Internal Transcribed Spacer Region Sequence Heterogeneity in Rhizopus microsporus : Implications for Molecular Diagnosis in Clinical Microbiology Laboratories

Abstract

Although internal transcribed spacer region (ITS) sequence heterogeneity has been reported in a few fungal species, it has very rarely been reported in pathogenic fungi and has never been described in Mucorales, causes of the highly fatal mucormycosis. In a recent outbreak investigation of intestinal mucormycosis due to Rhizopus microsporus infection in patients with hematological malignancies, PCR of the ITS of four of the 28 R. microsporus strains, P11, P12, D3-1, and D4-1, showed thick bands at about 700 bp. Direct sequencing of the purified bands showed frequent double peaks along all of the sequence traces and occasional triple peaks for P12, D3-1, and D4-1. The thick bands of the four R. microsporus strains were purified and cloned. Sequencing of 10 clones for each strain revealed two different ITS sequences for P11 and three different ITS sequences for P12, D3-1, and D4-1. Variations in ITS sequence among the different ribosomal DNA (rDNA) operons in the same strain were observed in only ITS1 and ITS2 and not the 5.8S rDNA region. One copy of P11, P12, and D4-1, respectively, and one copy of P11, P12, D3-1, and D4-1, respectively, showed identical sequences. This represents the first evidence of ITS sequence heterogeneity in Mucorales. ITS sequence heterogeneity is an obstacle to molecular identification and genotyping of fungi in clinical microbiology laboratories. When thick bands and double peaks are observed during PCR sequencing of a gene target, such a strain should be sent to reference laboratories proficient in molecular technologies for further identification and/or genotyping.Genes and intergenic regions of ribosomal DNA (rDNA) operons are the most widely used targets for molecular identification of bacteria and fungi in clinical microbiology laboratories. For bacterial identification, the 16S rDNA gene is the primary target to amplify and sequence (28), whereas for fungi, the 18S rDNA gene and internal transcribed spacer region (ITS) comprising the ITS1-5.8S-ITS2 rDNA gene cluster are commonly used, depending on the group of fungi being identified (4,10,23,27). Irrespective of the target, such a molecular identification technique usually involves PCR amplification of the target and purification and direct sequencing of the PCR product. Since most bacterial and fungal genomes contain more than one rDNA operon, the success of using this technology relies on sequence homogeneity in the various copies of targets in the rDNA operons within the genome of the bacterium or fungus.Interoperon heterogeneities for 16S rDNA genes have been reported in a number of bacteria (3,12). Recently, we reported rDNA operon heterogeneity in a novel genus and species of bacterium, Anaerospora hongkongensis, isolated from an intravenous drug user (25). When present, such rDNA operon heterogeneity will pose difficulties for direct sequencing of the PCR product for bacterial identification as double or multiple nucleotide peaks will be present in the sequence traces. Although ITS sequence heterogeneity has been reported ...

Simultaneous Detection and Identification of Aspergillus and Mucorales Species in Tissues Collected from Patients with Fungal Rhinosinusitis

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Advantages of Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry as a Rapid Diagnostic Tool for Identification of Yeasts and Mycobacteria in the Clinical Microbiological Laboratory

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Misidentification of Aspergillus nomius and Aspergillus tamarii as Aspergillus flavus: Characterization by Internal Transcribed Spacer, β-Tubulin, and Calmodulin Gene Sequencing, Metabolic Fingerprinting, and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

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