Culture-Independent Analysis of Mixed Microbial Communities - Mixed Fungal Analysis
Separation and Identification of Mixtures of Clinically Relevant Fungal Species
The application of molecular biological methods, especially based on
amplification and analysis of nucleic acids, has opened up new horizons for
diagnostic laboratories, especially in microbiology and mycology. As an example,
sequencing of internal transcribed spacer regions (ITS1 and 2) is used for rapid
identification of fungal species from clinical specimens which are otherwise
difficult or very slow to derive from culturing. However, this sequencing
approach is limited to single species because mixtures either cannot be resolved
by sequence analysis or a false consensus sequence is read that does not relate
to any of the species within the mixture.
Therefore a method is required to separate consensus PCR amplified fungal DNA
gene products prior to sequencing. This application note discusses a recently
published paper by Goldenberg and colleagues (2005) at the Charité –
Universitätsmedizin Berlin, Germany, who describe the use of denaturing high
performance liquid chromatography (DHPLC) for detection and identification of
Candida species from blood culture samples.
We also touch briefly on other recent relevant unpublished data from work at
Transgenomic and our collaborators.
Methods
A detailed description of the DHPLC method used has been published elsewhere
(Goldenberg et al., 2005) and is also included in the User Guide for the
Transgenomic MycoSeg™ Kit (Cat. No. 709018/19). Briefly, universal primers were
used to amplify ITS2 sequences from DNA preparations of single or mixed fungal
species. The amplified DNA fragments were separated on a DNASep® Cartridge, a
key component of the WAVE® Microbial Analysis System, using partially denaturing
conditions. The degree of separation depends on the size and sequence of the
respective fragments. After separation, DNA fragments were detected using UV or
fluorescence analysis. A fragment collector attached to the WAVE was used to
recover separated fragments for subsequent analysis such as re-amplification,
cloning or DNA sequencing.
Results
The amplified ITS2 genes from 12 Candida and one Cryptococcus species were
unequivocally separated on the WAVE Microbial Analysis System (Figure 1). A
mixture of those amplicons was used as a standard for the identification of
fungal species in 14 spiked and 66 ‘real’ clinical blood culture samples. All
species were correctly identified when compared to results from culture
analysis. Moreover, in a previously presumed pure culture, the WAVE System
resolved both C. albicans and C. glabrata present in that specimen.
The analysis of another sample revealed a peak which could not be assigned to
one of the marker species. Collection and sequencing of this peak revealed that
the sample contained Trichosporon asahii.
As the protocol proved to be useful for separation of yeast species, it was also
applied to the analysis of fungal DNA in stool samples of patients receiv- ing
antifungal treatment (Figure 2). The peaks generated were easily identified by
collecting and sequencing, showing the power of such a method to study the
ecology of fungi and to monitor changes in the community due to environmental
influences. Genera identified included Aureobasidium, Candida, Ganoderma,
Itersonilia, Malassezia and Saccharomyces.
Figure 1.
Shown is a mixture of PCR-amplified ITS2 DNA fragments from Candida membranaefaciens (peak 1),
C. tropicalis (peaks 2a and b), C. parapsilosis (peak 3), C. magnoliae (peak 4),
C. lusitaniae (peak 5), C. norvegensis (peak 6), C. dubliniensis (peak 7),
C. albicans (peak 8), C. inconspicua (peak 9), C. krusei (peak 10),
Cryptococcus neoformans (peak 11), Candida kefyr (peak 12), and C. glabrata (peak 13).
Figure 2.
Samples were taken from one patient receiving Amphotericin B (AmphB). Generated peaks from samples taken at day 0, 7 and 20 were collected and sequenced, and assigned to the following fungal species:
Candida lusitaniae (1), Saccharomyces cerevisiae (2), Aureobasidium pullulans (3),
C. inconspicua (4), Itersonilia perplexans (5), Ganoderma sp. (6),
Malassezia pachydermatis (7), and M. restricta (8).
Early detection and identification of invasive mycoses are also of great importance in immunosuppressed patients. Therefore, the Berlin protocol described above was tested by Transgenomic for its power to separate mixtures of fungi belonging to genera relevant to invasive mycoses:
Absidia, Aspergillus, Candida, Fusarium, Mucor,
Pseudallescheria and Rhizopus (Figure 3). Differences found in the retention times of the tested species demonstrated the ability of the WAVE System to identify fungal species involved in invasive mycoses. The separation power was enhanced by running the samples at different temperatures, e.g. 60 °C (Figure 3A) and 63 °C (Figure 3B). This study led to the development of the MycoSeg Kit containing all relevant reagents and guidelines needed to run this kind of analysis.
Finally, in a non-clinical setting, Mathias Hutzler and colleagues from the Section of Brewing Technology at the University of Technology in Munich, Germany, were able to separate beer contaminating yeasts by using the MycoSeg Kit and the WAVE System (31st EBC Congress Venice, 2007). They extended the range of species separated by using both the ITS1 and ITS2 sequences, giving them the ability to easily identify 29 different yeast species, including
Saccharomyces, Dekkera and Pichia species.
Figure 3. Differential retention times of fungal species known to cause invasive
mycoses in immunosuppressed patients. The ITS2 sequences of those species were
separately amplified and run on the WAVE System at 60 °C (A) and 63 °C (B).
Conclusions
Analysis of amplified ITS2 genes from fungal species on the WAVE Microbial Analysis System has been shown to be a powerful tool in the process of identifying single or mixed fungal species in clinical specimens such as blood cultures or feces. According to the desired application, the protocol can easily be adapted to a variety of genera such as
Candida, Aspergillus, Saccharomyces and Mucor.
The ability to couple the WAVE System with a fragment collector allows straightforward identification of separated gene fragments by post-analysis sequencing. The described method offers medium to high- throughput analysis of samples and can be automated. In the future, the WAVE System will be a key tool in clinical diagnostics labs to supplement time-consuming, and often difficult to interpret, culturing procedures.
Finally, the method is also applicable to fungal analyses outside the clinic, e.g. for quality control in the brewing industry.
References
- Use of denaturing high-performance liquid chromatography for rapid detection and identification of seven Candida species. Goldenberg, O., Herrmann, S., Adam, T., Marjoram, G., Hong, G., Göbel, U. and Graf, B. (2005)
J. Clin. Microbiol. 43, 5912-5915.
- PCR-DHPLC (Denaturing High Performance Liquid Chromatography): a potential novel method for rapid screening of mixed yeast populations.
Goldenberg, O. and Hutzler, M. (2007) 31st European Brewing Convention Congress, Venice, Italy: P86.
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