Dr Ben Jones

Abstract Ascaris species are soil-transmitted helminths that infect humans and livestock mainly in low and middle-income countries. Benzimidazole (BZ) class drugs have predominated for many years in the treatment of Ascaris infections, but persistent use of BZs has already led to widespread resistance in other nematodes, and treatment failure is emerging for Ascaris. Benzimidazoles act by binding to β-tubulin proteins and destabilising microtubules. Three mutations in the β-tubulin protein family are associated with BZ resistance. Seven shared β-tubulin isotypes were identified in Ascaris lumbricoides and A. suum genomes. Benzimidazoles were predicted to bind to all β-tubulin isotypes using in silico docking, demonstrating that the selectivity of BZs to interact with one or two β-tubulin isotypes is likely the result of isotype expression levels affecting the frequency of interaction. Ascaris β-tubulin isotype A clusters with helminth β-tubulins previously shown to interact with BZ. Molecular dynamics simulations using β-tubulin isotype A highlighted the key role of amino acid E198 in BZ-β-tubulin interactions. Simulations indicated that mutations at amino acids E198A and F200Y alter binding of BZ, whereas there was no obvious effect of the F167Y mutation. In conclusion, the key interactions vital for BZ binding with β-tubulins have been identified and show how mutations can lead to resistance in nematodes.

The efficacy of benzimidazole anthelmintics can vary depending on the target parasite, with Ascaris nematodes being highly responsive, and whipworms being less responsive. Anthelmintic resistance has become widespread, particularly in strongyle nematodes such as Haemonchus contortus in ruminants, and resistance has recently been detected in hookworms of humans and dogs. Past work has shown that there are multiple β-tubulin isotypes in helminths, yet only a few of these contribute to benzimidazole interactions and resistance. The β-tubulin isotypes of ascarids and soil-transmitted helminths were identified by mining available genome data, and phylogenetic analysis showed that the ascarids share a similar repertoire of seven β-tubulin isotypes. Strongyles also have a consistent pattern of four β-tubulin isotypes. In contrast, the whipworms only have two isotypes, with one of these clustering more basally and distinct from any other group. Key β-tubulin isotypes selected based on previous studies were the focus of in silico molecular docking simulations to look at the interactions with benzimidazoles. These showed that all β-tubulins had similar interactions with benzimidazoles and maintained the key bond with residue E198 in all species, indicating similar mechanisms of action. However, the interaction was stronger and more consistent in the strongyles and whipworms than it was in the ascarids. Alteration of β-tubulin isotypes with the common resistance-associated mutations originally identified in H. contortus resulted in similar interaction modeling for all species. In conclusion, ascarids, strongyles, and whipworms all have their own unique repertoire of β-tubulins, which could explain why benzimidazole resistance and susceptibility varies between these groups of parasites. These data complement recent work that has highlighted the roles of essential residues in benzimidazole drug binding and shows that there is a separation between strongyle parasites that frequently develop resistance and ascarid parasites, which have been much less prone to developing resistance

The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding β-tubulin proteins, the principal targets of BZ drugs. First, the β-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different β-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations. This study demonstrated the presence of at least seven β-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the β-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of β-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.

BackgroundDespite considerable recent reductions in antimicrobial use, the UK gamebird industry continues to struggle with production diseases during the rearing season, necessitating significant antibiotic use. This observational study investigated the presence of genes conferring resistance to beta-lactam antibiotics within industry-reared pheasants and red-legged partridges in the UK. MethodsDNA was extracted from 60 pooled caecal samples collected from gamebirds at routine postmortem examinations during the rearing season. Genes encoding extended-spectrum beta-lactamases (ESBL) were detected by PCR and the corresponding alleles were determined. ResultsOver half (53%) of the samples harboured genes encoding bla(TEM) resistance, with bla(SHV) identified in 20% of samples. The bla(TEM) gene was more common on sites with higher antibiotic use, whereas bla(SHV) was predominantly found in birds younger than 5 weeks. Genotyping of the identified resistance genes revealed the presence of bla(TEM-1), bla(SHV-1) and bla(SHV-11) alleles. LimitationsThis was a small-scale study conducted at four sites in southern England. ConclusionThis is the first report of the presence of ESBL genes in gamebirds, highlighting the need for further research into antimicrobial resistance in UK gamebirds.

Ascaris species are soil-transmitted helminths that infect humans and livestock mainly in low and middle-income countries. Benzimidazole (BZ) class drugs have predominated for many years in the treatment of Ascaris infections, but persistent use of BZs has already led to widespread resistance in other nematodes, and treatment failure is emerging for Ascaris. Benzimidazoles act by binding to β-tubulin proteins and destabilising microtubules. Three mutations in the β-tubulin protein family are associated with BZ resistance. Seven shared β-tubulin isotypes were identified in Ascaris lumbricoides and A. suum genomes. Benzimidazoles were predicted to bind to all β-tubulin isotypes using in silico docking, demonstrating that the selectivity of BZs to interact with one or two β-tubulin isotypes is likely the result of isotype expression levels affecting the frequency of interaction. Ascaris β-tubulin isotype A clusters with helminth β-tubulins previously shown to interact with BZ. Molecular dynamics simulations using β-tubulin isotype A highlighted the key role of amino acid E198 in BZ-β-tubulin interactions. Simulations indicated that mutations at amino acids E198A and F200Y alter binding of BZ, whereas there was no obvious effect of the F167Y mutation. In conclusion, the key interactions vital for BZ binding with β-tubulins have been identified and show how mutations can lead to resistance in nematodes.