Faecal pollution source tracking in the holy Bagmati River by portable 16S rRNA gene sequencing
Microbial community analysis
An overall microbial community analysis is presented as PCA plots and a dendrogram in Fig. 1. The cluster analysis showed good agreement between sample replicates, which clustered most closely. WWTP influent (i.e., untreated sewage) collected in the post-monsoon season clustered most closely with water samples from S4, S5 and S6 collected at same time, while WWTP influent from the monsoon season clustered with water samples from S6 collected in the same season. The WWTP effluent from both monsoon and post-monsoon season clustered together. The PCA plot with data from all the sampling times (Fig. 1b) generally showed a separation of downstream and WWTP influent water samples from the upstream and WWTP effluent samples along principal component 1, with only a few exceptions. Genera mostly found in the human gut microbiome15,26 like Streptococcus, Trichococcus, Lactobacillus, Enterococcus, Prevotella and Arcobacter, were highly prevalent in downstream and WWTP influent water samples, which separated these samples from the upstream water samples in the PCA. Among the three factors analysed (i.e., location, sampling time and water sample types), locations and sampling time had a significant effect on the similarity of the samples in the ANOSIM, although with relatively low R values (ANOSIM; Location: R = 0.29, p value = 0.001 and Sampling time: R = 0.16, p value = 0.01). ANOSIM further indicated no statistically significant differences between the microbial communities in water from locations S4, S5 and S6 and the wastewater influent (ANOSIM; (1) S4 and Inf: R = 0.0309, p value = 0.357: (2) S5 and Inf: R = 0.0617, p value = 0.369 and (3) S6 and Inf: R = 0.0123, p value = 0.3690).
Fig. 1: Cluster and PCA analysis at rank genus for 16S rRNA gene sequencing reads.
a Cluster analysis [all seasons], PCA plot b all seasons, c monsoon [June 2019] and d post monsoon [August 2019]. Arrows in the PCA plots indicate the ten variables with the highest loadings (vector lengths) in the PC1 and 2 space.
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An interesting picture emerged when a separate PCA (Fig. 1c, d) and cluster analysis (Supplementary Fig. 1) was conducted for water samples from the monsoon and the post-monsoon season. In both seasons, there were substantial, but seasonally distinct, contributions of genera found in the human gut microbiome to the variance among water samples along principal component 1: in the monsoon season Arcobacter, Aeromonas, Streptococcus and Prevotella had significant PC1 loadings; in the post-monsoon season Enterococcus, Acinetobacter, Streptococcus and Trichococcus had significant PC1 loadings. Separation of wastewater treatment plant effluent (WWTP Effluent) samples along PC1 away from the WWTP influent (WWTP Influent) samples in both sampling events signified the benefits of wastewater treatment, because human gut-associated genera became less predominant in treated wastewater microbiomes, as expected27. Accordingly, there was a clear separation of the most upstream water samples from the most downstream water samples along PC1 in both events, with the downstream water samples becoming more similar to WWTP Influent (Fig. 1c, d). Evidently, as the Bagmati River flowed into more densely populated areas, the characteristics of its water microbiome changed from a composition more similar to treated, to a composition more similar to untreated urban sewage, but the composition of the urban sewage was variable for the monsoon and post-monsoon season.
Abundance of human gut and putative pathogenic bacteria in the water microbiomes
A more detailed breakdown of the microbial community composition in the Bagmati River for the monsoon and post-monsoon season is reported in Table 1, and Supplementary Tables 1 and 2, which compare the total percentage relative abundance of putative human gut28 and pathogenic29 bacteria at genus and species level for different sampling sites in the Bagmati River, and the WWTP influent and effluent (Refer to Supplementary Tables 3–5 for more detailed lists of bacteria). Based on our previous findings24, species identities are not always reliable due to the limited read accuracy of the MinION sequencing reads, but the overall trends are nonetheless indicative of changes in microbial composition. For all sampling events, the water collected at the most upstream site S1 and S2 showed the lowest relative abundance for both human gut and putative pathogenic bacteria, whereas the highest relative abundance was observed in the water collected at the most downstream sites S4–S6 (Table 1, Supplementary Tables 1 and 2). The microbial water quality of water samples collected at site S1 can be considered as baseline data, as this watershed is distant from the densely populated Kathmandu Valley and has the minimal influence of human and urbanisation activities. Figure 2, and additional figures in Supplementary Information (Supplementary Fig. 2–9) show how the abundance of human gut and putative pathogenic genera changed in space and time along the Bagmati River. As the river flowed downstream, the abundance of some of these groups of bacteria increased, and the most drastic and significant increase was observed at the sites S4, S5 and S6 downstream of the Pashupatinath Temple as compared to site S1 (Two-sample t test, p value More
