Identification of Fungi Using Ribosomal Internal Transcribed Space DNA Sequences - Pharmaceutical Technology

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Identification of Fungi Using Ribosomal Internal Transcribed Space DNA Sequences
Identification of fungi, especially filamentous fungi, has been a very difficult task.

Pharmaceutical Technology

Advantages of ITS sequencing

More recently, ribosomal internal transcribed spacer (ITS) regions have been used for fungal systematics and classification. There are two ITS regions in the DNA genes encoding the fungal ribosomal RNA (rRNA) "operon." The first, ITS1, is found between the 18S and the 5.8S rRNA genes. The second, ITS2, is found between the 5.8S and the 28S rRNA genes. The entire rRNA "operon" is transcribed, but, after transcription, the two ITS sequences are excised and, therefore, not used for any functional purpose.

Because ITS sequences are important enough as spacer regions to be maintained by the cell, but not used for any functional purpose, they are allowed to accumulate mutations at a faster rate than the 5.8S, 18S, and 28S rRNA genes. It is this slightly increased rate of accumulated mutations that allows the ITS sequences to provide an improved level of resolution compared with the D2 sequence.

It is generally accepted to sequence the entire stretch of ITS1-5.8S–ITS2 for use in fungal classification. However, for the purposes of routine identification, our laboratory has found that ITS2 alone is usually sufficient for species-level identification.

As an example of the increased resolution of ITS2 sequences compared to D2 sequences, we will revisit the Komagataella problem. When comparing ITS2 DNA sequences of the three Komagataella species, we see many more nucleotide differences. There are 16 differences between K. pastoris and K. phaffii, and 18 differences between K. pastoris and K. pseudopastoris. This increased resolution offers a much more confident identification and allows for small strain-to-strain variability to ensure correct identification.

Phylogenetic analysis has offered fungal taxonomists the ability to reclassify thousands of species to create a more accurate taxonomic system. A huge advantage of DNA sequencing is its ability to classify an isolate based on DNA sequence alone, regardless of the accepted name of the organism or its official name.

Aspergillus brasiliensis is a newly described species that was, in part, created by the transfer of several existing Aspergillus niger strains to A. brasiliensis (6). Most significant in the creation of this new species to pharmaceutical microbiologists was the inclusion of Aspergillus niger ATCC 16404; this organism is cited in several US Pharmacopeia chapters as a quality control (QC) organism, including USP General Chapter <61> Microbial Limits Test—Enumeration and USP General Chapter <71> Sterility Test. Because of the number of pharmaceutical companies performing these tests, it is very important to correctly identify the QC organism. Unfortunately, this is another example where virtually all phenotypic tests, as well as D2 DNA sequencing, are unable to differentiate the two species. Phenotypically, it has always been difficult to differentiate between A. niger strains because of lack of diversity in morphological features, unstable phenotypic characters, and the significant influence of culture conditions on the phenotype (7). Furthermore, D2 sequences add no additional information because the DNA sequences for all observed A. niger and A. brasiliensis strains are identical.

A recent study by Houseknecht et al., looked at the justification for this recent classification (8). The study concluded that although A. niger and A. brasiliensis are very similar, there are ways to differentiate isolates of these species: through evaluation of conidia morphology under high magnification of an electron microscope (the differences are not observable using a light microscope), or through ITS DNA sequencing. The entire ITS1–5.8S–ITS2 DNA sequence shows five differences between the strains of A. niger and A. brasiliensis. When comparing ITS2 sequence alone, there is only one nucleotide difference between the species, but this difference has been shown to be extremely reproducible and is, therefore, considered a diagnostic indicator of the species.


The ability to accurately and reproducibility identify fungi—both yeasts and molds—has been greatly enhanced through comparative DNA sequencing. Fungal taxonomists have used DNA sequences for many years as a basis for reclassification of all fungal taxa and have more recently moved to ITS sequencing as the "gold standard." With the ability to use these powerful technologies as part of a comprehensive EM program, pharmaceutical microbiologists are given one more tool to ensure product safety. As made clear by FDA as part of its Pharmaceutical cGMPs for the 21st Century and process analytical technology initiatives, the agency encourages industry to use new rapid, and accurate technologies.

Michael Waddington is vice-president of business development at Accugenix, 223 Lake Drive, Newark, DE 19702, tel. 302.292.8888,


1. L. Jimenez, "Microbial Diversity in Pharmaceutical Product Recalls and Environments," PDA J. Pharm. Sci. Tech. , 61 (5), 383–399 (2007).

2. S. L. Baldauf and J. D. Palmer, "Animals and Fungi Are Each Other's Closest Relatives: Congruent Evidence From Multiple Proteins," PNAS , 90 (24) 11558–11562 (1993).

3. S.A. Meyer, R.W. Payne, and D. Yarrow, "Candida" in The Yeasts: A Taxonomic Study, C. P. Kurtzman and J. W. Fell, Eds (Elsevier Science B.V., Amsterdam, 1988) pp. 476–477.

4. P. Rozynek et al., "Quality Test of the MicroSeq D2 LSU Fungal Sequencing Kit for the Identification of Fungi," Int. J. Hyg. Environ. Health , 207 (3), 297–299 (2004).

5. L. Hall, S. Wohlfiel, and G. D. Roberts, "Experience with the MicroSeq D2 Large-Subunit Ribosomal DNA Sequencing Kit for Identification of Filamentous Fungi Encountered in the Clinical Laboratory," J. Clin. Microbiol. , 42 (2), 622–626 (2003).

6. J. Varga et al., "Aspergillus brasiliensis sp. Nov., ABiseriate Black Aspergillus Species with World-Wide Distribution," Int. J. Sys. Evol. Micr. , 57 (8) 1925–1932 (2007).

7. E. Rinyu, J. Varga, and L. Ferenczy, "Phenotypic and Genotypic Analysis of Variability in Aspergillus fumigatus," J. Clin. Microbiol., 33 (10), 2567–2575 (1995).

8. J. Houseknecht et al., "Reclassification of ATCC 16404 and ATCC 9642 as Aspergillus brasiliensis ," Pharm. Microbiol. Forum Newsletter, 14 (10), 2–8.


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