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kathleenseiber · 5 years

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Lab tests may misdiagnose 50% of diarrhea’s bacterial causes in kids

Conventional culture-based lab tests may misdiagnose as many as half of the microbial causes of diarrhea in children, a new study shows.

Further, the study, which used samples from Ecuadorian children, also found that a common strain of the E. coli bacterium may be more virulent than previously believed.

The research, which used multiple lines of evidence to determine the microbe causing changes in the gut microbiome during a diarrheal episode, may be significant for applying new diagnostic technologies that could enable personalized medical treatments of intestinal diseases.

The project is part of a study that integrates epidemiological, molecular, and metagenomic data to understand how enteric (food and waterborne) pathogens and the gut microbiome vary across an urban-rural gradient in Ecuador. Frequent illness can affect the growth and development of children during their critical early years.

The new findings appear in Applied and Environmental Microbiology.

When is a microbe not a pathogen?

“We wanted to understand where the illnesses are coming from and what the consequences are for the development of the children,” says Kostas Konstantinidis, a professor in the School of Civil and Environmental Engineering at Georgia Tech.

“Knowing more about the causative agent may allow us to prevent infections. This would be relevant not just for the developing world, but for children everywhere.”

Findings about the likely causative agent of enteric diseases raise questions about long-held definitions of pathogenic agents, says Karen Levy, an associate professor at the Emory Rollins School of Public Health.

Many so-called “pathogenic E. coli” infections are asymptomatic, not causing diarrhea, she notes. If health experts detect a microbe considered a pathogen in a stool sample, but the microbe doesn’t cause disease, is it a pathogen?

“The approach we used in this analysis helps to not only detect whether or not the pathogenic E. coli is present in the stool, but also if it is the likely cause of diarrhea experienced by study subjects,” Levy says.

“This holds promise that, in the future, we could use metagenomic approaches to diagnose not just the presence of the so-called ‘pathogenic E. coli,’ but also of the actual pathogenicity of these organisms.”

E. coli and diarrhea

Enteric infections often go undiagnosed in children, though they can in some circumstances be fatal. For the study, researchers collected more than 1,000 samples from infected children in Ecuador and analyzed them using traditional culture-based methods in which scientists place samples onto selective cell culture media, allow to grow, and then study them to determine the pathogen present.

Researchers sent a subset of 30 samples to the Konstantinidis lab at Georgia Tech, where his team has developed new culture-independent genomics testing that can identify microbes that standard culture techniques may not detect. The researchers examined the total gut microbiome and its shifts during diarrheal infections to assess the effects of three specific pathogenic genotypes of E. coli on the indigenous gut microbiota.

“We looked at the microbes in stool samples using culture-independent genomic techniques,” says Konstantinidis, who also has a faculty position in the School of Biological Sciences.

“We took the DNA out of the samples, sequenced it, and used bioinformatics tools to see what microorganisms were there. By looking at the entire microbiome, we can get more precise information about the pathogens that are causing disease and their effects on the commensal microbes of the gastrointestinal tract: the microbiome. We found that the effects were different for different E. coli pathogens, which is important for diagnosis and distinguishing among them.”

One major finding was that the metagenomics technique disagreed with the culture-based study. “In 50% of the cases where the lab suggested E. coli was the agent, we didn’t see evidence from metagenomics that this was the case,” Konstantinidis says. “We saw evidence that it was something else, potentially an enteric virus.”

Effect on growth and development

The researchers combined different diagnostic approaches in a way that offered a more complete picture of the microbiome, creating signatures that could potentially identify the microbial agent of the disease.

“Compared to the traditional approach, our technique uses multiple lines of evidence, including relative pathogen abundance, population clonality level and detection of virulence factors, and effects on the gut’s natural microbiome that haven’t been used together before,” Konstantinidis says.

The researchers also discovered that large amounts of co-eluted human DNA accompanied a strain known as diffuse adherent E. coli (DAEC) in the stool sample. Though scientists don’t generally consider DAEC a particularly virulent strain, the presence of human DNA suggests that the DAEC may have damaged epithelium cells in the gastrointestinal tract.

“Understanding changes in the gut microbiome can help us understand how different strains cause distinct pathogenicity and symptoms such as the elution of high amount of human DNA and changes in the abundance of commensal microbiota,” Konstantinidis says. “If infections like this happen often, that could have implications for a child’s growth and development.”

Innovation in diarrhea diagnosis and treatment

The metagenomics technique could help provide a foundation for personalized medicine that would select therapies based on the specifics of the microbe—including its virulence and antibiotic resistance profiles. The study shows that the technology works and can be done quickly, but trained personnel are needed to make it more widely available.

“In the next five or 10 years, we are going to see some big changes in which these new methodologies are adopted in the clinic, and that could lead, in the long term, to innovation in diagnosis and treatment,” Konstantinidis says.

Future work will examine how environmental factors—such as water quality, presence of animals in the home, and the chemical environment—affect the microbiomes of small children. In this cohort of children from different geographic areas, the team will examine additional enteric pathogens, and follow children for their first two years of life. This will offer the opportunity to understand whether infections are cleared from the body—or if they continue to lurk in low levels.

Additional coauthors are from Universidad San Francisco de Quito, Georgia Tech, and Emory. The National Institute of Allergy and Infectious Diseases funded the work. The contents are solely the responsibility of the author and do not necessarily represent the official views of the NIH.

Source: Georgia Tech

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kathleenseiber · 5 years

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As tundras warm, microbes could make climate change worse

Rising temperatures in the tundra of the Earth’s northern latitudes could affect microbial communities in ways likely to increase their production of greenhouse gases methane and carbon dioxide, a new study suggests.

About half of the world’s total underground carbon is stored in the soils of these frigid, northern latitudes. That is more than twice the amount of carbon currently found in the atmosphere as carbon dioxide, but until now most of it has been locked up in the very cold soil.

The new study, which relied on metagenomics to analyze changes in the microbial communities researchers experimentally warmed, could heighten concerns about how the release of this carbon may exacerbate climate change.

“We saw that microbial communities respond quite rapidly—within four or five years—to even modest levels of warming,” says corresponding author Kostas T. Konstantinidis, a professor in the School of Civil and Environmental Engineering and the School of Biological Sciences at Georgia Institute of Technology.

“Microbial species and their genes involved in carbon dioxide and methane release increased their abundance in response to the warming treatment. We were surprised to see such a response to even mild warming.”

The study provides quantitative information about how rapidly microbial communities responded to the warming at critical depths, and highlights the dominant microbial metabolisms and groups of organisms that are responding to warming in the tundra. The work underscores the importance of accurately representing the role of soil microbes in climate models.

Digging in the dirt

The research began in September 2008 at a moist, acidic tundra area in the interior of Alaska near Denali National Park. Researchers created six experimental blocks, and in each block, they constructed two snow fences about five meters apart in the winter to control snow cover. Thicker snow cover in the winter served as an insulator, creating slightly elevated temperatures—about 1.1 degrees Celsius (2 degrees Fahrenheit) in the experimental plots.

Other than the temperature difference, the soil conditions were similar in the experimental and control plots. Researchers took soil cores from the experimental and control plots at two different depths at two different times: 1.5 years after the experiment began, and 4.5 years after the start. Then, they extracted microbial DNA from the cores and sequenced it.

“Our analysis of the resulting data showed which species were there, in what abundances, which species responded to warming and by how much—and what functions they possessed related to carbon use and release,” says Eric R. Johnston, now a postdoctoral researcher at Oak Ridge National Laboratory, who conducted the study’s analysis as a PhD student.

Researchers compared cores from the experimental and control plots to assess the effects of the warming. They also sampled cumulative ecosystem respiration during the month following removal of the cores.

“The response we observed differed markedly between the two soil depths (15 to 25 centimeters and 45 to 55 centimeters) that were sampled for this study,” says Johnston. “Specifically, at the upper boundary of the initial permafrost boundary layer—45 to 55 centimeters below the surface—the relative abundance of genes involved in methane production (methanogenesis) increased with warming, while genes involved in organic carbon respiration—the release of carbon dioxide—became more abundant at shallower depths.”

Measurement of the community respiration showed increases in the rate of carbon dioxide and methane release in the plots that researchers warmed. “Similar measurements have also shown that these gases are being released at a greater rate throughout the entire region in recent years as a result of climate warming,” Johnston adds.

Remember microbes

The two soil depths correspond to an active layer near the surface that freezes during the winter but thaws during warmer months, exposing the carbon. The deeper measurements examined soil just above the permafrost that thaws for only a brief time each year. These variations create fundamental differences in the biology and chemistry at the two depths.

“We expected to observe warming responses that differed between the two sampling depths,” Johnston says. “Ongoing thaw of permafrost soil is being observed on the global scale, so we were particularly interested in evaluating microbiological responses to thawing permafrost.”

The research highlights the importance of microbial communities in contributing atmospheric methane and carbon dioxide to climate change, Konstantinidis says.

“Because of the very large amount of carbon in these systems, as well as the rapid and clear response to warming found in this experiment and other studies, it is becoming increasingly clear that soil microbes—particularly those in the northern latitudes—and their activities need to be represented in climate models,” he says. “Our work provides markers—species and genes—that can be used in this direction.”

The research appears in the Proceedings of the National Academy of Sciences.

The US Department of Energy and the National Science Foundation supported the research.

Researchers from the University of Oklahoma, Tsinghua University, Michigan State University, Lawrence Berkeley National Laboratory, and Northern Arizona University contributed to the study. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the sponsoring organizations.

Source: Georgia Tech

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