R Couronné

A Valladier

M Vidailhet

J C Corvol

S Lehéricy

S Durrleman

Longitudinal comparison of subjects with and without Sleep Disorders in Parkinson's Disease

Keywords: Longitudinal study, disease progression model, subgroup comparison, Parkinson's Disease

average scenario of progression for the 2 subgroups, relative to age. Abnormality thresholds (straight lines) are mapped to the corresponding age of occurrence (dotted lines). Note the major difference for the REM-quizz (chosen split criterion).

Introduction

Patients with Parkinson's Disease (PD) may have very different patterns of progression, corresponding to distinct disease subtypes. Here, we describe quantitatively the overall pattern of progression in subgroups of PD by using a Bayesian non-linear mixed effect model that describes the continuous progression of biomarkers at both population and individual level. This approach allows to model variability in progression patterns and disease stage between patients. We analyzed two subgroups of patients, with (RBD+) and without sleep disorders (RBD-), that are known to present different patterns of progression [START_REF] Postuma | How does parkinsonism start? Prodromal parkinsonism motor changes in idiopathic REM sleep behaviour disorder[END_REF]. We compared the two groups by extracting the ordering of abnormalities that occurred over the disease course, and by studying their disease onset and speed of progression.

Methods

Data

We used the Idiopathic PD patients of the PPMI Dataset. We considered eight biomarkers to describe the disease progression. First four clinical scores, the MDS-UPDRS part III, MoCA, 1 Corresponding author. Email: raphael.couronne@inria.fr SCOPA-AUT and REM-quizz, measuring respectively motor, cognitive, autonomy and sleep dysfunctions. Second, we included four measures from DatScan imaging, the Striatal Binging Ratios (SBR) of left and right side of both Putamen and Caudate. From the 362 idiopathic PD patients of the PPMI dataset, we excluded patients that had less than two visits of the biomarkers to obtain 349 patients. Patients that reached a REM-quizz score of 6 over 9 at least once over the course of the study were labeled as REM+ (94 patients), the others as REM-(249 patients).

Modeling approach

We used a multivariate non-linear Bayesian mixed-effect model, introduced in [2] and extended in [START_REF] Couronné | Learning disease progression models with longitudinal data and missing values[END_REF], to estimate the longitudinal progression of clinical and imaging scores. We estimated both population and individual parameters of patients, to obtain the average and individual trajectories, respectively. Subgroup trajectories were obtained by averaging individual parameters over the patient subgroups. Then, to assess the robustness of our method, accounting for both sampling bias and stochastic variations in the estimation algorithm, we performed a patient-wise repeated cross validation (10x5 folds) on our dataset.

Average progression RBD+ RBD-

With the methodology described above, we obtained 50 sub-population average trajectories for each group (RBD-and RBD+), shown in Figure 1. To build a hierarchy of arrival of abnormalities of PD biomarkers that may progress at very different speeds and occur at different ages, we introduced abnormality thresholds. For each biomarker independently, thresholds were computed from the data as the optimal cut-off for a balanced logistic regression between PD patients and controls at baseline. We then computed the times of intersection between the averaged trajectories of the subgroups and these abnormal thresholds to assess an abnormality timing density for each modality. Densities are shown as boxplots in Figure 2. times of intersection with threshold of abnormalities for each considered biomarker. Average age of conversion to PD is mostly the same for both groups.

Results and discussion

The ordering of abnormalities between modalities in the two groups was the same, at the exception of REM-quizz since it discriminated our 2 groups (Figure 2). The first abnormality was observed in the putamen, followed by MDS-UPDRS part III then, caudate, scopa and finally MoCA. Note that MDS-UPDRS abnormality occurred early, as even few points of MDS-UPDRS label patients as PD in our logistic regression. Thus, we could also consider the age at disease onset, mainly based on motor scores, to represent a more advanced stage of motor abnormality. Noticeably, the distance to PD average age at diagnosis differed between the two subgroups.

In RBD-patients, autonomic dysfunction occurred shortly after conversion to PD, whereas in RBD+, the SCOPA-AUT was already abnormal up to 4 years before conversion. To a lesser extent, MoCA abnormalities occurred earlier in RBD+ than in RBD-. Finally, considering only the individual parameters estimated by the model, we found that RBD+ were affected in average 6 years earlier than RBD-, and progressed 30% faster in average.

Conclusions

Using a longitudinal model, we were able to compare two sub-populations of a longitudinal cohort in several ways: progression speed, age at onset and ordering of abnormalities. RBD+ were affected 6 years earlier and progressed 30% faster. They also presented autonomic dysfunction up to 4 years before PD diagnosis whereas these symptoms occurred around PD onset in RBD-patients. These results stand as a proof of concept for longitudinal group comparison methodology, which could be applied to other types of diseases. Furthermore, this first subgroup analysis lay the foundations of a more precise subtyping of patterns of progression in PD.

Figure 2 :

 2 Figure 2: Boxplot of cross validated (10x5)times of intersection with threshold of abnormalities for each considered biomarker. Average age of conversion to PD is mostly the same for both groups.

Acknowledgements

The research leading to these results has received funding from the program "Investissements d'avenir" ANR-10-IAIHU-06. This work was funded in part by the French government under management of Agence Nationale de la Recherche as part of the "Investissements d'avenir" program, reference ANR-19-P3IA-0001 (PRAIRIE 3IA Institute).


L Castro-Oliveira

R.D.O. D O Carvalho M O O Silva

A Anchiêta

L J J Benevides

Gwénaël Jan

C J F J F Oliveira

P Figueiredo

R D O Carvalho

H C P Figueiredo

V A C Azevedo

S C Soares

Article Omics of probiotic bacteria: which features to seek?

Keywords: Omics approach, Probiotics, Metabiotics, Genomics, Culturomics Probiotic bacteria -omics approach

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Microorganisms responsible for food fermentation and to prevent putrefaction have been used by man during centuries [START_REF] Salque | Earliest evidence for cheese making in the sixth millennium bc in northern Europe[END_REF][START_REF] Yang | Selection of functional lactic acid bacteria as starter cultures for the fermentation of Korean leek (Allium tuberosum Rottler ex Sprengel.)[END_REF]. It is commonly accepted that the fermentation processes adapted by man appeared due to accidental contamination and appropriate climate and environment, resulting in widely used fermented products, such as kefir, leben and koumiss [START_REF] Hosono | Fermented milk in the orient[END_REF]. In addition to the possibility to store fermented foods, to their enhanced nutritional value and to their safety for consumption, fermented food has had great cultural importance, highlighted by citations of some of these products in the Holy Bible and sacred books of Hinduism [START_REF] Bibel | Elie Metchnikoff's Bacillus of Long Life[END_REF][START_REF] Hosono | Fermented milk in the orient[END_REF][START_REF] Shortt | The probiotic century: Historical and current perspectives[END_REF]. However, it was only after the 19 th century that the fermentation process was studied and that probiotic concepts were introduced by Louis Pasteur and Élie Metchnikoff (Johnson and [START_REF] Br | Impact of genomics on the field of probiotic research: historical perspectives to modern paradigms[END_REF], the fathers of microbiology and innate immunology, respectively. Later on, Bacillus bulgaricus (currently known as Lactobacillus delbrukeckii subsp. bulgaricus) was recovered from human feces and it was shown to reduce putrefaction toxins and help in colitis treatment [START_REF] Br | Impact of genomics on the field of probiotic research: historical perspectives to modern paradigms[END_REF].

In 1930, a Japanese microbiologist isolated a species from human feces that survived the gastrointestinal tract (GIT), identified as Lactobacillus casei, which was later used to develop the fermented milk product Yakult [START_REF] Shortt | The probiotic century: Historical and current perspectives[END_REF]. Besides the use of these probiotics in the production of fermented milk, other lactic acid bacteria (LAB) are also used in the preservation of vegetables, grains and meat [START_REF] Chaillou | The complete genome sequence of the meat-borne lactic acid bacterium Lactobacillus sakei[END_REF]. Recently, probiotics have been crossing barriers from the functional food market to pharmaceutical and therapeutic uses. This expansion is directly correlated with advances in the scientific and regulatory aspects of LAB related probiotics and the study of their protein delivery mechanisms [START_REF] Bolotin | The Complete Genome Sequence of the Lactic Acid Bacterium[END_REF][START_REF] Foligné | Probiotics from research to market: the possibilities, risks and challenges[END_REF].

Nowadays, probiotics are widely known for their use in the treatment of functional gastrointestinal disorders (FGID) including irritable bowel syndrome, Crohn's disease and ulcerative colitis, in conjunction with usual medical treatments [START_REF] Bibiloni | VSL#3 Probiotic-Mixture Induces Remission in Patients with Active Ulcerative Colitis[END_REF][START_REF] Sood | The Probiotic Preparation, VSL#3 Induces Remission in Patients With Mild-to-Moderately Active Ulcerative Colitis[END_REF][START_REF] Tursi | Treatment of Relapsing Mild-to-Moderate Ulcerative Colitis With the Probiotic VSL#3 as Adjunctive to a Standard Pharmaceutical Treatment: A Double-Blind, Randomized, Placebo-Controlled Study[END_REF]. This has occurred for several reasons, but mainly because there are few options of pharmaceutical treatments for FGID, and conventional treatment options have low efficacy and serious side effects [START_REF] Shen | Complementary and alternative medicine for treatment of irritable bowel syndrome[END_REF]. FGID are very common and are believed to be the cause or consequence of changes in gastrointestinal microbiota [START_REF] Porter | The Incidence and Gastrointestinal Infectious Risk of Functional Gastrointestinal Disorders in a Healthy US Adult Population[END_REF]. Consequently, probiotics have become a useful complement to the usual treatment of such diseases.

Probiotics have been found to be a favorable option not only against FGID, but also for a wide range of disorders, because they can reinforce the gut barrier function, conferring clinical benefits at distant sites through their immunomodulatory activities [START_REF] Bo | Probiotics for preventing ventilator-associated pneumonia[END_REF]. Some studies have shown the beneficial effects of probiotics in modulating inflammatory and autoimmune diseases, such as rheumatoid arthritis [START_REF] So | Lactobacillus casei suppresses experimental arthritis by downregulating T helper 1 effector functions[END_REF], type I diabetes [START_REF] Calcinaro | Oral probiotic administration induces interleukin-10 production and prevents spontaneous autoimmune diabetes in the non-obese diabetic mouse[END_REF], multiple sclerosis [START_REF] Lavasani | A Novel Probiotic Mixture Exerts a Therapeutic Effect on Experimental Autoimmune Encephalomyelitis Mediated by IL-10 Producing Regulatory T Cells[END_REF], atopic dermatitis [START_REF] Viljanen | Induction of inflammation as a possible mechanism of probiotic effect in atopic eczema-dermatitis syndrome[END_REF], and myasthenia gravis [START_REF] Chae | Prophylactic Effect of Probiotics on the Development of Experimental Autoimmune Myasthenia Gravis[END_REF]. Moreover, experimental results strongly suggest that selected strains of probiotics can help in the treatment of cancer, neurodegenerative diseases, metabolic syndrome and psychiatric illnesses, among other pathologies. Also, there is evidence for reduction of ventilator-associated pneumonia in intensive care units patients under mechanical ventilation [START_REF] Bo | Probiotics for preventing ventilator-associated pneumonia[END_REF]. However, one of the challenges in preclinical and clinical use of probiotics is how to select a strain with potent immune modulating properties [START_REF] Chae | Prophylactic Effect of Probiotics on the Development of Experimental Autoimmune Myasthenia Gravis[END_REF].

In view of this challenge, there is a growing interest in the study of probiotic bacteria through structural and functional genomics for the discovery of probiotic-related features. Because of the development of NGS, the bacterial whole-genome sequencing has become a low cost and suitable approach for fast and accurate screening of potential probiotic candidates for treatment of each type of disorder [START_REF] Didelot | Transforming clinical microbiology with bacterial genome sequencing[END_REF][START_REF] Senan | Feasibility of Genome-Wide Screening for Biosafety Assessment of Probiotics: A Case Study of Lactobacillus helveticus MTCC 5463[END_REF]. This approach allows researchers to detect and discard candidate strains that have potential risk factors, such as antibiotic resistance or virulence genes. It also facilitates the analysis and description of functional mechanisms, avoiding the difficulties of isolating and growing the microorganisms [START_REF] Papadimitriou | Discovering probiotic microorganisms: in vitro, in vivo, genetic and omics approaches[END_REF]. This new approach using NGS techniques to screen potential probiotic candidate strains makes it imperative that we understand the genomic features that should be prioritized and sought in new strains.

Probiotic related features

As a first step, the simplified definition of probiotic bacteria is originally related to live cultures that help in the maintenance of a healthy and balanced intestinal microbiota [START_REF] Cronin | Progress in genomics, metabolism and biotechnology of bifidobacteria[END_REF]. Specifically, for the GIT, probiotic features have been elucidated through the fusion of structural and functional genomics techniques. In this context, three main features of the mechanisms of probiotic action deserve attention: (i) survival through GIT passage (bile salts and gastric acidity) [START_REF] Bezkorovainy | Probiotics: determinants of survival and growth in the gut[END_REF], (ii) competitive exclusion and antimicrobial activity, such as microcin and hydrogen peroxide production [START_REF] Konuray | Potential Use of Bacillus coagulans in the Food Industry[END_REF] and (iii) modulation of the immune system of the host GIT (Johnson and Klaenhammer, 2014) (Figure 1). This includes molecular associated molecular patterns (MAMP) which may be recognized by host pattern recognition receptors (PRR) [START_REF] Lebeer | Host interactions of probiotic bacterial surface molecules: Comparison with commensals and pathogens[END_REF]. Those mechanisms will be further described in the next sections. Various bacterial genera and species are used as probiotics, for instance: Lacticaseibacillus casei, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium lactis, Propionibacterium freudenreichii, Bacillus subtilis, Bacillus cereus, E. coli and Enterococcus faecium. All of these species have therapeutic applications in prevention and treatment of intestinal disorders, such as diarrhea in newborns [START_REF] Ouwehand | Probiotics an overview of beneficial effects[END_REF].

The first widely known scientific report about probiotic bacteria dates back to 1907 and demonstrated the correlation between the ingestion of LAB and the increase in longevity of Bulgarians and other populations [START_REF] Howell | Metchnikoff and prolongation of life[END_REF]. Fermented foods produced with the use of many bacteria of the Lactobacillus genus have been widely employed as therapeutics for the prevention or treatment of diseases due to their beneficial properties, such as relief of lactose intolerance symptoms and decrease in diarrhea due to rotavirus [START_REF] Ouwehand | Probiotics an overview of beneficial effects[END_REF].

Recently, most of studies and utilization of probiotics involve LAB, especially Lactobacillus isolated from the GIT. When it comes to the administration of probiotic bacteria, a certain amount of bacteria are necessary to exert a desired feature and consequent host response, which may vary according to the strain, use, and formulation; it is generally recommended to consume at least 10 7 microorganisms per milliliter in a daily dose [START_REF] Corcoran | Life Under Stress: The Probiotic Stress Response and How it may be Manipulated[END_REF].

Bacterial safety aspects

Over time, there are changes in the content and in order of genetic information of organisms due to genomic plasticity and evolutionary pressure [START_REF] Soares | Plasticidade Genômica e Evolução Bacteriana[END_REF]. Genomic plasticity is the dynamic property of DNA that arises from genetic conversion and point mutations, rearrangements (through translocation and inversion, for example), deletions, and insertion of genetic material from other organisms (plasmids, transposons, bacteriophages, among others). These mechanisms alter the bacterial lifestyle, contributing to their adaptation to different environments and influencing their evolution [START_REF] Schmidt | Pathogenicity islands in bacterial pathogenesis[END_REF].

The detection and the analysis of phage regions plays a key role in the elucidation of the genomic plasticity of probiotic bacteria because they are used in fermented products for human consumption and therefore cannot harbor mobile elements that could be transferred to other bacteria. Phages are obligate parasites and most of them have a multiplication cycle that culminates in cellular lysis, through which hundreds of viral particles are released, ready to infect nearby cells [START_REF] Summers | Bacteriophage Research: Early History[END_REF]. Moreover, phages are widely distributed all over the world; it is possible to find up to 10 8 phages in just a drop of water from the ocean [START_REF] Wommack | Virioplankton: viruses in aquatic ecosystems[END_REF].

All industrial or biotechnological processes that require bacterial use for the production of fermented food products or of useful molecules could be rapidly interrupted by virulent phages. They are a primary cause of failure in the fermentative process during the industrial transformation of milk [START_REF] Garneau | Bacteriophages of lactic acid bacteria and their impact on milk fermentations[END_REF].

The first description of phages infecting dairy starter dates from 1935; since then, important improvements have been made, particularly in knowledge about bacterial ecology, phage genomics and resistance to environmental factors [START_REF] Brüssow | Phages of dairy bacteria[END_REF]. There are reports of phage regions in Lactococcus species, such as Lactococcus lactis [START_REF] Cavanagh | Phages of non-dairy lactococci: isolation and characterization of ΦL47, a phage infecting the grass isolate Lactococcus lactis ssp. cremoris DPC6860[END_REF]. However, even with all the advances in the area, phage contaminations still damage fermented milk products and reduce productivity [START_REF] Moineau | Control of Bacteriophages in Industrial Fermentations[END_REF]. Phages may can have various origins; therefore, it is very important to study all potential sources of contamination and their consequences for the production of fermented dairy products [START_REF] Garneau | Bacteriophages of lactic acid bacteria and their impact on milk fermentations[END_REF].

Another way that bacteria acquire genomic material is through genomic islands (GEI). GEI may be classified as pathogenicity islands (PAI), metabolic islands (MI), symbiotic islands (SI) and resistance islands (RI). They are large genomic regions acquired through horizontal gene transfer, harboring a large number of genes (encoding similar functions and operons) with the potential to allow the bacteria to evolve in leaps [START_REF] Soares | Plasticidade Genômica e Evolução Bacteriana[END_REF] Probiotic bacteria should be analyzed for PAI and RI, which contain a high concentration of virulence factors and antibiotic resistance genes, respectively, and could be transferred to other organisms, compromising the safety aspects of the bacteria. Probiotic bacteria should only contain natural resistance, with no trace of virulence factors or antibiotic resistance genes in unstable regions, such as GEI, phages, and plasmids [START_REF] Salminen | Demonstration of safety of probiotics -a review[END_REF].

There are some specific aspects that characterize probiotic action within the host GIT that will be discussed in the next section. Genomic islands related to interaction with the host and to persistence within its digestive tract have also been described by comparative genomics [START_REF] Kankainen | Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human-mucus binding protein[END_REF]. Their instability over time and in cultures evidenced the need for quality assurance and control measures targeting genome stability in probiotics [START_REF] Sybesma | Genome Instability in Lactobacillus rhamnosus GG[END_REF].

Tolerance towards digestive constraints, gastric juice and bile salts

One of the most important attributes of a probiotic microorganism is its ability to survive the GIT environment. A study with comparative genomics analyzed the niche-based stress-responsive genes of two Lactobacillus helveticus strains: MTCC 5463 (a potential probiotic) and DPC4571 (a cheese starter); 5463 apparently has many genes involved in stress response. This potential probiotic strain has a larger number of genes related to heat, osmotic, cold and oxidative stress resistance than DPC 4571 [START_REF] Senan | Comparative genome-scale analysis of niche-based stress-responsive genes in Lactobacillus helveticus strains[END_REF].

Functional genomics studies have been complementing and elucidating some questions related to stress response. Using transcriptomics and proteomics, a study of the probiotic Lacticaseibacillus rhamnosus GG analyzed the effects of bile stress and demonstrated that 316 transcripts changed in expression level and 42 proteins (intracellular and surface-exposed), were differentially abundant. The the changes were associated with the adaptation process of this bacterium [START_REF] Koskenniemi | Proteomics and Transcriptomics Characterization of Bile Stress Response in Probiotic Lactobacillus rhamnosus GG[END_REF]. Performing the same omics study on Bifidobacterium longum BBMN68, the expression level of 236 transcripts changed significantly and 44 proteins were differently abundant. A hypothesis involving the modification of cell membrane composition (cyclopropane fatty acid increases and transmembrane proteins decreases) was confirmed with a surface hydrophobicity assay [START_REF] An | Integrated Transcriptomic and Proteomic Analysis of the Bile Stress Response in a Centenarian-originated Probiotic Bifidobacterium longum BBMN68[END_REF].

Bacterial pathogens exclusion mechanisms

Competitive exclusion of pathogens is another criterion in the selection of probiotic bacteria. Among the mechanisms of action, there are, for instance, enhancement of the epithelial barrier, production of anti-microbial substances, competitive exclusion of bacterial pathogens, increased adhesion to intestinal mucosa and modulation of the immune system [START_REF] Bermudez-Brito | Probiotic Mechanisms of Action[END_REF]. Associated features may include surface compounds involved in aggregation, coaggregation, and adhesion, as well as specific biosynthesis pathways.

Adhesion-related genes

In addition to survival through the GIT, adhesion to the intestinal epithelium is another factor that may contribute to probiotic activity, through exclusion mechanisms. The interaction between microbe and host occurs via adhesion-related proteins that recognize and bind to specific receptor regions of the host cell, activating the innate response, promoting invasion or bacterial colonization. Adhesion may be mediated by pili or fimbriae extending out from the bacterial cell wall or Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) [START_REF] Soto | Bacterial Adhesins : Common Themes and Variations in Architecture and Assembly MINIREVIEW Bacterial Adhesins : Common Themes and Variations in Architecture and Assembly[END_REF].

Preliminary in vitro studies using intestinal epithelial cells revealed multiple probiotic Lactobacillus producing adhesins [START_REF] Chauviere | Adhesion of human Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells[END_REF][START_REF] Tuomola | Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures[END_REF] and Bifidobacterium spp. was shown to adhere to the human intestinal mucus [START_REF] He | Adhesion of Bifidobacterium spp. to human intestinal mucus[END_REF]. Access to the genomic sequence data combined with genomic techniques helped to elucidate the adhesion mediators. Most of these are secreted or bound to the cell wall in a sortase-dependent way, aiming to interact with the intestinal epithelium [START_REF] Vélez | Adherence factors of Lactobacillus in the human gastrointestinal tract[END_REF][START_REF] Lebeer | Impact of luxS and Suppressor Mutations on the Gastrointestinal Transit of Lactobacillus rhamnosus GG[END_REF]. Studying 43 Lactobacillus strains, [START_REF] Harris | Phylogenomics and comparative genomics of Lactobacillus salivarius, a mammalian gut commensal[END_REF] used Cluster of Orthologous Groups (COG) to identify at least one sortase A gene for each. Seven genomes among them have an extra sortase A, of which, five have a sortase C gene and a putative pilus operon.

In L. acidophilus NCFM, an in silico search on the genome allowed the identification of five cell surface adhesion proteins, including: one fibronectin binding protein (FbpA), one S-layer protein (SlpA), one mucin binding protein (Mub) and two homologous R28 proteins involved in Streptococcus adhesion [START_REF] Buck | Functional Analysis of Putative Adhesion Factors in Lactobacillus acidophilus NCFM[END_REF].

Through mutational analyses, FbpA, SlpA, and Mub were shown to contribute to adhesion to Caco-2 epithelial cells. Similarly, one stress response protein and one aggregation promoting factor (both surface proteins) were later found in other studies, which are also contribute to adherence to Caco-2 cells [START_REF] Goh | Functional roles of aggregation-promoting-like factor in stress tolerance and adherence of Lactobacillus acidophilus NCFM[END_REF][START_REF] Flaherty | The role and potential of probiotic bacteria in the gut, and the communication between gut microflora and gut/host[END_REF].

Slp genes may indeed be involved in adhesion to host cells in various probiotics such as lactobacilli [START_REF] Do Carmo | Mutation of the Surface Layer Protein SlpB Has Pleiotropic Effects in the Probiotic Propionibacterium freudenreichii CIRM-BIA 129[END_REF] and propionibacteria [START_REF] Do Carmo | Propionibacterium freudenreichii Surface Protein SlpB Is Involved in Adhesion to Intestinal HT-29 Cells[END_REF]. Inactivation of the corresponding Slp gene accordingly suppresses interaction with host cells in L. acidophilus [START_REF] Konstantinov | S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions[END_REF] and in P. freudenreichii [START_REF] Do Carmo | Propionibacterium freudenreichii Surface Protein SlpB Is Involved in Adhesion to Intestinal HT-29 Cells[END_REF].

Comparative genomics was used with two L. rhamnosus strains and revealed the presence of genomic islands; one of them, predicted in L. rhamnosus GG, harbors genes coding for three secreted proteins, sortase-dependent pili, encoded by a spaCBA operon that were later confirmed through experimental analyses as pili encoding genes [START_REF] Kankainen | Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human-mucus binding protein[END_REF]. Functional annotation was used to characterize the probiotic potential of Bacillus coagulans HS243, in which 11 genes were predicted as adhesion-related proteins, among them: enolase, fibronectin binding protein and flagellar hook associated proteins [START_REF] Kapse | Functional annotation of the genome unravels probiotic potential of Bacillus coagulans HS243[END_REF].

The adhesion mechanism is an important property to select a probiotic strain and using in silico analyses it is possible to determine more details of the adhesion sites, such as mucin and binding to fibronectin [START_REF] Papadimitriou | Discovering probiotic microorganisms: in vitro, in vivo, genetic and omics approaches[END_REF]

Antimicrobial peptides

The LAB action in the conservation of food is due to both medium acidification (pH 3.5 to 4.5) and the production of numerous bacterial agents, such as organic compounds and bacteriocins (Van de [START_REF] Van De Guchte | Production of growth-inhibiting factors by Lactobacillus delbrueckii[END_REF]. Bacteriocins are bacterial produced peptides, which act against other microorganisms and to which the producer has specific immunity mechanisms [START_REF] Cotter | Bacteriocins: developing innate immunity for food[END_REF].

The first work reporting the mechanism of action of bacteriocin mediated inhibition reported the discovery of antagonists among Escherichia coli strains [START_REF] Gratia | Sur un remarquable exemple d'antagonisme entre deux souches de colibacille[END_REF]. Although the use of bacteriocins was formally proposed later [START_REF] Hirsch | A note on the inhibition of an anaerobic sporeformer in Swiss-type cheese by a nisin-producing streptococcus[END_REF], it is probable that humans were already benefitting from bacteriocin production for ~8,000 years since the first production of cheese and fermented food [START_REF] Cotter | Bacteriocins: developing innate immunity for food[END_REF].

Bacteriocins were first classified in 1993 [START_REF] Klaenhammer | Genetics of bacteriocins produced by lactic acid bacteria[END_REF]; since then, modifications of this classification have been proposed [START_REF] Cotter | Bacteriocins: developing innate immunity for food[END_REF]. Bacteriocins are divided into classes I, II, III and IV. Class I harbors lantibiotics or thermostable peptides with a molecular weight below 5 kDa produced by gram-positive bacteria and which present atypical amino acids, such as lanthionine (Lan), methyl lanthionine (MeLan) and others [START_REF] Karpiński | Characteristic of bacteriocines and their application[END_REF]. The class II bacteriocins are represented by non-lanthionine bacteriocins; they are thermostable and have 10 kDa molecular weight, slightly heavier than class I. Due to differences in the structure of class II bacteriocins, they are divided into subclasses: pediocin (IIa), lactacin F (IIb), enterocin (IIc) and lactococcin A (IId) [START_REF] Cotter | Bacteriocins: developing innate immunity for food[END_REF][START_REF] Karpiński | Characteristic of bacteriocines and their application[END_REF]. Using comparative genomics to characterize the potential probiotic feature of L. plantarum ZJ316, a study showed that this strain is an important producer of bacteriocins, since it is capable of producing at least two classes of bacteriocins, IIb and IIc [START_REF] Li | Comparative genomic analysis of Lactobacillus plantarum ZJ316 reveals its genetic adaptation and potential probiotic profiles[END_REF].

Bacteriocins weighting more than 30 kDa are classified in class III. They are thermolabile and are mainly produced by gram-positive bacteria [START_REF] Van Belkum | Nonlantibiotic antibacterial peptides from lactic acid bacteria[END_REF]. Class III bacteriocins are also divided into subclasses, where a group is represented by bacteriolytic enzymes (bacterial lysins), which lyses sensitive strains, and the non-lytic group of antimicrobial proteins, represented by lysostaphin and enterolysin A [START_REF] Cotter | Bacteriocins: developing innate immunity for food[END_REF][START_REF] Karpiński | Characteristic of bacteriocines and their application[END_REF]. Using comparative genomics, a potential probiotic strain of Lactococcus lactis was analyzed for bacteriocins. Based on an annotated and curated genome, strain NCDO 2118 presented one bacteriocin for each of three classes, of which two were not previously predicted in the genome sequence (classes I and III) [START_REF] Oliveira | Analyses of the probiotic property and stress resistancerelated genes of Lactococcus lactis subsp. lactis NCDO 2118 through comparative genomics and in vitro assays[END_REF].

Class IV includes bacteriocins that require carbohydrates or lipids in their molecule to have a complete activity [START_REF] Jack | Bacteriocins of Gram-Positive Bacteria[END_REF]. Compared to the use of antibiotics in infection treatments, bacteriocins are more target-specific, have low or no toxicity to eukaryotic cells, and are active against antibiotic-resistant strains. However, there is still a lack of evaluation about their effect on the gut microbiota and also their role in probiotic effects in healthy animals [START_REF] Umu | The Potential of Class II Bacteriocins to Modify Gut Microbiota to Improve Host Health[END_REF].

Besides the gut, the skin and other mucosal tissues are in direct contact with external aggressive agents and consequently are continuously exposed to huge numbers of pathogenic microorganisms. To combat these pathogens, the epithelial/mucosal surface and the microbiota induce various mechanisms that directly kill or inhibit the growth of the pathogens [START_REF] Gallo | Epithelial antimicrobial defence of the skin and intestine[END_REF][START_REF] Dickson | The role of the bacterial microbiome in lung disease[END_REF][START_REF] Bao | Questions and challenges associated with studying the microbiome of the urinary tract[END_REF]. The bacteria of the microbiota also produce bacteriocins and these molecules are also essential for host protection in health and disease.

Immune development and function

Coevolution between microbes and mammals, including humans, has brought many mutual benefits, which are affected by the diversity and niches of these microbes, especially the gut bacteria, which aid in the prevention of many human diseases. One of the various benefits of this coevolution for humans is that the microbiota assist in the development of the human immune system [START_REF] Francino | Early Development of the Gut Microbiota and Immune Health[END_REF]. One of the clearest types ofevidence of this role is that germ-free animals, are severely affected deficiencies in the development of the immune system in the gut [START_REF] Kabat | Modulation of immune development and function by intestinal microbiota[END_REF]. Animals depleted of gut microbiota have smaller Peyer's patches, fewer antimicrobial peptides, antibodies and B cells, as well as other immunodeficiencies [START_REF] Round | The gut microbiota shapes intestinal immune responses during health and disease[END_REF]. Also, immune system development induced by the gut microbiota is associated with host protection against inflammatory disorders (Belkaid and Hand, 2014) and infectious diseases [START_REF] Duan | Microbial Colonization Drives Expansion of IL-1 Receptor 1-Expressing and IL-17-Producing γ/δ T Cells[END_REF].

Besides their role in the effective development of the immune system, probiotics present potent immunomodulatory and anti-inflammatory activities (as shown in Figure 1), acting in the prevention and treatment of inflammatory and autoimmune diseases. Because of these properties, various dairy products are popular and widely consumed, especially fermented milk (de Moreno de [START_REF] De Moreno De Leblanc | Importance of IL-10 Modulation by Probiotic Microorganisms in Gastrointestinal Inflammatory Diseases[END_REF].

An important strategy of mammals to maintain the homeostasis of the intestinal environment is to minimize contact between gut lumen microorganisms and the surfaces of intestinal epithelial cells (IEC) [START_REF] Foey | Probiotic Modulation of Innate Cell Pathogen Sensing and Signaling Events[END_REF]. Various types of pattern recognition receptors are expressed by intestinal immune cells, including Toll-like receptors (TLR), NOD-like (nucleotide oligomerization domain) receptors and G protein-coupled receptors (GPCR), which recognize microbial compounds (PAMPS, pathogen-associated molecular patterns); but this recognition or activation is limited [START_REF] Hill | Intestinal Bacteria and the Regulation of Immune Cell Homeostasis[END_REF][START_REF] Foey | Probiotic Modulation of Innate Cell Pathogen Sensing and Signaling Events[END_REF]. On the other hand, concerning microbiota and immunity, these live microorganisms are able to increase or modulate the activity of gut dendritic cells, monocytes, macrophages, natural killer cells and T cells, controlling the production/activity of chemokines, cytokines and antibodies [START_REF] Klaenhammer | The impact of probiotics and prebiotics on the immune system[END_REF][START_REF] Frei | Prebiotics, probiotics, synbiotics, and the immune system[END_REF][START_REF] Fata | Probiotics and the Gut Immune System: Indirect Regulation[END_REF].

Evaluating the probiotic potential of Lactobacillus jensenii TL2937 in pigs via the extracellular proteome, six proteins with potential immunogenic properties were found, incluiding chaperonic protease ClpB, Rpf protein (possesses a G5 protein family domainpresent in various extracellular peptidases, responsible for cleaving human IgA) [START_REF] Bateman | The G5 domain: a potential N-acetylglucosamine recognition domain involved in biofilm formation[END_REF][START_REF] Gilad | The extracellular proteome of Bifidobacterium animalis subsp. lactis BB-12 reveals proteins with putative roles in probiotic effects[END_REF].

In L. acidophilus, the surface layer protein SlpA was shown to mediate the key immunomodulatory effect of this probiotic by interacting with DC-SIGN receptors at the surface of dendritic cells [START_REF] Konstantinov | S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions[END_REF]. Propionibacteria, recently identified as human commensals with a key immunomodulatory role, mitigate intestinal inflammation. Accordingly, their presence in infant gut microbiota correlates with reduced incidence of necrotizing enterocolitis [START_REF] Colliou | Commensal Propionibacterium strain UF1 mitigates intestinal inflammation via Th17 cell regulation[END_REF]. In P. freudenreichii, these immunomodulatory properties were evidenced in vitro and in vivo [START_REF] Foligné | Promising immunomodulatory effects of selected strains of dairy propionibacteria as evidenced in vitro and in vivo[END_REF]. They rely on specific surface proteins (Le [START_REF] Maréchal | Surface proteins of Propionibacterium freudenreichii are involved in its anti-inflammatory properties[END_REF], are highly straindependent [START_REF] Foligné | Probiotics from research to market: the possibilities, risks and challenges[END_REF] and require a set of Slps [START_REF] Deutsch | Identification of proteins involved in the antiinflammatory properties of Propionibacterium freudenreichii by means of a multi-strain study[END_REF].

Based on these considerations and other published data, especially in the last five years, we may state that the microbiota is involved in the immune response to virtually all diseases already studied, whether they are of infectious origin or not. That is, dysbiosis of microbiota, whether in the intestine and/or in other tissues, is closely related to prevention and/or treatment of infections, inflammatory diseases, autoimmune diseases, cancers, neurodegenerative diseases, depression, anxiety, diet, trauma, metabolic syndrome and related diseases among other disorders [START_REF] Kang | Probiotics as an Immune Modulator[END_REF][START_REF] Sander | Use of probiotics for infection prevention in trauma patients[END_REF][START_REF] Westfall | Microbiome, probiotics and neurodegenerative diseases: deciphering the gut brain axis[END_REF][START_REF] Lee | Probiotics in human health and disease: from nutribiotics to pharmabiotics[END_REF].

OMICS APPLIED TO PROBIOTIC BACTERIA

Genomics

The first completely sequenced genome of the LAB group was L. lactis subsp. lactis IL1403 strain, published in 2001. This study revealed biosynthetic pathways, phages and some of the components that participate in aerobic metabolism [START_REF] Bolotin | The Complete Genome Sequence of the Lactic Acid Bacterium[END_REF].

In 2002, a program intended for the mass sequencing of LAB genomes was announced by Lactic Acid Bacteria Genome Sequencing Consortium [START_REF] Klaenhammer | Discovering lactic acid bacteria by genomics[END_REF]. Currently, more than 100 Lactococcus genomes are available on the NCBI database (National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/genome/genomes/156), of which 39 are complete genomes.

LAB have small genomes, approximately 2Mb in length on average, coding for approximately 2000 genes, but range from 1600 to 3000 genes, according to the species. This variation results from LAB evolution through gene loss, duplication, and acquisition [START_REF] Khalid | An overview of lactic acid bacteria[END_REF]. [START_REF] Bolotin | Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus[END_REF] showed that dairy streptococci have undergone reductive evolution, resulting in divergence between them and pathogenic species of streptococci. The most remarkable example is Streptococcus thermophilus, which diverged from other species of Streptococcus through the loss of virulence factors, such as those involved in adhesion and antibiotic resistance.

Many studies highlight the importance of genomic sequencing in the discovery of new features related to the LAB, such as the identification of genes encoding proteolytic enzymes (which participate in cheese maturation) in L. helveticus [START_REF] Smeianov | Comparative High-Density Microarray Analysis of Gene Expression during Growth of Lactobacillus helveticus in Milk versus Rich Culture Medium[END_REF]. The sequencing of the first Lactobacillus species: L. plantarum WCFS1 [START_REF] Kleerebezem | Complete genome sequence of Lactobacillus plantarum WCFS1[END_REF], Lactobacillus johnsonii NC533 [START_REF] David | The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533[END_REF][START_REF] Denou | Identification of genes associated with the long-gutpersistence phenotype of the probiotic Lactobacillus johnsonii strain NCC533 using a combination of genomics and transcriptome analysis[END_REF] and L. acidophilus NCFM [START_REF] Altermann | Complete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM[END_REF], revealed some interesting characteristics, such as lifestyle adaptation islands, lack of biosynthesis pathways, and unique structures named potential autonomic units (PAU).

Bioinformatics approaches have helped identify the citrate catabolic pathway in L. casei [START_REF] Deutsch | Lactobacillus casei metabolic potential to utilize citrate as an energy source in ripening cheese: A bioinformatics approach[END_REF], and other studies have identified genes responsible for decarboxylation of a branched-chain alpha-ketoacid of L. lactis [START_REF] De | Biochemical and molecular characterization of αketoisovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acids by Lactococcus lactis[END_REF][START_REF] Smit | Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain -Keto Acid Decarboxylase Involved in Flavor Formation[END_REF]. Genomic sequencing has also played a role in the elucidation of LAB probiotic effects; for instance in the study of antimicrobial compounds and immunomodulatory mechanisms of Lactobacillus reuteri [START_REF] Saulnier | Exploring Metabolic Pathway Reconstruction and Genome-Wide Expression Profiling in Lactobacillus reuteri to Define Functional Probiotic Features[END_REF], comparative analysis of pilus-associated genes and metabolic pathways in Lactobacillus rhamnosus and Lactobacillus casei [START_REF] Douillard | Comparative Genomic and Functional Analysis of Lactobacillus casei and Lactobacillus rhamnosus Strains Marketed as Probiotics[END_REF], and identification of adhesionassociated proteins (cwaA) in L. plantarum (Zhang et al., 2015).

Assessment of the probiotic properties of a selected microorganism requires many experiments in vitro and in vivo. This takes considerable time. The omics approach allows one to speed up these studies by enabling the identification of potential probiotic microbes. Recently, [START_REF] Salvetti | Comparative genomics reveals robust phylogroups in the genus Lactobacillus as the basis for reclassification[END_REF] studied 269 species of Lactobacillaceae and Leuconostocaceae with a phylogenetic approach. Twenty-nine ribosomal proteins and housekeeping genes were compared. They conclude that the Lactobacillus genus has various subclades, which may lead to reclassification of the lactobacilli. This grouping facilitates development of accurate molecular markers to help avoid problems sucha as the the misidentification of probiotic strains. More recently, 2459 high-quality whole genomes were analyzed and, based on a polyphasic phylogenetic approach, reclassification of the Lacatobacillus genus into 25 genera and fusion of the Leuconostocaceae and Lactobacillaceae families was proposed [START_REF] Zheng | A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae[END_REF].

Besides the genomic approach (Figure 2), other omics have been providing information to help understand the divergence and evolution many species over time [START_REF] Pfeiler | The genomics of lactic acid bacteria[END_REF]. Using an omics approach, it is possible, for example, to correlate protein data with survival within the host under stress conditions or secreted proteins that may exert a specific role in probiotic effects of certain strains, through analysis of bacterial-host interactions.

Metagenomics

Through metagenomic (Figure 2) analyses, it is possible to access physiological and genetic information about uncultured organisms, such as the human GIT microbiota, through the sequencing of 16S rRNA genes and by shotgun sequencing [START_REF] Handelsman | Metagenomics: Application of Genomics to Uncultured Microorganisms[END_REF]. This type of tool gives important genetic information for uncultured organisms, helping develop novel hypotheses of microbial function. Before metagenomics, the methodology consisted of cloning DNA from environmental sources, followed by functional expression screening [START_REF] Handelsmanl | Molecular biological access to the chemistry of unknown 05SP 09 soil microbes: a new frontier for natural products E4 + qfs[END_REF][START_REF] Handelsman | Metagenomics: Application of Genomics to Uncultured Microorganisms[END_REF], which is time consuming and limits the number of strains and species that can be studied.

A pioneer study in this area consisted in the large-scale metagenomics projects in in the Sargasso sea, in which a massive microbial population was characterized through 1,045 billion base pairs from seawater samples. This large number of sequences provided important information on the diversity, gene content and the relative abundance of the organisms [START_REF] Venter | Environmental Genome Shotgun Sequencing of the Sargasso Sea[END_REF].

The development of metagenomics, mainly with the advent of next-generation sequencing technologies (NGS), and the creation of the International Human Microbiome have both boosted the field and opened a new door in analyses of bacteria/host interactions. The culture-free methodology used by NGS technologies expanded the analyses of microbial composition and may now be used not only to predict new probiotics from a comparison of the microbiota from healthy and diseased individuals, but may also be used to analyze the composition of the microbiota before and after administration of a given probiotic bacterium [START_REF] Mcfarland | Use of probiotics to correct dysbiosis of normal microbiota following disease or disruptive events: a systematic review[END_REF].

In a study of the microbiota of genetically obese mice and their lean littermates, [START_REF] Turnbaugh | An obesity-associated gut microbiome with increased capacity for energy harvest[END_REF] demonstrated through metagenomics analyses that the obesity was associated with an abundance of two groups of bacteria: Bacteriodetes and Firmicutes. [START_REF] Nobutani | The effects of administration of the Lactobacillus gasseri strain CP2305 on quality of life, clinical symptoms and changes in gene expression in patients with irritable bowel syndrome[END_REF] administrated Lactobacillus gasseri strain CP2305 or a placebo to patients with irritable bowel syndrome. They identified 87 genera, among which 13 differed in frequency; the genera Dorea, Enterococcus, and Dialister decreased in the CP2305 group.

Culturomics

Some studies have demonstrated the usefulness of a culturomics approach (Figure 2) for probiotic analyses [START_REF] Dubourg | Culturomics and pyrosequencing evidence of the reduction in gut microbiota diversity in patients with broad-spectrum antibiotics[END_REF]. A culturomics approach consists in growing under multiple culture conditions, followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and 16 rRNA analysis for the identification of less representative species in the sample [START_REF] Lagier | Culture of previously uncultured members of the human gut microbiota by culturomics[END_REF][START_REF] Lagier | Culturing the human microbiota and culturomics[END_REF]. This technique has helped identify new organisms that are generally not found through metagenomics techniques [START_REF] Pfleiderer | Culturomics identified 11 new bacterial species from a single anorexia nervosa stool sample[END_REF][START_REF] Dubourg | Culturomics and pyrosequencing evidence of the reduction in gut microbiota diversity in patients with broad-spectrum antibiotics[END_REF].

As an aid to identify a larger number of organisms, culturomics can identify populations with a concentration of less than 10 3 CFU per mL, which is below what can be detected in large-scale metagenomics studies [START_REF] Lagier | Microbial culturomics: Paradigm shift in the human gut microbiome study[END_REF]. Culturomics was reborn with studies of environmental microbiologists. For instance, Bollman et al. (2007) created a new method that was able to isolate almost numerous microorganims present in a specific aquatic environmentthat were not detected with other methods.

The first study of the microbial composition of the gut microbiota using culturomics dates from 2012. [START_REF] Lagier | Microbial culturomics: Paradigm shift in the human gut microbiome study[END_REF] grew microorganisms under 212 different culture conditions and used mass spectrometry and 16S rRNA amplification and sequencing to help identify the colonies found. the culturomics analyses of the microbiome resulted in 31 new species in addition to more than 100 species never described from the human gut before.

Culturomics and metagenomics leverage the potential of identification of new species. They complement each other, providing greater knowledge and understanding of new and/or difficult-to-grow bacteria. A database was created in order to group several prokaryotic species associated with human beings (commensals or pathogens), highlighting the importance of culturomics and metagenomics. The 2172 species listed were classified into 12 different phyla, most of them being Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes [START_REF] Hugon | A comprehensive repertoire of prokaryotic species identified in human beings[END_REF]. More recently, it was reported that the number of species had increased, totalng 2776 species, due to culturomics techniques that facilitated the identification of new bacterial species [START_REF] Bilen | The contribution of culturomics to the repertoire of isolated human bacterial and archaeal species[END_REF].

Stool samples of malnourished and healthy patients from Senegal and Niger were analyzed using culturomics and metagenomics. Besides finding a decrease in the diversity of fecal microorganisms and an enrichment of potentially pathogenic bacteria, they identified some probiotic bacteria only in healthy children. Also, new species were identified, including Propionibacteriaceae and Bacillaceae species (Tidjani [START_REF] Alou | Gut Bacteria Missing in Severe Acute Malnutrition, Can We Identify Potential Probiotics by Culturomics?[END_REF].

Functional genomics Transcriptomics

The area of transcriptomics (Figure 2) may elucidate how genes are involved in adaptation for specific conditions. [START_REF] Van Der Meulen | Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism[END_REF] identified 375 novel regulatory mRNAs in L. lactis MG1363 involved in stress response and metabolic processes, such as internal promoters, operon structures and novel ORFs. Using probiotic preparations containing L. acidophilus, L. casei and L. rhamnosus, an in vivo experiment was performed with volunteers to analyze the gene-regulatory networks and pathways in the human mucosa. A significant variation was observed among the transcriptomics results of the volunteers; however, various factors, such as the resident microbiota, diet, genetic background and lifestyle can affect a probiotic response, [START_REF] Van Baarlen | Human mucosal in vivo transcriptome responses to three lactobacilli indicate how probiotics may modulate human cellular pathways[END_REF].

Functional genomics may also contribute to refine phylogenetic studies due to the high homology among some bacteria, such as L. acidophilus. Using core and transcriptomic data it was possible to identify small ORFs that are highly conserved and transcribed in various species of this group, highlighting new possibilities to characterize and present new probiotics to the market [START_REF] Crawley | Conserved Genome Organization and Core Transcriptome of the Lactobacillus acidophilus Complex[END_REF]. Studying the transcription profile of genes associated with adhesion and stress response of the probiotic L. acidophilus NCFM, [START_REF] Weiss | Transcriptional Analysis of Genes Associated with Stress and Adhesion in Lactobacillus acidophilus NCFM during the Passage through an in vitro Gastrointestinal Tract Model[END_REF] used specific conditions to mimic the GI tract in vitro. During gastric digestion, the genes encoding GroEL, ClpP and DnaK showed considerable up-regulation. The genes encoding mucin-binding and fibronectin-binding proteins were upregulated in the incubation process (duodenal juice and bile).

A major breakthrough in this field was in vivo transcriptomics. Introduction of a pure culture of a probiotic bacterium, constrained in a dialysis bag, within the colon of a rabbit, allows monitoring upregulation of genes related to digestive constraint adaptation. First described in an in vivo proteomic study of Bifidobacterium longum in rabbits [START_REF] Yuan | Analysis of Host-Inducing Proteome Changes in Bifidobacterium longum NCC2705 Grown in Vivo[END_REF], it was then adapted to an in vivo transcriptomic study of P. freudenreichii in the colon of pigs [START_REF] Saraoui | A unique in vivo experimental approach reveals metabolic adaptation of the probiotic Propionibacterium freudenreichii to the colon environment[END_REF]. This study revealed over expression of key genes involved in specific carbohydrate catabolisms, in alternative pathways to produce NADH, NADPH, ATP and precursors (utilizing of propanediol, gluconate, lactate, purine and pyrimidine and amino-acids), as well as genes specifically expressed during cell division.

Proteomics

Proteomics (Figure 2) allow the study of the expression of a large range of proteins from a specific organism. A proteomic analysis comparing a wild strain of L. plantarum with a mutant one under physiological and heat stress conditions showed an induction of proteins related to re-folding of proteins subject to cellular damage, elucidating the importance of CtsR regulon control in lactic acid bacteria [START_REF] Russo | Comparative proteomic analysis of Lactobacillus plantarum WCFS1 and ΔctsR mutant strains under physiological and heat stress conditions[END_REF]. Another study using proteomics comparison of three L. plantarum strains confirmed the bile resistance characteristics of L. plantarum 299V, already known as a probiotic. The analyses were made using strains with different levels of bile resistance and helped to understand how these strains modulate their metabolism to survive in stress environments [START_REF] Hamon | Comparative proteomic analysis of Lactobacillus plantarum for the identification of key proteins in bile tolerance[END_REF].

In a study of a long-chain carbohydrate known to be a prebiotic, called inulin, it was observed that L. plantarum was able to use this compound and an operon (fosRABCDXE) for inulin metabolism was identified in this genome [START_REF] Buntin | An inducible operon is involved in inulin utilization in Lactobacillus plantarum strains, as revealed by comparative proteogenomics and metabolic profiling[END_REF].

Proteomic analyses of Bifidobacterium longum, isolated from stool, was performed to evaluate protein expression under the effect of bile salts. Using different degrees of exposure to bile, it was possible to identify 34 different proteins differentially regulated, amongst them: general stress response chaperones and some enzymes of pyruvate and glycolysis catabolism [START_REF] Sánchez | Proteomic Analyis of Global Changes in Protein Expression during Bile Salt Exposure of Bifidobacterium longum NCIMB 8809[END_REF]. B. longum was also used in an in vivo proteomic study. Incubation of this probiotic bacterium within the colon of rabbits led to the induction of a set of specific proteins [START_REF] Yuan | Analysis of Host-Inducing Proteome Changes in Bifidobacterium longum NCC2705 Grown in Vivo[END_REF]. This set included proteins related to the metabolism, to translation, and to intestinal adaptation, such as EF-Tu which is a Bifidobacterium adhesin-like factor, and bile salt hydrolase (BSH).

Concerning probiotic propionibacteria, proteomics was used to reveal mechanisms induced in acid [START_REF] Jan | Changes in Protein Synthesis and Morphology during Acid Adaptation of Propionibacterium freudenreichii[END_REF], bile salts [START_REF] Leverrier | Susceptibility and Adaptive Response to Bile Salts in Propionibacterium freudenreichii: Physiological and Proteomic Analysis[END_REF] and thermal adaptation [START_REF] Leverrier | Mass spectrometry proteomic analysis of stress adaptation reveals both common and distinct response pathways in Propionibacterium freudenreichii[END_REF]. Some of these key stress proteins were also induced in a Swiss-type cheese matrix [START_REF] Gagnaire | Emmental Cheese Environment Enhances Propionibacterium freudenreichii Stress Tolerance[END_REF], in accordance with the protective role of this food matrix towards digestive constraints

Metabiotics and metabolomics

Another omic strategy recently applied to probiotics analysis is metabolomics (Figure 2). Through this approach it is possible to determine and quantify the metabolites present intracellularly [START_REF] Mozzi | Metabolomics as a tool for the comprehensive understanding of fermented and functional foods with lactic acid bacteria[END_REF]. Some metabolites promote health, and are named Metabiotics. They are metabolites from the structural components, metabolites or signaling molecules of probiotic bacteria, such as lactic acid, short chain fatty acids (SCFAs), linoleic acid, some glycoproteins/peptides and potentially carcinogenic metabolites. Metabiotics have beneficial bioactive substances that act in host-specific physiological functions, regulatory, metabolic and/or behavior reactions [START_REF] Shenderov | Metabiotics: novel idea or natural development of probiotic conception[END_REF][START_REF] Sharma | Metabiotics: One Step ahead of Probiotics; an Insight into Mechanisms Involved in Anticancerous Effect in Colorectal Cancer[END_REF]. Among these, SCFAs are the most studied, being a source of energy for colonocytes and the modulators of various metabolic activities [START_REF] Shenderov | Metabiotics: novel idea or natural development of probiotic conception[END_REF].

Metabiotics-producing bacteria include not only the well-known probiotic species of Lactobacillus, Escherichia, and Enterococcus, but also other species in the dominant human intestinal phyla (Bacteroides, Firmicutes, Proteobacteria, Actinobacteria, and Archae) for nutrition and medical purposes [START_REF] Shenderov | Metabiotics: novel idea or natural development of probiotic conception[END_REF].

Probiotics produce several bioactive substances with beneficial effects in combatting GIT diseases, which help in homeostasis and competitive exclusion of pathogens [START_REF] Verma | Administration of prebiotic inulin suppresses 1,2 dimethylhydrazine dihydrochloride induced procarcinogenic biomarkers fecal enzymes and preneoplastic lesions in early colon carcinogenesis in Sprague Dawley rats[END_REF]. More interestingly, the multifunctional SCFA acetate plays an important role in epithelial cell division, ileal motility and other functions [START_REF] Hong | Acetate and Propionate Short Chain Fatty Acids Stimulate Adipogenesis via GPCR43[END_REF]. Moreover, an increase in the colic content of propionate, another SCFA, as a result of probiotic propionibacteria consumption, was correlated with enhanced apoptotic depletion of colon cancer cells in human microbiota inoculated rats [START_REF] Lan | Increased induction of apoptosis by Propionibacterium freudenreichii TL133 in colonic mucosal crypts of human microbiota-associated rats treated with 1,2dimethylhydrazine[END_REF]. At the cell level, it was shown to induce apoptosis by acting on cancer cell mitochondria, triggering the intrinsic cell death pathway, leading to caspase activation [START_REF] Jan | Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria[END_REF] and increased susceptibility to induced cell death [START_REF] Cousin | The probiotic Propionibacterium freudenreichii as a new adjuvant for TRAIL-based therapy in colorectal cancer[END_REF].

The most widely studied metabiotic is SCFA butyrate, produced by Faecalibacterium prausnitzii and Eubacterium rectale in the gut [START_REF] Zhong | Emerging roles of lactic acid bacteria in protection against colorectal cancer[END_REF], which has the potential to differentiate between cancer and normal cells to exert epigenetic effects and inhibit the growth of cancer cells. Butyrate has been associated with the induction of apoptosis in colon cancer cells due to its ability to convert procaspase 3 to active caspase 3 [START_REF] Medina | Induction of caspase-3 protease activity and apoptosis by butyrate and trichostatin a (Inhibitors of histone deacetylase): Dependence on protein synthesis and synergy with a mitochondrial/cytochrome c-dependent pathway[END_REF].

Among beneficial metabolites produced by probiotic bacteria, recent studies have drawn attention to gamma-aminobutyric acid (GABA) in the context of the gut-brain axis and of altered brain function [START_REF] Janik | Magnetic resonance spectroscopy reveals oral Lactobacillus promotion of increases in brain GABA, N-acetyl aspartate and glutamate[END_REF][START_REF] Dinan | Microbes, Immunity and Behavior: Psychoneuroimmunology Meets the Microbiome[END_REF], and to trimethylamine/trimethylamine N-oxide (TMA/TMAO) in the context of cardiovascular disease [START_REF] Bu | Cross-Talk between Gut Microbiota and Heart via the Routes of Metabolite and Immunity[END_REF]. Some technologies are extremely useful for metabolomics studies; the most current and integrated methods related to separation and detection processes are liquid chromatography (LC: high-performance, HPCL and ultra performance, UPLC) and mass spectrometry (MS). HPLC technique is able to separate multiple compounds according to stationary phase and UPLC gives results similar to HPLC; however, this technology has greater capacity, resolution, sensitivity and higher speed [START_REF] Mozzi | Metabolomics as a tool for the comprehensive understanding of fermented and functional foods with lactic acid bacteria[END_REF]. Major progress has furthermore been achieved by the use of nuclear magnetic resonance spectroscopy [START_REF] Dunn | Metabolomics: Current Analytical Platforms and Methodologies[END_REF][START_REF] Sugahara | Probiotic Bifidobacterium longum alters gut luminal metabolism through modification of the gut microbial community[END_REF][START_REF] Janik | Magnetic resonance spectroscopy reveals oral Lactobacillus promotion of increases in brain GABA, N-acetyl aspartate and glutamate[END_REF].

Integrative omics and enrichment approaches

Identification of a single specific factor associated with a probiotic effect might have limited value for selecting bacterial strains and therapeutic rationales. In this case, a multi-omics approach compared to a single-omic analysis may offer greater advantages, as they cover a broader range of information through the identification of associated factors from different biological processes, such as gene expression, protein synthesis, posttranslational modifications and cellular metabolic processes [START_REF] Perakakis | Omics, Big Data and Machine Learning as Tools to Propel Understanding of Biological Mechanisms and to Discover Novel Diagnostics and Therapeutics[END_REF]. The integration of data from genomic, transcriptomic and metabolomic analysis can be a powerful tool to investigate and rapidly validate new molecular probiotic features. In this context, promising achievements have been made by studies investigating molecular resistance features towards stressful conditions that bacterial probiotic strains face during the manufacturing process. For example, [START_REF] Bianchi | A Combined Proteomics, Metabolomics and In Vivo Analysis Approach for the Characterization of Probiotics in Large-Scale Production[END_REF] identified altered amino acid production, as well as metabolic and health-promoting changes in probiotic strains of L. paracasei, Streptococcus thermophilus, and bifidobacteria as a result of different conditions of manufacture and formulation through an integrative approach of functional proteomics, metabolomics, and in vivo analyses.

Although promising, integrative omics currently presents great challenges such as a need to development and employ bioinformatics pipelines and algorithms to associate and harmonize large amounts of data generated by the different high-throughput platforms [START_REF] Jiménez-Pranteda | Food Omics Validation: Towards Understanding Key Features for Gut Microbiota, Probiotics and Human Health[END_REF]. Recently, feature-annotation enrichment analysis has been extensively used to identify biological processes by comparing de novo data with accumulated biological data deposited in public databases. This approach offers a rapid solution to systematically classify large feature lists into pathways, cellular localization and function categories [START_REF] Gandhi | Integrating Omics to Unravel the Stress-Response Mechanisms in Probiotic Bacteria: Approaches, Challenges, and Prospects[END_REF]. A plethora of enrichment tools have emerged in the last 20 years, and are being improved to become more suitable as data-mining exploration tools [START_REF] Huang | Bioinformatics Enrichment Tools: Paths toward the Comprehensive Functional Analysis of Large Gene Lists[END_REF].

CONCLUSIONS

Probiotic bacteria have been used by humans for a long time in the maturation of cheese and the production of fermented food. However, their importance has been only recently highlighted with the study of their safety aspects, exclusion mechanisms, survival through the host GIT and production of immunomodulatory and anti-inflammatory proteins. With the advent of NGS technologies for structural and functional genomics, coupled with whole proteomics analyses using mass spectrometry, there are several new possibilities for probiotic identification using metagenomics of GIT microbiota to investigate microbial changes under disease conditions and after probiotic administration. We can highlight the importance of auxiliary techniques, such as culturomics for the identification of bacteria not detected in metagenomics, for example. In addition, both approaches may be used to elucidate problems with identification of probiotic strains.

Omics will allow seeking for probiotic features in new and promising strains of probiotics. Based on the set of data acquired from established probiotic strains, omics will allow screening strains for expression of key molecules involved in beneficial interactions with the host. These include MAMPs such as Slps and pili, key metabolites such as GABA, as well as proteins involved in adaptation to the host gut.

Genomic studies may also be used in the analyses of genome plasticity of probiotic and non-probiotic related strains, for the identification of genes related to each of the probiotic features. Finally, transcriptomics and proteomics may help in the identification of differentially expressed genes in probiotic and non-probiotic species for the later elucidation of metabolic pathways and protein-protein interactions analyses. Future improvements in the area may involve the identification of probiotic-pathogenic and bacterial-host proteinprotein interactions from a wider systems biology perspective. The omics approach brought new paths and means to analyze characteristics of future potential probiotic bacteria and broaden our understanding of the different ways theh interact with the gut microbiota of the host; this was only possible with an integrative omics approach.

Figure 1 .

 1 Figure 1. After surviving gastric juice and bile salts, probiotic organisms can act through specific mechanisms: (a) adhesion and colonization, (b) modulation of the immune system, (c) enhancement of the epithelial barrier, (d) competitive exclusion, (e) production of anti-microbial substances.

Figure 2 .

 2 Figure 2. The goals of omics approaches.

Genetics and Molecular Research 19 (2): gmr18599 ©FUNPEC-RP www.funpecrp.com.br Probiotic bacteria -omics approach

Probiotic bacteria -omics approach

ACKNOWLEDGMENTS

The authors thank the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES), the Minas Gerais Research Foundation (FAPEMIG) and the National Council for Scientific and Technological Development (CNPq) for their support.

AUTHOR CONTRIBUTIONS

Images creation: LCO, MO, SCS; Writing -original draft: LCO, HCPF, VACA, SCS; Writing -review & editing: LCO, MO, AS, LJB, CJFO, GJ, RDOC, HCPF, VACA, SCS; Supervision: CJFO, HCPF, VACA, SCS

CONFLICTS OF INTEREST

The authors declare no conflict of interest.


R Couronné

A Valladier

M Vidailhet

J C Corvol

S Lehéricy

S Durrleman

Modeling the progression of Parkinson's Disease : comparison of subjects with and without Sleep Disorders

Introduction

Patients with idiopathic Parkinson's Disease (iPD) may have very different patterns of progression, corresponding to distinct disease subtypes. Here, we describe quantitatively the overall pattern of progression in subgroups of PD by using a Bayesian non-linear mixed effect model that describes the continuous progression of biomarkers at both population and individual level. This approach allows to model variability in progression patterns and disease stage between patients. We analyzed two subgroups of patients, with (iPD-RBD+) and without sleep disorders (iPD-RBD-), that are known to present different patterns of progression [START_REF] Postuma | How does parkinsonism start? Prodromal parkinsonism motor changes in idiopathic REM sleep behaviour disorder[END_REF]. We compared the two groups by extracting the ordering of abnormalities that occurred over the disease course, and by studying their disease onset and speed of progression.

Methods Data

We used the Idiopathic PD patients of the PPMI Dataset. We considered eight biomarkers to describe the disease progression. First four clinical scores, the MDS-UPDRS part III, MoCA, SCOPA-AUT and REM Questionnaire, measuring respectively motor, cognitive, autonomy and sleep dysfunctions. Second, we included four measures from DatScan imaging, the Striatal Binging Ratios (SBR) of left and right side of both Putamen and Caudate. From the 362 idiopathic PD patients of the PPMI dataset, we excluded patients that had less than two visits of the biomarkers to obtain 343 patients. Patients that reached a REM Questionnaire score of 6 over 13 at least once over the course of the study were labeled as iPD-RBD+ (192 patients), the others as iPD-RBD (151 patients).

Modeling

We used a multivariate non-linear Bayesian mixed-effect model, introduced in [START_REF] Schiratti | A Bayesian mixed-effects model to learn trajectories of changes from repeated manifold-valued observations[END_REF] and extended in [START_REF] Couronné | Learning disease progression models with longitudinal data and missing values[END_REF], to estimate the longitudinal progression of clinical and imaging scores. We estimated both population and individual parameters of patients, to obtain the average and individual trajectories, respectively. Sub-group trajectories were obtained by averaging individual parameters over the patient sub-groups. Then, to assess the robustness of our method, accounting for both sampling bias and stochastic variations in the estimation algorithm, we performed a patient-wise repeated cross validation (10x5 folds) on our dataset. Progression of patients with and without RBD With the methodology described above, we obtained 50 sub-population average trajectories for each group (iPD-RBD-and iPD-RBD+), shown in Figure 1. To build a hierarchy of arrival of abnormalities of PD biomarkers that may progress at very different speeds and occur at different ages, we introduced abnormality thresholds. For each biomarker independently, thresholds were computed from the data as the optimal cut-off for a balanced logistic regression between PD patients and controls at baseline. We then computed the times of intersection between the averaged trajectories of the sub-groups and these abnormal thresholds to assess an abnormality timing density for each modality. Densities are shown as boxplots in Figure 1.

Results and Discussion

The ordering of abnormalities between modalities in the two groups was the same, at the exception of sleep since it discriminated our two groups (Figure 1). The first abnormality was observed in the putamen, followed by MDS-UPDRS part III then, caudate, scopa and finally MoCA. Note that MDS-UPDRS abnormality occurred early, as even few points of MDS-UPDRS label patients as PD in our logistic regression. Thus, we could also consider the age at disease onset, mainly based on motor scores, to represent a more advanced stage of motor abnormality. Noticeably, the distance to PD average age at diagnosis differed between the two subgroups. In RBD-patients, autonomic dysfunction occurred shortly after conversion to PD, whereas in RBD+, the SCOPA-AUT was already abnormal up to 3.4 years before conversion. To a lesser extent, MoCA abnormalities occurred earlier in iPD-RBD+ than in iPD-RBD-. Finally, considering only the individual parameters estimated by the model, we found that RBD+ were affected in average 3.32 years earlier than RBD-, and progressed 26% faster in average. On the bottom, cross validated (10x5 folds) average scenario of progression for the 2 subgroups, relative to age. Lowest and highest points of the filled area represent quantiles at 5% and 95% of the biomarker's progression. Note the major difference for the REM Questionnaire, in green, as this biomarker was used as a criteria to split our subgroup. Thresholds of abnormalities for each score are represented as horizontal lines. On the top, boxplot of cross validated (10x5 folds) times of intersection with threshold of abnormalities for each considered biomarker.

Conclusions

Using a longitudinal model, we were able to compare two sub-populations of a longitudinal cohort in several ways: progression speed, age at onset and ordering of abnormalities. iPD-RBD+ were affected 3.32 years earlier and progressed 26% faster. They also presented autonomic dysfunction up to 4 years before PD diagnosis whereas these symptoms occurred around PD onset in iPD-RBD-patients. These results stand as a proof of concept for longitudinal group comparison methodology, which could be applied to other types of diseases. Furthermore, this first subgroup analysis lay the foundations of a more precise subtyping of patterns of progression in PD.

Figure 1 :

 1 Figure 1 : Progression of iPD-RBD is shown on the left plots, and iPD-RBD+ on the right. In each plot, average ages of conversion to PD for each split is shown in dark red.On the bottom, cross validated (10x5 folds) average scenario of progression for the 2 subgroups, relative to age. Lowest and highest points of the filled area represent quantiles at 5% and 95% of the biomarker's progression. Note the major difference for the REM Questionnaire, in green, as this biomarker was used as a criteria to split our subgroup. Thresholds of abnormalities for each score are represented as horizontal lines. On the top, boxplot of cross validated (10x5 folds) times of intersection with threshold of abnormalities for each considered biomarker.

Acknowledgements

The research leading to these results has received funding from the program "Investissements d'avenir" ANR-10-IAIHU-06. This work was funded in part by the French government under management of Agence Nationale de la Recherche as part of the "Investissements d'avenir" program, reference ANR-19-P3IA-0001 (PRAIRIE 3IA Institute). References