Location: Produce Safety and Microbiology Research
Project Number: 2030-42000-055-009-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Apr 1, 2018
End Date: Mar 31, 2023
Objective:
The long-term goal is to further understand emergence, spread and persistence of antimicrobial resistance (AMR) in agricultural ecosystems. To contribute to this long-term goal, in current project we will employ Campylobacter as model to achieve the following specific objective. 1) Identify mutations accruing upon progressive exposure of Campylobacter jejuni and C. coli to increasing amounts of erythromycin or gentamicin in vitro.; 2) identify erythromycin or gentamicin resistance mutations and additional persistence mutations accruing in chicks in vivo; 3) assess AMR spread via lateral gene transfer of mobile genetic elements-associated resistance determinants in vivo and in water microcosms; and 4) assessment of fitness costs to the bacteria that may impact AMR spread.
Approach:
The genomic changes that accrue during selection for erythromycin (Ery) and gentamicin (Gen) resistances will be determined by whole genome sequencing (WGS) for two C. jejuni strains and two C. coli strains. During stepwise selection of more resistant progeny in vitro, resistant isolates descending from each parent strain that have increased resistance will be characterized for resistance and sequenced to identify mutations. The same parent strains will be used to inoculate and establish colonization in chicks (in vivo). The chicks will then be treated with Ery, Gen or no antibiotic. Campylobacter will be enumerated every 3 days from freshly excreted fecal droppings and up to 10 isolates will be purified and preserved at each time point. Isolates will be characterized for antimicrobial resistance (AMR) profiles. Besides Ery and Gen, antimicrobials for MIC determinations will include members of other classes, to assess possible general impacts on AMR. The selected isolates from each time point will undergo WGS. Additionally, AMR can be introduced into Campylobacter via lateral transfer events of mobile genetic elements (MGE) possessing AMR genes. These events will be assessed in vivo by co-inoculation of Campylobacter strains possessing an MGE containing Genr and a strains that is Gen sensitive but resistant to another antibiotic. Strains recovered from the poultry will be selected with both antibiotics to identify and enumerate MGE transfer. Finally, the fitness costs of the AMR will be performed by competition experiments of the parent strain compared to a AMR descendent in vivo.
