Molecular evidence of widespread benzimidazole drug resistance in Ancylostoma caninum from domestic dogs throughout the USA and discovery of a novel isotype-1 β-tubulin benzimidazole resistance mutation.

Ancylostoma caninum is an important zoonotic gastrointestinal nematode of dogs worldwide and a close relative of human hookworms. We recently reported that racing greyhounds in the USA are infected with A. caninum that are commonly resistant to multiple anthelmintics. Benzimidazole resistance in A. caninum in greyhounds was associated with a high frequency of the canonical F167Y(TTC>TAC) isotype-1 {beta}-tubulin mutation. In this work, we show that benzimidazole resistance is remarkably widespread in A. caninum from domestic dogs across the USA. First, we identified and showed the functional significance of a novel benzimidazole isotype-1 {beta}-tubulin resistance mutation, Q134H(CAA>CAT). Several benzimidazole resistant A. caninum isolates from greyhounds with a low frequency of the F167Y(TTC>TAC) mutation had a high frequency of a Q134H(CAA>CAT) mutation not previously reported from any eukaryotic pathogen in the field. Structural modeling predicted that the Q134 residue is directly involved in benzimidazole drug binding and that the 134H substitution would significantly reduce binding affinity. Introduction of the Q134H substitution into the C. elegans {beta}-tubulin gene ben-1, by CRISPR-Cas9 editing, conferred similar levels of resistance as a ben-1 null allele. Deep amplicon sequencing on A. caninum eggs from 685 hookworm positive pet dog fecal samples revealed that both mutations were widespread across the USA, often at high frequency, with prevalences of 49.7% (overall frequency 54.0%) and 31.1% (overall frequency 16.4%) for F167Y(TTC>TAC) and Q134H(CAA>CAT), respectively. Canonical codon 198 and 200 benzimidazole resistance mutations were absent. The F167Y(TTC>TAC) mutation had a significantly higher prevalence and frequency in Western USA than in other regions, which we hypothesize is due to differences in refugia. This work has important implications for companion animal parasite control and the potential emergence of drug resistance in human hookworms. Author SummaryAlthough increasingly common in livestock, no reports of widespread anthelmintic resistance are confirmed in any companion animal or human gastrointestinal nematode parasite to date. The canine hookworm is a common intestinal zoonotic parasite of dogs with severe clinical impacts in young dogs, and for which control is dependent on regular anthelmintic use. We recently reported multiple anthelmintic drug resistance in A. caninum isolates from greyhounds derived from multiple locations in the USA likely caused by long standing intensive treatment regimens in kennels. In this study, we investigated benzimidazole resistance in A. caninum in pet dogs across the USA. We also identified and showed the functional significance of a novel benzimidazole isotype-1 {beta}-tubulin resistance mutation in A. caninum from greyhounds that has not been previously reported in the field for any organism. We then determined that this novel mutation, as well as a previously characterized resistance mutation, were present, often at high frequency, in many A. caninum populations across the USA. This study reports the first evidence of widespread drug resistance for any parasitic nematode of companion animals and illustrates the power of molecular approaches to rapidly assess anthelmintic resistance in a region.