Incidence of Vkorc1 gene mutations in brown rat (Rattus norvegicus) populations from downtown Madrid and their role in resistance to anticoagulant rodenticide: Implications for rodenticide management

The brown rat (Rattus norvegicus) is considered a pest and the use of anticoagulant rodenticides (ARs) has been considered a valuable method for their management. ARs inhibit the enzyme vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), preventing activation of coagulation factors and provoking death by internal bleeding. Resistance to ARs is associated to Vkorc1 gene mutations. In the present study, we aim to characterize the incidence of mutations in Madrid downtown by using a novel sampling approach, based on a systematic procedure. Madrid central core (approx. 42 km²) was divided in a grid with quadrants of one Km² each and 21 alternating quadrants were selected. Stool samples were collected (120 in total) for DNA extraction and sequencing of targeted genes. The Ser149Ile (S149I) mutation was more frequent in the south and the Glu155Lys (E155K, described for the first time in Spanish brown rats) in the north of the city while double mutants were found in the central area. A geospatial study was performed to relate population density, rat warnings, bromadiolone use and trash production with Vkorc1 mutation frequency. No statistically significant correlations were found, but the higher mutation frequency appeared in areas with higher trash production. Interestingly, the generated mutation frequency map will facilitate the effective management of ARs application by accounting for resistance evolution, serving as a valuable tool to enhance rodent control strategies. Docking analysis was done for comparing mutation effects on VKORC1 sensitivity to ARs showing differences among genotypes binding affinities towards vitamin K and ARs.