Enterotoxigenic and necrotizing Escherichia coli in human diarrhoea in Spain

Enterotoxigenic Escherichia coli (ETEC) strains of serotype 0153: K-:H45 CFA/I⁺ STa⁺ were associated with two outbreaks of neonatal diarrhoea that occurred in two different hospitals of Madrid, in one of which several children died. Two other outbreaks were associated with ETEC strains of serotypes 0159: K-: H21 (LT⁺) and 0159: K-: H4 (LT⁺ STa⁺) without CFA/I and CFA/II colonization factors. Necrotizing E. coli (NTEC) strains of serotype 06: K13, producing the cytotoxic necrotizing factor CNFI and -haemolysin, were also associated with two outbreaks of neonatal diarrhoea that occured in a hospital in Madrid and in a hospital in Talavera de la Reina. The results of the characterization of some ETEC and NTEC strains isolated from sporadic cases of diarrhoea are also discussed.Corresponding author.     

Comparison among. enterotoxigenic strains of escherichia coli isolated in Italy and Somalia

Nine strains of ETEC isolated in Italy have been compared with 13 isolates from Somalia with respect to toxin production, serotype and antimicrobial resistance pattern. None of the strains isolated from Italy belonged to any serotype or serogroups found among the strains from Somalia. Remarkable differences between the two groups of isolates were also observed with regard to the susceptibility to antimicrobials and the presence of R-plasmids. These findings suggest that ETEC strains isolated in Italy are not related to the strains widespread in Somalia and, generally, in developing countries.Corresponding author.     

Virulence factors and O groups of Escherichia coli isolates from patients with acute pyelonephritis, cystitis and asymptomatic bacteriuria

The relationship between the presence of bacterial virulence factors and the severity of urinary tract infection (UTI) was analized in this study. The production of -hemolysin (Hly), the expression of P-fimbriae and the mannose-resistant hemagglutination (MRHA) type IVa (associated with the presence of P-fimbriae), were all detected more frequently in Escherichia coli strains from acute pyelonephritis than in strains isolated from cystitis and asymptomatic bacteriuria. In contrast, the production of cytotoxic necrotizing factor type 1 (CNF1) and the expression of MRHA types III and IVb were distributed uniformly between strains causing different clinical categories of UTI. Thus 88% of the E. coli strains from acute pyelonephritis showed some of the virulence factors investigated in this study, whereas only 60% (p < 0.01) and 56% (p < 0.01) repectively of the strains isolated from cystitis and asymptomatic bacteriuria possessed virulence factors. There were no significant differences in the prevalence of virulence properties between strains isolated from patients with or without complicating factors. Only 16% (p < 0.001) of the fecal isolates from healthy individuals showed virulence factors. The virulence factors were concentrated in strains belonging to 10 (O1, O2, O4, O6, O7, O14, O18, O22, O75 and 083) of the 12 serogroups most frequently detected in uropathogenic E.coli strains. The majority of uropathogenic O4, O6, O14, O22, O75 and O83 E.coli strains were Hly⁺CNF1⁺ and expressed P-fimbriae or MRHA type III, whereas the strains of serogroup O18 were Hly⁺CNFI^– and P-fimbriated. Among O1 and O7 strains we found Hly^– CNF1^–strains that expressed P-fimbriae. Among O2 strains we found Hly⁺CNF1⁺ strains that expressed P-fimbriae or MRHA type III and other Hly^–CNF1^–strains that possessed P-fimbriae. We conclude that E.coli strains isolated from pyelonephritis show virulence factors more frequently than those from cystitis and asymptomatic bacteriuria, and that strains that cause urinary tract infections in Spain belong to the same serogroups as uropathogenic E.coli isolated in other areas of the world. Our results support the special pathogenicity theory and suggest that many cases of serious urogenital disease may be caused by a limited number of P-fimbriated E.coli strains that usually produce -hemolysin.     

Virulence genotypes and serotypes of verotoxigenic Escherichia coli isolated from cattle and foods in Argentina

Virulence factors of Verotoxin-producing Escherichia coli(VTEC) strains isolated from hamburgers and ground beef were studied in Argentina by PCR. Their virulence profiles were correlated with those corresponding to strains isolated from calves and adult cattle. Most virulent profiles (VTs⁺ eae ⁺Mp⁺) were present in E. colifrom healthy and diarrheic calves corresponding to O5:H-, O5:H27, O20:H?, O26:H11, O38:H?, O103:H-, O103:H2, O111:H-, O118:H16, O165:H-serotypes. The presence of the eaegene was significantly more frequent among VTEC strains isolated from calves (20/26; 76%) than from adult cattle (1/39; 2.5%) (p< 0.005). VT2⁺ eae ^? E. coliwas prevalent in foods and adult cattle at slaughterhouse. The prevalence of the eaegene was similar between VTEC strains isolated from meat (0/21) and adult cattle (1/39; 2.5%) which constitutes the main population processed at slaughterhouses in Argentina. Serotyping showed that VTEC strains were distributed among 31 serotypes, some of which (O20:H19, O91:H21, O113:H21, O116:H21, O117:H7, O171:H2, OX3:H21) were shared between bovine and food strains. These O serogroups have been isolated from cases of haemorrhagic colitis (HC) and haemolytic-uraemic syndrome (HUS) in humans in several continental European countries. This study confirms the role of cattle as a reservoir of many VTEC serotypes other than O157:H7 and represents a base for future diagnostic, prevention and control strategies of EHEC in this country. In addition, this study affirms the advantages of PCR-based screening of E. coliisolates given the finding of so many verotoxin-producing strains.     

浙江省舟山市269例海岛渔民病毒性肝炎型别分析

为探讨海岛渔民病毒性肝炎的病毒种类及其与临床的关系，对269例海岛渔民肝炎患者的型别进行了分析。研究对象为浙江省舟山市人民医院2001年1月至2004年12月住院的269例渔民病毒性肝炎患者，均为男性，年龄18～65岁，平均32．5岁。诊断标准根据2000年9月中华医学会传染病与寄生虫病学分会、肝病学分会联合修订的病毒性肝炎防治方案。患者人院后常规进行甲、乙、丙、丁、戊、庚型肝炎血清标志物检测，采用酶联免疫吸附法，HCV—RNA，HBV—DNA（采用PCR）。     

Contamination of potable water by enterotoxigenic Escherichia coli: qPCR based culture-free detection and quantification

Tourists visiting to endemic zones may acquire Enterotoxigenic Escherichia coli (ETEC) infection resulting into diarrhea due to consumption of contaminated potable waters. In this study, a qPCR assay (SYBR Green), targeting LT1 and ST1 genes was designed to quantify ETEC in potable waters derived from civic water supply. The assay could detect lowest 1 CFU/PCR targeting LT1/ST1 gene from ten-fold diluted culture of the reference strain (E. coli MTCC 723) and is ten-fold more sensitive than the conventional PCR. The quantification of the ETEC in potable waters collected from civic supply of a major city of the northern India exhibiting high flow of tourists reveals that all the sites that ran along sewage line were contaminated by the ETEC. Contamination was due to percolation of sewage. The assay could be used for the regular monitoring of potable water in places exhibiting heavy flow of tourists to prevent ETEC induced diarrhea.     

浙江省腹泻患者致泻性大肠埃希菌血清型分布及其鉴定效率的评价

目的 了解浙江省致泻性大肠埃希菌（DEC）血清型分布并探讨其血清学鉴定分类方法的鉴定效率。方法 对2009年7月至2013年6月浙江省腹泻症候群病原谱监测网络菌株库中的696株DEC菌株（通过毒力基因鉴定）开展血清学凝集试验,比较毒力基因和血清学鉴定分类的结果。结果 696株DEC中288株（41.4%）能明确O抗原型别,分属于35种O血清群。171株（24.6%）H血清凝集,分属于21种H型。肠集聚性大肠埃希菌（EAEC）、产肠毒素性大肠埃希菌（ETEC）、肠致病性大肠埃希菌（EPEC）和肠出血性大肠埃希菌（EHEC）凝集率分别为31.9%（130/408）、70.6%（127/180）、31.5%（29/92）和14.3%（2/14）,分属于30、18和15种O血清群,1株EHEC为O157∶H7。EAEC和EPEC血清群相对较多样化,而ETEC则相对集中,不同类型DEC可具有同一血清群/型。根据血清学结果可分类的75株DEC中,42株毒力基因和血清学分类结果一致,33株不一致。结论 浙江省DEC血清群/型种类多样,单纯采用血清学筛查可造成极大的漏检或误分,建议采用毒力基因鉴定分类。     

Serotypes of CNF1-producingEscherichia coli strains that cause extraintestinal infections in humans

The O:K:H serotypes of 137 necrotoxigenicEscherichia coli (NTEC) producing the cytotoxic necrotizing factor type 1 (CNF1) isolated from human extraintestinal infection were determined. Although NTEC producing CNF1 belonged to 58 different serotypes, only 10 of them accounted for 54% of strains. The most common serotypes, in order of frequency, were: O4:K?:H5, O6:K13:H1, O83:K1:H31, O75:K95:H5, O2:K1:H6, O2:K7:H-, O75:K1:H7, O2:K?:H1, O4:K12:H1 and O22:K13:H1. CNF1 strains of serotypes O2:K7:H- and O4:K12:H1 express P-fimbriae, whereas CNF1 strains of serotypes O2:K?:H1, O2:K1:H6 and O75:K95:H5 possess the adhesin responsible for MRHA type III. Among CNF1 strains of serotype O4:K?:H5 there exist some that express P-fimbriae and others that possess MRHA type III. Lastly, the majority of CNF1 strains of serotypes O6:K13:H1, O22:K13:H1, O75:K1:H7 and O83:K1:H31 do not express P-fimbriae nor the adhesin responsible to MRHA type III. Our results show that extraintestinal infections are caused by a limited number of virulent clones, as suggested by the theory of special pathogenicity.     

Impact of lead stress and adaptation in Escherichia coli

The growth rate of Escherichia coli was stimulated when cells were in media containing lead up to a concentration of 300 ppm. Higher concentrations inhibited growth. Metal analysis revealed that in the presence of lead E. coli concentrates 22.8 mg of lead per gram (dry weight) of cells. Analysis of cellular subfractions indicated that membrane fraction concentrated over 95% of the lead taken up by cells, of which a major portion was found to be associated with membrane lipids. Alterations in alkaline phosphatase, Ca2+-Mg2+ -ATPase activities and the carbohydrate and phospholipid contents in membrane fractions were also observed when cells were grown in the presence of lead. A time- and concentration-dependent increase in the release of carbohydrates by the cells was also evident. The results suggest that besides thriving in higher lead surroundings, E. coli possess a marked ability to concentrate substantial amount of inorganic lead.     

Antimicrobial resistance patterns and plasmids of enteropathogenic Escherichia coli isolated in Nigeria

In an epidemiological study of enteropathogenic Escherichia coli, 102 strains were isolated from patients seen at the University Teaching Hospital in Lagos. The most common serotype encountered was 055 followed by 026. Antimicrobial susceptibility testing and plasmid profiling of the strains were done. All the strains were sensitive to colistin, nalidixic acid, nitrofurantoin, cefotaxime, amikacin, and augmentin. Of the 102 strains, 47 (46%) were resistant to one or more of the following antimicrobial agents: Co-trimoxazole, tetracycline, ampicillin, streptomycin, sulphonamide and a combination of ampicillin with sulbactam. All the strains that were resistant to any antimicrobial agents were also resistant to tetracycline. Seventy-two strains (70.6%) harbored plasmid whose molecular weights ranged from 0.8 to 120 × 10⁶ daltons. The majority. of the plasmid were smaller than 6 × 10⁶; 90% of strains carrying plasmid ranging in size from 2 to 6 × 10⁶ daltons and 50 to 70 × 10⁶ daltons were resistant to one or more antimicrobial agents. Trasformation and conjugation experiment showed that about 57% of the resistant strains carried R plasmid. Plasmld-determined resistance to tetracycline, ampicillin, streptomycin and sulphonamide was found.     

An epidemiological study on Verotoxin-producing Escherichia coli (VTEC) infection among population of northern region of Iran (Mazandaran and Golestan provinces)

Faecal samples from 3268 randomly selected inhabitants of two provinces in the northern region of Iran were screened to detect Verotoxin-producing Escherichia coli (VTEC) using colony sweep polymyxin-B extraction method. Non-sorbitol fermentation phenotype and slide agglutination with O157 and H7 antisera used to detect this serotype. We found that 0.7% of the population were infected with VTEC, however none of the isolates belonged to O157:H7 serotype. We also found that children < 6 years of age were at highest risk of infection with VTEC (p < 0.05). Moreover, a significant association was found between the VTEC and diarrhoeal cases at the same age group (p < 0.001). Overall distribution of the VTEC isolates in the general population was found to be random, though a kind of clustered distribution could be noticed.     

Expression of enterotoxigenic Escherichia coli colonization factors in Vibrio cholerae

As a first step towards a vaccine against diarrhoeal disease caused by enterotoxigenic Escherichia coli (ETEC), we have studied the expression of several ETEC antigens in the live attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Colonization factors (CF) CFA/I, CS3, and CS6 were expressed at the surface of V. cholerae CVD 103-HgR. Both CFA/I and CS3 required the co-expression of a positive regulator for expression, while CS6 was expressed without regulation. Up-regulation of CF expression in V. cholerae was very efficient, so that high amounts of CFA/I and CS3 similar to those in wild-type ETEC were synthesized from chromosomally integrated CF and positive regulator loci. Increasing either the operon and/or the positive regulator gene dosage resulted in only a small increase in CFA/I and CS3 expression. In contrast, the level of expression of the non-regulated CS6 fimbriae appeared to be more dependent on gene dosage. While CF expression in wild-type ETEC is known to be tightly thermoregulated and medium dependent, it seems to be less stringent in V. cholerae. Finally, co-expression of two or three CFs in the same strain was efficient even under the control of one single regulator gene.     

Isolation in Italy of a verotoxin-producing strain of Escherichia coli 0157:H7 from a child with hemolytic-uraemic syndrome

Verotoxin-producing Escherichia coli 0157:117 was isolated for the first time in Italy from a child with hemolytic-uremic syndrome and his asymptomatic sister. Both parents remained asymptomatic, and neither had evidence of this infection. The source of the infection was not identified, but the children had eaten ground beef during the 15 days prior to the onset of symptoms.Corresponding author.     

山东省腹泻病人携带耶尔森菌HPI毒力岛大肠杆菌的调查

目的 了解携带耶尔森菌HPI毒力岛的大肠杆菌的易感人群、感染率、临床症状、分离率。方法 对4个医院1997年6～11月就诊泻患者,进行流行病学调查并收集大便标本,进行细菌分离和鉴定。结果 在671例腹泻病人中,共检出20个属种449株致病毒,志贺菌检出率为25.48%,致泻性大肠杆菌为15.05%,对于176株疑似产志贺样毒素且具侵袭大肠杆菌(ESIEC)菌株采用irp-2、irp-B基因探针进行菌落原位杂交,检出42株携带HPI毒力岛的大肠杆菌。与42株携带HPI毒力岛的大肠杆菌相关的腹泻病人所表现的临床症状为食欲不振、腹痛、腹泻、寒战、乏力、体温正常或低热,粪例多为粘液便或水样便,日腹泻次数超过6次以上者为33例,占78.57%。各年龄组均有感染,10岁以下儿童为主,占33.33%.结论 自671例腹泻病人的粪便标本中,分离到的志贺菌占首位,致泻性大肠杆菌次之;在致泻性大肠杆菌以携带耶尔森菌HPI毒力岛的大肠杆菌为主(6.26%)。     

The hemolysin of escherichia coli

Many strains of E. coli elaborate a hemolysin which is responsible for the zone of -hemolysis surrounding bacterial colonies on blood agar. The significance of this cytolysin as a determinant of bacterial pathogenicity has been established in animal models with the use of genetically engineered, isogenic bacterial strains. An analogous role in human infections has been inferred from the high association of hemolysin production with disease. Studies at a molecular genetical level have defined 4 genes that are required for the synthesis, post,translational modification and secretion of the hemolysin. The structural gene hlyA encodes for a 107-110 000 polypeptide, which must be modified in an unknown manner to its active form by the product of the neighboring hlyC gene. Genes hlyb and hlyD encode for proteins that export the molecule to the extracellular medium. The signal for secretion is contained in the C-terminal portion of the toxin molecule. The secreted hemolysin attacks plasma membranes of target mammalian cells by inserting as a monomer into the bilayer and generating hydrophilic transmembrane pores of approximately 2 nm effective diameter. The pores display a marked selectivity for cations over anions and pore-opening is dependent on the presence of a correct transmembrane potential. Binding to a membrane target does not require the presence of a specific receptor, and pores may be generated in planar lipid membranes consisting solely of phosphatidylcholine. Pore formation in nucleated cells can trigger secondary reactions such as stimulation of arachidonate metabolism with release of lipid mediators, probably initiated by passive influx of extracellular Ca²⁺. Perfusion of subcytolytic doses through isolated and ventilated rabbit lungs induces lung edema that is at least partially due to such secondary events. The capacity of E. coli hemolysin to damage human tissues via primary and secondary processes is consistent with the concept of its pathogenic role in human infections.[/p]Corresponding author.     

侵袭性大肠杆菌所致爆发型新生儿腹泻

本文报道1987年黄石市某市立医院一起产科婴儿室侵袭性大肠菌杆(EIEC)O152:K?和O112 ac:K66血清型所致的18例急性新生儿爆发型腹泻,死亡2例。对流行情况、病原学、生化、血清学试验、临床特征和耐药性进行了全面的讨论。作者认为,制定产科婴儿室管理制度和对产妇进行肠道病原菌的检查是十分重要的。     

Prevalence and characteristics of human and bovine verotoxigenic Escherichia coli strains isolated in Galicia (north-western Spain)

An epidemiological study was carried out to determine the incidence and the serotypes of verotoxigenic Escherichia coli (VTEC) that cause infections in Galicia (north-western Spain). Although, VTEC strains were isolated from 55 (14%) of the 387 calves sampled and the majority of bovine VTEC strains belonged to serotypes (026:H11 or H–, 091:H21, 0103:H2, 0105:H18, 0111:H–, 0113:H21, 0126:H–, 0128:H– and 0157:H7 or H–) previously associated with human haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) in other countries, VTEC are not a common cause of human infections in Spain. Thus, VTEC (026:H11 and 086:H10) were isolated from only 3 (0.6%) of the 482 children with diarrhoea investigated. We examined the 69 (3 humans and 66 bovines) VTEC strains that were initially isolated as E. coli producing a toxin cytotoxic to Vero and HeLa cells by polymerase chain reaction (PCR) using specific primers for VT1, VT2 and eae genes. PCR showed that 38 (55%) of VTEC strains carried VT1 genes, 18 (26%) possessed VT2 genes, and 10 (14%) carried both VT1 and VT2 genes. Three (one human and two bovine) strains which were formerly VTEC had lost the ability to produce verotoxins upon subculture and became negative for VT 1 and VT2 by PCR. In total 35 (51%) of 69 VTEC strains, including the two human VT1⁺ strains of serotype 026:H11, were positive for eae sequences when tested by PCR. Presence of the eae gene was significantly more frequent (100%; 21/21) among VTEC strains with serotypes (026:H11, 0111:H–, 0157:H–and 0157:H7) considered as enterohaemorrhagic E. coli (EHEC) than among VTEC strains with non-EHEC serotypes (29%; 14/48) (p < 0.001). Results obtained in this study indicate that cattle may be an important source of VTEC involved in human disease. However, severe clinical syndromes caused by VTEC, such as HC and HUS, are uncommon in Spain, in comparison with North America and the UK. In any case, VTEC disease can appear on the scene very suddenly, as occurred in the UK and North America in the 1980s.     

A systematic review of experimental infections with enterotoxigenic Escherichia coli (ETEC)

Volunteer challenge with enterotoxigenic Escherichia coli (ETEC) has been used for four decades to elucidate the pathogenesis and immune responses and assess efficacy of various interventions. We performed a systematic review of these studies and a meta-analysis of individual patient-level data (IPD) from a subset of studies using standard methodology.We identified 27 studies of 11 ETEC strains administered to 443 naive subjects at doses from 1 × 10⁶ to 1 × 10¹⁰ colony forming units (cfu). Diarrhea attack rates varied by strain, dose and enterotoxin. Similar rates were seen at doses of 5 × 10⁸ to 1 × 10¹⁰ cfu with the three most commonly used strains B7A, E24377A, H10407. In IPD analysis, the highest diarrhea attack rates were seen with strains B7A, H10407 and E24377A. The H10407 induced significantly higher stool output than the other strains. Additionally, the rate of output was different across strains.The risk of diarrhea, abdominal cramps, nausea and headaches differed significantly by ETEC strain. An increased risk of nausea, abdominal cramps and headaches was seen for females. Baseline anti-LT IgG titers appeared to be associated with a decrease risk of diarrhea outcomes, a trend not seen with anti-LT IgA or seen consistently with anti-colonization factor antibodies. Neither early antibiotic treatment nor diarrhea duration significantly affected the frequency or magnitude of serologic responses.These studies have served as an invaluable tool in understanding disease course, pathogenicity, innate immune responses and an early assessment of product efficacy. When designing and planning experimental ETEC infection studies in this age of increased ethical scrutiny and growing appreciation of post-infectious sequelae, better understanding of available data is essential.     

Characterisation of strains of enteroaggregative Escherichia coli isolated during the infectious intestinal disease study in England

Strains of Escherichia coli, hybridising with a DNA probe for enteroaggregative E. coli (EAggEC), were isolated from patients with infectious intestinal disease (IID) or gastro-enteritis, and healthy controls during the study of IID in England. Of 3506 cases presenting with an IID, 160 (4.6%) had faecal EAgg-EC as compared with 46 (1.7%) of 2772 healthy controls, 53% of EAggEC isolated from each of the case and the control groups adhered in a stacked-brick formation. Strains from cases and controls belonged to over 39 and 14 different serogroups respectively, and approximately half of the strains isolated did not react with antisera in the current somatic antigen serotyping scheme. Forty-nine cases with EAggEC (31%) had a known history of foreign travel. Over 50% of strains isolated from cases and controls were resistant to one or more of eight antimicrobials, and antimicrobial resistance was not statistically significantly more common among cases with a known history of foreign travel (p = 0.57). These data form part of the largest investigation carried out on these organisms in the UK to date and provide the most comprehensive analysis of strains of EAggEC isolated from the general population of England.     

Verocytotoxin-producing Escherichia coli in foodstuffs of animal origin

While much research effort has been targeted at the verocytotoxin-producing Escherichia coli (VTEC) serotype O157:H7, it is becoming more evident that other VTEC serotypes can also be associated with human foodborne disease. An increasing number of these non-O157 serotypes have been isolated from food sources and from humans suffering from haemolytic-uraemic syndrome and diarrhoea. The aim of our work was to investigate the prevalence of VTEC O157 and non-O157 in foodstuffs of animal origin using two rapid enzymatic procedures. Various types of food samples, 352 in total, were tested: 233 with the Premier EHEC, a screening test which directly detects the presence of verocytotoxin, regardless of serotype, while 119 of these with the Vidas ECO, which is a specific screening test for E. coli O157:H7, together with the Premier EHEC. Two samples were positive for VTEC, one of serogroup O126 and the other was non-serotypable. Another sample was positive in the test specific for E. coli O157:H7, but was not confirmed by culture. This study suggests that VTEC strains are not prevalent in Italy, and that the isolation of serogroup O157 is relatively infrequent. This leads us to conclude that there is little chance of exposure to pathogen for the average consumer in Italy.