Use of Ichip for High-Throughput In Situ Cultivation of “Uncultivable” Microbial Species

Abstract

One of the oldest unresolved microbiological phenomena is why only a small fraction of the diverse microbiological population grows on artificial media. The "uncultivable" microbial majority arguably represents our planet's largest unexplored pool of biological and chemical novelty. Previously we showed that species from this pool could be grown inside diffusion chambers incubated in situ, likely because diffusion provides microorganisms with their naturally occurring growth factors. Here we utilize this approach and develop a novel high-throughput platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments. We have designed and tested an isolation chip (ichip) composed of several hundred miniature diffusion chambers, each inoculated with a single environmental cell. We show that microbial recovery in the ichip exceeds manyfold that afforded by standard cultivation, and the grown species are of significant phylogenetic novelty. The new method allows access to a large and diverse array of previously inaccessible microorganisms and is well suited for both fundamental and applied research.It has been known for over a century that the overwhelming majority of microbial species do not grow on synthetic media in vitro and remain unexplored (13,32,37,39,40,43). The rRNA and metagenomics approaches demonstrated a spectacular diversity of these uncultivated species (11, 21, 25-27, 30, 36). Accessing this "missing" microbial diversity is of significant interest for both basic and applied sciences and has been recognized as one of the principal challenges for microbiology today (12,29,41). In recent years, technical advances in cultivation methodologies have recovered a diverse set of ecologically relevant species (1,3,5,7,15,20,24,28,33,42). However, by and large the gap between microbial diversity in nature and that in culture collections remains unchanged, and most microbial phyla still have no cultivable representatives (25,29). Earlier, we developed a novel method of in situ cultivation of environmental microorganisms inside diffusion chambers (15). The rationale for such an approach was that diffusion would provide cells inside the chamber with naturally occurring growth components and enable those species that grew in nature at the time of the experiment to also grow inside the diffusion chambers. Expectedly, this method yields a rate of microbial recovery many times larger than those of standard techniques. Even so, this method is laborious and does not allow an efficient, high-throughput isolation of microbial species en masse. This limits the method's applicability, for example, in the drug discovery effort. Here we transform this methodology into a high-throughput technology platform for massively parallel cultivation of "uncultivable" species. Capitalizing on earlier microfluidics methods developed for microbial storage and screening (4, 16), we have designed and tested an isolation chip, or ichip for short, which consists of hundreds of miniature ...

Antibiotic drug discovery

“…E. terrae was isolated with the help of a new cultivation technique, the so‐called iChip, which is a multichannel device composed of several hundred miniature diffusion chambers, each inoculated with a single environmental cell. The iChip then is placed back in the natural environment, which leads to a significantly increased colony count that subsequently can efficiently be cultivated under laboratory conditions (Nichols et al ., 2010). …”

Efficient total syntheses and biological activities of two teixobactin analogues

“…7 This is where the multichannel device, iChip, 8 has made a considerable contribution enabling the identification of molecules like teixobactin, some of which could be active against drug resistant bacteria. However, the iChip device has its limitations.…”

The race for antimicrobials in the multidrug resistance era

“…One illustrative example is the development and use of iChip technology (Nichols et al ., 2010) that permits the growth of uncultured microorganisms, which along with directed screenings against bacterial pathogens, have led to the identification of teixobactin, a novel antibiotic produced by the recently isolated Eleftheria terrae bacterium. This molecule binds to a highly conserved motif of lipid II and lipid III, arresting the cell wall synthesis (Ling et al ., 2015).…”