WASTEWATER RECEIVING PONDS IN RURAL INDIA AND THEIR IMPACT ON ANTIBIOTIC RESISTANCE AND GROUNDWATER QUALITY

Abstract

In several low- and middle-income countries, such as India, the rapid construction of toilets to combat open defecation has not been matched with adequate wastewater treatment, resulting in extreme fecal contamination of the receiving environments. The sewage-receiving surface waterbodies, typically near the residences, are a potential hotspot for disease transmission and antibiotic resistance. Water, soil, and sediment samples from seven wastewater receiving ponds (WRPs) were analyzed for water quality, chlorophyll-a, fecal contamination (yccT for Escherichia coli), 16S rRNA gene copies, and anthropogenic antibiotic resistance gene markers – sul1 and intI1. These WRPs were contrasted with two ponds (P-I and P-P; P-I: the pond, which is used for irrigation purposes and, P-P: the pond which is used for pisciculture purposes) that did not directly receive sewage. The water quality in the WRPs was comparable to raw sewage (BOD: 210-380 mg/L; COD: 350-630 mg/L;Total-N: 100-190 mg/L; and Total-P: 6-21 mg/L), and the relative levels of the DNA marker of E.coli were very high (yccT: 0.1 – ~100 % of total bacterial count) indicating extreme fecal contamination. The relative levels of sul1 and intI1 were 1 – 3 orders of magnitude higher in WRPs (sul1: 0.32 – 10 % of total bacterial count; and intI1: 0.2 – 5 % of total bacterial count) compared to the ponds that did not receive sewage directly. The relative levels of sul1 correlated with the DNA marker for the fecal indicator, E. coli (p -value<0.05; r=0.50; Spearman’s rank correlation), and poor water quality. After examining all these results, we focused on the seasonal variation to examine the response of these WRPs and ponds. Consequently, sampled the water, soil, and sediment from seven WRPs and two control ponds over three seasons {winter (December), summer (July), and monsoon (October)} to compare the water quality, chlorophyll-a, fecal contamination (yccT for Escherichia coli), anthropogenic antibiotic resistance gene marker – intI1 followed by the 16S metagenomics.The water quality in the polluted WRPs was comparable to raw sewage throughout the year: BOD ranged between 210 - 372 mg/L in winter, 250 - 350 mg/L mg/L in summer, and 260 – 375 mg/L in monsoon; the COD ranged from 350 - 610 mg/L in winter, 460 – 545 mg/L in summer, and 405 -630 mg/L in monsoon; NH4-N levels ranged from 130-180 mg/L in winter, 140 – 165 mg/L in summer, and 140 – 192 mg/L 130 – 160 mg/L in monsoon; and Total-P ranged from 11 – 20 mg/Lin summer is higher as compare to the 10-21 mg/L in winter and 13 – 19 mg/L in monsoon. The absolute levels of the DNA marker of E. coli were very high (106.25 – 108.2 gene copies/L) in summer as compared to the monsoon (106.6 – 107.6 gene copies/L) and winter (106.75 – 107.75 gene copies/L). The absolute levels of intI1 in summer (107.4 – 108.2 gene copies/L) were higher as compared to winter (106.75 – 107.75 gene copies/L) and monsoon season (106.6 – 107.6 gene copies/L). Using 16S rRNA gene amplicon sequencing facilitated the identification of pathogenic bacteria with elevated abundance, capable of infecting humans and animals. The analysis revealed that Arcobacter exhibited 41% and 9% prevalence during the monsoon and winter seasons, respectively, in the WRPs. Additionally, the prevalence of Pseudomonas was found throughout the year, accounting for 13% in monsoon, compared to 9% and 12% during the summer and winter, respectively, in WRPs. On the contrary, the pond which is used for irrigation purpose (P-I), the abundance of Clostridium during the monsoon season was recorded at 4%, and it also infects humans and animals due to its pathogenicity. The abundance of 15% and 13% of Bacillus genus and Pseudomonas genus, respectively were dominant in the monsoon in P-P. The study found a higher abundance of pathogenic genera such as Arcobacter, Synechococcus, Pseudomonas, Paracoccus, and Acinetobacter in the WRP compared to the P-I and P-P. This study found that the percentage of the abundance of the genus was generally high during summer and monsoon season. Some other genera, such as Cyanobium, Clostridium, Bacillus, and Pseudomonas, exhibited higher abundance percentages during specific seasons, including (15% in summer in P-I), (4% in monsoon in P-I and 6% in summer in P-P), (6% in summer and monsoon in P-I and 13% in monsoon in P-P), and (13% in monsoon in P-I and 17% in monsoon in P-P), respectively. The percentage of these genera indicates that the water used for fishing and irrigation purposes is also approaching a polluted state. Other than that, the different challenges such as groundwater pollution, concerns regarding human and animal health arise due to the existence of pathogenic microorganisms that exhibit resistance to disinfection procedures, and these microorganisms exhibit an exceptional level of resilience within wastewater, posing a significant risk to the well- being of rural communities and livestock.