT1 was detected in some animals, but faecal VT1 VTEC excretion persisted in spite of seroconversion. VT1 seroconversion was not associated with diarrhoea. A serological response to VT2 was not detected even in those animals excreting VT2 VTEC in the faeces.     

2007年浙江省衢州地区动物中产志贺毒素大肠埃希菌O157:H7感染状况

目的:了解2007年浙江省衢州地区产志贺毒素大肠埃希菌O157:H7在动物中的分布情况及其耐药性、PFGE分型及毒力基因携带状况。方法:按全国O157:H7监测方案于5~10月份肠道传染病高发季节,在衢州地区采集各种动物粪便/肛拭,用免疫磁珠富集后进行O157:H7分离培养、鉴定,可疑菌株以PCR法检测O、H抗原及志贺样毒素(SLT1和SLT2)、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,分析分离所得菌株的耐药状况。结果:共监测动物粪便标本300份,分离得产志贺毒素大肠埃希菌O157:H7菌株16株,分离率为5.33%。16株O157:H7菌株,毒力基因Hly、eaeA、SLT2均阳性,SLT1均阴性。脉冲场凝胶电泳分型显示,16株O157:H7菌株可分2个PFGE基因型,型间差异较小。耐药性分析显示这些菌株对红霉素、利福平的耐药率最高,达100.0%,对其他受试抗生素均敏感。结论:该地区动物中产志贺毒素大肠埃希菌O157:H7带菌率较高,所分离菌株主要携带SLT2基因,因此推测该地区存在发生产志贺毒素大肠埃希菌O157:H7感染暴发或流行的潜在危险,需增加对动物源性O157:H7的监测力度。     

Prevalence and characteristics of Escherichia coli serotype O157:H7 and other verotoxin-producing E. coli in healthy cattle.

From February to July of 1994, 328 faecal samples from 32 herds were collected and verotoxin-producing Escherichia coli (VTEC) found on 84% of the farms. The proportion of animals infected varied from 0-63%. VTEC were recovered from 52 (20%) of 257 cows and from 16 (23%) of 71 calves. Although the VTEC belonged to 25 different serogroups, 7 (O8, O20, O22, O77, O113, O126 and O162) accounted for 46% of strains. Nearly 45% of the strains. Nearly 45% of the 83 bovine VTEC strains belonged to serogroups associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. However, only 2 (2%) of 83 VTEC strains isolated from cattle belonged to enterohaemorrhagic E. coli (EHEC) serotypes (O26:H11 and O157:H7), and only 8 (10%) were positive for the attaching and effacing E. coli (eae) gene sequence. Polymerase chain reaction (PCR) showed that 17 (20%) of VTEC strains carried VT1 genes, 43 (52%) possessed VT2 genes, and 23 (28%) carried both VT1 and VT2 genes. Characterization of VTEC isolates revelated a heterogeneous population in terms of serogroup and toxin type in the positive herds. This study confirms that healthy cattle are a reservoir of VTEC, but, the absence of eae genes in most bovine VTEC strains suggests that they may be less virulent for humans than eae-positive EHEC.     

四川省大肠杆菌O157∶H7分子流行病学、耐药性及对消毒剂抗性研究

目的研究从四川省分离的大肠杆菌O157∶H7菌株流行病学特征。方法用多重PCR测定67株大肠杆菌O157∶H7的slt、eaeA、hly毒力基因;对不同来源的大肠杆菌O157∶H7进行质粒和PFGE分型;测定67株大肠杆菌O157∶H7的耐药性和对消毒剂的抗力。结果62.7%的株菌(42/67)携带有毒力基因,毒力图谱类型主要为slt1+slt2+eaeA+hly。67株菌共有6种质粒谱型和7种PFGE谱型。64.2%(43/67)的菌株分别对7种不同的抗生素耐药,其中有23、35、34株菌分别对氨苄青霉素、四环素和SMZ耐药。不同来源的大肠杆菌O157∶H7抗性谱不同,80.6%(54/67)的菌株对酒精和季铵盐产生了抗性,所有菌株对洗必泰敏感。结论四川省不同来源的大肠杆菌O157∶H7带毒率相似,但毒力基因谱型不完全相同。细菌有较高的耐药性。菌株对常用消毒剂有很高的抗性,在消毒灭菌时需要用较大剂量的消毒剂才能将其杀灭。不同来源的大肠杆菌O157∶H7耐药性、质粒和PFGE谱型有一定的相似性,提示菌株可能在人、动物、食品以及外环境之间相互传播并存在流行的可能。     

肠道致病菌暴发事件中分子分型技术的应用

目的探讨建立快速敏感的肠道病原菌的诊断分型方法。方法2002年7月用脉冲场电泳胶（PFGE）与噬菌体分型实验方法对发生在日本静冈县的4起暴发事件中29株宋内志贺菌（Shigella.sonnei）分离株与1999年口本静冈县某区现场分离的12株肠出血性大肠埃希菌（enterohemorrhagic Escherichia coli,EHEC）O157：H7菌株进行分型。结果4起事件中Shigetta．sonnei的PFGE结果显示,同起事件的DNA条带几乎一致;2起EHEC O157：H7暴发事件中,同起事件的PFGE条纹几乎一致,而3株散发菌株的PFGE条纹各异;12株EHEC O157：H7噬菌体分型结果也显示了比较好的分型力。结论PFGE具有分型能力强、重复性好等特点,能直观地判断肠道致病菌的亲缘关系,及时查明暴发流行的传染源,从而有效控制疫情的蔓延。     

Vero cytotoxin-producing Escherichia coli O157 outbreaks in England and Wales, 1995: phenotypic methods and genotypic subtyping.

Vero cytotoxin-producing Escherichia coli O157 belonging to four phage types (PTs) caused 11 outbreaks of infection in England and Wales in 1995. Outbreak strains of different PTs were distinguishable by DNA-based methods. Pulsed-field gel electrophoresis best discriminated among strains belonging to the same PT, distinguishing six of the seven PT2 outbreak strains and both PT49 outbreak strains.     

Distribution of Salmonella enteritidis phage types in Canada

The distribution of Salmonella enteritidis phage types in Canada is described; 606 of 674 strains examined were of human origin. Typable strains of all sources, constituted 99.6% (671/674) of all strains examined, and were representative of 15 different phage types. Five phage types (8, 13, 4, 13a and 1) accounted for 92.4% of the total. Phage type 8 consistently showed the highest incidence in human (69.96%) and non-human (72.05%) sources and appeared to be the most common in North America. Phage type 4, the most prevalent in the UK, is infrequent in Canada (38/674). The distribution of phage types showed regional variation among infrequent phage types, whereas the common type, 8, was observed in different frequencies in all provinces. Examination of 29 outbreaks of S. enteritidis representing 254 isolates for humans revealed 5 different phage types, the highest number of outbreaks (11) were type 8. A study of these outbreaks and the animal-host-associations of the common phage types, 8 and 13, indicated that contaminated poultry appeared to be the most common source of human infection in Canada.     

四川省大肠杆菌O157:H7分子流行病学、耐药性及对消毒剂抗性研究

目的研究从四川省分离的大肠杆菌O157：H7菌株流行病学特征。方法用多重PCR测定67株大肠杆菌O157：H7的sit、eaeA、hly毒力基因；对不同来源的大肠杆菌O157：H7进行质粒和PFGE分型；测定67株大肠杆菌O157：H7的耐药性和对消毒剂的抗力。结果62．7％的株菌（42／67）携带有毒力基因，毒力图谱类型主要为slt1＋slt2＋eaeA＋hly。67株菌共有6种质粒谱型和7种PFGE谱型。64．2％（43／67）的菌株分别对7种不同的抗生素耐药，其中有23、35、34株菌分别对氨苄青霉素、四环素和SMZ耐药。不同来源的大肠杆菌O157：H7抗性谱不同，80．6％（54／67）的菌株对酒精和季铵盐产生了抗性，所有菌株对洗必泰敏感。结论四川省不同来源的大肠杆菌O157：H7带毒率相似，但毒力基因谱型不完全相同。细菌有较高的耐药性。菌株对常用消毒剂有很高的抗性，在消毒灭菌时需要用较大剂量的消毒剂才能将其杀灭。不同来源的大肠杆菌O157：H7耐药性、质粒和PFGE谱型有一定的相似性，提示菌株可能在人、动物、食品以及外环境之间相互传播并存在流行的可能。     

Importance of culture confirmation of shiga toxin-producing Escherichia coli infection as illustrated by outbreaks of gastroenteritis--New York and North Carolina, 2005

Escherichia coli O157:H7 and other strains of E. coli that produce Shiga toxin are collectively known as Shiga toxin-producing E. coli (STEC). The current outbreak of STEC O157 infections associated with eating fresh spinach illustrates the importance of obtaining isolates to identify the source of the infections. Laboratory methods that do not require bacterial culture of stool specimens to identify STEC are being used increasingly by clinical diagnostic laboratories, sometimes without subsequent confirmation of a strain by isolating it in culture. This report describes findings from outbreaks of gastroenteritis in 2005 in New York and North Carolina in which clinical diagnostic laboratories initially used only non-culture methods to detect Shiga toxin (Stx). The findings highlight the importance of confirmation of Stx-positive stool specimens by bacterial culture for timely and reliable identification of STEC infections, including E. coli O157 and non-O157 STEC, to enable implementation of appropriate public health actions. An important part of that identification is determining the serotype of all STEC isolates and the subtype of STEC O157 strains so that outbreaks can be detected and traced back to sources.