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Improved characterization of medically relevant fungi in the human respiratory tract using next-generation sequencing.

Bittinger K, Charlson ES, Loy E, Shirley DJ, Haas AR, Laughlin A, Yi Y, Wu GD, Lewis JD, Frank I, Cantu E, Diamond JM, Christie JD, Collman RG, Bushman FD - Genome Biol. (2014)

Bottom Line: Comparison to clinical culture data documents improved detection after applying the filtering procedure.We analyze covariation of fungal and bacterial taxa, and find that oropharyngeal communities rich in Candida are also rich in mitis group Streptococci,a community pattern associated with pathogenic polymicrobial biofilms.Thus, using this approach, it is possible to characterize fungal communities in the human respiratory tract more accurately and explore their interactions with bacterial communities in health and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT

Background: Fungi are important pathogens but challenging to enumerate using next-generation sequencing because of low absolute abundance in many samples and high levels of fungal DNA from contaminating sources.

Results: Here, we analyze fungal lineages present in the human airway using an improved method for contamination filtering. We use DNA quantification data, which are routinely acquired during DNA library preparation, to annotate output sequence data, and improve the identification and filtering of contaminants. We compare fungal communities and bacterial communities from healthy subjects, HIV+ subjects, and lung transplant recipients, providing a gradient of increasing lung impairment for comparison. We use deep sequencing to characterize ribosomal rRNA gene segments from fungi and bacteria in DNA extracted from bronchiolar lavage samples and oropharyngeal wash. Comparison to clinical culture data documents improved detection after applying the filtering procedure.

Conclusions: We find increased representation of medically relevant organisms, including Candida, Cryptococcus, and Aspergillus, in subjects with increasingly severe pulmonary and immunologic deficits. We analyze covariation of fungal and bacterial taxa, and find that oropharyngeal communities rich in Candida are also rich in mitis group Streptococci,a community pattern associated with pathogenic polymicrobial biofilms. Thus, using this approach, it is possible to characterize fungal communities in the human respiratory tract more accurately and explore their interactions with bacterial communities in health and disease.

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Presence-absence analysis identifies fungal genera present more often in experimental samples relative to contamination controls.Aspergillus and Penicillium are present significantly more often in oropharyngeal and lung samples relative to controls, while Pichia and Saccharomyces are present more often only in OW relative to controls. Conversely, Wallemia likely derives solely from contamination sources.
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Fig5: Presence-absence analysis identifies fungal genera present more often in experimental samples relative to contamination controls.Aspergillus and Penicillium are present significantly more often in oropharyngeal and lung samples relative to controls, while Pichia and Saccharomyces are present more often only in OW relative to controls. Conversely, Wallemia likely derives solely from contamination sources.

Mentions: Because so many OTUs were eliminated by the application of a global abundance threshold (many of which may be authentic community members but cannot be definitively distinguished from environmental admixture), we wondered whether other methods might be able to identify less abundant genera legitimately present in OW or BAL samples. For genera where the PicoGreen-corrected abundance is not enough to call individual OTUs as authentically present in a particular sample, the frequency of occurrence can nevertheless reveal patterns across sample types. FigureĀ 5 shows the frequency of occurrence in OW, BAL, and contamination control samples for genera present in at least 10 OW or BAL samples. An expanded plot is included in Additional file 2.Figure 5


Improved characterization of medically relevant fungi in the human respiratory tract using next-generation sequencing.

Bittinger K, Charlson ES, Loy E, Shirley DJ, Haas AR, Laughlin A, Yi Y, Wu GD, Lewis JD, Frank I, Cantu E, Diamond JM, Christie JD, Collman RG, Bushman FD - Genome Biol. (2014)

Presence-absence analysis identifies fungal genera present more often in experimental samples relative to contamination controls.Aspergillus and Penicillium are present significantly more often in oropharyngeal and lung samples relative to controls, while Pichia and Saccharomyces are present more often only in OW relative to controls. Conversely, Wallemia likely derives solely from contamination sources.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4232682&req=5

Fig5: Presence-absence analysis identifies fungal genera present more often in experimental samples relative to contamination controls.Aspergillus and Penicillium are present significantly more often in oropharyngeal and lung samples relative to controls, while Pichia and Saccharomyces are present more often only in OW relative to controls. Conversely, Wallemia likely derives solely from contamination sources.
Mentions: Because so many OTUs were eliminated by the application of a global abundance threshold (many of which may be authentic community members but cannot be definitively distinguished from environmental admixture), we wondered whether other methods might be able to identify less abundant genera legitimately present in OW or BAL samples. For genera where the PicoGreen-corrected abundance is not enough to call individual OTUs as authentically present in a particular sample, the frequency of occurrence can nevertheless reveal patterns across sample types. FigureĀ 5 shows the frequency of occurrence in OW, BAL, and contamination control samples for genera present in at least 10 OW or BAL samples. An expanded plot is included in Additional file 2.Figure 5

Bottom Line: Comparison to clinical culture data documents improved detection after applying the filtering procedure.We analyze covariation of fungal and bacterial taxa, and find that oropharyngeal communities rich in Candida are also rich in mitis group Streptococci,a community pattern associated with pathogenic polymicrobial biofilms.Thus, using this approach, it is possible to characterize fungal communities in the human respiratory tract more accurately and explore their interactions with bacterial communities in health and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT

Background: Fungi are important pathogens but challenging to enumerate using next-generation sequencing because of low absolute abundance in many samples and high levels of fungal DNA from contaminating sources.

Results: Here, we analyze fungal lineages present in the human airway using an improved method for contamination filtering. We use DNA quantification data, which are routinely acquired during DNA library preparation, to annotate output sequence data, and improve the identification and filtering of contaminants. We compare fungal communities and bacterial communities from healthy subjects, HIV+ subjects, and lung transplant recipients, providing a gradient of increasing lung impairment for comparison. We use deep sequencing to characterize ribosomal rRNA gene segments from fungi and bacteria in DNA extracted from bronchiolar lavage samples and oropharyngeal wash. Comparison to clinical culture data documents improved detection after applying the filtering procedure.

Conclusions: We find increased representation of medically relevant organisms, including Candida, Cryptococcus, and Aspergillus, in subjects with increasingly severe pulmonary and immunologic deficits. We analyze covariation of fungal and bacterial taxa, and find that oropharyngeal communities rich in Candida are also rich in mitis group Streptococci,a community pattern associated with pathogenic polymicrobial biofilms. Thus, using this approach, it is possible to characterize fungal communities in the human respiratory tract more accurately and explore their interactions with bacterial communities in health and disease.

Show MeSH
Related in: MedlinePlus