If a Bear Spits in the Woods Do New Antibiotics Arise?

Scientists in Russia and the U.S. have combined ultrahigh-throughput (uHT) microfluidic technology with wild Siberian bear saliva to look for potential antibiotics against harmful bacteria. The technology makes it possible to rapidly test individual microbiota species—such as bacteria in bear saliva—against pathogenic bacteria, isolate the beneficial species with antimicrobial properties, and identify the antibiotic that is produced.

“It is tedious to look for bacteria that produce antibiotics by testing them on Petri dishes and looking at how they inhibit the growth of harmful bacteria,” says Konstantin Severinov, Ph.D., a principal investigator at the Waksman Institute of Microbiology and professor of molecular biology and biochemistry at Rutgers University-New Brunswick. “We swiftly determined the spectrum of the antibiotic activity in saliva from a Siberian bear.”  Dr. Severinov is co-author of the team’s published paper in the Proceedings of the National Academy of Sciences (PNAS), which is titled, “Ultrahigh-throughput functional profiling of microbiota communities.”

Studying the microbiomes of wild, captive, and domesticated animals is becoming a mainstream of modern microbiology, offering up new insights into longstanding biological issues, the authors write. Modern screening technologies and methods for analyzing the structure and function of individual species of microbiota at the single cell level mean it is now possible to characterize clones with antimicrobial, antifungal, and antiparasitic activity, as well as beneficial probiotic species, they continue.

The microbiota of wild species represents an “underestimated resource for this type of screening, the team adds. “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 probiotic strains.”

The researches adapted their ultrahigh-throughput microfluidic droplet platform to carry out functional screening of wild animal microbiota. The microfluidic double water-in-oil-in-water emulsion (MDE) technology involves encapsulating individual microbiota clones together with a target pathogen that produces a fluorescent reporter, in lipid droplets, and sorting those that demonstrate antibacterial activity.

For the reported studies the team collected saliva samples from the mouth of a wild Siberian brown bear, which they screened using their uHT microfluidic droplet platform to identify bacteria that inhibited the growth of pathogenic Staphylococcus aureus. Droplets with positive features were isolated by fluorescence-activated cell sorting (FACS) technology, if they conformed to three criteria simultaneously: a high initial S. aureus load and a low S. aureus count after in droplet cocultivation with the saliva-derived clone, and also 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,” the researchers state.

“The bear was chosen largely because it was captured way out in the wilderness where, it was assumed, microbes typical for the species and not affected by civilization are present,” Dr. Severinov comments. “The latter consideration is important since studies show that the diversity of microbiota depends on diet and decreases dramatically, for example, in zoo-kept animals or urban humans compared with people from indigenous tribes.”

Bear oral microbiota differs “dramatically” from human oral microbiota, and is more similar to human fecal microbiota, the authors note. The results from the uHT microfluidic droplet screen identified and isolated several bacterial clones with activity against S. aureus including Enterococcus casseliflavus, Weissella confusa, and Bacillus pumilus. Interestingly, these three strains hadn’t been identified through standard preliminary testing of bear’s microbiota.

The team concentrated further tests on the isolated B. pumilus 124 clone, which they had found to be the most effective a blocking S. aureus growth. Their analyses indicated that the isolated B. pumilus strain produced amicoumacin A (Ami), an antibiotic previously discovered in other Bacillus and Xenorhabdus species. Genome sequencing and mining indicated that the antibiotic was produced by a cluster of genes similar to biosynthetic clusters that have previously been shown to produce antibiotics including zwittermicin, paenilamicin, xenocoumacin, and olibactin. Subsequent studies identified the biochemical pathways that regulated Ami production in B. pumilus.

The researchers then devised a technique based on the MDE platform for uHT profiling of Ami activity in different microbiota sources, including clinical isolates, to see how the antibiotic impacted on the different bacterial species. “Placing single species of bacteria in droplets allows us to monitor their responses to various insults, such as antibiotics, while avoiding interactions in complex microbiomes, such as our own,” Dr. Severinov notes. Our method should allow us to test how our microbiome responds and changes when various drugs are administered.”

The method essentially involved cultivating individual cells from microbiota samples, together with an antibiotic, inside the MDE droplets. The droplets were then stained, selected using FACS, and genome sequences analyzed. “The spectrum of Ami activity was analyzed using different microbiomes and quantitative estimations of Ami activity on bacteria were made. We found Ami to be especially active against gram-positive bacteria,” the authors write. “… it is active against some bacteria relevant to dysbiosis, and we suppose that Ami-producing strains could be used for controlled microbiota remodeling.”

“Here we show how microfluidic uHT screening technologies could be applied for classical microbiological problems, i.e., antibiotic/probiotic selection and susceptibility/resistance testing,” they conclude. “…Finally, we assume that the demonstrated approach is not limited to bacterial communities and could be efficiently expanded to the deep functional profiling of eukaryotic cells in applications such as biomarker probing and chemotherapy resistance/efficiency screening.”