Frequent occurrence of mixed Enterocytozoon bieneusi infections in humans

Abstract

Enterocytozoon bieneusi (phylum Microsporidia) is a human pathogen with a broad host range. Following the sequencing of 3.8 Mb of the estimated 6-Mb E. bieneusi genome, simple sequence repeats (micro- and minisatellites) were identified. Sequencing of four such repeats from various human and animal E. bieneusi isolates identified extensive sequence polymorphism and enabled the development of a multilocus genotyping method to study the epidemiology of this pathogen. We genotyped E. bieneusi DNA extracted from 197 fecal samples originating from children with diarrhea who were residing in Kampala, Uganda. Three newly identified microsatellite markers and the internal transcribed spacer were PCR amplified, and multiple cloned amplicons for each marker were sequenced from each individual. Most microsatellite sequences were unique to the Ugandan population. Significantly, polymorphism not only was present among isolates but was also found within isolates. This observation suggests that infections with heterogeneous E. bieneusi populations are common in this region. However, the data do not exclude that some of the polymorphism originates from divergent paralogs within the genome. The frequent occurrence of multiple sequences within an isolate precluded the identification of multilocus genotypes. This observation raises the possibility that in a region in which the prevalence of E. bieneusi is high, sequencing of uncloned PCR products may not be adequate for multilocus genotyping.

Fig 1

Rarefaction analyses of MS1, MS3, and MS7 diversity from Uganda and other countries. (A) Analysis of Ugandan microsatellite genotypes and comparison with ITS sequence diversity. MS1 is significantly more diverse than other markers. (B) Diversity of Ugandan microsatellite sequences exceeds that observed in published sequences from a geographically diverse collection of sequences. Error bars indicate 95% confidence intervals.

Fig 2

Microsatellite and ITS sequence diversity in 25 Ugandan isolates. Diversity was calculated using the Shannon index and is plotted on the z axis. The shortest bars, as visible for isolate 7 MS3 and MS7, represent zero diversity (all sequences identical). Blank fields indicate no data.

Fig 3

Principal coordinate analysis for three microsatellite markers by country. Pairwise distances between sequences of cloned PCR products from Uganda and published sequences from seven countries were included in this analysis. The distances were calculated using two different models (onegap and eachgap) as detailed in Materials and Methods. A more dispersed topology in the eachgap analysis is consistent with most of the sequence differences being length polymorphisms.

Fig 4

Regression analysis of genetic diversity on geographic distance. According to the Mantel test, only the MS1 genetic distance is independent of geographical distance. Red, MS1; blue, MS3; green, MS7. Linear regression lines are dashed.