The human microbiome has been surveyed before, but typically in piecemeal fashion—the gut here, the skin there. Consequently, some of the body’s microbial niches came to be well represented in genomic databases, whereas others remained in the dark. But now the uncounted microbes and viruses that had never been recognized before are coming to light, thanks to a new, unbiased survey, one that has sampled circulating cell-free DNA from the blood.

The new survey, which effectively enabled sampling of the whole body, was undertaken by scientists based at Stanford University. These scientists report that by conducting a microbiome census that included both well-studied and previously ignored niches, they were able to show that our bodies contain vastly more diverse microbes than anyone previously understood. What's more, the overwhelming majority of those microbes have never been seen before, let alone classified and named.

Survey details appeared August 22 in the Proceedings of the National Academy of Sciences, in an article entitled “Numerous Uncharacterized and Highly Divergent Microbes Which Colonize Humans Are Revealed by Circulating Cell-Free DNA.” The article describes the Stanford team’s survey technique—massive shotgun sequencing—as well as the scope of sample collection—1351 blood draws from 188 patients.

After subjecting the samples to shotgun sequencing, the Stanford team assembled 7190 contiguous regions (contigs) larger than 1 kbp, of which 3761 are novel. According to the scientists, the novel contigs had little or no sequence homology in any existing database.

“The vast majority of these novel contigs possess coding sequences, and we have validated their existence both by finding their presence in independent experiments and by performing direct PCR amplification,” wrote the authors of the PNAS article. “When their nearest neighbors are located in the tree of life, many of the organisms represent entirely novel taxa, showing that microbial diversity within the human body is substantially broader than previously appreciated.”

“We found the gamut,” said Stephen Quake, Ph.D., a professor of bioengineering and applied physics, a member of Stanford Bio-X and the paper's senior author. “We found things that are related to things people have seen before, we found things that are divergent, and we found things that are completely novel.”

Of all the nonhuman DNA fragments the team gathered, 99% of them failed to match anything in existing genetic databases the researchers examined.

With that in mind, Mark Kowarsky, a graduate student in Dr. Quake's lab and the paper's first author, set about characterizing all of that mystery DNA.

The “vast majority” of it belonged to a phylum called Proteobacteria, which includes, among many other species, pathogens such as Escherichia coli and Salmonella. Previously unidentified viruses in the torque teno family, generally not associated with disease but often found in immunocompromised patients, made up the largest group of viruses.

“We've doubled the number of known viruses in that family through this work,” Dr. Quake noted. Perhaps more important, they've found an entirely new group of torque teno viruses. Among the known torque teno viruses, one group infects humans and another infects animals, but many of the ones the researchers found didn't fit in either group. “We've now found a whole new class of human-infecting ones that are closer to the animal class than to the previously known human ones, so quite divergent on the evolutionary scale,” he added.

The new survey was inspired by a curious observation Dr. Quake's lab made while searching for noninvasive ways to predict organ rejection. Ordinarily, it takes a tissue biopsy—meaning a large needle jabbed into one's side and at least an afternoon in a hospital bed for observation—to detect rejection.

The lab members figured there was a better way. In theory, they might be able to detect rejection by taking blood samples and looking at the cell-free DNA—bits and pieces of DNA circulating freely in blood plasma—contained therein. Apart from fragments of a patient's DNA, those samples would contain fragments of the organ donor's DNA as well as a comprehensive view of the collection of bacteria, viruses, and other microbes that make up a person's microbiome.

Over the course of several studies, the first of which was published in 2013, Dr. Quake, postdoctoral fellow Iwijn De Vlaminck, Ph.D., and others collected samples from 156 heart, lung, and bone marrow transplant recipients, along with 32 pregnant women. (Pregnancy, like immunosuppressant drugs taken by transplant patients, also changes the immune system, albeit in ways both more complicated and less well understood.)

The results of those earlier studies suggested there were identifiable changes to the microbiomes of people with compromised immune systems and that positive tests for the organ donor's DNA were a good sign of rejection.

Besides predicting organ rejection events, the technology deployed by the Stanford team could, with deeper sequencing and targeted sample collection, lead to the discovery of numerous new viral and bacterial species. The scrutiny of circulating nucleic acids of organisms could complement existing efforts to characterize the life within us.

“Novel taxa of microbes inhabiting humans, while of interest in their own right, also have potential consequences for human health,” the authors of the PNAS article concluded. “They may prove to be the cause of acute or chronic diseases that, to date, have unknown etiology and may have predictive associations that permit presymptomatic identification of disease.”

Going forward, Dr. Quake said the lab hopes to study the microbiomes of other organisms to see what's there: “There's all kinds of viruses that jump from other species into humans, a sort of spillover effect, and one of the dreams here is to discover new viruses that might ultimately become human pandemics.” Understanding what those viruses are could help doctors manage and track outbreaks.

“Work of this kind could arm infectious disease doctors with a whole set of new bugs to track and see if they're associated with disease,” Dr. Quake asserted. “That's going to be a whole other chapter of work for people to do.”








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