In vitro sensitivity of 30 anaerobic bacterial strains of the human intestinal core microbiota to antibiotics: Culture and LC-MS/MS approaches.

We have a vast knowledge on human intestinal microbiota but it can still be regarded incomplete. One of the objectives of scientists using so-called "omics" techniques is to be interested in the consequences that drugs can have on the composition of the intestinal microbiota and inversely. To date, few publications have reported the effects of drugs on the growth of bacteria composing this microbiota using a "culturomics" approach. We focused on antibiotics commonly prescribed for which the only published are the susceptibility of the pathogenic strains and not that of the commensal strains. The aim of our study was to determine the sensitivity of 30 strains considered to represent the intestinal core microbiota to 8 antibiotics and to study the possible modification of these molecules by bacteria. The 30 bacterial strains were cultured under anaerobic conditions in order to determine their sensitivity to the antibiotics. After 48 h of culture, the supernatants were also analyzed via UHPLC-MS/MS in order to determine if the antibiotics have been chemically modified. Under the current experimental conditions, cefpodoxime, metronidazole, erythromycin, sulfamethozaxole, trimethoprim and the trimethoprim/sulfamethozaxole combination have little impact on the core microbiota strain growth. On the contrary, moxifloxacin and amoxicillin inhibit the growth of numerous strains of our panel. Using UHPLC-MS/MS analyses, we have shown that some antibiotics can be modifed by the bacteria composing the intestinal core microbiome. The bacteria that make up the intestinal microbiota core are impacted by the antibiotics most commonly prescribed in clinics today and inversely.

Development of an in vitro Model of Human Gut Microbiota for Screening the Reciprocal Interactions With Antibiotics, Drugs, and Xenobiotics.

Altering the gut microbiota can negatively affect human health. Efforts may be sustained to predict the intended or unintended effects of molecules not naturally produced or expected to be present within the organism on the gut microbiota. Here, culture-dependent and DNA-based approaches were combined to UHPLC-MS/MS analyses in order to investigate the reciprocal interactions between a constructed Human Gut Microbiota Model (HGMM) and molecules including antibiotics, drugs, and xenobiotics. Our HGMM was composed of strains from the five phyla commonly described in human gut microbiota and belonging to Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, and Actinobacteria. Relevantly, the bacterial diversity was conserved in our constructed human gut model through subcultures. Uneven richness distribution was revealed and the sensitivity of the HGMM was mainly affected by antibiotic exposure rather than by drugs or xenobiotics. Interestingly, the constructed model and the individual cultured strains respond with the same sensitivity to the different molecules. UHPLC-MS/MS analyses revealed the disappearance of some native molecules in the supernatants of the HGMM as well as in those of the individual strains. These results suggest that biotransformation of molecules occurred in the presence of our gut microbiota model and the coupled approaches performed on the individual cultures may emphasize new bacterial strains active in these metabolic processes. From this study, the new HGMM appears as a simple, fast, stable, and inexpensive model for screening the reciprocal interactions between the intestinal microbiota and molecules of interest.

Comprehensive steroid profiling by liquid chromatography coupled to high resolution mass spectrometry.

A steroidomics workflow has been developed in the objective of monitoring a wide range (n >150) of steroids in urine. The proposed workflow relies on the optimization of an adequate SPE extraction step followed by an UHPLC-HRMS/MS simultaneous analysis of both free and conjugated forms of C18, C19 and C21 steroid hormones. On the basis of 44 selected steroids, representative of main classes of steroids constituting the steroidome, the performances of the developed workflow were evaluated in terms of selectivity, repeatability (< 13%) and linearity (R2> 0.985 in the concentration range [0.01-10 ng/mL]). As metabolites identification and characterization constitute the bottleneck of such profiling approaches, a homemade database was created encompassing a large number of characterized free and conjugated steroids (n> 150) for putative steroid-like biomarkers identification purposes. The efficiency of the workflow in highlighting fine modifications within the urinary steroidome was assessed in the frame of an anabolic treatment involving an intra-muscular administration of boldenone undecylenate (2 mg/kg) to veals (n=6) and the investigation of potential steroid biomarkers. Besides monitoring known phase II metabolites of boldenone in the bovine specie, namely, boldenone glucuronide and sulfate, the applied strategy also permitted to observe, upon boldenone administration, a modified profile of epiboldenone glucuronide. Furthermore, 31 signals corresponding to non-identified steroid species could also be highlighted as impacted upon the exogenous steroid treatment. This study is the first to simultaneously investigate both free and conjugated C18, C19 and C21 steroid hormones in their native form using UHPLC-HRMS/MS and allowing their comprehensive profiling. This strategy was probed in-vivo.

Impact of storage conditions on the urinary metabolomics fingerprint.

Urine stability during storage is essential in metabolomics to avoid misleading conclusions or erroneous interpretations. Facing the lack of comprehensive studies on urine metabolome stability, the present work performed a follow-up of potential modifications in urinary chemical profile using LC-HRMS on the basis of two parameters: the storage temperature (+4 °C, -20 °C, -80 °C and freeze-dried stored at -80 °C) and the storage duration (5-144 days). Both HILIC and RP chromatographies have been implemented in order to globally monitor the urinary metabolome. Using an original data processing associated to univariate and multivariate data analysis, our study confirms that chemical profiles of urine samples stored at +4 °C are very rapidly modified, as observed for instance for compounds such as:N-acetyl Glycine, Adenosine, 4-Amino benzoic acid, N-Amino diglycine, creatine, glucuronic acid, 3-hydroxy-benzoic acid, pyridoxal, l-pyroglutamic acid, shikimic acid, succinic acid, thymidine, trigonelline and valeryl-carnitine, while it also demonstrates that urine samples stored at -20 °C exhibit a global stability over a long period with no major modifications compared to -80 °C condition. This study is the first to investigate long term stability of urine samples and report potential modifications in the urinary metabolome, using both targeted approach monitoring individually a large number (n > 200) of urinary metabolites and an untargeted strategy enabling assessing for global impact of storage conditions.

Combining metabolomics and gene expression analysis reveals that propionyl- and butyryl-carnitines are involved in late stages of arbuscular mycorrhizal symbiosis.

The arbuscular mycorrhizal (AM) symbiosis is a widespread mutualistic association between soil fungi (Glomeromycota) and the roots of most plant species. AM fungi are obligate biotrophs whose development is partially under the control of their plant host. We explored the possibility to combine metabolomic and transcriptomic approaches to find putative mycorrhiza-associated metabolites regulating AM fungal development. Methanol extracts of Medicago truncatula roots colonized or not with the AM fungus Rhizophagus irregularis were analyzed and compared by ultra-high-performance liquid chromatography (UHPLC), high-resolution mass spectrometry (Q-TOF), and multivariate statistical discrimination. We detected 71 mycorrhiza-associated analytes exclusively present or at least 10-fold more abundant in mycorrhizal roots. To identify among these analytes those that could regulate AM fungal development, we fractionated by preparative and semi-preparative HPLC the mycorrhizal and non-mycorrhizal root extracts and established how the 71 analytes were distributed among the fractions. Then we tested the activity of the fractions on germinating spores of R. irregularis by quantifying the expression of 96 genes known for their diverse in planta expression patterns. These investigations reveal that propionyl- and butyryl-carnitines accumulated in mycorrhizal roots. The results suggest that these two molecules regulate fungal gene expression in planta and represent interesting candidates for further biological characterization.

Metabolite profiling of pea roots in response to phosphate availability.

The arbuscular mycorrhizal (AM) symbiosis is a mutualistic association between soil fungi (Glomeromycota) and roots of most plant species. A recent study showed that high phosphate fertilization could inhibit mycorrhizal colonization at a very early stage, before hyphopodium formation. The authors proposed that inhibiting and/or stimulatory compounds might be present in roots grown under high phosphate or low phosphate, respectively. To further address this question, we performed metabolite profiling analyses of extracts of pea roots grown under low and high phosphate concentrations. Ultra high performance liquid chromatography (UHPLC) was coupled with high resolution (HR) mass spectrometry (Q-TOF) and multivariate statistical analysis. This allowed the detection of 34 ions discriminating the two conditions. A majority (28 ions) were more abundant in roots grown under low phosphate concentration, and among them four were specific of this condition. The results suggest that the regulation of AM symbiosis by phosphate may involve the synthesis or accumulation of stimulatory compounds in roots grown under low phosphate.

Serum profile in preeclampsia and intra-uterine growth restriction revealed by iTRAQ technology.

In order to identify new protein markers modified in placental diseases, high-throughput analysis of proteins in the plasma of pregnant women was carried out for normal and pathological pregnancies (Preeclampsia and/or Intra-Uterine Growth Restriction) using iTRAQ technology. We could identify 166 proteins that were modified (p<0.05) and the technique used allowed the detection of previously undetected factors, such as various members of the SERPINA clade. The modifications of two proteins (C reactive protein and antichymotrypsin, SERPINA3) were validated on individual samples. Complement and coagulation cascades proteins were significantly enriched among modified protein clusters in the case of intra-uterine growth restriction (p<2.6.10(-11)). Several proteins were specifically enriched in isolated preeclampsia and depleted when preeclampsia was complicated by intra-uterine growth restriction. These findings suggest that the growth restricted foeto-placental unit is able to moderate some changes in maternal plasma composition. Overall, the use of iTRAQ technology, for the first time on this subject, enabled us to provide a new list of proteins modified in placental diseases, among which proteins expressed at a low level that were not accessible by other methods.