Diarrheal episodes with enterotoxigenicEscherichiacoli (ETEC) were prospectively monitored during the first 2 years of life in a cohort of 235 infants from Leo´n, Nicaragua. ETEC was an etiological finding in 38% (310 of 808) of diarrheal episodes and in 19% (277 of 1,472) of samples taken as asymptomatic controls at defined age intervals (P 5 <0.0001). The majority of diarrheal episodes (80%) occurred before 12 months of age. The major ETEC type was characterized by colonization factor CFA I and elaboration of both heat-labile enterotoxin and heat-stable enterotoxin (ST). The proportion of E. coli strains with CFA I was significantly higher in cases with diarrhea (P 5 0.002). The second most prevalent type showed putative colonization factor PCFO166 and production of ST. The prevalence of PCFO166 was approximately 20%, higher than reported before. Children with a first CFA I episode contracted a second ETEC CFA I infection 24% of the time, compared with 46% for ETEC strains of any subtype. Most of the ETEC episodes were of moderate severity, and only 5% (15 of 310) were characterized as severe. In conclusion, our results give valuable information for the planning of intervention studies using ETEC vaccines.
EnterotoxigenicEscherichiacoli (ETEC) has consistently been the predominant bacterial cause of diarrhea in many birth co- hort- and hospital-based studies conducted in Egypt. We evaluated the pathogenicity of ETEC isolates in a birth cohort of chil- dren living in a rural community in Egypt. Between 2004 and 2007, we enrolled and followed 348 children starting at birth until their second year of life. A stool sample and two rectal swabs were collected from children during twice-weekly visits when they presented with diarrhea and were collected every 2 weeks if no diarrhea was reported. From routine stool cultures, five E. coli- like colonies were screened for ETEC enterotoxins using a GM1 enzyme-linked immunosorbent assay (ELISA). The isolates were screened against a panel of 12 colonization factor antigens (CFAs) by a dot blot assay. A nested case-control study evaluated the association between initial or repeat excretion of ETEC and the occurrences of diarrhea. The pathogenicity of ETEC was esti- mated in symptomatic children compared to that in asymptomatic controls. ETEC was significantly associated with diarrhea (crude odds ratio, 1.37; 95% confidence interval [CI], 1.24 to 1.52). The distribution of ETEC enterotoxins varied between the symptomatic children (44.2% heat-labile toxin [LT], 38.5% heat-stable toxin [ST], and 17.3% LT/ST) and asymptomatic children (55.5% LT, 34.6% ST, and 9.9% LT/ST) (P < 0.001). The CFAs CFA/I (n ⴝ 61), CS3 (n ⴝ 8), CS1 plus CS3 (n ⴝ 24), CS2 plus CS3 (n ⴝ 18), CS6 (n ⴝ 45), CS5 plus CS6 (n ⴝ 11), CS7 (n ⴝ 25), and CS14 (n ⴝ 32) were frequently detected in symptomatic chil- dren, while CS6 (n ⴝ 66), CS12 (n ⴝ 51), CFA/I (n ⴝ 43), and CS14 (n ⴝ 20) were detected at higher frequencies among asymp- tomatic children. While all toxin phenotypes were associated with diarrheal disease after the initial exposure, only ST and LT/ ST-expressing ETEC isolates (P < 0.0001) were associated with disease in repeat infections. The role of enterotoxins and pathogenicity during repeat ETEC infections appears to be variable and dependent on the coexpression of specific CFAs.
The genetic diversity of 47 enterotoxigenicEscherichiacoli (ETEC) strains of serotypes O6:H16, O27:H7, O29:H21, O128ac:H12, and O153:H45, previously isolated from diarrheic patients in Brazil over a period of 15 years, was investigated by random amplification of polymorphic DNA (RAPD). Informative band arrays were obtained with three 10-mer primers with G 1 C contents of 50, 60, and 70%. Based on the combination of the band profiles generated by the three primers 22 RAPD types were detected, and 5 major clonal clusters, each one with at least 80% identical bands, were established. The clonal clusters corresponded to strains having the same serotype which, in most cases, also had the same virulence factors (colonization factors and toxin types) and outer membrane protein and lipopolysaccharide sodium dodecyl sulfate-polyacrylamide gel electrophore- sis profiles. The results suggested a correlation between phenotypic properties and genetic relatedness of ETEC isolates of human origin and indicated that a reduced number of clonally related strains are found in areas of ETEC endemicity in Brazil. Moreover, the RAPD technique revealed intraserotype-specific variations, unde- tectable by the combination of several phenotypic typing methods, among the ETEC strains analyzed. These results show that RAPD typing represents a useful tool for population genetics as well as for epidemiological studies of ETEC.
DNA amplification of lngA, the structural gene of longus type IV pilus produced by human enterotoxigenicEscherichiacoli (ETEC) was achieved by the use of specific oligonucleotide primers designed from the nucle- otide sequence of lngA. A 630-bp fragment representing the entire lngA gene was amplified in eight prototype strains previously characterized as longus positive. Five ETEC strains producing colonization factor antigen III (CFA III) (also a type IV pilus) were also positive by PCR, confirming the DNA homology between CFA III and longus. None of the non-ETEC and non-E. coli enteropathogens studied showed the 0.63-kbp amplicon. The procedure thus detected only ETEC strains harboring type IV pili genes with or without other colonization factors. Except for five lngA PCR-positive, probe-positive strains, all lngA PCR-positive strains produced the pilin as demonstrated by immunoblotting. To test the amplification procedure in a clinical setting, a collection of 264 fresh clinical E. coli strains isolated from 88 Mexican children with diarrhea was screened by PCR. Among 82 ETEC isolates found, 30 (36.5%) were lngA PCR-positive. Twenty-seven percent of the children shed ETEC that possessed lngA. In parallel with DNA probes or PCR protocols to detect enterotoxin genes, the lngA PCR method may prove useful for detection of ETEC harboring type IV pilus genes in epidemiological studies.
EnterotoxigenicEscherichiacoli (ETEC) is a common cause of diarrhea among children living in and among travelers visiting developing countries. Human ETEC strains represent an epidemiologically and phenotypi- cally diverse group of pathogens, and there is a need to identify natural groupings of these organisms that may help to explain this diversity. Here, we sought to identify most of the important human ETEC lineages that exist in the E. coli population, because strains that originate from the same lineage may also have inherited many of the same epidemiological and phenotypic traits. We performed multilocus sequence typing (MLST) on 1,019 ETEC isolates obtained from humans in different countries and analyzed the data against a backdrop of MLST data from 1,250 non-ETEC E. coli and eight ETEC isolates from pigs. A total of 42 different lineages were identified, 15 of which, representing 792 (78%) of the strains, were estimated to have emerged >900 years ago. Twenty of the lineages were represented in more than one country. There was evidence of extensive exchange of enterotoxin and colonization factor genes between different lineages. Human and porcine ETEC have probably emerged from the same ancestral ETEC lineage on at least three occasions. Our findings suggest that most ETEC strains circulating in the human population today originate from well-established, globally widespread ETEC lineages. Some of the more important lineages identified here may represent a smaller and more manageable target for the ongoing efforts to develop effective ETEC vaccines.
The prevalence of toxin types and colonization factors (CFs) of enterotoxigenicEscherichiacoli (ETEC) was prospectively studied with fresh samples (n ⴝ 4,662) obtained from a 2% routine surveillance of diarrheal stool samples over 2 years, from September 1996 to August 1998. Stool samples were tested by enzyme-linked immunoassay techniques and with specific monoclonal antibodies for the toxins and CFs. The prevalence of ETEC was 14% (n ⴝ 662), with over 70% of the strains isolated from children 0 to 5 years of age, of whom 93% were in the 0- to 3-year-old age range. Of the total ETEC isolates, 49.4% were positive for the heat-stable toxin (ST), 25.4% were positive for the heat-labile toxin (LT) only, and 25.2% were positive for both LT and ST. The rate of ETEC isolation peaked in the hot summer months of May to September and decreased in winter. About 56% of the samples were positive for 1 or more of the 12 CFs that were screened for. The coli surface antigens CS4, CS5, and/or CS6 of the colonization factor antigen (CFA)/IV complex were most prevalent (incidence, 31%), followed by CFA/I (23.5%) and coli surface antigens CS1, CS2, and CS3 of CFA/II (21%). In addition, other CFs detected in decreasing order were CS7 (8%), CS14 (PCFO166) (7%), CS12 (PCFO159) (4%), CS17 (3%), and CS8 (CFA/III) (2.7%). The ST- or LT- and ST-positive ETEC isolates expressed the CFs known to be the most prevalent (i.e., CFA/I, CFA/II, and CFA/IV), while the strains positive for LT only did not. Among children who were infected with ETEC as the single pathogen, a trend of relatively more severe disease in children infected with ST-positive (P < 0.001) or LT- and ST-positive (P < 0.001) ETEC isolates compared to the severity of the disease in children infected with LT only-positive ETEC isolates was seen. This study supports the fact that ETEC is still a major cause of childhood diarrhea in Bangladesh, especially in children up to 3 years of age, and that measures to prevent such infections are needed in developing countries.
EnterotoxigenicEscherichiacoli (ETEC) causes diarrhea in both humans and animals. The contaminated food and water are the most common vehicles for ETEC infection. The colonization factor antigen (CFA-1) is a fimbriae protein that promotes adherence of the ETEC strain to the epithelium of the small intestine of the host. In this study IgY proteins were produced against the CFA-1 of ETEC in immunized white leg horn chickens. The collection of antibody was found higher in 25% and 50% ammonium precipitated samples. The isolated CFA-1 protein (0.5 mg/ml) was purified by dialysis (0.42 mg/ml), gel-filtration (0.08 mg/ml), and ion-exchange chromatography (0.065 mg/ml). The CFA-1 protein obtained was submitted to pBLAST which matched with CFA-b with accession no.-AAC41415.1. The CFA-1 gene was cloned in pUC18 vector, and transformed into Escherichiacoli DH5α strain. In conclusion, we have successfully purified and characterized monoclonal antibodies (IgY proteins) against CFA-1 of enterotoxigenic E. coli that may be useful for diagnosis of widely occurring Escherichiacoli infections.
EnterotoxigenicEscherichiacoli (ETEC) is the most common bac- terial cause of diarrhoea in adults and children in developing coun- tries (Qadri 2005; Walker 2007). The annual incidence of this dis- ease is highest in young children and susceptibility to the disease declines with age. Children born in endemic regions are likely to experience two to three episodes of ETEC diarrhoea before their fifth birthday (Wennerås 2004). The practical difficulties associ- ated with making an accurate diagnosis of ETEC in low-resource settings mean that its significance has often been underestimated (Wennerås 2004; Qadri 2005). However, a review of microbiolog- ical studies conducted in endemic regions between 1992 and 2000 found that ETEC was the causative organism in approximately 25% of all diarrhoeal episodes in children aged between one and four years (Wennerås 2004). Many more children were shown to carry the organism asymptomatically in their gut (Walker 2007). A global burden of approximately 280 million clinical episodes and 380,000 deaths annually are estimated (WHO 2009). Person-to-person transmission of ETEC occurs via ingestion of faecally-contaminated food or water. In developed countries where sanitation standards are usually higher, ETEC infection is rare. However, it remains a major cause of ’travellers’ diarrhoea’ which occurs in people visiting or returning from ETEC-endemic regions (Qadri 2005; DuPont 2008; Widermann 2009). Epidemics of ETEC diarrhoea have also occurred during natural disasters, such as floods, where there has been an acute deterioration in the quality of drinking water and sanitation (Schwartz 2006; Harris 2008). The clinical illness is characterized by a profuse watery diarrhoea that lasts for several days and may be associated with abdominal cramp, malaise, vomiting, and a low grade fever. Without adequate treatment this can lead to dehydration. If people have a prolonged infection or are infected again, this can lead to malnutrition or growth inhibition in young children (Black 1984; Qadri 2005; Qadri 2007).
previously described (38). Primers were designed to amplify small fragments (500 bp to 1 kb) of putative ETEC virulence genes and are listed in Table S2 in the supplemental material. The leoA, tia, and tibC primers were designed based upon the E. coli H10407 sequence. clyA primers were designed to correspond to highly conserved regions of the gene which were determined from comparison of the clyA sequences available from different E. coli isolates (34). Primers specific to eatA were designed after inspection of a ClustalW multiple alignment of all the known serine protease autotransporter proteins. The presence or absence of the virulence gene was determined by PCR using two pairs of primers for each locus. Genomic comparisons. In an attempt to identify chromosomal genes potentially involved in enterotoxigenicity, genomic comparisons of the draft ETEC genome sequences from strains E24377A (GenBank accession number AAJZ01000001), B7A (GenBank accession numbers AAJT01000001 to AAJT01000198), and H10407 (available from http://www.sanger.ac.uk/Projects/E_coli_H10407) were performed. Orthologues were identified using the mutual best hits procedure, as previously described (7). Genes that had copies in all three ETEC genomes were further compared in the same manner with a representative set of complete and draft E. coli genome sequences (laboratory strain K-12 MG1655, GenBank accession number U00096; EHEC strain O157:H7 Sakai, GenBank accession number BA000007; uropathogenic strain CFT073, GenBank accession number AE014075; commensal strain HS, GenBank accession AAJY01000001; enteroinvasive E. coli strain 53638, GenBank accession numbers AAKB01000001 to AAKB01000119; EPEC1 strain E22, GenBank accession numbers AAJV01000001 to AAJV01000109; EPEC2 strain B171, GenBank accession numbers AAKB01000001 to AAKB01000119; and EAEC strain 042, available from http://www.sanger.ac.uk/Projects/Escherichia _Shigella).
The strains were analyzed for presence of known ETEC virulence factors. Strains E1777, E2265, and CE549 contained both LT and ST genes (Table 4). The ST structural gene (estA) was present in all strains except in strain CE516, while the LT structural gene (eltA) was present in all four genomes. In addition, genes clyA (cytolysin), eatA (serine protease autotranspor- ter), and ecpA (pilus subunit) were also present in all of the 4 ETEC strains, but genes leoA (accessory protein for LT secretion), tibA (autotransporter), and tia (surface protein) were absent in all genomes. Only CE549 contained the complete ~14 kb operon encod- ing longus known as a type IV pilus . The etpA gene, which mediates adhesion between ETEC flagella and host cells , was present only in CE549 but absent in other strains. These specific virulence factors present in CE549 may increase its virulence in humans, but their functional effects remain to be further determined.
Bacterial strains. The ETEC bacterial strains analyzed in this study were isolated from liquid stool samples of individuals being treated for severe cholera-like diarrhea at the International Centre for Diarrhoeal Disease Research, Mohakhali, Dhaka (http://ww- w.icddrb.org), Bangladesh, or the treatment center in the Mirpur district of Dhaka. Multiple lactose-fermenting colonies were se- lected from MacConkey agar culture plates and screened using multiplex PCR for genes encoding heat-labile toxin, as well as human and porcine heat-stable toxin (STh and STp), as previ- ously described (11). Isolated colonies of ETEC were then grown overnight in Luria-Bertani (LB) medium at 37°C with shaking and preserved as glycerol stocks stored at ⫺ 80°C. Included for com- parison in these studies were isolates from geographically and temporally disparate sources, including the ThroopD strain, iso- lated from a patient with severe cholera-like diarrhea in Dallas, TX, in 1975 (12), and several isolates (Juruá_18/11, Juruá_20/10, Envira_10/1, and Envira_8/11) obtained during ETEC outbreaks that caused severe diarrheal illness in two small villages, Juruá and Envira, in the Amazonia region of Brazil in 1998 (13) (see Table S1 in the supplemental material). A total of 208 new ETEC isolates were included in this study.
Microbial agents and target sequences. The targets for real-time PCR are presented in Table 1. The amplified regions of rotavirus, norovirus, sapovirus, astrovirus, and adenovirus were located in conserved genomic regions (21–25), and these assays have been used in our diagnostic labo- ratory for several years. Bacterial PCRs were developed with guidance from available publications with respect to suitable target regions (26–35), usually by adapting a traditional PCR method to real-time PCR (when this study was planned, suitable real-time PCR assays were lacking for most nonviral targets). Thus, established target regions were used, and primers and probes were designed with the aim of obtaining similar melting tem- peratures ( ⬃ 58 to 60°C for primers and ⬃ 68 to 70°C for probes). For enterotoxigenicEscherichiacoli (ETEC), both heat-labile toxin (eltB) and heat-stable toxin (estA) coding regions were targeted. Shigella was identi- fied by amplification of the invasion plasmid antigen H (ipaH) gene (which also may be present in enteroinvasiveE. coli [EIEC]), Campylobac- ter jejuni by the fibronectin-binding protein (cadF) gene, and Cryptospo- ridium parvum/hominis by the oocyst wall protein (OWP) gene.