Antibiotics are life-saving drugs, but they can also harm the beneficial microbes that live in the human gut, and this can put some patients at risk of developing inflammation or opportunistic infections such as Clostridiodes difficile.

As a potential strategy for reducing those risks, MIT engineers have developed a way to help protect the natural flora of the human digestive tract. The scientists took a strain of Lactococcus lactis bacteria that is safe for human consumption, and engineered it to produce an enzyme that breaks down a class of commonly used antibiotics called β-lactams. Given to mice in combination with antibiotics, this live engineered biotherapeutic product (eLBP) was shown to protect the animals’ gut microbiota, while allowing levels of antibiotics circulating in the bloodstream to remain high. The team also suggests that the approach won’t increase the potential for antibiotic resistance.

“This work shows that synthetic biology can be harnessed to create a new class of engineered therapeutics for reducing the adverse effects of antibiotics,” said James Collins, PhD, the Termeer Professor of Medical Engineering and Science in MIT’s Institute for Medical Engineering and Science (IMES) and Department of Biological Engineering. Collins is the senior author of the new study, which is published in Nature Biomedical Engineering.

“If the antibiotic action is not needed in the gut, then you need to protect the microbiota,” added first author Andrés Cubillos-Ruiz, PhD, a research scientist at IMES and the Wyss Institute for Biologically Inspired Engineering at Harvard University. This is similar to when you get an X-ray, you wear a lead apron to protect the rest of your body from the ionizing radiation. No previous intervention could offer this level of protection. With our new technology we can make antibiotics safer by preserving beneficial gut microbes and by reducing the chances of emergence of new antibiotic resistant variants.”

The researchers reported on their developments in a paper titled “An engineered live biotherapeutic for the prevention of antibiotic-induced dysbiosis,” in which they concluded, “Engineered live biotherapeutics that safely degrade antibiotics in the gut may represent a suitable strategy for the prevention of dysbiosis and its associated pathologies.”

Decades of research has found that the microbes in the human gut play important roles in not only metabolism but also immune function and nervous system function. “Throughout your life, these gut microbes assemble into a highly diverse community that accomplishes important functions in your body,” said Cubillos-Ruiz. “The problem comes when interventions such as medications or particular kinds of diets affect the composition of the microbiota and create an altered state, called dysbiosis. Some microbial groups disappear, and the metabolic activity of others increases. This unbalance can lead to various health issues.” As the authors noted, “Disruption of the ecological balance in gut microbial communities, termed dysbiosis, has been associated with a wide range of immunological and metabolic disorders such as allergies, autoimmunity and obesity.”

The global yearly use of antibiotics is about 77 billion doses, of which β-lactams constitute about 62%, the authors noted. This class of antibiotics includes penicillins, carapenems and cephalosporins. But while antibiotic therapy is essential for treating some bacteria infections, antibiotic-induced alterations in the gut microbiota may not only impact on metabolic and inflammatory diseases, but also increase the risk of secondary infections.

Repeated exposure of the gut microbiota to antibiotics may eliminate commensal bacteria from their intestinal niche, and this can open up opportunities for pathogens such as Clostridioides difficile to colonize and proliferate. C. difficile, a microbe that commonly lives in the gut, doesn’t usually cause harm. But when antibiotics kill off the strains that compete with C. difficile, these bacteria can take over and cause diarrhea and colitis. C. difficile infects about 500,000 people every year in the US, and causes around 15,000 deaths.

Antibiotic use is also linked with antibiotic resistance the authors pointed out. “Exposure to antibiotics may also support antimicrobial resistance through the enrichment of gut bacterial populations that carry antimicrobial resistance genes (ARGs), and these can be transferred to pathogenic bacteria through horizontal gene transfer (HGT),” they said. “Therefore, there is a pressing need for effective interventions that protect the gut microbiota while antibiotics systemically circulate in the body.”

Doctors sometimes prescribe probiotics (mixtures of beneficial bacteria) to people taking antibiotics, but those probiotics are usually also susceptible to antibiotics, and they don’t fully replicate the native microbiota found in the gut. “Standard probiotics cannot compare to the diversity that the native microbes have,” Cubillos-Ruiz said. “They cannot accomplish the same functions as the native microbes that you have nurtured throughout your life.”

As an alternative approach to protect the microbiota from antibiotics, the researchers decided to use modified bacteria. They engineered a strain of Lactococcus lactis – which is normally used in cheese production – to deliver a beta lactamase enzyme that breaks down β-lactam antibiotics. When these engineered bacteria are delivered orally, they transiently populate the intestines, where they secrete the enzyme β-lactamase. This enzyme then breaks down antibiotics that reach the intestinal tract. This same approach could also be used with antibiotics that are injected, as antibiotics administered by this route also end up reaching the intestine. Then, after their job is finished, the engineered β-lactamase-producing bacteria are excreted through the digestive tract.

The idea of using engineered bacteria that degrade antibiotics poses unique safety requirements. β-lactamase enzymes confer antibiotic resistance to harboring cells, and their genes can readily spread between different bacteria. “Clinical use of a food-associated bacterium that carries an antibiotic degradation trait requires robust safety features, namely the absence of a competitive advantage under antibiotic selection to preclude overgrowth and imperviousness of the trait to HGT,” the team pointed out.

To address this potential issue, the researchers used a synthetic biology approach to recode the way the bacterium synthesizes the enzyme. They broke up the gene for β-lactamase into two pieces, each of which encodes a fragment of the enzyme. These gene segments are located on different pieces of DNA, making it very unlikely that both gene segments would be transferred to another bacterial cell. The β-lactamase fragments are then exported outside the cell where they reassemble, restoring the enzymatic function. “Our eLBP incorporates multiple engineered features that ensure its clinical and environmental safety,” the team asserted.

Since the β-lactamase is now free to diffuse in the surrounding environment, its activity becomes a “public good” for the gut bacterial communities. This prevents the engineered cells from gaining an advantage over the native gut microbes.  “Our biocontainment strategy enables the delivery of antibiotic-degrading enzymes to the gut without the risk of horizontal gene transfer to other bacteria or the acquisition of an added competitive advantage by the live biotherapeutic,” Cubillos-Ruiz commented.

To assess the use of this approach, the researchers gave mice two oral doses of the engineered bacteria for every injection of ampicillin. They found that the engineered bacteria reached the intestine and began releasing β-lactamase. The researchers further confirmed that in mice given the engineered bacteria alongside antibiotic therapy the amount of ampicillin circulating the bloodstream was still as high as it was in the antibiotic-treated control mice did not receive the engineered bacteria.

The results also demonstrated that in antibiotic-treated mice given the engineered bacteria a much higher level of gut microbial diversity was maintained, when compared with the control mice that received only antibiotics. In the control mice microbial diversity levels dropped dramatically after they received ampicillin. Encouragingly, none of the mice that received the engineered bacteria developed opportunistic C. difficile infections, while all of the mice that received only antibiotics showed high levels of C. difficile in the gut.

“In a mouse model of parenteral ampicillin treatment, oral supplementation with the engineered live biotherapeutic minimized gut dysbiosis without affecting the ampicillin concentration in serum, precluded the enrichment of antimicrobial resistance genes in the gut microbiome and prevented the loss of colonization resistance against Clostridioides difficile,” the team wrote in summary. “This is a strong demonstration that this approach can protect the gut microbiota, while preserving the efficacy of the antibiotic, as you’re not modifying the levels in the bloodstream,” Cubillos-Ruiz further pointed out.

The researchers also found that eliminating the evolutionary pressure of antibiotic treatment made it much less likely for the microbes of the gut to develop antibiotic resistance after treatment. In contrast, they did find many genes for antibiotic resistance in the microbes that survived in mice who received antibiotics but not the engineered bacteria. Those genes might be passed to harmful bacteria, worsening the problem of antibiotic resistance. And as the authors further pointed out, “While we show that our strategy is effective at protecting microbial groups that determine colonization resistance against C. difficile, the same antibiotic-protecting effect may be beneficial for preserving the composition of the microbial groups that are associated with other dysbiosis-related pathologies.”

The use of β-lactamase enzymes to degrade β-lactam antibiotics was first described back in 2003, the investigators noted. Since then scientists have been working to develop encapsulated forms of purified β-lactamase that are released directly in the intestine, and clinical trials are now in progress. However, the MIT researchers believe the eLBP strategy will help to unlock strategies for using β-lactamases to protect the gut microbiota with respect to both manufacturing, and effectiveness. “First, the manufacture and scalability of a defined bacterial formulation is significantly easier and less costly than the production and purification of clinical-grade enzymes,” they wrote. “Second, our approach is likely to provide a more efficacious release of the active enzyme throughout the intestine, given the continuous metabolic activity of our eLBP.”

The researchers now plan to begin developing a version of the treatment that could be tested in people at high risk of developing acute diseases that stem from antibiotic-induced gut dysbiosis, and they hope that eventually, it could be used to protect anyone who needs to take antibiotics for infections outside the gut. They concluded in their paper, “… we envision that simple oral administration of our eLBP before parenteral antibiotic administration may significantly reduce the morbidity and mortality associated with antibiotic-related complications of gut dysbiosis.”

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