Circulation of enterotoxigenic Escherichia coli (ETEC) isolates expressing CS23 from the environment to clinical settings.

IMPORTANCE: The importance of clean water cannot be overstated. It is a vital resource for maintaining health and well-being. Unfortunately, water sources contaminated with fecal discharges from animal and human origin due to a lack of wastewater management pose a significant risk to communities, as they can become a means of transmission of pathogenic bacteria like enterotoxigenic E. coli (ETEC). ETEC is frequently found in polluted water in countries with a high prevalence of diarrheal diseases, such as Bolivia. This study provides novel insights into the circulation of ETEC between diarrheal cases and polluted water sources in areas with high rates of diarrheal disease. These findings highlight the Choqueyapu River as a potential reservoir for emerging pathogens carrying antibiotic-resistance genes, making it a crucial area for monitoring and intervention. Furthermore, the results demonstrate the feasibility of a low-cost, high-throughput method for tracking bacterial pathogens in low- and middle-income countries, making it a valuable tool for One Health monitoring efforts.

Conjugative transfer of multi-drug resistance IncN plasmids from environmental waterborne bacteria to Escherichia coli.

Watersheds contaminated with municipal, hospital, and agricultural residues are recognized as reservoirs for bacteria carrying antibiotic resistance genes (ARGs). The objective of this study was to determine the potential of environmental bacterial communities from the highly contaminated La Paz River basin in Bolivia to transfer ARGs to an Escherichia coli lab strain used as the recipient. Additionally, we tested ZnSO4 and CuSO4 at sub-inhibitory concentrations as stressors and analyzed transfer frequencies (TFs), diversity, richness, and acquired resistance profiles. The bacterial communities were collected from surface water in an urban site close to a hospital and near an agricultural area. High transfer potentials of a large set of resistance factors to E. coli were observed at both sites. Whole-genome sequencing revealed that putative plasmids belonging to the incompatibility group N (IncN, IncN2, and IncN3) were predominant among the transconjugants. All IncN variants were verified to be mobile by a second conjugation step. The plasmid backbones were similar to other IncN plasmids isolated worldwide and carried a wide range of ARGs extensively corroborated by phenotypic resistance patterns. Interestingly, all transconjugants also acquired the class 1 integron intl1, which is commonly known as a proxy for anthropogenic pollution. The addition of ZnSO4 and CuSO4 at sub-inhibitory concentrations did not affect the transfer rate. Metal resistance genes were absent from most transconjugants, suggesting a minor role, if any, of metals in the spread of multidrug-resistant plasmids at the investigated sites.

Molecular Epidemiology of Enteroaggregative Escherichia coli (EAEC) Isolates of Hospitalized Children from Bolivia Reveal High Heterogeneity and Multidrug-Resistance.

Enteroaggregative Escherichia coli (EAEC) is an emerging pathogen frequently associated with acute diarrhea in children and travelers to endemic regions. EAEC was found the most prevalent bacterial diarrheal pathogen from hospitalized Bolivian children less than five years of age with acute diarrhea from 2007 to 2010. Here, we further characterized the epidemiology of EAEC infection, virulence genes, and antimicrobial susceptibility of EAEC isolated from 414 diarrheal and 74 non-diarrheal cases. EAEC isolates were collected and subjected to a PCR-based virulence gene screening of seven virulence genes and a phenotypic resistance test to nine different antimicrobials. Our results showed that atypical EAEC (a-EAEC, AggR-negative) was significantly associated with diarrhea (OR, 1.62, 95% CI, 1.25 to 2.09, p < 0.001) in contrast to typical EAEC (t-EAEC, AggR-positive). EAEC infection was most prevalent among children between 7-12 months of age. The number of cases exhibited a biannual cycle with a major peak during the transition from warm to cold (April-June). Both typical and a-EAEC infections were graded as equally severe; however, t-EAEC harbored more virulence genes. aap, irp2 and pic were the most prevalent genes. Surprisingly, we detected 60% and 52.6% of multidrug resistance (MDR) EAEC among diarrheal and non-diarrheal cases. Resistance to ampicillin, sulfonamides, and tetracyclines was most common, being the corresponding antibiotics, the ones that are frequently used in Bolivia. Our work is the first study that provides comprehensive information on the high heterogenicity of virulence genes in t-EAEC and a- EAEC and the large prevalence of MDR EAEC in Bolivia.

Fecal Pollution Drives Antibiotic Resistance and Class 1 Integron Abundance in Aquatic Environments of the Bolivian Andes Impacted by Mining and Wastewater.

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 blaOXA-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.

Allele variants of enterotoxigenic Escherichia coli heat-labile toxin are globally transmitted and associated with colonization factors.

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of morbidity and mortality in the developing world. ETEC-mediated diarrhea is orchestrated by heat-labile toxin (LT) and heat-stable toxins (STp and STh), acting in concert with a repertoire of more than 25 colonization factors (CFs). LT, the major virulence factor, induces fluid secretion after delivery of a monomeric ADP-ribosylase (LTA) and its pentameric carrier B subunit (LTB). A study of ETEC isolates from humans in Brazil reported the existence of natural LT variants. In the present study, analysis of predicted amino acid sequences showed that the LT amino acid polymorphisms are associated with a geographically and temporally diverse set of 192 clinical ETEC strains and identified 12 novel LT variants. Twenty distinct LT amino acid variants were observed in the globally distributed strains, and phylogenetic analysis showed these to be associated with different CF profiles. Notably, the most prevalent LT1 allele variants were correlated with major ETEC lineages expressing CS1 + CS3 or CS2 + CS3, and the most prevalent LT2 allele variants were correlated with major ETEC lineages expressing CS5 + CS6 or CFA/I. LTB allele variants generally exhibited more-stringent amino acid sequence conservation (2 substitutions identified) than LTA allele variants (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-enzyme-linked immunosorbent assays (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 greater virulence potential for this genetic variant. Our data suggest that functionally distinct LT-CF variants with increased fitness have persisted during the evolution of ETEC and have spread globally.

Prevalence, seasonality and severity of disease caused by pathogenic Escherichia coli in children with diarrhoea in Bolivia.

The prevalence of infection caused by different categories of diarrhoeagenic E. 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.