Micalis Institute

This study examined the ability of four beneficial strains (Lactobacillus rhamnosus LbRE-LSAS, Bifidobacterium animalis subsp. lactis Bb12, and two yogurt starters TA040 and LB340) to ferment MRS or milk containing free linoleic acid (0, 0.5, or 1 mg/mL). The goal was to produce an enriched conjugated linoleic acid (CLA) isomers’ yogurt-type beverage. Linoleic acid (LA) at 0.5 mg/mL did not interfere with the growth of the assayed bacteria on de Man Rogosa and Sharpe broth (MRS) or milk. On the other hand, increasing the content of LA in the MRS or yogurt-type beverage to 1 mg/mL slightly inhibited all strains and prevented accumulating high biomasses. A gas chromatography analysis of the fatty acid profiles confirmed the bioconversion of LA. The yogurt starters TA040 and LB340 had the highest bioconversion rates in the yogurt-type beverages, whereas the probiotic Bb12 strain was the most interesting at converting LA into its active CLA. CLA from the MRS supernatants of TA040, Bb12, and LbRE-LSAS had maximum antibacterial activities against S. typhimurium, E. coli, and S. aureus, respectively. Whey from the Bb12 beverage showed an inhibitory effect against all pathogens. These results suggest that all strains could be used as starter cultures in the proposition of a yogurt-type beverage with a high CLA content and antibacterial potential.

Background: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated allergy without known biomarkers. We aimed to compare fecal biomarkers related to gut inflammation and immunity in children with FPIES, with resolved FPIES (tolerant), and in matched controls. Methods: Stools were collected from FPIES children on elimination diet, before and after an oral food challenge (OFC) performed to assess their natural tolerance, at the end of a follow-up in tolerant FPIES children, and in matched controls (1:1 ratio). Concentrations of calprotectin, EDN (eosinophilic derived neurotoxin), and secretory IgA (sIgA) underwent comparative paired analysis. Results: Thirty-eight patients were included (age: 1.3 years old, interquartile range: IQR [0.9 - 2.0]), of which 22 became tolerant during follow-up. Upon inclusion, allergic patients and controls had similar concentrations of calprotectin (38µg/g [8-85] vs 27µg/g [11-46], p=0.15) and EDN (504ng/g [275-1252] vs 516ng/g [215-844], p=0.86). However, concentrations of these inflammatory biomarkers increased transiently after a failed OFC (p<0.001 and p=0.01 respectively), without correlating with the severity of an allergic reaction. sIgA were higher in allergic than in tolerant patients: 2224µg/g [878-3529] vs 794µg/g [699-1767] (p<0.01). Calprotectin, EDN, and sIgA were comparable in tolerant patients and controls. sIgA less than 2637µg/g had a negative predictive value of 75.6% for the differentiation allergic patients from tolerant patients and controls (area under curve: 0.63, 95% CI: 0.52–0.74). Conclusion: A few days after an acute allergic reaction, there was no detectable chronic gut inflammation in FPIES. sIgA may be a useful tool for clinicians in timing OFC.

The efficiency of replication error repair is a critical factor governing the emergence of mutations. However, it has so far been impossible to study this efficiency at the level of individual cells and to investigate if it varies within isogenic cell populations. In addition, why some errors escape repair remains unknown. Here we apply a combination of fluorescent labelling of the Escherichia coli Mismatch Repair (MMR) complex, microfluidics, and time-lapse microscopy, to monitor in real-time the fate of >20000 replication errors. We show that i) many mutations result from errors that are detected by MMR but inefficiently repaired ii) this limited repair efficiency is due to a temporal constraint imposed by the transient nature of the DNA strand discrimination signal, a constraint that is likely conserved across organisms, and iii) repair capacity varies from cell to cell, resulting in a subpopulation of cells with higher mutation rate. Such variations could influence the fitness and adaptability of populations, accelerating for instance the emergence of antibiotic resistance.

Involuntary treatment may be a life-saving option for extremely severe anorexia nervosa (AN) in the context of life-threatening conditions and refusal of care. The long-term outcomes of patients undergoing involuntary treatment for AN are poorly understood. This study aims to explore quality of life, long-term outcomes and attitudes towards involuntary treatment in patients involuntarily treated for extremely severe AN. 23 patients involuntarily admitted for extremely severe AN (I-AN), and 25 voluntarily admitted patients (V-AN) were compared for body mass index (BMI), residual symptoms, quality of life, and attitudes towards treatment almost four years after discharge. In I-AN, clinical variables were also compared between admission and follow-up. At follow-up, weight restoration was higher in V-AN (p=0.01), while differences in quality of life, BMI, and mortality rates were not significant between I-AN and V-AN (p>0.05). In I-AN, BMI increased and weight-controlling strategies decreased at follow-up (p<0.05). Despite negative experiences of involuntary treatment, the perception of the necessity of treatment increased from admission to follow-up (p<0.01) and became comparable to V-AN (p>0.05). Involuntary treatment for AN does not appear to be a barrier to weight gain and clinical improvement, nor to long-term attitudes towards treatment.

Here we present a method to reduce the photobleaching of fluorescent proteins and the associated phototoxicity. It exploits a photophysical process known as reverse intersystem crossing, which we induce by near-infrared co-illumination during fluorophore excitation. This dual illumination method reduces photobleaching effects 1.5–9.2-fold, can be easily implemented on commercial microscopes and is effective in eukaryotic and prokaryotic cells with a wide range of fluorescent proteins.

Microbiomes have highly important roles for ecosystem functioning and carry out key functions that support planetary health, including nutrient cycling, climate regulation, and water filtration. Microbiomes are also intimately associated with complex multicellular organisms such as humans, other animals, plants, and insects and perform crucial roles for the health of their hosts. Although we are starting to understand that microbiomes in different systems are interconnected, there is still a poor understanding of microbiome transfer and connectivity. In this review we show how microbiomes are connected within and transferred between different habitats and discuss the functional consequences of these connections. Microbiome transfer occurs between and within abiotic (e.g., air, soil, and water) and biotic environments, and can either be mediated through different vectors (e.g., insects or food) or direct interactions. Such transfer processes may also include the transmission of pathogens or antibiotic resistance genes. However, here, we highlight the fact that microbiome transmission can have positive effects on planetary and human health, where transmitted microorganisms potentially providing novel functions may be important for the adaptation of ecosystems.

Abstract Membranes are a universal barrier to all cells. Phospholipids, essential bacterial membrane components, are composed of a polar head and apolar fatty acid (FA) chains. Most bacterial FA are synthesized by the FA synthesis pathway (FASII). In streptococcaceae, enterococci andLactococcus lactis, a unique feedback mechanism controls the FASII gene expression. FabT, encoded in the FASII main locus, is the repressor and it is activated by acyl-ACP. Many Streptococci,Enterococcus faecalis, but notL. lactis, possess two ACPS. AcpA encoding gene is within the FASII locus and is, to a certain extent, coregulated with the FASII genes. Acyl-AcpA is the end product of FASII. AcpB encoding gene is in operon withplsX. The role of AcpB as FabT corepressor is controversial.Streptococcus pyogenes, which causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections, possesses AcpB. In this study, we show by comparing gene repression of FASII genes in wild-type,fabTmutant andacpBmutant strains grown in the presence and the absence of exogenous FAs, that AcpB isS. pyogenesFabT main co-repressor. Also,acpBdeletion impacts the membrane FA composition and adhesion to eucaryotic cells, highlighting the role of AcpB.

Most lactobacilli produce extracellular polysaccharides that are considered to contribute to the probiotic effect of many strains. Lacticaseibacillus rhamnosus CNCM I-3690 is an anti-inflammatory strain able to counterbalance gut barrier dysfunction. In this study ten spontaneous variants of CNCM I-3690 with different EPS-production were generated and characterized by their ropy phenotype, the quantification of the secreted EPS and genetic analysis. Amongst them, two were further analysed in vitro and in vivo: an EPS over-producer (7292) and a low-producer derivative of 7292 (7358, with similar EPS levels than the wild type (WT) strain). Our results showed that 7292 does not have anti-inflammatory profile in vitro, and lost the capacity to adhere to the colonic epithelial cells as well as the protective effect on the permeability. Finally, 7292 lost the protective effects of the WT strain in a murine model of gut dysfunction. Notably, strain 7292 was unable to stimulate goblet cell mucus production and colonic IL-10 production, all key features for the beneficial effect of the WT strain. Furthermore, transcriptome analysis of colonic samples from 7292-treated mice showed a down-regulation of anti-inflammatory genes. Altogether, our results point out that the increase of EPS production in CNCM I-3690 impairs its protective effects and highlight the importance of the correct EPS synthesis for the beneficial effects of this strain.

ABSTRACTStreptococcus pyogenes prophage Φ1207.3 (formerly Tn1207.3) carries the mef(A)-msr(D) resistance genes, responsible for type M macrolide resistance. To investigate if Φ1207.3 is a functional bacteriophage, we transferred the element from the original S. pyogenes host in a prophage-free and competence-deficient S. pneumoniae strain. Pneumococcal cultures of the Φ1207.3-carrying lysogen were treated with mitomycin C to assess if Φ1207.3 enters the lytic cycle. Mitomycin C induced a limited phage burst and a growth impairment, resulting in early entrance into the stationary phase. To determine if Φ1207.3 is able to produce mature phage particles, we prepared concentrated supernatants recovered from a mitomycin C-induced pneumococcal culture by sequential centrifugation and ultracentrifugation steps. Negative-staining transmission electron microscopy (TEM) of supernatants revealed the presence of phage particles with an icosahedral, electron-dense capsid and a long, noncontractile tail, typical of a siphovirus. Quantification of Φ1207.3 was performed by quantitative PCR (qPCR) and semiquantitatively by TEM. PCR quantified 3.34 × 104 and 6.06 × 104 excised forms of phage genome per milliliter of supernatant obtained from the untreated and mitomycin C-treated cultures, respectively. By TEM, we estimated 3.02 × 103 and 7.68 × 103 phage particles per milliliter of supernatant. The phage preparations of Φ1207.3 infected and lysogenized pneumococcal recipient strains at a frequency of 7.5 × 10−6 lysogens/recipient but did not show sufficient lytic activity to form plaques. Phage lysogenization efficiently occurred after 30 min of contact of the phages with the recipient cells and required a minimum of 103 phage particles.IMPORTANCE Bacteriophages play an important role in bacterial physiology and genome evolution. The widespread use of genome sequencing revealed that bacterial genomes can contain several different integrated temperate bacteriophages, which can constitute up to 20% of the genome. Most of these bacteriophages are only predicted in silico and are never shown to be functional. In fact, it is often difficult to induce the lytic cycle of temperate bacteriophages. In this work, we show that Φ1207.3, a peculiar bacteriophage originally from Streptococcus pyogenes, which can lysogenize different streptococci and carries the macrolide resistance mef(A)-msr(D) gene pair, is capable of producing mature virions, but only at a low level, while not being able to produce plaques. This temperate phage is probably a partially functional phage, which seems to have lost lytic characteristics to specialize in lysogenization. While we are not used to conceiving phages separately from lysis, this behavior could actually be more frequent than expected.

The overarching biological impact of microbiomes on their hosts, and more generally their environment, reflects the co-evolution of a mutualistic symbiosis, generating fitness for both. Knowledge of microbiomes, their systemic role, interactions, and impact grows exponentially. When a research field of importance for planetary health evolves so rapidly, it is essential to consider it from an ethical holistic perspective. However, to date, the topic of microbiome ethics has received relatively little attention considering its importance. Here, ethical analysis of microbiome research, innovation, use, and potential impact is structured around the four cornerstone principles of ethics: Do Good; Don’t Harm; Respect; Act Justly. This simple, but not simplistic approach allows ethical issues to be communicative and operational. The essence of the paper is captured in a set of eleven microbiome ethics recommendations, e.g., proposing gut microbiome status as common global heritage, similar to the internationally agreed status of major food crops.