We describe natural variants of the heat stable toxin (STa) produced by enterotoxigenic Escherichia coli (ETEC) isolates collected worldwide. Previous studies of ETEC isolated from human diarrheal cases have reported the existence of three natural STa gene variants estA1, estA2 and estA3/4 where the first variant encodes STp (porcine, bovine, and human origin) and the two latter ones encode STh (human origin). We identified STa sequences by BLASTn and profiled ST amino acid polymorphisms in a collection of 118 clinical ETEC isolates from children and adults from Asia, Africa and, Latin America that were characterized by whole genome sequencing. Three novel variants of STp and STh were found and designated STa5 and STa6, and STa7, respectively. Presence of glucose significantly decreased the production of STh and STp toxin variants (p<0.05) as well as downregulated the gene expression (STh: p<0.001, STp: p<0.05). We found that the ETEC isolates producing the most common STp variant, STa5, co-expressed coli surface antigen CS6 and was significantly associated with disease in adults in this data set (p<0.001). Expression of mature STa5 peptide as well as gene expression of tolC, involved in ST secretion, increased in response to bile (p<0.05). ETEC expressing the common STh variant STa3/4 was associated with disease in children (p<0.05). The crp gene, that positively regulate estA3/4 encoding STa3/4, and estA3/4 itself had decreased transcriptional levels in presence of bile. Since bile levels in the intestine are lower in children than adults, these results may suggest differences in pathogenicity of ETEC in children and adult populations.

Pathogenic bacteria use specific host factors to modulate virulence and stress responses during infection. We found previously that the host factor bile and the bile component glyco-conjugated cholate (NaGCH, sodium glycocholate) upregulate the colonization factor CS5 in enterotoxigenic Escherichia coli (ETEC). To further understand the global regulatory effects of bile and NaGCH, we performed Illumina RNA-Seq and found that crude bile and NaGCH altered the expression of 61 genes in CS5 + CS6 ETEC isolates. The most striking finding was high induction of the CS5 operon (csfA-F), its putative transcription factor csvR, and the putative ETEC virulence factor cexE. iTRAQ-coupled LC-MS/MS proteomic analyses verified induction of the plasmid-borne virulence proteins CS5 and CexE and also showed that NaGCH affected the expression of bacterial membrane proteins. Furthermore, NaGCH induced bacteria to aggregate, increased their adherence to epithelial cells, and reduced their motility. Our results indicate that CS5 + CS6 ETEC use NaGCH present in the small intestine as a signal to initiate colonization of the epithelium.

The prevalence of infection caused by different categories of diarrhoeagenicE. coli(DEC) strains, including enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC) and enterohaemorrhagic (EHEC)E. coli, in children who suffered from diarrhoea (n = 3943) or did not have diarrhoea (n = 1026) were analysed in two areas in Bolivia over a period of 4 years. We also analysed the seasonality of DEC infections and severity of diarrhoea in children with DEC infection and compared antibiotic resistance in DEC strains isolated from children with and without diarrhoea. Stool samples were analysed for the presence of DEC by culturing followed by PCR. The most prevalent DEC categories in samples from the children were: EAEC (11.2 %); ETEC (6.6 %); EPEC (5.8 %); and EIEC and EHEC (<1 %). DEC strains were isolated significantly more often from diarrhoea cases (21.6 %) than from controls (17.6 %;P = 0.002). The number of children with diarrhoea associated with EAEC, EPEC and ETEC infections peaked in the Bolivian winter (April–September), although the proportion of DEC-positive stool samples was higher during the warm rainy season (October–March). High levels of antibiotic resistance were detected among the DEC strains. In particular, resistance to tetracycline and sulfamethoxazole–trimethoprim was significantly higher in strains isolated from individuals with diarrhoea than in samples from controls. The severity of disease in children infected with EAEC, EPEC and ETEC varied from mild to severe diarrhoea, although disease severity did not differ significantly between the different DEC categories. ETEC, EPEC and EAEC are commonly found in Bolivia and may cause severe disease in children.

An increased abundance of antibiotic resistance genes (ARGs) in aquatic environments has been linked to environmental pollution. Mining polluted sites with high concentration of metals could favor the in situ coselection of ARGs, whereas wastewater discharges release fecal antibiotic resistant bacteria in the environment. To study the effect of human fecal contamination and mining pollution, water and sediment samples affected by mining activities and sewage discharges were collected from three lakes in Bolivia, the pristine Andean lake Pata Khota, the Milluni Chico lake directly impacted by acid mine drainage, and the Uru-Uru lake located close to Oruro city and highly polluted by mining activities and human wastewater discharges. Physicochemical parameters, including metal composition, were analyzed in water and sediment samples. ARGs were screened for and verified by quantitative polymerase chain reaction (PCR) together with the mobile element class 1 integron (intl1), as well as crAssphage, a marker of human fecal pollution. The gene intl1 was positively correlated with sul1, sul2, tetA, and bla_OXA-2. CrAssphage was only detected in the Uru-Uru lake, and its tributaries and significantly higher abundance of ARGs were found in these sites. Multivariate analysis showed that crAssphage abundance, electrical conductivity, and pH were positively correlated with higher levels of intl1 and ARGs. Taken together, our results suggest that fecal pollution is the major driver of higher levels of ARGs and intl1 in environments contaminated by wastewater and mining activities.

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of morbidity and mortality in the developing world. ETEC-mediated diarrhea is orchestrated by heat-labile (LT) and heat-stable (STp and STh) toxins, acting in concert with a repertoire of more than 25 colonization factors (CFs). LT, the major virulence factor, induce fluid-secretion after delivery of a monomeric ADP-ribosylase (LTA) and its pentameric carrier B subunit (LTB). A study of Brazilian-derived ETEC isolates reported the existence of natural LT variants. Here, the analysis of predicted amino acid sequences showed that the LT amino acid polymorphisms is associated with a geographically and temporally diverse set of 192 clinical ETEC strains, identifying 12 novel LT variants. Twenty distinct LT amino acid variants were classified in the globally distributed strains and phylogenetic analysis showed these to be associated with different CF profiles. Notably, the most prevalent LT1 and LT2 allele variants were correlated with major ETEC lineages expressing CS1+CS3, CS2+CS3, and CS5+CS6, CFA/I, respectively. LTB allele variants generally exhibited more stringent amino acid sequence conservation (2 substitutions identified) than LTA (22 substitutions identified). The functional impact of LT1 and LT2 polymorphisms on virulence was investigated by measuring total toxin production, secretion and stability using GM1-ELISA and in silico protein modeling. Our data show that LT2 strains produce 5-fold more toxin than LT1 strains (P<0.001), which may suggest a greater virulence potential for this genetic variant. Our data suggests that functionally distinct LT - CF variants with increased fitness have persisted during evolution of ETEC and spread globally.