Surveillance of Carbapenem-resistant Gram-negative bacteria from animal sources in Mathura region, Uttar Pradesh, India

Abstract

A cross-sectional study was conducted to determine the prevalence of Carbapenem-resistant Gram-negative
bacteria (CR-GNB) in animals. The study involves one hundred eighty-four GNB isolates from 214 samples (feces, milk,
pus, and uterine discharge) from Buffalo (N=112), Cattle (N=50), and Dog (N=52). Healthy and diseased animals reported
to Veterinary Clinical Complex were sampled. Carbapenemase production was evaluated by phenotypic methods and
the presence of Metallo β-lactamase genes was assayed by PCR. We observed a 9.78% overall prevalence of CR-GNB in
animal sources. CR-GNB was more frequently recovered from companion animals (19.23%) when compared to livestock
(4.93%). IMP (44.4%), VIM (38.8%), and OXA-48 (16.66 %) were the main MBLs observed in the study.

Full text

Explor Anim Med Res,
Vol.12, Issue - 1, 2022, p. 91-98
DOI : 10.52635/eamr/12.1.91-98
ISSN 2277- 470X (Print), ISSN 2319-247X (Online)
Website: www.animalmedicalresearch.org
Research Article
SURVEILLANCE OF CARBAPENEM-RESISTANT GRAM-NEGATIVE BACTERIA
FROM ANIMAL SOURCES IN MATHURA REGION, UTTAR PRADESH, INDIA
A. Arun1, U. Jaiswal1, S. Tripathi1, A. P. Singh1*, S. Choudhury2, S. N. Prabhu3
Received 05 February 2022, revised 11 June 2022
ABSTRACT: A cross-sectional study was conducted to determine the prevalence of Carbapenem-resistant Gram-negative
bacteria (CR-GNB) in animals. The study involves one hundred eighty-four GNB isolates from 214 samples (faeces, milk,
pus, and uterine discharge) from Buffalo (N=112), Cattle (N=50) and, Dog (N=52). Healthy and diseased animals reported
to Veterinary Clinical Complex were sampled. Carbapenemase production was evaluated by phenotypic methods and
presence of metallo ββ
ββ
β-lactamase genes was assayed by PCR. We observed a 9.78% overall prevalence of CR-GNB in
animal sources. CR-GNB was more frequently recovered from companion animals (19.23%) when compared to livestock
(4.93%). IMP (44.4%), VIM (38.8%), and OXA-48 (16.66 %) were the main MBLs observed in the study.
Key words: Gram-negative bacteria, Carbapenem, Antibiotic resistance, Animals.
1Department of Veterinary Microbiology, 2Department of Veterinary Pharmacology & Toxicology, 3Department of Veterinary
Pathology, College of Veterinary Science and Animal Husbandry, U.P. Pandit Deen Dayal Upadhyayay Pashu Chikitsa
Vigyan Vishwavidhyalaya Ewam Gau Anusandhan Sansthan, Mathura, U.P., India.
*Corresponding author. e-mail: drajay_vet@yahoo.co.in
INTRODUCTION
Carbapenems are a beta-lactam class of antibiotics.
Carbapenems are stable to most beta-lactamase enzymes
mediated inactivation unlike other beta-lactam antibiotics
(Perrott et al. 2010). Carbapenems are clinicians’
preferred choice for the therapeutic management of
serious infections caused by MDR pathogens (Falagas
and Karageorgopoulos 2009). The ever-growing
dependence has led to the recent emergence of
carbapenem-resistant bacterial strains. Genes encoding
carbapenem-resistance are often associated with mobile
genetic elements leading to their spread across a variety
of carbapenem-resistant Gram-negative bacteria (CR-
GNB) (Schwaber et al. 2011).
Most clinically relevant carbapenem resistance appears
to have arisen and propagated because of its therapeutic
uses in humans (Poirel et al. 2014). CR-GNB has been
isolated predominantly from humans and environmental
samples. Presently, carbapenems are not authorized for
use in veterinary medicine in most parts of the world;
hence carbapenem resistance is not common in GNB
isolated from animals. Notwithstanding, CR-GNB has
been detected in livestock, companion animals, and their
environment by several workers across the globe in the
recent past (Wang et al. 2012, Woodford et al. 2014).
The overall carriage rate of CR-GNB has been on the
rise in food-producing animals and their environment in
India (Ghatak et al. 2013, Pruthvishree et al. 2017,
Nirupama et al. 2018). The colonization of CR-GNB in
livestock and companion animals has a potential
multiplier effect on rapid dissemination to humans
through close contact, environmental and, food-borne
transmission. The prevalence of CR-GNB has risen
significantly in animal healthcare settings over the past
few years, but the data on the population prevalence
among livestock and pet animals are scanty in India.
Therefore, it is necessary to include CR-GNB for routine
epidemiological investigations in animal population. The
present study aimed to predict the population prevalence
of CR-GNB from various animal sources and their
characterization.
MATERIALS AND METHODS
Bacterial Isolates
The carbapenem resistance surveillance includes 214
GNB samples (faces, milk, pus, and uterine discharge)
91

Exploratory Animal and Medical Research, Vol.12, Issue 1, June, 2022
collected between during May 2018 and April 2021 from
separate animals in and around the Mathura region, India,
without any inclusion or exclusion criteria. Sample details
given in Table 1. A total of 184 GNB were isolated, and
further identified by the standard microbiological
procedure (Barrow and Feltham 2004). Control strain
includes Klebsiella pneumoniae ATCC BAA 1705+KPC
and Klebsiella pneumoniae ATCC BAA 1706.
Antibiotic susceptibility test
Antimicrobial susceptibility analysis was carried out
by standard Kirby-Bauer disk diffusion method using
Mueller-Hinton agar (Sigma-Aldrich) following the CLSI
(2017) guidelines and interpretative criteria (Table S2).
The panel of antimicrobial agents consisted of 10 different
antimicrobial-impregnated disks: namely, ertapenem (10
µG), cefotaxime (30 µG), ceftazidime (30 µG),
gentamicin (10 µG), ampicillin (10 µG), amoxicillin-
clavulanate (10µg), ciprofloxacin (5 µg), cefoxitin (30
µg), ceftriaxone (30 µg) and cefpodoxime (10 µg). The
zone of inhibition was measured in mm and interpreted
as sensitive, intermediate, or resistant.
Minimum inhibitory concentration (MIC)
The minimum inhibitory concentrations (MICs) of
carbepenems (imipenem, ertapenem and meropenem)
were tested by the broth microdilution method (Wiegand
et al. 2008). Standardized bacterial inoculums were
prepared for each isolate to give a turbidity equivalent to
that of a 0.5 McFarland standard corresponding to 1 X108
cfu/ml. The final test concentration of the bacteria was
achieved by further diluting the adjusted suspension by
a factor of 1:100 to achieve approximately 5 × 105 cfu/
ml. The working antibiotic stock solution was prepared
by 1:10 dilution of antibiotic stock solution (potency
adjusted 1.28 mg/ml) in Muller Hinton Broth (MHB).
The plates were covered by sterile covers and incubated
at 37ºC for 18-24 h. The lowest concentration of the
antibiotics that did not have visible bacterial growth was
defined as the MIC.
Phenotypic and genotypic carbapenemase
identification
Carbapenemase activity was assessed by modified
Carba NP test (Rudresh et al. 2017), Carbapenemase
Inactivation Assay (Zwaluw et al. 2015) and Modified
Hodge test (Amjad et al. 2011). For genotypic detection,
DNA isolated by the snap chill method was subjected to
a target amplification of β-Lactamase genes using a panel
Fig. 1. Representative results obtained by Modified Carba
NP test. [b,c,d] represents results obtained by imipenem- non
susceptible strain where ‘A’ tube (red colour/control tube) is a
negative and tube ‘B’ (yellow colour) is a positive result i.e.,
carbapenemase-producing isolate. [a & e] represents results
obtained by imipenem-susceptible strain where both tubes
(A,B) are negative results i.e., carbapenemase-non producing
isolate. (a) Negative Control E. coli ATCC 25922 (b) Positive
Control Klebsiella pneumoniae ATCC BAA 1705 [1b]
Representative results obtained by mCIM: CIA (-) represents
negative result of ertapenem susceptible strain i.e., zone = 20
mm and CIA (-) represents positive result of ertapenem non-
susceptible strain i.e., no zone of inhibition around the
carbapenem disk. E. coli ATCC 25922 has been used as
reporter strain. [1c] Representative results obtained by
Modified hodge test: Isolates VA-41 and VA-52 were Modified
Hodge Test Negative, while isolates VA-19 was Modified
Hodge Test Positive.
Buffalo 1 52 33 26
(N=112)
Cattle 1 41 - 8
(N=50)
Dog
(N=52) - - - 52
Total
(N=214) 2 93 33 86
Fecal
sample
Mastitis
Milk
Uterine
discharge
Pus
Species
Sample type
Table 1. Details of samples collected.
92

of primers for detection of OXA-48, KPC, VIM, IMP,
and NDM genes by multiplex PCR (Dallenne et al. 2010).
A 25 µl reaction mixture containing 12.5 µl Dream Taq
Master Mix, variable number of primers and 2 µl of
isolated DNA template was used. Amplification was
carried involving initial denaturation at 940C for 10 min
and 30 cycles of denaturation at 940C for the 40s,
annealing at 550C for 40s, and extension at 720C for 1
min followed by final elongation step at 720C for 7 min.
A. The primer concentration and amplification
conditioned for the PCR reactions were used as per the
Table 1.
RESULTS AND DISCUSSION
Between May 2018 and April 2021, we processed 214
non-repeated samples comprised of faces, milk, uterine
swab, and pus. A total of 184 GNB isolates were obtained
on a MacConkey agar plate. A total of 18 CR-GNB
isolates including Escherichia coli (n=12), Klebsiella
pneumoniae (n=2), Citrobacter freundii (n=2),
Enterobacter cloacae (n=1), and Pseudomonas
aeruginosa (n=1) showed reduced susceptibility to
ertapenem, based on zone interpretative criteria. The
carbapenem resistance has been reported in E. coli (Zhang
et al. 2013), Klebsiella pneumonia (Diab et al. 2017),
Enterobacter, Citrobacter (Mollenkopf et al. 2017). MIC
of 18 CR-GNB isolates for imipenem, meropenem, and
ertapenem was shown in Table 2. MIC of 18 CR-GNB
isolates for imipenem, meropenem, and ertapenem were
detected in the range of 0.625 µg/ml to 64 µg/ml, 0.0625
µg/ml to 1 µg/ml, and 0.312 to 16 µg/ml, respectively
(Table 2). None of the CR-GNB isolates were resistant
to meropenem. All the isolates exhibited resistance to
amoxicillin-clavulanate, while various resistance rates
were observed for ceftazidime (83.3 %), cefotaxime (75
%), ceftriaxone (88.8 %), cefpodoxime (94.44 %), and
ciprofloxacin (91.6 %). The least frequent resistances
were against gentamicin (33.3%).
Out of eighteen carbapenem non-susceptible isolates,
thirteen (72.22 %) showed a positive reaction in the
carbapenemase biochemical test (Fig. 1) while PCR-based
identification revealed the presence of one or more
carbapenemase (IMP, VIM, and Oxa-48) in 12 isolates
(66.66%). Molecular testing showed the presence of IMP,
VIM, and OXA-48 MBLs in eight (44.44%), seven
(38.88%), and three isolates (16.66%), respectively (Fig.
2, Table 2). All three OXA-48 bearing isolates were
Fig. 2. [a] PCR amplification of blaOXA48: 281 bp, blaIMP: 139bp, Lane M: 100 bp DNA Ladder, Lane 1-9: Test
Isolates [b] PCR amplification of blaVIM : 390 bp , Lane M: 100 bp DNA Ladder, Lane 1-8: Test Isolates.
Fig. 3. Distribution of CRE-GNB isolates from various
sample sources.
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Surveillance of carbapenem-resistant gram-negative bacteria from animal sources in...

Exploratory Animal and Medical Research, Vol.12, Issue 1, June, 2022
VS-01
VS-02
VS-39
VS-51
VA-19
VA-52
VA-53
VA-55
VA-66
VA-77
VA-99
VA-100
VU-02
VU-03
VU-08
VU-14
VU-16
VU-17
Cow
Cow
Cow
Cow
Dog
Dog
Buffalo
Dog
Dog
Buffalo
Buffalo
Buffalo
Dog
Dog
Dog
Dog
Dog
Dog
Uterine
discharge
Uterine
discharge
Uterine
discharge
Uterine
discharge
Faecal
Faecal
Milk
Faecal
Faecal
Faecal
Faecal
Faecal
Faecal
Faecal
Faecal
Faecal
Faecal
Faecal
Pseudomonas
aeruginosa
E. coli
Enterobacter
cloacae
E. coli
E. coli
E. coli
Citrobacter
freundii
E.coli
Klebsiella
pneumoniae
Klebsiella
pneumonia
E. coli
E. coli
E. coli
Citrobacter
freundii
E. coli
E.coli
E.coli
E.coli
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, GM, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, GM, FOX, ETP
AMC, CPD, CRO,CTX, AM,
CIP, GM, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, GM, FOX, ETP
AMC, AM, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, FOX, ETP
AMC, CPD, CRO,CTX, CAZ,
AM, CIP, FOX, ETP
8 µg
1 µg
16 µg
4 µg
6 µg
2 µg
4 µg
8 µg
6 µg
0.25 µg
4 µg
16 µg
2 µg
4 µg
2 µg
6 µg
1 µg
1 µg
32 µg
32 µg
4 µg
64 µg
1 µg
0.125 µg
0.0625 µg
0.125 µg
1 µg
2 µg
1µg
8 µg
32 µg
32 µg
64 µg
1 µg
4 µg
16 µg
0.5 µg
0.5 µg
0.0625 µg
0.5 µg
1 µg
0.0625 µg
0.125µg
0.0625 µg
0.25 µg
0.125µg
0.25 µg
0.125 µg
0.0625 µg
0.0625 µg
0.0625 µg
0.125 µg
0.125 µg
0.125 µg
blaIMP,
blaVIM
blaIMP
blaIMP,
blaVIM
blaIMP,
blaVIM
blaOxa48
blaIMP
blaIMP
blaOxa48
-
blaVIM
-
blaIMP
blaIMP
blaOxa48
blaVIM
-
-
-
-
Isolate
No Source Sample
Origin Isolate Antibiotic resistant profile MIC MBL
gene
Ertapenem Imipenem Meropenem
(AMC: Amoxicillin–Clavulanic acid, CPD: Cefpodoxime; AMP: Ampicillin, CAZ: Ceftazidime, CRO: Ceftriaxone, CIP: Ciprofloxacin,
CTX: Cefotaxime, FOX: Cefoxitin, GM: Gentamicin, ETP: Ertapenem).
Table 2. Details on the tests performed using different carbapenam suspected isolates.
94

recovered from dogs. Three out of 18 isolates carried
both VIM and IMP, while the co-existence of IMP and
OXA-48 was found in one isolate. Previous reports
suggest rare prevalence of VIM and Oxa-48
carbapenemase genes from bacterial strains of animal
sources in India, however, recent report indicate more
frequent occurrence of OXA-48, VIM and IMP genotype
from fecal sample of the piglets, calves and dogs
(Nirupama et al. 2018, Murugan et al. 2019, Sankar et
al. 2021). The carbapenemase genes variants (KPC and
Companion
Animal
Dog
(n =52)
10
(19.23%)
Cattle
(n=50)
4
(8.00%)
Buffalo
(n =112)
4
(3.57%)
Total GNB isolates Livestock
Carbapenem resistant
GNB
Table 3. Number (% ) of Carbapenem resistant isolates
from animal source.
ββ
ββ
β-lactamase (s)
targeted
OXA-48-like
New Delhi metallo-
beta lactamase (NDM)
IMP
VIM
KPC
Primer name
OXA-48_for
OXA-48_rev
NDM_F
NDM_R
Multi IMP-F
Multi IMP-R
Multi VIM-F
Multi VIM-R
Multi KPC-F
Multi KPC-R
Sequence (5’-3’)
GCTTGATCGCCCTCGAT
GATTTGCTCCGTGGCCGAAA
GGTTTGGCGATCTGGTTTTC
CGGAATGGCTCATCACGATC
TTGACACTCCATTTACDGA
GATYGAGAATTAAGCCACYCTA
GATGGTGTTTGGTCGCATA
GATGGTGTTTGGTCGCATA
CATTCAAGGGCTTTCTTGCTGC
ACGACGGCATAGTCATTTGC
Primer concentration
(20 picomol)
20
20
20
20
25
25
25
25
10
10
Reference
Dallenne
et al. 2010
Table S1. Primers used in PCR analysis.
Amoxicillin & AMC-30 20/10 18-24 22 <=13 14-17 >=18
Clavulanic acid
Ampicillin AM 10 16-22 20 <=13 14-16 >=17
Cefotaxime CTX-30 30 29-35 34 <=14 15-22 >=23
Ceftazidime CAZ-30 30 25-32 29 <=14 15-17 >=18
Cefpodoxime CPD-10 10 23-28 25 <=17 18-20 >=21
Ceftriazone CRO-30 30 29-35 29 <=13 14-20 >=21
Cefoxitin FOX-30 30 23-29 24 <=14 15-17 >=18
Ciprofloxacin CIP-5 5 30-40 30 <=15 16-20 >=21
Gentamicin GM-10 10 19-26 17 <=12 13-14 >=15
Ertapenem ETP-10 10 29-36 32 <=15 16-18 >=19
Antibiotic Disc code Antibiotic
concentration
(µg)
Control
strain zone
diameter
(mm)
Control
diameter
observed
(mm) Resistant Intermediate Susceptible
Test zone diameters (mm)
Table S2. AST Zone diameters for control strain Escherichia coli (ATCC®25922™) and the test isolates used in this
assay (PSAST 2017).
95
Surveillance of carbapenem-resistant gram-negative bacteria from animal sources in...

Exploratory Animal and Medical Research, Vol.12, Issue 1, June, 2022
NDM) widely known for their rapid acquisition and
dissemination, were not found.
The prevalence of CRE in different species of animals
was recorded. Based on the results described herein, CR-
GNB appears to be having a significant prevalence (9.78
%) in cattle and dogs. The earlier studies showed the
varied incidence of CR-GNB ranging from 0.5 % to 25
% in different parts of the world. In the current study, we
found slightly higher prevalence rates of CR-GNB in
animals, than the rates reported by other researchers
(Stolle et al. 2013, Saheen et al. 2013, Reynolds et al.
2019). In absence of strict regulatory framework
governing the use of antimicrobials in animal production
system and irrational therapeutic usage of antibiotics in
veterinary practices in India, may have contributed for
higher prevalence of CR-GNB in animals. The recovery
of CR-GNB from cattle and dogs indicates a potential
future public health crisis (Abraham et al. 2014). We
recorded a higher prevalence of CR-GNB in dogs
(19.23%) in comparison to bovine (4.93%) (Table 3). A
significantly higher prevalence of CRE among companion
animals observed in our study is in agreement with
previous findings of Kock et al (2018) who inferred
higher prevalence rate (1-15%) among livestock and
companion animals in Asia.
We observed faecal samples 7.70% (14/184) were the
major source of CRE isolates followed by uterine samples
2.17% (4/184) (Fig. 3). Traditionally, bovine excrements
are used for mud-flooring, as manure in agricultural
farmland, and dung cake preparation in villages in India.
Human exposure to antibiotic-resistant bacteria present
in bovine excrements poses a health risk. The colonization
of CR-GNB in the animal gut microbiome is a concern
since it could be readily transmitted to pet owners,
veterinarians, farmers through close physical contact and
may result in community spread. The frequent use of beta-
lactams selects and maintains CR-GNB within the animal
population. The prevalence of CR-GNB in faecal samples
of dogs has been widely reported (González-Torralba et
al. 2016, Gentilini et al. 2018). The faecal carriage of
CR-GNB in dogs indicates the possible occurrence of
interspecies transmission between humans and
companion animals within the same household.
Industrialization and urban expansion of Indian cities
have resulted in an exponential rise in the stray dog
population in urban and peri-urban areas. Humans can
be exposed to CR-GNB through soil contaminated with
stray-dog faeces in densely populated urban
neighbourhoods inhabited by the low socio-income group.
CONCLUSION
The recovery of CR-GNB from livestock and
companion animals has significant public health
ramifications and this may be related to illegal
carbapenem use in veterinary practice. The dissemination
of carbapenem-resistant bacteria in livestock and the
environment potentially has a far-reaching effect.
Evidence of such transmission is the cause of concern
for public health experts and warrants strict vigil to limit
the species spillover cross-species transmission. Hence
continuous surveillance for antimicrobial-resistant must
include screening for CR-GNB in livestock and
companion animals.
ACKNOWLEDGMENT
The authors are also thankful to Vice-chancellor, U.P.
Pandit Deen Dayal Upadhyayay Pashu Chikitsa Vigyan
Vishwavidhyalaya Ewam Gau Anusandhan Sansthan
Mathura, 281001 (U.P.), for providing infrastructural
facility to review this study.
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