Determination of virulence and antimicrobial resistance genes of Clostridioides difficile in foals through whole genome sequencing.

• Grant number: 24/00964-4
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: August 01, 2024
• End date: November 30, 2027
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Clinics and Surgery
• Principal Investigator: Alexandre Secorun Borges
• Grantee: Fabricio Moreira Cerri
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated scholarship(s): 25/02776-3 - Whole-Genome Sequencing of Clostridioides difficile isolates recovered from horses to screen for Virulence factors and Antimicrobial Resistance Genes, BE.EP.DR

Abstract

Clostridioides difficile is a significant etiological agent of diarrhea in foals and represents a substantial problem for other animals and humans. This anaerobic bacterium harbors virulence genes responsible for its toxigenicity, adhesion, motility, sporulation, and antimicrobial resistance. Few studies have determined the importance of these genes in the development of diarrhea in foals and the relationship of isolates with those obtained from other animals, humans, and the environment. The objective of this project is to determine the virulence and antimicrobial resistance genes and the level of similarity among isolates of C. difficile obtained from foals (with and without diarrhea). The samples used in the project will come from a bank of fecal samples from foals (with and without diarrhea) and fecal samples to be collected from foals in stud farms in the Botucatu region with a history of diarrhea, subjected to C. difficile isolation. The collection of these new samples will continue until 50 toxigenic isolates are available for whole genome sequencing (WGS). Bacterial isolation will be performed by inoculation on non-selective medium supplemented with sodium taurocholate, followed by alcohol shock and seeding on plates of C. difficile Moxalactam Norfloxacin agar (CDMN) incubated under anaerobic conditions. Subsequently, bacterial DNA will be extracted from C. difficile colonies. Multiplex PCR will be performed for the detection of 16s RNA (constituent) genes, tcdA, tcdB, cdtA, and cdtB, ribotyping, and antimicrobial susceptibility testing. DNA libraries will be prepared for WGS (Illumina platform), contig assembly, and determination of MLST. The determination of virulence and antimicrobial resistance genes and alignment of genetic sequences with isolates already described in the literature will be carried out. This project will generate data that will contribute to understanding the epidemiology of C. difficile in foals and its implications one health.