Pascale Vonaesch

Mark Anderson

Philippe J Sansonetti

Pathogens, microbiome and the host: emergence of the ecological Koch's postulates

Even though tremendous progress has been made in the last decades to elucidate the mechanisms of intestinal homeostasis, dysbiosis and disease, we are only at the beginning of understanding the complexity of the gut ecosystem and the underlying interaction networks. We are also only starting to unravel the mechanisms that pathogens have evolved to overcome the barriers imposed by the microbiota and host to exploit the system to their own benefit. Recent work in these domains clearly indicates that the "traditional Koch's postulate", which state that a given pathogen leads to a distinct disease, are not valid for all "infectious" diseases, but that a more complete and complex interpretation of the Koch's postulate is needed in order to understand and explain them. This review summarizes the current understanding of what defines a healthy gut ecosystem and highlights recent progress in uncovering the interplay between the host, its microbiota and invading intestinal pathogens. Based on these recent findings, we propose a new interpretation of the Koch's postulate, that we term "ecological Koch's postulate".

One sentence summary

This review summarizes the current understanding of what defines a healthy gut ecosystem, highlights recent progress in uncovering the interplay between the host, its microbiota and invading intestinal pathogens and, based on the most recent findings in this field, proposes a new interpretation of the Koch's postulate, the "ecological Koch's postulates".

Introduction

Traditionally, pathogens have been viewed as armed warriors, fighting against the host [START_REF] Sansonetti | War and peace at mucosal surfaces[END_REF]). However, it is well established that not everyone that has ingested a typical infectious dose of a given pathogen, for example Salmonella Typhimurium, Helicobacter pylori or Campylobacter jejuni, will develop disease. There also is increasing evidence for asymptomatic carriage of enteric pathogens [START_REF] Kotloff | Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study[END_REF][START_REF] Breurec | Etiology and Epidemiology of Diarrhea in Hospitalized Children from Low Income Country: A Matched Case-Control Study in Central African Republic. Ryan ET[END_REF]Randremanana et al. 2016). In addition, it is also known that antibiotic use leads to an increased susceptibility to infection by enteropathogens (BOHNHOFF, DRAKE and MILLER 1954;MILLER, BOHNHOFF and DRAKE 1954;[START_REF] Barthel | Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host[END_REF][START_REF] Sekirov | Antibiotic-induced perturbations of the intestinal microbiota alter host susceptibility to enteric infection[END_REF]; Van der Waaij, Berghuis-de Vries and Lekkerkerk-van der Wees 2009).

These observations led to an extended concept that acknowledged the role of the microbiota in protecting the host against pathogens, referred to first as "microbial barrier" and later as "colonization resistance" (Ducluzeau and Railbaud 1989).. Bacteria can have an inhibitory effect on phylogenetically unrelated species/groups of bacteria ("interspecies barrier effect"). This was demonstrated as early as the late 1970s through work showing that precolonization of axenic mice with Escherichia coli could inhibit the colonization of Shigella flexneri [START_REF] Ducluzeau | Antagonistic effect of extremely oxygen-sensitive clostridia from the microflora of conventional mice and of Escherichia coli against Shigella flexneri in the digestive tract of gnotobiotic mice[END_REF]. Further, bacteria from the same species/group can have an inhibitory effect on the installation of their peers ("intra-species barrier effect"). This concept was shown in the 1980s in studies using closely related Clostridium species [START_REF] Corthier | Emergence in gnotobiotic mice of nontoxinogenic clones of Clostridium difficile from a toxinogenic one[END_REF]. Several studies have also assessed the role of the prokaryotic microbiota on 2/32 the susceptibility to viral infections. Indeed, susceptibility to rota-and norovirus seems to depend, at least in part, on the composition of a host's prokaryotic microbiota [START_REF] Rodríguez-Díaz | Relevance of secretor status genotype and microbiota composition in susceptibility to rotavirus and norovirus infections in humans[END_REF], as do sexually transmitted diseases, such as for example Human Immunodeficiency Virus (HIV, [START_REF] Nunn | Enhanced Trapping of HIV-1 by Human Cervicovaginal Mucus Is Associated with Lactobacillus crispatus-Dominant Microbiota[END_REF]), Cervical Human Papillomavirus (HPV, (Shannon et al. 2017b)) or Herpes Simplex Virus (Shannon et al. 2017a), in the context of a changed vaginal microbiota. For many years, the exact mechanisms underlying the phenomenon of colonization resistance remained unclear. However, work over the last decades has demonstrated that the microbiota forms a complex ecosystem and interacts with the host and invading pathogens in a dynamic manner.

The intestinal microbiota is composed of trillions of organisms belonging to hundreds of different species [START_REF] Eckburg | Diversity of the human intestinal microbial flora[END_REF][START_REF] Yatsunenko | Human gut microbiome viewed across age and geography[END_REF], reviewed in [START_REF] Cho | The human microbiome: at the interface of health and disease[END_REF]. While the majority belongs to the prokaryota (bacteria and archae), it also comprises viruses (including phages) and different eukaryotes, especially yeasts and protists [START_REF] Parfrey | Microbial eukaryotes in the human microbiome: ecology, evolution, and future directions[END_REF][START_REF] Ursell | Defining the human microbiome[END_REF][START_REF] Clemente | The impact of the gut microbiota on human health: an integrative view[END_REF][START_REF] Parfrey | Communities of microbial eukaryotes in the mammalian gut within the context of environmental eukaryotic diversity[END_REF][START_REF] Hamad | Repertory of eukaryotes (eukaryome) in the human gastrointestinal tract: taxonomy and detection methods[END_REF]. Members of the gram-negative Bacteroidetes and the gram-positive Firmicutes dominate the bacterial community residing in the gut. Less abundant groups include members of the Proteobacteria, the Verrucomicrobia, the Tenericutes, the Defferibacteres and the Fusobacteria [START_REF] Eckburg | Diversity of the human intestinal microbial flora[END_REF][START_REF] Yatsunenko | Human gut microbiome viewed across age and geography[END_REF]. In areas of the world without developed water supplies, the intestinal microbiota can also include multicellular organisms, for example helminths [START_REF] Giacomin | Helminths and Intestinal Flora Team Up to Improve Gut Health[END_REF].

Several different ecosystems formed by microbial communities in and on the body (e.g. the skin microbiota, the gastric microbiota, the vaginal microbiota), engage in a constant crosstalk with the human host [START_REF] Costello | Bacterial community variation in human body habitats across space and time[END_REF]. The microbes belonging to each of these communities are often specific and adapted to living in their particular environment (e.g. anaerobic bacteria in the gut). This review will focus on the interactions found in the intestine, relying mainly on data gathered in laboratory mice. Even though similar mechanisms are at work in all ecosystems found in and on the human host, a complete description would go beyond the scope of this review. In this review, we will summarize the current understanding of what defines a healthy gut ecosystem and highlight recent progress in uncovering the interplay between the host, its microbiota and invading intestinal pathogens.

The gut ecosystem-species abundance, chance, and the environment

It has been hypothesized that at least some of the gut microbes have co-evolved and/or co-speciated with mammals [START_REF] Groussin | Unraveling the processes shaping mammalian gut microbiomes over evolutionary time[END_REF]. However, co-occurrence of microbes and their host, even if they affect each others fitness, does not necessarily mean a shared evolutionary history but can also be forged by numerous other mechanisms, including unidirectional selection [START_REF] Moran | The Hologenome Concept: Helpful or Hollow?[END_REF]. Regardless of their evolutionary origin, most mammals have a distinct assembly of microorganisms organized into a complex social network, which is remarkably robust and resilient to aggressions from, for example, allogenic/pathogenic intruders.

Two major competing ecological theories have been proposed to explain how microbial communities are organized and maintained, i.e. how communities are assembled. The first, termed niche theory [START_REF] Chase | Ecological Niches: Linking Classical and Contemporary Approaches[END_REF], is based on deterministic processes and assumes that each species occupies a given realized niche (i.e. a particular position in the abiotic and biotic space) due to its species-specific properties that define its fundamental niche. The grounding of this theory for microbes lies in the work of Martinus Willem Beijerinck (1851-1931) and in the famous statement: "Everything is everywhere, but the environment selects" (O'Malley 2007).

On the other hand, the "neutral theory" is based only on stochastic processes and was proposed at the beginning of the century by Hubbell (Hubbell [2001. The Unified Neutral Theory of Biodiversity and Biogeography, Princeton University Press, Princeton, NJ]). The theory suggests that local communities are assembled independently of 3/32 species fitness differences, hence assuming that all individual have the same fitness. Further, the neutral theory claims that the fitness differences between species are not larger than the fitness differences within a given species. Competition is therefore not the driving factor of the observed community structure ("all species are equivalent; they have the same chance of immigration, extinction and speciation"). Of course, these two theories are not mutually exclusive and can act in parallel to drive microbial community assembly at (i) different spatial scales (e.g. at different locations in the intestine) or (ii) in different environments e.g. in different location on or in our body.

Testing the fit of both theories on intestinal microbial communities has been hampered by the inability to culture many of the present species and hence define the composition of the microbial communities. With the rise of advanced sequencing methodologies and the decreasing associated costs, large datasets have been put together that now allow testing of these theories on highly complex communities, for example the intestinal microbiota. In studies conducted in the past few years, evidence is emerging that the intestine is not only an ecosystem based on Hubbell's "neutral theory" where each member has the same fitness [START_REF] Costello | The Application of Ecological Theory Toward an Understanding of the Human Microbiome[END_REF]. Indeed the Human Microbiome Project, which screened several hundred stool samples from individuals residing in the United States, revealed that the microbiota of only one individual showed a composition structure consistent primarily with the "neutral theory" [START_REF] Li | Testing the Neutral Theory of Biodiversity with Human Microbiome Datasets[END_REF]. Instead, modelling confirmed that the microbiota is governed mainly by deterministic processes, including environmental factors [START_REF] Li | Testing the Neutral Theory of Biodiversity with Human Microbiome Datasets[END_REF]. This, however, does not exclude that both processes are shaping the communities at the same time and clearly more work is needed to elucidate these questions.

The microbiome is shaped by different deterministic forces, including maternal transmission at birth [START_REF] Dogra | Dynamics of infant gut microbiota are influenced by delivery mode and gestational duration and are associated with subsequent adiposity[END_REF][START_REF] Frese | Birth of the infant gut microbiome: moms deliver twice![END_REF][START_REF] Rutayisire | The mode of delivery affects the diversity and colonization pattern of the gut microbiota during the first year of infants' life: a systematic review[END_REF][START_REF] Stokholm | Cesarean section changes neonatal gut colonization[END_REF][START_REF] Edwards | Determinants and Duration of Impact of Early Gut Bacterial Colonization[END_REF], nutrition [START_REF] Turnbaugh | An obesity-associated gut microbiome with increased capacity for energy harvest[END_REF][START_REF] De Filippo | Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa[END_REF][START_REF] Muegge | Diet drives convergence in gut microbiome functions across mammalian phylogeny and within humans[END_REF][START_REF] David | Diet rapidly and reproducibly alters the human gut microbiome[END_REF][START_REF] Kashtanova | Association between the gut microbiota and diet: Fetal life, early childhood, and further life[END_REF][START_REF] Donovan | Human Milk Oligosaccharides Influence Neonatal Mucosal and Systemic Immunity[END_REF][START_REF] Edwards | Determinants and Duration of Impact of Early Gut Bacterial Colonization[END_REF][START_REF] Donovan | Introduction to the special focus issue on the impact of diet on gut microbiota composition and function and future opportunities for nutritional modulation of the gut microbiome to improve human health[END_REF][START_REF] Araújo | Impact of high-fat diet on the intestinal microbiota and small intestinal physiology before and after the onset of obesity[END_REF], host genetics [START_REF] Leamy | Host genetics and diet, but not immunoglobulin A expression, converge to shape compositional features of the gut microbiome in an advanced intercross population of mice[END_REF][START_REF] Camarinha-Silva | Host Genome Influence on Gut Microbial Composition and Microbial Prediction of Complex Traits in Pigs[END_REF], the use of different food additives or drugs [START_REF] Dethlefsen | The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing[END_REF][START_REF] Modi | Antibiotics and the gut microbiota[END_REF][START_REF] Chassaing | Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome[END_REF]2017;[START_REF] Namasivayam | Longitudinal profiling reveals a persistent intestinal dysbiosis triggered by conventional anti-tuberculosis therapy[END_REF][START_REF] Pourabedin | Mannan-and xylooligosaccharides modulate caecal microbiota and expression of inflammatory-related cytokines and reduce caecal Salmonella Enteritidis colonisation in young chickens. Nakatsu C[END_REF][START_REF] Uebanso | Effects of Low-Dose Non-Caloric Sweetener Consumption on Gut Microbiota in Mice[END_REF]) and infection [START_REF] Hoffmann | Community-wide response of the gut microbiota to enteropathogenic Citrobacter rodentium infection revealed by deep sequencing[END_REF][START_REF] Hill | Metagenomic analyses reveal antibiotic-induced temporal and spatial changes in intestinal microbiota with associated alterations in immune cell homeostasis[END_REF][START_REF] Braun | Fecal microbial characterization of hospitalized patients with suspected infectious diarrhea shows significant dysbiosis[END_REF]). The immune system's dynamic IgA host response to the microbiota [START_REF] Suzuki | Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut[END_REF][START_REF] Peterson | IgA response to symbiotic bacteria as a mediator of gut homeostasis[END_REF][START_REF] Slack | Functional flexibility of intestinal IgA -broadening the fine line[END_REF]Macpherson et al. 2012;[START_REF] Pabst | Secretory IgA in the Coordination of Establishment and Maintenance of the Microbiota[END_REF][START_REF] Moor | High-avidity IgA protects the intestine by enchaining growing bacteria[END_REF] and its action to hamper bacteria from interacting with the gut tissue ("immune exclusion") and to grow ("enchained growth", [START_REF] Moor | High-avidity IgA protects the intestine by enchaining growing bacteria[END_REF])), as well as other innate and adaptive immune mechanisms, also have an important role in controlling the microbiota and shaping the community structure [START_REF] Slack | Innate and adaptive immunity cooperate flexibly to maintain hostmicrobiota mutualism[END_REF][START_REF] Ivanov | Induction of intestinal Th17 cells by segmented filamentous bacteria[END_REF][START_REF] Macpherson | Innate and adaptive immunity in host-microbiota mutualism[END_REF][START_REF] Schnupf | Host interactions with Segmented Filamentous Bacteria: an unusual trade-off that drives the post-natal maturation of the gut immune system[END_REF][START_REF] Spasova | Blowing on embers: commensal microbiota and our immune system[END_REF][START_REF] Rescigno | Intestinal microbiota and its effects on the immune system[END_REF][START_REF] Kato | The role of the adaptive immune system in regulation of gut microbiota[END_REF][START_REF] Bang | The intestinal archaea Methanosphaera stadtmanae and Methanobrevibacter smithii activate human dendritic cells[END_REF][START_REF] Goto | Segmented filamentous bacteria antigens presented by intestinal dendritic cells drive mucosal Th17 cell differentiation[END_REF][START_REF] Dowds | Control of intestinal homeostasis through crosstalk between natural killer T cells and the intestinal microbiota[END_REF][START_REF] Atarashi | Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells[END_REF][START_REF] Furusawa | Commensal microbiota regulates T cell fate decision in the gut[END_REF][START_REF] Ivanov | Microbe Hunting Hits Home[END_REF].

The different environments that individuals and their gut ecosystem experience therefore select for a particular set of strains within the microbiota. However, a core bacterial genetic pool can be defined that is common to all individuals [START_REF] Ley | Microbial ecology: human gut microbes associated with obesity[END_REF][START_REF] Qin | A human gut microbial gene catalogue established by metagenomic sequencing[END_REF]. Scarce resources, such as nutrients and access to a given niche are limiting factors for growth in this confined ecosystem (reviewed in [START_REF] Stecher | Mechanisms controlling pathogen colonization of the gut[END_REF]). These thoughts were already, in part, formulated earlier in the "nutrient niche theory", a refined niche theory established by Rolf Freter in 1983(Freter et al. 1983a;1983b;1983c). The "nutrient niche theory" or "niche co-existence theory" stresses that ecological niches are defined by the available nutrients. Further, Freter asserted that a given species can only establish itself if it is using at least one of the limiting nutrients in the most efficient way. He also hypothesized that only a few nutrients are responsible for shaping the whole community as they limit the growth potential of the whole 4/32 ecological niche (Freter et al. 1983a;1983b;1983c). Although Freter's classic "nutrient niche theory" is useful to understand some of the mechanisms occurring in the gut ecosystem, it is not reflective of the entire complexity observed. Indeed, several cases of mixed-substrate utilization and metabolic flexibility have been described. For example, E. coli and Salmonella spp., can thrive on tetrathionate, nitrate, succinate, 1,2-propanediol, or ethanolamines among others, and therefore rapidly adapt to a changing environment in the intestine during inflammation [START_REF] Thiennimitr | Intestinal inflammation allows Salmonella to use ethanolamine to compete with the microbiota[END_REF]Winter et al. 2013;Rivera-Chávez et al. 2016a;[START_REF] Faber | Respiration of Microbiota-Derived 1,2-propanediol Drives Salmonella Expansion during Colitis[END_REF][START_REF] Spiga | An Oxidative Central Metabolism Enables Salmonella to Utilize Microbiota-Derived Succinate[END_REF]. There are also a few examples of nutritional cooperation between gut microbes described (Rakoff-Nahoum, Foster and Comstock 2016).

To date, the complexity of the gut ecosystem and the underlying interaction networks are only beginning to be understood. We are also only starting to unravel the mechanisms that pathogens have evolved to overcome the barriers imposed by the microbiota and host to exploit the system to their own benefit.

Hallmarks of homeostasis

Intestinal pathogens are mainly ingested through consumption of contaminated food or water. During their travel to their preferred "niche", they encounter several obstacles and barriers imposed by the members of the ecosystem, which impede colonization and invasion by intruders. Most pathogens must replicate in the gut lumen in order to elicit disease [START_REF] Ackermann | Self-destructive cooperation mediated by phenotypic noise[END_REF]. Below, we discuss the primary mechanisms in a healthy host that hamper pathogens from overcoming these obstacles and cause disease (see Figure 1).

Stomach acidity and bile acids

After ingestion and resisting salivary enzymes, the first major barrier to infection is the low pH environment of the stomach. Indeed, pharmacological perturbation of stomach acidity through the use of proton-pump inhibitors leads to an increased pH and greater susceptibility to enteric infections (reviewed in [START_REF] Eusebi | Proton pump inhibitors: Risks of long-term use[END_REF]). After surviving the stomach, the pathogen then enters the duodenum, where it is exposed to the massive influx of bile acids, that are produced by the liver and released by the gall bladder. The primary bile acids are involved in lipid absorption from food. They also show toxicity towards given groups of bacteria. For example, in rats treated with cholic acid, phylum-level alterations in the composition of the gut microbiota were observed with an increase in Firmicutes and a concomitant decrease in Bacteroidetes. Cholic acid feeding also led to a less complex composition of the microbiota with overrepresentation of members of the classes Clostridia and Erysipelotrichi [START_REF] Islam | Bile acid is a host factor that regulates the composition of the cecal microbiota in rats[END_REF].

Therefore, bile acids shape the resident bacterial community by promoting the growth of bile acid-metabolizing bacteria and by inhibiting in turn, the growth of bile-sensitive bacteria. Several studies in patients suffering from biliary obstruction, who display blocked bile flow into the small intestine, have shown an association with bacterial overgrowth and translocation of bacteria in the small intestine [START_REF] Clements | Role of the gut in the pathophysiology of extrahepatic biliary obstruction[END_REF]. It was also shown that this phenotype can be reversed by the administration of bile acids [START_REF] Lorenzo-Zúñiga | Oral bile acids reduce bacterial overgrowth, bacterial translocation, and endotoxemia in cirrhotic rats[END_REF]. Bile acids thus play an important role in regulating the microbiota in the small intestine.

Over the length of the intestinal tract, these primary bile acids are metabolized by the microbiota to over 50 different secondary bile acids. One of the possible transformations is deconjugation of bile acids through extracellular bile salt hydrolases (BSHs). BSHs are encoded by different members of the microbiota, especially in members of the Firmicutes, Bacteroidetes and some Actinobacteria. The deconjugated secondary bile acids show less toxicity towards the microbiota than the primary bile acids. Bile acids can also be oxidized and epimerized (transformation in between two stereoisomers) at specific hydroxyl-groups through transformation by 3-α, 7-α, or 12-α hydroxysteroid dehydrogenases. The secondary bile acids have an important role in gut homeostasis 5/32 by inhibiting inflammation (reviewed in [START_REF] Ridlon | Bile acids and the gut microbiome[END_REF][START_REF] Winston | Impact of microbial derived secondary bile acids on colonization resistance against Clostridium difficile in the gastrointestinal tract[END_REF][START_REF] Wahlström | Intestinal Crosstalk between Bile Acids and Microbiota and Its Impact on Host Metabolism[END_REF]. A recent study has shown that disuccinimidyl suberate (DSS)-mediated colitis was ameliorated in the presence of ursodeoxycholic acid or its taurine-or glycine-conjugated derivatives. Even though daily administration of these bile acids did not restore the full diversity of the microbiota to pre-DSS levels, specific species, such as Akkermansia muciniphila and the Clostridium cluster XIVa, were less depleted upon DSS treatment and the ratio of Firmicutes to Bacteroidetes remained normal [START_REF] Van Den Bossche | Ursodeoxycholic Acid and Its Taurine-or Glycine-Conjugated Species Reduce Colitogenic Dysbiosis and Equally Suppress Experimental Colitis in Mice. Elkins CA[END_REF]. Secondary bile acids have also been implicated in colony resistance, for example resistance against infection by Clostridium difficile [START_REF] Winston | Impact of microbial derived secondary bile acids on colonization resistance against Clostridium difficile in the gastrointestinal tract[END_REF][START_REF] Van Den Bossche | Ursodeoxycholic Acid and Its Taurine-or Glycine-Conjugated Species Reduce Colitogenic Dysbiosis and Equally Suppress Experimental Colitis in Mice. Elkins CA[END_REF]. Reducing the pool of secondary bile acids through antibiotic treatment relieves colonization resistance towards C. difficile and enhances spore germination. A later study demonstrated that the colonization resistance was mediated by a close relative, Clostridium scindens, through the production of the 7-αdehydroxylated bile acids lithocholic acid and deoxycholic acid [START_REF] Studer | Functional Intestinal Bile Acid 7α-Dehydroxylation by Clostridium scindens Associated with Protection from Clostridium difficile Infection in a Gnotobiotic Mouse Model[END_REF].

183 Recently, butyrate production by Clostridia species has been implicated in colonization resistance against S.

Short-chain fatty acids and other mechanisms of

Typhimurium (Rivera-Chávez et al. 2016b). The authors show that either through antibiotic treatment, or to a lesser extent, through Salmonella infection and the action of the Salmonella Type Three secretion system (T3SS) and associated virulence factors, a depletion of butyrate-producing Clostridia species occurs in the intestine of affected mice. This then leads to an increased epithelial oxygenation and subsequently aerobic expansion of S.

Typhimurium. The colonization resistance against Salmonella, alongside an anaerobic epithelial environment, could be restored through gavage of the mice with tributyrin, a butyrate metabolic precursor, clearly demonstrating the inhibitory role of butyrate on Salmonella invasion.

Colonization resistance and maintenance of the ecosystem through competition and cooperation

In addition to direct inhibition, invading bacteria must also contend with limited available nutrient resources. Nutrient availability in the gut varies with food intake and time of day. Therefore, the microbiota faces a constantly changing environment. In a healthy, fully established intestine, all nutrient niches are occupied and incoming species must utilize methods to displace resident species from their established niches in order to create their own niche. A recent study analysing the gene expression of co-occurring human gut microbes showed that for 41% of all cooccurring species the presence of one of the organisms was associated with an altered transcriptional profile in the other, the most affected genes being involved in nutrient uptake and anaerobic respiration [START_REF] Plichta | Transcriptional interactions suggest niche segregation among microorganisms in the human gut[END_REF].

Box 1-Colonization resistance

Colonization resistance describes a phenomenon, observed in the human gut as well as in many other ecosystems, that invading pathogens (and other organisms) face resistance against establishing themselves in this densely populated space. This "colonization resistance" is due to several factors, namely competition for nutrients or direct inhibition by chemical compounds or "molecular weapons", for example Type 6 secretion systems and translocated toxins or other toxins. Colonization resistance can be breached by interfering with the equilibrium of the ecosystem, for example by the administration of antibiotics or by infection.

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This suggests that nutrient niche partitioning is prevalent within the gut ecosystem.

Furthermore, early in development or after a destabilization of the equilibrium, e.g. by an infection or antibiotic treatment, the microbiota has to re-establish itself. In this context, a so called "priority effect" is observed, whereby the first re-colonizing species establish a large colony which hampers the subsequent re-establishment of otherwise fully adapted species to the other's niche [START_REF] Fukami | Community assembly: alternative stable states or alternative transient states?[END_REF] (reviewed in [START_REF] Pereira | Microbial nutrient niches in the gut[END_REF]). This leads to fierce competition for nutrients within the microbiota and by invading pathogens. It also suggests that most of the interactions in the gut are based on competition. To date, cooperation is a type of interaction only very rarely described and does not seem to be as successful as competition in a crowded and competitive environment such as the intestine [START_REF] Foster | Competition, not cooperation, dominates interactions among culturable microbial species[END_REF]. In one study of cooperation within the gut microbiota, Bacteroides ovatus was shown to produce two outer surface glycoside hydrolases, which digest complex carbohydrates, for example inulin. Despite the rare documented cases of cooperation, most microbial interactions in the intestine are indeed competitive.

The metabolic landscape is shaped by a few so-called "keystone species" or "keystone taxa", which have a large impact on the rest of the community by degrading initial substrates and making these accessible to many other species. Keystone species may only be detectable in very low relative abundance, however their outsized effect on the global microbial composition makes them a "keystone" of the microbiota (reviewed in [START_REF] Pereira | Microbial nutrient niches in the gut[END_REF].

One example of "keystone species" is Akkermansia muciniphila, which degrades secreted host mucus into products that are then accessible to other bacteria, such as B. vulgatus [START_REF] Png | Mucolytic bacteria with increased prevalence in IBD mucosa augment in vitro utilization of mucin by other bacteria[END_REF]. Hydrogen and sulfate/sulfite consuming species represent an example of a "keystone taxa" due to their regulatory effect on the fermentative activity of other species [START_REF] Carbonero | Microbial pathways in colonic sulfur metabolism and links with health and disease[END_REF][START_REF] Rey | Metabolic niche of a prominent sulfate-reducing human gut bacterium[END_REF]). Yet, some of these taxa, for example

256

Box 2-The concept of the nutritional/dietary niche A nutritional niche describes the nutrient sources which are available to, and usable by, a given set of organisms at a given time and space. The nutritional niche therefore defines if an organism is capable of establishing itself in a given place or not. The concept of the nutritional niche is a sub-definition of the ecological niche concept. The concept has been first proposed by Rolf Freter (Freter's nutritional niche theory) and has subsequently been adapted to take into account the instable flux of nutrients in given ecosystems, for example the intestine, and to take into account the co-existence of other microorganisms utilizing the same nutrients either at different geographic locations in the intestine or at different time points. The theory also has been expanded to take into account the metabolic flexibility and mixed-substrate utilization that most microorganisms exhibit.

The process by which a given organism changes its ecological niche is known as "niche construction". (See [START_REF] Pereira | Microbial nutrient niches in the gut[END_REF] for an extended review of the topic.)

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attaching to its o-glycosylated side-chains [START_REF] Johansson | The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host-microbial interactions[END_REF]. Mucus production is dynamic and depends on the presence of bacterial stimuli, especially LPS (lipopolysaccharide) and PGN (peptidoglycan) as reported in an elegant study using germ-free mice [START_REF] Petersson | Importance and regulation of the colonic mucus barrier in a mouse 29/32 model of colitis[END_REF]). It has also been known for a long time that SCFAs are involved in mucus secretion from goblet cells into the gut lumen [START_REF] Shimotoyodome | Short chain fatty acids but not lactate or succinate stimulate mucus release in the rat colon[END_REF][START_REF] Willemsen | Short chain fatty acids stimulate epithelial mucin 2 expression through differential effects on prostaglandin E(1) and E(2) production by intestinal myofibroblasts[END_REF]. Homeostasis of mucus production is regulated by two complementary bacteria, Bacteroides thetaiotaomicron (stimulating mucus production through increased goblet cell differentiation) and F. prausnitzii (inhibiting goblet cell proliferation and mucus glycosylation) [START_REF] Wrzosek | Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii influence the production of mucus glycans and the development of goblet cells in the colonic epithelium of a gnotobiotic model rodent[END_REF]. A preponderance of evidence shows that mucus composition and structure directly depends on the interplay between resident microbiota and epithelial tissues [START_REF] Jakobsson | The composition of the gut microbiota shapes the colon mucus barrier[END_REF]. Several studies have also linked reduced or aberrant O-glycosylation of mucin to the development of intestinal inflammation [START_REF] Fu | Loss of intestinal core 1-derived O-glycans causes spontaneous colitis in mice[END_REF][START_REF] Larsson | Altered O-glycosylation profile of MUC2 mucin occurs in active ulcerative colitis and is associated with increased inflammation[END_REF][START_REF] Sommer | Altered mucus glycosylation in core 1 O-glycan-deficient mice affects microbiota composition and intestinal architecture[END_REF].

Other studies have shown penetration of commensal bacteria into the inner mucus layer in the context of colitis [START_REF] Johansson | Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis[END_REF]. The mucus layer thickness is also related to nutrition, especially dietary intake, as it has been shown recently that low-fibre diets increase mucus-eroding bacteria communities, leading to greater access for pathogens at the epithelial surface and subsequently increased susceptibility to infection [START_REF] Desai | A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier and Enhances Pathogen Susceptibility[END_REF].

Furthermore, the attachment of the mucus layer to the epithelium is dependent on the microbiota. Meprin β, a hostderived zinc-dependent metalloprotease induced by the microbiota, is needed to detach the mucus in the small intestine and to subsequently release it into the intestinal lumen [START_REF] Schütte | Microbial-induced meprin β cleavage in MUC2 mucin and a functional CFTR channel are required to release anchored small intestinal mucus[END_REF].

A healthy mucus layer is therefore essential to protect the underlying epithelium from the dense bacterial population and to physically separate this population from immune cells in the underlying tissue in order to prevent exaggerated immune activation.

Mucosal immune system

Prokaryotic microbiota and the immune system

It is now well established that the immune system relies on the microbiota for proper maturation (reviewed in (Schnupf, Gaboriau- Mucosal IgA plays a crucial role in gut homeostasis [START_REF] Peterson | IgA response to symbiotic bacteria as a mediator of gut homeostasis[END_REF][START_REF] Macpherson | The functional interactions of commensal bacteria with intestinal secretory IgA[END_REF][START_REF] Pabst | Secretory IgA in the Coordination of Establishment and Maintenance of the Microbiota[END_REF]. It is known to protect the gut mucosa from access of bacteria ("immune exclusion") and to inhibit bacterial replication in the gut lumen ("enchained growth" [START_REF] Moor | High-avidity IgA protects the intestine by enchaining growing bacteria[END_REF]). Through these mechanisms, IgA regulates the composition and dynamics of the gut microbiota. IgA and the microbiota therefore regulate each other, leading to a delicate homeostatic balance between stimulation and control (reviewed in [START_REF] Peterson | IgA response to symbiotic bacteria as a mediator of gut homeostasis[END_REF][START_REF] Macpherson | The functional interactions of commensal bacteria with intestinal secretory IgA[END_REF][START_REF] Slack | Functional flexibility of intestinal IgA -broadening the fine line[END_REF]Macpherson et al. 2012;[START_REF] Pabst | Secretory IgA in the Coordination of Establishment and Maintenance of the Microbiota[END_REF]).

The microbiota's effect on the mucosal immune system is not limited to regulatory T cells and IgA secretion. It also plays a major role in inducing production of antimicrobial peptides (AMPs), which concentrate at the interphase between the thick, tightly formed, mucus layer and the more dispersed outer layer (reviewed in (Lehrer,

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Lichtenstein and Ganz 1993; [START_REF] Salzman | Enteric defensins are essential regulators of intestinal microbial ecology[END_REF])) [START_REF] Meyer-Hoffert | Secreted enteric antimicrobial activity localises to the mucus surface layer[END_REF][START_REF] Vaishnava | The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine[END_REF]. Antimicrobial peptides include the defensins, the Reg-protein family and several other proteins, which act directly on the bacteria by targeting the bacterial cell wall. In addition, other host factors with antimicrobial activity include lipocalin2/NGAL, which chelates bacterial siderophores involved in iron acquisition, as well as calprotectin, which leads to the chelation of two other essential trace elements, zinc and manganese. .

Eukaryotic microbiota and the immune system

The intestinal microbiota is not only composed of bacteria, but also harbours a whole array of archae, viruses and eukaryotes. It has been shown that the presence of helminths, through their effect on the host immune system [START_REF] Walsh | Infection with a helminth parasite attenuates autoimmunity through TGFbeta-mediated suppression of Th17 and Th1 responses[END_REF][START_REF] Finlay | Induction of regulatory cells by helminth parasites: exploitation for the treatment of inflammatory diseases[END_REF][START_REF] Finlay | Helminth Products Protect against Autoimmunity via Innate Type 2 Cytokines IL-5 and IL-33, Which Promote Eosinophilia[END_REF][START_REF] Ramanan | Helminth infection promotes colonization resistance via type 2 immunity[END_REF] [START_REF] Ramanan | Helminth infection promotes colonization resistance via type 2 immunity[END_REF]. For some of the observed immune changes, the helminth-induced changes seem to be mediated through the prokaryotic microbiota [START_REF] Zaiss | The Intestinal Microbiota Contributes to the Ability of Helminths to Modulate Allergic Inflammation[END_REF]. For other effects, they seem to be independent from the microbiota [START_REF] Osborne | Virus-helminth coinfection reveals a microbiota-independent mechanism of immunomodulation[END_REF], highlighting the complex interplay found within the gut ecosystem. Very recently, a member of the eukaryome, Tritrichomonas musculis, was shown to activate the epithelial inflammasome to induce protection against bacterial mucosal infection (Chudnovskiy et al.

2016).

Other protists, such as Giardia [START_REF] Barash | Giardia Alters Commensal Microbial Diversity throughout the Murine Gut. Appleton JA[END_REF]) and possibly Blastocystis [START_REF] Audebert | Colonization with the enteric protozoa Blastocystis is associated with increased diversity of human gut bacterial microbiota[END_REF][START_REF] Siegwald | Targeted metagenomic sequencing data of human gut microbiota associated with Blastocystis colonization[END_REF]) have also been shown to change the resident prokaryotic microbiota. However, data remains conflicting, pointing towards the fact that shifts in the microbiota through given eukaryotes might be strain-specific.

Due to incomplete databases, it is currently difficult to determine the exact strain of a eukaryotic organism by comparison against the database. More work in curating these databases is therefore needed to better appreciate the influence that specific eukaryotes might have on the prokaryotic microbiota. More work is also needed to unravel the triad of eukaryotes, prokaryotes, and the immune system to better understand the mutual interactions that take place.

Taken together, the microbiota and the development and proper function of the mucosal immune system are exquisitely intertwined. Thus, perturbations on either side of this ecosystem can deregulate the balance and leave the host open to infection or inflammatory diseases [START_REF] Clarke | Microbial programming of systemic innate immunity and resistance to infection[END_REF].

Friend or foe: pathogens facing the microbiota and the host

As discussed, the host and its microbiota have set-up a tightly regulated network of mutual control. Invading pathogens therefore have several barriers to overcome in order to establish themselves and cause disease (see Figure 2).

Within the last decade, much progress has been made in understanding the "ménage à trois" of the microbiota, the host, and pathogens. In this section we will discuss the mechanisms that have evolved in pathogens to overcome 9/32

the protective environment arising from homeostasis and summarize the complex interactions that take place between pathogens, the microbrobiota and their host.

Combatting the resident microbiota by direct killing or by inhibition

Small antibacterial toxins A number of small, mainly plasmid-encoded, antibacterial peptides termed bacteriocins or microcins, are produced and secreted by a broad range of bacteria, including the Bifidobacteria (reviewed in [START_REF] Martinez | Bacteriocin production by Bifidobacterium spp. A review[END_REF] Indeed, T6SS homologous have been described in up to 25% of all sequenced gram-negative genomes. While some bacteria use their T6SS to interfere with host processes [START_REF] Brodmann | Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape[END_REF] shown to be largely due to the T6SS [START_REF] Anderson | Shigella sonnei Encodes a Functional T6SS Used for Interbacterial Competition and Niche Occupancy[END_REF]. In B. fragilis this could also be observed at the strain level, where symbiotic, non-toxic B. fragilis was shown to outcompete a pathogenic strain in a T6SS-dependent manner [START_REF] Hecht | Strain competition restricts colonization of an enteric pathogen and prevents colitis[END_REF][START_REF] Chatzidaki-Livanis | Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species[END_REF].

The T6SS is also implicated in other important tasks that confer the bacterium harbouring it a selective advantage over the resident microbiota. These include iron acquisition [START_REF] Lin | A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition[END_REF] as well as different mechanisms to handle oxidative stress induced by the host [START_REF] Wang | Type VI Secretion System Transports Zn2+ to Combat Multiple Stresses and Host Immunity[END_REF][START_REF] Si | Manganese scavenging and oxidative stress response mediated by type VI 31/32 secretion system in Burkholderia thailandensis[END_REF][START_REF] Wan | Type VI secretion system contributes to Enterohemorrhagic Escherichia coli virulence by secreting catalase against host reactive oxygen species (ROS)[END_REF]. It has been shown

recently in marine bacteria that T6SS are horizontally shared between different species [START_REF] Salomon | Type VI Secretion System Toxins Horizontally Shared between Marine Bacteria[END_REF].

Furthermore, in Vibrio cholera, the T6SS was shown to be able to foster horizontal gene transfer (Borgeaud et al. 10/32

2015). Clearly, the last few years have seen strong advancements in the understanding of T6SS contributions to microbial life cycles [START_REF] Filloux | The rise of the Type VI secretion system[END_REF], while more work is needed in order to fully understand the full scope of T6SS functions.

Exploiting nutrients to gain a selective advantage over the resident microbiota

The most limiting nutrients in the gut are micronutrients, especially iron, as well as general energy sources, such as carbohydrates. Bacterial pathogens have evolved a number of strategies to selectively acquire nutrients over the resident microbiota in the race for these resources.

Iron scavenging and use of specific siderophores

The host has evolved sophisticated strategies termed "nutritional immunity" to limit the amount of available iron that the microbiota has access to, (reviewed in [START_REF] Cassat | Iron in infection and immunity[END_REF] and [START_REF] Kortman | Nutritional iron turned inside out: intestinal stress from a gut microbial perspective[END_REF]). Bacteria have responded by maximizing their ability to uptake iron through the secretion of iron-scavenging molecules, termed siderophores, which give them a selective advantage over strains lacking the scavenging capability [START_REF] Niehus | The evolution of siderophore production as a competitive trait[END_REF]). Lipocalin2 (NGAL in humans) is a potent antimicrobial factor secreted by the host whose function is to sequester iron bound siderophores [START_REF] Flo | Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron[END_REF]). Bacteria do not only have to compete with the host for iron, but also with the resident members of the microbiota. Indeed, many microbes encode species-specific siderophores that require specific re-uptake machinery to bind and import the iron-siderophore complex (reviewed in [START_REF] Miethke | Siderophore-based iron acquisition and pathogen control[END_REF]). Many enteropathogens have also evolved specific strategies to more efficiently scavenge any available iron. S. Typhimurium for example secretes Salmochelin, which is not recognized by lipocalin2/NGAL [START_REF] Raffatellu | Lipocalin-2 resistance confers an advantage to Salmonella enterica serotype Typhimurium for growth and survival in the inflamed intestine[END_REF]. Using Salmochelin, S. typhimurium is able to obtain enough iron to overcome iron-restriction, leading to a selective growth advantage over neighbouring bacteria. This proves especially important in the context of the inflamed intestine where Salmonella thrives amidst large quantities of Lipocalin2 secreted mainly by recruited neutrophils.

Use of alternative energy sources

S.

Typhimurium is a useful model pathogen to illustrate the basic mechanisms that pathogens employ to bypass the metabolic environment established by the microbiota. Initial replication of Salmonella in the yet undisturbed gut depends on hydrogen gas, an important intermediate in microbiota metabolism [START_REF] Maier | Microbiota-derived hydrogen fuels Salmonella typhimurium invasion of the gut ecosystem[END_REF]. Once Salmonella has invaded the gut mucosa and induced inflammation, other energy sources become available and are exploited by the pathogen. One of these is the aerobic and anaerobic respiration of 1, 2-propanediol generated by the resident microbiota (demonstrated in mono-associated mice using B. fragilis and B. thetaiotaomicron) through fermentation of fucose or rhamnose in the inflamed intestine [START_REF] Faber | Respiration of Microbiota-Derived 1,2-propanediol Drives Salmonella Expansion during Colitis[END_REF]. Salmonella can also thrive by oxidative respiration on succinate, which is released by the resident microbiota [START_REF] Spiga | An Oxidative Central Metabolism Enables Salmonella to Utilize Microbiota-Derived Succinate[END_REF]. As an alternative energy source, Salmonella is also capable of using galactarate and glucarate, generated by the microbiota after antibiotic treatment. This metabolic versatility, especially in an oxidative environment as is found in the inflamed intestine, is likely the basis of the long known condition of antibiotic-mediated Salmonella expansion [START_REF] Faber | Host-mediated sugar oxidation promotes post-antibiotic pathogen expansion[END_REF]. Another nutrient source is siacylic acid, liberated from the breakdown of sialyated mucins by B.

thetaiotaomicron and other microbiota members. B. thetaiotaomicron secretes a sialidase but lacks the ability to use the freed siacylic acid itself. In turn, this acid is metabolized by C. difficile and S. Typhimurium as an alternative nutrient source giving them a selective nutritional advantage over neighbouring bacteria [START_REF] Ng | Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens[END_REF][START_REF] Huang | Sialic acid catabolism drives intestinal inflammation and microbial dysbiosis in mice[END_REF]. Differential energy utilization has also been recently shown in some E. coli species, which use microbiota-derived formate as an alternative energy source to increase their advantage over other resident species 11/32 [START_REF] Hughes | Microbial Respiration and Formate Oxidation as Metabolic Signatures of Inflammation-Associated Dysbiosis[END_REF].

Several enteropathogens, including Citrobacter rodentium, Campylobacter jejuni and S. Typhimurium, induce acute intestinal inflammation through their virulence factors (reviewed in (Winter et al. 2010a)). The inflammation provides these pathogens with an advantage over the resident microbiota by transforming the intestine into an aerobic environment. Niche creation can be exemplified by the widely studied enteropathogen S. Typhimurium (reviewed in (Winter, Lopez and Under physiological conditions, the microbiota induces expression of PPARγ, resulting in an increase in β-oxidation of the colonocytes and hence an anoxic environment. In the context of antibiotic treatment, PPARγ-induction is inhibited and the colonocytes switch to anaerobic glucose oxidation. This then leads to increased availability of nitrate and allows for aerobic expansion of Enterobacteriaceae [START_REF] Byndloss | Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion[END_REF]. Inflammation also leads to outgrowth of other bacterial species, for example B. vulgatus [START_REF] Huang | Sialic acid catabolism drives intestinal inflammation and microbial dysbiosis in mice[END_REF]. Through its sialidase activity, B.

vulgatus releases sialic acid from the intestinal tissue, supporting the growth of E. coli. B. vulgatus thereby contributes to the occurrence of the "enterobacterial blooms" observed during inflammation. Conversely, enterobacterial blooms during infection are abrogated when sialidase inhibitors are administered [START_REF] Huang | Sialic acid catabolism drives intestinal inflammation and microbial dysbiosis in mice[END_REF]. Gut inflammation can also boost horizontal gene transfer, either between pathogenic and commensal enteropathogens or between dense populations of enteropathogens in the context of the so-called "enterobacterial booms" (see text above). This has been shown for the transfer of a colicin-carrying plasmid p2 [START_REF] Stecher | Gut inflammation can boost horizontal gene transfer between pathogenic and commensal Enterobacteriaceae[END_REF] as well as of temperate phages (Diard et al. 2017). Enterobacterial blooms can therefore contribute to pathogen evolution of some species.

Virulence genes

Once in contact with the host, pathogens have developed different strategies to influence the host and exploit it to their own benefit. This is mainly achieved through so called "virulence factors". unable to respond to the bile salts, leading to a competitive disadvantage compared to the wildtype strain in an infant mouse model of colonization. This colonization difference was abolished when a bile-salt sequestering resine, cholestyramine, was co-administered, confirming the crucial role of bile acids in the observed colonization defect [START_REF] Yang | Bile salt-induced intermolecular disulfide bond formation activates Vibrio cholerae virulence[END_REF].

A third example is the regulation of virulence genes in Enterotoxigenic E. coli (ETEC) and its close relative, the mouse pathogen C. rodentium in the intestine. A recent study has shown that the two neurotransmitters epinephrine and norepinephrine, that are produced by the endocrine cells localized in the intestine are needed for the full expression of virulence/colonization genes [START_REF] Moreira | Bacterial Adrenergic Sensors Regulate Virulence of Enteric Pathogens in the Gut[END_REF]).

Together, these observations show that pathogens have evolved varied and complex mechanisms to tightly regulate their virulence attributes. This helps pathogens avoid unnecessary energetic costs that may lead to a loss of fitness in the highly competitive gut environment (see [START_REF] Diard | Evolution of bacterial virulence[END_REF] for a recent review on the evolution of bacterial virulence).

Induction of pathogens's virulence genes by members of the microbiota

We previously discussed the cues from the host that lead to the induction of virulence genes expression in the invading pathogens. Beside this host-mediated induction, some virulence genes are also controlled by sensing metabolites derived from the microbiota, for example SCFAs (butyrate, actetate, lactate and propionate). A recent study has shown that microbiota-derived SCFAs modulate the expression of virulence genes in Campylobacter jejuni. The authors discovered that the gradient of SCFAs butyrate, acetate, and lactate along the intestinal tract guide expression of genes involved in virulence and commensalism of C. jejuni. Lactate, which is abundant in the 13/32

upper intestinal tract, suppresses production of C. jejuni virulence genes, while acetate and butyrate, two SCFA's that are mostly produced in the lower intestinal tract, activate virulence pathways [START_REF] Luethy | Microbiota-Derived Short-Chain Fatty Acids Modulate Expression of Campylobacter jejuni Determinants Required for Commensalism and Virulence[END_REF].

Expression of the Salmonella pathogenicity island 1 (SPI-1) is inhibited in the presence of propionate, a SCFA that is produced mainly by Lactobacilli and Bifidobacteria and is more abundant in the upper gastrointestinal tract.

Propionate acts through posttranslational modification on HilD, the master regulator of the Salmonella SPI-1 [START_REF] Hung | The intestinal fatty acid propionate inhibits Salmonella invasion through the post-translational control of HilD[END_REF]. This inhibition ensures that the coordinated expression of SPI-1 genes starts only in the distal small intestine, the main site of Salmonella infection. Another example of microbiota-mediated virulence gene expression is the enhancement of enterohemorraghic E. coli (EHEC) T3SS by B. thetaiotaomicron. B. thetaiotaomicron is an abundant member of the gut microbiota metabolizing complex polysaccharides into monosaccharides that can be further processed by a number of other bacteria. The presence of B. thetaiotaomicron leads to a local increase in the levels of succinate, which is sensed by the transcription factor Cra of EHEC. Cra activation leads to an increase in the expression of the genes encoding EHEC's T3SS while leaving the general growth of the pathogen unaffected [START_REF] Curtis | The gut commensal Bacteroides thetaiotaomicron exacerbates enteric infection through modification of the metabolic landscape[END_REF].

Induction of AMP's by pathogens to inhibit microbiota competition

Some pathogens have evolved mechanisms to exploit the host's own antibacterial defenses. By developing countermeasures against host AMPs, pathogens can rely on the host response to combat the resident microbiota and gain a selective advantage. Salmonella Typhimurium for example uses an alternative siderophore (Salmochelin, described above), which is resistant to chelation by Lipocalin2. Salmonella has also evolved strategies to resist against calprotectin-induced sequestration of zinc and manganese [START_REF] Liu | Zinc sequestration by the neutrophil protein calprotectin enhances Salmonella 27/32 growth in the inflamed gut[END_REF], giving Salmonella a selective advantage over the majority of microbiota members that do not have the necessary tools to survive in the inflamed gut.

Salmonella infection induces the cytokine IL-22, which in turn activates AMP's [START_REF] Behnsen | The cytokine IL-22 promotes pathogen colonization by suppressing related commensal bacteria[END_REF]. One of the proteins induced is the antimicrobial peptide Reg3beta. Previously, Stelter and colleagues showed that induction of this antimicrobial peptide inhibits the competing microbiota [START_REF] Miki | The bactericidal activity of the C-type lectin RegIIIβ against Gram-negative bacteria involves binding to lipid A[END_REF], while having no effect on the resistant S. Typhimurium [START_REF] Stelter | Salmonella-induced mucosal lectin RegIIIβ kills competing gut microbiota[END_REF]. In a new study, Miki and collaborators could show that Reg3beta extends gut colonization by S. Typhimurium through the prolonged induction of a pro-inflammatory environment and changes to the microbiota, especially an inhibition of Bacteroides species. The alteration of the microbiota also leads to profound changes in the metabolic landscape with metabolites of Vitamin B6 being the most affected. The authors could show that re-insertion of Bacteroides species or supplementation of Vitamin B6 alone was able to accelerate clearance of Salmonella from infected mice [START_REF] Miki | The Bactericidal Lectin RegIIIβ Prolongs Gut Colonization and Enteropathy in the Streptomycin Mouse Model for Salmonella Diarrhea[END_REF].

A recent study in Ixodes scacpularis ticks showed a similar mechanism whereby Anaplasma phagocytophilum infection induced Ixodes anti-freeze glycoprotein (Iafgp), which alters biofilm formation in the Ixodes gut to destabilize the resident microbiota and facilitate niche construction of A. phagocytophilum. It is likely that many pathogens have evolved similar mechanisms to take advantage of host defence mechanisms to facilitate niche construction and induce a host-derived selective advantage over the resident microbiota.

Evolutionary adaptions of pathogens to overcome homeostasis: penetrating the mucus layer

As described previously, the mucus layer constitutes a thick, protective layer between the gut lumen and the epithelium. To get access to the epithelium, pathogens have evolved strategies to penetrate the mucus layer and enter the underlying epithelium. Porphyromonas gingivalis, a pathogen found mostly in the oral cavity, secretes a cysteine protease (RgpB), which leads to Muc2 cleavage (van der [START_REF] Van Der Post | Site-specific O-glycosylation on the MUC2 mucin protein inhibits cleavage by the Porphyromonas gingivalis secreted cysteine protease (RgpB)[END_REF]. Mucinases also play an important role in the colonization and fitness of pathogenic E. coli [START_REF] Valeri | coli exploits SslE mucinase activity to translocate through the mucosal barrier and get access to host cells[END_REF], an E. coli strain associated 14/32

with Crohn's disease [START_REF] Gibold | The Vat-AIEC protease promotes crossing of the intestinal mucus layer by Crohn's disease-associated Escherichia coli[END_REF], and also eukaryotic pathogens such as Candida albicans [START_REF] Colina | Evidence for degradation of gastrointestinal mucin by Candida albicans secretory aspartyl proteinase[END_REF] or Entamoeba histolytica [START_REF] Lidell | Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel[END_REF]. It is likely that other enteropathogens also express mucinases to gain access to the underlying epithelium although more research is needed to fully address the scope of mucinase secretion and usage by pathogens.

Mechanisms established by the microbiota to clear off invading pathogen after infection

The microbiota not only plays an important role in preventing the colonization of pathogens, but also in the pathogen clearing from the gut upon resolution of the inflammation. The mechanisms underlying this process differ from those governing colonization resistance as the mucosa and the microbiota have both been affected and therefore need to return to homeostasis [START_REF] Endt | The microbiota mediates pathogen clearance from the gut lumen after nontyphoidal Salmonella diarrhea[END_REF]). In the case of S. Typhimurium infection it was shown that recovery is mainly mediated by the microbiota and was largely independent of the IgA pool [START_REF] Endt | The microbiota mediates pathogen clearance from the gut lumen after nontyphoidal Salmonella diarrhea[END_REF].

Mechanistic insights on the underlying causes of clearance were elucidated in a study on Vibrio cholera infection,

showing an increase in Ruminococcus obeum upon infection of mice with the pathogen. The same was also observed in a cohort of infected humans from Bangladesh recovering from the disease. In elegant mouse studies, the authors showed that Ruminococcus obeum, through the expression of the luxS gene (autoinducer-2 synthase), promotes quorum sensing-mediated restriction of virulence gene expression in V. cholerae, leading to a decrease in host symptoms [START_REF] Hsiao | Members of the human gut microbiota involved in recovery from Vibrio cholerae infection[END_REF])

Dysbiosis: Towards a new interpretation of Koch's postulate

It is now common knowledge that the gut is a complex ecosystem with different interacting entities and that infections must be understood in this context rather than isolated as a pathogen and a host. In consequence, for these complex diseases, neither Koch's postulate ("a pathogen, a disease") nor the molecular Koch's postulate as proposed by Stanley Falkow ("a virulence gene, a disease", [START_REF] Falkow | Molecular Koch's postulates applied to microbial pathogenicity[END_REF])) are sufficient. In a very recent review by Neville and collaborators, a third interpretation of the Koch's postulates, the commensal Koch's postulate, was proposed ("a beneficial microbe", "an ameliorated disease state"). The authors infer a new framework for establishing causation in microbiome studies were they use the commensal Koch's postulate to test if a given microorganism is able to ameliorate a disease state in a reproducible manner [START_REF] Neville | Commensal Koch's postulates: establishing causation in human microbiota research[END_REF].

As in the original Koch's postulate, they propose that for inferring causation, the "beneficial" commensals need to be isolated in pure cultures before they are re-introduced and tested in a host for their capacity to mitigate disease.

We propose yet another interpretation of Koch' postulate, which we have termed "ecological Koch's postulate ("a gut ecosystem state, a disease"). Underlying these postulates is the fact that the gut harbours a full ecosystem, rather than an isolated bacterium or pathogen. This means that rather than an isolated microorganism or group of microorganisms, a whole ecosystem, including the microbiota, the genetic make-up of the host as well as nutrition, age etc. form an entity, which can ultimately lead to disease (see Figure 3 and Box 3).

The ecological Koch's postulates are based on two major observations, both pointing towards the fact that the clear distinction between a pathogen and a commensal are probably too simple of a model to explain complex disease states.

15/32 (II) In the last years, several gastrointestinal diseases have emerged, which are not associated with an overt pathogen, but where the microbial community as a whole seems to mediate the disease. Examples are intestinal bowel disease, colorectal cancer, obesity, and different states of malnutrition. Indeed, depending on the microbial community pathogens find themselves in, bacteria, which normally do behave as commensals may become invasive and cause disease. For all of these diseases, a decrease in the composition complexity of the microbiota leads to dysbiosis and an oxidation of the gut environment as well as an increase in aeortolerant species such as Enterobacteriaceae (Rivera-Chávez, Lopez and Bäumler 2017). These disturbances in the ecosystem lead to a lowered resilience and increased susceptibility to pathogens and other, normally commensal, bacteria with potential harmful properties (often called "pathobionts"). Indeed, these diseases are characterized by the fact that the wrong bacteria are in wrong proportions, in wrong "company" [START_REF] Huang | Sialic acid catabolism drives intestinal inflammation and microbial dysbiosis in mice[END_REF] or in the wrong "place" [START_REF] Brown | Diet and specific microbial exposure trigger features of environmental enteropathy in a novel murine model[END_REF][START_REF] Tomas | High-fat diet modifies the PPAR-γ pathway leading to disruption of microbial and physiological ecosystem in murine small intestine[END_REF]. The presence of commensal bacteria near the epithelial surface has been put in relation with the breakdown of gut homeostasis and emergence of pathological states in the context of environmental enteropathy [START_REF] Brown | Diet and specific microbial exposure trigger features of environmental enteropathy in a novel murine model[END_REF] or in a pre-diabetic state [START_REF] Tomas | High-fat diet modifies the PPAR-γ pathway leading to disruption of microbial and physiological ecosystem in murine small intestine[END_REF]).

In the ecological Koch's postulate, a dysbiotic community, including or not "classical pathogens" or pathobionts therefore represents a disease. The entity of transmission is the complete dysbiotic microbiota rather than a pathogen (Koch's postulate), a virulence gene (molecular Koch's postulate) or a commensal (commensal Koch's postulate).

In accordance to the other Koch's postulate, the ecological Koch's postulate are proven through the fact that a given entity (here the dysbiotic microbiota) from a diseased individual can provoke disease in a formerly healthy individual. To prove this hypothesis, one hence has to transmit the microbiota from a diseased individual into a germfree individual/ mouse and this transplanted individual subsequently has to develop the signs of the disease.

This transfer can either be performed in (I) "standard" conditions, using a "wild type, normally fed" germ-free host (showing a direct effect of the microbiota on disease), or, else, in (II) a "pre-fragilised" host, as an example in a mouse exhibiting a mutation in a given gene or eating a specific chow. In syndromes, which do need a prefragilised host, the dysbiotic microbiota contributes to disease, without however being the only cause for it.

Box 3-Postulates for defining a disease-promoting ecosystem (dysbiosis) Ecological Koch's postulates 1 The dysbiotic microbiota is found in similar composition/ with similar characteristics in all affected individuals 2 The dysbiotic microbiota can be retrieved from the affected host 3 Gavaging of germ-free hosts with this retrieved microbiota leads, in combination with a similar environment (ex. genetic make-up of the host, nutrition, age), to similar symptoms as in the affected individual 4 The dysbiotic microbiota composition remains fairly stable in the newly affected host Original Koch's postulates 1 The microorganism must be present in all diseased individuals 2 The microorganism must be isolated from the diseased host and be grown in a pure culture 3 The re-inoculation of a naïve host with this pure culture must lead to the same disease as in the original host 4 The microorganism must be recovered from the newly diseased host

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As stated earlier, several inflammatory, gastrointestinal diseases can be explained through the ecological Koch's postulate, including obesity. Indeed, if a microbiota from obese mice is transplanted into lean mice, the transplanted mice showed increased fat deposition [START_REF] Turnbaugh | An obesity-associated gut microbiome with increased capacity for energy harvest[END_REF][START_REF] Ridaura | Gut microbiota from twins discordant for obesity modulate metabolism in mice[END_REF]. The same could also be proven for kwashiorkor, the oedematous form of acute undernutrition [START_REF] Smith | Gut microbiomes of Malawian twin pairs discordant for kwashiorkor[END_REF] as well as for environmental enteropathy [START_REF] Brown | Diet and specific microbial exposure trigger features of environmental enteropathy in a novel murine model[END_REF]. On the other hand, it is well known that mutations in nod2 facilitate and support the onset of IBD [START_REF] Cho | Update on the genetics of inflammatory bowel disease[END_REF]. Therefore, to prove the contribution of the microbiota in IBD, a susceptible host, mutated for nod2, should be transplanted with the dysbiotic microbiota and this transplanted microbiota should worsen the disease state.

An instructive example of a "pathological dysbiosis" and hence a disease following the ecological Koch's postulates is chronic and acute malnutrition coupled to associated environmental enteropathy. Several reports have shown evidence that children suffering from one of these two syndromes have an altered colonic microbiota [START_REF] Smith | Gut microbiomes of Malawian twin pairs discordant for kwashiorkor[END_REF][START_REF] Subramanian | Persistent gut microbiota immaturity in malnourished Bangladeshi children[END_REF][START_REF] Gough | Linear growth faltering in infants is associated with Acidaminococcus sp. and community-level changes in the gut microbiota[END_REF]Blanton et al. 2016a;2016b) and increased abundance of asymptomatic pathogen carriage, including enteroaggregative E. coli [START_REF] Havt | Prevalence and virulence gene profiling of enteroaggregative Escherichia coli in malnourished and nourished Brazilian children[END_REF], Campylobacter spp. and Giardia spp. [START_REF] Platts-Mills | Association between enteropathogens and malnutrition in children aged 6-23 mo in Bangladesh: a case-control study[END_REF]. Further, a recent study sequencing cultured microbes from two Bangladeshi children suffering or not of undernutrition showed that the B. fragilis strain found in the undernourished child is enterotoxic, while the strains found within the normally nourished child were not. When transplanting the native community into germfree mice and infecting with the enterotoxigenic B. fragilis strain, the authors could show that the enterotoxigenic strain causally led to malnutrition and associated pathophysiological disturbances only in its native community, but not when administered to mice harboring the microbiota of the healthy child [START_REF] Wagner | Effects of a gut pathobiont in a gnotobiotic mouse model of childhood undernutrition[END_REF]). These observations put forward the hypotheses that (I) even subclinical infections with enteropathogens can have negative effects on gut health and that (II) pathogens or pathobionts, depending on the community they dwell in, might have negative effects on host homeostasis or not. This indeed supports the concept of the "ecological Koch's postulates", stating that the whole ecosystem, rather than an isolated element contributes to morbidity.

Overall, we are only beginning to understand the complex relationships and interactions within the gut ecosystem and more research is needed in order to elucidate the origin and pathophysiological effect of the different dysbiotic communities and to understand the crosstalk they have between each other as well as with the host.

Conclusion

Infection biology has been moving in the last decades from the original Koch's postulate looking at pathogens, to molecular Koch's postulate looking at virulence factors, to the newly proposed ecological Koch's postulate looking at dysbiosis. Indeed, infection biology has shifted towards an integrated approach of systems biology, ecology, and evolution. This increasing complexity makes it more and more difficult to untangle the causative effects of disease states and asks for imaginative and sophisticated designs of experiments to explain the underlying pathophysiological mechanisms. Particularly, experiments need to take into account the physiological and pathophysiological state of the infected host, for example the microbiota and the exact nutrition the model animals are receiving. In recent years, several initiatives have been launched to standardize the microbiota of model animals [START_REF] Brugiroux | Genome-guided design of a defined mouse microbiota that confers colonization resistance against Salmonella enterica serovar Typhimurium[END_REF] or at least to meticulously report, not only the exact strain of pathogen used and the genetics of the mouse model, but also the microbiota composition, as well as the food composition of the animal models used [START_REF] Ma | Routine habitat change: a source of unrecognized transient alteration of intestinal microbiota in laboratory mice[END_REF][START_REF] Macpherson | Standardised animal models of host microbial mutualism[END_REF]. This will prove indispensable in the future in order to compare different studies and explain the pathophysiological mechanisms underlying the complex interplay between pathogens, the microbiota, and their host. To date, we are only at the beginning of understanding the 17/32

interactions within these ecosystems, the perturbations, which can be induced, and their effect on pathogen susceptibility and disease. The widely available techniques of sequencing, especially of metagenomics, metatranscriptomics and metabolomics, will prove essential in this endeavour. Special attention should also be paid to not forget that the microbiota harbours other organisms than prokaryotes, first and foremost viruses (including phages and prophages), and eukaryotes. Only an integrated view of the gut ecosystem, including the host, the pro-and eukaryome and virome as well as the pathogens will allow us to move forward in our understanding of which mechanisms are governing infection.

The scientific community is on the verge of experiencing another revolution in understanding the complex network of gut interactions. This will surely open the way for more targeted and personalized interventions to infectious diseases based on interference or corrections to the misbalances in the gut ecosystem and restoration of gut homeostasis. This could include siderophore-based immunization strategies [START_REF] Mike | Siderophore vaccine conjugates protect against uropathogenic Escherichia coli urinary tract infection[END_REF]Sassone-Corsi et al. 2016a), probiotic bacteria (e.g. E. coli strain Nissle) using similar iron-scavenging mechanisms than the invading pathogen [START_REF] Deriu | Probiotic Bacteria Reduce Salmonella Typhimurium Intestinal Colonization by Competing for Iron[END_REF], probiotic strains consuming H 2 and hence restricting the use of this energy source for invading pathogens [START_REF] Maier | Microbiota-derived hydrogen fuels Salmonella typhimurium invasion of the gut ecosystem[END_REF], the development of probiotic strains expressing bacteriocins or microcins targeting the pathogen [START_REF] Kommineni | Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract[END_REF][START_REF] Hegarty | Bacteriocin production: a relatively unharnessed probiotic trait?[END_REF]Sassone-Corsi et al. 2016b), expressing ironsequestering mechanisms to inhibit invading pathogens [START_REF] Vazquez-Gutierrez | High Iron-Sequestrating Bifidobacteria Inhibit Enteropathogen Growth and Adhesion to Intestinal Epithelial Cells In vitro[END_REF], siacylidase inhibitors [START_REF] Huang | Sialic acid catabolism drives intestinal inflammation and microbial dysbiosis in mice[END_REF] or inhibitors of anaerobic respiration (Winter and Bäumler 2014b) (see Table 1). There are certainly many other possible intervention strategies yet to be discovered. The generated knowledge will therefore prove very important in paving the way to propose other intervention strategies, which do not rely on antibiotics. In a world where antibiotic resistance is on a constant rise this aspect will be of utmost importance. In recent years, the original Koch's postulate ("a pathogen, a disease") have been extended to the molecular Koch's postulate (Stanley Falkow, "a virulence genes, a disease") and here to the ecological Koch's postulate ("a dysbiosis, a disease")

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Intervention strategy Evidence for intervention References

Siderophore-based immunization strategies

Mice immunized mice with siderophores conjugated to an immunogenic carrier protein were able to elicit a potent immune response and to protect against urinary tract infections.

Mice immunized with a cholera toxin β-siderophore conjugate show a potent immune response and are able to protect against infection with Salmonella Typhimurium. Microcin-producing E. coli Nissle is able to limit the growth of commensal E. coli, adherent-invasive E. coli and Salmonella enterica in the inflamed intestine. [START_REF] Kommineni | Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract[END_REF][START_REF] Hegarty | Bacteriocin production: a relatively unharnessed probiotic trait?[END_REF]Sassone-Corsi et al. 2016b)

Siacylidase inhibitors

Oral administration of sialidase inhibitors decreases outgrowth of E. coli as well as the severity of colitis in a mouse model. [START_REF] Huang | Sialic acid catabolism drives intestinal inflammation and microbial dysbiosis in mice[END_REF] Inhibitors of aerobic respiration Aerobic respiration is used by Salmonella spp and other Enterobacteriaceae to thrive in the inflamed intestine.

(Winter and Bäumler 2014b)

Sustaining PPAR-γ signaling PPAR-γ signaling in the homeostatic intestine leads to βoxidation in the colonocytes and hence an anoxic environment limiting nitrate availability and outgrowth of Enterobacteriaceae [START_REF] Byndloss | Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion[END_REF]

  colon, complex carbohydrates present in the food 186 or eaten in the form of prebiotics are metabolized by the 187 resident microbiota into short chain fatty acids (SCFA), 188 the three most abundant being acetate, propionate and 189 butyrate. Acetate and propionate are produced mainly by 190 members of the Lactobacilli and Bifidobacteriae. Butyrate 191 is produced by bacteria of the phylum Firmicutes, different members of the genus Clostridium (Ramirez-Farias et al. 2009; Rivière et al. 2016). Acetate triggers anti-inflammatory and anti-apoptotic responses in host epithelial cells, which leads to protection in the gut against colonization with pathogenic bacteria like Enterobacteriaceae and Clostridiae (Fukuda et al. 2011).

  Ruminococcus, have evolved to degrade mucin without giving other species access to the degradation products.The intramolecular transsialidase produced by R. gnavus releases 2,7-anhydro-Neu5Ac instead of sialic acid from mucin and other glycoproteins, a product that cannot by utilized by other species(Tailford et al. 2015b) (reviewed in(Tailford et al. 2015a)).This nutritional limitation represents a strong barrier for the niche establishment of invading species.243Mucus layer244The mucus layer forms a physical barrier to the 245 microbiota, preventing direct interaction with the 246 epithelium (reviewed in (McGuckin et al. 2011; 247 Pelaseyed et al. 2014)). In the gut, the goblet 248 cells are responsible for the secretion of the 249 mucin MUC2, which forms a disulfide cross-250 linked network. This network is comprised of an 251 inner layer, which is tightly attached to the 252 epithelium and mainly impenetrable to bacteria, 253 as well as a looser, outer layer. This outer layer 254 harbours a specific community of bacteria, 255 feeding on the mucus (Li et al. 2015) and

  ;[START_REF] Alvarez-Sieiro | Bacteriocins of lactic acid bacteria: extending the family[END_REF])), Lactobacilli[START_REF] Collins | Bacteriocin Gene-Trait matching across the complete Lactobacillus Pan-genome[END_REF], Enterococci[START_REF] Kommineni | Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract[END_REF]) and many more. It has been demonstrated that bacteriocin production leads to a niche advantage for the bacteria expressing them[START_REF] Riley | Bacteriocins: evolution, ecology, and application[END_REF][START_REF] Kommineni | Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract[END_REF]. Some of the bacteriocins and microcins have been shown to act against pathogenic strains[START_REF] Kommineni | Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract[END_REF] Sassone-Corsi et al. 2016b), thereby augmenting colonization resistance and mediating resistance to invading pathogens. One bacteriocin subclass produced byEnterobacteriaceae and termed colicins are encoded by several pathogenic strains including S. Typhimurium[START_REF] Nedialkova | Inflammation fuels colicin Ib-dependent competition of Salmonella serovar Typhimurium and E. coli in enterobacterial blooms[END_REF] and Shigella sonnei[START_REF] Anderson | Shigella sonnei Encodes a Functional T6SS Used for Interbacterial Competition and Niche Occupancy[END_REF][START_REF] Calcuttawala | Characterization of E-type colicinogenic plasmids from Shigella sonnei[END_REF]. It has been shown recently that S. Typhimurium expresses colicin Ib (ColIB), giving it a fitness advantage over the closely related E. coli, which blooms simultaneously in the gut upon Salmonella-induced intestinal inflammation. ColIb is regulated through the SOS-response and iron-limitation and upregulated in the context of inflammation[START_REF] Nedialkova | Inflammation fuels colicin Ib-dependent competition of Salmonella serovar Typhimurium and E. coli in enterobacterial blooms[END_REF]. Therefore, the carriage and induced upregulation of colicins seems to be an evolutionary adaption of enteropathogens in order to have a selective advantage in the ecological niche they share with commensal E. coli in the inflamed gut. Type 6 secretion system (T6SS) T6SS are encoded by a substantial number of gram-negative pathogens, as diverse as Helicobacter hepaticus[START_REF] Chow | A pathobiont of the microbiota balances host colonization and intestinal inflammation[END_REF], S. Typhimurium[START_REF] Sana | Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut[END_REF], S. sonnei[START_REF] Anderson | Shigella sonnei Encodes a Functional T6SS Used for Interbacterial Competition and Niche Occupancy[END_REF], Pseudomonas aeruginosa[START_REF] Mougous | A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus[END_REF], enteroaggregative E. coli[START_REF] Dudley | Proteomic and microarray characterization of the AggR regulon identifies a pheU pathogenicity island in enteroaggregative Escherichia coli[END_REF], Vibrio cholera[START_REF] Pukatzki | Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system[END_REF]) and B. fragilis[START_REF] Hecht | Strain competition restricts colonization of an enteric pathogen and prevents colitis[END_REF][START_REF] Chatzidaki-Livanis | Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species[END_REF] to name a few.

  First and foremost, pathogens express "classical" virulence factors, for example toxins and the T3SS and their associated effector proteins. The function and specific role of these virulence factors have been discussed in detail elsewhere ([START_REF] Puhar | Type III secretion system[END_REF][START_REF] Qiu | Legionella and Coxiella effectors: strength in diversity and activity[END_REF] and many others for specific pathogens). The expression of these virulence factors can be constitutive. However, with the immense fitness cost virulence gene expression imparts on the pathogen, virulence gene expression is often regulated by environmental cues. This means that the 12/32 bacterium only expresses the virulence genes once it is in close contact with the host and at the right location along the gastrointestinal tract.Induction of pathogen virulence genes by cues from the hostDifferent host cues allow invading pathogens to pinpoint their position within their host and to regulate virulence genes only once the appropriate location has been reached. Host cues for virulence regulation include bile acids(Antunes et al. 2012; Brotcke Zumsteg et al. 2014; Eade et al. 2016), pH (Behari, Stagon and Calderwood 2001), temperature(Elhadad et al. 2015; Nuss et al. 2015; Fraser and Brown 2017), nutrient availability (reviewed in (Porcheron, Schouler and Dozois 2016)), and oxygen levels((Marteyn et al. 2010; Fraser and Brown 2017) reviewed in(Marteyn et al. 2011; Marteyn, Gazi and Sansonetti 2012)). Oxygen levels are dynamic within the intestine and even within the anoxic colon an oxygen tension gradient is present in close proximity to the epithelial surface. Shigella flexneri has adapted to this gradient by repressing its T3SS in response to reduced oxygen levels encountered in the lumen of the intestine. The key outcome is to conserve metabolic resources in this energy and nutrient depleted environment. This regulation has the added benefit of more closely aligning the expression of virulence factors to the site of infection at the epithelial layer, where they are actually used. The suppression of the T3SS system is mediated by the oxygen sensitive regulator gene fnr. When oxygen pressure increases near the epithelium, the anaerobic block on the master regulators of the T3SS, spa32 and spa33, is released and the genes of the T3SS can be expressed(Marteyn et al. 2010).Another example of regulated virulence is the bile salt-mediated activation of virulence in Vibrio cholerae. It was recently shown through a series of elegant in vitro experiments in a tissue model of infection that virulence genes of V. cholerae are induced by the bile salt taurocholate, glyocholate and cholate, but not the deconjugated deoxycholate or chenodeoxycholate. This virulence activation is mediated through dimerization of the transcription factor TcpP by disulfide bond formation. Consequently, a V. cholerae strain mutated in the respective cysteine is

  be present and harbour the 605 virulence genes, but they also need to 606 express these genes and be able to 607 establish a niche for themselves within 608 the competitive environment of the 609 already established microbial community in order to replicate and then cause disease.

Figure 1 :

 1 Figure1: Homeostatic gut environment (A) versus a dysbiotic gut environment (B). Scheme of an intestine in homeostasis (left) and in dysbiosis upon invasion of a pathogen (depicted in red). Upon dysbiosis, the mucus layer is thinner, larger amounts of antimicrobial peptides are secreted (e.g. C-type lectins as Reg3γ) to prevent bacteria breach the barrier and get access to the underlying tissue. This leads to villous blunting, influx of inflammatory cells into the lamina propria and depletion of members of the microbiota, leading to a changed composition and lower diversity of the resident microbiota.

Figure 2 :

 2 Figure 2: Mechanisms evolved by pathogen to combat the resident microbiota and the host Pathogens have evolved several mechanisms to overcome the barrier imposed by the resident microbiota and the host. These include direct (bacteriocins, microcins, T6SS) and indirect (nutrient restriction) inhibition of members of the microbiota, the use of alternative energy sources as well as different mechanisms to overcome the barriers imposed by the hosts (mucinases, T3SS and other virulence factors acting on the host).

Figure 3 :

 3 Figure 3: The evolution of the Koch's postulate

(

  Escherichia coli strain Nissle 1917 reduces S. Typhimurium colonization in mouse models of acute colitis or chronic persistent infection. The observed probiotic activity depends on the iron-sequestering mechanisms of E. coli Nissle, which is highly similar to the one found in Salmonella Typhimurium.The two bifidobacterial strains Bifidobacterium pseudolongum PV8-2 (Bp PV8-2) and Bifidobacterium kashiwanohense PV20-2 (Bk PV20-2) are able to inhibit growth of S. Typhimurium and E. coli O157:H45 (EHEC) in in vitro co-culture experiments and are able to displace the pathogens on mucus-producing HT29-MTX cell lines. (Deriu et al. 2013) (Vazquez-Gutierrez et al. 2016),Probiotic strains consuming H 2 to prevent initial ecosystem invasionIn a non-inflamed intestine, S. Typhimurium relys on H 2 metabolisms for invasion. Introducing H 2 -consuming bacteria into the microbiota reduces hyb-dependent S. Typhimurium growth.[START_REF] Maier | Microbiota-derived hydrogen fuels Salmonella typhimurium invasion of the gut ecosystem[END_REF] Probiotic strains producing butyrate Oral gavage of mice with tributyrin reduces growth of S. Typhimurium in the inflamed intestine. with a bacteriocin-carrying E. faecalis strain defective for conjugation leads to clearance of vancomycin resistant enterococci.

  However, these two hydrolases are not necessary for B. ovatus to grow on inulin. Rather, other members of the microbiota, such as Bacteroides vulgatus, grow on the inulin breakdown products produced by B. ovatus. B. ovatus then uses products produced by B. vulgatus and both species flourish (Rakoff-Nahoum, Foster and Comstock 2016).

  , can have profound effects on the microbiota composition in the intestine[START_REF] Walk | Alteration of the murine gut microbiota during infection with the parasitic helminth Heligmosomoides polygyrus[END_REF][START_REF] Broadhurst | Therapeutic helminth infection of macaques with idiopathic chronic diarrhea alters the inflammatory signature and mucosal microbiota of the colon[END_REF][START_REF] Giacomin | Experimental hookworm infection and escalating gluten challenges are associated with increased microbial richness in celiac subjects[END_REF][START_REF] Mckenney | Alteration of the rat cecal microbiome during colonization with the helminth Hymenolepis diminuta[END_REF][START_REF] Ramanan | Helminth infection promotes colonization resistance via type 2 immunity[END_REF] Li et al. 2016;[START_REF] Guernier | Gut microbiota disturbance during helminth infection: can it affect cognition and behaviour of children?[END_REF] , reviewed in (Gause and Maizels 2016)) as well as on disease susceptibility within the intestine[START_REF] Reynolds | Enteric Helminths Promote Salmonella Coinfection by Altering the Intestinal Metabolome[END_REF]) and at distant sites, e.g. the respiratory system[START_REF] Mcfarlane | Enteric helminth-induced type I interferon signaling protects against pulmonary virus infection through interaction with the microbiota[END_REF]. However, alterations in the microbiota composition due to the presence of helminths are not generalisable, as changes were observed in a first study of 51 persons infected with

	Trichuris

trichiuria in Malaysia

[START_REF] Lee | Helminth colonization is associated with increased diversity of the gut microbiota[END_REF]

, but no changes were found in a second study in 97 children from Ecuador

[START_REF] Cooper | Patent human infections with the whipworm, Trichuris trichiura, are not associated with alterations in the faecal microbiota[END_REF]

nor in 8 persons living in Australia infected experimentally by Necator americanus

[START_REF] Cantacessi | Impact of experimental hookworm infection on the human gut microbiota[END_REF]

. Helminths have also been shown to attenuate the effect of intestinal bowel disease by restoring the number of goblet cells and preventing outgrowth of B. vulgatus in the context of Nod2 -/-mice and associated intestinal inflammation

  Bäumler 2013; Winter and Bäumler 2014a)). It is likely that similar mechanisms that have evolved in Salmonella have also evolved in other enteropathogens, for example E. coli and Shigella spp. and together contribute to the "enterobacterial bloom" that is observed in the inflamed gut[START_REF] Lupp | Host-Mediated Inflammation Disrupts the Intestinal Microbiota and Promotes the Overgrowth of Enterobacteriaceae[END_REF] Stecher et al. 2010) reviewed in(Winter and Bäumler 2014a)). Inflammation leads to the production of respiratory electron acceptors, for example nitrogen species and reactive oxygen species. These products are converted in the intestine to nitrate. Nitrate can be used by Salmonella spp., E. coli and potentially other facultative anaerobe members of the Enterobacteriaceae as an alternative electron acceptor, giving them a selective advantage over other resident bacteria, which rely mainly on anaerobic fermentation of carbohydrates[START_REF] Lopez | Phage-mediated acquisition of a type III secreted effector protein boosts growth of salmonella by nitrate respiration[END_REF][START_REF] Spees | Streptomycin-Induced Inflammation Enhances Escherichia coli Gut Colonization Through Nitrate Respiration[END_REF] Winter et al. 2013). Salmonella has also been shown to use other electron acceptors, for example Soxides, ethanolamine or tetrathionate, which are present in larger amounts in the inflamed intestine(Winter et al.

2010b;

[START_REF] Thiennimitr | Intestinal inflammation allows Salmonella to use ethanolamine to compete with the microbiota[END_REF]

. In a recent study, it could be shown that Salmonella T3SS activation leads to a depletion of Clostridium species, which in turn leads to a decrease in butyrate levels. Butyrate is the most important energy source of colonocytes and butyrate oxidation to carbon dioxide leads to the consumption of local oxygen and the generation of an anaerobic environment. The lack of butyrate therefore increases tissue oxygenation, generating a favourable niche for Salmonella's aerobic expansion in the gut lumen

(Rivera-Chávez et al. 2016b

).

Table 1 : Possible new intervention strategies intervening with the gut ecosystem to target diseases related to dysbioses-induced enteropathogenic blooms

 1

Acknowledgements

We would like to thank Pamela Schnupf, Kelsey Huus and Florent Mazel for fruitful discussions and critical reading of the manuscript. PV was supported by an Early.PostdocMobility Fellowship form the Swiss National Science Foundation, a Roux-Cantarini Postdoctoral Fellowship as well as a L'Oréal-UNESCO for Women in Science France Fellowship. Work in PJS's group is supported by ERC Grant No. 339579 (DECRYPT). PJS is a HHMI Senior Foreign Scholar and CIFAR scholar in the human microbiome consortium.