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.

Fecal Supernatant from Adult with Autism Spectrum Disorder Alters Digestive Functions, Intestinal Epithelial Barrier, and Enteric Nervous System.

Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders defined by impaired social interactions and communication with repetitive behaviors, activities, or interests. Gastrointestinal (GI) disturbances and gut microbiota dysbiosis are frequently associated with ASD in childhood. However, it is not known whether microbiota dysbiosis in ASD patients also occurs in adulthood. Further, the consequences of altered gut microbiota on digestive functions and the enteric nervous system (ENS) remain unexplored. Therefore, we studied, in mice, the ability offecal supernatant (FS) from adult ASD patients to induce GI dysfunctions and ENS remodeling. First, the analyses of the fecal microbiota composition in adult ASD patients indicated a reduced α-diversity and increased abundance of three bacterial 16S rRNA gene amplicon sequence variants compared to healthy controls (HC). The transfer of FS from ASD patients (FS-ASD) to mice decreased colonic barrier permeability by 29% and 58% compared to FS-HC for paracellular and transcellular permeability, respectively. These effects are associated with the reduced expression of the tight junction proteins JAM-A, ZO-2, cingulin, and proinflammatory cytokines TNFα and IL1ß. In addition, the expression of glial and neuronal molecules was reduced by FS-ASD as compared to FS-HC in particular for those involved in neuronal connectivity (ßIII-tubulin and synapsin decreased by 31% and 67%, respectively). Our data suggest that changes in microbiota composition in ASD may contribute to GI alterations, and in part, via ENS remodeling.

Faecal Microbiota Composition Varies between Patients with Breast Cancer and Healthy Women: A Comparative Case-Control Study.

The intestinal microbiota plays an essential role in many diseases, such as obesity, irritable bowel disease (IBD), and cancer. This study aimed to characterize the faecal microbiota from early-stage breast cancer (BC) patients and healthy controls. Faeces from newly diagnosed breast cancer patients, mainly for an invasive carcinoma of no specific type (HR+ and HER2-), before any therapeutic treatment and healthy controls were collected for metabarcoding analyses. We show that the Shannon index, used as an index of diversity, was statistically lower in the BC group compared to that of controls. This work highlights a reduction of microbial diversity, a relative enrichment in Firmicutes, as well as a depletion in Bacteroidetes in patients diagnosed with early BC compared to those of healthy women. A tendency towards a decreased relative abundance of Odoribacter sp., Butyricimonas sp., and Coprococcus sp. was observed. This preliminary study suggests that breast cancer patients may differ from healthy subjects in their intestinal bacterial composition.

Basolateral Secretion from Caco-2 Cells Pretreated with Fecal Waters from Breast Cancer Patients Affects MCF7 Cell Viability.

We hypothesized that the role of microbiota in breast cancer relates to its influence on gut lipid metabolism. This was tested in an in vitro model combining MCF-7 and Caco-2 cells. A total of 32 women newly diagnosed for breast cancer before any treatment and 28 healthy women provided their stools. Bacterial DNA was amplified by qPCR targeting 16s rRNA specific to Bacteroidetes and Firmicutes phyla, Lactobacillales sp., Clostridium cluster IV, Faecalibacterium prausnitzii, Clostridium cluster XIVa, Roseburia intestinalis, Blautia sp., Lactonifactor longoviformis, Bifidobacterium sp., Coriobacteriaceae, Eggertella lenta, Escherichia, and Shigella. Fecal waters (FW) were quantified for short chain fatty acids (SCFA). Caco-2 cells grown on filter inserts were incubated apically with 10% FW for 24 h, and LXR, apolipoproteins AIV, and E gene expression were estimated by real time (RT) qPCR. Then, MCF-7 cells were incubated with the whole basolateral medium for 24 h, and their viability was estimated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) test. Regression models were used to determine the correlation between MCF-7 viability and bacteria relative abundance, Caco-2 cells lipid metabolism gene expression and stool composition, as well as microbiota composition and short chain fatty acids. Logistic regression models established disease odds ratios (OR) for MCF-7 viability and Caco-2 gene expression. The OR of MCF-7 viability was 1.05 (1.01-1.10) (OR (5th-95th), p = 0.04), while that of apo AIV gene expression was 0.63 (0.39-1.01), p = 0.055). Viability correlated with % Bifidobacterium sp. (21.18 ± 7.66, p = 0.008) and valerate (-2.849 ± 1.048, p = 0.009) (ß ± s.d.). This study suggests that microbiota interacts with intestine cell lipid metabolism. Since these metabolites can reach breast cells by systemic circulation, we hypothesized that they may influence cancer disease.

Impact of pemetrexed chemotherapy on the gut microbiota and intestinal inflammation of patient-lung-derived tumor xenograft (PDX) mouse models.

Chemotherapy remains the gold standard for advanced cancer. Pemetrexed, a chemotherapeutic agent used in non-small cell lung cancer, can induce significant side effects in patients. Although microbiota's role in the efficacy and/or toxicity of chemotherapy agents has been demonstrated, the impacts of pemetrexed on the gut microbiota and on gastrointestinal inflammation remain unknown. The objective of this study was to evaluate the impact of pemetrexed and the tumor graft on the gut microbiota composition in immunodeficient mice. The faecal microbiota composition was studied with metabarcoding before, 24-h and one week after treatment. The colon epithelial barrier integrity was evaluated by histological examination, intestinal permeability measurement, and selected cytokines quantification. The tumor graft induced some variations in the microbiota composition. Pemetrexed further increased the relative abundance of Enterobacteriaceae and 3 families from the Firmicutes phylum: Enterococcaceae, Lactobacillaceae and Streptococcaceae. Pemetrexed also significantly altered the epithelial barrier integrity, which was associated with early inflammation. This pilot study shows that the association of a lung tumor graft with pemetrexed causes an alteration in the microbiota composition. Such information increases our knowledge about the impact of chemotherapy on the microbiota, which could help to minimize side effects and improve therapeutic effectiveness in the future.

Lean management and medical laboratory: application in transfusionnal immuno-hematology.

Despite a common use in industrial applications, only a few studies describe the lean management methods in medical laboratory. These tools have been evaluated in analysis laboratory of blood donors, especially in immuno-hematology sector. The aim was to optimize the organization and maintain team cohesion and strong staff involvement in a restructuring context. The tools used and the results obtained are presented in this study.

The oral cavity microbiota: between health, oral disease, and cancers of the aerodigestive tract.

Many studies show that the human microbiome plays a critical role in the chronic pathologies of obesity, inflammatory bowel diseases, and diabetes. More recently, the interaction between cancer and the microbiome has been highlighted. Most studies have focused on the gut microbiota because it represents the most extensive bacterial community, and the body of evidence correlating it with gut syndromes is increasing. However, in the strict sense, the gastrointestinal (GI) tract begins in the oral cavity, and special attention should be paid to the specific flora of this cavity. This study reviewed the current knowledge about the various microbial ecosystems of the upper part of the GI tract and discussed their potential link to carcinogenesis. The overall composition of the microbial communities, as well as the presence or absence of "key species", in relation to carcinogenesis is addressed. Alterations in the oral microbiota can potentially be used to predict the risk of cancer. Molecular advances and the further monitoring of the microbiota will increase our understanding of the role of the microbiota in carcinogenesis and open new perspectives for future therapeutic and prophylactic modalities.

16S rRNA gene pyrosequencing reveals shift in patient faecal microbiota during high-dose chemotherapy as conditioning regimen for bone marrow transplantation.

Gastrointestinal disturbances are a side-effect frequently associated with haematological malignancies due to the intensive cytotoxic treatment given in connection with bone marrow transplantation (BMT). However, intestinal microbiota changes during chemotherapy remain poorly described, probably due to the use of culture-based and low-resolution molecular methods in previous studies. The objective of our study was to apply a next generation DNA sequencing technology to analyse chemotherapy-induced changes in faecal microbiota. We included eight patients with non-Hodgkin's lymphoma undergoing one course of BMT conditioning chemotherapy. We collected a prechemotherapy faecal sample, the day before chemotherapy was initiated, and a postchemotherapy sample, collected 1 week after the initiation of chemotherapy. Total DNA was extracted from faecal samples, denaturing high-performance liquid chromatography based on amplification of the V6 to V8 region of the 16S ribosomal RNA (rRNA) gene, and 454-pyrosequencing of the 16 S rRNA gene, using PCR primers targeting the V5 and V6 hypervariable 16S rRNA gene regions were performed. Raw sequence data were screened, trimmed, and filtered using the QIIME pipeline. We observed a steep reduction in alpha diversity and significant differences in the composition of the intestinal microbiota in response to chemotherapy. Chemotherapy was associated with a drastic drop in Faecalibacterium and accompanied by an increase of Escherichia. The chemotherapy-induced shift in the intestinal microbiota could induce severe side effects in immunocompromised cancer patients. Our study is a first step in identifying patients at risk for gastrointestinal disturbances and to promote strategies to prevent this drastic shift in intestinal microbiota.

Emergence in Western African countries of MDR-TB, focus on Côte d'Ivoire.

Tuberculosis (TB) is responsible for a high mortality rate (2.5%) worldwide, mainly in developing countries with a high prevalence of human immunodeficiency virus (HIV). The emergence of multiresistant strains of TB poses an extreme risk for TB outbreaks and highlights the need for global TB control strategies. Among Western African countries, Côte d'Ivoire (CI) represents a specific example of a country with great potential to prevent TB. Specifically, CI has a promising healthcare system for monitoring diseases, including vaccination programs. However, military and political conflict in CI favors the spread of infectious diseases, TB being among the most devastating. Compilation of the studies identifying common causes of TB would be extremely beneficial for the development of treatment and prevention strategies. Therefore, the purpose of this comprehensive review is to evaluate the epidemiology of TB in CI, describe the factors involved in pathogenesis, and suggest simple and applicable prevention strategies.

Development of intestinal microbiota in infants and its impact on health.

Throughout the human lifetime, the intestinal microbiota performs vital functions, such as barrier function, metabolic reactions, trophic effects, and maturation of the host's innate and adaptive immune responses. Development of the intestinal microbiota in infants is characterized by rapid and large changes in microbial abundance, diversity, and composition. These changes are influenced by medical, cultural, and environmental factors such as mode of delivery, diet, familial environment, diseases, and therapies used. Thus, it is nearly impossible to define a universal standard for intestinal colonization and development of the intestinal microbiota. This review discusses recent data on the early colonization of the gut by microbial species, development of the intestinal microbiota, and its impact on health.

Application of denaturing high-performance liquid chromatography (DHPLC) for the identification of fish: a new way to determine the composition of processed food containing multiple species.

The identification of fish species in transformed food products is difficult because the existing methods are not adapted to heat-processed products containing more than one species. Using a common to all vertebrates region of the cytochrome b gene, we have developed a denaturing high-performance liquid chromatography (DHPLC) fingerprinting method, which allowed us to identify most of the species in commercial crab sticks. Whole fish and fillets were used for the creation of a library of referent DHPLC profiles. Crab sticks generated complex DHPLC profiles in which the number of contained fish species can be estimated by the number of major fluorescence peaks. The identity of some of the species was predicted by comparison of the peaks with the referent profiles, and others were identified after collection of the peak fractions, reamplification, and sequencing. DHPLC appears to be a quick and efficient method to analyze the species composition of complex heat-processed fish products.

Human intestinal microbiota gene risk factors for antibiotic-associated diarrhea: perspectives for prevention. Risk factors for antibiotic-associated diarrhea.

Antibiotic-associated diarrhea (AAD) is associated with altered intestinal microflora and other symptoms that may lead to possibly death. In critically ill patients, diarrhea increases rates of morbimortality. Assessing diarrhea risks is thus important for clinicians. For this reason, we conducted a hypothesis-generating study focused on AAD to provide insight into methods of prevention. We evaluated the hypothesis of predisposing factors within the resident intestinal microbiota in a cohort of outpatients receiving antibiotherapy. Among the pool of tested variables, only those related to bacterial 16S rRNA genes were found to be relevant. Complex statistical analyses provided further information: amid the bacteria 16S rRNA genes, eight were determined to be essential for diarrhea predisposition and characterized from the most important to the least. Using these markers, AAD risk could be estimated with an error of 2%. This molecular analysis offers new perspectives for clinical applications at the level of prevention.

Application of denaturing high-performance liquid chromatography for intestinal microbiota analysis of newborns.

AIMS: Neonatal microbiota development seems to play a key role in the early origins of health and disease. However, the analysis of this complex ecosystem is still difficult. The aim of this work was to investigate the feasibility of a new technique, denaturing high-performance liquid chromatography (dHPLC), to analyze newborn intestinal microbiota using genomic approaches. METHODS AND RESULTS: Eleven neonates were recruited among patients admitted for intestinal surgery to the neonatal intensive care unit. Preoperative samplings were obtained in each case. Three methodologies were compared for each sample: (i) dHPLC, (ii) temporal temperature gradient gel electrophoresis (TTGE), and (iii) conventional culture techniques. RESULTS: All samples were poorly colonized. In three samples, the microbiota was detected only with the dHPLC technique. Results obtained with culture and TTGE could be found with dHPLC. CONCLUSION: The results suggest that neonatal applications of the dHPLC technique, especially for gut microbiota analysis, appear to be a sensitive and promising analytical technique.

A revised method for estimating hepatitis B virus transfusion residual risk based on antibody to hepatitis B core antigen incident cases.

BACKGROUND: To take into account the transient nature of hepatitis B virus (HBV) antigenemia, the calculation of HBV residual risk (RR), based on the incidence/window period model, is adjusted by a correction factor that adds uncertainty to the RR estimates. STUDY DESIGN AND METHODS: This new method to estimate the RR for HBV is a weighted sum of the RR derived from hepatitis B surface antigen (HBsAg) incident cases and the one derived from antibody hepatitis B core antigen (HBc) incident cases. An anti-HBc incident case was defined as a donation from a blood donor who had made at least one anti-HBc-negative donation followed by a donation that was found positive with two different assays within a 3-year period and positive for at least one of the following markers: 1) antibody to hepatitis B e antigen or hepatitis B e antigen, 2) anti-HBc immunoglobulin M, 3) HBV DNA, 4) hepatitis B surface antibody without HBV vaccination history, or 5) HBV DNA retrospectively found in the previous donation. Five overlapping 3-year study periods between 2000 and 2006 were analyzed. RESULTS: The HBV RR estimated with the classical method ranged from 1.51 (2000-2002) to 0.69 per million donations in 2004 through 2006 with a decrease in 2002 through 2004 due to only two HBsAg incident cases reported in this period. By applying the revised model, the HBV RR ranged from 1.06 (2000-2002) to 0.49 per million donations (2004-2006), with a regular decrease. CONCLUSION: The new presented model provides HBV RR estimates that do not statistically differ from those obtained with the classical model; however, it provides more accurate data, especially in low endemic areas where the HBsAg incidence is low.