Characterization of Entercytozoon bieneusi and Drug Resistance-Associated Mutations Using the β-Tubulin Gene

OBJECTIVE: In the β-tubulin gene of Enterocytozoon bieneusi, five mutations: His6, Phe167, Glu198, Phe200, and Arg241 have been implicated in reducing albendazole efficacy, with Glu198 and Phe200 being particularly significant. The primary objective of this study was to characterize mutations in the β-tubulin gene of E. bieneusi associated with albendazole resistance. Specifically, the study focused on mutations at codons Glu198 and Phe200 using newly developed primers (EbBtu198/200) with DNA extracted from fecal samples of pigs and humans.METHODS: A total of 38 stored DNA samples, comprising 30 from different pigs and eight from different humans, were analyzed to evaluate the sensitivity of the newly designed primers and optimize the polymerase chain reaction (PCR) conditions. The encoded amino acid sequences were examined to identify the mutations at codons 198 and 200 in the β-tubulin gene of E. bieneusi. Additionally, a phylogenetic analysis was performed using the β-tubulin nucleotide sequences to determine the genetic relationships among different isolates.RESULTS: PCR-amplification of the β-tubulin gene yielded a 427 bp product, with a primer sensitivity rate of 94.74%. Sequencing of 18 gene products revealed that ten sequences from pigs corresponded to Haplotype A, while human samples showed four haplotypes: A, B, C, and D. Notably, a mutation resulting in the substitution of glutamic acid with glutamine at codon 198 (E198Q) was identified, uncovering a potential mechanism for albendazole resistance. Phylogenetic analysis applying the maximum likelihood method demonstrated that all β-tubulin sequences formed a monophyletic group, indicating low genetic diversity among the E. bieneusi isolates.CONCLUSION: This study underscores the significance of mutations in the β-tubulin gene, particularly at codon Glu198, as key factors potentially contributing to albendazole resistance in E. bieneusi. These findings may offer valuable insights for improving treatment strategies in patients harboring isolates with such mutations. Furthermore, the β-tubulin gene analysis revealed limited genetic diversity among E. bieneusi isolates, with distinct haplotypes detected in pig and human samples, suggesting possible host-specific adaptations or transmission patterns.