An untypeable enterotoxigenic Escherichia coli represents one of the dominant types causing human disease.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhoea in children below 5 years of age in endemic areas, and is a primary cause of diarrhoea in travellers visiting developing countries. Epidemiological analysis of E. coli pathovars is traditionally carried out based on the results of serotyping. However, genomic analysis of a global ETEC collection of 362 isolates taken from patients revealed nine novel O-antigen biosynthesis gene clusters that were previously unrecognized, and have collectively been called unclassified. When put in the context of all isolates sequenced, one of the novel O-genotypes, OgN5, was found to be the second most common ETEC O-genotype causing disease, after O6, in a globally representative ETEC collection. It's also clear that ETEC OgN5 isolates have spread globally. These novel O-genotypes have now been included in our comprehensive O-genotyping scheme, and can be detected using a PCR-based and an in silico typing method. This will assist in epidemiological studies, as well as in ETEC vaccine development

Publisher Correction: Identification and characterization of the novel colonization factor CS30 based on whole genome sequencing in enterotoxigenic Escherichia coli (ETEC).

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Expression of Colonization Factor CS5 of Enterotoxigenic Escherichia coli (ETEC) Is Enhanced In Vivo and by the Bile Component Na Glycocholate Hydrate

Enterotoxigenic Escherichia coli (ETEC) is an important cause of acute watery diarrhoea in developing countries. Colonization factors (CFs) on the bacterial surface mediate adhesion to the small intestinal epithelium. Two of the most common CFs worldwide are coli surface antigens 5 and 6 (CS5, CS6). In this study we investigated the expression of CS5 and CS6 in vivo, and the effects of bile and sodium bicarbonate, present in the human gut, on the expression of CS5. Five CS5+CS6 ETEC isolates from adult Bangladeshi patients with acute diarrhoea were studied. The level of transcription from the CS5 operon was approximately 100-fold higher than from the CS6 operon in ETEC bacteria recovered directly from diarrhoeal stool without sub-culturing (in vivo). The glyco-conjugated primary bile salt sodium glycocholate hydrate (NaGCH) induced phenotypic expression of CS5 in a dose-dependent manner and caused a 100-fold up-regulation of CS5 mRNA levels; this is the first description of NaGCH as an enteropathogenic virulence inducer. The relative transcription levels from the CS5 and CS6 operons in the presence of bile or NaGCH in vitro were similar to those in vivo. Another bile salt, sodium deoxycholate (NaDC), previously reported to induce enteropathogenic virulence, also induced expression of CS5, whereas sodium bicarbonate did not

Identification of ETEC Lineages and Novel Colonization Factors

Enterotoxigenic Escherichia coli (ETEC) infections are a common cause of diarrhea but little is known about the evolution and genomic composition of ETEC. The main aim of this thesis was to generate a large collection of whole genome sequenced ETEC isolates to study the evolution of such bacteria on a global level and to search for novel colonization factors (CFs) using both classical and cutting-edge genomic techniques. Using whole genome sequencing and epidemiological data of 362 human ETEC isolates collected during three decades from all over the world, we studied the population structure of ETEC. We could show that major ETEC lineages comprising isolates with specific virulence profiles, i.e. CFs and LT and/or ST toxins, are stable and spread worldwide. These findings suggest that the virulence genes have been acquired once and then spread through clonal expansion and that a vaccine based on the most prevalent CFs could be protective against a large proportion of ETEC diarrhea cases. At least 30% of all clinical ETEC isolates lack a known CF. Therefore, we examined whole genome sequences of 94 “CF negative” isolates with the aim to identify novel CFs using two different approaches. I) By comparative genomics we have characterized a novel CF, CS30, which is related to the porcine CF 987P (F6). The major subunit of CS30 is 18.5 kD in size and the assembly of the fimbriae is dependent on its expression. CS30-positive bacteria are heavily fimbriated, as shown by electron microscopy, which promotes binding to human intestinal (Caco-2) cells. Furthermore, CS30 expression is thermo-regulated. II) By means of phenotypic analyses (SDS-PAGE, Caco-2 adhesion assays and electron microscopy) we have identified a number of isolates that may harbor additional putative fimbrial and non-fimbrial novel CFs. Nine candidate isolates with putative novel CFs were identified from 35 isolates: these were shown to express thermo-regulated proteins of 12-25 kD by SDS-PAGE analyses indicating the presence of major subunits and these isolates were found to bind well to Caco-2 cells. Based on further analyses of these isolates, using comparative genomics to identify CF related genes/operons, four AFA/Dr/AAF-like operons and an operon related to the porcine CF K88, were identified. The findings in this thesis have improved the knowledge of ETEC genomics and will provide a basis for future studies of ETEC transmission and pathogenicity. Enterotoxigenic Escherichia coli (ETEC) infections are a common cause of diarrhea but little is known about the evolution and genomic composition of ETEC. The main aim of this thesis was to generate a large collection of whole genome sequenced ETEC isolates to study the evolution of such bacteria on a global level and to search for novel colonization factors (CFs) using both classical and cutting-edge genomic techniques. Using whole genome sequencing and epidemiological data of 362 human ETEC isolates collected during three decades from all over the world, we studied the population structure of ETEC. We could show that major ETEC lineages comprising isolates with specific virulence profiles, i.e. CFs and LT and/or ST toxins, are stable and spread worldwide. These findings suggest that the virulence genes have been acquired once and then spread through clonal expansion and that a vaccine based on the most prevalent CFs could be protective against a large proportion of ETEC diarrhea cases. At least 30% of all clinical ETEC isolates lack a known CF. Therefore, we examined whole genome sequences of 94 “CF negative” isolates with the aim to identify novel CFs using two different approaches. I) By comparative genomics we have characterized a novel CF, CS30, which is related to the porcine CF 987P (F6). The major subunit of CS30 is 18.5 kD in size and the assembly of the fimbriae is dependent on its expression. CS30-positive bacteria are heavily fimbriated, as shown by electron microscopy, which promotes binding to human intestinal (Caco-2) cells. Furthermore, CS30 expression is thermo-regulated. II) By means of phenotypic analyses (SDS-PAGE, Caco-2 adhesion assays and electron microscopy) we have identified a number of isolates that may harbor additional putative fimbrial and non-fimbrial novel CFs. Nine candidate isolates with putative novel CFs were identified from 35 isolates: these were shown to express thermo-regulated proteins of 12-25 kD by SDS-PAGE analyses indicating the presence of major subunits and these isolates were found to bind well to Caco-2 cells. Based on further analyses of these isolates, using comparative genomics to identify CF related genes/operons, four AFA/Dr/AAF-like operons and an operon related to the porcine CF K88, were identified. The findings in this thesis have improved the knowledge of ETEC genomics and will provide a basis for future studies of ETEC transmission and pathogenicity

Phenotypic CS5 levels in LB alone and LB supplemented with bile or individual bile salts.

<p>CS5 expression was quantified by inhibition ELISA after overnight culture to stationary phase of strains E1777, E1779, E1785, E2265, and E3003. CS5 surface expression tifwas induced by NaGCH, but not by the corresponding unconjugated bile salt NaCH or its tauro-conjugated counterpart TCA (representative data).</p

Dose-dependent induction of phenotypic CS5 expression by NaGCH.

<p>Expression of CS5 was determined by inhibition ELISA in strain E1777 after overnight culture to stationary phase in LB medium supplemented with NaGCH or crude bile. Bars indicate means and standard errors of the means of two measurements in one experiment.</p

Strains used in the study and results from culturing^a of clinical stool specimens.

a<p>Culture was performed on MacConkey agar plates to detect <i>E. coli</i> and on Taurocholate-tellurite-gelatin agar (TTGA) for detection of vibrios; all cultures were performed overnight at 37°C.</p>b<p>Department of Microbiology and Immunology, University of Gothenburg, enterotoxigenic <i>Escherichia coli</i> (ETEC) strain collection number.</p

Primers used for PCR and real-time Reverse Transcriptase PCR (rt RT-PCR).

a<p>CS5 minor subunit<b>;</b> GenBank accession number <u>AJ224079</u>.</p>b<p>CS6 structural subunit CssB; GenBank accession number <u>UO4846</u>.</p>c<p>Primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035827#pone.0035827-Nicklasson1" target="_blank">[44]</a>.</p>d<p><i>E. coli</i> D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH); GenBank accession number <u>U014639.1.</u></p