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J Clin Microbiol. 2010 January; 48(1): 208–214.
Published online 2009 November 11. doi:  10.1128/JCM.01750-09
PMCID: PMC2812305

Internal Transcribed Spacer Region Sequence Heterogeneity in Rhizopus microsporus: Implications for Molecular Diagnosis in Clinical Microbiology Laboratories [down-pointing small open triangle]

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 in a few fungal species (13, 15, 22), it has very rarely been reported in pathogenic fungi and has never been described in members of the order Mucorales, the etiological agents of the highly fatal mucormycosis (1, 18, 19). Recently, during the outbreak investigation of intestinal mucormycosis due to Rhizopus microsporus in patients with hematological malignancies, 28 strains of R. microsporus were subjected to ITS sequencing (5). Direct sequencing of the PCR products from the 28 strains showed unambiguous sequence in 24 of them (5). For the other four strains, double peaks were observed frequently in the sequence traces. We hypothesize that these four strains possess ITS sequence heterogeneity. To test this hypothesis, we cloned the PCR products of these four strains and sequenced 10 clones from each strain. In this article, we report this phenomenon of ITS sequence heterogeneity in R. microsporus. The implications for molecular diagnosis in clinical microbiology laboratories are also discussed.

MATERIALS AND METHODS

Strains.

The four strains of R. microsporus used in this study were isolated from two patients (P11 and P12) and two tablets of allopurinol (D3-1 and D4-1) during the outbreak investigation of intestinal mucormycosis in patients with hematological malignancies in Hong Kong (5). All four strains were identified to be R. microsporus by their morphological appearance and scanning electron microscopy (5).

DNA extraction.

Fungal DNA extraction was performed as described in our previous publications (24, 26). Briefly, DNA was extracted from 1 g of fungal cells in 10 ml of distilled water using a DNeasy plant minikit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The extracted DNA was eluted in 50 μl of kit buffer AE, the resultant mixture was diluted 10 times, and 1 μl of the diluted extract was used for PCR.

PCR, gel electrophoresis, and ITS sequencing.

PCR amplification and DNA sequencing of the ITS regions of the four strains of R. microsporus were performed according to published protocols (23, 27). Briefly, DNase I-treated distilled water and PCR Master Mix (which contains deoxynucleoside triphosphates [dNTPs], PCR buffer, and Taq polymerase) were used in all PCRs by adding 1 U of DNase I (Pharmacia, Sweden) to 40 μl of distilled water or PCR Master Mix and incubating the mixture at 25°C for 15 min and subsequently at 95°C for 10 min to inactivate the DNase I. The fungal DNA extract and controls were amplified with 0.5 μM primers (ITS1, 5′-TCCGTAGGTGAACCTGCGG-3′; ITS4, 5′-TCCTCCGCTTATTGATATGC-3′) (Gibco BRL, Rockville, MD). The PCR mixture (25 μl) contained fungal DNA, PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, and 0.01% gelatin), 200 μM each dNTP, and 1.0 U of Taq polymerase (Applied Biosystems, Foster City, CA). The mixtures were amplified in 40 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min, with a final extension at 72°C for 10 min in an automated thermal cycler (Applied Biosystem, Foster City, CA). R. microsporus strain P2 was used as the positive control, and DNase I-treated distilled water was the negative control (5). Ten microliters of each amplified product was electrophoresed in 1.5% (wt/vol) agarose gel, with a molecular size marker (Lambda AvaII digest; Fermentas, Ontario, Canada) in parallel. Electrophoresis in Tris-borate-EDTA buffer was performed at 100 V for 1.5 h. The gel was stained with ethidium bromide (0.5 μg/ml) for 15 min, rinsed, and photographed under UV light illumination.

The PCR products were gel purified using a QIAquick PCR purification kit (QIAgen, Hilden, Germany). Both strands of the purified PCR product for each strain were sequenced twice with an ABI Prism 3700 DNA Analyzer (Applied Biosystems, Foster City, CA) and using the PCR primers ITS1 and ITS4. In addition, the purified PCR products were also cloned into the pT-Adv vector (BD Biosciences) according to the manufacturer's instructions. Both strands of 10 of the clones for each strain were sequenced twice, using primers ITS1 and ITS4. The sequences of the cloned PCR products were compared with known ITS gene sequences of closely related species in the GenBank by multiple sequence alignment using ClustalX, version 1.83 (20).

Phylogenetic characterization.

Phylogenetic tree construction was performed using the neighbor-joining method with ClustalX, version 1.83. The trees were constructed by the neighbor-joining method using a Jukes-Cantor correction. A total of 737 nucleotide positions were included in the analysis.

Nucleotide sequence accession numbers.

The ITS sequences of the four strains of R. microsporus have been deposited in the GenBank under accession numbers GQ502275 to GQ502285.

RESULTS

Direct ITS sequencing.

PCR of the ITS regions of the four R. microsporus strains, P11, P12, D3-1, and D4-1, showed thick bands at about 700 bp (Fig. (Fig.1).1). Direct sequencing of the purified bands showed double peaks frequently along all of the sequence traces (Fig. (Fig.2).2). For the sequence traces of P12 D3-1 and D4-1, occasional triple peaks were also observed (Fig. 2B, C, and D).

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 ...
FIG. 2.
Sequence traces from direct sequencing of the purified bands of R. microsporus shown in Fig. Fig.1:1: 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 ...

Sequencing of cloned PCR products.

The thick bands of the four R. microsporus strains were purified and cloned into the pT-Adv vector. Sequencing of 10 clones for each strain revealed two different ITS sequences for strain P11 (Fig. (Fig.3A)3A) and three different ITS sequences for strains P12, D3-1, and D4-1 (Fig. 3B, C, and D). For strain P11, 2 of the 10 sequences were of one type (Fig. (Fig.3A,3A, copy 1) and 8 were of a second type (Fig. (Fig.3B,3B, copy 2); for strain P12, 5 of the 10 sequences were of one type (Fig. (Fig.3B,3B, copy 1), 4 were of a second type (Fig. (Fig.3B,3B, copy 2), and 1 was of a third type (Fig. (Fig.3B,3B, copy 3); for strain D3-1, 3 of the 10 sequences were of one type (Fig. (Fig.3C,3C, copy 1), 3 were of a second type (Fig. (Fig.3C,3C, copy 2), and 4 were of a third type (Fig. (Fig.3C,3C, copy 3); and for strain D4-1, 6 of the 10 sequences were of one type (Fig. (Fig.3D,3D, copy 1), 2 were of a second type (Fig. (Fig.3D,3D, copy 2), and 2 were of a third type (Fig. (Fig.3D,3D, copy 3). For strain P11, there were 141 (19.1%) nucleotide differences between copies 1 and 2 (Fig. (Fig.3A).3A). For strain P12, there were 13 (1.7%) nucleotide differences between copies 1 and 2, 142 (19.2%) nucleotide differences between copies 1 and 3, and 144 (19.4%) nucleotide differences between copies 2 and 3 (Fig. (Fig.3B).3B). For strain D3-1, there were 121 (16.7%) nucleotide differences between copies 1 and 2, 69 (9.5%) nucleotide differences between copies 1 and 3, and 52 (7.2%) nucleotide differences between copies 2 and 3 (Fig. (Fig.3C).3C). For strain D4-1, there were 4 (0.6%) nucleotide differences between copies 1 and 2, 142 (19.1%) nucleotide differences between copies 1 and 3, and 143 (19.2%) nucleotide differences between copies 2 and 3 (Fig. (Fig.3D).3D). The sequence of copy 2 of strain P11 was identical to sequences of copy 1 of strain P12 and copy 1 of D4-1, and the sequence of copy 1 of strain P11 was identical to that of copy 3 of strain P12, copy 3 of strain D3-1, and copy 3 of strain D4-1 (Fig. (Fig.44).

FIG. 3.FIG. 3.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 ...

DISCUSSION

We report the first evidence of ITS sequence heterogeneity in Mucorales. In this study and the recent outbreak investigation of intestinal R. microsporus infections (5), four (14%) of the 28 R. microsporus strains isolated were found to possess ITS sequence heterogeneity. Since a major component of the heterogeneous nature of the ITS was due to DNA insertion/deletion, small differences in the lengths of the PCR products were generated during amplification of the ITS regions of the R. microsporus strains. This gave rise to the thick bands observed in agarose gel electrophoresis (Fig. (Fig.1).1). Furthermore, double and occasionally triple peaks were observed when the PCR products were directly sequenced because two or more kinds of PCR products were sequenced simultaneously (Fig. (Fig.2).2). Cloning the PCR products and sequencing 10 clones from each of the four R. microsporus strains confirmed ITS sequence heterogeneity in all four R. microsporus strains isolated. This is different from the observation described in Pneumocystis jiroveci, for which direct PCR sequencing of its ITS regions in clinical samples showed heterogeneous sequences, which probably represent different strains of P. jiroveci infecting the same patient instead of different ITS sequences in the same strain (8).

Variations in ITS sequence among the different rDNA operons in the same strain of R. microsporus were observed only in ITS1 and ITS2 but not the 5.8S rDNA region. The mature 28S, 5.8S, and 5S rRNA, assembled with the many ribosomal proteins, form the larger subunit of the ribosome in eukaryotes. Since the 5.8S rRNA is an essential functional component of only approximately 160 nucleotides in length, minimal variations in its sequence among the different rDNA operons is expected. In fact, its sequence is relatively conserved among fungi of different species. On the other hand, for ITS1 and ITS2, although they have a role in the development of functional RNA, sequence variations among fungi of different species and even among different strains of the same fungal species are much more common. This phenomenon is also present in the four strains of R. microsporus with ITS sequence heterogeneity in the present study. As shown in Fig. Fig.3,3, all the sequence variations were observed in ITS1 and ITS2 for all four strains of R. microsporus. It is notable that copy 2 of strain P11, copy 1 of strain P12, and copy 1 of strain D4-1 showed identical sequences; and copy 1 of strain P11, copy 3 of strain P12, copy 3 of strain D3-1, and copy 3 of strain D4-1 also showed identical sequences (Fig. (Fig.4).4). This implies that hot spots of insertion/deletion in ITS1 and ITS2 may be present.

ITS sequence heterogeneity is an obstacle to molecular identification and genotyping of pathogenic fungi in clinical microbiology laboratories. Accurate identification of pathogenic fungi is the cornerstone to prescribing antifungal treatment (2, 7, 9, 17); for example, identification of R. microsporus will necessitate the prescription of a combination treatment of posaconazole, amphotericin B, and caspofungin as posaconazole has been shown to have synergistic effects with amphotericin B and caspofungin against the Mucorales (6, 16). For molecular identification of fungal pathogens, the ITS is one of the most commonly used targets because its length and sequence are relatively conserved for the same fungal species but often different in different fungal species (4, 23). On the other hand, due to variation in ITS sequence among different strains in some fungal species, it has also been used for fungal genotyping (11, 14, 21). For example, in the recent outbreak of intestinal mucormycosis, eight alleles were observed among 24 strains of R. microsporus, thereby confirming multiple strain involvement in the outbreak (5). This number of different alleles was large compared to that for the gene loci used in multilocus sequence typing schemes (MLST) of molds. For example, in our recently published highly discriminatory MLST scheme for Penicillium marneffei, only 5 to 11 alleles were observed among 44 strains of P. marneffei for each of the five individual gene loci despite the very high evolutionary rates of all the five gene loci (24). Not only is the cloning of PCR fragments labor-intensive and time-consuming, but the technology is also often not available in most clinical microbiology laboratories. Therefore, variations of ITS sequences in different rDNA operons within the same strain of fungus will make identification and typing of such a strain by ITS sequencing very difficult 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.

Acknowledgments

This work is partly supported by the Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Disease for the Department of Health of the Hong Kong Special Administrative Region of China, a Research Grant Council Grant, the University Development Fund, a Committee for Research and Conference grant, and an Outstanding Young Researcher Award from The University of Hong Kong.

Footnotes

[down-pointing small open triangle]Published ahead of print on 11 November 2009.

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