Development of a Multilocus Sequence Typing Tool for High-Resolution Genotyping of Enterocytozoon bieneusi

Abstract

ABSTRACTThus far, genotyping ofEnterocytozoon bieneusihas been based solely on DNA sequence analysis of the internal transcribed spacer (ITS) of the rRNA gene. Both host-adapted and zoonotic (human-pathogenic) genotypes ofE. bieneusihave been identified. In this study, we searched for microsatellite and minisatellite sequences in the whole-genome sequence database ofE. bieneusiisolate H348. Seven potential targets (MS1 to MS7) were identified. Testing of the seven targets by PCR using two human-pathogenicE. bieneusigenotypes (A and Peru10) led to the selection of four targets (MS1, MS3, MS4, and MS7). Further analysis of the four loci with an additional 24 specimens of both host-adapted and zoonoticE. bieneusigenotypes indicated that most host-adapted genotypes were not amplified by PCR targeting these loci. In contrast, 10 or 11 of the 13 specimens of the zoonotic genotypes were amplified by PCR at each locus. Altogether, 12, 8, 7, and 11 genotypes of were identified at MS1, MS3, MS4, and MS7, respectively. Phylogenetic analysis of the nucleotide sequences obtained produced a genetic relationship that was similar to the one at the ITS locus, with the formation of a large group of zoonotic genotypes that included mostE. bieneusigenotypes in humans. Thus, a multilocus sequence typing tool was developed for high-resolution genotyping ofE. bieneusi.Data obtained in the study should also have implications for understanding the taxonomy ofEnterocytozoonspp., the public health significance ofE. bieneusiin animals, and the sources of humanE. bieneusiinfections.