NEW YORK—Using a pipette as a makeshift rolling pin, Raymond Schuch spent some of his lab time last summer pressing the guts out of earthworms that he had collected, fresh from Manhattan soil. For his efforts, The Rockefeller University microbiologist extracted what looked like just a small pile of dirt, but was actually a microcosm teeming with phages—viruses that infect bacteria. Schuch was on the hunt for phages that could kill anthrax and become anti-anthrax therapies, but what he discovered were viruses that enable this deadly bacteria to grow and survive when the going gets tough.

Scientists have suspected for decades that some phages have a hand at helping the growth of anthrax, Bacillus anthracis, and its less deadly cousins in the Bacillus genus. Then, four years ago, Schuch, along with Vincent Fischetti, a professor of bacteriology at Rockefeller, found a direct link—a type of phage that made anthrax resistant to an antibiotic commonly produced by other bacteria in soil, such as Streptomyces.

But how anthrax manages to persist in soil for hundreds of years despite environmental extremes, including wind and rain, and even go undetected during outbreaks in humans and livestock has puzzled scientists. In the earthworm intestines Schuch spotted a group of lysogenic phages—viruses that insert their genetic material into anthrax's genome. Instead of killing the pathogen the phages spur its ability both to grow and persist in the soil.

"It's known that there are lysogenic phages—they're very common," Schuch says. "The unusual thing is seeing what dramatic effects they could have on these anthracis strains."

Like other scientists studying anthrax, Schuch and Fischetti assumed that the bacteria's options were limited when it was not inside a host, often herbivores such as cattle or sheep. Although anthracis cells flourish when they are infecting a mammal, they tend to otherwise starve and transform into dormant spores in the soil, where they await their next victim. Typically, spores infect animal hides and human skin, causing infections that are rarely fatal. But inhaling or ingesting them can be deadly in up to 75 percent and 60 percent of cases, respectively.

The phages that Schuch and Fischetti discovered can prompt anthrax cells to remain in a growing, or vegetative, state even when they are in soil. In this condition the cells can form sticky biofilms that resist harsh wind and rain. Moreover, the researchers found that these lysogenic phages bestow anthracis with the ability to colonize earthworm guts, which Schuch calls "a safe haven" for the bacteria. He and Fischetti published their findings of new anthrax phages in the August 2009 issue of PLoS ONE.

"The most surprising [finding] is that anthracis can be grown outside the mammalian host," says Agnès Fouet, a microbiologist at the Institut Pasteur (Pasteur Institute) and the Centre National de la Recherche Scientifique (National Center for Scientific Research), both in Paris. Because of the thinking that anthracis is only in spore form in the soil, scientists typically heat soil samples to try to separate it from the vegetative bacteria that are killed by the heat treatment. "People were just not looking for it," Fouet says, "People were looking for spores." Instead, Schuch says, "If you want to look for anthrax in endemic areas, you look for earthworms in that area." In addition, he says that scientists can turn to heat-free methods to find vegetative anthrax-causing bacteria growing free in the soil.

Survival skills
To date, scientists have only found a handful of phages that can infect anthracis and, other than the case of the phage that bestows the bacteria with antibiotic resistance, few clues exist as to how viral infection affects anthracis. Because previous investigations have uncovered phages even in areas where there was no anthrax—in sewage, soil and water—Schuch looked for new phages in samples that were anthracis-free—fern roots and commercial potting soil.



Schuch also included earthworm guts in his search due to reading a biography about Louis Pasteur. The 19th-century microbiologist was the first to observe that areas with anthrax-infected carcasses were crawling with earthworms, although no one had actually proved that anthracis lived in these invertebrates. If earthworms do make a good habitat for anthracis, Schuch reasoned that they might also contain viruses that infect this and related Bacillus bacteria.

From their phage hunt, Schuch and Fischetti found eight new viruses, including two that they isolated from earthworm guts. When the researchers exposed a lab strain of anthracis to each of these phages separately, they found that, rather than bursting open and killing the bacteria, as lytic phages usually do, the viruses boosted the survival of their bacterial host: For the infected anthracis, the number of cells in test tube cultures of dirt and water remained constant for at least six months. In contrast, the population of phage-free anthracis dropped in half by 2.5 months, and was nonexistent after six months.

Although all of the phage types gave anthracis a survival advantage, they accomplished this effect through differing means. Three of the eight phages could speed the bacteria's transition from growing cells to spores when conditions were unfavorable for growth—low nutrient conditions or low temperatures (24 degrees Celsius instead of 30).

On the other hand, five of the phages triggered anthracis in nutrient-poor media to form biofilms, which are aggregates of vegetative cells encased in sugar-based matrices. Biofilms are the preferred state for other types of soil bacteria, helping them adhere to surfaces. Schuch and Fischetti found that these phages contain a gene whose protein activates the expression of a cluster of bacterial genes that are involved in growth and in sensing the environment. Unlike other bacteria in the Bacillus genus, however, whose genes get made into proteins, anthracis has a mutation that would normally prevent it from expressing this cluster. Anthracis is "just waiting for phages to turn on these genes," Fischetti says.

So far, the researchers have focused on the effect of one phage at a time—whether spore-promoting or growth- and biofilm-promoting—on laboratory strains of anthracis. But Fischetti says that more than one phage can infect anthracis simultaneously, and the researchers are planning to look at the interplay between different types of phages.

Worming out of detection
In addition to enhancing soil survival, Schuch and Fischetti found that the phages are critical for anthracis to colonize earthworms, which have been a suspected hideout since Pasteur's observation 130 years ago. Similar to how the viruses enhance the bacteria's survival in soil cultures, both spore- and biofilm-promoting phages kept the bacteria alive inside earthworms, without harming the invertebrates, for at least six months. But "if anthrax is in the earthworm and it loses the phage, it goes right out in the soil," where conditions are less favorable for the bacteria, Fischetti says.



The ability of anthracis to grow in earthworms has been "one of those puzzles that people have talked about for years, but no one's really taken the trouble to answer," says Nicholas Bergman, a bacteriologist at the National Biodefense Analysis and Countermeasures Center (NBACC) at Fort Detrick in Frederick, Md.

In lab-grown microcosms of earthworms and soil, the researchers also found that infected bacteria shed viruses both in the earthworm guts and soil, suggesting that anthracis can encounter and become infected with new viruses in both environments.

In future studies, Schuch says that he and Fischetti plan to import earthworms from areas where anthrax is endemic, which include sub-Saharan Africa, parts of Canada and western regions of the U.S., and examine whether they are colonized with anthracis. Scientists already know that related Bacillus bacteria live in wild earthworms. Schuch thinks that the invertebrates could provide a reservoir in which anthracis can grow out of season, when the soil is too cold and dry.

Ghosts of anthracis past
In the late 1800s cowboys on horseback drove cattle from Texas ranches to railroad stations in Kansas, Oklahoma, New Mexico and Colorado so that the cattle could be transported farther north and out west. Today, 120 years after the last cattle drives, trails of soil contaminated with anthrax spores still remain, left from where infected cows fed and rested.

"Normal weather processes and things of that nature really ought to diminish the spore population [in the soil]," says Bergman of the NBACC. Anthracis spores have no means to keep from being washed or blown away. But, "if phages are directing them to vegetative growth," Bergman adds, "they might be able to form a biofilm and produce something that would help them stick."

Another example of anthracis's puzzling persistence are the outbreaks that occurred in the early 1990s after the Soviet Union dissolved and unfurrowed land was farmed for the first time in about 80 years. Anthrax started infecting and killing the livestock, probably because the soil was still contaminated from agricultural activity four scores earlier.

Bergman points out that scientists are now using historical land surveys and town records to help them predict areas that might be harboring anthracis. "Mostly they tend to be areas that have had outbreaks before, or had high volume of either containment or trafficking of livestock," he says.



Schuch says that two of the ways that scientists could look for anthrax in these areas would be to use polymerase chain reaction or antibodies that recognize anthrax. As opposed to heating the soil, both of these methods would detect anthrax growing there. Following up on Schuch's idea to look for the bacteria not just in the soil but also in earthworms would be as simple as squeezing out the intestinal contents, as Schuch does, and letting the bacteria in the mix grow on a Petri dish. Anthracis would reveal itself because the bacterial cells grow as characteristic flat and colorless colonies, Schuch says.

Unfortunately, methods to prevent anthracis from persisting and causing disease are not so straightforward. "I don't know if we know enough yet about how to control these [phages]," Fischetti says. Among the next steps, he plans to examine if the phages that he and Schuch discovered influence bacterial genes that are involved in infecting mammals.

New phage frontier
Schuch and Fischetti have no reason to think that the eight phages they discovered complete the inventory of anthracis viruses. Fischetti estimates that there are one million bacteria in one gram of soil, and 10 times more phages. He suspects that other phages infect anthracis and probably have additional roles such as controlling the disease's virulence. "Phages may be doing more for bacterial survival on Earth than we thought," he says. "I think they are in control."

The new anthracis phages probably originated in related Bacillus bacteria because, as Schuch points out, these cousins of anthracis are more widespread in soil and earthworm intestines. But, combined with anthracis's particular genome that harbors disease-causing genes, these phage genes have unique effects, such as inducing expression of growth and biofilm formation.

"The remarkable thing about phages is that they expand the genetic diversity of the host that they infect," says Anca Segall, a phage biologist at San Diego State University. Segall, who calls Schuch and Fischetti's work to uncover the role of new anthracis phages "absolutely spectacular," started sequencing the DNA of phages from marine Bacilli several years ago. Some of the viruses she found induce the aquatic bacteria to sporulate.

Fischetti suspects that efforts to sequence the bacteria in the human gut, which are currently underway, will also reveal that phages control this diverse microcosm.

As the researchers work to understand the interplay between anthracis and phages, Schuch says that they will still be on the lookout for phage genes with anti-anthrax properties. It is possible that some of their eight new viruses contain genes that, when activated, kill the bacteria. In earlier work, Fischetti's team found a phage gene that encodes an enzyme that destroys anthracis cell walls. It is currently in development as a clinical therapy. "It's envisioned mostly as a treatment—as a replacement for or an adjunct to antibiotics—in people who have been exposed to [antibiotic-resistant] anthrax strains," Schuch says. "It might be better to use mixtures rather than single enzymes, just because then you'd be sure to kill every strain."