Metagenomic analysis reveals differential effects of sewage treatment on the microbiome and antibiotic resistome in Bengaluru, India

Abstract


Introduction
Antimicrobial resistance (AMR) is a complex global health challenge involving the transfer of bacteria and genes between humans, animals, and the environment.The resistance is mediated by antimicrobial resistance genes (ARGs), also known as "the resistome" which circulate among microbiomes representing different sectors of the One Health Concept.Antibiotics are the main preventive agents for bacterial infectious diseases in human and veterinary medicine due to their high e cacy and safety.
However, the extent and scale of AMR are largely shaped by selection pressure resulting from antibiotic usage.The environment plays a signi cant role in the ecology, evolution, and transmission of antimicrobial resistance among pathogenic and non-pathogenic bacteria, and transmission occurs in multiple directions.For instance, AMR developed in animal reservoirs can spill over to humans through food, water, or other environmental routes, or through reverse zoonotic transmission of AMR [1].
Therefore, it is necessary to develop an understanding of the major pathways and drivers of antimicrobial resistance in the environment.
Environmental (sewage) surveillance (ES) is an important part of the One Health approach.Urban sewage, particularly in low-resource settings, is a major source for the dissemination of ARGs in various environments [2][3][4][5].ES is an effective tool for collecting reliable data to assess the spatiotemporal patterns of ARGs diversity at the community level.Many antibiotics currently used to treat infections, as well as ARGs acquired by human pathogens originate from the environment [6].Large quantities of antibiotics are discharged into municipal wastewater through to incomplete metabolism in humans or the disposal of unused antibiotics [7].Sewage treatment plants (STPs), like gut microbiomes, provide environments with abundant ecological opportunities and niche availability, which promote antibiotic persistence and adaptation among community members.Eukaryotes such as protozoa, fungi, parasites, and algae play vital roles in enhancing the e ciency of STP processes [8].STPs are critical environments where high microbial density and diverse ARGs are exposed to selective agents such as antibiotics, disinfectants, and heavy metals.STPs act as melting pots where selective agents facilitate horizontal gene transfer (HGT) through mobile genetic elements (MGE), resulting in the emergence of antibioticresistant bacteria [9,10].Wastewater treatment typically involves various mechanical, biological, physical, and chemical processes, that can potentially impact the fate of antibiotics, antibiotic-resistant bacteria, and ARGs as well as the spread of resistance in the environment, thereby increasing the risk of transmission back to humans [5].
Climate change and health are closely linked to urban wastewater.Water security is a pressing issue in India.Water scarcity and reduced availability of agricultural water have spurred increased interest in the use of recycled irrigation water.Currently, only 28% of wastewater in India is treated and reused [11].Wastewater is a valuable source of nutrients (nitrogen, phosphorus, potassium).Therefore, reusing treated wastewater for irrigation can have economic and environmental bene ts.However, there is limited tracking of treated wastewater parameters such as pH (5-5.9), total suspended solids (TSS: 20 mg/l), nitrogen (10 mg/l), phosphorus (1 mg/l), biological oxygen demand (BOD:10 mg/l), chemical oxygen demand (COD: 50 mg/l), and fecal coliform levels (less than 100 MPN/100 ml) and regular monitoring usually does not include a complete pro ling of pathogens [12].It is crucial to identify and address any water quality issues associated with the use of recycled water including the possible presence of bacterial, viral, and protozoan pathogens.Furthermore, there is concern that continuous addition of antibiotics through treated wastewater for irrigation poses a signi cant risk to soil and plant quality and health.This includes antibiotic uptake and accumulation by crops as well as the potential selection of antibiotic resistance from consuming contaminated vegetables [13].Even low concentrations of antibiotics in plants could promote the selection of ARGs in bacterial ecosystems and contribute to antibiotic resistance in the environment [14].
In India, obtaining representative data on AMR for healthy human and animal populations is challenging.
Until 2010, India was the largest consumer of antibiotics [15], with antibiotics easily accessible without a prescription [16].The rise in AMR among bacterial pathogens is primarily attributed to the acquisition of ARGs or the accumulation of spontaneous mutations in the bacterial genome.Six bacterial pathogens, namely Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa (ESKAPE), are the leading cause of death worldwide.
In the Indian context, studies have demonstrated the presence of diverse ARGs in wastewater treatment plants, river basins or estuaries.These include ARGs coding for resistance against tetracyclines, betalactams, aminoglycosides, and macrolides [e.g., [17][18][19].However, these studies have been cross-sectional with limited spatio-temporal resolution and lack associated data on microbial communities.To address this gap, we designed a comprehensive longitudinal study in the urban sewage network of Bengaluru city to understand the overall microbiological water quality and the role of sewage digestion processes, which potentially affect the prevalence and abundance of AMR genes in the environment.Bengaluru (12.9716°N, 77.5946° E, Karnataka, India) is the third largest city (~11 million inhabitants) in India with an e cient sewage network that processes ~1142.5 million litres per day (MLD) of wastewater.
Each STP follows a water treatment technology depending on the quality of raw sewage to make the treated water reusable (https://bwssb.karnataka.gov.in/info-1/About+BWSSB/en).The most common sewage treatment method used in Bengaluru is the Activated Sludge Process (ASP), which employs aeration and microbial ocs or granules to remove C, N, P, micropollutants (for example, toxins, pesticides, hormones, and pharmaceuticals), pathogens [20], Biochemical Oxygen Demand (BOD), fecal coliform levels, turbidity etc.There are two other variations of ASP used: Extended Aeration, Sequential Batch Reactor (SBR) and Up ow Anaerobic Sludge Blanket Reactor (UASB).Extended Aeration is better suited for treating low sewage loads and is often used in apartment complexes and residential areas.SBR, on the other hand, can remove nutrients like nitrogen from sewage, allowing treated water to be directly released into lakes.Bengaluru has the largest water footprint compared to any other Indian city and is the second-largest city after Mexico where treated wastewater is used not only for agriculture but also for recharging groundwater in drought-prone regions outside the city.For example, in the Kolar district of Karnataka, one of the worst-affected districts in terms of drought and climate change, treated wastewater pumped from Bengaluru has helped improve groundwater quality and ensure the availability of water regardless of weather and climate patterns.While it is essential to minimize the public health risks before using treated wastewater, it is important to understand the effectiveness of treatment plants in removing harmful parasites and whether there is spatial and temporal segregation in AMR and related microbial diversity.To explore this, we used amplicon-based sequencing of the 16S ribosomal RNA gene (16S rRNA) and an AMR panel targeting 478 ARGs to characterize the spatio-temporal variations in the antibiotic resistome and corresponding bacterial community.We are speci cally interested in understanding the following: 1) How does bacterial diversity change from the inlet to the outlet in STPs based on season and processing methods?
2) Is there spatial and temporal heterogeneity in ARG diversity?
3) Is there a shift in differential abundance across bacterial and ARGs from the inlet to the outlet of the treatment?4) What type of co-occurrence networks exist in the microbial community and ARGs between the inlet and the outlet?4) How stable is the microcosm in the inlet and outlet community (equalization analysis)?

Sample collection and processing
Raw sewage (inlet) and treated (outlet) wastewater samples were collected from 26 STPs in Bengaluru.
The samples were collected between August 2021 and July 2022 (Fig. 1).A total of 86 grab samples were collected in 250 mL plastic bottles, tightly sealed upon collection, and stored at 4°C in the eld.The samples were stored at -20°C until further processing.

DNA extraction, ampli cation, and sequencing
Samples were thawed at 4°C and processed in a Biosafety Level-2 laboratory.First, each sample was pre ltered using a sterile muslin cloth to remove large debris.After the debris was removed, the sample was ltered using 0.22 µm membranes (MF-Millipore® Membrane Filter).DNA was isolated from the lter paper using the Qiagen DNeasy PowerWater DNA Isolation Kit.A negative control was included with each extraction batch.DNA samples with a nucleic acid ratio of 260/280, greater than 1.7 nm and a concentration greater than 4.5 ng/µL were considered for sequencing and stored at -20°C.

16S ribosomal RNA (rDNA) V4 sequencing by MiSeq Illumina
Universal bacterial primers, the forward primer 338F and reverse primer 802R by Klindworth et al. [21], tagged with an Illumina adaptor sequence, were used to amplify the V3-V4 hypervariable region of the 16S rDNA gene through polymerase chain reaction (PCR).The PCR reaction mix (25µL) contained 12.5 µL of KAPA HiFi HotStart ReadyMix (Roche), 1.0 µL (5 µM) of the forward and reverse primer, 2.0 µL of template DNA (5 ng/µL), and 8.5 µL of Ambion nuclease-free water (Invitrogen).Each PCR plate included a negative control to detect potential contamination.The PCR conditions included an initial denaturation step at 95°C for 3 min, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and a nal extension at 72°C for 30 s. Ampli cation success was con rmed through gel electrophoresis, using a 2% agarose gel.PCR products were puri ed using bead puri cation with Agencourt AMPure XP (Beckman Coulter, USA).Samples were normalized, then multiplexed with the Nextera XT Index kit (96 indexes) (Illumina) and sequenced on an Illumina MiSeq owcell using a V3 sequencing chemistry kit (2 × 350) at the NCBS NGS facility.

16S rDNA read processing and taxonomy assignments
Raw reads were processed in R (version 3.4.3)(R Development Core Team 2008) using a modi ed version of the Divisive Amplicon Denoising Algorithm 2 (DADA2) pipeline [22] and online tutorials v1.6 and work ow for big data v1.4 (benjjneb.github.io/dada2/tutorial.html).The lterAndTrim function was used to remove the low-quality reads.Error rates were calculated for each amplicon dataset.The identical reads were dereplicated, and a sequence variant inference algorithm was applied to each dataset.Tables of ASV sequences per sample within each run were then combined, and chimera detection using all pooled samples was performed (see Table S2 for the number of reads retained across each step).Taxonomy assignment from Kingdom to Genus was performed using the RDP classi er and MiDAS 4 [23] reference database formatted for DADA2 (benjjneb.github.io/dada2/training.html),using the assignTaxonomy function.All ASV tables produced by the pipelines were converted into phyloseq objects using the phyloseq package [24].

Antibiotic resistant genes sequencing
Wastewater samples were sequenced for ARGs using the AmpliSeq Illumina AMR panel.The PCR conditions were adapted from Illumina's AmpliSeq for Illumina On-Demand, Custom, and Community Panels reference guide.Brie y, the total reaction volume was 20 µL, consisting of 12.5 µL of KAPA 2G Fast Multiplex Mix (Roche).Two PCRs were set up simultaneously as the panel offers 2 pools (Pool 1: 408 amplicons, Pool 2: 407 amplicons).The primer volume for individual runs was 5.0 µL each, template DNA was 1.0 µL (5ng/µL), and nuclease-free water (Invitrogen) was added.The ARGs PCR conditions used included an initial denaturation step at 99°C for 2 min, followed by 20 cycles of denaturation at 99°C for 15 s, annealing at 60°C for 4 min, and a hold at 4°C.A negative control was included in each PCR.Samples showing positive ampli cation for ARGs were considered for sequencing.Amplicon libraries for the Illumina platform were prepared using the AmpliSeq Library PLUS for Illumina kit (Cat. No. 20019102).Library quality was assessed using a 2100 Bioanalyzer with a DNA high sensitivity assay kit (Agilent CA, USA).Sequencing was carried out using an Illumina MiSeq system with a MiSeq reagent kit v2 500 cycles (2 ×150) paired-end chemistry.

Microbial functional and antibiotic resistance analyses
To identify ARGs, we analyzed raw reads using the CLC Genomics Workbench 21.0.5 Microbial Genomics Module (CLC MGM) and the Find Resistance with ShortBRED (FRSB) tool (similar to ShortBRED) [25].Within the FRSB tool (CLC Microbial Genomics Module), we used DIAMOND v0.9.31 to match queried sequences against the QMI-AR Peptide Marker database (released 2019-11).We retained all default settings in the FRSB tool except for the "more sensitive search" parameter, which allowed us to run DIAMOND in its highest sensitivity mode.We cross-checked the identi ed AMR genes against entries in the Comprehensive Antibiotic Resistance Database (CARD) [26].It is important to note that our methodology can only detect ARGs that have been annotated in CARD.Therefore, some novel types of ARGs present in the samples may be missed since the analysis is based on a similarity search.

Statistical analyses of metagenomics data
All analyses were conducted using R software v.3.4.3.Rare ed operational taxonomic unit (OTU) tables were generated by performing multiple rarefactions on bacterial reads using the phyloseq package.The OTU tables were rare ed to the sample with the lowest number of sequences, with a threshold of >10,000 sequences (any samples with less than 10,000 sequences were excluded from the analyses before the rarefaction step; see Fig. S1).Rarefaction curves were generated using the rarefy_even_depth function in the phyloseq package, to equalize sequencing depth among samples.
The unweighted UniFrac distances between samples were calculated using the rare ed OTU abundance tables in the phyloseq package.We assessed alpha diversity (Observed richness, Shannon diversity), conducted beta diversity analyses using Principal Coordinate Analysis (PCoA) and performed permutational multivariate analysis of variance (PERMANOVA) using the adonis2 function.Additionally, Non-parametric Multidimensional Scaling (NMDS) and ANOSIM tests were used to assess the differences in communities based on inlet versus outlet, season and STP processing.All statistical analyses associated with sample grouping were performed using the vegan package [27].Relative abundance plots were constructed using the ggplot2 package [28].

Co-occurrence gene network construction analysis
To identify potential interactions between ARGs and bacteria, a gene co-occurrence network was created.The correlation matrix between the ARG pairs in the inlet and outlet samples was calculated using a pairwise Spearman's correlation rank test [29].The values in the matrix indicated the co-occurrence correlation of the ARGs, with positive value indicating a positive correlation and negative values indicating a negative interaction.These matrix values were input into Gephi 0.10.1 [30] to plot the gene network using built-in functions.The resulting network included interactions between ARGs, ARG and species, and species and species using the Force Atlas layout format.To create a clear network, only statistically signi cant values with a p-value < 0.01 and a correlation coe cient >0.6 for inlet samples and >0.5 for outlet samples were plotted in the constructed network for an easier interpretation [31][32][33].
Additionally, the node size was scaled based on the abundance of ARGs in the data.
Using the hypervariable region of the 16S rRNA, we classi ed OTUs at the species level, primarily identifying bacterial groups in the context of human health.Water-borne bacteria such as Mycobacterium and Legionella contributed exclusively between 0.01% and 0.37% in outlet samples.Leptospira and Vibrio contributed less than 0.001% only in outlet samples.The major represented genera that contributed to bacterial diversity were Arcobacter, Blautia, Catenibacterium, Ligilactobacillus, Megasphaera and Acinetobacter, Streptococcus (environmental bacteria) (Fig. 2).Among these, enteric bacteria such as Clostridium, Escherichia/Shigella, and Klebsiella were detected with a relative abundance of 0.03% (Fig. 3C).Among the top 10 species are Catenibacterium mitsuokai, Lactobacillus ruminis, Megamonas funiformis, Megasphaera indica, and Streptococcus parasuis (Fig. 2).
We rst examined the alpha diversity measures across inlet and outlet samples by season.While the taxa diversity (observed: F = 43.56,P < 0.001; Shannon diversity: F = 38.25,P < 0.001) was higher in outlet samples than in inlet samples (Fig. 3A-B).However, there was no change in diversity across seasons except in October to December probably due to low sample size.To determine whether the overall microbial composition of inlet and outlet samples was different, we examined β-diversity measures by season and sample types (inlet and outlet).In PCoA plots, these groups were not clustered together by season, indicating that there were no differences (PERMANOVA: R 2 = 0.04, P > 0.37; Fig. 3C).Similar results were observed in NMDS plots (ANOSIM: R = 0.02, P > 0.24; Fig. 3D).
We identi ed six resistance mechanisms that which did not show a signi cant difference in the mean between inlet and outlet samples (Fig. 7).Two mechanisms, the e ux pump complex or subunit conferring antibiotic resistance and the gene involved in antibiotic sequestration, showed a reduction in the outlet samples.In Fig. 7, a total of 16 drug classes were retrieved from the 86 samples identi ed.We found a downward shift in drug classes from the inlet to the outlet samples except for aminoglycosides, beta-lactams, MLSB, and Tetracycline.

Linking antibiotic resistome with bacterial phylogeny in urban sewage
Network analysis was used to explore the co-occurrence pattern of ARGs and bacteria and identify the potential hosts of ARGs in inlet and outlet urban wastewater.Each node represents a bacterial phylum and ARG, and a connection (edge) represents a correlation between the ARGs and the phylum.The size of a node here is proportional to the number of connections formed by that ARG or phylum.There are 6 and 25 modularity classes displayed in the inlet and outlet networks, respectively (Fig. 8).
In general, there were more complex and dense correlations between ARG-ARG, ARG-bacteria and bacteria-bacteria in inlet samples.For example, ARG-ARG pairs, such as bla TEM (Pseudomonas aeruginosa) and bla NDM (Klebsiella pneumoniae), as well as ARG-bacteria pairs, such as CAT (Campylobacter coli) and bla CMY−1 (Klebsiella pneumoniae), showed strong positive correlations.
In contrast, outlet samples showed stronger and positive correlations between ARG-ARG pairs and bacterial assemblages, with very few interactions between ARG-bacteria pairs.

Equalization Analysis Between ARG and Bacterial Community Structures
Under treated sewage conditions, the observed species ratio of ARG/16S decreased signi cantly at the outlet (Fig. 9A).The correlation between Shannon diversity indices of ARG diversity and bacterial diversity increased in both the inlet (r = 0.57, p < 0.005) and the outlet (r = 0.74, p < 0.005; Fig. 9B).The Chao1 and Ace indices of the bacterial community showed non-signi cant correlation with those of the ARG (Fig. 9C&D).These ndings suggest that the overall equalization of the bacterial and ARG communities was affected by both the water treatment plants and other environmental factors, thereby indicating the major contributors to the change in the microbial equilibrium.The variation in the richness of microbial species was the principal cause for the change in the microbial equilibrium.

Discussion
Studying the micro ora of wastewater is of great importance to public health and ecology.The microbial diversity in wastewater is constantly changing and highly dynamic, in uenced by various factors.Our large-scale urban sewage sampling in Bengaluru revealed that there is a shift in microbial diversity and the antibiotic resistome from the inlet to the outlet of a treatment plant.This study represents the most comprehensive longitudinal research on Bengaluru wastewater to date, signi cantly enhancing our understanding of the potential variations in the risk of exposure to bacterial pathogens and antibiotic resistance genes originating from diverse sewage processing methods used in urban STPs.Our data con rms that conventional STPs effectively eliminate a signi cant number of bacterial cells, along with their associated resistance genes.
We found signi cant bacterial diversity in the inlet and outlet wastewaters.At the phylum level, Firmicutes (40%), Actinobacteriota (18%), Proteobacteria (18%), and Patescibacteria were the most dominant in the inlet samples.These major bacterial phyla are found in the gut microbiota of healthy human beings [34] and are dominant in the gut microbiome of Indian communities [35].Firmicutes showed a downward shift in the outlet and were replaced by Proteobacteria and Patescibacteria.The environmental bacteria genera Acinetobacter, Aeromonas, and Pseudomonas were present in both the inlet and outlet samples, contributing between 0.74% to 0.01%.These three genera have shown strains that become multi-resistant and should receive special attention.For example, Zhang et al. [36] showed an increase in antibioticresistant Acinetobacter spp. in a wastewater treatment plant.At the phylum level there were two distinct clusters based on the inlet and outlet speci c bacterial communities, which showed no signi cant differences by season or processing methods.It is possible that abiotic parameters such as oxygen concentration, as well as competition among different bacterial species with different metabolic characteristics, could be driving these compositional differences in the bacterial community between the inlet and outlet.
We found a similar downward shift from the inlet to the outlet at species and genus level.We recorded six pathogens-Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Streptococcus pneumoniae, Pseudomonas aeruginosa and Enterobacter spp.(ESKAPE) -in inlet samples, albeit in small proportions, which showed a further decrease in the outlet samples.This matches ndings from studies in other parts of the world, which show that sewage treatment is not able to remove the total burden of microbial diversity but minimizes the effect of pathogenic bacteria [37,38].Catenibacterium mitsuokai was dominant and has been positively associated with obesity-related insulin resistance.Lactobacillus ruminis, a commensal motile lactic acid bacterium living in the intestinal tract of humans and animals contributed to the top 10 bacterial species.Streptococcus parasuis (S. parasuis), a close relative of Streptococcus suis and a potential opportunistic zoonotic pathogen, was also recorded.Megasphaera indica, an obligate anaerobic bacterium isolated from human feces and the genus Subdoligranulum, which was associated with autoantibody development, were present.The genus Arcobacter has been considered an emergent enteropathogen and a potential zoonotic agent [39,40].We found Arcobacter midas_s_2255, a potential pathogen commonly abundant in the in uent wastewater with high relative abundance in both the inlet and outlet sewage water.Kristensen et al. [41] reported similar results from Danish wastewater treatment plants and attributed the main reason for the high abundance of this genus since Arcobacter cells do not occulate and attach well to the activated sludge ocs, thus remaining in high concentration in the water phase.
An equalization analysis using alpha diversity indices revealed a signi cant increase in the ARG/16S ratio suggesting strong competition between ARG diversity and 16S, which could be detrimental for horizontal gene transfer in a dynamic microbial community in both the inlets and outlets.This further suggests that the microbial equilibrium of the bacterial and fungal communities is regulated by species richness and diversity [42].
Bengaluru sewage harboured a wide range of ARGs including clinically relevant CTX-M, qnr, sul-1, and NDM-1 which confer resistance to six major classes of antibiotics.Higher richness of ARGs was detected in the sewage inlets compared to the outlets, and there was signi cant variation in the clustering of ARGs by STPs but not by sewage processing methods.This is in contrast to studies conducted on a continental scale in Europe [43] and China [44], where the absolute abundance of ARGs did not exhibit biogeographical patterns in STP e uents.Our data showed spatial structuring of ARGs by STPs, suggesting that there might be environmental factors such as temperature, catchment area size, or number of hospitals that could in uence the patterns at a city scale.Hospitals are major contributors of ARGs in wastewater [45].It is possible that hospitals in the catchment areas have an impact on the e uents of STPs, which requires further investigation.Among the nine antibiotic classes examined, aminoglycosides and beta-lactams were the most dominant.Resistance genes found in many clinical isolates tend to spread beyond the boundaries of hospital catchments.For example, there was an increase prevalence of NDM-1 in river sediment following a pilgrimage in the upper Ganges [46].
Aminoglycosides are used against bacteria that are already resistant to beta-lactams and uoroquinolones, which further suggests the presence of aminoglycoside-resistant genes in multidrugresistant (MDR) bacteria, and pan-drug resistant bacteria, particularly in the hospital environment [47].The Indian healthcare system lacks strict regulations on over-the-counter access to antibiotics [16].
Furthermore, there are no regulatory provisions for the use of antimicrobials in livestock, including pigs and poultry raised for domestic consumption.Therefore, it is di cult to determine the overall rates of antibiotic prescription or how antibiotic usage varies by season.We observed a signi cant high in ARG abundance in April to June, which coincides with the opening of schools and an increase in public movement, as well as a decrease in remote working.This led to a huge surge in SARS-CoV-2 infections, as re ected by an increasing viral load in wastewater in June 2022 in Bengaluru city [48].Overall, there was a decrease in ARG abundance from July to October, which could be attributed to a decrease in SARS-CoV-2 infections, as observed through a decreasing viral load in wastewater.It is important to understand if the high abundance of ARGs could be used as a proxy for a seasonal increase in antibiotic usage in the city.Lu et al. [49] demonstrated a positive correlation between the concentration of tetracyclines and the abundance of total tetracycline-resistant genes in environmental samples.However, there are contrasting ndings regarding the signi cant (p >0.05) correlation between the absolute concentrations of selected genes and antibiotics.Liyanage et al. [50] found that resistance genes were detected even in the absence of tetracycline and penicillin in water.It is important to highlight that the type of environmental samples and sampling design such as a longitudinal study across the sewage network in an urban environment or fecal samples, may better capture these dynamics better than an exposed river basin or coastal environments where the prevalence of ARGs is caused by the persistence of these genes even in the absence of selection pressure [51].Nonetheless, our data indicate that uctuating abundance of ARGs could be driven by selective pressure from either microbial communities or antibiotics [52].
One of the striking ndings was the high prevalence of Tetracycline in urban sewage.India accounts for about 3% of the global consumption of antimicrobials in food animals [53].Poultry is one of the most widespread food industries in India.A large variety of antimicrobials is used to raise poultry, and Tetracycline class of antibiotics is especially used in animal feed to prevent diseases caused by Mycoplasma, Pasteurella multocida and Escherichia coli.The growth enhancement properties of antibiotics such as chlortetracycline and oxytetracycline also contribute to their increased commercial use in animal feeds, particularly for broiler chickens [54].TetA and tetB are the commonly found tetracycline resistance genes in livestock associated Enterobacteriaceae [55].There is reported tetracycline resistance in poultry even without the administration of this antibiotic [56].
Our goal is to quantify the differential effects of water treatment mechanisms on ARG diversity and abundance in the outlet of four STPs in Bengaluru where treated water is used for groundwater recharging and agriculture purposes [11].Using a combination of metagenomic approaches, our preliminary ndings based on 273 ARGs screened in wastewater showed 54 dominant genes with a downward shift in drug class from inlet to outlet samples, except for aminoglycosides, beta-lactams, MLSB and tetracycline.
According to United Nations statistics, it has been estimated that up to 90% of perishable vegetables consumed in cities globally are provided by 200 million city dwellers engaged in urban farming.The signi cant role of urban agriculture in contributing to the proper and su cient availability of food and poverty alleviation has been recognized and promoted [57].Therefore, as part of environmental surveillance, it is important to know the diversity of the microbial community and their role in the digestion process.This knowledge can help improve the performance of sewage treatment plants and the total abundance of ARGs in environmental samples [58].Bengaluru has the second-largest treated water system used for agriculture in the world.Further research is needed to understand the dynamics that occur downstream of the STPs and to improve water treatment mechanisms in four key STPs that supply treated water for agriculture and groundwater recharging in peri-urban areas.Extending this study to Bengaluru peri-urban areas (e.g., Kollar, Chikkaballapura, Tumkuru and Kollar) and pro ling ARGs, bacterial, and fungal diversity to evaluate the health impact of treated water in the areas while working with the sewage board in developing a policy and strategy for water quality issues associated with the use of recycled water (e.g., possible persistence of bacterial, viral, protozoan pathogens) will be crucial for mitigating the impact of wastewater on the health of the ecosystem.

Limitations of the study
This study does not represent the complete metagenome of the STP.We used an AMR panel designed to screen only select ARGs.It is possible that the diversity of genes in the wastewater is larger than we sequenced.Our data will serve as a baseline study that could aid in quantifying of genes from both culturable and nonculturable taxa.We have a very low representation of genes that confer resistance to last resort antibiotics.Nonetheless, this was an improvement over PCR-based approaches which may have greater sensitivity to low-abundance ARGs due to targeted ampli cation but are limited in the ARGs they can detect.
Going forward, shotgun metagenomics can help alleviate some of the drawbacks of amplicon-based sequencing.The inherent connections between microbial ecology and environmental biotechnology (of which wastewater treatment is an important aspect) have recently been emphasized.A longitudinal study that combines antibiotic levels in wastewater and ARGs could help us understand the impact of treated wastewater on the microbial community.(D) by STP processing method (see Supplementary Table 1 for details on processing method).

Figure 1 Map
Figure 1

Figure 2 Relative
Figure 2

Figure 3 A
Figure 3

Figure 5 Alpha
Figure 5