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Cycle 7 (2018 Deadline)

Monitoring of antimicrobial resistance of bacteria for a better health of animals in Tunisia

PI: Lilia Messadi (, Ecole nationale de médecine vétérinaire de Sidi Thabet
U.S. Partner: Charlene Jackson, U.S. National Poultry Research Center
Dates: January 2019 - December 2020

Project Overview:

International organizations such as the World Health Organization, the World Organization for Animal Health, and the UN Food and Agriculture Organization consider antimicrobial resistance (AMR) as a major risk for human, animal, and environmental health. Therapeutic failures are witnessed, with an increasing risk of moving towards a "post-antibiotic era" where common infections could kill humans and animals. Scientists have predicted that by 2050, more than 10 million people could die each year because of AMR. A very recent study shows that between 2000 and 2015, Tunisia ranked as the world's second highest country for consumption of antibiotics, which is very worrying because there is a direct relationship between antibiotic consumption and AMR. Tunisia does not yet have a national strategy for monitoring the AMR of bacteria isolated from animals. Some preliminary studies have been conducted on a few bacterial species, mainly targeting commensal bacteria of healthy animals. It appears that the observed AMR is important in some animal species, especially among poultry, with a risk of transfer of resistance genes or resistant bacteria to consumers and to people in contact with pets or livestock.

The objective of this project is to survey AMR of bacteria at the animal/human/environment interface by targeting animals, food of animal origin, and pests (rats and cockroaches), according to the "One Health" concept. The epidemiological study of resistance and the identification of risk factors in husbandry practices and in veterinary use of antibiotics should results in suggested corrective measures to reduce AMR. For livestock, the team will focus primarily on smallholder farmers with limited income, to provide them with added value through health education messages in order to improve hygiene and prevent infectious diseases of animals. These livestock promotion measures are in line with USAID's development goals. They will target women as much as possible because they are often involved in small farms and are more receptive to educational messages on hygiene. The USG-supported partners will assist by developing laboratory methods for the monitoring of AMR and characterization of resistance genes, and also for training and education programs. Practical training of laboratory technicians and students will improve monitoring methods. Workshops and seminars for students and veterinarians will improve the use of antibiotics to reduce the frequency of resistant bacteria. Consequently, the trained students will be able to develop sensitization messages for breeders and in particular women, who are generally more concerned about hygiene measures. One of the main difficulties would be to convince veterinarians to change their practices regarding antibiotics. The main results of the study will be sent to the decision makers of the Ministry of Agriculture to enable the establishment of a national strategy to fight against this threat.

Summary of Recent Activities

During this quarter, the PI and her team worked on collecting biological samples from several animal species (broilers, cattle, sheep, cockroaches). Feces from healthy or diarrhea animals and milk for cows with mastitis represent the samples collected. These samples are then analyzed in their laboratory by standardized methods to isolate Escherichia coli, indicator or sentinel bacterium; this is very important to monitor because it is very favorable to the acquisition of resistance by chromosomal mutation (vertical transfer) or by acquisition of mobile resistance genes (horizontal transfer). The isolation of this bacterium follows enrichment in buffered peptone water with or without cefotaxime (3rd generation cephalosporin) followed by the use of McConkey agar with or without cefotaxime. Antibiotic resistance of isolated strains is then investigated using the antibiogram or disc method, following EUCAST / SFM recommendations. In total, they tested 21 antibiotics from 8 families, choosing molecules that are widely used in veterinary medicine or that are of interest for public health. The protocol uses a special disposal of antibiotics disks to visualize extended-spectrum beta-lactamases (ESBL) thanks to the synergy image in champagne cork.

A collaboration was developed with Ceva pharmaceutical laboratories (Tunisia) to provide training for some veterinary practitioners, with a view to setting up proximity laboratories specialized in bovine mastitis. It is expected that their laboratory will perform in the future quality control tests to verify their proper functioning and that the antimicrobial sensitivity of isolated strains be tested according to standardized methods, to detect emerging phenomena of resistance.

Relationships have been established with the National Committee Against Antimicrobial Resistance, in association with WHO, to finalize the national plan of action for control. A meeting was held with the Directorate General of Veterinary Services (Ministry of Agriculture) to discuss the National Action Plan to combat antibiotic resistance in the veterinary field.

In the  next 3-6 months, field visits will continue to collect samples (feces, milk) from healthy or sick animals (diarrhea, mastitis...) of various species (horses, dogs, cats, chickens, dromedaries) to complete the database. Laboratory analysis will be continued to enrich the strains library with Escherichia coli strains and to study the main resistance phenomena using phenotypic (antibiogram) and molecular techniques (study of the main resistance genes). The results will be regularly inserted into the database.

On the one hand, students will receive practical training (methods of isolation and identification of bacteria, realization and reading of antibiograms, statistical analysis, and PCR techniques for students in masters or PhD). On the other hand, they will carry out actions to raise awareness about the proper use of antibiotics in the context of our teaching or seminars. They also plan to conduct an antibiogram training workshop for veterinary biologists, in partnership with colleagues in medical microbiology. A seminar will be organized on the occasion of World Antimicrobial Resistance Day.

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