Studies in gnotobiotic piglets on non-O157:H7 Escherichia coli serotypes isolated from patients with hemorrhagic colitis

A number of verotoxin-producing Escherichia coli strains isolated from sporadic cases of hemorrhagic colitis in the United States over the last 5 yr were shown to belong to serogroups other than O157:H7-the serotype originally implicated in this disease. Experimental infection of gnotobiotic piglets with five such strains (0111:NM, 0145:NM, 045:H2, 04:NM, and Ound:NM) caused diarrhea resulting from mucosal lesions in the cecum and colon that were indistinguishable from those previously described in piglets infected with E. coli O157:H7. This suggests that, as with other categories of pathogenic E. coli, several serotypes cause hemorrhagic colitis in humans. The five E. coli strains that were compared with one O157:H7 strain and with an enteropathogenic calf strain (serotype 05:NM) caused a spectrum of disease ranging from moderate diarrhea (O157:H7) to severe illness (including septicemia and death) (0111:NM). Characteristic lesions, which were identical for all seven pathogenic strains, included bacterial attachment, effacement of the microvillus border, and dissolution of the cell membranes of surface and glandular epithelium, resulting in complete cell destruction. Some piglets exhibited neurologic signs of convulsions and ataxia. It is concluded that a number of E. coli serotypes, in addition to O157:H7, fulfill the present limited criteria for enterohemorrhagic E. coli, which include association with hemorrhagic colitis, production of one or more verotoxins, possession of a large plasmid (50-70 megadaltons), and induction of distinct mucosal lesions in the large bowel of gnotobiotic piglets.     

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.     

Escherichia coli O114:nonmotile as a pathogen in an outbreak of severe diarrhea associated with a day care center

Five infants from a day care center developed severe diarrhea associated with enteropathogenic Escherichia coli O114:nonmotile (EPEC O114:NM) and required hospitalization. Five additional cases of diarrhea associated with EPEC O114:NM subsequently occurred, four in hospital contacts of the patients and one in a household contact. Biochemically, all EPEC O114:NM isolates were sorbitol nonfermenters. All isolates produced low concentrations of cytotoxin with a mean of 10(1.23) CD50/mg of protein. Cytotoxin was not neutralized with antibody to Shiga-like toxin I or II. Heat-labile and heat-stable enterotoxins were not present by gene probe analysis. Stool isolates from 9 of 10 hospitalized infants were positive for EPEC adherence factor by colony blot DNA probe analysis. The severity of the disease, sorbitol nonfermentation, and presence of enteroadherence are unusual features of this organism.     

The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor

Isolates of the most common O serogroups of enteropathogenic Escherichia coli (EPEC) associated with infant diarrhea (designated class I) adhere to Hep-2 cells; the genes for this adhesin, termed EPEC adherence factor (EAF), are located on plasmids 50-70 MDa in size. Volunteers ingested 10(10) organisms of an O127:H6 Hep-2-adhesive class I strain (E2348/69) or its plasmid-minus, nonadhesive derivative. Diarrhea occurred in nine of 10 volunteers who ingested the parent strain (mean, 1,178 ml) but in only two of nine who took the plasmid-minus variant (mean, 433 ml; P less than .006). All volunteers ill from strain E2348/69 mounted serum IgA and IgG responses to a 94-kDa plasmid-associated outer membrane protein of E2348/69; this protein was found in other class I EPEC but not in enterotoxigenic or meningitic strains. The 50-70-MDa EAF plasmid seems necessary for full expression of pathogenicity in EPEC that exhibit Hep-2 adhesiveness. EPEC isolates of certain other, less common, O serogroups (O44, O86, and O114) are rarely Hep-2 adhesive. These EPEC, designated class II, possess distinct 50-70 MDa plasmids lacking EAF genes. Diarrhea was caused by 10(8) or 10(10) organisms of an O114:H2 class II EPEC strain (mean, 1,156 ml) in six of 11 volunteers. This result confirmed that class II EPEC are pathogenic by a mechanism not involving Hep-2 adhesiveness.     

Acquisition of stcE,a C1 esterase inhibitor-specific metalloprotease,during the evolution of Escherichia coli O157:H7

Escherichia coli O157:H7 is a source of foodborne illness, causing diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. E. coli O157:H7 secretes, via the etp type II secretion system, a metalloprotease, StcE, that specifically cleaves the serpin C1 esterase inhibitor. We determined by hybridization techniques the prevalence of stcE and etpD, a type II secretion gene, among diarrheagenic E. coli strains. stcE and etpD are ubiquitous among the O157:H7 serotype and are found in some enteropathogenic E. coli O55:H7 strains but are absent from other diarrheagenic E. coli. stcE was acquired on a large plasmid early in the evolution of E. coli O157:H7, before the inheritance of the Shiga toxin prophage. Other plasmidborne virulence factors, such as ehxA, katP, and espP, were acquired later by the enterohemorrhagic E. coli 1 complex in a stepwise manner. These data refine the sequential model of E. coli O157:H7 evolution proposed elsewhere.     

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.     

Characterization of a new putative colonization factor (CS17) from a human enterotoxigenic Escherichia coli of serotype O114:H21 which produces only heat-labile enterotoxin

Enterotoxigenic Escherichia coli (ETEC) of serotype O114:H21, which produced only heat-labile enterotoxin (LT), gave mannose-resistant hemagglutination (MRHA) with bovine erythrocytes. One strain, E20738A, was shown to possess fimbriae of approximately 7.5 nm diameter. On SDS-PAGE two possible fimbrial polypeptides of molecular masses 17.5 and 15.5 kDa were seen; the 17.5-kDa band was the most prominent. Loss of LT and MRHA together from strain E20738A was associated with loss of a 100-MDa plasmid. An absorbed anti-strain E20738A serum reacted specifically with the 17.5- and 15.5-kDa polypeptides and bound to the intact fimbriae. This antiserum reacted positively in an ELISA with LT-positive E. coli strains of serogroups O8, O15, O48, O114, and O146. The antiserum did not react with ETEC carrying known colonization factors. The term coli-surface-associated antigen (CS) 17 has been used to describe the fimbriae.     

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。     

Molecular epidemiology of unrelated clusters of multiresistant strains of Haemophilus influenzae.

Three epidemiologically unrelated clusters of Haemophilus influenzae resistant to ampicillin, chloramphenicol, and tetracycline were studied. The biotypes and cell-envelope protein patterns were determined for 17 nonencapsulated strains, 6 from Dundee and 11 from Cheltenham, and for 6 type b encapsulated strains from Guildford. After mobilization by conjugation, large 32- to 36-MDa plasmids were purified from all the strains. The restriction fragment patterns of the plasmids were determined by ethidium bromide staining of digested purified plasmid or by Southern hybridization of digested total cellular DNA of the parent strains, probed with purified plasmid. Evidence is presented for a chromosomal location of the plasmids in the parent strains, the spread in nature of a plasmid between distinguishable strains of H. influenzae, the person-to-person spread of a strain within a cluster, and a high degree of sequence homology between distinguishable plasmids, implying their close relatedness.     

Serotype-specific prevalence of Escherichia coli strains with EPEC adherence factor genes in infants with and without diarrhea in S?o Paulo, Brazil

To examine interrelationships of classic enteropathogenic Escherichia coli (EPEC) serotypes, EPEC adherence factor (EAF) genes, and diarrheal disease, E. coli were studied from stools of 500 infants less than 1 y of age with acute diarrhea and 500 age-matched controls. EAF-containing (EAF+) E. coli of three common classic EPEC serotypes (O111:H-, odds ratio [OR] 36.0; O111:H2, OR 55.0; O119:H6, OR 3.7) were individually strongly associated with diarrhea, as were EAF+ strains of less common classic serotypes combined (OR 5.3). Among EPEC serogroups, neither EAF+ strains of nonclassic serotypes (OR 1.8) nor EAF-strains of classic (OR 2.2) or nonclassic (OR 1.4) serotypes were significantly associated with diarrhea. At least one EAF+ non-EPEC serogroup serotype (O88:H25) may represent an unrecognized EPEC serotype. Serotype-specific variation in the association of EAF+ E. coli with diarrhea suggests that other factors are also important in determining virulence; thus, both EAF detection and E. coli serotyping are desirable in studying the etiology of diarrheal disease.     

霍乱弧菌毒素协同调节茵毛基因tcpA的克隆及序列分析

目的以新疆分离0139霍乱弧菌XJ93006株的DNA为模板，自行设计引物克隆毒素协同调节菌毛亚单位A(tcpA)基因(包括侧序列)并构建重组载体。方法以0139霍乱弧菌新疆分离株XJ93006基因组DNA作为模板；根据0l群ElTor型霍乱弧菌N16961全基因组序列设计tcpA PCR引物，高保真酶扩增tcpA片段：限制性内切酶切PCR产物和pNEB193空质粒，T4DNA连接酶连接酶切产物，重组质粒转化大肠什菌TB1工程菌，蓝白斑菌落试验筛选阳性克隆；提取质粒经酶切鉴定后测序并利用生物信息数据库对该序列进行序列分析，比较.结果DNA限制性内切酶可从重组质粒pNEB193-tcpA的EcoRI和Sal I位点之间切出一个1037bp的DNA片段，测序结果与01群ETTor型霍乱弧菌N16961、O139霍乱弧菌标准株M045的tcpA同源性均达到99.9％以上。结论成功构建0139霍乱弧菌新疆分离株XJ93006毒素协同调节菌毛亚单位A(tcpA)基因的重组质粒pNEB193-tcpA；序列分析表明霍乱弧菌毒素协同调节菌毛亚单位A(tcpA)基因住01群ETTor型霍乱弧菌和0139霍乱弧菌中是一类高度保守的原核基因，可作为霍乱弧菌疫苗的候选基因。另外为研究0139霍乱弧菌的来源奠定基础。     

A DNA probe to identify enterohemorrhagic Escherichia coli of O157:H7 and other serotypes that cause hemorrhagic colitis and hemolytic uremic syndrome

Enterohemorrhagic Escherichia coli (EHEC) cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), make potent cytotoxins (Verotoxins [VT] or Shiga-like toxins), and possess a plasmid (approximately 60 megadaltons) that encodes a new fimbrial antigen and promotes attachment to epithelial cells. We evaluated the use of a DNA probe, prepared from a 3.4-kilobase segment of the EHEC plasmid, to detect EHEC. The probe hybridized with 106 (99%) of 107 O157:H7 and 34 (77%) of 44 O26:H11, VT-positive strains from patients with colitis, HUS, and diarrheal disease and hybridized with 21 (81%) of 26 VT-positive E. coli of serotypes other than O157:H7 or O26:H11 from patients with hemorrhagic colitis and HUS. We examined 601 other strains, including 18 serotype O26 isolates of H types other than H11, 306 enteropathogenic E. coli, 60 enteroinvasive E. coli, 119 enterotoxigenic E. coli, and 20 isolates from the urinary tract and 77 isolates from the normal intestinal flora; only one (O127:H-) was positive (specificity, 99.8%). Serotype O26:H11, previously considered a classic enteropathogenic E. coli serotype, is now shown to be EHEC.     

Isolation of Escherichia coli O128:HNM harboring stx2f gene from diarrhea patients

Shiga-like-toxin-producing Esherichia coli O128:HNM were isolated from feces of a one-year-old boy with diarrhea and abdominal pain on July, 2002, and a 11-month-old girl with diarrhea and fever on June, 1997. None of other enteropathogenic bacteria including Salmonella were isolated. E. coli O128:HNM isolates from both patients carry stx2f and eaeA gene, but not stx1, stx2, aggR, bfpA, esth, estp, invE, astA, ureC and hlyA gene. As far as we know, this may be the first report indicating that E. coli O128:HNM carrying stx2f gene were isolated from patients in Japan.     

Cloning and surface expression of Pseudomonas aeruginosa O antigen in Escherichia coli.

As a step toward developing recombinant oral vaccines, we have explored the feasibility of expression of O polysaccharide antigens from Pseudomonas aeruginosa by Escherichia coli. We cloned in E. coli HB101 a 26.2-kilobase DNA fragment from P. aeruginosa strain PA103 that specifies the production of the O polysaccharide of Fisher immunotype 2 (IT-2) strains. The recombinant organism incorporated the P. aeruginosa IT-2 O polysaccharide onto the core of the E. coli lipopolysaccharide (LPS). Transfer of the recombinant plasmid to three LPS-rough strains of P. aeruginosa resulted in synthesis of IT-2 O antigen, and two of these transconjugant strains also synthesized a second O polysaccharide, presumably representing expression of a repressed, or an incomplete, set of genes for an endogenous O polysaccharide. Rabbits injected with the purified recombinant LPS made antibody specific for P. aeruginosa IT-2 O side chains, as did mice fed the recombinant E. coli strain. Expression of P. aeruginosa O antigens by enteric bacteria makes it possible to study these recombinant strains as oral vaccines to prevent P. aeruginosa infections.     

Toxin genotypes and plasmid profiles as determinants of systemic sequelae in Escherichia coli O157:H7 infections

In 1987, 93 Escherichia coli O157:H7 isolates were collected during routine surveillance for this pathogen in the state of Washington. Toxin genotypes and plasmid profiles were correlated with the clinical sequelae of illness in 88 of the 93 patients from whom these strains were isolated. Thirteen plasmid patterns were observed among the 88 tested isolates; four patterns accounted for 82% of the isolates. Genetic probing for Shiga-like toxins (SLT) I and II demonstrated the presence of both genes in 67 (76%), SLT I alone in three (3%), and SLT II alone in 18 (20%). The hemolytic uremic syndrome or thrombotic thrombocytopenic purpura developed in seven (39%) of 18 patients infected with isolates having only the SLT II gene, while these complications occurred in only four (6%) of 70 patients infected with isolates having the other two genotypes (relative risk, 6.8; 95% confidence interval, 1.9, 26.4). This study shows that E. coli O157:H7 isolates systematically collected from a single geographic region over a defined time period exhibit considerable diversity in plasmid content and toxin genotype and that the toxin genotype of the infecting strain may influence the risk of developing microangiopathic sequelae.     

Tissue culture-adherent Escherichia coli in infantile diarrhea.

To determine the association of tissue culture-adherent Escherichia coli with diarrhea, serotyped E. coli strains isolated in a yearlong case-control study of infantile diarrhea in Bangkok, Thailand, were examined for adherence to HeLa cells and for hybridization with the enteropathogenic E. coli adherence factor, the F1845, and the enteroaggregative E. coli (EAggEC) DNA probes. E. coli that adhered to HeLa cells in a localized adherence (LA) pattern (LA E. coli) was isolated from 26 of 509 infants with diarrhea (cases) and 9 of 509 age-matched controls (P = .006); E. coli with diffuse or aggregative adherence (DA or AA) to HeLa cells or that hybridized with the F1845 or EAggEC probes was not associated with infantile diarrhea. LA E. coli of classical enteropathogenic E. coli (EPEC) serotypes was isolated from 11 cases and 1 control (P = .003). EPEC O44:H18 that adhered to HeLa cells in a DA pattern and hybridized with the F1845 DNA probe was the predominant E. coli (five of five colonies tested) isolated from a 5-month-old girl with diarrhea in whom no other enteric infections were identified. Although LA E. coli was highly associated with infantile diarrhea, the role of DA and AA E. coli was uncertain in this setting.     

DNA polymorphisms among Escherichia coli isolated from bacteriuric women

Restriction fragment length polymorphism (RFLP) patterns, plasmid profiles, and antimicrobial susceptibility patterns of paired sequential Escherichia coli isolates from antibiotic-treated and untreated elderly women were analyzed. Isolates from 26 of 27 subjects who were treated successfully with antibiotics but became reinfected differed by RFLP analysis, whereas 10 of 12 subjects who failed treatment and 11 of 14 untreated subjects had paired isolates with identical RFLP banding patterns. Only 40 of the 53 pairs of isolates could be analyzed by plasmid profiles; 36 of these 40 were concordant with RFLP analysis. Antimicrobial susceptibility patterns showed poor concordance with RFLP analysis. This study demonstrates the utility of RFLP analysis and indicates that isolation of E. coli from elderly women after sterilization of the urinary tract usually results from introduction of a new strain; elderly women who fail antibiotic therapy or receive no therapy may remain persistently infected with the same E. coli strain.     

Plasmids of human strains of Yersinia enterocolitica: molecular relatedness and possible importance for pathogenesis

One-hundred fifteen strain of Yersinia enterocolitica were examined for plasmids and plasmid-mediated pathogenic properties. Human strains of serotypes O:3 and O:9 harbored plasmids of 46 and 44 megadaltons, respectively, with 90% homology of DNA sequences. The plasmid-mediated properties were calcium dependence, survival in human serum, conjunctivitis provocation in guinea pigs, and O agglutinogens. One strains of serotype O:8 harbored a 42-megadalton plasmid with 75% sequence homology with plasmids of serotypes O:3 and O:9. An additional plasmid-mediated property was lethality for white mice. Filter hybridization of restriction endonuclease-digested plasmid DNA indicated that a 5.6-megadalton fragment of the plasmid of serotype O:8 had virtually no sequence homology with plasmid DNA of serotypes O:3 and O:9 and therefore may be associated with the lethal factor for mice.     

Four serotypes highly positive for pathogenic factor-related genes found among Escherichia coli isolates from sporadic diarrheal cases in Fukui Prefecture

To evaluate the prevalence of pathogenic factor-related genes in 964 O-serotype Escherichia coli isolated from sporadic diarrhea cases at 2 hospitals in Fukui during 1997 to 2001, we checked all of these strains for H-serotype and examined them for 4 pathogenic factor-related genes (LT, ST, stx and invE) by PCR. Of these strains, 409 except for most of 9 serotypes which are usually low prevalence of pathogenic factor-related genes, and additional 16 strains isolated from other hospitals in Fukui Prefecture were also examined for 3 pathogenic factor-related genes (eaeA, astA and aggR) by PCR. It was found that 4 serotypes, O6:H16, O25:HNM, O111:H21 and O126:H27, were highly positive for pathogenic factor-related genes; O6:H16 (11/12 strains) positive for LT, ST, astA, O 25:HNM (10/14 strains) positive for ST and astA, O111:H21 (22/22 strains) positive for astA or aggR, and O126:H27 (8/9) positive for astA and aggR genes. According to the drug susceptibility test, these serotypes as O6:H16, O25:HNM, O111:H21 and O126:H27 showed a significant resistance to some drugs in 7/12, 4/14, 21/22 and 9/9 strains, respectively. Such results indicate that both O111 and O126-serotypes are highly positive for pathogenic factor-related genes and also drug-resistant, namely this fact suggests that the O-serotyping as laboratory screening is one of the useful measures of clinical management. Additionally, the pulsedfied gel electrophoresis patterns found in each of the 4 serotypes mentioned revealed that a part of E. coli in Fukui might be derived from the same source of infection.     

福建省O157大肠杆菌的rfb及fliC基因的研究

目的　对福建省分离的O15 7大肠杆菌的O抗原编码基因 (rfb基因 )及H抗原编码基因 (fliC基因 )进行研究分析 ,以明了福建省O15 7大肠杆菌的抗原特征。方法　应用聚合酶链反应 (PCR) ,以O15 7抗原编码基因 (rfbO157基因 )、H7抗原编码基因 (fliCH7基因 )为靶基因进行检测。将rfbO157基因PCR扩增产物进行DNA测序 ,使用NCBIgenBank进行DNA序列相似性比较。结果　rfbO157基因PCR扩增产物经DNA测序 ,片段序列与O15 7∶H7大肠杆菌国际参考株EDL933完全一致。福建省分离的 80株O15 7大肠杆菌的rfbO157基因均为阳性。fliCH7基因仅 6 2 5 % (5 / 80 )阳性。结论　福建省分离的O15 7大肠杆菌少部分 (6 2 5 % )为O15 7∶H7,并携带毒力基因 ;大部分为O15 7非H7,且未检测到毒力基因。