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Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.

ABSTRACT: Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10-49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n=2), Pichia (Candida) norvegensis (n=2), Candida tropicalis (n=1) and Saccharomyces cerevisiae (n=1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC4632790 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.

Zhao Ying Y Tsang Chi-Ching CC Xiao Meng M Cheng Jingwei J Xu Yingchun Y Lau Susanna K P SK Woo Patrick C Y PC

International journal of molecular sciences 20151022 10

Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the ...[more]

PMID: 26506340

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Project description:BackgroundMicrosporidia are spore-forming obligate intracellular parasites that include both emerging pathogens and economically important disease agents. However, little is known about the genetic diversity of microsporidia. Here, we investigated patterns of geographic population structure, intraspecific genetic variation, and recombination in two microsporidian taxa that commonly infect cladocerans of the Daphnia longispina complex in central Europe. Taken together, this information helps elucidate the reproductive mode and life-cycles of these parasite species.MethodsMicrosporidia-infected Daphnia were sampled from seven drinking water reservoirs in the Czech Republic. Two microsporidia species (Berwaldia schaefernai and microsporidium lineage MIC1) were sequenced at the internal transcribed spacer (ITS) region, using the 454 pyrosequencing platform. Geographical structure analyses were performed applying Fisher's exact tests, analyses of molecular variance, and permutational MANOVA. To evaluate the genetic diversity of the ITS region, the number of polymorphic sites and Tajima's and Watterson's estimators of theta were calculated. Tajima's D was also used to determine if the ITS in these taxa evolved neutrally. Finally, neighbour similarity score and pairwise homology index tests were performed to detect recombination events.ResultsWhile there was little variation among Berwaldia parasite strains infecting different host populations, the among-population genetic variation of MIC1 was significant. Likewise, ITS genetic diversity was lower in Berwaldia than in MIC1. Recombination signals were detected only in Berwaldia.ConclusionGenetic tests showed that parasite populations could have expanded recently after a bottleneck or that the ITS could be under negative selection in both microsporidia species. Recombination analyses might indicate cryptic sex in Berwaldia and pure asexuality in MIC1. The differences observed between the two microsporidian species present an exciting opportunity to study the genetic basis of microsporidia-Daphnia coevolution in natural populations, and to better understand reproduction in these parasites.

Dataset Information

Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.

Publications

Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.

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