The objective of the present study was to analyze the diversity structure of E. coli & K. pneumonia strains isolated from the River Yamuna & other water bodies traversing though the National Capital Territory of Delhi (India) and understand the molecular mechanisms underlying ?-lactamases mediated antibiotic resistance as well as co-resistance to one of the other important classes of antibiotics viz (fluro) quinolones, if any.· Water samples were collected from different Fifteen geographical sites of Delhi.· The results of this study show that E. coli is more affected by the acquisition and stable integration of resistance genes in an aquatic environment than Klebsiella. · Total Ninety-six E.
coli & Eighty-four K. pneumoniae isolates were obtained from different-2 Geographical sites, Seven different -2 sites of Yamuna River, Three different-2 sites of Sewage water, Two different -2 sites of Ground water, One sites of Canal water & Two different -2 sites of Other water bodies Faridabad waste water and Hindon River.· The isolated E. coli & K. pneumoniae strains when tested for their tolerance to AMR exhibited similar resistant patterns both in liquid and solid medium.· All the biochemical tests were same for different environmental strains of E. coli and K. pneumoniae.
From this collection of E. coli & K. Pneumonia a group of strains representing all phylogroups was used further studies.
· Total Ninety-six E. coli & Eighty-four K. pneumoniae isolates were obtained from different water samples, the isolated E. coli & K. pneumoniae strains when tested for their tolerance to AMR exhibited similar resistant patterns both in liquid and solid medium.· All the biochemical tests were same for different environmental strains of E.
coli and K. pneumoniae.· From this collection of E. coli & K. Pneumonia a group of strains representing all phylogroups was used further studies.
The Forty-eight E. coli & Forty-two K. pneumonia strains were CONFIRMED by sequencing of their 16s ribosomal RNA (rRNA genes).· Correlation between antibiotic and AMR in collected AMR strains was examined, resistance pattern towards Ten different antibiotics i.e.
Ceftazidime (30mcg), cefotaxime (30mcg), Cefpodoxim (10mcg), Azithromycin (15mcg), Metronidazole (4mcg), (Meropenem (10mcg), Amikacin (30mcg), Ampicilin (10mcg), Ceftriaxone (30mcg), and Ciprofloxacin (5mcg).· All the selected strains were resistant in E. coli strains almost Ampicillin (100%), Cefotaxime (72.91%), Ceftriaxone (70.83%), Ciprofloxacin (100%), Cefpodoxim(56.25%), Ceftazidime (83.
33%), Metronidazole (70.83%), Meropenem (31.25%), Azithromycin (60.
41%) and Amikacin (12.5%) & K. pneumonia strains to Ampicillin (100 %), Cefotaxime (76.
19 %), Ceftriaxone (80.95 %), Ciprofloxacin (100%), Cefpodoxim (88.095 %), Ceftazidime (85.71 %), Metronidazole (78.57%), Meropenem (50 %), Azithromycin (71.42 %) and Amikacin (23.80 %) & Ampicillin (100%).
· Phenotypic Detection of ESBL to Cefotaxime, Ceftazidime, Cefepime Cefepirome alone and with Clavulanic acid. Almost all 76/90 (84%) of the E. coli (46) & K. pneumonia (30) were showed ESBL & PMQR Positive.· We were detect the presence of protein in the strains of E. coli & K. pneumonia.· All the seventy six strains were screened for the presence of Protein.
Following protein isolation, all the selected strains showed the presence of protein when subjected on SDS-PAGE.· All the seventy six strains were screened for the presence of plasmid. Following plasmid DNA isolation, all the selected strains showed the presence of plasmid when visualized on 1% agarose gel.
· Our main effort was to characterize ESBL & PMQR genes from different isolates so that we could find out the divergence between them and then they can be further used for bioremediation of drug. Having established the plasmid borne nature of ESBL & PMQR gene in the selected strains, PCR amplification of TEM, CTX-M, qnrS and aac (6′)-lb-cr) genes were carried using specific primers, the expected length of PCR products for TEM gene corresponding to 780 bp was obtained from E. coli (46) 65.21 % & K. pneumonia (30) 66.66 % strains, CTX-M gene corresponding to 530 bp was obtained from E.
coli (46) 34.7 % & K. pneumonia (30) 36.
66 % strains, qnrS gene corresponding to 456bp was obtained from E. coli (46) 30.43 % and K. pneumonia (30) 19.56 % wild type isolated strains and aac(6′)-lb-cr) gene corresponding to 482bp FQRB respectively was obtained from only wild type isolated E. coli (46) 19.
56 % & K. pneumonia (30) 23.33 % when checked on 1% agarose gel. Other PMQR gene such as qnrC were detected less frequently. Additionally no qnrD, gene was observed in this study.· DNA sequencing was performed in order to check the diversity and distribution of ESBL & PMQR genes. In the present study we found that all the nucleotide sequences for CTX-M gene fragments from selected strains were showing a great diversity (74-84%) but the sequences for all the selected TEM gene fragments showed a high level of similarity (upto 99%) and qnrS gene strain showed upto (94%) with other already reported sequences of that gene.
· A phylogenetic tree showed a different data with other already reported sequences of that gene. · Yamuna water appeared to be a reservoirof ESBL & PMQR that could become endemic threat to population.· 57 million people live off the Yamuna asa source of water it accounts for ever seventy percent of New Delhi watersupply, however many religious practices. · Preventionof Yamuna river pollution cannot be achieved without people’s participation.
Therefore it’s important to create awareness among the people regarding the wayriver pollution is occurring and its related consequences. People should betaught various means to be adopted to reduce the increasing pollution levels inthe river. · InDelhi, state government has formed 10 feet high wire barricades along allYamuna bridge under the Yamuna Action Plan I with signboard messages to preventpeople from tossing things into the Yamuna.
· Despitethat almost everyday people throw flowers and religious materials in the river,which creates pollution. Due to chemicals imbalance that includes chemicalssuch as phosphate due to Diarrohea and Pneumonia are very common diseases, whenthese chemicals are adjusted by the people. This solution we can work together.· Helpof different volunteer organizations and NGO’s should be taken to launchcampaign and create awareness. · Printmedia and electronic media can play a major role in creating awareness andurging people not to throw garbage and other pollutants in rivers. · E.coli& K.
pneumonia are two of themain causative agents of most of the infectious diseases. These are the commonbacteria to produce ESBL & PMQR is derived from the beta lactamases and hasthe ability to hydrolyze the beta lactam drugs. · We conclude that multidrug resistant E.
coli & K. pneumonia are emerging and that necessitates appropriatetherapeutic intervention as regard to selection and use of antibiotics. Ourresistant shows that E.
coli & K. pneumonia strains are more resistantto common antibiotics. · The results of this study show that K.pneumonia is more affected by the acquisition and stable integration of resistancegenes in an aquatic environment than E. coli in ESBL, where as E. coli is more affected by the acquisitionand stable integration of resistance genes in an aquatic environment than K.
pneumonia in PMQR. · In conclusion, in thisstudy a high prevalence of ESBL (TEM) more than PMQR positive E. coli & K. pneumonia has been observed insamples collected from Yamuna River, Hindon River, Sewage water, Canal water& Ground water. Samples of Faridabad waste water, ISBT Kashmiri Gate water of Sarai Kale khan the high prevalence of PMQR genes were more thanESBL positive E. coli & K.
pneumonia strains.· The blaCTX?Mwas found to be the dominant ESBL & PMQR encoding gene in this study,while qnrS, aac-lb cr, bla-TEMand bla-CTXM were the dominant PMQR genes in the ESBL positive E. coli . pneumonia.· To our knowledge, this is coexistence of the qnrS,aac(6?)-Ib-cr, bla-TEM and bla-CTX-M genes in one E. coli .
pneumonia strain. · ESBL strain frequency are increasingsignificantly according to PMQR during time and it is a major cause of concernand deservs special attention.· One of our aim was to provideinformation for laboratories and policy makers which enables them to makeinformed decisions about the best methods available for detecting newlyemergent strains of antibiotic resistant E.coli & K.
pneumonia. · The PCR confirms that it is possible andfeasible to perform a simultaneous amplification of the virulence ESBL genes of antibiotic resistant E.coli & K. pneumonia and this technique can be applied to the ESBL& PMQR genes characterization of antibiotic resistant E. coli & K. pneumonia. · Knowledge of the pathogenic mechanism ofE. coli & K.
pneumonia pathotypes hasled to the development of rational interventions for the treatment andprevention of E. coli & K. pneumonia induced diseases. Continuousresearch and investigation into E. coli& K.
pneumonia virulence areproviding us with useful insights into the origins and evolution of thisversatile bacterial pathogen. · However, the ability of various E. coli & K. pneumonia virulence factors to affect such a wide range ofcellular functions has produced an unexpected high level of complexity in orderto develop an effective vaccine.
Because of its natural habitat and its abilityin subvert, circumvent and/or evade the immune defenses, the surviving of thesebacteria is safeguarded in nature. The acquisition of different virulenttraits, the continuous exchange of genetic elements and the expression of virulencegenes generally regulated by environmental factors probably will revealdifferent strategies shared by E. coli& K. pneumonia strains. Some of other future prospectof this research include:Ø Avoid throwing flowers, sweets, Puja materials into a Yamuna River. Ø It will degrade the quality of water.
The river won’t be happy with this.Ø Avoid throwing dead bodies in a river. This will ultimately landing inthe mouth of dogs, vultures & other animals. Ø Never dump anything into the water bodies.
Ø Avoid use of weedicides.Ø In case of improper exploitation of the outputwater can contaminate other environmental sections, suchas water resources, and result in theemission of these contaminants. Urgent measures need to be takento minimize the effects ofreleasing wastewater into water resources.· More studies should becarried out in the future in order to ensure that the ESBL & PMQR geneswere located on the same plasmid or not.
· Molecular investigation of E. coli & K. pneumonia strains for confirmation.· Cloning and expression of ESBL &PMQR gene regulon and characterization of produced protein.