Location: Arthropod-borne Animal Diseases Research
Project Number: 3020-32000-018-012-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2020
End Date: Jul 31, 2025
Objective:
(1) To determine the effects of abiotic factors such as operation size (heads of cattle), location, time, weather, manure management practices, pest management, etc. on the abundance, prevalence, richness and diversity of antimicrobial-resistant (AMR) bacteria in flies and environmental samples (manure, water, feed). (2) To characterize genotype and antimicrobial resistance genes (ARGs) from enteric strains that are shared among flies and environmental samples through whole genome sequencing (WGS).
Approach:
Sites (n=6) are small KS feedlots. Samples (n=10 each) of male and female flies, manure, feed and water will be collected 2x/month when flies are present (~April-October). Abiotic variables (e.g., date, weather data, location, animal diet, manure and pest management and proximity to other livestock) will be collected. Homogenized samples will be replica plated on MacConkey agar (or equivalent) with antibiotics used in cattle production, e.g. florfenicol, tetracycline, enrofloxacin and ceftiofur, and grown at 37C for 24-48 h to enumerate non-fastidious enteric, AMR isolates. Morphotypes will be identified to species by molecular methods (16S, or other taxonomic marker, PCR and sequencing). A subset of isolates (e.g. those that are multidrug resistant) that are present across flies and environmental samples will be selected for genotype and ARG analysis via whole-genome sequencing (WGS). Isolates will be sequenced to a depth of 60-100x coverage using 150x150 paired end reads on an Illumina NovaSeq. Reads will be quality filtered using Trimmomatic and de novo assembled using SPAdes. Identification of AMR genes will be performed using automated gene annotation on the RAST server, CARD (comprehensive antibiotic resistance database), and manual curation. Multivariate models will be used to determine the effect of fly sex, date, operation size, location and other abiotic variables on (1) AMR bacterial abundance within sample type; (2) prevalence of AMR phenotypes and genotypes among sample types; and (3) correlative properties between these metrics (fixed effects and response variables).
