Stephen J. Savarino

Enteroaggregative Escherichia coli elaborate a heat-stable enterotoxin demonstrable in an in vitro rabbit intestinal model.

Enteroaggregative Escherichia coli (EAggEC) have been associated with persistent diarrhea in young children, but little is known about its pathogenesis. We assayed for enterotoxic activity in culture filtrates (CF) of EAggEC strains in Ussing chambers mounted with rabbit ileal mucosa. CF from strain 17-2, a prototype Chilean EAggEC strain, caused a greater rise in potential difference and short circuit current (SCC) than that seen in HB101 control, and this effect was abolished by protease pretreatment and partially stable after heat treatment. Ultrafiltration of 17-2 CF preparations localized the active moiety to the 2-5 kD Mr size range. CF from HB101 transformed with the 17-2 plasmid showed Ussing chamber activity. less than 10-kD CF fractions from five of six other EAggEC strains screened in Ussing chambers gave SCC responses of similar magnitude to 17-2. The 17-2 CF activity was not neutralized after pretreatment with polyclonal anti-STa antibody. Additionally, all of the seven EAggEC strains studied were nonreactive by heat-stable enterotoxin variant STa ELISA, were negative in the suckling mouse assay, and failed to hybridize with heat-stable enterotoxin variant STh and STp DNA probes. In summary, our data indicate that 17-2 produces a low molecular weight, partially heat-stable, protease-sensitive enterotoxin which appears to be plasmid associated, and genetically and immunologically distinct from E. coli STa. Preliminary screening suggests that this tox+ phenotype may be common among EAggEC.

A systematic review of ETEC epidemiology focusing on colonization factor and toxin expression.

INTRODUCTION Vaccine development for enterotoxigenic Escherichia coli (ETEC) is dependent on in-depth understanding of toxin and colonization factor (CF) distribution. We sought to describe ETEC epidemiology across regions and populations, focusing on CF and toxin prevalence. METHODS We conducted a systematic review of the published literature, including studies reporting data on ETEC CF and toxin distributions among those with ETEC infection. Point estimates and confidence intervals were calculated using random effects models. RESULTS Data on 17,205 ETEC isolates were abstracted from 136 included studies. Approximately half of the studies (49%) involved endemic populations, and an additional 17% involved only travel populations. Globally, 60% of isolates expressed LT either alone (27%) or in combination with ST (33%). CFA/I-expressing strains were common in all regions (17%), as were ETEC expressing CFA/II (9%) and IV (18%). Marked variation in toxins and CFs across regions and populations was observed. DISCUSSION/CONCLUSIONS These results demonstrate the relative importance of specific CFs in achieving target product profiles for a future ETEC vaccine. However, heterogeneity across time, population, and region, confounded by variability in CF and toxin detection methodologies, obfuscates rational estimates for valency requirements.

A commensal gone bad: Complete genome sequence of the prototypical enterotoxigenic escherichia coli strain H10407

In most cases, Escherichia coli exists as a harmless commensal organism, but it may on occasion cause intestinal and/or extraintestinal disease. Enterotoxigenic E. coli (ETEC) is the predominant cause of E. coli-mediated diarrhea in the developing world and is responsible for a significant portion of pediatric deaths. In this study, we determined the complete genomic sequence of E. coli H10407, a prototypical strain of enterotoxigenic E. coli, which reproducibly elicits diarrhea in human volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains, revealing that the chromosome is closely related to that of the nonpathogenic commensal strain E. coli HS and to those of the laboratory strains E. coli K-12 and C. Furthermore, these analyses demonstrated that there were no chromosomally encoded factors unique to any sequenced ETEC strains. Comparison of the E. coli H10407 plasmids with those from several ETEC strains revealed that the plasmids had a mosaic structure but that several loci were conserved among ETEC strains. This study provides a genetic context for the vast amount of experimental and epidemiological data that have been published.

Comparative Analyses of Phenotypic and Genotypic Methods for Detection of Enterotoxigenic Escherichia coli Toxins and Colonization Factors

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of childhood diarrhea in developing countries and in travelers. However, this pathogen has often not been reported in surveys of diarrheal pathogens, due to lack of simple standardized methods to detect ETEC in many laboratories. ETEC expresses one or both of two different enterotoxin subtypes: heat-stable toxins, a heat-labile toxin (LT), and more than 22 different colonization factors (CFs) that mediate adherence to the intestinal cell wall. Here we compare established phenotypic and genotypic detection methods and newly developed PCR detection methods with respect to sensitivity, specificity, positive predictive value, and ease of performance. The methods include GM1-enzyme-linked immunosorbent assay and dot blot techniques using specific monoclonal antibodies (MAbs) for phenotypic detection of the toxins and CFs, respectively, as well as different PCR and DNA/DNA hybridization techniques, including new PCR assays, for genotypic identification of the toxin and CF genes, respectively. We found very good general agreement in results derived from genotypic and phenotypic methods. In a few strains, LT and CFs were identified genetically but not phenotypically. Based on our analyses, we recommend initial screening for ETEC in clinical samples by multiplex toxin gene PCR. Toxin-positive strains may then be analyzed by dot blot tests for detection of the CFs expressed on the bacterial surface and by PCR for determination of additional CFs for which MAbs are currently lacking as well as for strains that harbor silent CF genes.

Evolutionary and Functional Relationships of Colonization Factor Antigen I and Other Class 5 Adhesive Fimbriae of Enterotoxigenic Escherichia coli

ABSTRACT Colonization factor antigen I (CFA/I) is the archetype of eight genetically related fimbriae of enterotoxigenic Escherichia coli (ETEC) designated class 5 fimbriae. Assembled by the alternate chaperone pathway, these organelles comprise a rigid stalk of polymerized major subunits and an apparently tip-localized minor adhesive subunit. We examined the evolutionary relationships of class 5-specific structural proteins and correlated these with functional properties. We sequenced the gene clusters encoding coli surface antigen 4 (CS4), CS14, CS17, CS19, and putative colonization factor antigen O71 (PCFO71) and analyzed the deduced proteins and the published homologs of CFA/I, CS1, and CS2. Multiple alignment and phylogenetic analysis of the proteins encoded by each operon define three subclasses, 5a (CFA/I, CS4, and CS14), 5b (CS1, CS17, CS19, and PCFO71), and 5c (CS2). These share distant evolutionary relatedness to fimbrial systems of three other genera. Subclass divisions generally correlate with distinguishing in vitro adherence phenotypes of strains bearing the ETEC fimbriae. Phylogenetic comparisons of the individual structural proteins demonstrated greater intrasubclass conservation among the minor subunits than the major subunits. To correlate this with functional attributes, we made antibodies against CFA/I and CS17 whole fimbriae and maltose-binding protein fusions with the amino-terminal half of the corresponding minor subunits. Anti-minor subunit Fab preparations showed hemagglutination inhibition (HAI) of ETEC expressing homologous and intrasubclass heterologous colonization factors while anti-fimbrial Fab fractions showed HAI activity limited to colonization factor-homologous ETEC. These results were corroborated with similar results from the Caco-2 cell adherence assay. Our findings suggest that the minor subunits of class 5 fimbriae may be superior to whole fimbriae in inducing antiadhesive immunity.

Phenotypic Profiles of Enterotoxigenic Escherichia coli Associated with Early Childhood Diarrhea in Rural Egypt

ABSTRACT 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.

Safety and Immunogenicity of an Oral, Killed Enterotoxigenic Escherichia coli-Cholera Toxin B Subunit Vaccine in Egyptian Adults

Enterotoxigenic Escherichia coli (ETEC) is the leading cause of bacterial diarrhea in young children in developing countries. The safety and immunogenicity of a killed, oral ETEC vaccine consisting of whole cells plus recombinantly produced cholera toxin B subunit (rCTB) was evaluated in Egypt, which is endemic for ETEC diarrhea. Seventy-four healthy Egyptian adults (21-45 years old) were randomized and received two doses of the ETEC/rCTB vaccine (E003) or placebo 2 weeks apart. The frequency of adverse events after either dose did not differ by treatment group, and no severe adverse events were reported. After vaccination, peripheral blood IgA B cell responses to CTB (100%) and to vaccine colonization factor antigens CFA/I (94%), CS4 (100%), CS2 (81%), and CS1 (69%) were significantly higher than response rates for the placebo group. These favorable results in Egyptian adults indicate that the ETEC/rCTB vaccine is a promising candidate for evaluation in younger age groups in this setting.

An Outbreak of Foodborne Illness Caused by Escherichia coli O39:NM, an Agent Not Fitting into the Existing Scheme for Classifying Diarrheogenic E. coli

An outbreak of gastrointestinal illness with clinical and epidemiologic features of enterotoxigenic Escherichia coli (ETEC) occurred among patrons of a restaurant during April 1991. Illnesses among several groups of patrons were characterized by diarrhea (100%) and cramps (79%-88%) lasting a median of 3-5 days. Median incubation periods ranged from 50 to 56 h. A nonmotile strain of E. coli (E. coli O39), which was negative for heat-labile (LT) and heat-stable (STa, STb) ETEC toxins, was isolated only from ill patrons. This organism produced enteroaggregative E. coli heat-stable enterotoxin 1 and contained the enteropathogenic E. coli gene locus for enterocyte effacement; it did not display mannose-resistant adherence, but produced attaching and effacing lesions in the absence of mannose on cultured HEp-2 cells. E. coli that are not part of highly characterized but narrowly defined groups may be important causes of foodborne illness.

Oral, Inactivated, Whole Cell Enterotoxigenic Escherichia coli plus Cholera Toxin B Subunit Vaccine: Results of the Initial Evaluation in Children

Two randomized, double-blinded trials assessed the safety and immunogenicity of an oral, killed enterotoxigenic Escherichia coli (ETEC) plus cholera toxin B subunit vaccine in Egyptian children. Two doses of vaccine or E. coli K-12 were given 2 weeks apart to 105 6- to 12-year-olds and 97 2- to 5-year-olds. Safety was monitored for 3 days after each dose. Blood was collected before immunization and 7 days after each dose to measure immune responses. Few children reported postdosing symptoms, with no differences in the frequency of symptoms between treatment groups. Most vaccinees had an IgA antibody-secreting cell response against colonization factor antigen I (100%, 6-12 years; 95%, 2-5 years), coli surface antigen 2 (92%, 6-12 years; 83%, 2-5 years), and coli surface antigen 4 (93%, 6-12 years). Vaccination evoked a >/=4-fold rise in antitoxic IgA and IgG titers in 93% and 81% of children, respectively. In conclusion, the oral ETEC vaccine was safe and immunogenic in 2- to 12-year-old children, justifying further evaluation in infants.

Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli

Adhesion pili (fimbriae) play a critical role in initiating the events that lead to intestinal colonization and diarrheal disease by enterotoxigenic Escherichia coli (ETEC), an E. coli pathotype that inflicts an enormous global disease burden. We elucidate atomic structures of an ETEC major pilin subunit, CfaB, from colonization factor antigen I (CFA/I) fimbriae. These data are used to construct models for 2 morphological forms of CFA/I fimbriae that are both observed in vivo: the helical filament into which it is typically assembled, and an extended, unwound conformation. Modeling and corroborative mutational data indicate that proline isomerization is involved in the conversion between these helical and extended forms. Our findings affirm the strong structural similarities seen between class 5 fimbriae (from bacteria primarily causing gastrointestinal disease) and class 1 pili (from bacteria that cause urinary, respiratory, and other infections) in the absence of significant primary sequence similarity. They also suggest that morphological and biochemical differences between fimbrial types, regardless of class, provide structural specialization that facilitates survival of each bacterial pathotype in its preferred host microenvironment. Last, we present structural evidence for bacterial use of antigenic variation to evade host immune responses, in that residues occupying the predicted surface-exposed face of CfaB and related class 5 pilins show much higher genetic sequence variability than the remainder of the pilin protein.