Poster by Tushar Kumar Dey, Bibek Shome, Ram Pratim Deka, Rajeswari Shome and Johanna Lindahl presented at the 16th International Symposium of Veterinary Epidemiology and Economics, Halifax, Canada, 9 August 2022.

1. RESEARCH POSTER PRESENTATION
DESIGN © 2015
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• Food safety is a great concern in low- and middle-income countries. India is the
largest milk producer in the world and milk is consumed by the most people.
• Antimicrobial resistance (AMR) is a serious threat to the health of both humans
and animals, and resistant bacteria can also be spread through food
• Therefore, the risk of exposure to antibiotic resistant bacteria and antibiotic
residues in milk needs serious attention.
• This study was designed to investigate the presence of AMR bacteria and
antibiotic residues in milk in two states in India, Assam and Haryana.
Introduction
• Milk samples were collected from two Indian states during 2016-18.
• 729 samples (285 from Assam and 444 from Haryana) were analyzed
for microbiological studies
• 730 milk samples (330 from Assam and 400 from Haryana) were
analyzed for antibiotic residues.
• Bacteria were isolated as per standard protocol and subjected to
antibiotic disc diffusion testing
• Gram-positive bacteria were tested against cefoxitin (30µg),
methicillin (5µg) and oxacillin (1µg) for screening methicillin resistance
• Gram-negative bacteria were tested against cefoxitin (30µg), cefotetan
(30µg), cefotaxime (30µg), ceftazidime (30µg), imipenem (10µg) and
meropenem (10µg) was used for screening beta-lactamases (extended
spectrum beta-lactamase, metallo-beta-lactamase, AmpC betalactamase).
• The isolates resistant by disc diffusion test were screened for methicillin
resistance genes and beta-lactamases (ESBL, MBL, AmpC) genes using
polymerase chain reaction
• The collected samples were also analyzed for antibiotic residue testing
using spore-based assay and Charm Rosa test.
Materials & Methods
• In total, 256 resistant Gram-positive isolates were obtained by disc diffusion testing, most were
identified as genus Staphylococcus, and 7% (17/256) of phenotypically resistant staphylococci
harbored methicillin resistance mecA / mecC genes by genotyping.
• The 17 methicillin resistant staphylococci identified, carried mecA (15) and mecC (2) genes.
• Of the resistant Gram-negative isolates (148) obtained by disc diffusion testing, by genotyping
29% (n=43/148) were beta-lactamase isolates among E.coli, Klebsiella spp., Shigella spp. and
other gram-negative bacteria.
• ESBL - 16% (7/43), AmpC - 60% (26/43), MBL - 14% (6/43).
• Some bacteria also had combinations of resistance genes: MBL+AmpC - 7% (3) and ESBL+
AmpC - 2% (1).
• The prevalence of antibiotic residues in milk of Haryana was 8% (32/400) and in Assam was 5%
(16/330 ).
• The antibiotic residues detected were novobiocin 46% (22/48), macrolides 27% (13/48), sulfa
drugs 10% (5/48), tetracycline 10% (5/48), and beta-lactam 6% (3/48).
• Antibiotic resistant bacteria and residues were found in milk samples from both the states, but
more commonly in Haryana compared to Assam.
Results
Acknowledgements
The authors gratefully acknowledge International Livestock Research Institute (ILRI), Kenya for funding the research studies, National Institute of Veterinary Epidemiology and Disease
Informatics (NIVEDI), Bangalore, India for providing the laboratory facility to conduct the bench work, National Dairy Research Institute, (NDRI) Haryana, India for the residue analysis.
1International Livestock Research Institute, Kenya; 2Department of medical biochemistry and microbiology, Uppsala University, Sweden;
3National Institute of Veterinary Epidemiology and Disease Informatics, Bangalore, India
Tushar Kumar Dey1,2,3, Bibek Shome3, Ram Pratim Deka1, Rajeswari Shome3, Johanna Lindahl1,2
Molecular characterization of antibiotic resistant bacteria and assessment of
antimicrobial residues in the dairy milk in India
Conclusions
• The study found both the occurrence of 7% methicillin resistance genes among isolated
Staphylococcus spp, as well as 29% ESBL, MBL and AmpC producers among the isolated
E.coli, Klebsiella and Shigella spp.
• Also, antibiotic residues were found in 7% of the milk samples.
• Since the milk sampled were all at the point of sales, these resistant bacteria could point to
risks for human health, and even if the milk was boiled before consumption, the antibiotic
residues could contribute to the risks for humans.
• Hence there is need for intensified awareness creation and surveillance on the occurrence
of antimicrobial resistance and antibiotic residues in animals and food chain of animal
origin to device strategies for preventing their spread.
Senior Scientist, International Livestock Research Institute,
Box 30709, Nairobi, Kenya
Department of Medical Biochemistry & Microbiology, Uppsala
University, Box 256, 751 05 Uppsala, Sweden
Name: Johanna Lindahl
Affiliation: ILRI & Uppsala University
j.lindahl@cgiar.org
Fig 1: Map depicting the sampling states from India
7%
35%
29%
6%
6%
6%
6%
6%
6%
0% 10% 20% 30% 40%
Methicliin resistant
S. epidermidis
S. aureus
S. scuiri
S. pseudoxylosis
S. saprophyticus
S. arlettae
S. warneri
E. gallinarum
Molecular identification of staphylococci and methicillin
resistance genes
Methicillin resistance markers in genus
Staphylococcus & mecA by duplex PCR
Detection of ESBL/MBL/AmpC genes in Gram
-negative bacteria by mPCR
56%
14%
26%
5%
29%
0% 10% 20% 30% 40% 50% 60%
Other GNB
Klebsiella spp
Shigella spp
E.coli
Beta-lactamases
Beta-lactamases
6%
10%
10%
27%
46%
5%
8%
0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50%
Beta-lactam
Tetracycline
Sulfa-drugs
Macrolides
Novobiocin
Antibiotic residues in Assam
Antibiotic residues in Haryana
Antibiotic residue prevalence in Assam and Haryana, and the proportional distribution of the detected
antibiotics