Whole-Genome Sequencing of Clostridioides difficile isolates recovered from horses to screen for Virulence factors and Antimicrobial Resistance Genes

• Grant number: 25/02776-3
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: August 15, 2025
• End date: February 15, 2026
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Clinics and Surgery
• Principal Investigator: Alexandre Secorun Borges
• Grantee: Fabricio Moreira Cerri
• Supervisor: Luis G Arroyo
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Institution abroad: University of Guelph, Canada
• Associated to the scholarship: 24/00964-4 - Determination of virulence and antimicrobial resistance genes of Clostridioides difficile in foals through whole genome sequencing., BP.DR

Abstract

Clostridioides difficile is a pathogen associated with diarrhea in horses of all ages, representing a significant concern for animal health. This condition is exacerbated by the increasing antimicrobial resistance, particularly against widely used drugs such as metronidazole and vancomycin. Recent studies have identified multidrug-resistant isolates in various countries, underscoring the need to further investigate into the origin and dissemination of these resistance genes. The project aims to implement advanced techniques for the isolation and genomic characterization of C. difficile in horses. Fecal samples will be collected from both diarrheic and healthy foals (n=100) and mares (n=100). Approximately 1 g of feces will be suspended in sterile saline solution, followed by plating on chromogenic medium and incubation under anaerobic conditions at 37°C for 48 hours. Isolate identification will be based on morphological characteristics specific to the culture medium, alongside bacterial DNA extraction. The extracted DNA will be processed for library preparation using the NEBNext Ultra II FS DNA Library Prep Kit, targeting fragment sizes of 800-1000 base pairs. Sequencing will be performed on the Illumina HiSeq2500 platform, generating high-quality paired-end reads. Genome assembly will be conducted using the Unicycler software. Multilocus sequence typing (MLST) will be performed using the PubMLST database, while single nucleotide polymorphism (SNP)-based phylogenetic analysis will incorporate C. difficile isolates from horses, humans, and other species available in genomic databases. Antimicrobial resistance will be assessed using the Resistance Gene Identifier (CARD) tool, applying stringent criteria to identify resistance genes.