Technological advances have helped spur the frequently tedious and timeconsuming efforts necessary to understand the complexity of drug resistance.
June 10, 2014 - Jane L. Depriest or Holly Whetstone
The evolution of antibiotic resistance and its impact on the environment is highly complex, and understanding it requires extensive time and energy. But thanks to modern-day advances in technology, some parts of the process move at what comparatively seems like lightning speed. High-capacity tools for sequencing whole genomes, including those in nature, along with analytical methods for the enormous amount of data produced have significantly accelerated the rate of the research.
James Tiedje is the director of the MSU Center for Microbial Ecology, which was founded 25 years ago as one of the first National Science Foundation Science and Technology Centers in the United States. Work at the center focuses on the competitiveness, diversity and function of microorganisms in their natural and managed habitats. Tiedje said the benefits acquired through technology have helped to improve scientists’ understanding of microbes and how they affect their surroundings.
“The information gained through these technologies is allowing insight into the microbial world around us — in soil, water and even air — that was previously unimaginable,” said Tiedje, an MSU AgBioResearch scientist.
One of the latest breakthroughs is the ability to detect hundreds of genes at a time, rather than just one or two as in the past. This technology has been particularly helpful for Tiedje and other researchers who are working to determine the extent to which antibiotic use in agriculture may be contributing to the growing antibiotic resistance problem in pathogens.
Though technological advances have helped to expedite research on antibiotic resistance, researchers say that working collaboratively is critical as well.
“Collaborations allow researchers to do things that, alone, none of us could have done,” said MSU AgBioResearch environmental and soil scientist Stephen A. Boyd. “The results can be obtained and examined from several viewpoints— experimental, spectroscopic, computational and analytical — instead of relying on a single method or area of expertise. In the end, the results from all these sources must all be consistent, which makes the conclusions far more convincing.”
Today, much of the research at the MSU Center for Microbial Ecology is focused on the microbiome — communities of microbes that are nearly everywhere, including those living with larger organisms such as plants and humans.
“These studies have led to a new field of ecological genomics with two components: the high-capacity technologies and devices that provide the data about biological systems, and the gene databases and computational analysis tools to make sense of the huge amounts of data generated,” explained Tiedje, a University Distinguished Professor in the Department of Plant, Soil and Microbial Sciences.
As an example of this work, Tiedje and a multidisciplinary MSU and Chinese research team used a new high-capacity DNA technology and a sophisticated database to determine the levels of antibiotic-resistant genes — ARGs — on commercial pig farms in China. ARGs reduce the ability of antibiotics to fend off diseases in humans and animals and can reach the general population through food crops, drinking water and interactions with farm workers (see diagram on page 7).
The database for the foundation of the work was developed by Robert Stedtfeld and Syed Hashsham, both from the MSU Department of Civil and Environmental Engineering, in collaboration with Benli Chai and James Cole, bioinformatics specialists in the Center for Microbial Ecology.
“By going through DNA databases in public references and looking at other research done on antibiotic resistance, we developed an extensive database for ARGs,” Stedtfeld explained. “Then we designed primers, which are pieces of DNA sequence specific to each targeted gene. These primers aid in the detection of specific genes present in the sample being tested.”
The database with the primers is one of the most comprehensive for detecting ARGs.
Manure, compost and soil samples from the Chinese pig farms were screened by a high-throughput tool used for sensitive detection of hundreds of genetic signatures in multiple samples simultaneously. These capabilities are available commercially in technologies from WaferGen (SmartChip) and Life Technologies (OpenArray), both life science companies offering genomic solutions.
“The capabilities of tools offered by WaferGen and Life Technologies are advanced techniques for performing thousands of screening reactions in parallel, revealing gene presence and abundance,” Stedtfeld said.
Testing by Tiedje and his research groups has been with laboratory instruments, but researchers are working to put the most important genes from the database on a hand-held analyzer that can be used in the field.
“The continuing developments of these kinds of technologies can help us to better track genes of interest in the environment,” Tiedje said. “They also can help us learn more about the critical points for intervention to minimize the growing problem of multiple-drug-resistant pathogens.”
Tiedje also worked on a controlled study with collaborators at the National Animal Disease Laboratory in Ames, Iowa. The findings showed that antibiotics used as growth promoters in feed increased the number of ARGs in the gastrointestinal tract of pigs compared with those found in littermates not fed antibiotics.
“Daily exposure to antibiotics allows microbes carrying ARGs to thrive,” Tiedje explained. “In some cases, these antibiotic-resistant genes can be highly mobile, meaning they can be transferred to other bacteria, some of which could cause illnesses in humans. That’s a growing concern because the infections those bacteria cause can no longer be treated with antibiotics.”
Soil and surface waters are other important areas of ARG investigation. MSU AgBioResearch soil chemist Hui Li in collaboration with Boyd and Brian Teppen, an MSU AgBioResearch scientist, researches soil contaminants, including antibiotics. A key instrument in this work is a liquid chromatograph with tandem mass spectrometers (LC-MS/MS), which gives specific analytical information and has a higher throughput analysis than gas chromatography, another laboratory technique for the separation of mixtures. The LC-MS/MS, purchased with funds from MSU AgBioResearch and other sources for Li’s lab, helps to identify antibiotics and many other pharmaceuticals in the environment and measure their quantities in water and soil.
“With the LC-MS/MS we are able to get the big picture,” said Li, an associate professor of plant, soil and microbial sciences. “We can bring samples back to the lab and do extractions to measure the antibiotics in soil and water.”
Li has focused on emerging organic contaminants in soil and water, especially identifying and measuring antibiotics in the environment. Tetracyclines, broadspectrum antibiotics used in the treatment of numerous infections and also in animal feeding operations, are the focus of much of his research.
“If tetracyclines get into the soil or water, they can exert selective pressure on native bacterial populations, which can cause the development of antibiotic resistance in bacteria,” Li said. “However, our research has shown that if there is more calcium or magnesium in the water, the antibiotic response is much lower than in water without these minerals. This could reduce the bioavailability and hence selective pressure of tetracyclines.”
Bioavailability describes the degree and rate at which a substance from an environmental matrix is absorbed into a living system and is a key factor linking environmental exposure with the development of ARGs.
Other factors affecting bioavailability are environmental conditions, such as wet, dry or moist soils.
“Tetracyclines become more bioavailable to bacteria particularly in moist conditions,” Li said. “In soil that is moist but not watery, tetracyclines become highly bioavailable. If tetracyclines are bound to soil particles suspended in water, however, they will not become bioavailable.”
Those that are more available to bacteria would be expected to exert more selective pressure, he added. Li notes that antibiotic resistance has always existed in the environment and that bacteria will pick up resistant genes, if needed.
“If you have selective pressure from chemicals or other antibiotics, exposed bacteria will self-protect. They will grab resistant genes into their bodies, and they can transfer these genes to other bacteria. All of that adds to the environmental sources of antibiotic resistance.”
Li agrees with Boyd that, in addition to innovative tools and equipment, collaborations are an important part of the work.
“For me as a soil chemist, I don’t know much about microbiology, for example,” Li said. “By working with researchers in other fields, such as James Tiedje, we can obtain more information that can spur our own research.”
New molecular tools that allow researchers to measure the proliferation of ARGs in soil have helped to advance the work of Boyd, an MSU University Distinguished Professor of Plant, Soil and Microbial Sciences. His research focuses specifically on the environmental fate and effects of organic contaminants and pesticides in soil.
“We can use engineered bacteria as very sensitive detectors of pharmaceuticals such as tetracyclines, and we don’t have to work in really clean systems, as once was the case. That has been a big step forward,” Boyd explained. “On the analytical side, the LC-MS/MS has enabled us to work with more complex mixtures and detect them at lower concentrations, which is important in dealing with pharmaceuticals.”
His research, which began in the 1980s, has recently expanded to include pharmaceuticals in the soil.
“Pharmaceuticals — everything from codeine to tetracyclines to antidepressants — are showing up in the environment, especially when you look at surface water,” Boyd said.
Pharmaceuticals in soil and water are considered emerging contaminants because they are not highly regulated.
“For example, there are no drinking water standards or tolerances for them in soils,” Boyd explained, “but industrial organic compounds and pesticides are regulated to limit their occurrence in soils and groundwater.”
In collaboration with Li and Teppen, Boyd is working on ways to make the soil sequester contaminants, whether industrial or pharmaceutical organic molecules, and use bioavailability as a way of managing the risks posed by contamination in soils and sediments.
“The basic idea is what we call sorbent amendments,” Boyd explained. “We want to see if contaminant molecules that are sequestered by geosorbents in soils and sediments display reduced bioavailability to a target organism, whether a bacterium or a human being. Does a molecule have to escape from the geosorbent before it can enter the cells of bacteria, plants or humans? Those are the kinds of basic questions we are trying to answer because they have big practical implications.”
The geosorbents that Boyd and his collaborators are most interested in are chars – in essence, burnt pieces of plant material. Some char occurs naturally in soils from fires, but it can be produced as a byproduct in biofuels production. They also are attractive as geosorbents because they are an effective way of sequestering carbon. In addition, chars can be beneficial to soil productivity in terms of crop growth.
“The bottom line is to use chars or some other sorbent amendment to reduce the bioavailability of chemicals that occur as contaminants in soils and sediments,” Boyd said. “This reduces risks associated with these contaminants and may let us safely relax cleanup criteria. This might allow, for example, more contaminated sites to be remediated using the limited funds available.”
Any given antibiotic in the environment can take many chemical forms at the same time. For example, some are attached to dissolved compounds in the water, and some are attached to soil particles. Teppen tries to understand this complexity.
“We call it chemical speciation,” said Teppen, a professor of plant, soil and microbial sciences. “We use computational tools to try to model the complex distribution of antibiotics among all their forms.”
First, experimental data is collected under a variety of controlled conditions.
“Then we use chemical models to help extract all the information we can from the data. One goal is to understand exactly which chemical forms of a given antibiotic induce resistance in the bacteria,” Teppen explained. “Perhaps then we would be able to suggest management schemes that would minimize the antibiotic-resistance selection pressure in fields where manures are applied.”
Even with ongoing advances in technology and equipment that aid ARG research, solutions are not easy. Li and other researchers working on antibiotic resistance issues believe that antibiotics in animal feeding operations have to be better managed and controlled.
“But, at the same time, we need to develop effective treatment procedures to reduce the effect of antibiotics in the environment,” Li explained. “For example, we need more advanced treatments in the animal waste facilities to deal with antibiotics and their resistance genes (ARGs), but that is difficult to do right now. If we knew more about fundamental processes in the environment and how they affect microbial communities, perhaps we could figure out better ways to mitigate the antibiotic resistance effect.”
For Tiedje, the ultimate goal is to minimize the rate of development of multidrugresistant pathogens. Research using new technologies can identify points of intervention where new or altered practices reduce this risk.
“It is extremely difficult to find new antimicrobial drugs, so we must protect the effectiveness of the ones we have for as long as we can,” Tiedje said. “One principle has been not to use antibiotics important in human use on animals.”
The idea is that, if resistance developed in microbes for these animal drugs, it would not affect the effectiveness of the human drugs.
“Our research, however, consistently shows that that principle is not sound because of co-selection,” Tiedje explained. “In other words, the development of antibiotic resistance to one drug also results in resistance to other drugs, including ones important for human use. This is because the resistance genes have become linked on the same piece of DNA and are transferred together to new microbes. When that recipient of the transfer is a human pathogen, a multidrug-resistant pathogen is born.”