Using Plate-Wash PCR and High-Throughput Sequencing to Measure Cultivated Diversity for Natural Product Discovery Efforts

Abstract

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.

Particle-associated bacteria in seawater dominate the colony-forming microbiome on ZoBell marine agar

“…The characterization of CFU microbiomes by Illumina-based partial 16S rRNA gene amplicon sequencing has recently more often been applied, that is, with CFUs from a raccoon (Junkins and Stevenson 2021 ) and from tropical sediment (Demko et al 2021 ). We used the technology to cover the (whole) diversity on plates and to use the method´s sensitivity to identify bacteria of small colonies that were not visible to the naked eye.…”

Microbial diversity in tropical marine sediments assessed using culture‐dependent and culture‐independent techniques

“…Given the opportunities afforded by next‐generation sequencing, it is surprising that CD and CI approaches are seldom combined. Recent exceptions include studies of river (Pédron et al ., 2020) and lake sediment (Elfeki et al ., 2018), seawater (Rygaard et al ., 2017), cheese (Perin et al ., 2017), lungs (Dickson et al ., 2014) and raccoon microbiomes (Junkins and Stevenson, 2021). Here, we used both techniques to explore microbial diversity in marine sediments and to estimate culturing efficiency by sequencing bacteria directly from agar plates, in effect a type of enrichment culture, as opposed to the more traditional approach of strain isolation or colony counting.…”

“…One surprising result was the detection of cultured genera that were not detected using CI methods. Similar results were reported from Mediterranean water samples (Crespo et al ., 2016), recent work in a freshwater system (Pédron et al ., 2020) and a comparative study on raccoon microbiomes (Junkins and Stevenson, 2021). Almost all genera unique to the CD methods are likely members of the rare biosphere.…”

“…Nonetheless, these values were determined empirically and removed approximately 50% of the low abundance ASVs detected on both media. Interestingly, a recent study comparing plate‐washed cultures to CI, oral and gut microbiome sequences found that >50% of the cultured ASVs were absent from the CI analyses (Junkins and Stevenson, 2021). Thus, culturing efficiency can vary widely depending on sample type, with microbial interactions on agar plates likely playing important and largely undefined roles in bacterial culturability.…”

Environmental structure impacts microbial composition and secondary metabolism

“…Samples were stored at −20 °C until extractions could be processed at once. For community characterization, a conserved region of the SSU rRNA gene of most bacteria, archaea, and eukarya was amplified using primers 515F-Y and 926R [ 41 ] following a previously published protocol [ 42 ]. The amplified fragments were purified using Sera-Mag magnetic beads (ThermoFisher, Waltham, MA, USA) with the AmPureXP (Beckman Coulter, Indianapolis, IN, USA) protocol at a final concentration of 1.8× v/v.…”