Human-Driven Microbiological Contamination of Benthic and Hyporheic Sediments of an Intermittent Peri-Urban River Assessed from MST and 16S rRNA Genetic Structure Analyses

Abstract

Rivers are often challenged by fecal contaminations. The barrier effect of sediments against fecal bacteria was investigated through the use of a microbial source tracking (MST) toolbox, and by Next Generation Sequencing (NGS) of V5-V6 16S rRNA gene (rrs) sequences. Non-metric multi-dimensional scaling analysis of V5-V6 16S rRNA gene sequences differentiated bacteriomes according to their compartment of origin i.e., surface water against benthic and hyporheic sediments. Classification of these reads showed the most prevalent operating taxonomic units (OTU) to be allocated to Flavobacterium and Aquabacterium. Relative numbers of Gaiella, Haliangium, and Thermoleophilum OTU matched the observed differentiation of bacteriomes according to river compartments. OTU patterns were found impacted by combined sewer overflows (CSO) through an observed increase in diversity from the sewer to the hyporheic sediments. These changes appeared driven by direct transfers of bacterial contaminants from wastewaters but also by organic inputs favoring previously undetectable bacterial groups among sediments. These NGS datasets appeared more sensitive at tracking community changes than MST markers. The human-specific MST marker HF183 was strictly detected among CSO-impacted surface waters and not river bed sediments. The ruminant-specific DNA marker was more broadly distributed but intense bovine pollution was required to detect transfers from surface water to benthic and hyporheic sediments. Some OTU showed distribution patterns in line with these MST datasets such as those allocated to the Aeromonas, Acinetobacter, and Pseudomonas. Fecal indicators (Escherichia coli and total thermotolerant coliforms) were detected all over the river course but their concentrations were not correlated with MST ones. Overall, MST and NGS datasets suggested a poor colonization of river sediments by bovine and sewer bacterial contaminants. No environmental outbreak of these bacterial contaminants was detected.

Keywords: benthic and hyporheic sediments; fecal contamination; high throughput sequencing (HTS); microbial community; peri-urban river.

Figure 1

Map showing the sites sampled along the Chaudanne river. Sites are further detailed in Table 1. (A) Analyzed segment of the Chaudanne River; Yzeron river outlet is about 20 km downstream of point 1. (B) Scheme of the Chaudanne river section showing the different compartments and the vertical distribution of ruminant and human specific Bacteroidales MST markers and genera inferred from NGS 16S rRNA gene analyses. SW, Surface Water; BS, Benthic Sediment; HS, Hyporheic Sediment. Acido, Acidobacteria; Aero, Aeromonas; Acineto, Acinetobacter; Albi, Albidiferax; Arco, Arcobacter; Flavo, Flavobacterium; Gai, Gaiella; Hal, Haliangium; Thermo, Thermoleophilum; Pelo, Pelomonas; Pseudo, Pseudomonas; Vario, Variovorax. Underlined genera showed higher relative counts in wastewater than in surface water.

Figure 2

Relative counts of fecal bacteria and MST host-markers expressed over total Bacteroidales concentrations measured along the Chaudanne River watercourse and bed sediments. See Table 1 for a description of the sampling sites. Datasets used for the computations are shown in Table S5. SW, surface water, BS, benthic, and HS, hyporheic sediments.

Figure 3

Correspondence analysis computed from OTU 16S rRNA gene contingency scores at the level of phyla. The 50K dataset (5670 reads per sample) containing 16S rRNA gene sequences from surface waters (SW), benthic (BS), and hyporheic sediments (HS), and wastewater (WW), sampled along the Chaudanne river, were used (see text). Only the most significant phyla in terms of relative weight are shown (in red). The intensity of the filled circles indicates the absolute contribution of each set of phyla patterns.

Figure 4

Heatmap illustrating the significance of some genera inferred from the 16S rRNA gene dataset recovered from river and wastewater samples. The 50K dataset (5670 reads per sample) containing 16S rRNA gene sequences from surface waters (SW), benthic (BS), and hyporheic sediments (HS), and wastewater (WW), sampled along the Chaudanne river, were used (see text). Genera representing less than 0.4% of the full dataset were merged and grouped into the term “Other.”

Figure 5

Ascendant Hierarchical Classification (A) and Non Metric Multi-Dimensional Scaling (B) analyses of 16S rRNA gene OTU. The 50K dataset (5670 reads per sample) containing 16S rRNA gene sequences from surface waters (SW), benthic (BS), and hyporheic sediments (HS), and wastewater (WW), sampled along the Chaudanne river was used (see text). Vertical lines in (A) are indicative of the proximity between the OTU distribution patterns.