The Antibiotic Resistome of Farmed Rainbow Trout Filets Using Smartchip Real-time PCR

Abstract

National audienceIntroduction: The role of food in the routes of transmission of resistant bacteria and antimicrobial resistance genes (ARG) is yet to be explored. There can be a risk of ARG presence on farmed fish filets because of their environmental exposures (animal farms, human activities, aqueous environment). Consequently, it is essential to determine the farmed fish antibiotic resistome. To analyse fresh fish filet resistomes, where bacterial load is known to be low, a highly efficient method like high-throughput qPCR arrays, able to detect and quantify hundreds of selected ARGs in a single run, can be useful. Purpose: The objective was to evaluate the significance of Smartchip RT-PCR technology to analyse the antibiotic resistome profile of rainbow trout filets. Methods: The analyses were performed on both fresh and spoiled rainbow trout filets. The filets were inoculated with antimicrobial-resistant bacterial strains (n=6) at various concentrations (104 to 108 CFU/g) to assess the capacity to detect a specific gene among the microbial communities of the filet. The ARG were detected and quantified using a 245 primer pair set. The set was chosen after bibliography analyses and In Silico verification. The amplification was realised thanks to the Smartchip Real-Time PCR technology (Takara). Results: The ARG detection threshold was determined to be 3.52 log CFU/g. Some ARG were detected at Ct values around 25 on fresh filets (tetL, tetB, sul1, qacEΔ1). In spoiled filets, the quantification of some ARG was enhanced (Ct around 18 to 20). Other genes such as tetS, tetB, strB, mexE, mexF were only detectable in spoiled filets. Significance: The Smartchip real-time PCR technology allows detecting ARG at different presence levels in the bacterial communities of fish filets. It may be interesting to use this tool to investigate and describe the resistome patterns of rainbow trout filets