Both now on their way of combating these

Both developed and developing countries are suffering from different forms of water pollution. Developed countries that have water pollution problem due to industrial proliferation and modernized agricultural technologies are now on their way of combating these problems through improved technologies of waste treatment. But developing countries like India, with limited financial resources, lack of technical knowledge and with weak implementation of environmental policies, are still facing severe problems. In most of these countries including India, water in many rivers receives a heavy flux of sewage, industrial effluents, domestic agricultural wastes (Cullen and Reimer, 1989).

Being an extremely heterogeneous species which harbour strains ranging from gastrointestinal commensals to various intestinal and extra-intestinal pathogens, the need to study diversity of waterborne Escherichia coli & K. pneumonia especially, in the urban aquatic environments is of utmost importance, as it is directly related to public health. Further, with ?-lactams representing the most widely and successfully 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 and dissemination of resistance mediated by the expression of ?-lactamases (Bradford et al 2001), it is important to identify the strains’ susceptibility profiles and understand the molecular basis of such resistance. The need to perform such studies with strains isolated from urban aquatic environments have been well-established to serve as an ideal reservoir an disseminator of antibiotic resistant bacteria as well as the resistance genes (Baquero et al . 2008; Zhang et al. 2009; Tacao et al. 2012).

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The major problem of multidrug resistance among infection causing organisms represents one of the greatest challenges worldwide. With increased antimicrobial usage, complexities in the resistance mechanisms have become more advanced. Densely populated centers with improper water supplies and inconsistent sanitation contribute significantly to acquisition and dissemination of resistance determinants among microbial inhabitants of water bodies. The Yamuna River, which originates from the Yamunotri glacier in the lower Himalayas (38°59?N 78°27?E), is the major source of water to urban areas in Delhi. Although the proportion of the river catchment area in Delhi is small (~2%), this area contributes more than 50% of pollutants that it receives through sewage from urban effluents, with high levels of antimicrobials in addition to toxic compounds being discharged by industries (Sharma and Kansal, 2012; Sehgal et al., 2012; Mutiyar and Mittal, 2014). The acquisition and transmission of resistance genes from microflora of human and animal origin discharged as part of sewage can substantially influence the pattern of resistance among the microbial inhabitants of the aquatic ecosystem (Amos et al., 2014).

Increasing incidences of ESBL & PMQR producing bacteria that showed a drastic shift in recent years in environmental settings are of serious concern. The contribution of selection to the acquisition and as such spread of resistance among bacteria against major classes of antibiotics is alarming due to their higher dissemination rate. As such, the high prevalence of ESBL & PMQR producing isolates in natural water bodies like rivers, sewage, canal and ground water has 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 water samples from upper ranges of Ganges River till its tributary Yamuna that stretches in Delhi and beyond.

Recently, Anas et al. (2017) also reported high prevalence of the ESBL among E. coli.

Antimicrobial Resistance Pattern

Shortly after the prescription of antibiotics became widespread in the 1940s, the pathogenic bacteria began to demonstrate resistance towards these drugs. Enhanced selection pressures caused by the inappropriate or careless use of antibiotics tend to increase the prevalence of resistant populations microorganisms;

Plasmid-mediated quinolone resistance mechanisms play an important role in the expansion of quinolone and fluoroquinolone resistance among ESBL & PMQR producing E. coli & K. pneumonia (Du J. et al. 2014). Infections caused by such resistant isolates can be difficult to treat (Varela et al. 2016). Over the past 10 years, PMQR determinants have appeared as an important issue (Rodriguez et al. 2015).

Antibiotics reduce mortality in severe infections. Efficient antibiotics are a prerequisite for modern health care. New antibiotic classes against gram-negative infections are not to be expected in the near future, why the currently available must be used in a rational way, considering the growing challenge from antibiotic resistance. 

Prescribing patterns vary worldwide and mirror the level of antimicrobial resistance. In Europe, high-prescribing countries have a higher prevalence of antimicrobial resistance and vice versa. Greece showed the highest prescription rates of antibiotics in the primary care sector during 2014, 34.0 daily delivered doses (DDD) per 1000 inhabitants and per day. This is more than three times as much as the lowest prescribing country, the Netherlands, with a prescription rate of 10.6 DDD per 1000 inhabitants and per day. In Sweden, the rate was 13.0 DDD per 1000 inhabitants and per day in 2014 (http://ecdc.europa.eu). Increased use of broad-spectrum antibiotics due to the high prevalence of MDR doubtlessly leads to the further selection of resistant strains. The regional differences in prescribing patterns are substantial even within Sweden, where Stockholm Country 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 to 352 per year and 1000 inhabitants (Concise, e-health Authority, www.ehalsomyndigheten.se), still, over-prescription can be further reduced.

In a study, Center for Disease Dynamics, Economics & Policy (CDDEP) reported an increase in the percentage of carbapenems resistance from 10% in 2008 to 13% in 2013 among E. coli and 29% in 2008 to 57% in 2014 in isolates of K. pneumonia from India Center for Disease Dynamics Economics and Policy (CDDEP),. Besides strengthening the existing knowledge of their prevalence, the presence of MDR, XDR, and PDR bacteria in the natural environment endorses them for the potential threat that they possess for the mankind.

Accordingly, investigations of ESBL & PMQR genes (blaTEM and blaCTX-M) & (qnrS & aaclb-cr) among bacteria that harbor both multidrug resistance are thought to provide useful information regarding their epidemiology in human influenced polluted environments.

The present study was undertaken to analyze the diversity of E. coli & K. pneumonia strains isolated from the River Yamuna and other water bodies traversing through the metropolitan city 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, where maximum antibiotic-resistant bacteria has been reported even in drinking water (Mandal et al., 1996). In its entire stretch through the city, the river Yamuna receives 950 million gallons of sewage per day making it one of the most polluted river in the world. It, therefore, represents an ideal ecological niche for conducting studies on diversity of E. coli & K. pneumonia and resistance to clinically important antibiotics. In addition to this, this study also aimed for the determination of homology or divergence in antibiotic resistance genes among the selected strains.

Thus, a total of 180 strains comprising E. coli (96) & K. pneumonia (84) strains, were isolated from water samples collected along different points of river Yamuna & other water bodies traversing through Delhi.

All the selected strains of wild-type E. coli & K. pneumonia strains were further confirmed by biochemical test and the results of all these tests were similar for all the strains.

Further studies were carried out using a collection of 90 {E. coli (48) & K. pneumonia (42)} strains selected from a total 180 antimicrobial resistant environmental E. coli (96) & K. pneumoniae (84) strains. The identification of these strains were further confirmed by the gold standard method of sequencing of their 16S ribosomal RNA (rRNA) genes.

           Antimicrobial susceptibility test:

Entire analysis of collected samples showed the resistance against following antibiotics: In the present study, all of the isolated 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%).

High levels of resistance to 7 to 8 antimicrobial agents were observed in the presence of multidrug-resistant ESBL & PMQR-producing E. coli & K. pneumoniae, while on the other hand, the resistance level was low for Amikacin and Meropenem.

Of the 90 E. coli (48) & K. pneumonia (42) isolates that were positive in the screening test, ESBL production was confirmed by PDCT in 76 (84.3%) isolates, which included 46 (95.83 %)  E. coli and 30 (71.42 %)  K. pneumoniae, indicating that the prevalence of ESBL 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 India SENTRY surveillance, the prevalence of ESBL production was reported to be 84% (Manoharan et al. (2011). 

These results clearly indicate that the incidence of antimicrobial resistance was widespread and probably resulted from either the intensive use of antibiotics or the uncontrolled availability of them. However multiple drug resistance in enteric organisms like E. coli & K. pneumoniae is also known to be associated 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 were integrated. Globally disseminated Tn21 like transposons which carry class1 integron (typically conjugated plasmids) might also account for the high incidence of antibiotic resistance among commensal, environmental and clinical bacterial isolates (Zuhlsdorf and Wiedmann, 1992).

Subsequently, considering that the co-existence of ?-lactamase mediated resistance and resistance to quinolones in the same strains represents a major health hazard, the study investigated the molecular mechanisms conferring quinolone co-resistance to the ESBL producing E. coli & K. pneumonia strains 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 resistance genetic elements do not reside on the same genomic platforms as the plasmid-mediated genes for ESBLs, the co-expression of resistance to both quinolones and  ?-lactam classes of antibiotics in the waterborne E. coli & K. pneumonia consistently being subjected to anthropogenic exposures through river waters & other water bodies represents serious public health concerns which, if manifested in the human infections would be extremely difficult to treat with the limited choice of remaining therapeutics. It is known that protein play an important role in regulation of the biofilm phenotype ((Oosthuizen et al., 2002). Different classes of extracellular proteins have been described as part of an adaptive response to a change in the environment (Tjalsma et al., 2000).

This active drug transport is involved in low intrinsic susceptibility, cross-resistance to chemically unrelated classes of molecules, and selection/acquisition of additional mechanisms of resistance.

We have also attempted to detect the presence of differential expression of total proteins in the strains of E. coli & K. pneumonia using SDS-PAGE.

Differences were observed in the expressed proteins but the identity of the proteins was not investigated further in this study. The changes in the protein expression may probably play an important role to resensitize bacteria towards antibiotics (Chaudhary and Payasi, 2012).

In our study, more than 93% of ESBL Gram negative isolates were observed to harbor blaTEM, blaCTX-M. These variants were showing similarity to those reported by Wattal et al. (2010); and Rastogi et al. (2010), during their studies on ESBL production among clinical isolates (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

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The later, in fact, was first reported from the Indian isolates (Karim et al. 2001), and since then has been reported to encode the most widely disseminated CTX-M ESBL (Poirel et al. 2002b). It is in concordance with the previous reports that documented the possible emergence of blaCTX-M genes from Kluyvera sp. (Sarria et al., 2001; Humeniuk et al., 2002; Bonnet et al, 2004; Munday et al., 2004; Mc Gettigan et al., 2009; Zhang et al., 2009).

Continuous threat posed by resistant organisms to human health has necessitated the need for further studies to improve understanding of their resistance mechanisms.

As shown in the present study, a greater contribution of the substitutions in chromosomal QRDRs than the presence of PMQR genes has been reported earlier in conferring high levels of quinolone resistance 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 in E. 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 supported by previous studies demonstrating the frequent occurrences of these genes in environmental isolates (Zhang et al. 2012).

The qnrS-type genes seem to be the most commonly identified acquired qnr genes in the environment (Cattoir et al. 2012). They have been mainly identified from waterborne species and in particular Aeromonas spp. (Picao et al. 2015). Aac (60)Ib-cr is widespread geographically and stable over time (Varela et al. 2015). It has often been more common than qnr alleles.

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 in the development and spread of antibiotic resistance.

In this study, the prevalence of the genes aac(60)-Ib-cr and qnrS was significantly higher in samples of Yamuna water samples than other water samples. However, there was no significant difference between Yamuna water samples and other water samples.

Moreover, qnr genes have been found to exist in many extended-spectrum beta-lactamase producing Enterobacteriaceae (Galvin et al. 2011), suggesting their co-selection with other resistance elements.

A resistance gene reservoir not only allows resistant genes to stably exist, but also facilitates transfer of these genes to other species in a natural state. Hence, blaTEM and blaCTX-M type ESBL genes were the most common genotypes in this study.

Besides the chromosomally-encoded mechanisms of the E. coli & K. pneumonia strains also harbored the plasmid-mediated quinolone resistance genes viz. qnrS and aac(6′)-lb-cr, respectively. Additionally, the qnrS gene was also observed in the remaining ESBL producing E. coli & K. pneumonia strains which carried the 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 did not contribute to high resistance in majority of the strains, their presence nevertheless deserves a notable mention. This is especially, because presence of the PMQR genes in E. coli & K. pneumonia has been reported to provide a very favourable background for the selection and enrichment of chromosomally-mediated (fluro) quinolones resistance (Poirel et al. 2006; Rodriguez-Martinez et al. 2007). The strains carrying PMQR genes may therefore serve as an important reservoir for the eventual increase and spread of resistance to quinolones. 

From these data, it is clear that the potential impact of E. coli & K. pneumoniae infections on humans is significant. Prevalence of resistance determinants for the front line choice of drugs threatens the future continued clinical use of antibiotics. Therefore, the breadth of resistome, monotony of resistance mechanisms, evolution and emergence of antibiotic resistant populations needs to be clearly understood to allow the discovery of new therapeutic drugs. In cases where such phenotypic resistance was not significantly observed, the ability conferred on the strains carrying ESBL & PMQR genes to serve as a crucial reservoir for the accumulation of potential resistance-associated mutations and their efficient spread cannot be under-mined. This again highlights the need to truly appreciate the potential of aquatic environments in emergence and spread of antibiotic resistance, and underscores the need for routine surveillance and measures to contain their spread.

               Prevention And Control

For better of management of Infection health care workers (both hospital and community based) should undertake practices known to reduce the spread of ESBLs.

These fall into two broad groups

            1) Good hand hygiene and cleanliness

2) A restrictive approach to antibiotic prescribing, especially in the limitation of third                 

    generation cephalosporin and quinolone use.

a)      These simple interventions can have a major influence on the impact of ESBLs in health care setting.

b)      Appropriate use of antibiotics will greatly reduce the selection pressure for colonization and infection with ESBLs:

Antibiotics must be prescribed according to the Antimicrobial Policy and detailed Antimicrobial Prescribing Guidelines.

Where there is more than one case on the ward, the prescriber should consider avoiding cephalosporin use altogether in other patients on the ward.

In an outbreak situation, the Infection Control Doctor (ICD), a Consultant Medical Microbiologist and the Antimicrobial Pharmacist will suggest interim alternative antibiotic prescribing guidelines on a ward /unit.

Limitations of resistance to antimicrobials may be achieved by:

·         Avoidance of indiscriminate use by ensuring that the indication for a dose of and duration of treatment are appropriate.

·         Using antimicrobial combinations in selected circumstances.

·         Constant monitoring of resistance patterns in a hospital or community.

·         Restricting control 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 so long as the currently used drugs are effective.

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