Gut Microbiome Integration in Drug Discovery and Development of Small Molecules.

Human microbiomes, particularly in the gut, could have a major impact on the efficacy and toxicity of drugs. However, gut microbial metabolism is often neglected in the drug discovery and development process. Medicen, a Paris-based human health innovation cluster, has gathered more than 30 international leading experts from pharma, academia, biotech, clinical research organizations, and regulatory science to develop proposals to facilitate the integration of microbiome science into drug discovery and development. Seven subteams were formed to cover the complementary expertise areas of 1) pharma experience and case studies, 2) in silico microbiome-drug interaction, 3) in vitro microbial stability screening, 4) gut fermentation models, 5) animal models, 6) microbiome integration in clinical and regulatory aspects, and 7) microbiome ecosystems and models. Each expert team produced a state-of-the-art report of their respective field highlighting existing microbiome-related tools at every stage of drug discovery and development. The most critical limitations are the growing, but still limited, drug-microbiome interaction data to produce predictive models and the lack of agreed-upon standards despite recent progress. In this paper we will report on and share proposals covering 1) how microbiome tools can support moving a compound from drug discovery to clinical proof-of-concept studies and alert early on potential undesired properties stemming from microbiome-induced drug metabolism and 2) how microbiome data can be generated and integrated in pharmacokinetic models that are predictive of the human situation. Examples of drugs metabolized by the microbiome will be discussed in detail to support recommendations from the working group. SIGNIFICANCE STATEMENT: Gut microbial metabolism is often neglected in the drug discovery and development process despite growing evidence of drugs' efficacy and safety impacted by their interaction with the microbiome. This paper will detail existing microbiome-related tools covering every stage of drug discovery and development, current progress, and limitations, as well as recommendations to integrate them into the drug discovery and development process.

Missense Mutations in the CrrB Protein Mediate Odilorhabdin Derivative Resistance in Klebsiella pneumoniae.

NOSO-502 is a preclinical antibiotic candidate of the Odilorhabdin class. This compound exhibits activity against Enterobacteriaceae pathogens, including carbapenemase-producing bacteria and most of the Colistin (CST)-resistant strains. Among a collection of CST-resistant Klebsiella pneumoniae strains harboring mutations on genes pmrAB, mgrB, phoPQ, and crrB, only those bearing mutations in gene crrB were found to be resistant to NOSO-502.CrrB is a histidine kinase which acts with the response regulator CrrA to modulate the PmrAB system, which finally induces the restructuring of the lipopolysaccharide present on the outer membrane and thus leading to CST resistance. Moreover, crrB mutations also enhance the transcription of neighboring genes such as H239_3063, an ABC transporter transmembrane region; H239_3064, a putative efflux pump also known as KexD; and H239_3065, a N-acetyltransferase.To elucidate the mechanism of resistance to NOSO-502 induced by CrrB missense mutations in K. pneumoniae, mutants of NCTC 13442 and ATCC BAA-2146 strains resistant to NOSO-502 and CST with single amino acid substitutions in CrrB (S8N, F33Y, Y34N, W140R, N141I, P151A, P151L, P151S, P151T, F303Y) were selected. Full susceptibility to NOSO-502 was restored in crrA or crrB deleted K. pneumoniae NCTC 13442 CrrB(P151L) mutants, confirming the role of CrrAB in controlling this resistance pathway. Deletion of kexD (but no other neighboring genes) in the same mutant also restored NOSO-502-susceptibility. Upregulation of the kexD gene expression was observed for all CrrB mutants. Finally, plasmid expression of kexD in a K. pneumoniae strain missing the locus crrABC and kexD significantly increased resistance to NOSO-502.

A standardized gnotobiotic mouse model harboring a minimal 15-member mouse gut microbiota recapitulates SOPF/SPF phenotypes.

Mus musculus is the classic mammalian model for biomedical research. Despite global efforts to standardize breeding and experimental procedures, the undefined composition and interindividual diversity of the microbiota of laboratory mice remains a limitation. In an attempt to standardize the gut microbiome in preclinical mouse studies, here we report the development of a simplified mouse microbiota composed of 15 strains from 7 of the 20 most prevalent bacterial families representative of the fecal microbiota of C57BL/6J Specific (and Opportunistic) Pathogen-Free (SPF/SOPF) animals and the derivation of a standardized gnotobiotic mouse model called GM15. GM15 recapitulates extensively the functionalities found in the C57BL/6J SOPF microbiota metagenome, and GM15 animals are phenotypically similar to SOPF or SPF animals in two different facilities. They are also less sensitive to the deleterious effects of post-weaning malnutrition. In this work, we show that the GM15 model provides increased reproducibility and robustness of preclinical studies by limiting the confounding effect of fluctuation in microbiota composition, and offers opportunities for research focused on how the microbiota shapes host physiology in health and disease.

Gut microbiota diversity after autologous fecal microbiota transfer in acute myeloid leukemia patients.

Acute myeloid leukemia (AML) intensive chemotherapy combined with broad-spectrum antibiotics, leads to gut microbiota dysbiosis promoting pathological conditions and an increased incidence of complications. Here we report findings from a phase II single-arm, multicenter study evaluating autologous fecal microbiota transfer (AFMT) in 25 AML patients treated with intensive chemotherapy and antibiotics (ClinicalTrials.gov number: NCT02928523). The co-primary outcomes of the study are to evaluate the efficacy of AFMT in dysbiosis correction and multidrug-resistant bacteria eradication. The main secondary outcomes are to define a dysbiosis biosignature, to evaluate the effect of dysbiosis correction on patient clinical status, to assess the short and mid-term safety of AFMT in this immunocompromised population, and to evaluate the feasibility of the AFMT procedure and acceptability by the patient. Intensive induction chemotherapy induces a dramatic decrease of α-diversity indices, and a microbial dysbiosis with a significant shift of the microbial communities and domination of pro-inflammatory families. After AFMT treatment, α-diversity indices return to their initial mean levels and the similarity index shows the restoration of microbial communities. The trial meets pre-specified endpoints. AFMT appears to be safe and may be effective for gut microbiota restoration in AML patients receiving intensive chemotherapy and antibiotics, with an excellent gut microbiota reconstruction based on both richness and diversity indices at the species level.

Impact on the Gut Microbiota of Intensive and Prolonged Antimicrobial Therapy in Patients With Bone and Joint Infection.

There is a growing interest in the potentially deleterious impact of antibiotics on gut microbiota. Patients with bone and joint infection (BJI) require prolonged treatment that may impact significantly the gut microbiota. We collected samples from patients with BJI at baseline, end of antibiotics (EOT), and 2 weeks after antibiotic withdrawal (follow-up, FU) in a multicenter prospective cohort in France. Microbiota composition was determined by shotgun metagenomic sequencing. Fecal markers of gut permeability and inflammation as well as multi-drug-resistant bacteria (MDRB) and Clostridioides difficile carriage were assessed at each time point. Sixty-two patients were enrolled: 27 native BJI, 14 osteosynthesis-related BJI, and 21 prosthetic joint infections (PJI). At EOT, there was a significant loss of alpha-diversity that recovered at FU in patients with native BJI and PJI, but not in patients with osteosynthesis-related BJI. At EOT, we observed an increase of Proteobacteria and Bacteroidetes that partially recovered at FU. The principal component analysis (PCoA) of the Bray-Curtis distance showed a significant change of the gut microbiota at the end of treatment compared to baseline that only partially recover at FU. Microbiota composition at FU does not differ significantly at the genus level when comparing patients treated for 6 weeks vs. those treated for 12 weeks. The use of fluoroquinolones was not associated with a lower Shannon index at the end of treatment; however, the PCoA of the Bray-Curtis distance showed a significant change at EOT, compared to baseline, that fully recovered at FU. Levels of fecal neopterin were negatively correlated with the Shannon index along with the follow-up (r 2 = 0.17; p < 0.0001). The PCoA analysis of the Bray-Curtis distance shows that patients with an elevated plasma level of C-reactive protein (≥5 mg/L) at EOT had a distinct gut microbial composition compared to others. MDRB and C. difficile acquisition at EOT and FU represented 20% (7/35) and 37.1% (13/35) of all MDRB/C. difficile-free patients at the beginning of the study, respectively. In patients with BJI, antibiotics altered the gut microbiota diversity and composition with only partial recovery, mucosal inflammation, and permeability and acquisition of MDRB carriage. Microbiome interventions should be explored in patients with BJI to address these issues.

A comprehensive evaluation of binning methods to recover human gut microbial species from a non-redundant reference gene catalog.

The human gut microbiota performs functions that are essential for the maintenance of the host physiology. However, characterizing the functioning of microbial communities in relation to the host remains challenging in reference-based metagenomic analyses. Indeed, as taxonomic and functional analyses are performed independently, the link between genes and species remains unclear. Although a first set of species-level bins was built by clustering co-abundant genes, no reference bin set is established on the most used gut microbiota catalog, the Integrated Gene Catalog (IGC). With the aim to identify the best suitable method to group the IGC genes, we benchmarked nine taxonomy-independent binners implementing abundance-based, hybrid and integrative approaches. To this purpose, we designed a simulated non-redundant gene catalog (SGC) and computed adapted assessment metrics. Overall, the best trade-off between the main metrics is reached by an integrative binner. For each approach, we then compared the results of the best-performing binner with our expected community structures and applied the method to the IGC. The three approaches are distinguished by specific advantages, and by inherent or scalability limitations. Hybrid and integrative binners show promising and potentially complementary results but require improvements to be used on the IGC to recover human gut microbial species.

An Oral FMT Capsule as Efficient as an Enema for Microbiota Reconstruction Following Disruption by Antibiotics, as Assessed in an In Vitro Human Gut Model.

Fecal microbiota transplantation (FMT) is an innovative therapy already used in humans to treat Clostridioides difficile infections associated with massive use of antibiotics. Clinical studies are obviously the gold standard to evaluate FMT efficiency but remain limited by regulatory, ethics, and cost constraints. In the present study, an in vitro model of the human colon reproducing medically relevant perturbation of the colonic ecosystem by antibiotherapy was used to compare the efficiency of traditional FMT enema formulations and a new oral capsule in restoring gut microbiota composition and activity. Loss of microbial diversity, shift in bacterial populations, and sharp decrease in fermentation activities induced in vivo by antibiotherapy were efficiently reproduced in the in vitro model, while capturing inter-individual variability of gut microbiome. Oral capsule was as efficient as enema to decrease the number of disturbed days and bacterial load had no effect on enema performance. This study shows the relevance of human colon models as an alternative approach to in vivo assays during preclinical studies for evaluating FMT efficiency. The potential of this in vitro approach could be extended to FMT testing in the management of many digestive or extra-intestinal pathologies where gut microbial dysbiosis has been evidenced such as inflammatory bowel diseases, obesity or cancers.

Caecal microbiota compositions from 7-day-old chicks reared in high-performance and low-performance industrial farms and systematic culturomics to select strains with anti-Campylobacter activity.

There is growing interest in exploring the chickens' intestinal microbiota and understanding its interactions with the host. The objective is to optimize this parameter in order to increase the productivity of farm animals. With the goal to isolate candidate probiotic strains, specific culturomic methods were used in our study to culture commensal bacteria from 7-days old chicks raised in two farms presenting long history of high performance. A total of 347 isolates were cultured, corresponding to at least 64 species. Among the isolates affiliated to the Firmicutes, 26 had less than 97% identity of their partial 16S sequence with that of the closest described species, while one presented less than 93% identity, thus revealing a significant potential for new species in this ecosystem. In parallel, and in order to better understand the differences between the microbiota of high-performing and low-performing animals, caecal contents of animals collected from these two farms and from a third farm with long history of low performance were collected and sequenced. This compositional analysis revealed an enrichment of Faecalibacterium-and Campylobacter-related sequences in lower-performing animals whereas there was a higher abundance of enterobacteria-related sequences in high-performing animals. We then investigated antibiosis activity against C. jejuni ATCC 700819 and C. jejuni field isolate as a first phenotypic trait to select probiotic candidates. Antibiosis was found to be limited to a few strains, including several lactic acid bacteria, a strain of Bacillus horneckiae and a strain of Escherichia coli. The antagonist activity depended on test conditions that mimicked the evolution of the intestinal environment of the chicken during its lifetime, i.e. temperature (37°C or 42°C) and oxygen levels (aerobic or anaerobic conditions). This should be taken into account according to the stage of development of the animal at which administration of the active strain is envisaged.

MAVS deficiency induces gut dysbiotic microbiota conferring a proallergic phenotype.

Prominent changes in the gut microbiota (referred to as "dysbiosis") play a key role in the development of allergic disorders, but the underlying mechanisms remain unknown. Study of the delayed-type hypersensitivity (DTH) response in mice contributed to our knowledge of the pathophysiology of human allergic contact dermatitis. Here we report a negative regulatory role of the RIG-I-like receptor adaptor mitochondrial antiviral signaling (MAVS) on DTH by modulating gut bacterial ecology. Cohousing and fecal transplantation experiments revealed that the dysbiotic microbiota of Mavs-/- mice conferred a proallergic phenotype that is communicable to wild-type mice. DTH sensitization coincided with increased intestinal permeability and bacterial translocation within lymphoid organs that enhanced DTH severity. Collectively, we unveiled an unexpected impact of RIG-I-like signaling on the gut microbiota with consequences on allergic skin disease outcome. Primarily, these data indicate that manipulating the gut microbiota may help in the development of therapeutic strategies for the treatment of human allergic skin pathologies.

Lactobacillus paracasei feeding improves immune control of influenza infection in mice.

Respiratory tract infections such as flu cause severe morbidity and mortality and are among the leading causes of death in children and adults worldwide. Commensal microbiota is critical for orchestrating tissue homeostasis and immunity in the intestine. Probiotics represent an interesting source of immune modulators and several clinical studies have addressed the potential beneficial effects of probiotics against respiratory infections. Therefore, we have investigated the mechanisms of protection conferred by L. paracasei CNCM I-1518 strain in a mouse model of influenza infection. Notably, local myeloid cells accumulation is generated in the lungs after seven days feeding with L. paracasei prior to viral infection. L. paracasei-fed mice showed reduced susceptibility to the influenza infection, associated with less accumulation of inflammatory cells in the lungs, faster viral clearance and general health improvement. Interestingly, Allobaculum was significantly increased in L. paracasei-fed mice 7 days after influenza infection, even if the gut microbiota composition was not altered overall. L. paracasei-purified peptidoglycan partially recapitulated the protective phenotype observed with the entire bacteria. Collectively, our results demonstrate that oral consumption of L. paracasei CNCM I-1518 modulates lung immunity was associated with an improved control of influenza infection. These results further extend the beneficial role for certain lactobacilli to alleviate the burden of respiratory tract infections.

Identification of Nascent Memory CD8 T Cells and Modeling of Their Ontogeny.

Primary immune responses generate short-term effectors and long-term protective memory cells. The delineation of the genealogy linking naive, effector, and memory cells has been complicated by the lack of phenotypes discriminating effector from memory differentiation stages. Using transcriptomics and phenotypic analyses, we identify Bcl2 and Mki67 as a marker combination that enables the tracking of nascent memory cells within the effector phase. We then use a formal approach based on mathematical models describing the dynamics of population size evolution to test potential progeny links and demonstrate that most cells follow a linear naive→early effector→late effector→memory pathway. Moreover, our mathematical model allows long-term prediction of memory cell numbers from a few early experimental measurements. Our work thus provides a phenotypic means to identify effector and memory cells, as well as a mathematical framework to investigate their genealogy and to predict the outcome of immunization regimens in terms of memory cell numbers generated.