MONITORING THE ANITMICROBIAL RESISTANCE PROFILE OF BACTERIAL FOOD-BORNE PATHOGENS IN HUMANS, FOOD ANIMALS AND RETAIL MEAT IN INDIA

• Sponsoring Institution: Cooperating Schools of Veterinary Medicine
• Project Status: TERMINATED
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 1005898
• Project No.: NCV-VMAH-56534
• Project Start Date: Jan 29, 2015
• Project End Date: Sep 30, 2015

Project Director
Thakur, SI.

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
College of Vet Medicine

Non Technical Summary
Foodborne diseases are increasingly recognized as a major public health problem both nationallyand globally. The burden of illnesses associated with bacterial foodborne pathogens iscompounded by the increase in rise of infections caused by pathogens that are resistant tomultiple antimicrobials. The problem of antimicrobial resistance (AR) is primarily seen inenteric bacterial pathogens including Salmonella, Campylobacter, Escherichia coli andEnterococcus. Antimicrobial use in humans and animals has been shown to be stronglyassociated with the development of resistance in known foodborne pathogens. The developedcountries have specific organizations that monitor the use of antimicrobials and trends inresistance development, in humans and agriculture. However, no such monitoring/surveillancesystems are available in many developing countries, in particular in the South East Asia region.This is concerning since in developing countries the use of antimicrobials is not regulated as it isin the US or other developed countries. It is imperative that we conduct studies in India todetermine the status of AR in bacterial pathogens in humans, food animals and retail meat.Many international and national organizations have called for increased surveillance ofantimicrobial use in humans and animals and prevalence of AR in human pathogens andindicator bacteria in developing countries. The main objective of our proposal is to conduct afield based study to determine the prevalence and AR profile of Salmonella, Campylobacter,Escherichia coli and Enterococcus in humans, food animals and retail meat. We will also collectrelevant antimicrobial use data using questionnaires from the hospitals and farm owners todetermine attribution of antimicrobial use in AR development.Specific objectives include: 1) Perform systematic review of existing data and information,which is relevant to our project, collected by national agencies and researchers in India; 2)Establish the prevalence of Salmonella, Campylobacter, Escherichia coli and Enterococcus inhumans, food animals and retail meats in four distinct metropolitan regions of India; 3)Determine the antimicrobial susceptibility profiles of the above pathogens against a panel ofantimicrobials representing different classes of antimicrobials, and; 4) Conduct analysis todetermine the attribution of antimicrobial use in humans and food animals on AR development inthe above pathogens.We have established a team of investigators from the US and India who will partner in acollaborative effort to work towards meeting the above objectives. The information created inthis study will aid us in meeting the demand for global surveillance of antimicrobial use andresistance development to improve public health. Through the study we will also conduct a needsassessment for capacity building including training of pertinent personnel and establishment of alaboratory-based monitoring/surveillance system for antimicrobial resistant foodborne pathogensin India. We are confident that the data generated in the study will provide the groundwork toconduct future quantitative studies on understanding the burden of antimicrobial resistantpathogens and factors that lead to the development of AR in these pathogens in humans, foodanimals and retail meat in India.

Animal Health Component

25%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3820	1160	50%
712	3299	1160	50%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3299 - Poultry, general/other; 3820 - Goats, meat, and mohair;

Field Of Science
1160 - Pathology;

Keywords

india

foodborne disease

public health

Goals / Objectives
Specific objectives include: 1) Perform systematic review of existing data and information,which is relevant to our project, collected by national agencies and researchers in India; 2)Establish the prevalence of Salmonella, Campylobacter, Escherichia coli and Enterococcus inhumans, food animals and retail meats in four distinct metropolitan regions of India; 3)Determine the antimicrobial susceptibility profiles of the above pathogens against a panel ofantimicrobials representing different classes of antimicrobials, and; 4) Conduct analysis todetermine the attribution of antimicrobial use in humans and food animals on AR development inthe above pathogens.

Project Methods
Participant recruitment and survey methodology from 20 hosptials and food animal farms (poultry and goat) will be recruited for answering a questionnaire. Farm Management and Antimicrobial Use Data: At the farm, we will collect information onthe antimicrobial use during production phase, animal flow, stocking density, barn hygiene, herdhealth, production performance, information on concurrent infections, waste disposal andmanagement system, feed type, temperature on day of sampling and trucking facility.Biosecurity: Information on the biosecurity measures at the farm including facilities for handwashing before entering the farm, access to a restroom, personal hygiene practices for farmworkers, and proximity to other vertebrates and invertebrates will be collected.We aim to sample human, food animals and retail meat samples from different humanhospitals, animal farms and retail meat (poultry and goat meat) from supermarkets/butcher shopslocated in four metropolitan regions (Chandigarh, Haldwani, Pantnagar and Bareilly) of India.The human population of Chandigarh is 9.6 million, Haldwani is 0.5 million, Pantnagar is 40,000and Bareilly is 1 million. Both these regions have numerous broiler and goat farms which caterto supermarkets and butcher shops for selling the retail meat products. A total of 3000 samplesincluding 1000 samples each from humans, food animals (broiler and goat) and retail meat(chicken and goat meat) will collected in the study. All the sampling will be completed over aperiod of 18 months which will amount to collecting approximately 30 samples each fromhumans, food animals and retail meat per month. Pathogen isolation and characterization will bedone simultaneously. We will attempt to match human samples collection with farm animals andretail meat samples during the sampling phase.Antimicrobial susceptibility testing will be performed at the National Salmonellalaboratory (NSL) located in the Division of Veterinary Public Health at the Indian VeterinaryResearch Institute, Izatnagar, India.Salmonella, Campylobacter, E. coli and Enterococcus prevalence from the differentsources (humans, food animals and retail meats) will be compared using the likelihood ratio χ2test, adjusting for dependencies within the sampling scheme (e.g., within farm, plot) using multilevelmixed logistic regression models (Stata 12.0, College Station, TX) and exact methodswherever applicable.

Progress 01/29/15 to 09/30/15

Outputs
Target Audience:World Health Organization (sponsor); India Changes/Problems:Multiple challenges were faced in procurring supplies and shipping to collaborating centers in India. Delays were also due to setting up sub-contract agreements between NCSU and the collaborating centers and procuring the right transport permits to ship and receive the samples within India. What opportunities for training and professional development has the project provided?Opportunity for graduate students and laboratory technicians in India to collect samples and perform detailed characterization of the isolates. Contributed to graduate student research work. How have the results been disseminated to communities of interest?Report submitted to World Health Organization (sponsor). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Using conventional isolation procedures involving enrichment in Bolton broth supplemented with 5% sheep blood followed by plating in mCCDA medium, a total of 56 isolates were obtained. All the 56 putative Campylobacter isolates possessed characteristic small (1-2mm), circular, smooth, watery and spreading in nature, flat to slightly raised, greyish coloured colonies, which were easily recognized on mCCDA plates after incubation for 48 hrs. The Biochemical tests, viz., oxidase, catalase, urease, nitrate reduction, H2S production on TSI, indoxyl acetate hydrolysis, hippurate hydrolysis, performed during the course of present study showed characteristic reaction. All the 56 isolates were positive for oxidase, catalase, nitrate reduction and indoxyl acetate hydrolysis tests and negative for urease and H2S production on TSI. Campylobacter genus confirmation using latex agglutination test was performed on all 56 isolates as per the recommendations of OIE terrestrial manual (2008). All the isolates produced visible agglutination along with positive control, confirming Campylobacter as the genus. Amplification and separation of DNA from Campylobacter isolates yielded two bands, one specific for the genus (857 bp) and other species specific either of 589 bp and 462 bp for C. jejuni and C. coli, respectively. The primers used here were also specific for respective species of Campylobacter as they did not produce any amplification for Salmonella and E. coli. On the basis of mPCR analysis, 50 isolates were differentiated into C. jejuni (35) and C. coli (15) species. Six isolates are still being processed for identification. Majority of the C. jejuni isolates were resistant to Cephalothin (99%), Tetracycline (96%), Suphamethoxazole (96%), and very few were resistant to Gentamicin (5%), Chloramphenicol (7%) and Erythromycin (1%). All C. coli isolates were resistant to Cephalothin, Nalidixic acid, Norfloxacin and tetracycline but susceptible to Chloramphenicol, Erythromycin and Gentamicin. There was a significant difference between the rate of resistance to macrolides of C. coli and C. jejuni isolated in poultry, which amounted to 23% and 3%, respectively. Three genes viz. invA, stn and sopB of Salmonella isolates were targeted for amplification through PCR with specific reported primers of 244 bp, 617 bp and 220 bp and confirmed that isolates underhand were all of Salmonella spp. All 9 confirmed Salmonella serovars amplified a specific primer fragment of 244 bp which is a conserved sequence of invA gene. All the confirmed Salmonella isolates were subjected to PCR for amplification of 617bp length Salmonella enterotoxin (stn) gene and all the isolates amplified the fragment successfully. Nine isolates yielded 220 bp product after amplification of sopB gene. The suspected Salmonella isolates were subjected to multiplex-PCR using Salmonella specific stn and sopB primers, respectively. Multiplex-PCR yield diagnostic amplified DNA bands of molecular size marker at 617 bp and 220 bp respectively in tested Salmonella cultures. The antibiotic discs (Hi-media, Mumbai)) used were consisted of Ciprofloxacin (5 mcg), Norfloxacin (10 mcg), Ampicillin (10 mcg), Gentamicin (10 mcg), Cefalexin (30 mcg), Tetracycline (30 mcg), Nalidixic acid (30 mcg), Chloremphenicol (30 mcg), Sulphamethazine (300 mcg), Furazolidone (50 mcg), Tobramycin (10mcg), Streptomycin (10mcg) and Amikacin (30mcg). Strains were evaluated as susceptible, intermediate or resistant. The two antibiotics, Chloramphenicol and Gentamicin were found to be 100% effective, whereas varying degree of resistance was shown by other antibiotics: Streptomycin and Furazolidone (66% each), Norfloxacin and Amikacin (50% each), Tetracycline and Ampicillin (22% each), Tobramycin and Ciprofloxacin (11.11% each) and Nalidixic acid (5%). The isolates were resistant to the extent of 100% to Cefalexin and Sulphamethizole or Sulphamethazine.

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