TY - JOUR
T1 - Using Plate-Wash PCR and High-Throughput Sequencing to Measure Cultivated Diversity for Natural Product Discovery Efforts
AU - Junkins, Emily N.
AU - Stevenson, Bradley S.
N1 - Funding Information:
We thank H. Nunn and the team of Mammalian Microbiome undergraduate researchers (A. Byrd, J. Dewberry, B. Hansen, C. Hauger, and J. Jacobs) for sample processing during cultivation. We also thank A. K. Dunn for helpful comments during the writing process. The isolates used for the pathogen panel were kindly provided by Robert Cichewicz, Institute for Natural Products and Research Technologies (INPART), University of Oklahoma. Funding. This research was partially supported by the US National Aeronautics and Space Administration Exobiology program (Grant No. 80NSSC19K0479).
Funding Information:
This research was partially supported by the US National Aeronautics and Space Administration Exobiology program (Grant No. 80NSSC19K0479).
Publisher Copyright:
© Copyright © 2021 Junkins and Stevenson.
PY - 2021/7/20
Y1 - 2021/7/20
N2 - Molecular techniques continue to reveal a growing disparity between the immense diversity of microbial life and the small proportion that is in pure culture. The disparity, originally dubbed “the great plate count anomaly” by Staley and Konopka, has become even more vexing given our increased understanding of the importance of microbiomes to a host and the role of microorganisms in the vital biogeochemical functions of our biosphere. Searching for novel antimicrobial drug targets often focuses on screening a broad diversity of microorganisms. If diverse microorganisms are to be screened, they need to be cultivated. Recent innovative research has used molecular techniques to assess the efficacy of cultivation efforts, providing invaluable feedback to cultivation strategies for isolating targeted and/or novel microorganisms. Here, we aimed to determine the efficiency of cultivating representative microorganisms from a non-human, mammalian microbiome, identify those microorganisms, and determine the bioactivity of isolates. Sequence-based data indicated that around 57% of the ASVs detected in the original inoculum were cultivated in our experiments, but nearly 53% of the total ASVs that were present in our cultivation experiments were not detected in the original inoculum. In light of our controls, our data suggests that when molecular tools were used to characterize our cultivation efforts, they provided a more complete and more complex, understanding of which organisms were present compared to what was eventually detected during cultivation. Lastly, about 3% of the isolates collected from our cultivation experiments showed inhibitory bioactivity against an already multidrug-resistant pathogen panel, further highlighting the importance of informing and directing future cultivation efforts with molecular tools.
AB - Molecular techniques continue to reveal a growing disparity between the immense diversity of microbial life and the small proportion that is in pure culture. The disparity, originally dubbed “the great plate count anomaly” by Staley and Konopka, has become even more vexing given our increased understanding of the importance of microbiomes to a host and the role of microorganisms in the vital biogeochemical functions of our biosphere. Searching for novel antimicrobial drug targets often focuses on screening a broad diversity of microorganisms. If diverse microorganisms are to be screened, they need to be cultivated. Recent innovative research has used molecular techniques to assess the efficacy of cultivation efforts, providing invaluable feedback to cultivation strategies for isolating targeted and/or novel microorganisms. Here, we aimed to determine the efficiency of cultivating representative microorganisms from a non-human, mammalian microbiome, identify those microorganisms, and determine the bioactivity of isolates. Sequence-based data indicated that around 57% of the ASVs detected in the original inoculum were cultivated in our experiments, but nearly 53% of the total ASVs that were present in our cultivation experiments were not detected in the original inoculum. In light of our controls, our data suggests that when molecular tools were used to characterize our cultivation efforts, they provided a more complete and more complex, understanding of which organisms were present compared to what was eventually detected during cultivation. Lastly, about 3% of the isolates collected from our cultivation experiments showed inhibitory bioactivity against an already multidrug-resistant pathogen panel, further highlighting the importance of informing and directing future cultivation efforts with molecular tools.
KW - 16S rRNA gene sequencing
KW - cultivation
KW - cultivation efficiency
KW - drug discovery
KW - high-throughput sequencing
KW - plate-wash PCR
KW - road-kill
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U2 - 10.3389/fmicb.2021.675798
DO - 10.3389/fmicb.2021.675798
M3 - Article
C2 - 34354680
AN - SCOPUS:85111946194
VL - 12
JO - Frontiers in Microbiology
JF - Frontiers in Microbiology
SN - 1664-302X
M1 - 675798
ER -