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

Jingwei Cheng,Yingchun Xu,Patrick Woo,Susanna Lau,Meng Xiao,Ying Zhao,Chi-Ching Tsang

doi:10.3390/ijms161025067

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.

Highlights

Molecular identification of fungal pathogens in clinical microbiology laboratories most commonly involves polymerase chain reaction (PCR) amplification of genes and/or intergenic regions in the fungal genome and direct nucleotide sequencing of the purified PCR products
All patients suffered from invasive yeast infections, including peritonitis, mediastinitis, as well as lung abscess and empyema, with the yeasts recovered from the corresponding clinical specimens
We report and describe the first evidence of intra-genomic Internal transcribed spacer region (ITS) sequence heterogeneity in a variety of yeast species recovered from clinical specimens

Summary

Introduction

Molecular identification of fungal pathogens in clinical microbiology laboratories most commonly involves polymerase chain reaction (PCR) amplification of genes and/or intergenic regions in the fungal genome and direct nucleotide sequencing of the purified PCR products. Since most fungi possess more than one nrDNA operon in their genomes [3,4], whether molecular characterization can be used to achieve fungal identification depends very much on the perfect intra-genomic sequence homogeneity in the multiple copies of the nrDNA operons of fungi. Intra-genomic ITS sequence heterogeneity has rarely been described in pathogenic fungi. When present, such ITS sequence heterogeneity within a single genome makes fungal identification based on direct sequencing of the PCR product difficult, because multiple PCR products will result and double or multiple nucleotide peaks will be observed in the sequencing electropherograms. During the process of performing ITS sequencing on 71 isolates of yeasts recovered from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them (unpublished data)

Methods

Results

Conclusion