Both developed and developingcountries are suffering from different forms of water pollution. Developedcountries that have water pollution problem due to industrial proliferation andmodernized agricultural technologies are now on their way of combating theseproblems through improved technologies of waste treatment. But developingcountries like India, with limited financial resources, lack of technicalknowledge and with weak implementation of environmental policies, are stillfacing severe problems. In most of these countries including India, water inmany rivers receives a heavy flux of sewage, industrial effluents, domesticagricultural wastes (Cullen and Reimer, 1989). Being an extremely heterogeneousspecies which harbour strains ranging from gastrointestinal commensals tovarious intestinal and extra-intestinal pathogens, the need to study diversityof waterborne Escherichia coli & K. pneumonia especially, in the urbanaquatic environments is of utmost importance, as it is directly related topublic health.

Further, with ?-lactams representing the most widely andsuccessfully used antibiotics for treatment of infections caused by E. coli & K. pneumonia (Herniques et al.2006) and treatment failures being largely attributed to the emergence anddissemination of resistance mediated by the expression of ?-lactamases(Bradford et al 2001), it isimportant to identify the strains’ susceptibility profiles and understand themolecular basis of such resistance. The need to perform such studies withstrains isolated from urban aquatic environments have been well-established toserve as an ideal reservoir an disseminator of antibiotic resistant bacteria aswell as the resistance genes (Baquero etal . 2008; Zhang et al. 2009; Tacao et al. 2012).

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The major problem ofmultidrug resistance among infection causing organisms represents one of thegreatest challenges worldwide. With increased antimicrobial usage, complexitiesin the resistance mechanisms have become more advanced. Densely populatedcenters with improper water supplies and inconsistent sanitation contributesignificantly to acquisition and dissemination of resistance determinants amongmicrobial inhabitants of water bodies. The Yamuna River, which originates fromthe Yamunotri glacier in the lower Himalayas (38°59?N 78°27?E), is the majorsource of water to urban areas in Delhi. Although the proportion of the rivercatchment area in Delhi is small (~2%), this area contributes more than 50% ofpollutants that it receives through sewage from urban effluents, with highlevels of antimicrobials in addition to toxic compounds being discharged byindustries (Sharma and Kansal, 2012; Sehgal et al., 2012; Mutiyar and Mittal, 2014). The acquisition andtransmission of resistance genes from microflora of human and animal origindischarged as part of sewage can substantially influence the pattern ofresistance among the microbial inhabitants of the aquatic ecosystem (Amos et al., 2014).

Increasing incidencesof ESBL & PMQR producing bacteria that showed a drastic shift in recentyears in environmental settings are of serious concern. The contribution ofselection to the acquisition and as such spread of resistance among bacteriaagainst major classes of antibiotics is alarming due to their higherdissemination rate. As such, the high prevalence of ESBL & PMQR producingisolates in natural water bodies like rivers, sewage, canal and ground waterhas drawn concern regarding increased spread of resistance in the environment(Upadhyay and Joshi, 2015).Recently, Bajaj et al. (2015) and Ahmad et al.(2014) also reported high prevalence of the several ?–lactamase genes (TEM,SHV, CTX-M, AmpC, and NDM-1) among E.coli and other coliform bacterial species screened in collected watersamples from upper ranges of Ganges River till its tributary Yamuna thatstretches in Delhi and beyond.

Recently, Anas et al. (2017) also reported highprevalence of the ESBL among E. coli.Antimicrobial Resistance PatternShortly after theprescription of antibiotics became widespread in the 1940s, the pathogenicbacteria began to demonstrate resistance towards these drugs. Enhancedselection pressures caused by the inappropriate or careless use of antibioticstend to increase the prevalence of resistant populations microorganisms; Plasmid-mediated quinolone resistancemechanisms play an important role in the expansion of quinolone andfluoroquinolone resistance among ESBL & PMQR producing E. coli & K. pneumonia(Du J.

et al. 2014). Infections caused bysuch resistant isolates can be difficult to treat (Varelaet al. 2016). Over the past 10 years, PMQR determinants have appeared as animportant issue (Rodriguezet al. 2015). Antibiotics reducemortality in severe infections. Efficient antibiotics are a prerequisite formodern health care.

New antibiotic classes against gram-negative infections arenot to be expected in the near future, why the currently available must be usedin a rational way, considering the growing challenge from antibioticresistance.  Prescribing patternsvary worldwide and mirror the level of antimicrobial resistance. In Europe,high-prescribing countries have a higher prevalence of antimicrobial resistanceand vice versa.

Greece showed the highest prescription rates of antibiotics inthe primary care sector during 2014, 34.0 daily delivered doses (DDD) per 1000inhabitants and per day. This is more than three times as much as the lowestprescribing country, the Netherlands, with a prescription rate of 10.6 DDD per1000 inhabitants and per day. In Sweden, the rate was 13.

0 DDD per 1000inhabitants and per day in 2014 (http://ecdc.europa.eu). Increased use ofbroad-spectrum antibiotics due to the high prevalence of MDR doubtlessly leadsto the further selection of resistant strains. The regional differences inprescribing patterns are substantial even within Sweden, where StockholmCountry has one of the country’s highest prescriptions rates of antibiotics. However,between 2009 and 2015 the number of prescriptions decreased by 18%, from 430 to352 per year and 1000 inhabitants (Concise, e-health Authority,www.

ehalsomyndigheten.se), still, over-prescription can be further reduced.In a study, Center forDisease Dynamics, Economics & Policy (CDDEP) reported an increase in thepercentage of carbapenems resistance from 10% in 2008 to 13% in 2013 among E.

coli and 29% in 2008 to 57% in 2014in isolates of K. pneumonia fromIndia Center for Disease Dynamics Economics and Policy (CDDEP),. Besidesstrengthening the existing knowledge of their prevalence, the presence of MDR,XDR, and PDR bacteria in the natural environment endorses them for thepotential threat that they possess for the mankind.

Accordingly, investigationsof ESBL & PMQR genes (blaTEM and blaCTX-M) & (qnrS & aaclb-cr) among bacteria that harbor both multidrugresistance are thought to provide useful information regarding theirepidemiology in human influenced polluted environments.The present study wasundertaken to analyze the diversity of E.coli & K. pneumonia strainsisolated from the River Yamuna and other water bodies traversing through themetropolitan city of Delhi (India) and understand the molecular mechanismsunderlying ?-lactamases mediated antibiotic resistance as well as co-resistanceto one of the other important classes of antibiotics viz, (fluro) quinolones,if any, where maximum antibiotic-resistant bacteria has been reported even indrinking water (Mandal et al., 1996).

In its entire stretch through the city, the river Yamuna receives 950 milliongallons of sewage per day making it one of the most polluted river in theworld. It, therefore, represents an ideal ecological niche for conductingstudies on diversity of E. coli &K. pneumonia and resistance toclinically important antibiotics. In addition to this, this study also aimedfor the determination ofhomology or divergence in antibiotic resistance genes among the selectedstrains.

Thus, a total of 180 strainscomprising E. coli (96) & K. pneumonia (84) strains, were isolatedfrom water samples collected along different points of river Yamuna & otherwater bodies traversing through Delhi.All the selectedstrains of wild-type E. coli & K.

pneumonia strains were furtherconfirmed by biochemical test and the results of all these tests were similarfor all the strains.Further studies were carried outusing a collection of 90 {E. coli(48) & K. pneumonia (42)} strainsselected from a total 180 antimicrobial resistant environmental E. coli (96) & K. pneumoniae (84) strains. The identification of these strainswere further confirmed by the gold standard method of sequencing of their 16Sribosomal RNA (rRNA) genes.            Antimicrobialsusceptibility test:Entire analysis ofcollected samples showed the resistance against following antibiotics: In the present study, all of theisolated Ninety strains were resistant to approximately 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%) in E.coli strains & in K. pneumonia Cefotaxime (76.19%),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%). Highlevels of resistance to 7 to 8 antimicrobial agents were observed in the presenceof multidrug-resistant ESBL & PMQR-producing E. coli &K. pneumoniae, while on theother hand, the resistance level was low for Amikacin and Meropenem.

Of the 90 E. coli (48)& K. pneumonia (42) isolates thatwere positive in the screening test, ESBL production was confirmed by PDCT in76 (84.3%) isolates, which included 46 (95.83 %)  E. coli and30 (71.42 %)  K.

pneumoniae, indicating that the prevalence ofESBL production is 95.83% in E. coli (ESBL-EC)and 71.42% in K.

pneumoniae (ESBL-KP). In a multi-centric study conducted as part of IndiaSENTRY surveillance, the prevalence of ESBL production was reported to be 84% (Manoharan et al. (2011).  These results clearlyindicate that the incidence of antimicrobial resistance was widespread andprobably resulted from either the intensive use of antibiotics or theuncontrolled availability of them. However multiple drug resistance in entericorganisms like E. coli & K.

pneumoniae is also known to beassociated with integron. Integron generally contain an integrase gene (int1)(Martinez and Cruz 1988) and a cassette integration site (att1) (Strokes et al ., 1997) into which antibiotic resistance gene cassettes wereintegrated. Globally disseminated Tn21 like transposons which carry class1integron (typically conjugated plasmids) might also account for the highincidence of antibiotic resistance among commensal, environmental and clinicalbacterial isolates (Zuhlsdorf and Wiedmann, 1992). Subsequently,considering that the co-existence of ?-lactamase mediated resistance andresistance to quinolones in the same strains represents a major health hazard,the study investigated the molecular mechanisms conferringquinolone co-resistance to the ESBL producing E.

coli & K. pneumoniastrains isolated from the Yamuna River, Hindon River, Hasanpur Village sewage,Rohini Sewage, Punjabi Bagh sewage, Faridabad waste water, Rohini Canal water& Ground water. Here, it is important to note that even if these resistancegenetic elements do not reside on the same genomic platforms as the plasmid-mediatedgenes for ESBLs, the co-expression of resistance to both quinolones and  ?-lactam classes of antibiotics in the waterborneE. coli & K. pneumonia consistently being subjected to anthropogenicexposures through river waters & other water bodies represents seriouspublic health concerns which, if manifested in the human infections would beextremely difficult to treat with the limited choice of remaining therapeutics.

It is known that protein play an important role in regulation of thebiofilm phenotype ((Oosthuizen et al.,2002). Different classes of extracellular proteins have been described as partof an adaptive response to a change in the environment (Tjalsma et al., 2000).Thisactive drug transport is involved in low intrinsic susceptibility,cross-resistance to chemically unrelated classes of molecules, andselection/acquisition of additional mechanisms of resistance.

Wehave also attempted to detect the presence of differential expression of total proteinsin the strains of E. coli & K. pneumonia using SDS-PAGE.Differenceswere observed in the expressed proteins but the identity of the proteins wasnot investigated further in this study. The changes in the protein expression mayprobably play an important role to resensitize bacteria towards antibiotics (Chaudharyand Payasi, 2012).In our study, more than 93% ofESBL Gram negative isolates were observed to harbor blaTEM, blaCTX-M.

These variants wereshowing similarity to those reported by Wattal et al. (2010); and Rastogi et al.(2010), during their studies on ESBL production among clinicalisolates (Rastogi et al., 2010; Wattal et al., 2010).Following PCR-based detection, the blaTEM, blaCTX-M genes were identified TEM in E.

coli (30/46) 65.21 % & K.pneumonia (20/30) (66.66 %) and in CTX-M E. coli (16/46) 34.7 % & K.pneumonia (11/30) 36.

66 % strains  4    5     M M   3  3   2  2   1    .    Thelater, in fact, was first reported from the Indian isolates (Karim et al. 2001), and since then has beenreported to encode the most widely disseminated CTX-M ESBL (Poirel et al. 2002b). It is in concordance with the previousreports that documented the possible emergence of blaCTX-M genesfrom Kluyvera sp.

 (Sarriaet al., 2001; Humeniuket al., 2002; Bonnetet al, 2004; Mundayet al., 2004; Mc Gettiganet al., 2009; Zhanget al., 2009).Continuous threat posed by resistantorganisms to human health has necessitated the need for further studies toimprove understanding of their resistance mechanisms.

As shown in the present study, a greater contribution of the substitutionsin chromosomal QRDRs than the presence of PMQRgenes has been reported earlier in conferring high levels of quinoloneresistance to E. coli strains(Rodriguez-Martinez et al. 2007);Dobiasova et al.2013). Following PCR-based detection, the qnrS, aac-lb cr genes were identified inE. coli (14/46) 30.

43 % & K. pneumonia (12/30) 40% and aac(60)-lb-cr in the E. coli (9/46)19.56 % & K. pneumonia (7/30) 23.33 % strains. This conclusion was supportedby previous studies demonstrating the frequent occurrences of these genes inenvironmental isolates (Zhanget al. 2012).

TheqnrS-type genes seem to be the mostcommonly identified acquired qnrgenes in the environment (Cattoiret al. 2012).They have been mainly identified from waterborne species and in particularAeromonas spp. (Picaoet al. 2015). Aac(60)Ib-cr is widespread geographically and stable over time (Varelaet al. 2015).It has often been more common than qnralleles.

Noticeably,some studies identified a high prevalence of other PMQR genes such as qnrD or qnrB in aquatic environments (Varela et al. 2015; and Cattoir et al. 2008).

Factors,such as bacterial species and microbial habitat, may play important roles inthe development and spread of antibiotic resistance.Inthis study, the prevalence of the genes aac(60)-Ib-cr and qnrS was significantly higher in samples of Yamuna water samplesthan other water samples. However, there was no significant difference betweenYamuna water samples and other water samples.

Moreover,qnr genes have been found to exist inmany extended-spectrum beta-lactamase producing Enterobacteriaceae (Galvin et al. 2011), suggestingtheir co-selection with other resistance elements.A resistance gene reservoir notonly allows resistant genes to stably exist, but also facilitates transfer ofthese genes to other species in a natural state. Hence, blaTEMand blaCTX-M type ESBL genes were the most common genotypesin this study.Besides the chromosomally-encodedmechanisms of the E. coli & K. pneumonia strains also harbored theplasmid-mediated quinolone resistance genes viz.

qnrS and aac(6′)-lb-cr, respectively. Additionally, the qnrS gene was also observed in theremaining ESBL producing E. coli . pneumonia strains which carriedthe wild type sequences of the QRDRs of chromosomal gyrase and topoisomerase?V, and did not show high resistance to (fluro) quinolones. Although, the PMQR genes per se didnot contribute to high resistance in majority of the strains, their presencenevertheless deserves a notable mention. This is especially, because presenceof the PMQR genes in E.

coli & K. pneumonia has been reported toprovide a very favourable background for the selection and enrichment ofchromosomally-mediated (fluro) quinolones resistance (Poirel et al. 2006; Rodriguez-Martinez et al. 2007). The strains carrying PMQRgenes may therefore serve as an important reservoir for the eventual increaseand spread of resistance to quinolones.  From these data, it is clear thatthe potential impact of E. coli . pneumoniae infections on humans issignificant.

Prevalence of resistance determinants for the front line choice ofdrugs threatens the future continued clinical use of antibiotics. Therefore,the breadth of resistome, monotony of resistance mechanisms, evolution andemergence of antibiotic resistant populations needs to be clearly understood toallow the discovery of new therapeutic drugs. In cases where such phenotypicresistance was not significantly observed, the ability conferred on the strainscarrying ESBL & PMQR genes to serve as a crucial reservoir for theaccumulation of potential resistance-associated mutations and their efficientspread cannot be under-mined.

This again highlights the need to trulyappreciate the potential of aquatic environments in emergence and spread ofantibiotic resistance, and underscores the need for routine surveillance andmeasures to contain their spread.               Prevention And Control For better of management ofInfection health care workers (both hospitaland community based) should undertake practices known to reduce the spread ofESBLs. These fall into two broad groups             1) Good hand hygiene and cleanliness2) A restrictive approach toantibiotic prescribing, especially in the limitation of third                    generation cephalosporin and quinolone use.a)     Thesesimple interventions can have a major influence on the impact of ESBLs inhealth care setting.

b)     Appropriateuse of antibiotics will greatly reduce the selection pressure for colonizationand infection with ESBLs: Antibiotics must be prescribedaccording to the Antimicrobial Policy and detailed Antimicrobial PrescribingGuidelines. Where there is more than one caseon the ward, the prescriber should consider avoiding cephalosporin usealtogether in other patients on the ward. In an outbreak situation, theInfection Control Doctor (ICD), a Consultant Medical Microbiologist and the AntimicrobialPharmacist will suggest interim alternative antibiotic prescribing guidelineson a ward /unit.Limitations of resistance toantimicrobials may be achieved by:·        Avoidanceof indiscriminate use by ensuring that the indication for a dose of and durationof treatment are appropriate.·        Usingantimicrobial combinations in selected circumstances.

·        Constantmonitoring of resistance patterns in a hospital or community.·        Restrictingcontrol of drug use, which involves an agreement between clinicians and microbiologists,e.g. by limiting the use of the newest member of a group of antimicrobials solong as the currently used drugs are effective.