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

 4

 

 5

 

  M M

 

3  3

 

2  2

 

 

.   

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.