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.     

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.     

急性腹泻患者致泻性大肠埃希菌流行情况及耐药性分析

目的 了解致泻性大肠埃希菌（DEC）在急性腹泻患者中的分布及对常用抗菌药物的敏感性,为疾病的防控和治疗提供依据。方法 收集2014年1月至2015年12月期间就诊于浙江大学医学院附属第一医院急性腹泻患者粪便标本,采用常规病原菌检验流程对常见肠道致病菌进行分离培养鉴定,分离到的疑似大肠埃希菌采用多重PCR和单重PCR进行鉴定、分型,采用纸片扩散法（K-B法）对各型DEC进行18种常用药物敏感试验。结果 从1 019例急性腹泻患者标本中分离到396株病原菌,其中DEC 230株,分离率为22.6%,占所有分离菌株的58.1%,居病原菌首位。230株DEC中,ETEC占56.5%,EAEC占30.4%,EPEC占8.7%,STEC占0.4%,混合型DEC占0.9%,未发现EIEC。各型DEC全年均有检出,其中夏季（6~8月）分离率最高,达24.0%,患者主要分布在19~45岁,男女检出率分别为21.8%和23.2%,差异无统计学意义。DEC对氨苄西林的耐药率最高,达49.3%,对头孢唑林、复方新诺明、氨苄西林/舒巴坦的耐药率分别为46.4%、35.7%、30.9%,其余抗菌药物耐药率均低于15.0%,未出现亚胺培南耐药株。207株DEC中,产ESBLs菌株占41.1%,多重耐药菌株占44.9%。结论 DEC是本地区急性腹泻最常见的细菌性病原,是临床肠道感染中不可忽视的病原之一,尤其是同时携带多型别毒力基因菌株及产ESBLs菌株和多重耐药菌。长期持续监测其分布及耐药趋势,对疾病的预防、治疗具有重要意义。     

Prevalence and characteristics of cytolethal distending toxin-producing Escherichia coli from children with diarrhea in Japan

In the present study, we examined the prevalence and characteristics of CTEC among diarrheal children in Japan during a year-long surveillance study. A PCR-RFLP assay for the detection and differentiation of five types of E. coli cdtB gene (types I through V) was developed, and 362 stool specimens collected from patients reporting to pediatric departments in two hospitals were analyzed. Of the 35 samples (9.7%) that were positive for the cdtB gene, 21 were positive for cdt-I , three for cdt-II , four for cdt-III , three for cdt-IV and four samples were positive for cdt-V , as determined by different molecular techniques. The recovery of CTEC having cdt alleles was a little less, which included 19 with cdt-I , one cdt-II , three cdt-III, three cdt-IV and four with cdt-V . Among 30 CTEC strains isolated, the majority of them (43%) belonged to serogroup O2. The other virulence genes such as astA , cnf1 , eaeA , cnf2 and bfpA genes were detected in 14 (47%), 11 (37%), four (13%), three (10%) and one (3.3%) strains of CTEC, respectively. However, the other common virulence-associated genes specific for DEC were not detected in these strains. Interestingly, an untypable cdt gene was detected by PCR-RFLP in Providencia alcalifaciens . Our data indicate that CTEC may be associated with diarrheal children in Japan and most of them do not belong to a conventional enteropathogenic pathovar and thus differ from strains isolated in developing countries.     

Pathogenic enteric Escherichia coli in children with and without diarrhea in Maputo, Mozambique

A study was conducted on the circulation of potentially diarrheagenic Escherichia coli in two groups of children, both under the age of seven. The first group (548 children) suffered from mild diarrhea and attended the Xipamanine Health Center of Maputo, in Mozambique. The second group (380 children) included randomly chosen, asymptomatic, children from the same population. A total of 503 E. coli strains were isolated from the two groups of children (n=375 and 128, respectively). All E. coli strains were genotypically and phenotypically screened. The presence of virulence-associated genes was assessed by a set of multiplex PCR specific for st and lt genes of enterotoxic Escherichia coli (ETEC), eae and bfpA genes of enteropathogenic E. coli (EPEC), stx(1) and stx(2) of enterohemorrhagic E. coli (EHEC), ial of enteroinvasive E. coli (EIEC) and the species-specific gene uidA. Adhesion and citotoxicity of isolated E. coli were evaluated in vitro on different cell cultures. A total of 37 isolates harbored virulence-associated genes: 18 were classified as ETEC, (15 from symptomatic, and three from asymptomatic children), 16 as EPEC (respectively, 13 and 3) and three EIEC in the symptomatic group. No stx(1) or stx(2) genes, associated with enterohemorrhagic E. coli were found. On the basis of the adhesion pattern on HeLa cells, 167 E. coli were classified as diffusely adhering, (125 in patients and 42 in controls) and 67 as enteroaggregative, (50 and 17, respectively). To the best of our knowledge, this is the first report in the literature on the circulation of potentially diarrheagenic E. coli in Mozambique.     

The Relative Contribution of Immigration or Local Increase for Persistence of Urban Schistosomiasis in Salvador,Bahia, Brazil

Urbanization is increasing across the globe, and diseases once considered rural can now be found in urban areas due to the migration of populations from rural endemic areas, local transmission within the city, or a combination of factors. We investigated the epidemiologic characteristics of urban immigrants and natives living in a neighborhood of Salvador, Brazil where there is a focus of transmission of Schistosoma mansoni. In a cross-sectional study, all inhabitants from 3 sections of the community were interviewed and examined. In order to determine the degree of parasite differentiation between immigrants and the native born, S. mansoni eggs from stools were genotyped for 15 microsatellite markers. The area received migrants from all over the state, but most infected children had never been outside of the city, and infected snails were present at water contact sites. Other epidemiologic features suggested immigration contributed little to the presence of infection. The intensity and prevalence of infection were the same for immigrants and natives when adjusted for age, and length of immigrant residence in the community was positively associated with prevalence of infection. The population structure of the parasites also supported that the contribution from immigration was small, since the host-to-host differentiation was no greater in the urban parasite population than a rural population with little distant immigration, and there had been little differentiation in the urban population over the past 7 years. Public health efforts should focus on eliminating local transmission, and once eliminated, reintroduction from distant migration is unlikely.     

Genetic relationship of diarrheagenic Escherichia coli pathotypes among the enteropathogenic Escherichia coli O serogroup

The genetic relationship among the Escherichia coli pathotypes was investigated. We used random amplified polymorphic DNA (RAPD) data for constructing a dendrogram of 73 strains of diarrheagenic E. coli. A phylogenetic tree encompassing 15 serotypes from different pathotypes was constructed using multilocus sequence typing data. Phylogram clusters were used for validating RAPD data on the clonality of enteropathogenic E. coli (EPEC) O serogroup strains. Both analyses showed very similar topologies, characterized by the presence of two major groups: group A includes EPEC H6 and H34 strains and group B contains the other EPEC strains plus all serotypes belonging to atypical EPEC, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). These results confirm the existence of two evolutionary divergent groups in EPEC: one is genetically and serologically very homogeneous whereas the other harbors EPEC and non-EPEC serotypes. The same situation was found for EAEC and EHEC.     

Racial differences in anthropometric traits in school children of Bahia,Brazil

Two thousand four hundred and forty-four Brazilian school children were classified as Light, Medium and Dark according to a subjective judgment of the proportion of Negro admixture, and studied for weight, height, span and upper-segment/lower-segment (US/LS) ratio. The mean US/LS ratio decreases significantly as the proportion of Negro admixture increases giving an intermediate value for those children belonging to the Medium group for race.     

Draft genome sequences of the diarrheagenic Escherichia coli collection

We report the draft genome sequences of the collection referred to as the Escherichia coli DECA collection, which was assembled to contain representative isolates of the 15 most common diarrheagenic clones in humans (http://shigatox.net/new/). These genomes represent a valuable resource to the community of researchers who examine these enteric pathogens.     

Prevalence of HEp-2 cell-adherent Escherichia coli and characterisation of enteroaggregative E. coli and chain-like adherent E. coli isolated from children with and without diarrhoea, in Londrina, Brazil

A total of 919 Escherichia coli isolates from 125 children with diarrhoea (cases) and 98 controls were assayed for adherence to HEp-2 cells. Localised adherence was found only in isolates from cases. Diffuse, aggregative (AA), chain-like adherence (CLA) and variants of the AA pattern were found in both cases and controls. The AA isolates were tested for gene sequences associated with enteroaggregative E. coli (EAEC). Only 25% of the isolates hybridised with the EAEC probe, and the aafA, astA and pet gene sequences were found in 7.9%, 44.7% and 7.9% of the isolates, respectively. The aggA gene was not found, although 7.9% were positive for aggC. The CLA isolates reacted with the EAEC probe (55.6%), and the aggC, astA and pet gene sequences were found in 66.7%, 33.3% and 11.1%, respectively. The aggR (55.6%), aspU (55.6%), shf (33.3%) and she (22.2%) genes were also found in CLA isolates.     

Curli fimbria production among Escherichia coli strains isolated from children with diarrhea and healthy children

E. coli curli fimbria are produced during starvation and at room temperature. In the study curli synthesis among E. coli strains isolated from children with diarrhea and healthy children was investigated at human body temperature and in the variable atmosphere conditions: aerobic, supplemented with CO2 and anaerobic. The ability of curli synthesis was estimated using qualitative and quantitative methods basing on Congo red binding by curli. Curli production of all E. coli strains examined was observed at temperature 37 degrees C during anaerobic cultivation, confirming their role in colonization of intestinal mucosa.     

Antibacterial activity evaluation of microcin J25 against diarrheagenic Escherichia coli

The inhibitory activities of known microcins were evaluated against some diarrheagenic Escherichia coli strains. Some antibacterial properties of microcin J25, the most active one, were studied. A rapid two-step purification was performed. The MIC and the minimum bactericidal concentration of J25 against E. coli O157:H7 were 1 and 100 microg ml(-1), respectively. A 10(4)-CFU ml(-1) contamination by this strain was destroyed in milk and meat extract by 6.25 microg of J25 ml(-1) and in half-diluted egg yolk by 50 microg of J25 ml(-1).     

A prospective study on Shiga toxin-producing Escherichia coli in children with diarrhoea in Paraná State, Brazil

Aims:  To examine stool specimens from children with diarrhea from Paraná State, southern Brazil, for presence of STEC.
Methods and Results:  A PCR screening assay for stx genes was used to examine a loopful of confluent colonies of 306 stool samples cultures. In six (1.96%) of them, DNA fragments of the expected size were observed, and the presence of stx was confirmed by DNA sequencing. Then up to 100 single colonies from each of the six stool cultures were analyzed using the same PCR protocol. However, stx -positive colonies were found only in two of the cultures. The E. coli strains belonged to serotypes O69:H11 and O178:H19, and presented genotypes stx ₁ eae ehxA and stx ₁ respectively. Shiga toxin production was confirmed using the VTEC Screen Seiken. Except ampicillin, they were susceptible to all the antimicrobials tested.
Conclusions:  These results show that STEC may be an important cause of diarrhea in children of Paraná State, and that they are present in low numbers in stools. The strains belonged to serotypes not commonly found associated with STEC and probably present low virulence.
Significance and Impact of Study:  These results indicate that molecular methods are required to diagnosis of STEC infections.     

Sylvatic vectors invading houses and the risk of emergence of cases of Chagas disease in Salvador, State of Bahia, Northeast Brazil

During the last twenty years, several adults of Triatoma tibiamaculata infected with Trypanosoma cruzi have been spontaneously caught by inhabitants, inside their houses in the new habitational district of Pitua?u of Salvador, Bahia. In this communication the authors call attention to the necessity of studies about the possibility of occurrence of new human cases of Chagas disease, to clarify the obscure origin of some positive blood donors in Salvador.