Detection with synthetic oligonucleotide probes of nucleotide sequence variations in the genes encoding enterotoxins of Escherichia coli.

We examined variations in the genes encoding heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) in 88 strains of Escherichia coli isolated from individuals with traveler's diarrhea to find suitable sequences for use as oligonucleotide probes. Four oligonucleotide probes of the gene encoding ST of human origin (STIb or STh), one oligonucleotide probe of the gene encoding ST of porcine origin (STIa or STp), and three oligonucleotide probes of the gene encoding LT of human origin (LTIh) were used in DNA colony hybridization tests. In 15 of 22 strains possessing the STh gene and 28 of 42 strains producing LT, the sequences of all regions tested were identical to the published sequences. One region in the STh gene examined with a 18-mer probe was relatively well conserved and was shown to be closely associated with the enterotoxicity of the E. coli strains in suckling mice. This oligonucleotide, however, hybridized with strains of Vibrio cholerae O1, V. parahaemolyticus, and Yersinia enterocolitica that gave negative results in the suckling mouse assay.     

Blinded, two-laboratory comparative analysis of Escherichia coli heat-stable enterotoxin production by using monoclonal antibody enzyme-linked immunosorbent assay, radioimmunoassay, suckling mouse assay, and gene probes

Heat-stable enterotoxin (ST)-producing enterotoxigenic Escherichia coli (ETEC) can be identified by a variety of assays, including the suckling mouse assay (SMA), radioimmunoassay (RIA), polyclonal or monoclonal antibody enzyme-linked immunosorbent assay (ELISA), and DNA hybridization with STh and STp gene probes. To compare the sensitivity and reliability of these assays, 100 coded ETEC and non-ETEC isolates were blindly tested in two independent laboratories. SMA, RIA, and monoclonal ELISA were performed in Cincinnati, Ohio, while gene probe analysis was performed in Baltimore, Md. The method of storage of organisms had a profound effect on the stability of plasmids in certain strains. Hybridization experiments to determine the presence or absence of the enterotoxin gene showed that strains stored on Dorset egg medium at room temperature better retained their plasmids than strains stored frozen in skim milk. Forty-four of the 100 organisms obtained from the skim milk stock were found to produce STa in liquid culture by the RIA, SMA, and monoclonal ELISA (100% agreement). However, 50 of 54 of the strains stored on Dorset egg medium which were originally classified as STa+ or ST+ LT+ (positive for both heat-stable and heat-labile [LT] enterotoxins) were found to produce STa and retain the plasmid by each of these assays. Three additional strains were found which harbored the plasmid but did not elaborate STa by any of the assays (3% discrepancy). The monoclonal antibody ELISA appears to be highly reliable for determination of STa production by ETEC and can be easily scored visually even by untrained personnel. Furthermore, when this STa assay is coupled with a polyclonal antibody assay, it is possible to predict the genotype of STh- and STp-producing organisms.     

Molecular Characterization of Enterotoxigenic Escherichia coli Isolates Recovered from Children with Diarrhea during a 4-Year Period (2007 to 2010) in Bolivia

Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood diarrhea. This study aimed to characterize ETEC strains isolated from Bolivian children aged <5 years according to enterotoxin profile, colonization factors (CFs), suggested virulence genes, and severity of disease. A total of 299 ETEC isolates recovered from children with diarrhea and 55 ETEC isolates from children without diarrhea (controls) were isolated over a period of 4 years. Strains expressing heat-labile toxin (LT) or heat-stable toxin (ST) alone were about equally common and twice as common as ETEC producing both toxins (20%). ETEC strains expressing human ST (STh) were more common in children aged <2 years, while ETEC strains expressing LT plus STh (LT/STh) were more frequent in 2- to 5-year-old children. Severity of disease was not related to the toxin profile of the strains. CF-positive isolates were more frequently identified in diarrheal samples than in control samples (P = 0.02). The most common CFs were CFA/I and CS14. CFA/I ETEC strains were more frequent in children aged <2 years than CS1+CS3 isolates and CS14 isolates, which were more prevalent in 2- to 5-year-old children. The presence of suggested ETEC virulence genes (clyA, eatA, tia, tibC, leoA, and east-1) was not associated with disease. However, east-1 was associated with LT/STh strains (P < 0.001), eatA with STh strains (P < 0.001), and tia with LT/STh strains (P < 0.001). A minor seasonal peak of ETEC infections was identified in May during the cold-dry season and coincided with the peak of rotavirus infections; this pattern is unusual for ETEC and may be important for vaccination strategies in Bolivia.     

Cloning and nucleotide sequence of a heat-stable enterotoxin gene from Vibrio cholerae non-O1 isolated from a patient with traveler''s diarrhea.

We determined the nucleotide sequence of the heat-stable enterotoxin (STa) gene of the Vibrio cholerae non-O1 strain NRT36 isolated from a patient with traveler's diarrhea. The gene is chromosomally encoded and the presumed product is 78 amino acids in length, with a molecular weight of 8,814, though the genes of Escherichia coli STa(s) (STh and STp) are encoded in plasmid DNA and both products are 72 amino acids in length. The first 18 amino acids at the NH2 terminus were hydrophobic, suggesting that this region of the polypeptide acts as a signal sequence for the toxin. The last 17 amino acids at the COOH terminus were identical to those deduced from the toxin (NAG-ST) produced by V. cholerae non-O1 strain A-5 isolated from a frozen shrimp. The deduced amino acid sequence of the NAG-ST precursor had 50 and 46% homology to those of E. coli STh and STp, respectively. The hydropathy plot analysis of each predicted protein revealed similar profiles between them, suggesting that the NAG-ST precursor has structural similarity to those of E. coli STa(s).     

Enterotoxigenic Escherichia coli in a population of infants with diarrhea in Chile.

The incidence of enterotoxigenic Escherichia coli (ETEC) was investigated in 95 E. coli strains isolated from 48 infants with diarrhea in Santiago, Chile. By using standard biological assays and DNA-DNA hybridization procedures, ETEC was found in 31.2% of the cases: 14 strains produced heat-stable enterotoxin (ST) only, three strains produced heat-labile enterotoxin (LT) and ST, and two strains produced LT only. DNA probes detected all enterotoxin producers except one ST-producing strain. The ST strains hybridized with one or both of the human ST probes (ST Ib and ST A2). Two of the LT-ST strains hybridized with the ST Ia and ST Ib probes, and the third strain did not hybridize with any of the ST probes. Only the ST group expressed multiple resistance (85.7%) and colonization factor antigen I (CFA I) (92.8%); CFA II was found in two of three LT-ST strains. The O153:H45 serotype was found in 10 of 14 ST strains, and O6:K15:H16 was found in one LT strain and in two LT-ST strains. These findings suggest that ETEC, especially strains that produce ST, may be an important cause of diarrhea among Chilean infants.     

Characterization of Immunological Cross-Reactivity between Enterotoxigenic Escherichia coli Heat-Stable Toxin and Human Guanylin and Uroguanylin

Enterotoxigenic Escherichia coli (ETEC) expressing the heat-stable toxin (ST) (human-type [STh] and porcine-type [STp] variants) is among the five most important enteric pathogens in young children living in low- and middle-income countries. ST mediates diarrheal disease through activation of the guanylate cyclase C (GC-C) receptor and is an attractive vaccine target with the potential to confer protection against a wide range of ETEC strains. However, immunological cross-reactivity to the endogenous GC-C ligands guanylin and uroguanylin is a major concern because of the similarities to ST in amino acid sequence, structure, and function. We have investigated the presence of similar epitopes on STh, STp, guanylin, and uroguanylin by analyzing these peptides in eight distinct competitive enzyme-linked immunosorbent assays (ELISAs). A fraction (27%) of a polyclonal anti-STh antibody and an anti-STh monoclonal antibody (MAb) cross-reacted with uroguanylin, the latter with a 73-fold-lower affinity. In contrast, none of the antibodies raised against STp, one polyclonal antibody and three MAbs, cross-reacted with the endogenous peptides. Antibodies raised against guanylin and uroguanylin showed partial cross-reactivity with the ST peptides. Our results demonstrate, for the first time, that immunological cross-reactions between ST and the endogenous peptides can occur. However, the partial nature and low affinity of the observed cross-reactions suggest that the risk of adverse effects from a future ST vaccine may be low. Furthermore, our results suggest that this risk may be reduced or eliminated by basing an ST immunogen on STp or a selectively mutated variant of STh.     

Disease burden due to enterotoxigenic Escherichia coli in the first 2 years of life in an urban community in Bangladesh

A cohort of 321 children was followed from birth up to 2 years of age to determine the incidence of enterotoxigenic Escherichia coli (ETEC) in Bangladesh. The average number of diarrheal days and incidence rates were 6.6 and 2.3/child/year, respectively. ETEC was the most common pathogen and was isolated in 19.5% cases, with an incidence of 0.5 episode/child/year. The prevalence of rotavirus diarrhea was lower (10%). ETEC expressing the heat-stable enterotoxin (ST) was predominant. Strains isolated from diarrheal cases were positive for colonization factors (CFs) in higher frequency (66%) than from healthy children (33%) (P < 0.001). The heat-labile toxin (LT)-positive strains from healthy children were more often CF negative (92%) than those isolated from children with diarrhea (73%) (P < 0.001). In children with symptomatic or asymptomatic infections by CFA/I, CS1 plus CS3, CS2 plus CS3, or CS5 plus CS6 strains, a repeat episode of diarrhea or infection by the homologous CF type was uncommon. Repeat symptomatic infections were noted mostly for LT- and ST-expressing ETEC. ETEC diarrhea was more prevalent in children in the A and AB groups than in those in the O blood group (P = 0.032 to 0.023). Children with ETEC diarrhea were underweight and growth stunted at the 2-year follow-up period, showing the importance of strategies to prevent and decrease ETEC diarrheal morbidity in children.     

Escherichia coli in acute diarrhea in a population of infants

The incidence of Escherichia coli EPEC and ETEC had been studied on 555 infants from Tunis area. 193 of them do not present any diarrhea. Frequency of EPEC is 9.14% in infants with diarrhea and 3.10% in the group without diarrhea. Analysis of 87 Escherichia coli strains using genetic probes showed that 4 EPEC strains present effacing attachment factor (EAF) and one EPEC strain produce verotoxin. The frequency of ETEC isolated is 18.05%, the majority of them produced ST enterotoxin. Only colonization factor antigen I (CFAI) was found in ETEC strains.     

Bacteriological and epidemiological characteristics of enterotoxigenic Escherichia coli isolated in Tokyo, Japan, between 1966 and 2009

Enterotoxigenic Escherichia coli (ETEC) caused 131 outbreaks in Tokyo, Japan, between 1966 and 2009. The major serogroups were O6, O27, O148, and O159. The incidence of serogroups O25 and O169 recently increased. Heat-stable enterotoxin (ST) subtyping revealed that E. coli of serogroups O6, O15, O25, and O159 possessed the STh gene, whereas those serotyped as O27 and O169 possessed the STp gene.     

Production of Escherichia coli STa-like heat-stable enterotoxin by Citrobacter freundii isolated from humans.

Citrobacter species are often present in the stools of children and are generally considered a normal component of the intestinal microflora. Previous reports suggested that they might act as enteric pathogens. Aiming at defining the role of Citrobacter species in inducing diarrhea, we looked for their presence in the stools of 328 children with diarrhea and in 108 controls. Citrobacter strains were isolated from 46 patients (14%) and 7 controls (6.5%) (P less than 0.05). All isolates were tested for heat-stable (ST) and heat-labile (LT) enterotoxin. No LT-producing organisms were found. Three C. freundii strains, all isolated from children with diarrhea, elaborated an enterotoxin detected by the suckling mouse assay. A crude extract was prepared by acetone precipitation and a sequential ultrafiltration technique. The enterotoxin was heat stable, and its estimated molecular weight was between 2,000 and 10,000. Citrobacter enterotoxin was soluble in methanol and stable at acid and neutral pHs but not above pH 8, and its activity was destroyed by treatment with 2-mercaptoethanol. Citrobacter enterotoxin was inactive in the 18-h rabbit ileal loop test. All these characteristics closely resemble STa produced by Escherichia coli. The time course of Citrobacter enterotoxin-induced intestinal secretion in suckling mice was similar to that of E. coli STa. The enterotoxin produced by C. freundii cross-reacted with monoclonal antibodies raised against E. coli STa. These results suggest that C. freundii is capable of inducing diarrhea through the production of an E. coli-like STa, and its presence in the stools of patients with diarrhea should be considered as that of a possible etiologic agent.     

Detection of enterotoxigenic Escherichia coli by colony hybridization with biotinylated enterotoxin probes.

By using biotinylated enterotoxin DNA probes, a method to detect enterotoxigenic Escherichia coli by colony hybridization was developed. The treatment of colonies on nitrocellulose membrane filters with proteinase K and Triton X-100 was essential for obtaining the specific hybridization. A total of 200 E. coli strains isolated from travelers with diarrhea were tested for colony hybridization by using a probe encoding heat-labile toxin (LT) type h. All strains (86 of 86) that produced LT, but none of the non-LT producers, hybridized with 32P-labeled and biotinylated LT type h probes. A total of 36 strains chosen randomly from the 200 isolates were tested for colony hybridization by using heat-stable enterotoxin (ST) probes. All but two strains that hybridized with the 32P-labeled ST type Ia probe also hybridized with the biotinylated ST type Ia probe. All strains that hybridized with the 32P-labeled ST type Ib probe also hybridized with the biotinylated ST type Ib probe. Thus, almost all E. coli strains tested were judged to be the same by colony hybridization with biotinylated or 32P-labeled enterotoxin probes. These results demonstrate that the biotinylated enterotoxin probes are useful in the diagnosis of enterotoxigenic E. coli strains by colony hybridization.     

Identification of enterotoxigenic Escherichia coli harboring longus type IV pilus gene by DNA amplification

DNA amplification of lngA, the structural gene of longus type IV pilus produced by human enterotoxigenic Escherichia coli (ETEC) was achieved by the use of specific oligonucleotide primers designed from the nucleotide 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.     

Prospective Cohort Study of Enterotoxigenic Escherichia coli Infections in Argentinean Children

In a follow-up study, enterotoxigenic Escherichia coli (ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children (P = 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872; P = 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea (P = 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors (P = 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81; P = 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6; P = 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea.     

Controlled study of Escherichia coli diarrheal infections in Bangladeshi children.

Diarrheal diseases are highly prevalent in Bangladesh. However, the relative contribution of diarrheagenic Escherichia coli organisms--those that are enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroinvasive, enterohemorrhagic, enteroaggregative, and diffuse adherent--to diarrhea in Bangladeshi populations is not known. With DNA probes specific for these diarrheagenic E. coli strains, we analyzed fecal E. coli from 451 children up to 5 years of age with acute diarrhea seeking treatment at a Dhaka hospital and from 602 matched control children without diarrhea from July 1991 to May 1992. Enteroinvasive E. coli was not isolated from any children; enterohemorrhagic E. coli was not isolated from any diarrheal children but was isolated from five control children; enteroaggregative and diffuse adherent E. coli strains were isolated with similar frequencies from children with and without diarrhea, thereby showing no association with diarrhea; ETEC was significantly associated with diarrhea in the diarrheal children as a whole and especially in the age groups of 0 to 24 months and 37 to 48 months (further analysis suggests an association with diarrhea for the heat-stable toxin only and for both heat-labile- and heat-stable-toxin-producing ETEC only); and EPEC was significantly associated with diarrhea in the diarrhea group as a whole and particularly in infants up to 1 year of age. Further analysis suggested that EPEC strains of only the traditional serogroups were significantly associated with diarrhea. ETEC and EPEC infections peaked during warm months. Our data thus suggest that EPEC and ETEC are important causes of acute diarrhea in children in this setting.     

Prevalence and association of the longus pilus structural gene (lngA) with colonization factor antigens, enterotoxin types, and serotypes of enterotoxigenic Escherichia coli.

Human enterotoxigenic Escherichia coli (ETEC) produces a plasmid-encoded type IV pilus termed longus (for long pilus). Regardless of the geographic origins of ETEC strains, the longus structural gene lngA was found to have the highest level of association with ETEC producing colonization factor antigen (CFA) CFA/II, followed by ETEC producing CFA/I and CFA/IV. ETEC bearing the less prevalent CFA/III and putative colonization factors and ETEC negative for CFA and putative colonization factor also contained lngA-related sequences. lngA was found in a considerable number of ETEC serotypes and was more often associated with ETEC producing heat-stable enterotoxins than with ETEC producing both heat-labile and heat-stable enterotoxins or heat-labile enterotoxin alone. lngA was found more often in strains isolated from children with diarrhea than in strains from healthy children, suggesting an association with intestinal disease. We conclude that longus is a widely distributed antigenic determinant in ETEC that is highly associated with known plasmid-encoded virulence factors, namely, CFAs and enterotoxins. A longus-specific probe may be a helpful epidemiological tool to assist in the identification of ETEC.     

Phenotypic profiles of enterotoxigenic Escherichia coli associated with early childhood diarrhea in rural Egypt

Enterotoxigenic Escherichia coli (ETEC) causes substantial diarrheal morbidity and mortality in young children in countries with limited resources. We determined the phenotypic profiles of 915 ETEC diarrheal isolates derived from Egyptian children under 3 years of age who participated in a 3-year population-based study. For each strain, we ascertained enterotoxin and colonization factor (CF) expression, the O:H serotype, and antimicrobial susceptibility. Sixty-one percent of the strains expressed heat-stable enterotoxin (ST) only, 26% expressed heat-labile enterotoxin (LT) alone, and 12% expressed both toxins. The most common CF phenotypes were colonization factor antigen I (CFA/I) (10%), coli surface antigen 6 (CS6) (9%), CS14 (6%), and CS1 plus CS3 (4%). Fifty-nine percent of the strains did not express any of the 12 CFs included in our test panel. Resistance of ETEC strains to ampicillin (63%), trimethoprim-sulfamethoxazole (52%), and tetracycline (43%) was common, while resistance to quinolone antibiotics was rarely detected. As for the distribution of observed serotypes, there was an unusually wide diversity of O antigens and H types represented among the 915 ETEC strains. The most commonly recognized composite ETEC phenotypes were ST CS14 O78:H18 (4%), ST (or LTST) CFA/I O128:H12 (3%), ST CS1+CS3 O6:H16 (2%), and ST CFA/I O153:H45 (1.5%). Temporal plots of diarrheal episodes associated with ETEC strains bearing common composite phenotypes were consistent with discrete community outbreaks either within a single or over successive warm seasons. These data suggest that a proportion of the disease that is endemic to young children in rural Egypt represents the confluence of small epidemics by clonally related ETEC strains that are transiently introduced or that persist in a community reservoir.     

Genotypic and Phenotypic Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Peruvian Children

Enterotoxigenic Escherichia coli (ETEC) is a major cause of childhood diarrhea. The present study sought to determine the prevalence and distribution of toxin types, colonization factors (CFs), and antimicrobial susceptibility of ETEC strains isolated from Peruvian children. We analyzed ETEC strains isolated from Peruvian children between 2 and 24 months of age in a passive surveillance study. Five E. coli colonies per patient were studied by multiplex real-time PCR to identify ETEC virulence factors. ETEC-associated toxins were confirmed using a GM1-based enzyme-linked immunosorbent assay. Confirmed strains were tested for CFs by dot blot assay using 21 monoclonal antibodies. We analyzed 1,129 samples from children with diarrhea and 744 control children and found ETEC in 5.3% and 4.3%, respectively. ETEC was more frequently isolated from children >12 months of age than from children <12 months of age (P < 0.001). Fifty-two percent of ETEC isolates from children with diarrhea and 72% of isolates from controls were heat-labile enterotoxin (LT) positive and heat-stable enterotoxin (ST) negative; 25% and 19%, respectively, were LT negative and ST positive; and 23% and 9%, respectively, were LT positive and ST positive. CFs were identified in 64% of diarrheal samples and 37% of control samples (P < 0.05). The most common CFs were CS6 (14% and 7%, respectively), CS12 (12% and 4%, respectively), and CS1 (9% and 4%, respectively). ST-producing ETEC strains caused more severe diarrhea than non-ST-producing ETEC strains. The strains were most frequently resistant to ampicillin (71%) and co-trimoxazole (61%). ETEC was thus found to be more prevalent in older infants. LT was the most common toxin type; 64% of strains had an identified CF. These data are relevant in estimating the burden of disease due to ETEC and the potential coverage of children in Peru by investigational vaccines.Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children from developing countries and in adult travelers from industrialized countries to the developing world (16, 21). According to the World Health Organization (WHO), ETEC is the second most common cause of diarrhea after rotavirus in children less than 5 years of age and is therefore an important target for vaccine development (11). Diarrhea due to ETEC develops between 8 and 72 h after initial infection, usually due to the ingestion of contaminated food and water (21). The disease varies from a mild illness to one of great severity, usually without leukocytes or fecal blood but often with vomiting and, potentially, dehydration (10).The ability of ETEC to adhere to and colonize the human intestinal mucosa has been correlated with the presence of specific antigenic fimbriae called colonization factors (CFs), which have been designated colonization factor antigens (CFAs), coli surface antigens (CSs), or putative colonization factors (PCFs), followed by a numeric designation. The CFs are mainly fimbrial or fibrillar proteins, although some are not fimbrial in structure (21). To date, over 25 human ETEC CFs have been described. In turn, these CFs have been divided into different families: (i) a CFA/I-like group including CFA/I, CS1, CS2, CS4, CS14, and CS17; (ii) a CS5-like group including CS5, CS7, CS18, and CS20; and (iii) a unique group including CS3, CS6, and CS10 to CS12 (8, 21, 33).Following CF-mediated mucosal adhesion, ETEC elaborates one or both of two enterotoxins: heat-labile toxin (LT), a protein multimer which shares many features with cholera toxin and which binds to intracellular adenylylcyclase, leading to increased cyclic AMP levels, and/or heat-stable toxin (ST), a small-peptide molecule that similarly activates guanylylcyclase and which produces increased intracellular cyclic GMP. For both toxins, the increased chloride secretion resulting from these toxins produces a watery diarrhea (10, 16). Both of these virulence factors are plasmid encoded. ST is encoded by two different genes: estA and st1, which produce STh (originally isolated from ETEC in humans) and STp (originally from a pig isolate), respectively. LT toxin is encoded by the eltA and eltB genes (12). The diagnosis of ETEC infection relies upon the detection of either the genes themselves or their gene products in clinical specimens.Currently, derivatives of LT and the CFs are targets for the development of vaccines against ETEC. However, the great variability of ETEC CFs requires determination of the CF types prevalent in different geographic locations (21, 33). The aims of this study were (i) to determine the clinical and epidemiological characteristics of ETEC diarrhea in Peruvian children, (ii) to determine the presence of ST and LT, (iii) to determine the presence and distribution of colonization factors in these strains, and (iv) to determine the antibiotic susceptibilities of these strains.     

Characterization of heat-stable enterotoxin from a hypertoxigenic Escherichia coli strain that is pathogenic for cattle.

An enterotoxigenic Escherichia coli (ETEC) strain isolated from a calf with clinical scours was found to produce over 17- to 60-fold more heat-stable enterotoxin (STa) than four laboratory-adapted bovine ETEC strains. The purified STa of this strain was identical to those produced by other ETEC strains. A severe form of scours was induced in 5- to 15-day-old colostrum-fed calves and in 1- to 2-week-old piglets by oral administration of the purified STa. This study demonstrates that STa is a mediator of diarrhea in newborn calves and piglets and that under identical growth conditions diverse strains of bovine ETEC may produce variable amounts of homologous STa's.     

Relation between antibiotic resistance and number of plasmids in enterotoxigenic and non-enterotoxigenicEscherichia coli strains

We have investigated the relation between antibiotic resistance and the number of plasmids contained in 44 enterotoxigenic (ETEC) and 69 non-enterotoxigenic (non-ETEC) Escherichia coli strains. Both ETEC and non-ETEC strains were resistant to one or more antibiotics at a similar rate, but multiple resistance in resistant non-ETEC strains was higher than in resistant ETEC strains, showing a significant difference (P less than 0.001) when subjected to Student's t test. Among the ETEC strains, those producing both LT and STa enterotoxins were more sensitive to antibiotics than strains producing only a single toxin type. On the other hand, resistant ETEC strains possessed a higher number of plasmids per strain than the resistant non-ETEC strains (P less than 0.05 by Student's t test). Consequently, resistance to antibiotics could not be related to the possession of a higher number of plasmids. Furthermore, our results support the hypothesis that there is a generalized incompatibility process between plasmids which code for antibiotic resistance and plasmids which code for enterotoxin production in ETEC strains, at least in those strains producing both LT and STa enterotoxins.     

Detection of enterotoxigenic Escherichia coli in stool samples by using nonradioactively labeled oligonucleotide DNA probes and PCR.

The detection of heat-labile enterotoxin LT-A and heat-stable enterotoxin ST Ia and ST Ib genes from enterotoxigenic Escherichia coli (ETEC) by using oligonucleotide DNA probes and the PCR was evaluated in reconstruction experiments and by testing stool specimens from 29 healthy subjects and from 50 patients with diarrhea who had returned from the (sub)tropics. ETEC strains were detected in concentrations ranging from 10(6) to 10(8) CFU/g of feces when oligonucleotide probes were applied to colony blots from five randomly picked E. coli-like colonies from CLED (cystine lactose electrolyte deficient) agar plates inoculated with the feces. When these probes were applied to blots from whole stool cultures collected from the agar plates (sweep blot), the detection limit was 10(6) CFU/g of feces. PCR of the sweep material could detect toxin genes when the concentration of ETEC strains was 10(2) CFU/g of feces. Results obtained with stool specimens from 29 healthy control subjects were negative. Testing stool specimens from 50 patients confirmed the observation that the number of samples containing ETEC enterotoxin genes was higher when PCR of sweeps was used than when oligonucleotide DNA probe hybridization of either sweep blots or colony blots was used. Furthermore, PCR of sweeps is an easy and rapid method which does not require DNA extraction and purification from fecal specimens.