Characterization of an engineered live bacterial therapeutic for the treatment of phenylketonuria in a human gut-on-a-chip.

Engineered bacteria (synthetic biotics) represent a new class of therapeutics that leverage the tools of synthetic biology. Translational testing strategies are required to predict synthetic biotic function in the human body. Gut-on-a-chip microfluidics technology presents an opportunity to characterize strain function within a simulated human gastrointestinal tract. Here, we apply a human gut-chip model and a synthetic biotic designed for the treatment of phenylketonuria to demonstrate dose-dependent production of a strain-specific biomarker, to describe human tissue responses to the engineered strain, and to show reduced blood phenylalanine accumulation after administration of the engineered strain. Lastly, we show how in vitro gut-chip models can be used to construct mechanistic models of strain activity and recapitulate the behavior of the engineered strain in a non-human primate model. These data demonstrate that gut-chip models, together with mechanistic models, provide a framework to predict the function of candidate strains in vivo.

Nutrition, Microbiota and Role of Gut-Brain Axis in Subjects with Phenylketonuria (PKU): A Review.

The composition and functioning of the gut microbiota, the complex population of microorganisms residing in the intestine, is strongly affected by endogenous and exogenous factors, among which diet is key. Important perturbations of the microbiota have been observed to contribute to disease risk, as in the case of neurological disorders, inflammatory bowel disease, obesity, diabetes, cardiovascular disease, among others. Although mechanisms are not fully clarified, nutrients interacting with the microbiota are thought to affect host metabolism, immune response or disrupt the protective functions of the intestinal barrier. Similarly, key intermediaries, whose presence may be strongly influenced by dietary habits, sustain the communication along the gut-brain-axis, influencing brain functions in the same way as the brain influences gut activity. Due to the role of diet in the modulation of the microbiota, its composition is of high interest in inherited errors of metabolism (IEMs) and may reveal an appealing therapeutic target. In IEMs, for example in phenylketonuria (PKU), since part of the therapeutic intervention is based on chronic or life-long tailored dietetic regimens, important variations of the microbial diversity or relative abundance have been observed. A holistic approach, including a healthy composition of the microbiota, is recommended to modulate host metabolism and affected neurological functions.

Clostridium ventriculi Infection in a Child with Phenylketonuria.

Disabled individuals may be at risk for common and rare infections. We report on a 13-year-old female who had a diagnosis of phenylketonuria (PKU). The child received a percutaneous endoscopic gastrostomy (PEG) feeding tube at five years of age for the supplementation of her specialized formula. After eight years, she no longer required the gastrostomy tube for formula supplementation, and she presented for the closure of the gastrocutaneous fistula tract. The histological examination revealed acute and chronic inflammation and colonization by gram-positive bacteria with a characteristic tetrad packet arrangement known as Clostridium ventriculi (formerly Sarcina ventriculi). A review of the literature evidenced the rarity of this infection in children. This patient is the 11th case of such infection in literature, and the first patient affected with PKU. Physically and mentally disabled children are particularly vulnerable to infection because of their different feeding abilities, toilet needs, and sanitary arrangements.

Phenylketonuric diet negatively impacts on butyrate production.

BACKGROUND AND AIMS: Phenylalanine (Phe) restricted diet, combined with Phe-free l-amino acid supplementation, is the mainstay of treatment for phenylketonuria (PKU). Being the diet a key factor modulating gut microbiota composition, the aim of the present paper was to compare dietary intakes, gut microbiota biodiversity and short chain fatty acids (SCFAs) production in children with PKU, on low-Phe diet, and in children with mild hyperphenylalaninemia (MHP), on unrestricted diet. METHODS AND RESULTS: We enrolled 21 PKU and 21 MHP children matched for gender, age and body mass index z-score. Dietary intakes, including glycemic index (GI) and glycemic load (GL), and fecal microbiota analyses, by means of denaturing gradient gel electrophoresis (DGGE) and Real-time PCR were assessed. Fecal SCFAs were quantified by gas chromatographic analysis. RESULTS: We observed an increased carbohydrate (% of total energy), fiber and vegetables intakes (g/day) in PKU compared with MHP children (p = 0.047), as well a higher daily GI and GL (maximum p < 0.001). Compared with MHP, PKU showed a lower degree of microbial diversity and a decrease in fecal butyrate content (p = 0.02). Accordingly, two of the most abundant butyrate-producing genera, Faecalibacterium spp. and Roseburia spp., were found significantly depleted in PKU children (p = 0.02 and p = 0.03, respectively). CONCLUSION: The low-Phe diet, characterized by a higher carbohydrate intake, increases GI and GL, resulting in a different quality of substrates for microbial fermentation. Further analyses, thoroughly evaluating microbial species altered by PKU diet are needed to better investigate gut microbiota in PKU children and to eventually pave the way for pre/probiotic supplementations.

The assay on a defined medium of the effects of beta-2-thienylalanine on the growth of anaerobic bacterial isolates from phenylketonuric patients.

Faecal samples were taken from three diet-managed phenylketonuric children to determine effects of beta-2-thienylalanine (beta-2-t) on indigenous bacteria. From sample swabs, 127 anaerobes were identified and tested for beta-2-t inhibition on a phenylalanine (Phe)-free medium, Anaerobe Inhibition Test (AIT) agar. Of the isolates, 77.9% grew sufficiently to assay reactions on at least 25% of AIT plates. Using Phe-containing Columbia agar, 86.5% of the strains could be assayed. None of 28 Bacteroides cultures was inhibited by beta-2-t on AIT. Of the genera, Bifidobacterium, Eubacterium, Lactobacillus, Peptostreptococcus, and Propionibacterium, no isolates which would grow on AIT were inhibited. At least one isolate of each of the genera Peptococcus, Fusobacterium, and Clostridium was inhibited. Of 127 total isolates, only nine were inhibited by beta-2-t on AIT, and inhibition was abolished on Columbia agar. Thirty-nine "aerobes" were isolated from the same patients. Strains of the genera tested reacted similarly to previously tested strains from non-PKU sources. Also, anaerobically isolated Excherichia coli were inhibited, while Streptococcus faecalis cultures were not, confirming results on aerobically-isolated non-PKU cultures of the same species. These studies, the first dealing with beta-2-t and anaerobic bacteria, suggest that little change in intestinal bacterial populations might be expected during in vivo beta-2-t treatment.