Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.     

腹泻犊牛源大肠杆菌对小鼠的致病性

[背景]建立细菌性动物腹泻模型是研究细菌性腹泻机制及抗腹泻药机理的常用方法.[目的]从临床腹泻犊牛粪便中分离出5株不同血清型大肠杆菌(Escherichia coli),经口灌服小鼠建立与临床犊牛腹泻症状近似的小鼠腹泻模型.[方法]72只昆明小鼠随机分为6组,每组12只,分为正常对照组(NC)、E.coli O1干预组...     

Correlation between the presence of aggregative fibria and a type of Escherichia coli adhesion

Enteroaggregative strains of E. coli (EAEC) are an important agents possessing among many virulence factors, aggregative fimbria AAF hemagglutinating in the presence of mannose human group A or/and rats erythrocytes. The aim of the study was to determine the correlation between the presence of AAF fimbria and pattern of adherence in vitro. Tested strains of E. coli were obtained from children with diarrhea (133 strains) and healthy children (105 strains). Among strains of E. coli from children with diarrhea 81 (61%) showed the presence of AAF fimbria and 19 (23%) were adhering in aggregative pattern. In the group of strains of E. coli isolated from healthy children 31 (30%) were AAF positive and 8 (25.8%) of them presented aggregative adherence. Examination of AAF fimbria only dose not allow to distinguish EAEC strains. The data showed the participation of EAEC strains in diarrhea of children below 3 years old.     

Passive protective effect of egg-yolk antibodies against enterotoxigenic Escherichia coli K88+ infection in neonatal and early-weaned piglets

The protective effects of egg-yolk antibodies obtained from hens immunized with fimbrial antigens from a local strain (Escherichia coli K88+ MB, Manitoba, Canada) of K88+ piliated enterotoxigenic E. coli (ETEC) were evaluated in 3- and 21-day-old piglets in which ETEC diarrhea was induced and also in early-weaned piglets in a commercial farm. The results demonstrated that the E. coli K88+ MB-induced diarrhea in 3-day-old piglets was cured 24 h after treating with egg-yolk antibodies while those treated with egg-yolk powder from conventional hens continued to have diarrhea and 62.5% of them died of severe diarrhea. For 21-day-old weaned piglets, those fed egg-yolk antibodies had transient diarrhea, positive body weight gains and 100% survival during the period of the experiment, whereas control piglets that were treated with placebo had severe diarrhea and dehydration and some died within 48 h after infection. In the field trial, the incidence and severity of diarrhea of 14-18-day-old weaned piglets fed egg-yolk antibodies were much lower than in those fed a commercial diet containing an antibiotic. These results indicate that the neonatal and early-weaned piglets that received the egg-yolk antibodies were protected against ETEC infection.     

腹泻仔猪大肠埃希菌分离株ST4214的全基因组测序分析

分析腹泻仔猪大肠埃希菌(E.coli)分离株血清型、毒力因子及耐药基因。

本课题组前期研究从腹泻仔猪粪便样本中分离到64株E.coli, 本研究取其中含有毒力因子最多的1株进行全基因组测序分析。

E.coli分离株基因组序列为5.5 Mb, 预测蛋白质编码序列数为5 743, 与已知致病菌E.coli O145的基因重合率较低, 为64.53%。经多位点序列分型分析, 确定分离株为E.coli ST4214, 血清型为O3∶H45。与病原菌O157∶H7、O145∶H28和O104∶H4的毒力基因比较表明: 4株菌共同含有的毒力基因为354个, E.coli ST4214含有64种独特的毒力基因、编码鞭毛蛋白、Ⅳ型分泌蛋白、Ⅱ型分泌蛋白、溶血素、菌毛、肠毒素、荚膜多糖相关蛋白质。耐药基因E.coli分离株含有氨基香豆素抗性基因、磺胺类耐药基因、β-内酰胺抗性基因、多粘菌素耐药基因、肽抗生素抗性基因和抗生素耐药基因外排泵等多重耐药基因。

腹泻仔猪E.coli ST4214分离株是一株新菌株, 具有较多毒力因子及耐药基因, 可能与仔猪腹泻发病相关。

     

Incidence and some characteristics of fimbriae FY and 31A of Escherichia coli isolates from calves with diarrhea in Japan

Escherichia coli isolates from calves with diarrhea (1 day to 8 weeks old, 140 individuals) were surveyed for the three immunologically distinct fimbrial adhesins FY, 31A, and K99. Of a total of 1,370 strains isolated, 96 (7.0%), 34 (2.5%), 75 (5.5%), and 13 (0.9%) were identified as FY+, 31A+, FY+.31A+, and K99+, respectively. The K99+ strains also manifested heat-stable enterotoxin production (ST+), while FY+, 31A+, and FY+.31A+ strains were ST-. Expression of FY and 31A was repressed at lower temperatures or poor aeration. The FY+ and 31A+ E. coli showed mannose-resistant hemagglutinating activity with bovine erythrocytes. Electron microscopy revealed that FY is a gently curled fimbria with a mean diameter of 4.2 nm, and 31A is a fimbria with a mean diameter of 5.1 nm. The molecular mass of protein subunits was found to be approximately 20 kilodaltons (Kd) and 19 Kd for FY and 31A, respectively. Lethal diarrhea of neonatal calves was induced by challenge with the combination of a K99+.ST+ E. coli strain and either a 31A+ E. coli strain or a 31A+ E. coli strain plus an FY+ E. coli strain under the experimental conditions in which lethal diarrhea was not induced by challenge with a K99+.ST+ E. coli strain alone.     

Enteropathogens associated with diarrheal disease in infants of poor urban areas of Porto Velho, Rondônia: a preliminary study

One hundred and thirty cases of diarrhea and 43 age-matched controls, 0 to 5 years old, were studied in a pediatric outpatient unit from a poor peri urban area of Porto Velho, Rond?nia. Eighty percent of diarrheal cases were observed in the groups under 2 years of age. Rotavirus (19.2%) was the most frequent enteropathogen associated with diarrhea, followed by Shigella flexneri (6.15%) and S. sonnei (1.5%) and Salmonella sp. (6.9%). Four cases of E. coli enterotoxigenic infections (3.1%), E. coli enteropathogenic (EPEC)(2.3%) one case of E. coli enteroinvasive infection (0.8%) and one case of Yersinia enterocolitica (0.8%) were also identified. Mixed infections were frequent, associating rotavirus, EPEC and Salmonella sp. with Entamoeba histolytica and Giardia lamblia.     

Studies on diarrheagenic Escherichia coli isolated from children with diarrhea in Myanmar

Escherichia coli isolates from 217 children in Myanmar with diarrhea were investigated for the presence of virulence genes related to diarrhea by colony hybridization and PCR. The genes examined were lt, stI, stII, stx1, stx2, eae, bfp, pCVD (which is the representative gene of plasmid of pCVD of EAEC), and ial (which is invasion-associated locus of the invasion plasmid found in EIEC). Isolates from 47 of 217 children (21.7%) possessed virulence genes characteristic of diarrheagenic E. coli. No instance was found of co-existence of different E. coli strains with different virulence genes in the same patient. Diarrheagenic E. coli are currently classified into five categories based on their virulence markers: ETEC, EHEC, EPEC, EAEC, and EIEC. Of the 47 isolates examined, 30 were EAEC, 12 were EPEC and 5 were ETEC. Susceptibility tests for antimicrobial agents showed that almost all diarrheagenic isolates were resistant to penicillin, tetracycline and streptomycin. However, the majority of strains were sensitive to cephalexin, nalidixic acid and norfloxacin. In particular, 42 of the 47 isolates were sensitive to norfloxacin, which is a fluoroquinolone. This study shows EAEC and EPEC are responsible for sporadic diarrhea in Myanmar and fluoroquinolones appear to be effective in the treatment of these patients.     

新生儿病区产ESBLs大肠埃希菌整合子及其耐药性分析

目的 了解新生儿病区产ESBLs大肠埃希菌整合子的携带情况及其耐药性.方法 采用K-B琼脂扩散法对56株产ESBLs大肠埃希菌进行药敏试验;应用PCR法检测Ⅰ类、Ⅱ类和Ⅲ类整合子;以肠杆菌科重复序列-聚合酶链式反应(ERIC-PCR)进行基因分型.结果 56株产ESBLs大肠埃希菌的Ⅰ类整合子检出率为60.7％,未检出Ⅱ类和Ⅲ类整合子;菌株对庆大霉素、环丙沙星、左氧氟沙星、复方新诺明、头孢唑林、氨曲南、头孢他啶的耐药率差异有统计学意义(P＜0.05),阳性菌株的耐药率高于阴性菌株;56株大肠埃希菌分为45种基因型.结论 Ⅰ类整合子广泛存在于新生儿病区产ESBLs大肠埃希菌并与其耐药性相关.     

Fimbriae in Escherichia Coli Isolated from the Small Intestine of Piglets

Ninety E. coli strains, isolated from piglets which had died from neonatal diarrhea, were tested for the presence of K88, K99, 987P and type 1 fimbriae. Two or more types of fimbriae were demonstrated in 14 of the strains, a single fimbria! type in 44 strains while in 32 strains no fimbriae were detected. Of the 14 E. coli strains with more than 1 type of fimbriae, 12;, 10, 8 and 4 strains showed K88, K99, 987P and type 1, respectively. Twelve E. coli strains were isolated from piglets which had died in the neonatal period without showing signs of neonatal diarrhea at necropsy. One strain showed 987P and 3 strains showed type 1 fimbriae, while the remaining 8 strains were unfimbriated. Sixteen fimbriated E. coli strains were subcultured in order to examine the stability of fimbrial expression in the strains. The K88 and the type 1 fimbriae were regularly expressed, while the K99 and 987P were inconsistently demonstrated.     

2009~2012年黑龙江省细菌性腹泻症候群病原菌检测结果分析

为了解黑龙江省细菌性腹泻病原谱构成,探讨主要病原体的变异和流行变迁规律,提高监测实验室腹泻病原实验室诊断、监测预警、突发细菌性腹泻疫情的处置能力,并为进一步防治工作提供科学依据。收集哨点医院肠道门诊的细菌感染性腹泻患者粪便,采用分离培养、生化鉴定和血清分型的方法,检测沙门氏菌、志贺氏菌、致泻大肠杆菌等肠道致病菌。检测细菌性腹泻症候群患者标本537份,检出率为36.69%,其中,主要致病菌为致泻性大肠杆菌、志贺氏菌、副溶血弧菌、沙门氏菌、类志贺邻单胞菌、嗜水气单胞菌、小肠结肠炎耶尔森菌、霍乱弧菌、空肠弯曲菌、大肠埃希氏菌、检出率分别为9.31%、6.89%、1.30%、3.35%、0.56%、0.19%、0.37%、0.74%、5.40%、16.95%;性别差异不显著,各年龄组病原菌检出率和各月病原菌检出率差异显著,病原菌检出率存在季节性差异,其中6、7、8、9月份病原菌检出率高,检出率之和占总检出率的93.40%,0～10岁年龄组患病人数和检出率均高于其他年龄组。 分析发现:黑龙江省夏秋两季是细菌感染性腹泻高发季节,主要致病菌为大肠杆菌埃希氏菌、志贺氏菌、沙门氏菌、空肠弯曲菌。     

Human colostrum contains IgA antibodies reactive to colonization factors I and II of enterotoxigenic Escherichia coli

Diarrhea is an important cause of morbidity and mortality amongst infants of low socio-economic levels in developing countries and in travelers who visit such areas. Enterotoxigenic E. coli strains express two sets of virulence-associated factors: enterotoxins (heat-stable toxins or heat-labile toxins) and colonization factors. Studies have shown that breast-feeding protects infants against infectious diseases, such as diarrhea, as it presents a great variety of immunological components. The aim of this study was to analyze the reactivity of immunoglobulin A from human colostrum to colonization factor antigens I and II. The colostrum ability in preventing enterotoxigenic E. coli adhesion to Caco-2 cells was also evaluated. Colostrum samples were collected from 32 healthy women, and a human colostrum pool was prepared. Enterotoxigenic E. coli strains expressing colonization factor antigens I and II were utilized. The colostrum pool and individual samples showed variable antienterotoxigenic E. coli immunoglobulin A titers, that were reactive with colonization factor antigen I and CS1/CS3 (colonization factor antigen II). The human colostrum pool and individual samples inhibited enterotoxigenic E. coli colonization factor antigen I and II adhesion to Caco-2 cells, at variable levels, and this ability was a result of immunoglobulin A antibodies reactive to these colonization factors. The immunoglobulin A-depleted pool lost this inhibitory ability. As bacterial adhesion is the initial mechanism of enterotoxigenic E. coli infection, breast-feeding could protect the offspring against diarrhea caused by this agent.     

Bacterial, viral and parasitic enteric pathogens associated with acute diarrhea in hospitalized children from northern Jordan

To determine the etiology of acute diarrhea in Jordanian children under 5 years of age, we examined stool samples from 265 children admitted to the pediatric ward at Princess Rahma Hospital for Children, Irbid, Jordan, for parasites, rotavirus and enteric bacteria. Using both traditional and molecular diagnostic techniques, we detected enteropathogens in 66.4% of patients with diarrhea. A single enteric pathogen was detected in 50.9% of the children, and multiple pathogens were detected in 15.5%. The prevalence of enteropathogens identified was as follows: rotavirus (32.5%), enteropathogenic Escherichia coli (12.8%), enteroaggregative E. coli (10.2), enterotoxigenic E. coli (5.7%), Shigella spp. (4.9%), Entamoeba histolytica (4.9%), Salmonella spp. (4.5%), Campylobacter jejuni/coli (1.5%), Cryptosporidium spp. (1.5%), enteroinvasive E. coli (1.5%), eae-, Ehly-positive E. coli (0.8%), Giardia lamblia (0. 8%) and Yersinia enterocolitica (0.4%). No Vibrio cholerae, Shiga toxin-producing E. coli, microsporidia, adenovirus or small round virus were detected. Findings from this study demonstrate that rotavirus and several types of diarrheagenic E. coli, which are not screened for during routine examinations of stool samples in public health laboratories, were the most frequently detected enteropathogens in these children. Our findings highlight the value of using a combination of traditional and molecular techniques in the diagnosis of diarrheal disease in this population.     

Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia coli O86:HNM isolated from patients

We characterized two Shiga toxin-producing Escherichia coli (STEC) O86:HNM isolates from a patient with hemolytic uremic syndrome (HUS) or bloody diarrhea. Both of them did not possess the eaeA gene. However, the isolate from a HUS patient carried genetic markers of enteroaggregative E. coli (EAEC) and showed aggregative adherence pattern to HEp-2 cells. The other isolate from bloody diarrhea, which was negative with EAEC markers, was diffusely adhered to HEp-2 cells. The stx2 gene in both E. coli O86:HNM strains was encoded in each infectious phage, which was partially homologous to that of strain EDL933, a STEC O157:H7. These results will help to explain the genotypic divergences of STEC.     

The whole Shebang: the gastrointestinal tract, Escherichia coli enterotoxins and secretion

This review focuses on diarrhea caused by toxins released by enterotoxigenic Escherichia coli. These bacteria are known to produce toxins that have adverse effects on the intestinal tissue in Man and animals. E. coli is contracted through the ingestion of water or food contaminated by this bacterium. Generally, E. coli colonizes the intestinal mucosa where it multiplies and causes damage to the target cells or interferes with the homeostasis that prevails in the gastrointestinal tract. Enteropathogens such as E. coli are only able to exhibit their effects after colonization of the intestinal mucosa from where they release their toxins. These bacteria mainly affect chloride ions secretion through second messenger pathways resulting in secretory diarrhea. In this review, the association of bacteria with the gastrointestinal tract as pathogens and the resulting effects on the various systems of the intestine, including the nervous system and mediators leading to secretion and diarrhea are examined.     

Escherichia coli STb toxin and colibacillosis: knowing is half the battle.

Expression of both adherence and enterotoxin expression are required for enterotoxigenic Escherichia coli (ETEC) strains to cause colibacillosis. ETEC strains are responsible for diarrhea in humans and animals by production of various enterotoxins. For many years, the role of the heat-stable E. coli enterotoxin STb as a diarrhea-causing toxin in animals, and in particular in swine, has been controversial. In fact, although the presence of STb-positive E. coli strains and diarrhea in animals is frequently observed, the difficulty of reproducing the pathology in an animal model was interpreted as a lack of toxicity. Recently, new light was shed on the activity of STb in intestinal ligated loops and in pigs orally inoculated with STb-positive E. coli strains. This minireview revisits the effects of STb on the intestinal epithelium and enlightens the significance of STb in swine colibacillosis. The interaction of STb toxin with other E. coli enterotoxins and dual ETEC/enteropathogenic E. coli or ETEC/attaching effacing E. coli infections are also discussed.     

Virulence gene profiling of enteroaggregative Escherichia coli heat-stable enterotoxin 1-harboring E. coli (EAST1EC) derived from sporadic diarrheal patients

Between 2007 and 2009, a total of 2168 Escherichia coli strains derived from diarrheal patients, defined as putative diarrheagenic E. coli (DEC), were collected from medical institutions in Akita prefecture, Japan. Thirty five of the strains lacked typical pathogenic determinants of DEC other than astA, which encodes enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1). These E. coli strains are referred to as EAST1EC. Several studies have suggested a role of EAST1 in diarrhea; however, the correlation between diarrhea and the presence of astA remains inconclusive. To investigate whether EAST1EC strains derived from diarrheal patients shared pathogenic factors other than EAST1, virulence gene profiling of 12 virulence genes - iha, lpfA, ldaG, pilS, pic, pet, irp2, daa, aah, aid, cdtB and hlyA - was carried out. PCR analysis revealed that four of the 35 EAST1EC strains harbored only astA, 24 harbored genes associated with adhesins and intestinal colonization, three strains harbored the gene for α-hemolysin, and 24 strains harbored the gene for a siderophore. These results indicated that some EAST1EC strains harbor various virulence genes associated with distinct E. coli pathotypes, primarily enterohemorrhagic E. coli and EAggEC, which may represent additional pathogenic determinants of EAST1EC.     

Localized, diffuse, and aggregative-adhering Escherichia coli from infants with acute diarrhea and matched-controls.

Of 126 infants under 2 years, enrolled in a study on the etiology of acute diarrhea in Recife, Brazil, we selected 37 from whom no recognized enteropathogens, except classic enteropathogenic Escherichia coli, were identified. For comparison, we also examined 37 matched-control infants without diarrhea seen at the same hospital setting. This paper had the purpose to determine the prevalence of localized, diffuse, and aggregative-adhering E. coli strains in both groups. Three to five fecal E. coli colonies, of each case and control, were tested individually for adherence to HeLa cells by using the one step 3-h incubation assay. Strains of E. coli showing localized adherence were found significantly more often in patients (37.8%) than in controls (13.5%), p < 0.02, and they were practically confined to EPEC serovars 055:H-, 0111:H2, and 119:H6. In contrast, E. coli isolates exhibiting the diffuse or aggregative patterns of adherence were restricted to non-EPEC serogroups and were more frequently encountered among controls.     

Response of nursery pigs to a synbiotic preparation of starch and an anti-Escherichia coli K88 probiotic

Postweaning diarrhea in pigs is frequently caused by enterotoxigenic Escherichia coli K88 (ETEC). The aim of this study was to test the efficacy of E. coli probiotics (PRO) in young pigs challenged with E. coli K88. We also tested the synbiotic interaction with raw potato starch (RPS), which can be used as a prebiotic. Forty 17-day-old weaned piglets were randomly assigned to four treatments: treatment 1, positive-control diet (C), no probiotics or RPS but containing in-feed antibiotics; treatment 2, probiotic (PRO), no feed antibiotics plus a 50:50 mixture of probiotic E. coli strains UM-2 and UM-7; treatment 3, 14% RPS, no antibiotics (RPS); treatment 4, 14% RPS plus a 50:50 mixture of probiotic E. coli strains UM-2 and UM-7, no antibiotics (PRO-RPS). The pigs were challenged with pathogenic E. coli K88 strains on day 7 of the experiment (24-day-old pigs) and euthanized on day 10 of the experiment (35-day-old pigs). Probiotic and pathogenic E. coli strains were enumerated by selective enrichment on antibiotics, and microbial community analysis was conducted using terminal restriction length polymorphism analysis (T-RFLP) of 16S rRNA genes. The combination of raw potato starch and the probiotic had a beneficial effect on piglet growth performance and resulted in a reduction of diarrhea and increased microbial diversity in the gut. We conclude that the use of E. coli probiotic strains against E. coli K88 in the presence of raw potato starch is effective in reducing the negative effects of ETEC in a piglet challenge model.     

Succinate accumulation in pig large intestine during antibiotic-associated diarrhea and the constitution of succinate-producing flora

Succinate was the major organic acid detected in the hindgut content of pigs suffering from antibiotic-associated diarrhea. Antibiotic-associated diarrhea was induced by an oral dose of polymyxin B sulfate (3,000,000 units/day) or an intramuscular injection of enrofloxacin (0.6 g enrofloxacin/day). In the large intestine of enrofloxacin-treated pigs, Gram-negative facultative anaerobic rods phylogenetically related to Escherichia coli and Gram-positive facultative anaerobic non-spore-forming rods phylogenetically related to Lactobacilli were isolated as succinate producers. Succinate-producing Lactobacilli were only isolated as the succinate producer in polymyxin B sulfate-treated pigs. In contrast to antibiotic-associated diarrhea pigs, bacteria belonging to Bacteroidaceae, Fusobacteria, and Enterobacteriaceae were detected as succinate producers in a non-treated pig. In antibiotic-associated diarrhea conditions, antibiotic-resistant Enterobacteria, E. coli in particular, and Lactobacilli may contribute to an abnormal succinate accumulation and may affect water absorption in the hindgut that relates to an expression of antibiotic-associated diarrhea.