J Antimicrob Chemother 2012; 67: 857 859

doi:10.1093/jac/dkr564 Advance Access publication 19 January 2012

Identification of extended-spectrum b-lactamase and quinolone

resistance genes in Escherichia coli isolated from hospital
wastewater from central India
Vishal Diwan1,2*, Salesh P. Chandran3,4, A. J. Tamhankar5, Cecilia Stalsby Lundborg2 and Ragini Macaden3
1
Department of Public Health and Environment, R. D. Gardi Medical College, Agar Road, Ujjain, India; 2Department of Public Health
Sciences, Division of Global Health/IHCAR, Karolinska Institutet, Nobels vag 9, SE 17177, Stockholm, Sweden; 3Infectious Disease Unit,
St Johns Research Institute, Bangalore, India; 4Department of Microbiology, St Johns Medical College, Bangalore, India; 5Indian Initiative
for Management of Antibiotic Resistance (IIMAR), Department of Environmental Medicine, R. D. Gardi Medical College, Agar Road,
Ujjain, India

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*Corresponding author. Tel: +91-7368-261231; Fax: +91-7368-261235; E-mail: vishaldiwan@hotmail.com

Both the authors contributed equally.

Received 2 August 2011; returned 10 August 2011; revised 7 December 2011; accepted 8 December 2011
Objectives: To investigate the presence of antibiotic resistance genes (ARGs), related to commonly used
b-lactams and quinolones, in Escherichia coli present in hospital wastewater in central India.
Methods: Cefotaxime- and ciprofloxacin-resistant E. coli isolates from hospital-associated wastewater samples
were collected from two tertiary care hospitals in the Ujjain district of India during 2008 09. The presence of
blaCTX-M, blaTEM, blaSHV, qnrA, qnrB, qnrS, aac(6 )-Ib-cr and qepA genes was detected by PCR and sequencing.
Results: Twenty-five E. coli isolates were extended-spectrum b-lactamase (ESBL) positive. blaCTX-M-15 and
blaTEM-1 genes were identified in 21 and 16 ESBL-positive isolates by PCR, respectively. Amongst 30 fluoroquinolone-resistant E. coli isolates, aac(6 )-Ib-cr was detected in 27 isolates, qnrA in 1 isolate, qnrB in 2 isolates and
qepA in 3 isolates.
Conclusions: blaCTX-M-15, blaTEM-1, aac(6 )-Ib-cr, qnrA, qnrB and qepA genes are present in E. coli occurring in
hospital wastewater in central India.
Keywords: blaCTX-M, blaTEM, blaSHV, qnr genes

Introduction

Materials and methods

Antibiotic resistance is not confined to clinically important bacteria but is also present in bacteria in the aquatic environment,
which might be an important contributory factor in the spread
of resistance.1 Further, antibiotic contamination of wastewater2
might also lead to a selective pressure on bacteria that could
additionally promote antibiotic resistance.1 It is important, therefore, to document the occurrence and types of antibiotic resistance genes (ARGs) in various aquatic environments, particularly
those that are prone to antibiotic contamination. There is a
need to understand the details of this, in order to plan for
suitable interventions. In view of this, we identified and documented ARGs related to commonly used antibiotics, i.e.
expanded-spectrum cephalosporins and fluoroquinolones, in
hospital wastewater in central India, where we are conducting
studies on microbial and antibiotic contamination of hospital
wastewater.

This study was conducted in two private tertiary care hospitals located in
the Ujjain district of Madhya Pradesh, India. During the period 200809,
276 samples were collected from wastewater outlets of both the hospitals.
The sampling and bacterial culturing procedure was as described by Diwan
et al.2 Five non-mucoid lactose-fermenting colonies isolated on MacConkey
agar were selected randomly. These were identified biochemically as
Escherichia coli. The first 32 confirmed E. coli isolates from 32 different
water samples that were resistant to expanded-spectrum cephalosporins
or fluoroquinolones by the KirbyBauer disc diffusion method (performed
according to CLSI guidelines3) were included in the study. Resistance to
the antibiotics was confirmed by determining the MICs of cefotaxime
and ciprofloxacin, the most commonly used expanded-spectrum cephalosporin and fluoroquinolone antibiotics in our hospitals, by using the agar
dilution method according to the CLSI breakpoints.3
E. coli reference strain ATCC 25922 was used for quality control.
Total bacterial DNA from isolates was extracted using the alkaline lysis
method.4

# The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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857

Diwan et al.

Table 1. Resistance and genetic analysis of E. coli isolates from hospital wastewater
Cefotaxime

Isolate
no.

Ciprofloxacin

PMQR

Location

Phylogenetic
group

disc
diffusion
test

MIC
(mg/L)

phenotypic
ESBL
detection

blaCTX-M
PCR

blaTEM
PCR

blaSHV
PCR

disc
diffusion
test

MIC
(mg/L)

qnrA,
qnrB,
qnrS

H1
H1
H1
H1
H1
H1
H1
H2
H2
H2
H2
H2
H2
H1
H1
H2
H2
H2
H1
H1
H2
H2
H1
H1
H2
H2
H2
H2
H2
H1
H1
H1

B1
B2
D
D
B1
B1
B1
B2
B2
B2
B2
B2
B2
B1
B1
B2
B2
B2
B2
B2
B2
B2
B2
A
B1
A
B2
B2
B2
B2
B2
B2

Ra
R
R
R
S
S
S
R
R
R
R
R
R
R
R
R
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R

2a
256
.512
256
1
0.125
0.5
.512
8
8
64
16
32
128
.512
.512
1
.512
.512
.512
.512
256
256
.512
64
32
.512
.512
256
.512
.512
.512

+
+
+
+
NA
NA
NA
+
+
+
+
+
+
+
+
+
NA
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

2
2
+
+
NA
NA
NA
+
+
2
2
+
+
+
+
+
NA
+
+
+
+
+
+
2
2
2
+
+
+
+
+
+

+
+
+
+
NA
NA
NA
2
2
+
+
2
+
2
2
2
NA
2
+
+
+
2
+
+
2
2
+
+
+
+
+
+

2
2
2
2
NA
NA
NA
2
2
+
+
2
2
2
2
2
NA
2
2
2
2
2
2
+
2
+
+
2
2
2
2
2

R
R
R
R
R
R
R
R
R
R
S
R
R
R
R
R
R
R
R
R
R
R
R
R
S
R
R
R
R
R
R
R

16
.128
32
32
128
16
8
.128
64
0.5
0.125
32
64
.128
128
32
32
32
.128
64
32
64
64
32
0.125
.128
.128
.128
.128
4
64
.128

qnrB
2
2
2
2
2
2
2
2
2
NA
2
2
2
2
2
2
2
2
2
2
2
2
qnrA
NA
2
2
2
2
2
2
qnrB

aac(6 )Ib-cr
qepA
2
+
+
+
2
+
+
+
+
2
NA
+
+
+
+
+
+
+
+
+
+
+
+
+
NA
+
+
+
+
+
+
+

2
2
2
2
2
2
2
2
2
2
NA
2
2
2
2
2
2
2
2
2
2
2
2
2
NA
2
+
+
+
2
2
2

H1, hospital 1; H2, hospital 2; NA, not applicable; S, susceptible; R, resistant.

a
MIC value - intermediate (CLSI 2010).

Extended-spectrum b-lactamases (ESBLs) were detected phenotypically by the combined disc diffusion method.3 Amplification and identification of b-lactamase-encoding genes (blaCTX-M, blaTEM and blaSHV) and
plasmid-mediated quinolone resistance [PMQR; qnrA, qnrB, qnrS,
aac(6 )-Ib-cr and qepA] genes was performed using previously described
primers.5,6 After PCR amplification, all the PCR products were visualized
using a gel documentation system. The purified PCR products were
sequenced using an ABI 3730XL (Applied Biosystems, USA) sequencer.
The nucleic acid sequences were analysed using the Basic Local Alignment Search Tool available in the National Center for Biotechnology database.7 The nucleic acid sequences were submitted to GenBank (GenBank
accession numbers JN636752 JN636795). All the E. coli isolates were
phylogrouped by multiplex PCR based on chuA, yjaA and TspE4C2 genes
as previously described.8

858

Ethics approval
The Ethics Committee of the R. D. Gardi Medical College, Ujjain, approved
the study.

Results and discussion

MICs of cefotaxime and ciprofloxacin are presented in Table 1.
Twenty-seven out of 32 E. coli isolates were resistant to cefotaxime. Out of the 27, 25 isolates were ESBL-positive and were confirmed by the presence of blaCTX-M-15 only (n 20) or blaSHV-12
only (n 4). Isolate 27 had both blaCTX-M-15 and blaSHV-12.
blaTEM-1 (which does not encode an ESBL enzyme) was

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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32

b-Lactamase determinants

ESBL and quinolone resistance in hospital wastewater, India

Acknowledgements
We wish to thank the management of the R. D. Gardi Medical College,
Ujjain, and St Johns Research Institute, Bangalore, for facilitating
this work. Vivek Parashar and Dinesh Damde gave assistance during
sampling and Senia Rosales gave constructive comments on the
manuscript.

Funding
This study was supported by the Swedish Research Council (K200770X-20514-01-3) and Asia Link (348-2006-6633). V. D. is a recipient of
scholarships from Erasmus Mundus External Cooperation Window India
Lot 15 and the Swedish Institute.

Transparency declarations
None to declare.

References
1 Zhang XX, Zhang T, Fang HH. Antibiotic resistance genes in water
environment. Appl Microbiol Biotechnol 2009; 82: 397 414.
2 Diwan V, Tamhankar AJ, Khandal RK et al. Antibiotics and
antibiotic-resistant bacteria in waters associated with a hospital in
Ujjain, India. BMC Public Health 2010; 10: 414.
3 Clinical and Laboratory Standards Institute. Performance Standards for
Antimicrobial Susceptibility Testing: Twentieth Informational Supplement
M100-S20. CLSI, Wayne, PA, USA, 2010.
4 Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory
Press, 1989.
5 Dallenne C, Da Costa A, Decre D et al. Development of a set of
multiplex PCR assays for the detection of genes encoding important
b-lactamases in Enterobacteriaceae. J Antimicrob Chemother 2010; 65:
4905.
6 Kim HB, Park CH, Kim CJ et al. Prevalence of plasmid-mediated
quinolone resistance determinants over a 9-year period. Antimicrob
Agents Chemother 2009; 53: 639 45.
7 National Center for Biotechnology. Basic Local Alignment Search Tool.
http://blast.ncbi.nlm.nih.gov/Blast.cgi (15 March 2011, date last
accessed).
8 Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of
the Escherichia coli phylogenetic group. Appl Environ Microbiol 2000; 66:
4555 8.
9 Reinthaler FF, Feierl G, Galler H et al. ESBL-producing E. coli in Austrian
sewage sludge. Water Res 2010; 44: 1981 5.
10 Kummerer K. Antibiotics in the aquatic environmenta reviewpart I.
Chemosphere 2009; 75: 417 34.

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also detected in 16 of these isolates. Out of the 30

fluoroquinolone-resistant E. coli isolates, 90% carried the
aac(6 )-Ib-cr (n 27) gene, 20% carried other PMQR determinant
genes such as qnrA (n1), qnrB (n 2) and qepA (n 3) and
none carried the qnrS gene. The presence of both blaCTX-M-15
and aac(6 )-Ib-cr was observed in 21 isolates. One isolate that
was positive for blaCTX-M-15, blaTEM-1 and blaSHV-12 by PCR also
carried aac(6 )-Ib-cr and qepA genes (Table 1). Phylogenetic
grouping revealed that 21 E. coli isolates belonged to the phylogenetic group B2, which most often denotes virulent extraintestinal strains, 7 isolates belonged to B1 and 2 each to group A
(denotes commensal isolates) and group D.
To our knowledge, this is the first report on the presence of
blaCTX-M, blaSHV, blaTEM, qnrA, qnrB, qnrS, aac(6 )-Ib-cr and qepA
genes in E. coli present in hospital wastewater in central India.
Although the number of isolates was relatively small, detection
of such a large number of E. coli resistant to expanded-spectrum
cephalosporin and fluoroquinolone antibiotics in the hospital
wastewater samples is alarming. Most of the blaCTX-M-15-positive
E. coli isolates also showed high MICs of cefotaxime (MIC50
.512 mg/L; MIC90 .512 mg/L, ranging from 8 to .512 mg/L)
and ciprofloxacin (MIC50 64 mg/L; MIC90 .128 mg/L, ranging
from 4 to .128 mg/L). Isolates 1, 2 and 25 showed MICs of cefotaxime of 2, 256 and 64 mg/L, respectively (Table 1). CTX-M and
SHV genes were not amplified in isolates 1, 2 and 25; it is possible
that other, non-detected ESBL-encoding genes may be involved
in these cases. The presence of ESBL-producing isolates along
with resistance genes is a matter of concern. ESBL-positive plasmids are known to carry more than one b-lactamase gene and
can transfer resistance.9 Further, plasmids carrying blaCTX-M and
qnr genes occur as self-transmissible plasmids carrying transportable ARGs, which are capable of transferring and replicating
in different hosts.1 Significant quantities of antibiotics find their
way into the aquatic environment and have been detected previously in the wastewater of the hospitals mentioned in this
study.2 Such antibiotic residues can further accelerate the development of ARGs in bacteria. In addition, elimination of such resistant bacteria or ARGs from wastewater is difficult.10
It is likely that we could have detected a higher number of
resistant bacteria by using a selective medium, as the vast
majority of coliforms in the wastewater are predictably susceptible and resistant colonies may not be present within the five
colonies that were picked up by us. However, the results of this
study indicate that bacteria carrying ARGs are present in hospital
wastewater in India. Wastewater treatment and management is
not commonly practised in many hospital settings in India. This
will lead to a situation where bacteria carrying ARGs may enter
the community through contaminated water sources. Therefore,
decontaminating wastewater from hospitals appears to be
the need of the hour. This will in turn reduce the spread of
multidrug-resistant bacteria in a given geographical area.

JAC