mBiosphere

Diagnosing a 700-Year-Old Infection

Shortly after Mark Pallen, PhD, and colleagues described recovering tuberculosis genomes from the lung tissue of a 215-year-old mummy from Hungary in the New England Journal of Medicine last summer, news of his ability to conduct metagenomic analyses on historical samples spread, and materials started to flow in.

Italian anthropologist Raffaella Bianucci asked Pallen, a professor of microbial genomics at Warwick Medical School in Coventry, England, if he would look for pathogens in archaeological samples from Belgium and Sardinia, an island off the coast of Italy, and he agreed. The relationship led to recovering a genome of the bacterium Brucella melitensis from a 700-year-old skeleton found in the ruins of a Medieval Italian village.

Reporting this week in mBio®, the authors describe using a technique called shotgun metagenomics to sequence DNA from a calcified nodule in the pelvic region of a middle-aged male skeleton excavated from the Sardinian settlement of Geridu, thought to have been abandoned in the late 14th century. Shotgun metagenomics allows scientists to sequence DNA without looking for a specific target.

Skeleton and calcium deposits -- courtesy Mark Pallen, Warwick Medical School

From this sample, the researchers recovered the genome of
, which causes an infection called brucellosis in livestock and humans. In humans, brucellosis is usually acquired by ingesting unpasteurized dairy products or from direct contact with infected animals. Symptoms include fevers, arthritis and swelling of the heart and liver. The disease is still found in the Mediterranean region.

“Normally when you think of calcified material in human or animal remains you think about tuberculosis, because that’s the most common infection that leads to calcification,” Pallen says. “We were a bit surprised to get Brucella instead.”

The skeleton contained 32 hardened nodules the size of a penny in the pelvic area, though Pallen says it’s unclear if they originated in the pelvis, or higher up in the chest or other body part. The team took care to sample the interior of a nodule, to eliminate the risk of contamination from soil.

In additional experiments, the research team showed that the DNA fragments extracted had the appearance of aged DNA – they were shorter than contemporary strands, with only 100 base pairs, and had characteristic G-A or C-T mutations at the ends. They also found that the medieval Brucella strain, which they called Geridu-1, was closely related to a recent Brucella strain called Ether, identified in Italy in 1961, and two other Italian strains identified in 2006 and 2007. They confirmed their findings by comparing the distribution of genetic insertions and deletions located in Geridu-1 with those found in other Brucella strains.

The study “confirms that whole-genome sequences from bacterial pathogens can be recovered from human remains by metagenomics hundreds or even thousands of years postmortem,” Pallen says.

Brucella melitensis -- credit: CDC

Pallen’s team is now testing shotgun metagenomics on a range of additional samples, including historical material from Hungarian mummies; Egyptian mummies; a Korean mummy from the 16th or 17th century; and lung tissue from a French queen from the Merovingian dynasty, which ruled France from the 5th to 8th centuries; as well as contemporary sputum samples from the Gambia in Africa.

“Metagenomics stands ready to document past and present infections, shedding light on the emergence, evolution and spread of microbial pathogens,” Pallen says. “We’re cranking through all of these samples and we’re hopeful that we’re going to find new things.”

-- Karen Blum

Fecal Transplants Really Do Work

Fecal microbiota transplantation might seem off-putting to the average person. But the technique has been successful in helping many patients recover from dangerous Clostridium difficile infections (CDI), and a study published in mBio® this week suggests why.

Fecal transplants work by restoring healthy bacteria and functioning to the gut, study authors found. The work provides insight into the structural and potential metabolic changes that occur following the transplants, which may aid in the development of new treatment methods for CDI.

C. difficile. Credit: CDC

“The bottom line is fecal transplants work, and not by just supplying a missing bug but a missing function being carried out by multiple organisms in the transplanted feces,” says senior study author Vincent B. Young, MD, PhD, an associate professor in the Department of Internal Medicine/Infectious Diseases and the Department of Microbiology & Immunology at the University of Michigan in Ann Arbor. “By restoring this function, C. difficile isn’t allowed to grow unchecked, and the whole ecosystem is able to recover.”

CDI has significantly increased during the past decade, Young says. Previous studies have estimated more than 500,000 cases in the United States annually, with health care costs ranging from $1.3 billion to $3.4 billion, he says, and up to 40 percent of patients suffer from recurrence of disease following standard antibiotic treatment. Fecal transplants, which have been successful at curing more than 90 percent of recipients, have been used successfully since the 1950s, says Young, though it hasn’t been clear how they worked.

Historically, Young says, fecal transplants date back thousands of years. In fact, they were first documented in 4^th century China, where physicians fed patients “yellow soup,” a mixture of fecal matter and water. The elder physicians may have become interested by observing sick animals try to self-medicate by eating healthy animals’ feces, says Young: “It’s an old idea. Even in ‘modern medicine’ it still dates back over 50 years.” With the recent re-emergence of CDI associated with the appearance of a current epidemic strain known as B1/NAP1/027, interest in the technique has resurfaced.

Young and colleagues used 16S rRNA-encoding gene sequencing to study the composition and structure of fecal microbiota in stool samples from 14 patients before and two to four weeks after fecal transplant. In 10 of the patients, researchers also compared stool samples before and after transplant to samples from their donors. All transplant patients, treated at the Essentia Health Duluth Clinic in Minnesota, had a history of at least two recurrent C. difficile infections following an initial infection and failed antibiotic therapy.

Studying families of bacteria in the samples, investigators found marked differences among donor, pre-transplant and post-transplant samples. However, those from the donors and post-transplant patients were most similar to each other, indicating that the transplants at least partially returned a diverse community of healthy gut bacteria to the recipients. While not as robust as their donors, the bacterial communities in patients after transplant showed a reduced amount of Proteobacteria, which include various infectious agents, and an increased amount of Firmicutes and Bacteroidetes typically found in healthy individuals, compared to their pre-transplant status.

Then, using predictive software, researchers analyzed the relationship between the community structure of the micoorganisms and their function, presumably involved in maintaining resistance against CDI.

They identified 75 metabolic/functional pathways prevalent in the samples. The samples taken from patients before transplant had decreased levels of several modules related to basic metabolism and production of chemicals like amino acids and carbohydrates, but were enriched in pathways associated with stress response, compared to donor samples or post-transplant samples. 

Further identification of the specific microorganisms and functions that promote resistance of bacterial colonization, or growth, may aid in the development of improved CDI treatments, Young says: “If we can understand the functions that are missing, we can identify supplemental bacteria or chemicals that could be given therapeutically to help restore proper gut function.”

-- Karen Blum

Virulent Fish Disease Linked to Asian Source

A bacterium causing life-threatening disease among catfish farms in the southeastern United States is closely related to organisms found in diseased grass carp in China, a study published this week in mBio® has found, suggesting that the virulent U.S. fish epidemic emerged from an Asian source.

An emerging aggressive strain of Aeromonas hydrophila since 2009 has severely impacted catfish farming in Alabama, Mississippi and Arkansas by causing Aeromonas septicemia. The disease, with clinical signs including blood loss and ulcerative skin lesions, can cause death in as little as 12 hours. To date, disease outbreaks have been responsible for an estimated loss of more than $12 million in catfish aquaculture operations in the southeastern United States.

Credit: iStockphoto

When the initial outbreak occurred in western Alabama, scientists noticed something unusual. Normally, A. hydrophila, which can be found in fresh or brackish water usually in warm water temperatures, only affects fish that are stressed or injured. This time, even healthy mature fish that were not obviously stressed were getting sick. In fact, “thirty to forty percent of fish were going belly up,” said Mark Liles, PhD, an associate professor in the Department of Biological Sciences at Auburn University and senior author of the study. When initial tests of the diseased fish showed A. hydrophila was responsible, said Liles, scientists “didn’t believe it at first, because the signs didn’t match the more typical opportunistic infections in stressed fish that we associate with A. hydrophila.”

The U.S. isn’t the only country to see these problems. Several episodes of epidemic outbreaks caused by A. hydrophila have been reported in farmed carp in China starting in the late 1980s.

Liles and colleagues at three other institutions studied the molecular epidemiology of the epidemic-causing A. hydrophila to try to trace its evolution. They compared samples of the bacteria to 264 known Aeromonas strains in an international database. Only one virulent strain came close to matching the one sampled from Alabama: ZC1, isolated from a diseased grass carp in China’s Guangdong Province. ZC1 was isolated from fish that had experienced an epidemic outbreak atypical of Aeromonas infections.

Researchers also identified a related but less aggressive A. hydrophila strain called S04-690, taken in 2004 from a diseased catfish in a commercial aquaculture pond in Mississippi. That strain caused a serious Aeromonas septicemia outbreak that killed thousands of catfish but did not result in an epidemic on neighboring farms.

Next, the scientists evaluated the evolutionary relationships of additional Chinese carp bacteria samples from epidemics in the Hubei Province in China. But getting samples of ZC1 to study provided additional challenges. Liles contacted some Chinese researchers, who politely declined. Then one of Liles’ Chinese graduate students inquired, and also was turned down. Only when Liles traveled to China and was able to contact Ye Xing of the Pearl River Fisheries Research Institute was his request granted, and only then after he filed paperwork with the federal Centers for Disease Control and Prevention here and arranged for export from China.

The researchers found that all of the Chinese carp bacteria samples, including ZC1, group together in one clade with the recent U.S. epidemic samples of bacteria in catfish, suggesting that a common ancestor is responsible for the virulent A. hydrophila strains causing fish disease in both China and the United States. S04-690, from the Mississippi fish, also was found to be in the same group, related to both the U.S. catfish and Asian carp bacteria. However, it was genetically distinct from the other two strains. By contrast, A. hydrophila strains that do not cause epidemics were more heterogeneous. Additional experiments found that isolated bacteria from diseased catfish in America and diseased carp in China shared alternate forms of 10 key ‘housekeeping’ genes required for basic cellular function.

It’s not clear how the bacterium was introduced in America, Liles said. It could be from importing Asian carp to America over the past several decades for aquatic weed control, or from transporting ornamental fish or contaminated processed seafood products from Asia. The spread of disease among farms also is not fully understood but could result from birds that move from pond to pond eating catfish, or from harvesting equipment that may be insufficiently sanitized between uses.

Liles and other researchers are investigating means to control the spread of illness, including developing vaccines against the bacteria, and using medicated feed and/or probiotics. Meanwhile, he said, U.S. farmed catfish are safe to eat and pose no disease threat to humans due to strict standards regarding harvesting and processing of sick animals.

-- Karen Blum

Researchers Find Unique Avian Influenza in Antarctic Penguins

When influenza researcher Aeron Hurt heard that INACH, the Chilean Antarctic Institute, would be studying mite and tick infection in a group of Antarctic penguins, he had a thought: As long as the penguins were already being tested for those infections, might they also be examined for presence of avian influenza?

This collaboration led to the exciting discovery of a novel avian influenza virus in Adélie penguins in Antarctica that was determined to be unlike any other circulating avian flu. The virus was described in a study published this week in mBio.

Hurt with Penguin. Credit: Aeron Hurt, WHO Collaborating Centre for Reference and Research on Influenza

While other research groups have taken blood samples from penguins before and detected influenza antibodies, no one had detected actual live influenza virus in penguins or other birds in Antarctica previously, says Hurt, a senior research scientist at the WHO Collaborating Centre for Reference and Research on Influenza in Melbourne, Australia.

Hurt and his Chilean colleagues collected swabs from the windpipes and posterior openings of 301 Adélie penguins, and blood from 270 of those penguins, from two locations on the Antarctic Peninsula: Admiralty Bay and Rada Covadonga. The samples were collected during January and February 2013.

Using real-time reverse transcription-PCR, the researchers found avian influenza virus (AIV) RNA in eight (2.7%) samples, six from adult penguins and two from chicks. Seven of the samples were from Rada Covadonga. The researchers were able to culture four of these viruses, demonstrating that live infectious virus was present. On further analysis, the investigators determined that all viruses were H11N2 influenza viruses that were highly similar.

But when the researchers compared the full genome sequences of four of the collected viruses to all available animal and human influenza virus sequences in public databases, “we found that this virus was unlike anything else detected in the world,” says Hurt. “When we drew phylogenetic trees to show the evolutionary relationships of the virus, all of the genes were highly distinct from contemporary AIVs circulating in other continents in either the Northern or Southern Hemisphere.”

Four of the gene segments were most closely related to North American avian lineage viruses from the 1960s to 1980s. Two genes showed a distant relationship to a large number of South American AIVs from Chile, Argentina and Brazil. Using a molecular clock to incorporate the evolutionary rate of each AIV gene segment, the researchers estimated that the virus has been evolving for the past 49 to 80 years without anyone knowing about it. Whether this evolution has occurred exclusively in Antarctica is currently unknown, Hurt says.

Additional experiments found that 16% of penguins (43 of 270) had influenza A antibodies in their blood, and that the newly identified virus is likely to be exclusive to birds, as it did not readily infect a group of ferrets used as a test to see if the virus could infect mammals.

While the virus did not cause illness in the penguins, the study shows that “avian influenza viruses can get down to Antarctica and be maintained in penguin populations,” Hurt says. “It raises a lot of unanswered questions,” including how often AIVs are being introduced into Antarctica, whether it is possible for highly pathogenic AIVs to be transferred there, what animals or ecosystems are maintaining the virus, and whether the viruses are being cryopreserved during the winters.

Hurt says he hopes to continue studying the penguins, to see if AIVs will be detected in subsequent years, if new viruses emerge, and if viruses will be found in other locations in Antarctica.

-- Karen Blum