Oxygen mediated mobilization and co-occurrence of antibiotic resistance in lab-scale bioreactor using metagenomic binning.

Sub-lethal levels of antibiotic stimulate bacteria to generate reactive oxygen species (ROS) that promotes emergence and spread of antibiotic resistance mediated by mobile genetic elements (MGEs). Nevertheless, the influence of dissolved oxygen (DO) levels on mobility of antibiotic resistance genes (ARGs) in response to ROS-induced stress remains elusive. Thus, the study employs metagenomic assembly and binning approaches to decipher mobility potential and co-occurrence frequency of ARGs and MGEs under hyperoxic (5.5-7 mgL- 1), normoxic (2.5-4 mgL- 1), and hypoxic (0.5-1 mgL- 1) conditions in lab-scale bioreactor for 6 months. Among 163 high-quality metagenome-assembled genomes (MAGs) recovered from 13 metagenomes, 42 MAGs harboured multiple ARGs and were assigned to priority pathogen group. Total ARG count increased by 4.3 and 2.5% in hyperoxic and normoxic, but decreased by 0.53% in hypoxic conditions after 150 days. On contrary, MGE count increased by 7.3-1.3% in all the DO levels, with only two ARGs showed positive correlation with MGEs in hypoxic compared to 20 ARGs under hyperoxic conditions. Opportunistic pathogens (Escherichia, Klebsiella, Clostridium, and Proteus) were detected as potential hosts of ARGs wherein co-localisation of critical ARG gene cassette (sul1, dfr1,adeF, and qacC) were identified in class 1 integron/Tn1 family transposons. Thus, enhanced co-occurrence frequency of ARGs with MGEs in pathogens suggested promotion of ARGs mobility under oxidative stress. The study offers valuable insights into ARG dissemination and hosts dynamics that is essential for controlling oxygen-related stress for mitigating MGEs and ARGs in the environment.

Molecular advances in mycoremediation of polycyclic aromatic hydrocarbons: Exploring fungal bacterial interactions.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous high global concern environmental pollutants and tend to bioaccumulate due to hydrophobic properties. These xenobiotics, having variable concentrations along different matrices, gradually undergo various physical, chemical, and biological transformation processes. Myco-remediation aids accelerated degradation by effectively transforming complex ring structures to oxidized/hydroxylated intermediates, which can further funnel to bacterial degradation pathways. Exploitation of such complementing fungal-bacterial enzymatic activity can overcome certain limitations of incomplete bioremediation process. Furthermore, high-throughput molecular methods can be employed to unveil community structure, taxon abundance, coexisting community interactions, and metabolic pathways under stressed conditions. The present review critically discusses the role of different fungal phyla in PAHs biotransformation and application of fungal-bacterial cocultures for enhanced mineralization. Moreover, recent advances in bioassays for PAH residue detection, monitoring, developing xenobiotics stress-tolerant strains, and application of fungal catabolic enzymes are highlighted. Application of next-generation sequencing methods to reveal complex ecological networks based on microbial community interactions and data analysis bias in performing such studies is further discussed in detail. Conclusively, the review underscores the application of mixed-culture approach by critically highlighting in situ fungal-bacterial community nexus and its role in complete mineralization of PAHs for the management of contaminated sites.

Resolution of inter/intraspecies variation in Weissella group requires multigene analysis and functional characterization.

Weissella confusa and Weissella cibaria strains isolated from the human- gut are considered as potential probiotics, but remain under-explored owing to their ambiguous taxonomic assignment. The present study assesses the taxonomic resolution of 11 strains belonging to W. confusa and W. cibaria species and highlights the inter- and intraspecies variations using an array of phenetic and molecular methods. Remarkable genomic variability among the strains was observed by phylogenetic analysis using concatenated housekeeping genes (pheS, gyrB, and dnaA) along with 16S rRNA gene sequence, suggesting intraspecies variations; which is also supported by the phenetic data. Analysis showed that 16S rRNA gene sequence alone could not resolve the variation, and among the tested marker genes, signals from pheS gene provide better taxonomic resolution. The biochemical and antibiotic susceptibility tests also showed considerable variations among the isolates. Additionally, 'quick' identification using mass spectroscopy-based matrix-assisted laser desorption/ionization-time of flight mass spectra was accurate up to genus only, and not species level, for the Weissella group. The study highlights need for inclusion of functional, phenetic, and multigene phylogenetic analysis in addition to 16S rRNA gene-based identification for the Weissella group, to provide better resolution in taxonomic assignments, which is often a prerequisite for the selection of potential strains with biotechnological applications.

Phylogenomic analysis of Citrobacter sp. strain AAK_AS5 and its metabolic capabilities to support nitrogen removal behavior.

Despite the ubiquity of the genus Citrobacter in clinical, industrial, and environmental scenarios, a large number of Citrobacter strains have not been explored at the genome-scale level. In this study, accurate taxonomic assignment of strain AAK_AS5 isolated from activated sludge was achieved by in-silico genomic comparison using Overall Genome-based Relatedness Indices (ANI(OAT): 97.55%, ANIb:97.28%, and ANIm: 97.83%) that indicated its closest identity to the related strain Citrobacter portucalensis A60T . Results were consistent with a digital DNA-DNA hybridization value of 80% with C. portucalensis A60T which was greater than the species boundary value >70% for delineating closely related bacterial species. Gene mining through Kyoto Encyclopedia of Genes and Genomes (KEGG), and annotation using rapid annotation subsystem technology (RAST) revealed the notable gene contents for nitrogen metabolism and other pathways associated with nitrate/nitrite ammonification (28 genes), ammonia assimilation (22 genes), and denitrification pathways (14 genes). Furthermore, the strain AAK_AS5 also exhibited a high soluble chemical oxygen demand (sCOD), NH4 + -N, and NO3 - -N removal efficiency of 91.4%, 90%, and 93.6%, respectively thus validating its genetic capability for utilizing both (NH4 )2 SO4 and KNO3 as the nitrogen source. The study provided deeper insights into the phylogenomics and the genetic potential of Citrobacter, sp. strain AAK AS5 associated with nitrogen metabolism thus signifying the potential application of the isolate for treating nitrogen-rich wastewaters.

Harnessing taxonomically diverse and metabolically versatile genus Paracoccus for bioplastic synthesis and xenobiotic biodegradation.

The genus Paracoccus represents a taxonomically diverse group comprising more than 80 novel species isolated from various pristine and polluted environments. The species are characterized as coccoid-shaped Gram-negative bacteria with versatile metabolic attributes and classified as autotrophs, heterotrophs and/or methylotrophs. The present study highlights the up-to-date global taxonomic diversity and critically discusses the significance of genome analysis for identifying the genomic determinants related to functional attributes mainly bioplastic synthesis and biodegradation potential that makes these isolates commercially viable. The analysis accentuates polyphasic and genomic attributes of Paracoccus spp. which could be harnessed for commercial applications and emphasizes the need of integrating genome-based computational analysis for evolutionary species and functional diversification. The work reflects on the underexplored genetic potential for bioplastic synthesis which can be harnessed using advanced genomic methods. It also underlines the degradation potential and possible use of naturally-occurring pollutant-degrading Paracoccus isolates for the development of a biodegradation system and efficient removal of contaminants. The work contemplates plausible use of such potent isolates to establish the plant-microbe interaction, contributing toward contaminated land reclamation. Overall, the work signifies the need and application of genome analysis to identify and explore the prospective potential of Paracoccus spp. for environmental application toward achieving sustainability.

Complete genome sequence of Paracoccus sp. strain AK26: Insights into multipartite genome architecture and methylotropy.

The present study reports the functional annotation of complete genome of methylotrophic bacterium Paracoccus sp. strain AK26. The 3.6 Mb genome with average GC content of 65.7% was distributed across five replicons; including chromosome (2.7 Mb) and four extrachromosomal replicons pAK1 (471Kb), pAK2 (189Kb), pAK3 (129Kb) and pAK4 (84 Kb). Interestingly, nearly 23% of the Cluster of Orthologous Group (COG) of proteins were annotated on extrachromosomal replicons and 185Kb genome content was attributed to segregated 19 genomic island regions. Among the four replicons, pAK4 was identified as essential and integral part of the genome, as supported by codon usage, GC content (66%) and synteny analysis. Comparative genome analysis for methylotrophy showed mechanistic variations in oxidation and assimilation of C1 compounds among closely related Paracoccus spp. Collectively, present study reports the functional characterization and genomic architecture of strain AK26 and provides genetic basis for quinone and isoprenoid based secondary metabolites synthesis using strain AK26.

Myco-remediation of Chlorinated Pesticides: Insights Into Fungal Metabolic System.

Synthetic chemicals including organochlorine pesticides pose environment and health hazard due to persistent and bio-accumulation property. Majority of them are recognized as endocrine disruptors. Fungi are ubiquitous in nature and employs efficient enzymatic machinery for the biotransformation and degradation of toxic, recalcitrant pollutants. This review critically discusses the organochlorine biotransformation process mediated by fungi and highlights the role of enzymatic system responsible for biotransformation, especially distribution of dehalogenase homologs among fungal classes. It also explores the potential use of fungal derived biomaterial, mainly chitosan as an adsorbing biomaterial for pesticides and heavy metals removal. Further, prospects of employing fungus to over-come the existing bioremediation limitations are discussed. The study highlights the potential scope of utilizing fungi for initial biotransformation purposes, preceding final biodegradation by bacterial species under environmental conditions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00940-8.

Phylogenomic Framework for Taxonomic Delineation of Paracoccus spp. and Exploration of Core-Pan Genome.

The taxonomic classification of metabolically versatile Paracoccus spp. has been so far performed using polyphasic approach. The topology of single gene phylogenies, however, has highlighted ambiguous species assignments. In the present study, genome based multi-gene phylogenies and overall genome related index were used for species threshold assessment. Comprehensive phylogenomic analysis of Paracoccus genomes (n = 103) showed concordant clustering of strains across multi-gene marker set phylogenies (nMC = 0.08-0.14); as compared to 16S rDNA phylogeny (nMC = 0.37-0.42) suggesting robustness of multi gene phylogenies in drawing phylogenetic inferences. Functional gene content distribution across the genus showed that only 1.7% gene content constitutes the core genome highlighting the significance of extensive genomic variability in the evolution of Paracoccus spp. Further, genome metrics were used to validate characterized strains, identifying classification anomalies (n = 13), and based on this, genome derived taxonomic amendments were notified in present study. Conclusively, validated metric tools can be employed on whole genome sequences, including draft assemblies, for the assessment and assignment of uncharacterized strains and species level ascription of newly isolated Paracoccus strains in future.

Understanding SARS-CoV-2: Genetic Diversity, Transmission and Cure in Human.

As the SARS-CoV-2 virus race around the world across the different population, there needs to be a consolidated effort to understand the divergence of demographically distributed strains. The emerging trends in SARS-CoV-2 genome data show specific mutation and genetic diversity, which could provide the basis to develop a cocktail of vaccine and may also be used to develop the region-specific diagnostic tool, thus decreasing the chances of testing failures in fields. Since the transmission of SARS-CoV-2 is subject to the extent of human interaction, the insights from the correlation of genetic diversity with epidemiological parameter would give paramount information to tackle this transmission. Previously, studies have also correlated the epidemiological data with gut microbiome and its role in immunomodulation for maintaining health status, and such information could be generated from recovered individuals from different demographic regions. It will help in designing a probiotic-based diet for modulation of the gut microbiome, and that could be another plausible prophylactic treatment option. The genomics data suggest that a specific variant of SARS-CoV-2 gets enriched with the specific demographic region. Overall, demographic data suggests that host influences mutation and expression of the virus. Hence, the experiences from the clinical intervention for that region should be considered in control and treatment strategies.

Microbial taxonomy in the era of OMICS: application of DNA sequences, computational tools and techniques.

The current prokaryotic taxonomy classifies phenotypically and genotypically diverse microorganisms using a polyphasic approach. With advances in the next-generation sequencing technologies and computational tools for analysis of genomes, the traditional polyphasic method is complemented with genomic data to delineate and classify bacterial genera and species as an alternative to cumbersome and error-prone laboratory tests. This review discusses the applications of sequence-based tools and techniques for bacterial classification and provides a scheme for more robust and reproducible bacterial classification based on genomic data. The present review highlights promising tools and techniques such as ortho-Average Nucleotide Identity, Genome to Genome Distance Calculator and Multi Locus Sequence Analysis, which can be validly employed for characterizing novel microorganisms and assessing phylogenetic relationships. In addition, the review discusses the possibility of employing metagenomic data to assess the phylogenetic associations of uncultured microorganisms. Through this article, we present a review of genomic approaches that can be included in the scheme of taxonomy of bacteria and archaea based on computational and in silico advances to boost the credibility of taxonomic classification in this genomic era.

Simultaneous Biodegradation of Polyaromatic Hydrocarbons by a Stenotrophomonas sp: Characterization of nid Genes and Effect of Surfactants on Degradation.

A polyaromatic hydrocarbon degrading bacterium was isolated from a petroleum contaminated site and designated as Stenotrophomonas sp. strain IITR87. It was found to utilize pyrene, phenanthrene and benzo(a)pyrene as sole carbon source, but not anthracene, chrysene and fluoranthene. Gas chromatography and mass spectroscopy analysis resulted in identification of pyrene metabolites namely monohydroxypyrene, 4-oxa-pyrene-5-one, dimethoxypyrene and monohydroxyphenanthrene. Southern hybridization using naphthalene dioxygenase gene (nidA) as probe against the DNA of strain IITR87 revealed the presence of nidA gene. PCR analysis suggests dispersed occurrence of nid genes in the genome instead of a cluster as reported in a PAH-degrading Mycobacterium vanbaalenii PYR-1. The nid genes in strain IITR87, dioxygenase large subunit (nidA), naphthalene dioxygenase small subunit (nidB) and aldehyde dehydrogenase gene (nidD) showed more than 97 % identity to the reported nid genes from Mycobacterium vanbaalenii PYR-1. Most significantly, the biodegradation of PAHs was enhanced 25-60 % in the presence of surfactants rhamnolipid and Triton X-100 due to increased solubilization and bioavailability. These results could be useful for the improved biodegradation of high-molecular-weight PAHs in contaminated habitats.

Isolation and characterization of a novel Gram-negative bacterium Chromobacterium alkanivorans sp. nov., strain IITR-71T degrading halogenated alkanes.

The taxonomic position of a Gram-stain negative, non-violaceinpigmented bacterium isolated from an insecticide-contaminated site was characterized by a polyphasic approach. The bacterium was able to grow on three different halogenated compounds namely 1-hlorobutane, 1-hloropropane and 1,2-ichloroethane. As a critical step in the degradation of these haloalkanes, stoichiometric amounts of dechlorination were estimated. Based on selective enrichment method for three months, using a highly contaminated mixed chemical soil, a bacterium was obtained and designated as IITR-71T. Its versatility and novelty led us to further characterize it by polyphasic taxonomy. The 16S rRNA gene sequence (1446 bases) comparison showed highest similarity with those of members of the genus Chromobacterium with the most closely related species to strain IITR-71T being Chromobacterium aquaticum (99.3 %) followed by Chromobacterium haemolyticum (98.6 %) and Chromobacterium piscinae (97.1 %). The major ubiquinone was Q-8. Predominant polar lipids are phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG). The DNA G+C content of IITR-71T was estimated to be 61.2 mol%. The genotypic and phenotypic distinctiveness of IITR-71T and its phylogenetic relationships indicate that IITR-71T represents a novel species, for which the name Chromobacterium alkanivorans sp. nov. is proposed. The type strain is IITR-71T (=MTCC 11059T=JCM 30068T=KCTC 52433T).

Taxonomic description and genome sequence of Rheinheimera mesophila sp. nov., isolated from an industrial waste site.

A novel Gram-staining-negative gammaproteobacterium, designated IITR-13T, was isolated from a pesticide-contaminated soil and characterized using a polyphasic approach. On the basis of 16S rRNA gene sequence analysis, the strain showed the closest similarity (98.7 %) to Rheinheimera tangshanensis JA3-B52T followed by Rheinheimera texasensis A62-14BT (97.7 %) and Rheinheimera soli BD-d46T (97.3 %). The 16S rRNA gene sequence similarity of the novel strain to other members of the genus Rheinheimera was < 97.3 %. However, DNA-DNA hybridization between strain IITR-13T and the type strains of R. tangshanensis, R. texasensis and R. soli was 47.5 ± 0.6, 42.4 ± 0.4 and 39.8 ± 0.3 %, respectively; these values are less than 70 %, a threshold value for delineation of a novel species. The strain had C12 : 0 3-OH, C16 : 0, C17 : 1ω8c, summed feature 3 (C16 : 1ω6c/C16 : 1ω7c) and C18 : 1ω6c as the major fatty acids. The major isoprenoid quinones detected for strain IITR-13T were ubiquinone Q-8 and menaquinone MK-7.The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and seven unknown phospholipids. Based on phenotypic and chemotaxonomic characteristics and analysis of the 16S rRNA gene sequence, the novel strain should be assigned to a novel species, for which the name Rheinheimera mesophila sp. nov. is proposed, with the type strain IITR-13T ( = MTCC 12064T = DSM 29723T). Also, we report the draft genome sequence of Rheinheimera mesophila IITR-13T; the draft genome sequence includes 3 749 903 bases and comprises 3449 predicted coding sequences, with a G+C content of 47.8 %. It consists of 102 contigs (>1000 bp).

Bacillus mesophilum sp. nov., strain IITR-54T, a novel 4-chlorobiphenyl dechlorinating bacterium.

The taxonomic position of a Gram-positive, endospore-forming bacterium isolated from soil sample collected from an industrial site was analyzed by a polyphasic approach. The strain designated as IITR-54T matched most of the phenotypic and chemical characteristics of the genus Bacillus and represents a novel species. It was found to biodegrade 4-chlorobiphenyl through dechlorination and was isolated through enrichment procedure from an aged polychlorinated biphenyl-contaminated soil. Both resting cell assay and growth under aerobic liquid conditions using 4-chlorobiphenyl as sole source of carbon along with 0.01% yeast extract, formation of chloride ions was measured. 16S rRNA (1,489 bases) nucleotide sequence of isolated strain was compared with those of closely related Bacillus type strains and confirmed that the strain belongs to the genus Bacillus. Strain IITR-54T differs from all other species of Bacillus by at least 2.1% at the 16S rRNA level, and the moderately related species are Bacillus oceanisediminis (97.9%) followed by Bacillus infantis (97.7%), Bacillus firmus (97.4%), Bacillus drentensis (97.3%), Bacillus circulans (97.2%), Bacillus soli (97.1%), Bacillus horneckiae (97.1%), Bacillus pocheonensis (97.1%) and Bacillus bataviensis (97.1%), respectively. The cell wall peptidoglycan contained meso-diaminopimelic acid and the major isoprenoid quinone was MK-7. Major fatty acids are iso-C15:0 (32.4%) and anteiso-C15:0 (27.4%). Predominant polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The results of physiological and biochemical tests allowed the genotypic and phenotypic distinctiveness of strain IITR-54T with its phylogenetic relatives and suggest that the strain IITR-54T should be recognized as a novel species, for which the name Bacillus mesophilum sp. nov. is proposed. The type strain is IITR-54T (=MTCC 11060T=JCM 19208T).

Deterministic effect of oxygen level variation on shaping antibiotic resistome.

An increase in acquisition of antibiotic resistance genes (ARGs) by pathogens under antibiotic selective pressure poses public health threats. Sub-inhibitory antibiotics induce bacteria to generate reactive oxygen species (ROS) dependent on dissolved oxygen (DO) levels, while molecular connection between ROS-mediated ARG emergence through DNA damage and metabolic changes remains elusive. Thus, the study investigates antibiotic resistome dynamics, microbiome shift, and pathogen distribution in hyperoxic (5-7 mg L-1), normoxic (2-4 mg L-1), and hypoxic (0.5-1 mg L-1) conditions using lab-scale bioreactor. Composite inoculums in the reactor were designed to represent comprehensive microbial community and AR profile from selected activated sludge. RT-qPCR and metagenomic analysis showed an increase in ARG count (100.98 ppm) with enrichment of multidrug efflux pumps (acrAB, mexAB) in hyperoxic condition. Conversely, total ARGs decreased (0.11 ppm) under hypoxic condition marked by a major decline in int1 abundance. Prevalence of global priority pathogens increased in hyperoxic (22.5%), compared to hypoxic (0.9%) wherein major decrease were observed in Pseudomonas, Shigella, and Borrelia. The study observed an increase in superoxide dismutase (sodA, sodB), DNA repair genes (nfo, polA, recA, recB), and ROS (10.4 µmol L-1) in adapted biomass with spiked antibiotics. This suggests oxidative damage that facilitates stress-induced mutagenesis providing evidence for observed hyperoxic enrichment of ARGs. Moreover, predominance of catalase (katE, katG) likely limit oxidative damage that deplete ARG breeding in hypoxic condition. The study proposes a link between oxygen levels and AR development that offers insights into mitigation and intervention of AR by controlling oxygen-related stress and strategic selection of bacterial communities.

Meta-analysis of wastewater microbiome for antibiotic resistance profiling.

The microbial composition and stress molecules are main drivers influencing the development and spread of antibiotic resistance bacteria (ARBs) and genes (ARGs) in the environment. A reliable and rapid method for identifying associations between microbiome composition and resistome remains challenging. In the present study, secondary metagenome data of sewage and hospital wastewaters were assessed for differential taxonomic and ARG profiling. Subsequently, Random Forest (RF)-based ML models were used to predict ARG profiles based on taxonomic composition and model validation on hospital wastewaters. Total ARG abundance was significantly higher in hospital wastewaters (15 ppm) than sewage (5 ppm), while the resistance towards methicillin, carbapenem, and fluoroquinolone were predominant. Although, Pseudomonas constituted major fraction, Streptomyces, Enterobacter, and Klebsiella were characteristic of hospital wastewaters. Prediction modeling showed that the relative abundance of pathogenic genera Escherichia, Vibrio, and Pseudomonas contributed most towards variations in total ARG count. Moreover, the model was able to identify host-specific patterns for contributing taxa and related ARGs with >90% accuracy in predicting the ARG subtype abundance. More than >80% accuracy was obtained for hospital wastewaters, demonstrating that the model can be validly extrapolated to different types of wastewater systems. Findings from the study showed that the ML approach could identify ARG profile based on bacterial composition including 16S rDNA amplicon data, and can serve as a viable alternative to metagenomic binning for identification of potential hosts of ARGs. Overall, this study demonstrates the promising application of ML techniques for predicting the spread of ARGs and provides guidance for early warning of ARBs emergence.

Bacterial remediation of pesticide polluted soils: Exploring the feasibility of site restoration.

For decades, reclamation of pesticide contaminated sites has been a challenging avenue. Due to increasing agricultural demand, the application of synthetic pesticides could not be controlled in its usage, and it has now adversely impacted the soil, water, and associated ecosystems posing adverse effects on human health. Agricultural soil and pesticide manufacturing sites, in particular, are one of the most contaminated due to direct exposure. Among various strategies for soil reclamation, ecofriendly microbial bioremediation suffers inherent challenges for large scale field application as interaction of microbes with the polluted soil varies greatly under climatic conditions. Methodically, starting from functional or genomic screening, enrichment isolation; functional pathway mapping, production of tensioactive metabolites for increasing the bioavailability and bio-accessibility, employing genetic engineering strategies for modifications in existing catabolic genes to enhance the degradation activity; each step-in degradation study has challenges and prospects which can be addressed for successful application. The present review critically examines the methodical challenges addressing the feasibility for restoring and reclaiming pesticide contaminated sites along with the ecotoxicological risk assessments. Overall, it highlights the need to fine-tune the available processes and employ interdisciplinary approaches to make microbe assisted bioremediation as the method of choice for reclamation of pesticide contaminated sites.

Antibiotic resistance in wastewater: Indian scenario.

The surge of Antibiotic Resistant Bacteria (ARB) in the environment is poised to be the next health threat. World Health Organisation's (WHO's) Global Antimicrobial Surveillance System (GLASS) report indicates that developing countries may be at a greater risk. Among various factors, the major driver here could be untreated wastewater and poor sanitation. Bacteria are extremely adaptable to their surroundings and develop Antimicrobial Resistance (AMR) when exposed to antibiotics and other pollutants that cause microbial stress. Thus, untreated domestic wastewater drains could easily become hotspots for the occurrence of ARBs. This study reports surveillance of sewage-carrying drains across four urban cities in India and demonstrated the presence of ARBs in the bacterial community against 7 classes of antibiotics, namely, ß-Lactams, Chloramphenicol, Glycopeptides, Macrolides, Tetracycline, Third Generation Cephalosporin, and Quinolones. Untreated domestic wastewater flowing in target drains was collected twice a month, for a period of six months and the microbial community was subjected to Antibiotic Susceptibility Testing (AST) by plate assays. The zone of inhibition was recorded and interpreted as per the interpretive chart of The Clinical & Laboratory Standards Institute (CLSI) & The European Committee on Antimicrobial Susceptibility Testing (EUCAST). The total number of samples showing resistance against antibiotics was used to define an Antibiotic Resistance Index (ARI), calculated for all 20 sampling sites (drains). Results demonstrated that the highest ARI was observed in Delhi and Mumbai, ranging from 0.81 to 0.92 in Delhi and 0.49-0.56 in Mumbai. This surveillance study reveals the antibiotic resistance pattern of the representative bacterial community in the drains and goes beyond few targeted bacterial species. The alarming presence of antibiotic resistant bacterial community highlights the concern of ARBs being the next looming health threat. This report aims to demonstrates the importance of considering sewage surveillance on routine basis by state authorities.