Ultrahigh-throughput functional profiling of microbiota communities

September 9, 2018  16:31

Analyzing complex microbial communities is the milestone of modern microbiology, calling for “deep functional profiling” techniques. While next generation sequencing revolutionized our understanding of microbiota communities, we still lack high-throughput technologies to precisely determine their functionality. Here we show how cultivation of individual bacteria inside droplets of microfluidic double water-in-oil-in-water emulsion enables us to isolate the clones with a desired activity. This approach allows us not only to select the potent antibiotic producer but also to discover a distinct mechanism of self-resistance as well as assess its efficiency on entire microbiomes. The outcome of this methodology shows that it could be effectively transferred to numerous applications in microbiology and biotechnology.

Microbiome spectra serve as critical clues to elucidate the evolutionary biology pathways, potential pathologies, and even behavioral patterns of the host organisms. Furthermore, exotic sources of microbiota represent an unexplored niche to discover microbial secondary metabolites. However, establishing the bacterial functionality is complicated by an intricate web of interactions inside the microbiome. Here we apply an ultrahigh-throughput (uHT) microfluidic droplet platform for activity profiling of the entire oral microbial community of the Siberian bear to isolate Bacillus strains demonstrating antimicrobial activity against Staphylococcus aureus. Genome mining allowed us to identify antibiotic amicoumacin A (Ami) as responsible for inhibiting the growth of S. aureus. Proteomics and metabolomics revealed a unique mechanism of Bacillus self-resistance to Ami, based on a subtle equilibrium of its deactivation and activation by kinase AmiN and phosphatase AmiO, respectively. We developed uHT quantitative single-cell analysis to estimate antibiotic efficacy toward different microbiomes and used it to determine the activity spectra of Ami toward human and Siberian bear microbiota. Thus, uHT microfluidic droplet platform activity profiling is a powerful tool for discovering antibiotics and quantifying external influences on a microbiome.

The latest insights into microbiome revealed close links between the spectra of coexisting bacteria and progression of several pathologies in human hosts. Microbiome spectra turned out to be a viable marker of behavior and habits of Homo sapiens and Neanderthals, as well as the environmental and historical conditions they lived in. The bactericide properties of microbiome spectra and bacterial coexistence are becoming a hallmark of present-day biomedical investigations in humans. Scrutinizing the microbiota of a plethora of organisms is becoming a mainstream of modern microbiology. The microbiota of wild, captive, and domesticated animals as well as birds, reptiles, and gene-modified animal models (6⇓⇓⇓–10) were analyzed, offering valuable answers to the long-standing issues of biology. Microbiomes have also come into the limelight of evolutionary studies.

The revolution in screening technologies complemented by that of functional and structural analyses of large arrays of microbiota species on a single-cell level allows us to isolate and characterize clones with different activities, such as microbial killers, antifungi and antiparasite drugs, as well as probiotic bacterial strains. Microbiota of wild animals is an underestimated resource for this type of screenings. The ability of wild animals to thrive while surrounded by aggressive microorganisms may be partially mediated by their microbiota, making this kind of microbiota a potentially attractive niche for a targeted screening of antibiotics and prospective probiotic strains. In this work, we adjusted our ultrahigh-throughput (uHT) microfluidic droplet platform to perform functional screening of wild-animal microbiota. Our platform allows for packaging the individual bacterial clones in double emulsion droplets and screening these microcompartments by FACS. This technique enables functional screening of the cell libraries with an enormous biodiversity displaying productivity reaching 108 variants per hour. The critical advantage of the system lies in the possibility of microbiome functional profiling on a single-cell level.

Here we present the analyses of the microbiome collected from East Siberian brown bear (Ursus arctos collaris) obtained immediately after capture in the taiga. We aimed to screen this microbiome resource and search for probiotics and physiologically active compounds. A Bacillus pumilus strain producing an unstable antibiotic amicoumacin A (Ami) was isolated, enabling us to identify the B. pumilus Ami biosynthetic gene cluster and discover a crucial role of Ami kinase/phosphatase in the regulation of Ami production. We than applied uHT screening to quantify the external influence on the microbiome diversity, thus obtaining a detailed description of Ami activity spectra toward human and Siberian bear microbiota.

The saliva samples collected from an oral cavity of the Siberian brown bear were screened for bacteria inhibiting the growth of pathogenic Staphylococcus aureus cells using a microfluidic platform. It is based on cocultivation of oral microbiota members with the target S. aureus strain producing GFP reporter in droplets of microfluidic double water-in-oil-in-water emulsion (MDE).

The combination of three independent fluorescent signals was used to isolate MDE droplets by FACS. The isolated droplets had to conform to the following criteria simultaneously; they exhibited a high initial S. aureus load and a low S. aureus count after in droplet cocultivation accompanied by the presence of live, metabolically active cells. The overall throughput of this platform was estimated to embrace 30,000 droplets per second, which enabled deep probing of microbial community based on anti-S. aureus activity. Several bacterial clones with anti-S. aureus activity were selected and identified as Enterococcus casseliflavus, Weissella confusa, and B. pumilus. These strains were not selected during the preliminary standard testing of bear’s microbiota. This indicates a substantial enrichment of bacteria displaying antagonistic properties against S. aureus after uHT screening in MDE droplets. In what follows, we focus on the isolated B. pumilus, as it was the most efficient inhibitor of S. aureus growth in a plate overlay assay.

The notable peculiarity of these clusters is the presence of a gene coding the specific peptidase, activating cognate preantibiotics during the export process. There is, however, an additional gene encoding N-acetyltransferase AmiS inactivating Ami in the case of Ami-like gene cluster from X. bovienii. The fact that amiS homolog is absent in both B. pumilus 124 and B. subtilis 1779 strains gave rise to an idea that Bacilli should have an alternative mechanism of self-resistance. Hashimoto et al. have reported the presence of a phosphorylated Ami, produced by B. pumilus, and demonstrated that the phosphorylated Ami is inactive. We found two genes, amiN and amiO, encoding a putative kinase and an alkaline phosphatase, respectively, adjacent to the core biosynthetic genes of the Ami cluster and hypothesized that their products may contribute to both self-resistance and Ami biosynthesis. The tandem-mass spectral analysis was used to find molecular weight of Ami derivates, namely the phosphorylated AmiA-P and AmiB-P (Fig. 2D). It was shown that these compounds have exactly the same mass value as described by Hashimoto et al. where the site of Ami phosphorylation by NMR was determined.

The Regulation of Ami Production in B. pumilus.
The essential role of genes amiA–amiM in Ami biosynthesis was previously demonstrated through a homologous B. subtilis 1779 cluster; yet the mechanism was not elucidated. Here we observe Ami production to be inducible and regulated at several additional levels in B. pumilus. Ami production was induced while cultivated in a thin layer without shaking. Cultivation with limited aeration and shaking resulted in more than a 20-fold decrease in Ami production, which was insufficient to inhibit Staphylococcus Меtabolomic analysis of B. pumilus 124 cultivated in “activated” (F) and “inactivated” (N) conditions revealed that the inactive phosphorylated Ami derivatives were present exclusively inside the bacterial cells, and absent in the culture medium. A dramatic difference in Ami phosphorylation balance between F and N states was also manifested, suggesting that Ami was efficiently inactivated via phosphorylation in N conditions.

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