S.Yadry.A Kapley Science of the Total Enyt t62201911155-116 robes.and accession numbe Acces rainM743 strain Mo10 7258 F电5.C-RAST metabolite gene encoding cluster in both activated sludee metagenome pathogenic and bacteria (.1). asmid pB8 we ed in both sludge met m Resistance type Accession no. 7etlandantibiodl Fuet ation of MRB to the presence of In mid n the unlimit number of genetic ding tothe red bi e strain MO10 3.4.Secondary metabolites encoding potential in the activated sludge eighty years,the role prolife tion of A be ent pathways: .and our abi 1.Vertical evolution:due tom nds is a priority.According to Blackbox theory.99 e antibiotic tolerance is utten.2014). ofantim ng for thes ( ompatibility g hyl-gamma ulanic acid,an h 0 o-slu e metag D et al 20 ntly it has pec Tezuka and hnishi.01 ry genes that may find use in environmental bic Te py SoOm ermreo Phenazinesare nitrl activity against MRBs (Kapleyt 95%confidence intervals 98e-8 0 983e8 Phages.Prophage 3,29e-6 0013 0.0 774-10 5 10 15 Proportion (% Difference between proportions (% Comparative analysis of mobile genetic elements abundance in the two metagenome Fig 4.Comparative analysis of mobile genetic elements abundance both activated sludge metagenome using STAMPmicrobes acts as the crucial factor in the evolution and maintenance of the resistance (Melnyk et al. 2015). Resistance can evolve through two different pathways: 1. Vertical evolution: due to mutations enhance antibiotic tolerance is acquired and transmitted to the next progeny. 2. HGT: bacteria acquire resistant genes from neighboring bacteria via genetic exchange by the process conjugation, transformation, and transduction. Extrachromosomal genetic elements belonging to incompatibility (IncP) group which can replicate in harmful gram-negative bacteria were also present in the two-sludge metagenome. These plasmids en￾code for the essential functional genes responsible for DNA replication and accessory genes that may find use in environmental bioremedia￾tion. These accessory genes often encode for the resistance to broad￾spectrum antibiotics and heavy metals. They are found in the strain of pathogenic and opportunistic bacteria (Fuente-Núñez et al., 2014). In the present study, IncP-1 plasmid, pKJK5, and IncP-1beta multi￾resistance plasmid pB8 were detected in both sludge metagenome. These broad-host-range IncP-1 plasmids carry resistance to heavy metals and antibiotics (Fuente-Núñez et al., 2014; Huijbers et al., 2015; Seiler and Berendonk, 2012). It has been found that the dissemi￾nation of MRBs in a diverse niche like soil, water, and WWTPs occurs due to the presence of IncP-1 plasmid (Miller et al., 2016; Osińska et al., 2016). The presence of IncP-1 plasmid and transposons may result in the unlimited number of genetic rearrangements leading to the emergence of the new resistance, which will be difficult to control (Klumper et al., 2015). 3.4. Secondary metabolites encoding potential in the activated sludge metagenome Since the last eighty years, the role of microbes in the field of medi￾cine and agriculture was tremendous. But with the rapid emergence and proliferation of AMRs, the situation is becoming worse, and our abil￾ity to treat diseases is declining. Therefore, the search for new antimi￾crobial compounds is a priority. According to Blackbox theory, 99% bacterium and 95% fungi are yet to be cultured (Bardgett and van der Putten, 2014), which may act as bioresource of the discovery of antimi￾crobial compounds (Bernard et al., 2018; Crits-Christoph et al., 2018). Metagenomic mining for the secondary metabolites targeting bacterial cytostatic, differentiation factors, and antibiotics indicated the presence of three types of the biosynthetic gene for 2-isocapryloyl-3R￾hydroxymethyl-gamma-butyrolactone, Clavulanic acid, and Phenazine (Fig. 5). Gamma-butyrolactone induces secondary metabolism in the Streptomyces species (Du et al., 2011; Tezuka and Ohnishi, 2014). Re￾cently it has been found in the draft genome of “Alcaligenes sp. HPC 1271,” having antimicrobial activity against MRBs (Kapley et al., 2016). Phenazines are nitrogen-containing aromatic compounds of bacteria and archaea origin. Table 3a List of annotated acquired antibiotic resistant genes, microbes, and accession number present in A2_S27_assembly sludge metagenome. Bacteria Resistance type Accession no. Pseudomonas aeruginosa Aminoglycoside X60321 Plasmid pMON234 Beta-Lactam J03427 Salmonella enterica subsp. Colistin KY807921 Salmonella enterica subsp. Colistin KY807922 Salmonella enterica subsp. Colistin KY807923 Klebsiella pneumoniae strainM743 Fluoroquinolone EF636461 Escherichia coli aacA4 Phenicol AB212941 Acinetobacter baumannii strain 6 Rifampicin HQ141279 Acinetobacter baumannii strain 7 Rifampicin HQ141280 Vibrio cholerae strain MO10 Sulphonamide AY034138 Klebsiella pneumoniae Sulphonamide DQ143913 Klebsiella pneumoniae Sulphonamide DQ143914 K. aerogenes plasmid R751 Trimethoprim X72585 Table 3b List of annotated acquired antibiotic resistant genes, microbes, and accession number present in AKR012_contigs sludge metagenome. Bacteria Resistance type Accession no. Pseudomonas aeruginosa strain K7 Aminoglycoside JQ414041 Escherichia coli plasmid RSF1010 Aminoglycoside M28829 Pseudomonas aeruginosa Aminoglycoside AF024602 Pseudomonas aeruginosa plasmid pMLH50 Beta-lactam AY027589 Photobacterium damselae subsp. Macrolide AB571865 Escherichia coli aacA4 Phenicol AB212941 Acinetobacter baumannii strain 6 Rifampicin HQ141279 Uncultured bacterium clone riv1 Sulphonamide MG649393 Vibrio cholerae strain MO10 Sulphonamide AY034138 Klebsiella pneumoniae Sulphonamide DQ143913 Pseudomonas sp. plasmid Tetracycline AF133140 Klebsiella aerogenes Trimethoprim X72585 Fig. 5. Secondary metabolite gene encoding cluster in both activated sludge metagenome using MG-RAST. Fig. 4. Comparative analysis of mobile genetic elements abundance both activated sludge metagenome using STAMP. S. Yadav, A. Kapley / Science of the Total Environment 692 (2019) 1155–1164 1161