Diarrheal bacterial pathogens and multi-resistant enterobacteria in the Choqueyapu River in La Paz, Bolivia

Abstract

Water borne diarrheal pathogens might accumulate in river water and cause contamination of drinking and irrigation water. The La Paz River basin, including the Choqueyapu River, flows through La Paz city in Bolivia where it is receiving sewage, and residues from inhabitants, hospitals, and industry. Using quantitative real-time PCR (qPCR), we determined the quantity and occurrence of diarrheagenic Escherichia coli (DEC), Salmonella enterica, Klebsiella pneumoniae, Shigella spp. and total enterobacteria in river water, downstream agricultural soil, and irrigated crops, during one year of sampling. The most abundant and frequently detected genes were gapA and eltB, indicating presence of enterobacteria and enterotoxigenic E. coli (ETEC) carrying the heat labile toxin, respectively. Pathogen levels in the samples were significantly positively associated with high water conductivity and low water temperature. In addition, a set of bacterial isolates from water, soil and crops were analyzed by PCR for presence of the genes blaCTX-M, blaKPC, blaNDM, blaVIM and blaOXA-48. Four isolates were found to be positive for blaCTX-M genes and whole genome sequencing identified them as E. coli and one Enterobacter cloacae. The E. coli isolates belonged to the emerging, globally disseminated, multi-resistant E. coli lineages ST648, ST410 and ST162. The results indicate not only a high potential risk of transmission of diarrheal diseases by the consumption of contaminated water and vegetables but also the possibility of antibiotic resistance transfer from the environment to the community.

Fig 1. Quantification results for diarrheal bacterial pathogens in water samples from the La Paz River basin.

a) enterobacteria-gapA b) ETEC-eltB c) ETEC- estA1 d) ETEC- estA2-4 e) EPEC/EHEC-eae f) EAEC-aggR g) Shigella spp./EIEC-ipaH. The bars show the number of copies per pathogen gene per 100 ml of river water (N° gc/100mL H₂O) per month (April to March 2013–14, January results are absent) and per site obtained by qPCR absolute quantification analysis. All sampling points along the La Paz River basin are listed and compared. SP1: un-impacted site close to a water reservoir, SP2: site located in the Choqueyapu River and in the urban area, directly downstream of hospitals, SP3: agricultural area where river water is used for irrigation of crops and SP4: tributary river inside the urban area of La Paz city. Statistical significant differences between sampling points are indicated with stars, p≤0.05 (*), p≤0.01 (**) and p≤0.001 (***). Statistical significance analyzed by One-Way ANOVA, Post-Hoc Test: Tukey in SPSS.

Fig 2. Linear association between conductivity (μS/cm) and DNA concentration (ng/μL) obtained from water samples from the La Paz River basin.

Figure obtained from Linear Regression Analysis in SPSS version 22, p<0.01.

Fig 3. Number of pathogen gene copies in samples from the agricultural area Mecapaca (SP3) during the year of sampling (2013–14) obtained by qPCR absolute quantification analysis.

a) soil samples and b) vegetables (lettuce and chard). Results are expressed in number of genes copies per gram of soil fresh weight (N° gc/g SFW) or vegetable fresh weight (N° gc/g VFW). Number of gene copies is expressed in logarithmic scale; only values greater than zero are plotted. Samples from January 2014 were not included in this study.