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.

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Water samples were collected from different
Fifteen geographical sites of Delhi.

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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. 

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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.

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 The isolated E. coli & K. pneumoniae
strains when tested for their tolerance to AMR exhibited similar resistant
patterns both in liquid and solid medium.

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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.

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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.

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All the biochemical tests were same for different
environmental strains of E. coli and K. pneumoniae.

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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).

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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).

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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%).

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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.

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We were detect the presence of protein
in the strains of E. coli & K. pneumonia.

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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.

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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.

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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.

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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. 

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A
phylogenetic tree showed a different data with other already reported sequences
of that gene.