Microbes that are used for health, agricultural, or other applications need to be able to withstand extreme conditions and, ideally, the manufacturing processes used to make tablets for long-term storage. MIT researchers have now reported on a method for making microbes hardy enough to withstand these extreme conditions.
Their approach involves mixing bacteria with food and drug additives from a list of compounds that the FDA classifies as “generally regarded as safe.” The researchers identified formulations that help to stabilize several different types of microbes, including yeast, gram-negative and gram-positive bacteria, and they showed that these formulations could withstand high temperatures, radiation, and industrial processing that can damage unprotected microbes.
In an even more extreme test, some of the microbes recently returned from a trip to the International Space Station, coordinated by Space Center Houston Manager of Science and Research Phyllis Friello, and the researchers are now analyzing how well the microbes were able to withstand those conditions.
“What this project was about is stabilizing organisms for extreme conditions,” said Giovanni Traverso, PhD, an associate professor of mechanical engineering at MIT, and a gastroenterologist at theBrigham and Women’s Hospital. We’re really thinking about a broad set of applications, whether it’s missions to space, human applications, or agricultural uses.”
Senior author Traverso, together with lead author Miguel Jimenez, PhD, a former MIT research scientist and now an assistant professor of biomedical engineering at Boston University, and colleagues reported on their results in Nature Materials, in a paper titled “Synthetic extremophiles via species-specific formulations improve microbial therapeutics.” In their report the team concluded, “… these synthetic extremophiles stand to transform our capacity to disseminate bioactive organisms across human applications, from shelves across the globe to fields for agricultural practices to shuttles for space exploration.”
Microorganisms typically used to produce food and pharmaceuticals are now being explored as medicines and agricultural supplements, the authors wrote. “Microorganisms have been central to human technological progress and continue to be key in wide-ranging fields from food production (for example, baked goods) to biologics manufacturing (for example, synthetic insulin.” And for these types of application “… the microbial cells are kept alive only during the manufacturing process and are destroyed, deactivated or removed from the final product.” In contrast, the team continued, “… the pharmaceutical, agricultural and space health fields have now turned to developing live microorganisms as the final product to cure disease, to enhance crop production and for on-demand bioproduction…Critical to these new microbial technologies is the maintenance of high cell viability throughout the entire life cycle of the product.” An ideal solution, they suggested, would be dry, microbial formulations that are easy to package, ship and use.
About six years ago, with funding from NASA’s Translational Research Institute for Space Health (TRISH), Traverso’s lab began working on new approaches to make helpful bacteria such as probiotics and microbial therapeutics more resilient. As a starting point, the researchers analyzed 13 commercially available probiotics and found that six of these products did not contain as many live bacteria as the label indicated. “… when we surveyed the viable cell counts (colony-forming units, CFUs) across a range of probiotics … we found only 7 in 13 products contained viable cell counts at or higher than the promised amount on the label … with a mean (geometric) viability of ~21% of that promised,” they wrote.
“What we found was that, perhaps not surprisingly, there is a difference, and it can be significant,” Traverso explained. “So then the next question was, given this, what can we do to help the situation?” For their reported experiments, the researchers focused on three bacteria and one yeast. The bacterium Escherichia coli Nissle 1917 is a probiotic. Ensifer meliloti, is a bacterium that can fix nitrogen in soil to support plant growth. The bacterium Lactobacillus plantarum is used to ferment food products. The yeast Saccharomyces boulardii is also used as a probiotic.
For medical or agricultural applications microbes are usually dried into a powder through a process called lyophilization However, they cannot normally be made into forms such as a tablet or pill because this process requires exposure to an organic solvent, which can be toxic to the bacteria. The MIT team set out to find additives that could improve the microbes’ ability to survive this kind of processing. “In designing our approach, our overarching requirement was regulatory and industrial translatability,” they explained. Their strategy was to apply material stabilizers rather than apply genetic changes that would ‘add regulatory burden.’” Similarly, they pointed out, rather than develop new synthetic materials, we designed what they described as a material library composed primarily of materials generally recognized as safe by FDA.
“We developed a workflow where we can take materials from the ‘generally regarded as safe’ materials list from the FDA, and mix and match those with bacteria and ask, are there ingredients that enhance the stability of the bacteria during the lyophilization process?” Traverso noted.
The approach allows them to mix microbes with one of about 100 different ingredients and then grow them to see which survive the best when stored at room temperature for 30 days. These experiments revealed different ingredients, mostly sugars and peptides, that worked best for each species of microbe.
The researchers then picked one of the microbes, E. coli Nissle 1917, for further optimization. This probiotic has been used to treat “traveller’s diarrhea,” a condition caused by drinking water contaminated with harmful bacteria. The researchers found that if they combined caffeine or yeast extract with a sugar called melibiose, they could create a very stable formulation of E. coli Nissle 1917. This mixture, which the researchers called formulation D, allowed survival rates greater than 10 percent after the microbes were stored for six months at 37° Celsius=. In contrast, a commercially available formulation of E. coli Nissle 1917 lost all viability after only 11 days under those conditions. “Both melibiose and formulation D outperformed the commercial product Mutaflor (E. coli Nissle 1917) by over 3.5 orders of magnitude when stored at room temperature for one month,” the investigators noted.
Formulation D was also able to withstand much higher levels of ionizing radiation, up to 1,000 grays. “At this radiation level a liquid suspension of the same bacteria lost all measurable viability,” the scientists wrote. The typical radiation dose on Earth is about 15 micrograys per day, and in space, it’s about 200 micrograys per day.
Their formulation was in addition compatible with pharmaceutical processing and tableting. “We also showed that these synthetic extremophiles withstand pharmaceutical manufacturing workflows enabling the production of controlled release microbial dosage forms,” they stated. The researchers don’t know exactly how their formulations protect bacteria, but they hypothesize that the additives may help to stabilize the bacterial cell membranes during rehydration.
The investigators in addition showed that these microbes can not only survive harsh conditions, they also maintain their function following such exposures. After Ensifer meliloti were exposed to temperatures up to 50° Celsius, the researchers found that they were still able to form symbiotic nodules on plant roots and convert nitrogen to ammonia. They also found that their formulation of E. coli Nissle 1917 was able to inhibit the growth of Shigella flexneri, one of the leading causes of diarrhea-associated deaths in low- and middle-income countries, when the microbes were grown together in a lab dish. “We further show, in live animals and plants, that synthetic extremophiles remain functional against enteric pathogens and as nitrogen-fixing plant supplements even after exposure to elevated temperatures.”
Last year, several strains of these extremophile microbes were sent to the International Space Station, which Jimenez described as “the ultimate stress test. He said “Even just the shipping on Earth to the preflight validation, and storage until flight are part of this test, with no temperature control along the way.” The samples recently returned to Earth, and Jimenez’ lab is now analyzing them. He plans to compare samples that were kept inside the ISS to others that were bolted to the outside of the station, as well as control samples that remained on Earth.
Concluding on their reported work, the team stated, “We develop a high-throughput pipeline to define species-specific materials that enable survival through drying, elevated temperatures, organic solvents and ionizing radiation…This synthetic, material-based stabilization enhances our capacity to apply microorganisms in extreme environments on Earth and potentially during exploratory space travel.”
Further improvements in microbial stability and dosage form design may involve genetic approaches, or single-cell encapsulation techniques, they wrote. “Our bulk material stabilization approach is orthogonal to and could be combined with these approaches to make microbial materials with more advanced robustness and pharmacokinetic release profiles.”
