Journal of Environmental Chemical Engineering 8 (2020) 102088

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Journal of Environmental Chemical Engineering

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Sewage mediated transfer of antibiotic resistance to River Yamuna in Delhi, T

India
Manisha Lamba, T.R. Sreekrishnan, Shaikh Ziauddin Ahammad*
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, India

ARTICLE INFO ABSTRACT

Keywords: Increase in antibiotic resistance (AR) in environment is a global health concern as the emergence of AR is
Drains limiting the treatment options. Inappropriate and indiscriminate uses of antibiotics are known causes for the
River yamuna spread of AR but lack of access to safe drinking water and poor sanitation aggravate the problem. The present
Antibiotic resistance study aims to estimate the abundance of coliforms, carbapenem and ESBL resistant bacteria and respective
β-lactam
resistant genes in 20 sewer drains and 5 locations of the river Yamuna across New Delhi over two seasons.
Proliferation
Significant positive correlations were observed between FC levels and carbapenem (R = 0.90,p = 0.002,n = 40)
and ESBL resistant bacteria (R = 0.93,p = 0.005,n = 40) levels across all the samples, indicating possible as-
sociation of resistant bacteria with faecal matter. However, higher abundances of coliforms, resistant bacteria
and genes were observed in winter relative to summer season. Poor/lack of connectivity between drains and
sewage treatment plants (STPs) and thus discharge of treated/untreated sewage at different locations of the river
Yamuna is the major reason for increasing the pollution load of the river. Microbial culturing indicated presence
of “critical pathogen” listed by WHO in urgent need of new antibiotics in the collected samples. Overall, release
of untreated sewage into the river Yamuna carried by the sewer drains poses a high risk to the people residing in
the city and highlights an urgent need to treat the entire sewage. Complete treatment of the entire sewage
generated using treatment techniques such as advanced oxidation processes (80–90%) could reduce the in-
troduction of these emerging pollutants in the environment.

1. Introduction
Antibiotics are extensively used to prevent or to treat bacterial in-
fections in aquaculture, humans, and animals. Antibiotics consumed by
humans and animals are not always metabolized completely and are
thus discharged into the environment along with their excreta [1].
Presence of these residual antibiotics in the environment creates se-
lection pressure and increases the chance of proliferation of antibiotic
resistant bacteria (ARB). Most often, the antibiotic resistant genes
(ARGs) present in the antibiotic resistant bacteria (ARB) are associated
with integrons and transposons present in the plasmids which further
increases the risk of spread of antibiotic resistance (AR) mediated by
horizontal gene transfer [2]. The rapid proliferation of resistance
among pathogenic and non-pathogenic bacteria results in increased
abundance of drug-resistant bacteria and subsequent failure of treat-
ment options [3]. AR also leads to prolonged illness and increased
mortality. The pace of discovery of new antibiotics is not fast enough to
tackle the problem of proliferation and emergence of new AR in the
environment [4]. Prevalence of AR in the environment has a major
impact on human health. In 2013, only in USA 20,49,442 illness cases
and 23,000 deaths have been reported due to antibiotic resistance [4].
Dissemination of AR in the environment includes soils, water
bodies, plants, animals and human beings. Among these, water bodies
such as rivers and lakes play a significant role in the proliferation of
ARB and ARGs in the environment. The ARB and ARGs from its dif-
ferent sources such as discharges from hospitals, industries and
household, run-off from agricultural sites and discharges from livestock
farming are either released into the drains or the wastewater treatment
plants (WWTPs). Finally, it flows into the river as either direct dis-
charge from the drains or after getting treated in the WWTPs. Most of
the existing WWTPs are designed for removing only conventional pol-
lutants such as organic carbon, nitrogen and phosphorous from was-
tewater using biological, chemical and physicochemical treatment op-
tions. As the treatment plants are not designed for treating emerging
hazards like antibiotic resistance, most often these contaminants flow
through the treatment systems without getting any treatment. The final
effluent discharged by the treatment plants carry these emerging ha-
zards and contaminates the water bodies. In fact, WWTPs are

⁎
Corresponding author.
E-mail address: zia@iitd.ac.in (S.Z. Ahammad).

https://doi.org/10.1016/j.jece.2017.12.041
Received 3 July 2017; Received in revised form 14 December 2017; Accepted 18 December 2017
Available online 20 December 2017
2213-3437/ © 2017 Elsevier Ltd. All rights reserved.
M. Lamba, et al.
considered as the reservoir for the proliferation of AR [5–7]. The pre-
sence of diverse bacterial population and high nutrient content creates
a suitable environment for the spread of resistance in the treatment
plants [5]. Thus, the treated and untreated discharge from different
drains and WWTPs into the river increases the abundance of these
pollutants in the river.
AR have been reported in bacteria found in surface water [8],
marine water and estuaries [9,10]. Significant positive correlation be-
tween resistant bacteria in rivers and urban effluent discharge into the
river have been reported [11]. In another study, human and agri-
cultural activities were shown to be the primary sources for the in-
creased level of ARGs in the river sediments [12]. ARB have been found
in the seepage and tap water samples from New Delhi [13]. Strains
carrying New Delhi metallo-β-lactamase (blaNDM-1) were also detected
in the river Ganges and Yamuna [14]. A group of researchers have
documented the presence of 24 ARGs and their active expression
against eight classes of antibiotics including β-lactams, tetracycline,
trimethoprim, glycopeptides, sulfonamides, aminoglycosides, macro-
lides, and chloramphenicols in surface water samples [15]. Researchers
have reported the presence of tet(W) and tet(O) in treated drinking
water and recycled wastewater [16]. A recent study detected thirteen
ARGs, three antibiotics, six heavy metals, and seventeen other organic
pollutants at a few sites along the River Almendares [17].
The river is considered as one of the main repositories for these
contaminants, as it receives treated/untreated discharges from various
sources. Discharges containing ARB and ARGs contaminate the river
water. Since rivers are also one of the primary sources of water, such
contamination impacts the river water quality and the health of their
users. A report by Central Pollution Control Board (CPCB), a statutory
organization established to maintain water quality standards at a na-
tional level in India, reported that the estimated sewage generation in
Delhi is 3800 million liters per day (MLD) and the installed treatment
capacity is 2330 MLD. But approximately 1470 MLD (∼60%) of the
installed capacity is actually used for treatment, thus large amounts of
untreated sewage are being discharged into the river [18,19]. Only 40%
of the wastewater (sewage) generated in Delhi is treated whereas the
remaining 60% is discharged directly into the river without any treat-
ment at different locations in the river [18,19]. This data indicates a
possibility of getting huge AR contributed by the sewage to the river.
Therefore, efforts were made to estimate the abundance of ARB and
ARGs in major sewer drains in Delhi and to access their contribution in
polluting the river Yamuna. The abundance of integrons associated with
the proliferation of AR was also estimated. Efforts were made to iden-
tify different sources of pollution responsible for the spread of re-
sistance in the drains. β-lactam antibiotics are the most commonly used
antibiotics in India [20]. Hence, ARB and ARGs resistant to two im-
portant representatives (ESBL and carbapenem) of the β-lactam class of
antibiotics were selected for the present work. Understanding the
causes/sources of ARB and ARGs proliferation in the aquatic environ-
ment is essential for developing appropriate mitigation strategies for
treating these pollutants. Establishing a link between the source of
pollution and the abundance of ARB/ARGs will help in developing ef-
fective mitigation strategies to reduce the burden of ARB/ARGs in the
environment.
2. Materials and methods
2.1. Study sites and sample collection
Samples were collected from 20 major sewer drains and five pro-
minent sites in river Yamuna in New Delhi (Fig. S1). Drains were chosen
based upon the design discharges (a range of 10–300 m3/s was chosen)
and locations of the drains. The reason for choosing the location as one
of the factors was to study the contribution by the residents/users of
different areas such as residential areas, unauthorized settlements, in-
dustrial areas and agricultural areas to the spread of resistance. Details
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Journal of Environmental Chemical Engineering 8 (2020) 102088
of the drains studied are given in Table S1. Samples collected from
major drains in Delhi include D1, D2, D3, D4, D5 and D6 covering
South West Delhi; D7 from West Delhi, D8, D9, D10, D11 and D12
covering North Delhi; D13, D14, D15, D16, D17 and D18 covering
North East and East Delhi; D19 and D20 from South Delhi. Out of all the
20 drains only 5 major drains including D1 (283 m3/s, 57000 m), D9
(19 m3/s, 20000 m), D13 (157 m3/s, 6000m), D19 (71 m3/s, 2800 m)
and D20 (59 m3/s, 1300 m) discharge their contents directly into the
river Yamuna.
The Yamuna (also called as Jamuna) is the largest tributary of
Ganga in the Northern India. The river Yamuna covers a stretch of
22 km from Wazirabad to Kalindi Kunj in Delhi. Five sites were selected
at almost equidistant locations to cover the entire 22 km stretch of the
river in Delhi. The five sampling sites are Y1, Wazirabad
(28∘76′83.74″N 77∘24′10.47″E), Y2, ITO (28∘62′77.88″N
77∘25′33.03″E), Y3, Nizamuddin (28∘60′10.28”N 77∘26′34.85”E), Y4,
Ohkla (28∘56′78.69″N 77∘29′48.53″E), and Y5, Kalindi Kunj
(28∘54′39.21″N 77∘31′33.93″E). Replicates of water samples (YW) were
collected from all sampling sites of drains and river. Sediment samples
(YS) were also collected from all the five sites of the river. All samples
were collected in sterile 500-mL containers, transported to the labora-
tory under refrigeration and stored at +4 °C for further analysis.
Microbial analysis was performed within 12 h of sampling and DNA
extraction was performed within 24 h of sampling. Samples were col-
lected in both summer and winter seasons to check the seasonal var-
iation of antibiotic resistance abundance. Temperature, pH, total dis-
solved solids (TDS) and conductivity were measured in-situ using a
hand-held multi-parameter water quality meter (Hanna Instruments,
USA). Water quality data obtained from various locations are given in
Table S2.
2.2. Microbial analysis
Faecal Coliform (FC), Total Coliform (TC), ESBL resistant bacteria
(ESBL) and carbapenem resistant bacteria (KPC) plate counts were
performed using selective media obtained from Himedia (India). All the
samples were serially diluted in sterile phosphate buffer solution (PBS)
and plated in triplicates on Rapid HiColiform Agar (TC), HiCrome KPC
Agar (carbapenem resistant bacteria), HiCrome ESBL Agar (ESBL re-
sistant bacteria), and incubated at 37 °C for 24 h. FC plate counts were
performed using M-FC Agar (FC) and the plates were incubated at 45 °C
for 24 h. Bacteria resistant to ESBL and carbapenem (KPC) were isolated
from the respective plates based on their morphology. From each
HiCrome ESBL and KPC agar plates, 40–50 strains were isolated and
identified. DNA from the isolated strains was extracted using Fast DNA
Spin kit for soil (MP Biomedicals, India) according to manufacturer’s
instructions and stored at −20 °C for further analysis. To identify the
bacterial strains, DNA from the isolates were amplified using bacterial
338F and 1046R primers following the program mentioned in Table S3.
The conserved 16S rRNA gene sequences were analyzed using Sanger
sequencing and BLAST search was done to know the possible identity of
these strains. The identity of the strains was also verified using
Biochemical Test Kits (Himedia, India) as per manufacturer’s instruc-
tions.
2.3. DNA extraction and quantification of ARGs using quantitative
polymerase chain reaction (qPCR)
DNA was extracted from all the water and sediment samples using
the FastDNA Spin kit for soil (MP Biomedicals, USA) according to
manufacturer’s instructions. Before DNA extraction, the samples were
either filtered through sterile 0.22 μm membrane disc filters (Pall Life
Science, USA) or pelleted (centrifugation at 13,000 rpm for 30 mins)
and the concentrated samples were used for DNA extraction. The con-
centration of the extracted DNA was measured using spectrophotometer
(NanoDrop 2000c; Thermo Fisher Scientific, USA). The extracted DNA
M. Lamba, et al.
Fig. 1. Abundance of TC, FC, ESBL and KPC resistant bacteria in the water samples collected from the outfall of major drains and the river Yamuna in Delhi in summer and winter seasons.
(YW- water samples of the river Yamuna).
was stored at −20 °C for further analysis. The DNA was diluted (1:100)
using nuclease free water to obtain inhibition free qPCR analysis and.
The diluted DNA was used to quantify the key genes (ARGs and in-
tegron genes) using qPCR. Specific genes targeted were 16S-rRNA gene,
β-lactamases genes (blaTEM, blaOXA, blaCTX) and carbapenemase (blaNDM-
1) class 1 integron (int1), class 2 integron (int2) and class 3 integron
(int3). All genes were quantified in triplicates using a BioRad CFX
C1000 System (BioRad, Hercules, CA USA). All the reactions were run
with serially diluted DNA standards and DNA free negative controls.
Correlation coefficients for the calibration curves were > 0.99 and all
the log gene abundance values were in the linear range of detection.
The program and the reaction conditions used for quantifying different
genes are mentioned in Table S3.
2.4. Treatment technology
Samples collected from different drains were treated using activated
sludge process (ASP), ozonation and a combination of ASP and ozo-
nation to estimate the efficiencies of these treatment technologies in
reducing the load of ARB and ARGs. All the reactors (ASP, ozonations,
and ASP followed by ozonation) were run for 20 HRTs.
2.4.1. Activated sludge process (ASP)
Aerobic biological treatment process using Activated Sludge Process
(ASP) is commonly used for the treatment of sewage. ASP is a system in
which sewage from the primary clarifier is aerated to support the
growth of microbes that ultimately consume the organics present in the
sewage. The treated effluent from the aeration basin goes to the sec-
ondary clarifier for clarification, and the clarified effluent is finally
discharged. Part of the sludge from the secondary clarifier is recycled
back to the aeration basin, and a part of it is wasted. The volume of the
aeration tank is 4 liters (working volume 3.5 L) and that of the clarifier
is 5 liters. The residual dissolved oxygen (DO) in the aeration basin was
1.5 mg/L and pH was 6.9. The solid retention time of 11 days and hy-
draulic retention time of 8 hrs was maintained in the aeration tank. The
aeration tank was initially inoculated with aerobic sludge collected
from the return activated sludge line (RAS) from Mehrauli sewage
treatment plant, Delhi. The reactor was run in the batch reactor for one
week after which it was run in continuous mode.
2.4.2. Ozone treatment unit
Ozone was generated using ‘OZ-AIR® ISM 10′ ozone generator
3
Journal of Environmental Chemical Engineering 8 (2020) 102088
supplied by Creative Oz-Air (I) Pvt. Ltd (India). Different concentration
of ozone and time of exposure were tested to estimate the optimized
exposure time required for complete removal of the microbes. The
optimized concentration of ozone for complete removal of microbes
was estimated as 90 ppm with a retention time of 20 min.
2.5. Data analysis
All the data analysis was done using Excel 2007 (Microsoft Office
2007, Microsoft Corp., USA) and SPSS ver. 19.0 (IBM Inc, USA). All the
data was log-transformed before the analysis and the statistical sig-
nificance was defined by 95% confidence intervals (p < .05). The bi-
variate correlation analysis was performed using the Spearman’s non-
parametric methods to obtain correlation coefficients (r) and p-values.
3. Results and discussion
3.1. Abundance of coliforms (total and faecal) in drains and river Yamuna
Coliforms are commonly used as an indicator of water contamina-
tion and possible presence of pathogenic microbes. Faecal coliforms
particularly originate from the intestinal tract of warm-blooded animals
and are mostly associated with sewage (faecal matter). Most of the
drains receive household waste, agricultural wastes, hospital wastes but
the substantial part of it is sewage. The presence of coliforms was es-
timated in the samples collected from the drains and the water and
sediment samples collected from the river Yamuna (Fig. 1). The abun-
dance of faecal coliform was almost similar in the samples collected
from all the five major drains directly discharging their contents into
the river Yamuna which indicates the contamination of river Yamuna
with the sewage discharges. But, the abundance of total coliforms was
higher in the samples collected from drains D1 and D13 compared to
the samples collected from D9, D19 and D20. However, the presence of
total coliform is not the best indicator of faecal contamination since
total coliform estimation can sometimes show high counts due to pre-
sence of coliform bacteria in soil and water [21]. The faecal coliform
was absent in the water sample collected from first site of Yamuna river
(Y1) which is due to lack of sewage contamination in this pristine site.
However faecal coliform bacteria was present in the samples collected
from all other sites of river Yamuna (Y2-Y5) with water samples from
Y2 showing the highest abundance. The river Yamuna does not have
substantial flow when it flows through the city as most of the water
M. Lamba, et al.
(∼1100 MLD) is withdrawn from the river for treatment in a drinking
water treatment plant situated before Wazirabad barrage (before Y2)
where the river enters the city [22]. The lack of water in the river after
the Wazirabad barrage is mostly compensated by the discharges from
different drains. The presence of faecal coliform in the samples col-
lected from sites Y2 to Y5 and its absence in the sample collected from
Y1 signifies the discharge of sewage contaminated water in the river
Yamuna through various drains.
A similar scenario was observed in summer and winter seasons with
a higher abundance of coliforms in the winter season compared to
summer. This is possibly because of lower per capita water consump-
tion in winter compared to summer which resulted in less diluted
sewage in all the drains in winter. Higher total solid content (3 fold) of
the sewage in winter also indicates the same. The river Yamuna is a
perennial river and it gets water from Yamunotri glacier. The volu-
metric flow rate of river Yamuna is higher (almost double) in summer
season than winter due to higher ice melt. In winters, the water in the
river Yamuna (river stretch within Delhi city) is mostly composed of
undiluted sewage discharged by the drains. Lower water flow rate in
winter results in lower dilution of pollutants which could be a possible
reason for getting higher coliform abundance in the river Yamuna in
winter [14].
3.2. Abundance of ESBL and carbapenem resistant bacteria in drains and
river yamuna
Humans and animals use antibiotics and as a result, they develop
resistant bacteria in their guts. There is a high chance of getting residual
antibiotics, ARB and ARGs in the sewage through the excreta of the
humans and animals consuming antibiotics. Abundances of ESBL and
carbapenem resistant bacteria were estimated in the collected samples.
Two prominent representatives of the β-lactam group of antibiotics
were chosen for the present study as β-lactams are the highest con-
sumed drugs in India [20]. Higher abundances of ARB were observed in
D1, D13 and D20 as compared to D9 and D19 in both the seasons
(Fig. 1). Among all the three (D1, D13, and D20) major drains, D20 is
the smallest contributor with respect to its flow and the wastewater in
D20 is mostly composed of domestic sewage from posh residential
areas. The high abundance of ESBL and carbapenem resistant bacteria
in this drain is possibly due to high anthropogenic activities and greater
usage of advanced antibiotics in such posh areas. Previous studies have
also demonstrated that the use of advanced antibiotics, antibiotic
misuse and self-medication is more in urban areas compared to the
rural areas. Improper use of antibiotics for an inadequate period of time
can exert selection pressure and lead to the proliferation of resistance
[23]. Studies conducted in Nigeria and Nepal have also reported that
people residing in posh areas have a high tendency to harbour resistant
bacteria compared to the people living in rural or economically-de-
prived areas [24]. The higher abundances of ARB observed in D13 are
possibly because of the discharge of 5 other drains (D14, D15, D16,
D17, and D18) into D13. D13 and its five other contributing drains
mostly receive waters from domestic sewage, small industries and slum
areas. One of the contributing drains (D14) receives wastewater from
nearby fruit and fish markets and cancer hospital. Usually, strong an-
tibiotics are used in such clinical settings and effluent from such clinical
settings may influence development and proliferation of ARB. Hospital
effluents are considered as one of the major promoters for the pro-
liferation of resistance [25,26]. Presence of various ARB and ARGs have
been reported in the hospital effluents [27]. Another contributing drain
(D16) to D13 receives wastewater from cattle farms, nearby villages
and textile dyeing units. High antibiotic usage in cattle farms is a well-
known practice worldwide [28]. Discharges from the cancer hospital,
cattle farms and domestic sewage in D13 resulted in high abundance of
ARB in samples collected from D13.
D1 is considered as the major contributing drain to pollute river
Yamuna [22]. D1 receives wastewater from residential complexes,
4
Journal of Environmental Chemical Engineering 8 (2020) 102088
small houses, slums and economically deprived areas, small-scale in-
dustries, villages and agricultural areas. D1 is impacted by different
sources such as residential area and industries and is influenced by both
city area and village throughout its flow from south-west Delhi to the
point it meets the river Yamuna. As the wastewater in the drain is
primarily composed of sewage, higher abundance of coliforms and ARB
were observed in the samples collected from this drain. Sewage is a
potential contributor of residual antibiotics [29], ARB [30] and ARGs
[31] to the environment.
D9 receives contributions from D10 and D11 before discharging its
contents into the river Yamuna. The drains receive domestic sewage
from the village. D19 also receives domestic sewage from rural and
semi-urban localities. The possibility of use of expensive/advanced
antibiotics by people living in such rural areas is less as compared to the
people residing in the posh areas. Lack of medical treatment facility is
very common in rural areas and people residing in these areas may not
get appropriate medications. Therefore the use of expensive drugs/an-
tibiotics is not very common in such rural areas. Use of less potent
antibiotics and incomplete dosage of medicines can contribute towards
the spread of resistance towards less potent antibiotics by creating se-
lection pressure for the bacterial population [32]. Additionally, in
contrast to the posh areas, economically deprived areas such as villages
are not organized in terms of sewage collection and treatment due to
the lack of infrastructural development. Presence of different pollutants
in the untreated waste creates stress to the microbial population present
in the waste stream and further enhances the proliferation of ARB.
Samples collected from river Yamuna also showed higher abun-
dances of ARB (Fig. 1). In site Y1, the abundance of ESBL resistant
bacteria in the water samples was almost negligible but significant
abundances of carbapenem resistant bacteria were observed in the
summer season. Whereas considerable levels of ESBL resistant bacteria
along with the increased levels of carbapenem resistant bacteria were
observed in winter season relative to summer in the water samples. Site
Y2 shows the highest abundance of ARB in both the seasons. This site
receives discharge of hospital wastes, sewage, discharges from public
office places and major bus station near the site. The composite was-
tewater received (primarily from hospitals) in this site resulted in in-
creased ARB and coliform abundances in the samples collected at Y2.
Moreover, D1 discharges its content before this site, and hence higher
abundance of ARB were expected in the water sample obtained from
this site. D1 is considered as the major drain for increasing the pollution
load to river Yamuna. D1 carries a large amount of raw sewage and
treated sewage from various STPs to the river Yamuna. The discharge of
D1 in river Yamuna is approximately 2000 MLD (million liters per day)
and the BOD load contributed by D1 in the river varies from 80 t/d to
195 t/d. D1 alone is responsible for contributing 50% of the hydraulic
load and 25% of the organic load to the river Yamuna in Delhi [33]. In
site Y2, the abundance of ARB increased by almost 10 folds in winter.
Samples collected from site Y3, Nizamuddin, also showed high abun-
dance of resistant bacteria which may be contributed by the waste
streams from a crematorium near the site. Discharge of untreated
wastewater from the local population and railway station located near
this site are major factors to increase the abundance of ARB in Y3.
Location near site Y4, Okhla is mostly an industrial hub and it receives
discharge from D13 drain. The high abundance of coliform and ARB
observed in site Y4 is a direct contribution of the discharge from D13
which mostly contains discharge from hospitals, cattle farms and do-
mestic sewage. Site Y5, Kalindi Kunj, represents the exit point of river
Yamuna from Delhi. Discharge of wastewater from D19 and D20 drain
before site Y5 resulted in the high abundance of ARB and coliform in
the water sample collected from this site.
In all the collected samples, the abundances of ARB were higher in
the winter season compared to the summer season. Generally, usage of
antibiotics is more in winters compared to summer [20,34]. Higher use
of antibiotics in winter season relative to summers is a crucial factor for
getting higher ARB abundances in sewage in winter. Higher use of
M. Lamba, et al. Journal of Environmental Chemical Engineering 8 (2020) 102088

Fig. 2. Abundance of TC, FC, ARB (ESBL and KPC) and relative abundances of ARGs (blaNDM-1/16S rRNA, blaCTX/16S rRNA, blaOXA/16S rRNA, blaTEM/16S rRNA) in the sediment samples
collected from river Yamuna in both the seasons. (YS- sediment samples of the river Yamuna).

antibiotics results in the discharge of more residual antibiotics to the
environment. Increased stress caused by the residual antibiotics and
subsequently elevated abundance of ARB in the environment is ob-
served elsewhere [35]. Furthermore, the increased abundance of ARB
probably resulted from lower flow rates and lower dilution of sewage in
winter [14].
The abundance of ARB in the sediments was higher than the
abundance of ARB in water samples collected from all the five sites
which is similar to the result shown elsewhere [36] (Fig. 2). Further, in
sediment samples, no substantial difference in the abundance of coli-
forms and ARB was observed in winter relative to summer. From the
data, it can be inferred that partially treated and untreated discharge
from the drains has increased the pollution load to the river Yamuna
substantially.
Resistant strains isolated from antibiotics containing plates were
identified using 16S rRNA sequence analysis (Sanger shotgun sequen-
cing) and biochemical test kits. Resistant strains isolated from drains
include gram-negative bacteria such as Escherichia spp., Klebsiella spp.,
Aeromonas spp., Acinetobacter spp., Pseudomonas spp. and Enterobacter
spp. Shigella spp. from HiCrome ESBL agar plates and Escherichia spp.,
Shigella spp., Klebsiella spp., Enterobacter spp., Pseudomonas spp. and
Acinetobacter spp. from carbapenem containing plate. ESBL resistant
strains isolated from river water include Escherichia spp., Aeromonas
spp., Pseudomonas spp. and Enterobacter spp. Except for Aeromonas spp.,
all other ESBL resistant isolates were found in sediment samples as well.
Carbapenem resistant strains isolated from river water include
Escherichia spp., Klebsiella spp., and Pseudomonas spp. Additionally,
Aeromonas spp. and Acinetobacter spp. were isolated from sediment
samples. Most of these pathogens are on the WHO “priority pathogen”
list in urgent need of new antibiotics [37].
3.3. Sewage – the possible contributor of ARB
Drains receive discharges from various nonpoint sources along with
the sewage. To estimate the linkage/contribution obtained from
sewage, the correlation was performed between the indicator organism
(FC) and ARB. Significant correlations between carbapenem resistant
bacteria and FC were observed in the samples obtained from drains
(R = 0.90, p = 0.001, n = 20) and Yamuna river (R = 0.92, p = 0.004,
n = 20). Similar correlations were also found between ESBL resistant
bacteria and FC in the drain (R = 0.92, p = 0.009, n = 20) and river
samples (R = 0.96, p = 0.002, n = 20). This observation indicates a
possible association of ARB with FC which is generally present in the
faecal matter. Thus, sewage can be considered as the major contributor
of ARB in the environment. The possible association of ARB with the
faecal matter highlights the need for proper treatment of sewage gen-
erated in the city before its disposal into the river.
3.4. Estimation of ARGs, integrons and 16S rRNA gene
Along with ARB, 16S rRNA gene, ARGs including blaNDM-1, blaCTX,
blaOXA and blaTEM as well as int1, int2 and int3 genes were quantified
using qPCR. High levels of all the ARGs were observed in samples
collected from all the drains. In most of the drains, the levels of blaCTX
and blaTEM genes were higher as compared to the levels of blaNDM-1 and
blaOXA genes in both the seasons, indicating that there is a larger influx
of blaCTX and blaTEM genes compared to blaNDM-1 and blaOXA genes
(Fig. 3). Contrary to the results of ARB where a significant difference in
the abundance of ARB was observed among different drains, a minimal
difference of ARGs abundance was observed among all the drains. This
situation of reduced ARB and increased ARGs raises the possible chance
of creating multi-drug resistance among the ARB. The abundance of 16S
rRNA gene was higher in D1 and D20 in summer season compared to
other drains. In the winter season, the abundance of 16S rRNA gene in
Najafgarh drain increased substantially (∼ 1.5 folds) whereas increase
in the abundance of 16S rRNA gene was less for all other drains. This is
possibly because of the discharge of wastewater from various sources
into D1 relative to other drains which increases the microbial load in
the sample collected from D1 compared to other drains. Moreover, D1
is the largest drain in terms of flow rate and its length. The discharge of
a large amount of waste primarily consisting of sewage in D1 results in
high microbial load and thus higher abundance of 16S rRNA gene.
The high abundance of ARGs was also observed in both the water
and sediment samples collected from the five sites of the river Yamuna.
Among all the ARGs, blaCTX and blaTEM genes also showed higher
abundance compared to blaNDM-1 and blaOXA genes in the water and
sediment samples collected from all the five sites of river Yamuna in
both the seasons. The higher abundances of blaCTX and blaTEM genes are
contributed by the drains as the water flows from drains into the
mainstream river. The abundance of ARGs was higher in the sediments
as compared to water samples suggesting that river sediment is an
important ARGs reservoir. Moreover, the river does not have

5
M. Lamba, et al.
Fig. 3. Relative abundance of different ARGs (blaNDM-1/16S rRNA, blaCTX/16S rRNA, blaOXA/16S rRNA, blaTEM/16S rRNA) in the water samples collected from the outfall of major drains
and river Yamuna in Delhi. (YW- water samples of the river Yamuna).
substantial flow when it flows through the city as most of the water is
withdrawn from the river. The lack of water in the river after the
Wazirabad barrage is mostly compensated by the discharges from dif-
ferent drains. Lack of flow of the river is also a major reason for de-
position of solids on the river bed due to sedimentation of solids present
in the wastewater discharged from the drains. This could be another
reason for getting enhanced abundance of ARB and ARGs in the river
sediment.
To understand the relative abundance of ARGs within the bacterial
population, ARG abundance data (blaNDM-1, blaCTX, blaOXA and blaTEM
genes copy numbers) was normalized with respect to 16S rRNA gene
levels (Fig. 3). This normalization provides a comparison of ARGs as a
proportion of total bacterial population. The data indicated greater
carriage of ARGs by the microbes in the summer season compared to
winter season. Though the absolute abundance data showed higher
abundances of ARGs in the winter season compared to the summer
season (Fig. S2) the normalized data (Fig. 3) showed higher tendency of
the bacteria to harbour more ARGs in the summer season compared to
the winter season.
High levels of integrons were observed in the samples collected from
the drains. Class 1 integron showed the highest abundance followed by
class 3 and class 2 in summer season (Fig. S3). However, in the winter
season, higher abundance of class 3 integron was observed relative to
class 1 integron. In the water and sediment samples collected from the
river, the levels of class 1 integron were found to be higher followed by
class 3 and class 2 integrons in both the seasons with a slightly higher
abundance in winter. The high abundance of ARGs and integrons were
observed in winter compared to summer in both drains and the water
and sediment samples collected from river Yamuna. The high abun-
dance of ARGs and integron genes observed in winters is a direct con-
sequence of increased ARB abundance in winters. Higher ARB abun-
dance ultimately controls the transmission of the concerned antibiotic
resistant genes and the integron genes.
To find the integron genes responsible for increasing the prolifera-
tion of ARGs in the environment, efforts were made to find a correlation
between the abundance of ARGs and different integron genes.
Significant correlation was obtained for all the ARGs with both class 1
integron and class 3 integron (Table S4). However, the correlation of
ARGs with class 2 integron was insignificant. Previous studies have
shown the widespread presence of integrons in the ARB and have also
shown that the presence of integrons leads to the development of multi-
drug resistance [38]. Hence, the high abundance of ARGs and
6
Journal of Environmental Chemical Engineering 8 (2020) 102088
integrons, along with a significant correlation of ARGs with integrons
further increase the chance of dissemination of ARGs to other resistant
and non-resistant microbes and generation of multi-drug resistant
bacteria.
3.5. Treatment technologies
Biological and physicochemical treatment schemes [Activated
sludge process (ASP), ozonation and ASP followed by ozonation] were
used in laboratory scale reactors to evaluate their efficiency in re-
moving AR present in the wastewater collected from various drains.
Coliforms and ARB (ESBL and KPC) were used as markers. Fig. 4 shows
the performance of the treatment schemes used for removing AR. It was
observed that biological treatment alone (only ASP) resulted
in ∼30–40% reduction of coliforms and ARB whereas only physico-
chemical treatment (Ozonation) and biological followed by physico-
chemical treatment resulted in ∼80–90% reduction of coliforms and
ARB. No reduction in the abundance of 16S rRNA and ARGs was ob-
served after the biological treatment alone. However, the abundance of
16S rRNA gene and ARGs got reduced by 40% after only physico-
chemical treatment (Ozonation) and biological followed by physico-
chemical treatment. Thus, using advanced oxidation techniques for
treating the sewage along with the commonly used secondary biological
treatment techniques (ASP) can help in reducing the AR in the waste-
water present in drains. Previous studies have also reported similar
results [39,40].
4. Conclusion
The rapid emergence of the antibiotic resistance (AR) in developing
countries is posing a greater health risk and increasing the global dis-
ease burden. Lack of access to safe drinking water, poor sanitation and
inadequate sewage treatment facilities in the developing countries are
exaggerating the problem associated with the emergence of AR. The
present study indicated that sewage is a prominent source of ARB and
ARGs in the river Yamuna. The results indicated higher abundances of
coliforms, ARB and ARGs in the river in the winter season relative to the
summer season. Significant positive correlations were observed be-
tween FC and ARB levels across all the collected samples, indicating the
possible association of ARB with the faecal matter. Significant positive
correlations were also observed between integron gene (int1 and int3)
abundances and ARGs abundances, indicating a potential for higher AR
M. Lamba, et al.
proliferation in the water bodies. The presence of critical drug resistant
pathogens listed by WHO in ‘urgent need of new antibiotics’ in the river
water samples creates a serious health concern and would further in-
crease the disease burden in India. The high abundance of ARB and
ARGs released by the sewer drains in the river Yamuna is alarming and
poses a serious threat to the use of river water for various purposes due
to a high possibility of the spread of resistance to humans and animals
through the use of river water. Appropriate interventions such as im-
proving the connectivity between drains and STPs for proper sewage
collection and complete treatment of the entire volume of sewage
generated in Delhi can limit the release of untreated sewage from drains
to river Yamuna. This would ultimately help in improving the quality of
river water and result in its healthy use.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.jece.2017.12.041.
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