Probiotics are live bacteria that many people now take as dietary supplements to help improve gut health, but new research in mouse models has shown how these organisms can also evolve in the gastrointestinal system. The studies, reported by a Washington University School of Medicine-led team found that probiotic bacteria may in some cases adapt to the selective pressures of their environment to become either ineffective, or even potentially harmful to health.
The scientists studied a strain of Escherichia coli known as E. coli Nissle (EcN) 1917, which is marketed in Europe as an antidiarrheal probiotic. Tests in mice demonstrated how these bacteria evolved in the animals’ intestines within just a few weeks. In some instances—depending on mouse diet and composition of the animals’ existing gut microbial community—the probiotic EcN strain even gained the ability to damage the protective layer of the intestine. Destruction of this layer has been linked to irritable bowel syndrome.
“If we’re going to use living things as medicines, we need to recognize that they’re going to adapt, and that means that what you put in your body is not necessarily what’s going to be there even a couple hours later,” commented senior author Gautam Dantas, PhD, a professor of pathology and immunology, molecular microbiology, and biomedical engineering. “There is no microbe out there that is immune to evolution. This isn’t a reason not to develop probiotic-based therapies, but it is a reason to make sure we understand how they change and under what conditions.”
The researchers reported their findings in Cell Host & Microbe, in a paper titled, “Adaptive strategies of the candidate probiotic E. coli Nissle in the mammalian gut.”
The healthy gut microbiome comprises a diverse community of bacteria and other microorganisms that help to digest food, but which can also provide vitamins, modulate inflammation, and stop pathogenic organisms from taking hold. Probiotics in dietary supplements and in some foods are marketed to help support a strong microbiome and associated benefits.
It is also proving possible to expand the scope of probiotics by genetically engineering the microorganisms, the authors wrote. Preclinical work has shown that such modified bacteria can help to treat infectious and metabolic diseases. Probiotics are being developed as treatments for medical conditions such as inflammatory bowel disease, phenylketonuria (PKU), neurological damage, and necrotizing enterocolitis in infants.
Such opportunities mean that engineered probiotics represent “exciting platforms” for in situ drug synthesis and delivery, but “provided they maintain appropriate abundance and activity at their target site,” the team pointed out. Unlike conventional drugs, which are “abiotic,” probiotic bacteria, whether wild-type, or engineered, replicate in the gut system, and so are subjected to natural selection, which could adversely impact on their intended therapeutic effect and safety profile. The situation is further complicated, because both the ability of probiotics to colonize the gut and their effectiveness will also vary between the individuals who take them, and this variation will at least in part depend upon their microbiome diversity and diet. As the authors commented, “While probiotics have been broadly used in humans for decades, engineering probiotics for therapeutic applications in humans will only be acceptable if it is demonstrated that probiotics exhibit long-term safety … Clinical use of probiotics, especially genetically engineered probiotics, therefore, will benefit from a thorough understanding of their in vivo evolutionary trajectories under diverse schemes of microbiome complexity and host diet.”
To look in more detail at selective pressures acting on probiotics in vivo, the team studied adaptations gained by the probiotic bacterium E. coli Nissle 1917, administered orally to mouse models under varied dietary conditions and different microbiome complexities. Some of the mice were germ-free (and so had no pre-existing microbiota), others had a limited diversity of gastrointestinal bacterial species, which was characteristic of an unhealthy gut, a third group had normal gut microbiomes, and a fourth group of mice had a normal microbiome after antibiotic therapy.
The animals were given the NcE probotic, and then their diets were varied so that they were fed on either a normal, high-fiber mouse chow, or a high-fat, high-sugar diet typical of Western-type diets, or a Western diet plus fiber. After five weeks bacteria taken from the mouse gastrointestinal tracts were analyzed. The results indicated that the NcE organisms had evolved in the gut, but their adaptations were dependent upon the background microbiota and the animals’ diets. Notably, some of the bacteria accumulated genetic mutations that altered how they metabolized different carbohydrates, as well as mutations that impacted on
stress response pathways, and adhesion to mucin-producing epithelial cells in the gastrointestinal tract. Evolution in the bacteria was thus directed “to gain competitive fitness,” the authors wrote.
Fewer genetic changes were evident when the EcN colonized the gut systems of animals with an already highly diverse microbiome. In contrast, low microbiome diversity was associated with adaptations to utilize the available carbon sources “… in the context of low microbiome diversity, the greatest selective pressure on EcN is carbon source limitation,” the authors commented.
Their observations of mutations in stress response pathways were also suggestive that low gut diversity, which is known to be pathologic to the host, “can also subject the present bacteria to stress, which might exacerbate genomic instability.” Interestingly, some of the bacteria administered to mice that had received streptomycin antibiotic therapy weeks previously still developed antibiotic resistance genes. “These results could indicate residual streptomycin present in the mouse gut …” the authors stated.
In a final set of experiments, the researchers generated an engineered strain of EcN as a potential therapeutic approach to resolving PKU, a condition in which individuals can’t metabolize the amino acid phenylalanine (Phe). They inserted the gene for the enzyme phenylalanine ammonia lyase 2 (PAL2) into the NcE organism and then gave the engineered bacteria to a mouse model of PKU. Within 24 hours phenylalanine levels in some of the mice had dropped by half. A reduction in serum Phe levels, depending on strain design and the extent of hyperphenylalaninemia, indicates the opportunity for personalized engineered probiotic therapies,” the team stated. Encouragingly, the engineered EcN strains were genetically stable over the course of a week, “thereby validating EcN’s utility as a chassis for engineering.”
The authors suggest that their studies represent both a generalizable framework for developing and regulating “living therapeutics”, and a first step towards the goal of demonstrating the safety of probiotics. They also advocate that future studies should include “a systematic assessment of probiotic efficacy and adverse effects in relation to personalized microbiome features prior to treatment.”