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 reservoir
of ESBL & PMQR that could become endemic threat to population.
57 million people live off the Yamuna as
a source of water it accounts for ever seventy percent of New Delhi water
supply, however many religious practices.
of Yamuna river pollution cannot be achieved without people’s participation.
Therefore it’s important to create awareness among the people regarding the way
river pollution is occurring and its related consequences. People should be
taught various means to be adopted to reduce the increasing pollution levels in
Delhi, state government has formed 10 feet high wire barricades along all
Yamuna bridge under the Yamuna Action Plan I with signboard messages to prevent
people from tossing things into the Yamuna.
that almost everyday people throw flowers and religious materials in the river,
which creates pollution. Due to chemicals imbalance that includes chemicals
such as phosphate due to Diarrohea and Pneumonia are very common diseases, when
these chemicals are adjusted by the people. This solution we can work together.
of different volunteer organizations and NGO’s should be taken to launch
campaign and create awareness.
media and electronic media can play a major role in creating awareness and
urging people not to throw garbage and other pollutants in rivers.
& K. pneumonia are two of the
main causative agents of most of the infectious diseases. These are the common
bacteria to produce ESBL & PMQR is derived from the beta lactamases and has
the ability to hydrolyze the beta lactam drugs.
We conclude that multidrug resistant E. coli & K. pneumonia are emerging and that necessitates appropriate
therapeutic intervention as regard to selection and use of antibiotics. Our
resistant shows that E. coli & K. pneumonia strains are more resistant
to common antibiotics.
The results of this study show that K.
pneumonia is more affected by the acquisit
ion and stable integration of resistance
genes in an aquatic environment than E. coli in ESBL, where as E. coli is more affected by the acquisition
and stable integration of resistance genes in an aquatic environment than K.
pneumonia in PMQR.
In conclusion, in this
study a high prevalence of ESBL (TEM) more than PMQR positive E. coli & K. pneumonia has been observed in
samples collected from Yamuna River, Hindon River, Sewage water, Canal water
& Ground water. Samples of Faridabad waste water, ISBT Kashmiri Gate &
Ground water of Sarai Kale khan the high prevalence of PMQR genes were more than
ESBL positive E. coli & K. pneumonia strains.
was found to be the dominant ESBL & PMQR encoding gene in this study,
while qnrS, aac-lb cr, bla-TEM
and bla-CTXM were the dominant PMQR genes in the ESBL &
PMQR positive E. coli &
To our knowledge, this is coexistence of the qnrS,
aac(6?)-Ib-cr, bla-TEM and bla-CTX-M genes in one E. coli &
K. pneumonia strain.
ESBL strain frequency are increasing
significantly according to PMQR during time and it is a major cause of concern
and deservs special attention.
One of our aim was to provide
information for laboratories and policy makers which enables them to make
informed decisions about the best methods available for detecting newly
emergent strains of antibiotic resistant E.
coli & K. pneumonia.
The PCR confirms that it is possible and
feasible to perform a simultaneous amplification of the virulence ESBL &
PMQR 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 of
E. coli & K. pneumonia pathotypes has
led to the development of rational interventions for the treatment and
prevention of E. coli & K. pneumonia induced diseases. Continuous
research and investigation into E. coli
& K. pneumonia virulence are
providing us with useful insights into the origins and evolution of this
versatile bacterial pathogen.
However, the ability of various E. coli & K. pneumonia virulence factors to affect such a wide range of
cellular functions has produced an unexpected high level of complexity in order
to develop an effective vaccine. Because of its natural habitat and its ability
in subvert, circumvent and/or evade the immune defenses, the surviving of these
bacteria is safeguarded in nature. The acquisition of different virulent
traits, the continuous exchange of genetic elements and the expression of virulence
genes generally regulated by environmental factors probably will reveal
different strategies shared by E. coli
& K. pneumonia strains.
Some of other future prospect
of 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 in
the 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 output
water can contaminate other environmental sections, such
as water resources, and
result in the
emission of these contaminants. Urgent measures need to be taken
minimize the effects of
releasing wastewater into water resources.
More studies should be
carried out in the future in order to ensure that the ESBL & PMQR genes
were 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.