Genotypic characterization of multi-drug resistant coliform bacteria: Insights into their mechanisms of antibiotic resistance using Whole Genome Sequencing

Anthropogenically polluted water is a potential reservoir for pathogenic microorganisms and micro-contaminants like antibiotics. Due to the selective pressure of antibiotics, resident bacteria tend to acquire resistance mechanisms through mutations, genome rearrangements and horizontal gene transfer. (Darvinism: Survival of the fittest!). This study aimed to isolate and characterize multi-drug resistant coliform bacteria from natural water bodies of Pune city and to analyse whole genome sequences for identification of genomic alterations possibly responsible for multi-drug resistance (MDR). The isolates were identified by next generation sequencing. Sequence type of isolates was determined by Multilocus sequence typing (MLST). Genes responsible for antibiotic resistance were identified using Comprehensive Antibiotic Resistance Database (CARD). The isolates were found resistant to third and fourth generation cephalosporins and carbapenems which is very alarming as these are the antibiotics of last resort. The mechanisms of resistance developed by isolates were efflux pump mediated drug resistance and β- lactamase production. Mutation rate was found higher when set of genes responsible for efflux pump mediated drug resistance (mdt A, mdt B, mdt C, mex J, mex K, opr N) was analysed. Mutation leading to change in single amino acid (Arg-235 to Lys) was detected in Pseudomonas aeruginosa ST- 635 for the gene blaPDC-3 . Escherichia coli ST-410 and ST- 617 were found single amino acid variants for the gene blaCMY- 47 (Pro-121 to Ser). Mutations observed in CMY-47 and PDC-3 are indicative of rapid evolution of AmpC β- lactamases. Indiscriminate use of antibiotics has resulted into emergence and dissemination of MDR leading to antibiotic- driven adaptive bacterial evolution

Molecular typing of antibiotic resistant bacteria isolated and identified as ESBL producers from polluted water reservoirs

Anthropogenic polluted reservoirs are the mating hub for antibiotic resistant genes and multidrug resistant bacteria (MDR). The rapid emergence of this MDR is the consequence of mutations in the genes as well as the horizontal gene transfer of mobile elements carrying the resistant genes. Current study focuses on isolation and characterization of Extended spectrum β-lactamase (ESBL) producers from diverse water resources of Pune city and to understand the genetic modifications responsible for multidrug resistance using whole genome sequencing (Next Generation sequencing-Illumina sequencing). The identified isolates were Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae and Stenotrophomonas maltophilia. Mechanism of resistance developed by all isolates was efflux pump as per the genes (adeL, macA, macB, ros B) identified by Comprehensive Antibiotic Resistance Database. Primary phenotypic detection of isolates as ESBL producers and AmpC hyper producers was supportive as identified genes were resistant to all antibiotics including last resorts like carbapenems, peptide antibiotics. Rapid emergence of antibiotic resistance was seen in one isolate due to presence of additional 19 antibiotic resistant genes (blaI, exo bet lactamase, PDC 9, CMY-83, mec I, etc.). The investigation alarms the deadly pollution of reservoirs due to haphazard use of antibiotics which pressurizes rapid emergence and persistence of MDR