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.

FIG. 1.

DNA products from PCR of ITS in R. microsporus. Lane M, molecular marker Lambda AvaII digest; lane 1, strain P11; lane 2, strain P12; lane 3; strain D3-1; lane 4, strain D4-1; lane 5, strain P2 (positive control); lane 6, negative control containing DNase I-treated distilled water.

FIG. 2.

Sequence traces from direct sequencing of the purified bands of R. microsporus shown in Fig. 1: strain P11 (A), strain P12 (B), strain D3-1 (C), and strain D4-1(D). Examples of triple peaks in strains P12, D3-1, and D4-1 are indicated by arrows.

FIG. 3.

Multiple alignment of ITS sequences of R. microsporus. (A) Strain P11. (B) Strain P12. (C) Strain D3-1. (D) Strain D4-1. ITS1 and ITS2 are shaded in gray.

FIG. 3.

Multiple alignment of ITS sequences of R. microsporus. (A) Strain P11. (B) Strain P12. (C) Strain D3-1. (D) Strain D4-1. ITS1 and ITS2 are shaded in gray.

FIG. 3.

Multiple alignment of ITS sequences of R. microsporus. (A) Strain P11. (B) Strain P12. (C) Strain D3-1. (D) Strain D4-1. ITS1 and ITS2 are shaded in gray.

FIG. 4.

Phylogenetic tree showing the relationship of the four strains of R. microsporus. The tree was inferred from ITS sequence data (737 nucleotide positions) by the neighbor-joining method and was rooted using Absidia blakesleeana (AY944894). The scale bar indicates the estimated number of substitutions per 200 bases. Numbers at nodes indicate levels of bootstrap support calculated from 1,000 trees.