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

Abstract

Ancylostoma caninum is an important zoonotic gastrointestinal nematode of dogs worldwide and a close relative of human hookworms. We recently reported that racing greyhound dogs 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 β-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 β-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 β-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, with prevalences of 49.7% (overall mean frequency 54.0%) and 31.1% (overall mean 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.

Unambiguous identification of Ancylostoma caninum and Uncinaria stenocephala in Australian and New Zealand dogs from faecal samples

“…Our small‐scale study demonstrated absence of detectable alleles in A. caninum and U. stenocephala associated with benzimidazole resistance. Recently Venkatesan et al 11 demonstrated widespread β‐tubulin F167Y(TTC > TAC) mutation present in 50% (156/314) dogs infected with A. caninum using the ‘nemabiome’ approach 11 and Leutenegger et al 10 in 11% (54/511) using A. caninum allele‐specific qPCR tests. In AU6 A. caninum , the persistent shedding of A. caninum , despite repeated treatment reported by the owner, is therefore attributed to re‐infection or non‐compliance.…”

“…Using a locally curated reference database with ITS‐2 sequences for A. caninum , A. ceylanicum , A. braziliense , Ancylostoma duodenale , Nectar americanus and U. stenocephala , we assigned species to ASVs with the aid of phylogenetic analysis in CLC Main Workbench. For isotype‐1 ‐tubulin, ASVs were assembled against a reference A. caninum isotype‐1 β‐tubulin sequence (DQ459314) in CLC Main Workbench and frequency of mutations Q134H(CAA > CAT), F167Y(TTC > TAC), E198A(GAA > GCA), E198L(GAA > TTA), E198V(GAA > GTA) and F200Y(TTC > TAC) identified according to Venkatesan et al 11 The data are available at LabArchives (https://doi.org/10.25833/91fj-cq08). Raw FastQ sequence data were deposited at SRA NCBI BioProject: PRJNA957571.…”

“…9 Recently two studies investigated the extent of benzimidazole resistance across the United States and one found over half of the A. caninum isolates had resistance allele frequency higher than 50% of that of the isotype-1 β-tubulin F167Y. 10,11 Besides A. caninum, Australian dogs are hosts for three other hookworms, including the tropical hookworms Ancylostoma ceylanicum and Ancylostoma braziliense, and the cooler climate hookworm Uncinaria stenocephala. Dogs in New Zealand are traditionally considered to be only infected by U. stenocephala, because A. caninum was assumed to be restricted to racing greyhounds.…”

“…In Australia, A. caninum resistance to pyrantel embonate was experimentally confirmed already in 2008 and the authors urged “for greater vigilance and more judicious use of anthelmintics in small animal practice” 9 . Recently two studies investigated the extent of benzimidazole resistance across the United States and one found over half of the A. caninum isolates had resistance allele frequency higher than 50% of that of the isotype‐1 ‐tubulin F167Y 10,11 . Besides A. caninum , Australian dogs are hosts for three other hookworms, including the tropical hookworms Ancylostoma ceylanicum and Ancylostoma braziliense , and the cooler climate hookworm Uncinaria stenocephala .…”

Mapping resistance-associated anthelmintic interactions in the model nematodeCaenorhabditis elegans

“…Validated resistance mechanisms in parasitic nematodes are restricted to mutations in the cytoskeletal targets of the benzimidazoles [5], but genetic tools in the model nematode Caenorhabditis elegans have helped to identify anthelmintic resistance mechanisms beyond drug target mutations [20]. These include mutations that affect the ability of drugs to accumulate within the worm by altering drug uptake, distribution, efflux, or metabolism [14,16,21,22].…”