IMPROVING THE DIAGNOSTIC METHODS AND PROCESSES FOR THE IDENTIFICATION AND CHARACTERIZATION OF BRACHYSPIRA
Date
2021-05-28
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Thesis
Degree Level
Doctoral
Abstract
There are no standardized antimicrobial susceptibility test methods or interpretative criteria for Brachyspira spp. and laboratories rely heavily on in-house methods that yield highly variable test results. Despite advances in diagnostic technologies, treatment choices for swine dysentery are most often empiric, which can lead to increasing levels of antimicrobial resistance. The objectives of this study were (1) to develop a standardized agar dilution method and describe the antimicrobial susceptibility of a collection of Brachyspira isolates, (2) to develop a feasible methodology for antimicrobial susceptibility testing to include in the diagnostic workflow; including an analysis of accuracy and cost for different diagnostic approaches, (3) to develop assay selection decision trees based on the diagnostic objectives and (4) to investigate the genetic determinants of resistance to protein synthesis inhibitory drugs. To obtain consistent minimum inhibitory concentrations (MICs) between replicates in susceptibility testing for all Brachyspira spp. the media (TSA based), temperature (42°C), and the starting inoculum size (1-2 × 10⁸ CFU/ ml) were determined. A standard antibiotic susceptibility test (agar dilution) with good reproducibility (80-100%) was developed. According to the test results B. hyodysenteriae and B. hampsonii isolates were profoundly susceptible to pleuromutilin drugs while some of B. pilosicoli isolates indicated high MICs (MIC > 8 µg/ml) for pleuromutilin drugs. Moreover, highly variable susceptibility test results for tylosin, tylvalosin, and lincomycin were observed amongst all species. The material and technical cost for all the diagnostic tests performed in the WCVM diagnostic laboratory were calculated, including a modified (miniaturized) agar dilution test. Further, decision trees were developed to select the most cost-effective test bundle based on the diagnostic objective. Using whole genome sequencing, isolates with higher MICs to macrolides were found to carry either a resistant gene called lnuC or have SNPs (A2058G or A2059G both) while isolates with higher MICs for pleuromutilin drugs were found to have the tvaB gene. None of these resistance genes or SNPs were identified in isolates with lower MICs for those drugs. A multiplex PCR was developed to screen the two resistance genes (lnuC and tvaB) and predict the phenotypic resistance of clinical isolates in future. In conclusion, overall findings and novel test methods that were developed will help to improve the diagnostic workflow for the identification of Brachyspira-associated diseases including swine dysentery and support veterinarians for rapid diagnosis and evidence-based selection of antimicrobials to improve the treatment outcome.
Description
Keywords
Brachyspira, antimicrobial susceptibility testing
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Veterinary Microbiology
Program
Veterinary Microbiology