Wanted, Alive: Microbes

Unlike mammals, microbes don’t have pulses; determining whether they are alive or dead takes some scientific creativity.

AsianScientist (Sep. 29, 2017) – Deep beneath the floor of the Pacific Ocean lie ancient organisms that, against the odds, are still breathing—just barely. In 2012, scientists reported the discovery of microbial communities in 86-million-year-old sediment cores from 30 meters below the North Pacific Gyre, an area one thousand kilometers north of Hawaii.

Incredibly, the microbes were alive, despite having last seen sunlight and fresh nutrients during the Jurassic period, when dinosaurs still roamed the earth. They subsisted on the oxygen and organic material trapped in the surrounding sediment, metabolizing it at an extraordinarily slow rate to ration it out over millennia.

These aren’t the only long-interred microbes to have been found alive. In 2015, scientists discovered microbes some 2.5 kilometers beneath the ocean floor off the northeast coast of Japan that still feed on coal and release methane.

Studies such as these tell us how microorganisms can survive in the most unlikely of environments, as well as inform the search for life on other planets. But they also bring to the forefront a question that is deceptively simple: How do you tell if a microbe is dead or alive—and why does it matter?


Living or dead? It matters

Scientists used to culture bacteria to identify them; today, they are more likely to sequence their genomes instead. High-throughput DNA sequencing and bioinformatics have been used to study microbial communities from just about everywhere—guts, skin, pondwater, seawater, bathroom floors, subway stations and espresso machines, just to name a few locations.

Genomic data tells us in unprecedented detail what bacteria are present in a community, as well as what metabolic functions they could be performing. But unlike good old microbial culture, what it can’t tell us is whether they are alive or dead—DNA from dead bacteria can hang around in the environment for long enough to get sequenced as well.

This uncertainty poses an important problem, say the authors of a new review of laboratory methods for distinguishing live microbes from dead ones. Relying on DNA sequencing alone will likely overestimate the number of live bacterial taxa in a sample, resulting in an inaccurate picture of the microbial community being studied.

This isn’t just a question of scholarly accuracy, or of whether your yogurt has as many live cultures as it claims. The stakes are higher: When investigating a disease outbreak, for example, pinning the blame on the wrong microbe—the dead one—could delay public health containment measures; whether a toxic algal bloom is alive, dead or dying will, as the review’s authors point out, have important implications for the environment.


We’ve got a live one here

If you wanted to determine whether a mammal is alive or dead, you might listen for a pulse or watch for movement. Unfortunately, that doesn’t work for microbes.

While culturing is the traditional method—and often still the gold standard—for determining viability, many microbes are difficult or extremely tedious to grow under laboratory conditions.

Thus, scientists have developed a host of other methods to test for viability, including using dyes to check if cell membranes are intact; measuring various aspects of cellular metabolism such as respiration and protein translation; and molecular approaches that only amplify DNA from live cells.

One recently published method, an algorithm known as iRep, makes use of microbial sequence data itself to determine if bacteria are actively replicating (and hence alive). It takes advantage of the fact that bacterial genomes are replicated from a single starting point or origin, from which copying proceeds in both directions along the DNA.

When an actively replicating bacterial population is sequenced, we expect to see a bias—more sequences will be recovered from regions closer to the origin. In contrast, slowly replicating bacteria will show a relatively even sequence coverage across the genome. The iRep algorithm quantifies this difference, providing a measure of how active the population is.

Microbiomes are now recognized to have a major impact on human health—perturbations of the normal gut flora have been linked to a host of systemic conditions, including autoimmune, metabolic and mental health disorders.

Still, the mechanisms behind how the presence or absence of particular groups of bacteria may contribute to human disease remain largely unknown. Figuring these out will require not just sequencing firepower, but also the ability to distinguish live from dead.


This article is from a monthly column called The Bug Report. Click here to see the other articles in this series.

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Copyright: Asian Scientist Magazine; Photo: Shutterstock.
Disclaimer: This article does not necessarily reflect the views of AsianScientist or its staff.

Shuzhen received a PhD degree from the Johns Hopkins Bloomberg School of Public Health, USA, where she studied the immune response of mosquito vectors to dengue virus.

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