Trends The gut microbiota contains trillions of bacteria belonging to hundreds, possibly thousands, of species and is crucial for optimal maintenance of host physiological processes. The microbiota protects against infections and other pathologies by directly inhibiting invading microbes or by orchestrating appropriate immune responses; conversely, metabolites produced by some gut commensals can promote a variety of diseases such as atherosclerosis or cancer. Antibiotics alter the microbiota composition, resulting in an increased risk of disease, secondary infections, allergy, and obesity. In addition, they promote the spread of drug-resistant pathogens, making the search for alternative clinical approaches imperative. Novel strategies are being developed to substitute or complement antibiotic therapies, attempting either to selectively target pathogens without perturbing the microbiota and/or to re-establish commensal communities together with the protective and beneficial effects they confer to the host.

The gut microbiota is a key player in many physiological and pathological processes occurring in humans. Recent investigations suggest that the efficacy of some clinical approaches depends on the action of commensal bacteria. Antibiotics are invaluable weapons to fight infectious diseases. However, by altering the composition and functions of the microbiota, they can also produce long-lasting deleterious effects for the host. The emergence of multidrug-resistant pathogens raises concerns about the common, and at times inappropriate, use of antimicrobial agents. Here we review the most recently discovered connections between host pathophysiology, microbiota, and antibiotics highlighting technological platforms, mechanistic insights, and clinical strategies to enhance resistance to diseases by preserving the beneficial functions of the microbiota.

microbiota (see 1 Qin J.

et al. A human gut microbial gene catalogue established by metagenomic sequencing. 2 Walter J.

Ley R. The human gut microbiome: ecology and recent evolutionary changes. 3 Brestoff J.R.

Artis D. Commensal bacteria at the interface of host metabolism and the immune system. 4 Buffie C.G.

Pamer E.G. Microbiota-mediated colonization resistance against intestinal pathogens. Box 1 The Gut Microbiota: A Structural Overview 129 Rajilic-Stojanovic M.

de Vos W.M. The first 1000 cultured species of the human gastrointestinal microbiota. 2 Walter J.

Ley R. The human gut microbiome: ecology and recent evolutionary changes. + aerobic and anaerobic bacteria. Prominent members are Clostridia strains, whose activities range from beneficial and protective (e.g., C. scindens, clusters IV–XIVa) to pathogenic (e.g., C. difficile, C. perfrigens). Potentially pathogenic streptococci, enterococci, and staphylococci are also Firmicutes. Bacteroidetes are Gram− bacteria that are extremely well adapted to the intestinal environment. Here they ferment otherwise indigestible carbohydrates producing SCFAs, molecules that have been implicated in a plethora of important processes. Actinobacteria are Gram+ bacteria generally considered to be beneficial, such as the Bifidobacterium genus, and which are included in many probiotic preparations. The Proteobacteria phylum contains Gram− bacteria, most notably the family of Enterobacteriaceae, including E. coli and K. pneumoniae. These are not very abundant under normal conditions, but tend to expand upon dysbiosis. The human gut microbiota consists of an estimated 100 trillion bacteria belonging to several hundreds of different species []. These fall into four major phyla covering more than 90% of the total bacterial population, namely Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria, and include many additional minor phyla such as Verrucomicrobia and Fusobacteria. The representation of these groups changes along the GI tract, influenced by distinct microenvironments and nutrient availability []. The Firmicutes phylum is composed mainly by Gramaerobic and anaerobic bacteria. Prominent members are Clostridia strains, whose activities range from beneficial and protective (e.g., C. scindens, clusters IV–XIVa) to pathogenic (e.g., C. difficile, C. perfrigens). Potentially pathogenic streptococci, enterococci, and staphylococci are also Firmicutes. Bacteroidetes are Grambacteria that are extremely well adapted to the intestinal environment. Here they ferment otherwise indigestible carbohydrates producing SCFAs, molecules that have been implicated in a plethora of important processes. Actinobacteria are Grambacteria generally considered to be beneficial, such as the Bifidobacterium genus, and which are included in many probiotic preparations. The Proteobacteria phylum contains Grambacteria, most notably the family of Enterobacteriaceae, including E. coli and K. pneumoniae. These are not very abundant under normal conditions, but tend to expand upon dysbiosis. 130 Ley R.E.

et al. Obesity alters gut microbial ecology. 131 Nguyen T.L.

et al. How informative is the mouse for human gut microbiota research?. 132 Xiao L.

et al. A catalog of the mouse gut metagenome. 133 Seedorf H.

et al. Bacteria from diverse habitats colonize and compete in the mouse gut. 55 Hsiao A.

et al. Members of the human gut microbiota involved in recovery from Vibrio cholerae infection. 65 Ridaura V.K.

et al. Gut microbiota from twins discordant for obesity modulate metabolism in mice. 134 Blanton L.V.

et al. Gut bacteria that prevent growth impairments transmitted by microbiota from malnourished children. 135 Kau A.L.

et al. Functional characterization of IgA-targeted bacterial taxa from undernourished Malawian children that produce diet-dependent enteropathy. Notably, the majority of the studies of the microbiota have been performed in mice, even though the human and mouse microbiota differ in genus representation []. Some genera such as Prevotella, Faecalibacterium, and Ruminococcus are abundant in humans, while others, namely Lactobacillus, Alistipes, and Turicibacter, are highly prevalent in mice []. However, a core of common taxa can be identified, and mouse and human intestinal metagenomes appear to be remarkably similar if analyzed from a functional perspective [i.e., representation of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which depict the overall metabolic potential of a community] []. Most importantly, GF animals can be efficiently reconstituted with microbial communities isolated from other species, including humans, reproducing effects observed in a donor in the recipient host []. Reconstitution of GF mice with stool samples from obese or malnourished subjects is sufficient to phenocopy patient defects in energy harvest or growth [], demonstrating that despite interspecies divergences, the mouse model is a valuable tool to study the human microbiota. In the past two decades the gut(see Glossary ) has been recognized as a fundamental player orchestrating host physiology and pathology ( Box 1 ). Trillions of bacteria inhabit the gastrointestinal (GI) tract of complex metazoans including humans, greatly expanding the host genetic repertoire []. This translates into the possibility for the host to perform functions that are not encoded by its own genome: commensals protect from pathogen invasion, extract additional energy from food, and synthesize key molecules for tissue development in a way that is highly specialized with respect to their location along the GI tract [].

5 Goyal M.S.

et al. Feeding the brain and nurturing the mind: linking nutrition and the gut microbiota to brain development. 6 Marsland B.J.

Salami O. Microbiome influences on allergy in mice and humans. 7 Caballero S.

Pamer E.G. Microbiota-mediated inflammation and antimicrobial defense in the intestine. Although the physiology of virtually all organs is influenced by the microbiota [], the intestinal mucosa and its immune components are most affected by this symbiosis []. We first review recent findings elucidating the impact of the microbiota on the immune system. Second, we discuss the involvement of gut commensals in the pathogenesis of disease. Third, we examine the role of antibiotics in perturbing or driving these processes. Finally, we discuss the mechanisms of antibiotic resistance development and spread, as well as the proposed approaches to overcome the drawbacks of antibiotic therapy.

Beneficial Roles of the Microbiota 8 O’Hara A.M.

Shanahan F. The gut flora as a forgotten organ. short-chain fatty acids (SCFAs) that feed enterocytes and modulate immune functions [ 2 Walter J.

Ley R. The human gut microbiome: ecology and recent evolutionary changes. 3 Brestoff J.R.

Artis D. Commensal bacteria at the interface of host metabolism and the immune system. 9 Sommer F.

Backhed F. The gut microbiota–masters of host development and physiology. 10 Kelly C.J.

et al. Crosstalk between microbiota-derived short-chain fatty acids and intestinal epithelial HIF augments tissue barrier function. 11 Reinhardt C.

et al. Tissue factor and PAR1 promote microbiota-induced intestinal vascular remodelling. 9 Sommer F.

Backhed F. The gut microbiota–masters of host development and physiology. 12 Luna R.A.

Foster J.A. Gut brain axis: diet microbiota interactions and implications for modulation of anxiety and depression. 13 Mayer E.A.

et al. Gut/brain axis and the microbiota. 14 Mu C.

et al. Gut microbiota: the brain peacekeeper. 15 Collins S.M.

et al. The interplay between the intestinal microbiota and the brain. The gut microbiota exerts many beneficial functions for the host, to a level that it can be considered an additional organ []. For example, commensal bacteria convert primary bile acids into secondary bile acids and they also produce vitamins of the B and K groups, and ferment otherwise indigestible plant-derived fibers producing(SCFAs) that feedand modulate immune functions []. Furthermore, the microbiota drives intestinal development by promoting vascularization, villus thickening, mucosal surface widening, mucus production, cellular proliferation, and maintenance of epithelial junctions []. Notably, the influence of the microbiota is not limited to the intestine, and affects the physiology of most host organs, even the brain []. One of the most prominent roles of the gut microbiota is to promote the development and education of the immune system, both locally and systemically, as described below. Education of the Immune System dextran sodium sulfate (DSS)-induced colitis in mice by depleting microbial ligands that normally signal through Toll-like receptors (TLRs) and function to ensure expression of tissue homeostasis and repair mediators [ 16 Rakoff-Nahoum S.

et al. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Box 2 Microbiota and the Immune System: Strategies to Ensure a Tricky Coexistence 136 Schluter J.

Foster K.R. The evolution of mutualism in gut microbiota via host epithelial selection. 137 Pickard J.M.

et al. Rapid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness. As a first challenge, the host must maintain in the gut lumen beneficial strains without allowing overproliferation of non-beneficial rapid growers. The epithelium has been proposed to act as a selectivity amplifier, causing major rearrangements in the composition of the microbial community through secretion of nutrients and reduced quantities of AMPs, and acting mainly on microbes that are closer to the mucosa []. Provision of nutrients is maintained at all cost. For instance, upon starvation–a natural reaction of animals following infection–a pathway signaling through DCs, ILCs, and IECs induces massive fucosylation of the intestinal epithelium in mice, thus providing nutrients to commensal bacteria and maintaining colonization resistance to C. rodentium infection []. 12 colony forming units (CFU)/ml, and the host immune system is well suited to react. Indeed, breakdown of the intestinal barrier upon infection or DSS treatment of mice has been shown to result in a complete effector and memory immune response against commensal flagellin [ 138 Hand T.W.

et al. Acute gastrointestinal infection induces long-lived microbiota-specific T cell responses. As a second challenge, immune recognition of commensal antigens and cues must be blunted. The luminal bacterial load, a few micrometers in distance, is as high as 10colony forming units (CFU)/ml, and the host immune system is well suited to react. Indeed, breakdown of the intestinal barrier upon infection or DSS treatment of mice has been shown to result in a complete effector and memory immune response against commensal flagellin []. 139 Johansson M.E.

et al. Normalization of host intestinal mucus layers requires long-term microbial colonization. +CD11c+ mononuclear phagocytes in the intestine, thus reducing transport of bacterial antigens to the MLNs and preventing triggering of adaptive immune responses against luminal antigens, a tolerogenic mechanism that can be broken by antibiotic treatment [ 140 Diehl G.E.

et al. Microbiota restricts trafficking of bacteria to mesenteric lymph nodes by CX 3 CR1hi cells. 141 Hepworth M.R.

et al. Innate lymphoid cells regulate CD4+ T-cell responses to intestinal commensal bacteria. 142 Hepworth M.R.

et al. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4+ T cells. 141 Hepworth M.R.

et al. Innate lymphoid cells regulate CD4+ T-cell responses to intestinal commensal bacteria. 142 Hepworth M.R.

et al. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4+ T cells. 142 Hepworth M.R.

et al. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4+ T cells. Under normal physiological conditions, mucus creates a physical barrier that keeps most bacteria at a safe distance from the epithelium, mainly through diffusion of AMPs and IgA in the small intestine and the formation of a tight, impenetrable structure in the large intestine []. At steady-state, commensal microbes provide signals that dampen the activity of CX3CR1CD11cmononuclear phagocytes in the intestine, thus reducing transport of bacterial antigens to the MLNs and preventing triggering of adaptive immune responses against luminal antigens, a tolerogenic mechanism that can be broken by antibiotic treatment []. Moreover, type 3 ILCs in the intestine blunt the immune response against the microbiota by inducing apoptosis of commensal-specific T cells through an MHCII-dependent antigen presentation process that resembles thymic negative selection []. Deletion of the MHCII gene on ILCs has been shown to result in a T cell-dependent inflammatory disease modeling IBD colitis, which could be ameliorated by antibiotic treatment []. Of note, human ILC3s have also been found to express MHCII. ILC3s from pediatric patients with Crohn's disease (IBD) were found to present reduced HLA-DR expression, and HLA-DR levels negatively correlated with the number of Th17 cells in colon biopsies from these patients []. 143 Fanning S.

et al. Bifidobacterial surface-exopolysaccharide facilitates commensal–host interaction through immune modulation and pathogen protection. 99 Cullen T.W.

et al. Antimicrobial peptide resistance mediates resilience of prominent gut commensals during inflammation. Finally, commensals can also adopt strategies to ‘hide’. Some abundant strains of bifidobacteria alternatively produce two variants of cell surface-associated exopolysaccharide, decreasing their chances of being recognized by the immune system []. B. thetaiotaomicron reduces the negative charge of LPS through phosphatase activity, thus acquiring resistance to positively-charged AMPs []. Figure 1 Roles of the Microbiota in the Development and Maintenance of the Intestinal Immune System. The gut microbiota is separated from the intestinal epithelium by a thin layer of mucus that is secreted by Goblet cells in a microbiota- and NLRP6-dependent manner. The mucus layer has a different structure in small and large intestine (not depicted in the figure). Microbe-associated molecular patterns (MAMPS) can be sensed by intestinal epithelial cells (IECs), as well as by myeloid cells in the lamina propria (LP), and induce a variety of effects including tissue repair and the production of antimicrobial peptides (AMPs) and proteins such as RegIIIγ in intestinal epithelial and Paneth cells through a dendritic cell (DC)–innate lymphoid cell (ILC) axis. Luminal ATP and serum amyloid A (SAA)/IL-1β produced by IECs and DCs in response to adhesion of segmented filamentous bacteria (SFB) promote type 17 T helper cell (Th17) development. Antigens presented during this process are largely derived from SFB. Regulatory T cell (Treg) induction is also regulated by bacterial cues. Clostridia of the IV and XIVa groups induce Tregs in a TGF-β-dependent manner. Short-chain fatty acids (SCFA) promote Treg differentiation by acting directly on T cells and indirectly on DCs. Macrophages (MΦ) in the LP are involved in a pathway that includes also ILCs (not shown), resulting in the production of IL-10 and retinoic acid (RA), and also sustaining the expansion of Tregs. All pathways illustrated in the figure were shown to be affected by the use of antibiotics, leading to a lack of homeostasis, increased sensitivity to infection, and an increase in the severity of various conditions, as in the case of allergy. Figure 2 Antibiotic-Mediated Microbiota Depletion Causes Disease in Multiple Organs. Antibiotics act on the gut microbiota by decreasing its density and modifying its composition in a long-lasting fashion. This causes reduced signaling to the intestinal mucosa and peripheral organs, which results in impaired functioning of the immune system. Depicted are examples of diseases that were shown to arise or be worsened as a consequence of antibiotic treatment in mouse models (see main text). MΦ, macrophages; NK, natural killer cells. The close proximity of dense microbial populations to host tissues poses risks of invasion, and the immune system must thoroughly monitor bacteria present in the gut lumen ( Box 2 ). Nonetheless, the microbiota is allowed to prosper on the surface of the intestinal mucosa, orchestrating the overall physiology of the tissue lying underneath. This concept was established with the observation that antibiotic treatment worsens the severity ofin mice by depleting microbial ligands that normally signal through(TLRs) and function to ensure expression of tissue homeostasis and repair mediators [] ( Figure 1 Figure 2 ). Paneth cells. RegIII-γ is not detected in germ-free (GF) mice [ 17 Cash H.L.

et al. Symbiotic bacteria direct expression of an intestinal bactericidal lectin. 18 Brandl K.

et al. Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits. +CD11b+ dendritic cells (DCs) in the lamina propria (LP) contributes to maintenance of RegIII-γ expression. Upon TLR5 signaling, DCs produce IL-23, promoting IL-22 release by innate lymphoid cells (ILCs), and therefore RegIII-γ expression in intestinal epithelial cells (IECs) [ 19 Kinnebrew M.A.

et al. Bacterial flagellin stimulates Toll-like receptor 5-dependent defense against vancomycin-resistant Enterococcus infection. 20 Kinnebrew M.A.

et al. Interleukin 23 production by intestinal CD103+CD11b+ dendritic cells in response to bacterial flagellin enhances mucosal innate immune defense. All branches of the immune system rely on this tonic signaling to properly function ( Figure 1 ). Microbiota-derived lipopolysaccharide (LPS) maintains basal level expression of RegIII-γ (a bactericidal C-type lectin) in intestinal epithelial cells (IECs) and. RegIII-γ is not detected in], and even short-term antibiotic treatment impairs its expression, rendering mice susceptible to vancomycin-resistant enterococcus (VRE) infection, a defect that can be reverted by oral administration of LPS []. Similarly, commensal flagellin sensing by TLR5 on CD103CD11b(DCs) in the(LP) contributes to maintenance of RegIII-γ expression. Upon TLR5 signaling, DCs produce IL-23, promoting IL-22 release by(ILCs), and therefore RegIII-γ expression in intestinal epithelial cells (IECs) []. 22 Clarke T.B.

et al. Recognition of peptidoglycan from the microbiota by Nod1 enhances systemic innate immunity. 23 Deshmukh H.S.

et al. The microbiota regulates neutrophil homeostasis and host resistance to Escherichia coli K1 sepsis in neonatal mice. 22 Clarke T.B.

et al. Recognition of peptidoglycan from the microbiota by Nod1 enhances systemic innate immunity. 23 Deshmukh H.S.

et al. The microbiota regulates neutrophil homeostasis and host resistance to Escherichia coli K1 sepsis in neonatal mice. type 2 T helper cell (Th2)–IL-4–IgE pathway, which leads to exacerbated allergic syndromes [ 24 Hill D.A.

et al. Commensal bacteria-derived signals regulate basophil hematopoiesis and allergic inflammation. Granulocytes also receive commensal cues, which they sense while residing in the bone marrow (BM). NOD1-mediated sensing of meso-diaminopimelic acid (DAP) promotes neutrophil-mediated killing of pathogens such as Staphylococcus aureus and Streptococcus pneumoniae []. In GF mice reconstituted with Escherichia coli, DAP was detected in the blood and BM over the course of 3 days, showing that bacterial ligands from the intestinal lumen can have systemic distribution, and therefore a systemic effect []. Moreover, perinatal antibiotic exposure alters neutrophil number and functions by impairing G-CSF and IL-17 production, predisposing neonates to increased risk of E. coli or Klebsiella pneumoniae-induced sepsis []. Similarly, antibiotic treatment perturbs the basophil compartment in the blood and BM of mice by acting through a(Th2)–IL-4–IgE pathway, which leads to exacerbated allergic syndromes []. inflammasome-dependent manner [ 25 Ichinohe T.

et al. Microbiota regulates immune defense against respiratory tract influenza A virus infection. Professional antigen-presenting cells (APCs) also rely on the microbiota to orchestrate the immune response. For instance, DC migration and IL-1β/IL-18 production have been shown to be impaired in antibiotic-treated mice infected with influenza virus in an-dependent manner []. Immunoglobulin levels, T cell numbers, and IFN-γ production were consequently affected, resulting in increased viral titers. However, rectal stimulation with TLR agonists restored lung immunity in this study, indicating that microbial signals from the intestine can modulate and re-establish systemic immunity. + T cell expansion, as well as in IFN-γ/TNF-α and IgG production [ 26 Abt M.C.

et al. Commensal bacteria calibrate the activation threshold of innate antiviral immunity. 27 Ganal S.C.

et al. Priming of natural killer cells by nonmucosal mononuclear phagocytes requires instructive signals from commensal microbiota. 27 Ganal S.C.

et al. Priming of natural killer cells by nonmucosal mononuclear phagocytes requires instructive signals from commensal microbiota. Antibiotics have been shown to impair immunity against lymphocytic choriomeningitis virus (LCMV) in mice by lowering the expression of antiviral genes such as Ifnb and Irf7 in lung macrophages, resulting in defects in CD8T cell expansion, as well as in IFN-γ/TNF-α and IgG production []. Another study reported that splenic macrophages from GF and antibiotic-treated mice also failed to prime natural killer (NK) cells because of reduced chromatin accessibility in the promoter region of genes encoding cytokines such as type I IFNs (α, β), IL-6, and TNF-α []. As a result, antibiotic-treated mice failed to control infection with mouse cytomegalovirus (MCMV) []. With regard to adaptive immune cells, the activity of T lymphocytes is severely impaired by disruption of the dialogue between the immune system and the microbiota, as in the case of antibiotic exposure. + T lymphocytes in lymphoid organs, and an increased ratio of Th2 cells to type 1 T helper cells (Th1) [ 28 Mazmanian S.K.

et al. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. 28 Mazmanian S.K.

et al. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. type 17 T helper cells (Th17) is particularly interconnected with the activity of luminal bacteria. Indeed, Th17 differentiation in the small intestine (SI) LP depends on the microbiota; GF mice have a reduced Th17 compartment that is restored by introduction of fecal material from conventional mice [ 29 Ivanov I.I.

et al. Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. 30 Atarashi K.

et al. ATP drives lamina propria T H 17 cell differentiation. + bacilli residing in the terminal ileum of some mouse strains [ 31 Ivanov I.I.

et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. 32 Gaboriau-Routhiau V.

et al. The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses. +-targeting antibiotics impairs Th17 development in mice [ 29 Ivanov I.I.

et al. Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. 29 Ivanov I.I.

et al. Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. GF mice exhibit aberrant spleen architecture, decreased numbers of CD4T lymphocytes in lymphoid organs, and an increased ratio of Th2 cells to(Th1) []. Reconstitution of mice with Bacteroides fragilis, an abundant commensal, reverts these defects via immunogenic presentation of the zwitterionic polysaccharide A (PSA) and priming of splenic Th1 cells []. The biology of(Th17) is particularly interconnected with the activity of luminal bacteria. Indeed, Th17 differentiation in the small intestine (SI) LP depends on the microbiota; GF mice have a reduced Th17 compartment that is restored by introduction of fecal material from conventional mice []. Of note, ATP, likely of commensal origin, contributes to the conversion of naïve T cells into Th17 in the intestinal LP []. Prominent inducers of Th17 differentiation are segmented filamentous bacteria (SFB), spore-forming anaerobic Grambacilli residing in the terminal ileum of some mouse strains []. Accordingly, treatment with Gram-targeting antibiotics impairs Th17 development in mice []. Mice reconstituted with SFB-lacking microbiota develop a reduced intestinal Th17 compartment and are therefore more susceptible to Clostridium rodentium challenge, a model for human enterohemorrhagic Escherichia coli infections []. 21 Yang Y.

et al. Focused specificity of intestinal TH17 cells towards commensal bacterial antigens. 33 Sano T.

et al. An IL-23R/IL-22 circuit regulates epithelial serum amyloid a to promote local effector Th17 responses. 34 Atarashi K.

et al. Th17 cell induction by adhesion of microbes to intestinal epithelial cells. Commensal bacteria are detected by T cells, and most mouse LP Th17 T cells recognize SFB-derived antigens []. Physical interactions between SFB and enterocytes, to which the bacteria are tightly anchored, are required for the induction of Th17-polarizing molecules [serum amyloid A (SAA), IL-1β] []. Regulatory T cells (Tregs) have a fundamental protective role against autoimmune and chronic inflammatory diseases, such as inflammatory bowel disease (IBD). Commensal sensing by LP-resident phagocytes promotes release of retinoic acid (RA) and IL-10 in mice, leading to the generation and expansion of Tregs, a mechanism that is important in establishing tolerance to food antigens [ 35 Mortha A.

et al. Microbiota-dependent crosstalk between macrophages and ILC3 promotes intestinal homeostasis. (Tregs) have a fundamental protective role against autoimmune and chronic inflammatory diseases, such as inflammatory bowel disease (IBD). Commensal sensing by LP-resident phagocytes promotes release of retinoic acid (RA) and IL-10 in mice, leading to the generation and expansion of Tregs, a mechanism that is important in establishing tolerance to food antigens []. 36 Atarashi K.

et al. Induction of colonic regulatory T cells by indigenous Clostridium species. 37 Atarashi K.

et al. Treg induction by a rationally selected mixture of clostridia strains from the human microbiota. 38 Arpaia N.

et al. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. 39 Furusawa Y.

et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. 40 Smith P.M.

et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. 38 Arpaia N.

et al. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. 39 Furusawa Y.

et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. 40 Smith P.M.

et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. 38 Arpaia N.

et al. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. 39 Furusawa Y.

et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. 40 Smith P.M.

et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. cathelicidin-related antimicrobial peptide (CRAMP) production in pancreatic β cells in mice, promoting a regulatory fate in macrophages, which enhanced the priming of Tregs [ 41 Sun J.

et al. Pancreatic beta-cells limit autoimmune diabetes via an immunoregulatory antimicrobial peptide expressed under the influence of the gut microbiota. non-obese diabetic (NOD) mice ameliorated diabetes, while antibiotic treatment favored priming of diabetogenic T cells, thus promoting disease progression. Female NOD mice displayed lower SCFA production than did male NOD mice, and their CRAMP levels could be restored by transfer of microbiota from males, resulting in protection from diabetes. This falls in line with findings from a previous report showing that microbiota from males could partially protect NOD females from diabetes by inducing high levels of circulating testosterone, glycerophospholipids, and sphingolipids [ 42 Markle J.G.

et al. Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity. 43 Fox H.S. Androgen treatment prevents diabetes in nonobese diabetic mice. 44 Roden A.C.

et al. Augmentation of T cell levels and responses induced by androgen deprivation. 45 Kissick H.T.

et al. Androgens alter T-cell immunity by inhibiting T-helper 1 differentiation. Commensal clostridia belonging to clusters IV and XIVa have been shown to promote accumulation of Tregs in the colon by increasing TGF-β production in IECs, thus protecting mice from DSS-induced colitis and strengthening oral tolerance []. A follow up study described a protective consortium of 17 clostridia strains isolated from a human fecal sample, a community with therapeutic potential for allergic or inflammatory diseases such as colitis []. Recently, three different laboratories have identified microbiota-derived SCFAs (particularly propionate and butyrate) to be responsible for Treg differentiation/accumulation []. Indeed, SCFAs were present at reduced concentrations in the fecal pellets of GF or antibiotic-treated mice, and oral administration of SCFAs protected mice from T cell-induced colitis by inducing immunosuppressive Tregs []. SCFAs were found to act directly on T cells via the receptor GPCR43, thus enhancing FoxP3 expression through DNA acetylation and, on DCs, by conferring a higher capacity to drive naïve T cell differentiation into Tregs []. Notably, microbiota-produced SCFAs can also promote tolerance in non-intestinal tissue. SCFAs were reported to drive-related antimicrobial peptide (CRAMP) production in pancreatic β cells in mice, promoting a regulatory fate in macrophages, which enhanced the priming of Tregs []. In this study, CRAMP treatment ofameliorated diabetes, while antibiotic treatment favored priming of diabetogenic T cells, thus promoting disease progression. Female NOD mice displayed lower SCFA production than did male NOD mice, and their CRAMP levels could be restored by transfer of microbiota from males, resulting in protection from diabetes. This falls in line with findings from a previous report showing that microbiota from males could partially protect NOD females from diabetes by inducing high levels of circulating testosterone, glycerophospholipids, and sphingolipids []. Androgens are in fact known to limit diabetes [], possibly by reducing T cell proliferation and polarization towards a Th1 cell fate []. 46 Trompette A.

et al. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Finally, SCFAs can also promote T cell tolerance in allergic diseases models. Mice fed a high-fiber diet have shown increased levels of circulating SCFAs; in a model of intranasal challenge with house dust mite (HDM) extract, the SCFAs prevented lung allergic inflammation by acting on the propionate receptor GPR41, promoting the development of phagocytes with reduced Th2-polarizing capacity []. Collectively, these murine studies show that commensal bacteria contribute to the maintenance of immune tolerance in the host, principally through induction of regulatory T cells. 25 Ichinohe T.

et al. Microbiota regulates immune defense against respiratory tract influenza A virus infection. 26 Abt M.C.

et al. Commensal bacteria calibrate the activation threshold of innate antiviral immunity. B1 cells in mouse spleen and for the maintenance of basal levels of circulating IgM, which exert a protective role in the cecal ligation and puncture (CLP)-induced sepsis model [ 47 Proietti M.

et al. ATP-gated ionotropic P2X7 receptor controls follicular T helper cell numbers in Peyer's patches to promote host-microbiota mutualism. B cell-mediated immunity is also affected by perturbation of the microbiota. In fact, immunoglobulin titers are decreased in mice treated with antibiotics before viral infection []. Furthermore, sensing of commensal-derived LPS has been shown to be necessary for the development ofin mouse spleen and for the maintenance of basal levels of circulating IgM, which exert a protective role in the cecal ligation and puncture (CLP)-induced sepsis model []. unadjuvanted vaccines were reported to induce IgG and IgM production in a TLR5-dependent manner through the microbiota in mice and, possibly, humans, as suggested by the correlation between leukocyte TLR5 expression levels and anti-influenza antibody titers in cohorts of vaccinees [ 48 Oh J.Z.

et al. TLR5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination. −/−) mice exhibited defective antibody production, but administration of the TLR5 agonist flagellin restored the humoral response. TLR5 signaling had an effect on short-lived plasma cells, enhancing survival/differentiation and antibody production, as well as on lymph node (LN) medullary cord macrophages, leading to the production of plasma cell-sustaining factors such as TNF-α, IL-6, and APRIL. Surprisingly, gut microbes are capable of modulating vaccination outcomes. In a recent study, TIV (influenza trivalent inactivated) and otherwere reported to induce IgG and IgM production in a TLR5-dependent manner through the microbiota in mice and, possibly, humans, as suggested by the correlation between leukocyte TLR5 expression levels and anti-influenza antibody titers in cohorts of vaccinees []. TIV-challenged antibiotic-treated (and TLR5-deficient, Tlr5) mice exhibited defective antibody production, but administration of the TLR5 agonist flagellin restored the humoral response. TLR5 signaling had an effect on short-lived plasma cells, enhancing survival/differentiation and antibody production, as well as on lymph node (LN) medullary cord macrophages, leading to the production of plasma cell-sustaining factors such as TNF-α, IL-6, and APRIL. Box 3 The Gut Microbiota Can Modulate the Efficacy of Anticancer Therapeutics + T cell responses, as well as the production of reactive oxygen species (ROS) by myeloid cells [ 144 Iida N.

et al. Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment. Recent publications have suggested an unexpected role for commensal bacteria in modulating the effectiveness of anticancer drug-based and antibody-based therapies. For instance, antibiotic treatment has been shown to impair responses to intratumoral CpG-oligodeoxynucleotide (ODN) injection or oxaliplatin administration in mice by reducing the induction of necrosis (via TNF-α), the magnitude of CD8T cell responses, as well as the production of reactive oxygen species (ROS) by myeloid cells []. Commensal taxa have been positively (e.g., Alistipes and Ruminocuccus) or negatively (e.g., Lactobacillus) associated with anticancer responses in this model. 145 Viaud S.

et al. The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide. Along the same lines, the antitumor alkylating agent cyclophosphamide (CTX) has been shown to cause intestinal damage and augmented epithelial permeability in mice, thus promoting translocation of commensals to MLNs and spleen (particularly L. johnsonii and E. hirae) []. In this study, IFN-γ-producing Th17 cells (pTh17) responsive to these bacteria were generated in CTX-treated (but not control) mice. Transfer of these cells rescued immune responses in vancomycin-treated CTX-treated mice that were otherwise unable to arrest tumor growth, suggesting that commensal-specific pTh17 generated as a consequence of CTX treatment were largely responsible for the anticancer effect of this drug. 146 Sivan A.

et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. + T cell response. In another study, checkpoint blockade anti-PDL1 therapeutic efficacy was improved by the presence or transfer of Bifidobacterium (but not Lactobacillus) in a mouse model of melanoma []. In this model, bacterial translocation was not observed, but the commensal bacteria improved the functionality of DCs, upregulating the expression of cytokines as well as T cell activation-related genes (e.g., antigen presentation, cross-presentation, and costimulatory molecules), thus inducing a more potent anti-tumor CD8T cell response. 147 Vetizou M.

et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. 147 Vetizou M.

et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Similarly to PDL1, the efficacy of CTLA4 blockade (ipilimumab), was strongly impaired in GF or antibiotic-treated mice, with reduced T cell proliferation and IFN-γ/TNF-α production in several cancer models (melanoma, sarcoma, colon carcinoma) []. Anti-CTLA4 treatment induced T cell-mediated intestinal damage and altered the microbiota by increasing B. thataiotaomicron, B. uniformis, and Burkholderiaceae representation []. GF mice reconstituted with these but not other species (E. coli, L. plantarum, B. diastonis, E. hirae) recovered full responses to anti-CTLA4 administration as a result of the generation of bacterium-specific IFN-γ-producing T cells. Notably, T cells with the same bacterial specificity could be recovered from patients, and melanoma tumor-ridden mice reconstituted with human stool enriched in protective species exhibited a high incidence of tumor regression. 147 Vetizou M.

et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. 148 Dubin K.

et al. Intestinal microbiome analyses identify melanoma patients at risk for checkpoint-blockade-induced colitis. 148 Dubin K.

et al. Intestinal microbiome analyses identify melanoma patients at risk for checkpoint-blockade-induced colitis. Of note, development of colitis upon ipilimumab administration, a common adverse effect observed in cancer patients undergoing treatment, was reduced in tumor-bearing mice reconstituted with B. fragilis and B. crepacia []. Accordingly, a recent analysis of a cohort of patients undergoing ipilimumab treatment identified members of the Bacteroidetes phylum as being significantly increased in subjects who did not develop colitis []. Polyamine transport and B vitamin biosynthesis modules were also enriched in these subjects, and were successfully used as high-sensitivity and -specificity molecular markers to predict the risk of a patient to develop colitis. This analysis thus provided a potentially useful microbiota-based diagnostic tool []. Overall, these works demonstrate that the microbiota contributes to the efficacy of anticancer treatment and the development of adverse reactions. However, whether antibiotic use in the context of cancer treatment affects outcome, remains an important albeit unanswered question. The described studies provide substantial demonstration of a strong influence of the gut microbiota on the immune response. This connection is currently being investigated in the context of different pathological conditions and therapeutic approaches. In this regard it is of particular interest that the gut microbiota has been recently proposed to modulate the efficacy of anticancer therapies ( Box 3 ), rapidly becoming an area of active research. Colonization Resistance colonization resistance, which can be severely impaired by antibiotic treatment [ 4 Buffie C.G.

Pamer E.G. Microbiota-mediated colonization resistance against intestinal pathogens. antimicrobial peptides [AMPs]) [ 18 Brandl K.

et al. Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits. 19 Kinnebrew M.A.

et al. Bacterial flagellin stimulates Toll-like receptor 5-dependent defense against vancomycin-resistant Enterococcus infection. Nod-like receptor (NLR) protein–and increased susceptibility to oral C. rodentium infection in mice [ 49 Wlodarska M.

et al. Antibiotic treatment alters the colonic mucus layer and predisposes the host to exacerbated Citrobacter rodentium-induced colitis. 50 Wlodarska M.

et al. NLRP6 inflammasome orchestrates the colonic host-microbial interface by regulating goblet cell mucus secretion. The microbiota can confer protection against pathogens, a phenomenon referred to as, which can be severely impaired by antibiotic treatment []. Colonization resistance takes place through direct (not requiring host involvement) or indirect mechanisms (ultimately mediated by the host response, as mentioned above for the modulation of) []. Mucus production is another mediator of indirect colonization resistance. Treatment with metronidazole, which selectively depletes anaerobes, but not streptomycin, reduced the thickness of the inner mucus layer in the large intestine (LI)–a process that depends on the NLRP6and increased susceptibility to oral C. rodentium infection in mice []. 51 Fan D.

et al. Activation of HIF-1alpha and LL-37 by commensal bacteria inhibits Candida albicans colonization. Another example of colonization resistance is that mediated by commensal anaerobes such as Blautia producta and B. thetaiothaomicron, that confer trans-kingdom resistance to C. albicans in a mouse model of oral infection by inducing expression of the hypoxia inducible factor HIF1-α and production of the antimicrobial peptide CRAMP (LL-37) []. 52 Fukuda S.

et al. Bifidobacteria can protect from enteropathogenic infection through production of acetate. Bifidobacterium longum has been shown to protect mice from enterohemorrhagic E. coli O157 infection through the production of SCFAs that prevent IEC apoptosis and inflammation, thus preserving gut epithelium integrity, and reducing the spread of Stx2 toxin into the bloodstream, ultimately improving host survival []. 53 van Nood E.

et al. Duodenal infusion of donor feces for recurrent Clostridium difficile. 54 Buffie C.G.

et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. 54 Buffie C.G.

et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. In humans, recurrent infection with C. difficile occurs in hospitalized patients, in particular those undergoing antibiotic treatment, causing diseases ranging from mild diarrhea to deadly toxic megacolon. Colonization resistance against C. difficile presents itself in healthy individuals, and transfer of healthy microbiota is currently being performed to treat patients with recurrent infections []. Of note, the commensal bacterium C. scindens can inhibit growth of C. difficile through the generation of secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) []. Reconstitution with C. scindens alone or within a bacterial consortium protected antibiotic-treated mice from C. difficile intestinal colonization. In this study, C. scindens was also associated to protection from C. difficile colonization in patients receiving allogeneic hematopoietic stem-cell transplantation []. 55 Hsiao A.

et al. Members of the human gut microbiota involved in recovery from Vibrio cholerae infection. bacteriocin bac-21 were shown to outcompete VRE in a model where colonization was established upon continuous administration of the pathogen in drinking water to mice harboring an intact microbiota [ 56 Kommineni S.

et al. Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract. By combining the study of microbiota recovery in human samples from infected patients, and GF mouse reconstitution, Ruminococcus obeum was found to provide direct colonization resistance against Vibrium cholerae (responsible for cholera). Through the LuxS-based AI-2 quorum-sensing signaling system, whose expression is enhanced in the presence of V. cholerae, R. obeum induces downregulation of virulence and colonization factors in the pathogen, promoting its clearance []. Moreover, enterococci carrying a plasmid encodingbac-21 were shown to outcompete VRE in a model where colonization was established upon continuous administration of the pathogen in drinking water to mice harboring an intact microbiota []. Thus, taken from these examples, it is clear that commensals can directly or indirectly protect from pathogens, and antibiotic use severely impairs this function, which in turn increases the risk of host infection.

Gut Microbiota and Pathogenesis The microbiota can contribute to a variety of diseases through different mechanisms, including the production of noxious catabolites and the capacity to overgrow, to sustain inflammation, or to provide support for pathogens. Many factors, such as diet, underlying inflammation, and dysbiosis, can modulate such pathogenic potential. In the next section we discuss recent literature on the role of gut commensal bacteria in disease pathogenesis. The Microbiota in Diet-Promoted Disease 57 David L.A.

et al. Diet rapidly and reproducibly alters the human gut microbiome. 58 Suez J.

et al. Artificial sweeteners induce glucose intolerance by altering the gut microbiota. microbiome in modulating glycemic responses has been recently demonstrated with the development of an effective machine-learning algorithm to predict postprandial glucose levels in humans based on several personal as well as biochemical parameters, including the composition of the microbiota in healthy individuals [ 59 Zeevi D.

et al. Personalized nutrition by prediction of glycemic responses. Diet profoundly influences gut microbiota composition and functions [], an effect that can promote disease. Consumption of artificial sweeteners in mice and humans has been shown to lead to deregulation of the intestinal microbiota, with altered glycan degradation capacity promoting decreased glucose tolerance, a pre-diabetic condition []. An important role for thein modulating glycemic responses has been recently demonstrated with the development of an effective machine-learning algorithm to predict postprandial glucose levels in humans based on several personal as well as biochemical parameters, including the composition of the microbiota in healthy individuals []. In this study, personalized dietary interventions adopted on the basis of algorithm predictions significantly altered microbiota composition while improving glycemic control, suggesting that modulation of microbiome configuration through diet could be used to prevent diabetes-predisposing conditions. 60 Karlsson F.H.

et al. Gut metagenome in European women with normal, impaired and diabetic glucose control. 61 Qin J.

et al. A metagenome-wide association study of gut microbiota in type 2 diabetes. 62 Forslund K.

et al. Disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota. 63 Lewis J.D.

et al. Inflammation, antibiotics, and diet as environmental stressors of the gut microbiome in pediatric Crohn's disease. A correlation between particular bacterial taxa and type 2 diabetes has been proposed based on analyses of human gut metagenomes []. However, a recent study suggested that patient treatment with the drug metformin, a variable that subjects had not been stratified for previously, constituted the main determinant of microbial signatures in diabetic cohorts []. This finding demonstrates that multiple variables can influence gut microbiota composition in disease []. conventionalization (i.e., microbiota transfer by feeding fecal material) [ 64 Turnbaugh P.J.

et al. An obesity-associated gut microbiome with increased capacity for energy harvest. 65 Ridaura V.K.

et al. Gut microbiota from twins discordant for obesity modulate metabolism in mice. Gut microbiota plays a key role in the pathogenesis of obesity. Indeed, experiments have shown that the condition can be transferred from obese mice or humans to healthy mice via(i.e., microbiota transfer by feeding fecal material) []. These studies strongly suggest that diet is responsible for the selection of commensal strains with enhanced energy-harvesting capacity. 66 Yoshimoto S.

et al. Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. 67 Belcheva A.

et al. Gut microbial metabolism drives transformation of MSH2-deficient colon epithelial cells. Obese mice harbor an altered microbiota enriched in clostridia that produces high quantities of DCA, contributing to liver inflammation and predisposing the host to an increased risk of developing hepatic cancer []. Low-fat diet, antibiotic treatment, or pharmacological inhibition of microbial conversion of primary to secondary bile acids has had a protective effect in this model. Along similar lines, in a mouse model of colorectal cancer, microbiota-derived butyrate enhanced intestinal epithelial cell proliferation as well as the occurrence of intestinal polyps and tumor development []. In this report, low-carbohydrate diet or treatment with selected antibiotics could prevent polyp formation. 68 Koeth R.A.

et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. 69 Wang Z.

et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. 70 Tang W.H.

et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. −/− mice, and disease was ameliorated by antibiotic treatment [ 68 Koeth R.A.

et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. 69 Wang Z.

et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. 68 Koeth R.A.

et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. 70 Tang W.H.

et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. 68 Koeth R.A.

et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. 71 Wang Z.

et al. Non-lethal Inhibition of gut microbial trimethylamine production for the treatment of atherosclerosis. Furthermore, a series of recent studies [] revealed a link between microbiota and atherogenesis. Dietary phosphatydilcholine (PC), a lipid broadly present in foods, and L-carnitine (LC), a molecule abundant in red meat, are transformed by the microbiota into pro-atherogenic trimethylamine (TMA). LC- or PC-supplemented diets were found to induce higher atheroma formation and wider aortic lesions in Apoemice, and disease was ameliorated by antibiotic treatment []. Also in humans, PC or LC administration augmented TMA plasma levels and increased the risk for cardiovascular disease; notably, antibiotic treatment dramatically reduced TMA levels in these subjects []. Vegan or vegetarian subjects exhibited a reduced or non-existent capacity to produce TMA upon LC supplementation, and it was proposed that bacteria, presumably selected through the diet of the host, were incapable of performing this biochemical conversion []. Overall, these studies convincingly demonstrate a prominent role for the gut microbiota in diet-driven pathogenesis of atherosclerosis. Interestingly, a follow-up study showed that feeding mice an analog of choline, which inhibits TMA production by blocking microbial TMA lyases, could prevent atherosclerosis in mice []. Thus, the microbiota represents a promising target for pharmacological therapeutic intervention in atherosclerosis. 72 Haiser H.J.

et al. Predicting and manipulating cardiac drug inactivation by the human gut bacterium Eggerthella lenta. Commensal bacteria can also impair drug activity. Eggerthella lenta, a human commensal, reduces digoxin, a drug used to prevent heart failure and arrhythmia, thus destroying its biological activity. This activity is enhanced in the presence of other commensals, but is repressed by arginine, which can be supplemented through a high-protein diet []. The Microbiota in Infection, Inflammation, and Aberrant Immunity 73 Elinav E.

et al. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. A strong link between the microbiota and intestinal inflammation has been established. For instance, it has been shown that NLRP6-deficient mice harbor a microbiota that is more prone to induce colitis upon DSS treatment, a phenotype that could be transferred to healthy wild-type mice upon cohousing, which promotes microbiota transfer []. 74 Winter S.E.

et al. Host-derived nitrate boosts growth of E. coli in the inflamed gut. 74 Winter S.E.

et al. Host-derived nitrate boosts growth of E. coli in the inflamed gut. +Ly6 Chi monocytes via release of the hemolysin A toxin [ 75 Seo S.U.

et al. Distinct commensals induce interleukin-1beta via NLRP3 inflammasome in inflammatory monocytes to promote intestinal inflammation in response to injury. 76 Ayres J.S.

et al. Lethal inflammasome activation by a multidrug-resistant pathobiont upon antibiotic disruption of the microbiota. 76 Ayres J.S.

et al. Lethal inflammasome activation by a multidrug-resistant pathobiont upon antibiotic disruption of the microbiota. Upon dysbiosis or inflammation, pathobionts lodged among healthy commensal species can promote disease. For instance, like other Enterobacteriaceae, E. coli can utilize nitrate as an electron acceptor for respiration, which confers a selective growth advantage following iNOS (Nos2) gene activation upon DSS-induced inflammation in mice []. This has been proposed to be a leading cause of inflammation-related dysbiosis []. As another example, DSS-induced intestinal damage has been demonstrated to allow the human and mouse commensal Proteus mirabilis to engage the NLRP3 inflammasome on mouse CCR2Ly6 Cmonocytes via release of the hemolysin A toxin []. Consequently, NLRP3 activation leads to production of IL-1β, fostering inflammation and inducing colitis which can be ameliorated by antibiotic treatment. By contrast, antibiotic treatment has itself been reported to promote sepsis in mice in at least one model of DSS-induced colitis, by inducing overgrowth of the antibiotic resistant commensal strain of E. coli O21:H+, which carries a virulence gene cluster []. Upon expansion and DSS-induced epithelial damage, this pathobiont could enter the circulation and induce IL-1β production by macrophages, thus promoting sepsis []. 77 Pacheco A.R.

et al. Fucose sensing regulates bacterial intestinal colonization. 77 Pacheco A.R.

et al. Fucose sensing regulates bacterial intestinal colonization. 78 Ng K.M.

et al. Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens. gnotobiotic mice, B. thetaiothaomicron-liberated sialic acid can support the expansion of pathogens such as C. difficile or S. enterica upon oral infection. Accordingly, pathogen burden is reduced upon infection of mice monocolonized with a sialidase-deficient B. thetaiothaomicron strain [ 78 Ng K.M.

et al. Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens. 78 Ng K.M.

et al. Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens. The microbiota can favor or amplify the activity of pathogens, and antibiotics play an important role in this process. Enterohemorrhagic E. coli carries a fucose-sensing system (FusKR) that regulates virulence gene expression and therefore competition with commensals []. In vitro, B. thetaiotaomicron can cleave off fucose from mucin and enhance virulence of E. coli. In vivo, E. coli lacking FusKR are outcompeted by a wild-type strain []. These data suggest that, in vivo, B. thetaiotaomicron liberates fucose, thus fueling pathogenic E. coli growth. In addition, B. thetaiotaomicron also carries sialidases that can cleave sialic acid from mucus, although the bacterium lacks the enzymatic machinery necessary to catabolize such sugar []. In mono-associated, B. thetaiothaomicron-liberated sialic acid can support the expansion of pathogens such as C. difficile or S. enterica upon oral infection. Accordingly, pathogen burden is reduced upon infection of mice monocolonized with a sialidase-deficient B. thetaiothaomicron strain []. In conventional mice, antibiotics reduce the amount of commensals competing for sugars liberated before treatment, increasing nutrient availability and favoring pathogen expansion through a limited time-window (peaking at day 1 and ending at day 3 after antibiotic treatment for this study) []. +CD103+ DCs to adipose tissues rather than to mesenteric lymph notes (MLNs) [ 79 Fonseca D.M.

et al. Microbiota-dependent sequelae of acute infection compromise tissue-specific immunity. 79 Fonseca D.M.

et al. Microbiota-dependent sequelae of acute infection compromise tissue-specific immunity. Commensal bacteria have also been implicated in long-term sequelae from resolved Yersinia pseudotuberculosis infection in conventional mice, such as those resulting in leakage of gut-draining lymphatic vessels and deviated trafficking of CD11bCD103DCs to adipose tissues rather than to(MLNs) []. This ‘immunological scar’ has been found to impair mucosal immune responses as well as oral tolerance to food antigens. DC migration was not impaired in GF mice, and was restored in conventional mice upon antibiotic treatment, indicating a role for the microbiota in promoting scar formation []. 80 Horai R.

et al. Microbiota-dependent activation of an autoreactive T cell receptor provokes autoimmunity in an immunologically privileged Site. 80 Horai R.

et al. Microbiota-dependent activation of an autoreactive T cell receptor provokes autoimmunity in an immunologically privileged Site. Finally, the gut microbiota has also been associated with the induction of autoimmunity. For instance, R161H mice bear transgenic T cells that recognize the retinal photoreceptor RBP3 and spontaneously develop autoimmune uveitis over time. However, because RBP3 is confined to the eye, which is an immune-privileged site, it has been unclear how autoreactive T cells could encounter the cognate antigen and be activated before migrating to the eye and promoting inflammation. Recent data have emerged to suggest that crossreactivity to unidentified commensal antigens triggers an autoimmune response in this model []. Indeed, effector autoreactive T cells could also be detected in R161H mice lacking RBP3, especially in the intestinal LP, suggesting a non-endogenous source for cognate peptide. Autoreactive T cells responded to stimulation with cecal content. Importantly, the onset of uveitis in R161H mice could be delayed upon antibiotic treatment []. Collectively, the discussed data demonstrate that the gut microbiota, especially with dysbiosis, can promote a variety of pathological conditions. In some of these cases, targeted treatments to selectively deplete the involved pathogenic species may be considered as a promising therapeutic approach.

Antibiotic Treatment Induces Long-Lasting Changes in the Microbiota that Correlate with Disease 81 Jernberg C.

et al. Long-term ecological impacts of antibiotic administration on the human intestinal microbiota. 82 Buffie C.G.

et al. Profound alterations of intestinal microbiota following a single dose of clindamycin results in sustained susceptibility to Clostridium difficile-induced colitis. Numerous studies have confirmed that antibiotics have a tremendous impact on the composition and functionality of the human microbiota. One study documented that healthy volunteers treated for 1 week or less with antibiotics reported effects on their bacterial flora that persisted 6 months to 2 years after treatment, including a dramatic loss in diversity as well as in representation of specific taxa, insurgence of antibiotic-resistant strains, and upregulation of antibiotic resistance genes (ARGs) []. Antibiotic treatment in mice recapitulates the impact and long-term shifts in human gut communities. For example, a single dose of clindamycin has been shown to induce profound changes in mouse microbiota composition and, consequently, to confer long-lasting susceptibility to C. difficile infection []. − commensals can be depleted by vancomycin, which is a Gram+-targeting drug [ 83 Ubeda C.

et al. Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes bloodstream invasion in humans. 84 Lewis B.B.

et al. Loss of microbiota-mediated colonization resistance to Clostridium difficile infection with oral vancomycin compared with metronidazole. One potential explanation for the magnitude and duration of antibiotic effects in vivo is the remarkable interdependence of different bacterial taxa. For instance, Gramcommensals can be depleted by vancomycin, which is a Gram-targeting drug []. As a consequence of its profound effect on gut autochthonous (native) communities, vancomycin has been shown to cause long-lasting susceptibility to a variety of secondary infections in both humans and mice []. Microbiota development during early life stages of humans and mice is crucial, and its perturbation predisposes to disease in later infancy or adulthood, particularly in the case of allergic and metabolic syndromes. 85 Dominguez-Bello M.G.

et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. 85 Dominguez-Bello M.G.

et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. 86 Dominguez-Bello M.G.

et al. Partial restoration of the microbiota of cesarean-born infants via vaginal microbial transfer. For instance, whereas vaginally delivered neonates acquire bacteria through the birth canal, the microbiota of infants delivered by C-section resembles that populating the skin of adults, with streptococci being a dominant genus []. It has been posited that this could represent a factor predisposing individuals to subsequent infections []. Notably, swabbing newborns delivered by C-section with gauze incubated in the vagina has been shown to partially restore a normal microbiota, albeit with effects that remain to be ascertained []. 87 Kozyrskyj A.L.

et al. Increased risk of childhood asthma from antibiotic use in early life. 88 Risnes K.R.

et al. Antibiotic exposure by 6 months and asthma and allergy at 6 years: findings in a cohort of 1,401 US children. 89 Korpela K.

et al. Intestinal microbiome is related to lifetime antibiotic use in Finnish pre-school children. 90 Russell S.L.

et al. Early life antibiotic-driven changes in microbiota enhance susceptibility to allergic asthma. vertical acquisition of microbiota [ 91 Cox L.M.

et al. Altering the intestinal microbiota during a critical developmental window has lasting metabolic consequences. Surveys on thousands of children have highlighted a link between the use of antibiotics during the first year of life and development of asthma by years 6–7 []. Early use of macrolides in Finnish children was found to generate a distinct microbial profile characterized by a loss of Actinobacteriaceae and an increase in Bacteroidetes and Proteobacteria, an induction of ARGs, and a decrease in bile salt hydrolases. This profile positively correlated with either a later development of asthma or an increase in body mass index []. Concordant with these findings, studies in mice have shown that neonatal (from pre-birth) but not adult exposure to antibiotics resulted in exacerbated asthma following intranasal challenge with ovalbumin []. Along similar lines, low-dose penicillin (LDP) administration was shown to induce stronger physiological alterations if administered from the beginning of gestation, rather than at weaning, confirming a crucial role forof microbiota []. In particular, following early LDP administration, body mass and fat content were increased in adulthood, while the expression of intestinal immune genes coding for proteins such as RegIII-γ, β-defensins, and IL-17 was decreased. Therefore, antibiotic exposure, even for short periods of time and especially during infancy, has long-lasting effects on the microbiota, and this can predispose the host to a variety of diseases, some of which remain to be potentially identified. This evidently represents a matter of crucial importance for public health.

Generation of Antibiotic Resistance: The Driving Forces 92 Levy S.B.

et al. High frequency of antimicrobial resistance in human fecal flora. 93 Sommer M.O.

et al. Functional characterization of the antibiotic resistance reservoir in the human microflora. 94 Santiago-Rodriguez T.M.

et al. Gut microbiome of an 11th Century A.D. pre-Columbian Andean mummy. 95 D’Costa V.M.

et al. Antibiotic resistance is ancient. 96 Bhullar K.

et al. Antibiotic resistance is prevalent in an isolated cave microbiome. 95 D’Costa V.M.

et al. Antibiotic resistance is ancient. 96 Bhullar K.

et al. Antibiotic resistance is prevalent in an isolated cave microbiome. Antibiotic-resistant pathogens are a major public health burden. However, ARGs are highly represented not only in such pathogens but also among human commensal bacteria. An early survey suggested that a sizable fraction of the anaerobe compartment within the microbiota of healthy subjects is resistant to one or multiple antibiotics, with the proportion of such bacteria increasing following antibiotic treatment []. A more recent metagenomic analysis of the gut microbiota obtained from two healthy subjects estimated that multidrug-resistant species accounted for 20% of total bacteria []. As discussed below, exogenous antibiotics can contribute considerably to such accumulation of ARGs. However, antimicrobial molecules and resistance mechanisms are abundant in any bacterial community, and play an important evolutionary and regulatory role. Accordingly, ARGs were identified in microbiota from an 11th century AD mummy [], in a 30 000 year-old permafrost sediment [], as well as in a cave in New Mexico that had been isolated for millions of years []; none of these environments could have been influenced by the presence of modern-day drugs. Surprisingly, both the DNA sequences and the structural organization within AR operons identified in these ancient bacteria were found to exhibit high similarity to those carried by currently circulating microbes []. Furthermore, antibiotic resistance to multiple classes (up to 14) of antibiotics, even to semi-synthetic molecules, was also identified in these prehistoric samples []. 97 Modi S.R.

et al. Antibiotics and the gut microbiota. 98 Oldenburg M.

et al. TLR13 recognizes bacterial 23S rRNA devoid of erythromycin resistance-forming modification. 99 Cullen T.W.

et al. Antimicrobial peptide resistance mediates resilience of prominent gut commensals during inflammation. 99 Cullen T.W.

et al. Antimicrobial peptide resistance mediates resilience of prominent gut commensals during inflammation. Figure 3 Generation and Spread of Antibiotic Resistance. (Left) Antibiotic resistance (ABX) is generated by mutations that can be induced by several driving forces. From the top: competition of bacteria (inter- or intra-species, here depicted as intersections between circles representing population niches) mainly mediated by bacteriocins, induces a selective pressure that favors development of resistance. Some Toll-like receptors (TLRs) or host-derived antimicrobial peptides (AMPs) target bacterial molecules that can undergo mutations and provide resistance to clinically-relevant antibiotics. Exogenous antibiotic pressure through medical or industrial practices (e.g., antibiotic use in livestock) promotes the generation and selection of resistant strains that can rapidly diffuse. (Right) Antibiotic resistance genes (ARGs) can be exchanged among bacteria also of different species (not shown) through horizontal gene transfer, in other words conjugation, transduction, or transformation. Notably, these three mechanisms are all enhanced upon antibiotic exposure, resulting in a faster and more efficient spread of ARGs in the gut as well as in the environment. It is recognized that at least three major forces drive the development and spread of antibiotic resistance in the host or in the environment: (i) immune recognition and response, (ii) bacterial competition within communities, and (iii) exogenous antibiotic pressure [] ( Figure 3 ). Antibiotic resistance can arise in bacteria that inhabit animal body sites as a means to escape host defense strategies. For instance, erythromycin resistance gained by mutation of the 23s rRNA gene was found to concurrently confer resistance to recognition by TLR13, which also happens to bind the same molecular target, and is likely to exert selective pressure in vivo []. The commensal B. thethaiotaomicron is highly resistant to the cationic peptide polymyxin B, which is considered a model for mammalian AMPs. Transposon mutation libraries in B. thethaiotaomicron have led to the identification of the bacterial gene lpxF, which encodes a phosphatase acting on LPS to diminish its negative charge, and consequently impairing polymyxin B ligation to the bacterium []. Notably, upon DSS- or C. rodentium-induced inflammation, lpxF-deficient bacteria were outcompeted by wild-type strains in mouse intestines, suggesting that bacterial resistance can be both induced and maintained by the immune response []. 95 D’Costa V.M.

et al. Antibiotic resistance is ancient. 96 Bhullar K.

et al. Antibiotic resistance is prevalent in an isolated cave microbiome. 100 Koch G.

et al. Evolution of resistance to a last-resort antibiotic in Staphylococcus aureus via bacterial competition. 2+ concentration medium, which favors biofilm formation, has been shown to induce the rapid generation of a mutated strain (W) that attempts to outcompete the wild-type strain (O) through secretion of surfactant molecules and the bacteriocin Bsa [ 100 Koch G.

et al. Evolution of resistance to a last-resort antibiotic in Staphylococcus aureus via bacterial competition. 100 Koch G.

et al. Evolution of resistance to a last-resort antibiotic in Staphylococcus aureus via bacterial competition. The development of antibiotic resistance in natural environments, as a result of competition among different bacterial taxa, is a well-documented event []. Intra-species competition, however, is sufficient to drive the acquisition of resistance []. Growth of methicillin-resistant S. aureus (MRSA) in a high Mgconcentration medium, which favors biofilm formation, has been shown to induce the rapid generation of a mutated strain (W) that attempts to outcompete the wild-type strain (O) through secretion of surfactant molecules and the bacteriocin Bsa []. Increased Bsa concentrations drive in turn the selection and expansion of a resistant S. aureus strain (Y) and, because Bsa and vancomycin bind to a common target within the cell wall, the Y strain acquires resistance to the antibiotic, resulting in greater virulence in infected mice []. 101 Looft T.

et al. In-feed antibiotic effects on the swine intestinal microbiome. 102 Forsberg K.J.

et al. The shared antibiotic resistome of soil bacteria and human pathogens. Shotgun sequencing through an ARG-dedicated platform (PARFuMS) has in fact led to the identification of DNA cassettes conferring resistance to seven different classes of antibiotics in soil bacteria, exhibiting perfect identity in coding and non-coding regions to genes encoded by common human pathogens such as Pseudomonas aeruginosa and Salmonella enterica. Some cassettes were flanked by transposable elements, strongly suggesting horizontal gene transfer (HGT) between soil-resident and pathogenic bacteria [ 102 Forsberg K.J.

et al. The shared antibiotic resistome of soil bacteria and human pathogens. Box 4 Broad Use and Misuse of Antibiotics 110 Costello E.K.

et al. The application of ecological theory toward an understanding of the human microbiome. 116 Laxminarayan R. Antibiotic effectiveness: balancing conservation against innovation. Since the beginning of their commercial distribution in the 1940s, antibiotics have proved to be an invaluable weapon against infectious agents, saving millions of lives. However, their use has broadened to a level that raises many concerns. In fact, it is estimated that a sizable proportion of the human population (1–3%) makes use of antibiotics every day []. The overall administration of medical antibiotics has increased by more than 30% in the decade 2001–2011, and last-resort antibiotic use is alarmingly frequent. This has ignited a vicious circle in which the augmented use of antibiotics and the development of antibiotic resistance fuel one another, leading to the return of previously well-controlled threats such as gonorrhea and Enterobacteriaceae infection []. 149 Chang Q.

et al. Antibiotics in agriculture and the risk to human health: how worried should we be?. 150 Meek R.W.

et al. Nonmedical uses of antibiotics: time to restrict their use?. 151 Kennedy D. Time to deal with antibiotics. 150 Meek R.W.

et al. Nonmedical uses of antibiotics: time to restrict their use?. Antibiotics are broadly employed in farming practices at low doses with the main aim of enhancing animal growth []. Livestock account for the vast majority of antibiotic production and use in the USA (80% in 2013), resulting in the selection of resistant bacteria that can either infect humans or horizontally transfer resistance to pathogens. The use of antibiotics for livestock has been widely criticized and the FDA has responded by introducing Guidance #213 in 2013, which recommends avoidance of unnecessary antibiotics. This remains, however, a voluntary policy. Of note, it has been calculated that the ban of this procedure would not significantly increase costs for producers or consumers []. Finally, and most importantly, exogenous administration of antibiotics to a host can induce rapid expansion of ARGs ( Box 4 ). In one study, pigs were fed a diet supplemented with a growth-enhancing antibiotic cocktail for 2 weeks. Subsequently, their microbiota showed increased expression of ARGs, conferring tolerance even to drugs that were not administered during the study []. That type of occurrence is of particular concern because drug-resistance genes can be horizontally transferred from soil bacteria to pathogens [].through an ARG-dedicated platform (PARFuMS) has in fact led to the identification of DNA cassettes conferring resistance to seven different classes of antibiotics in soil bacteria, exhibiting perfect identity in coding and non-coding regions to genes encoded by common human pathogens such as Pseudomonas aeruginosa and Salmonella enterica. Some cassettes were flanked by transposable elements, strongly suggesting horizontal gene transfer (HGT) between soil-resident and pathogenic bacteria []. β-lactams being the most widely prescribed antibiotic to patients in the same period and region [ 103 Hu Y.

et al. The abundance of antibiotic resistance genes in human guts has correlation to the consumption of antibiotics in animal. Interestingly, a correlation was suggested between the increased use of tetracycline in Danish livestock and the prevalence of tetracycline-resistance genes in Danish human subject microbiomes, despitebeing the most widely prescribed antibiotic to patients in the same period and region []. 104 Field W.

Hershberg R. Alarmingly high segregation frequencies of quinolone resistance alleles within human and animal microbiomes are not explained by direct clinical antibiotic exposure. 105 Moore A.M.

et al. Gut resistome development in healthy twin pairs in the first year of life. 106 Lu N.

et al. DNA microarray analysis reveals that antibiotic resistance-gene diversity in human gut microbiota is age related. A detailed analysis of dozens of metagenomic datasets obtained from hosts (humans, animals) or environments (water, soil, etc.) has identified housekeeping alleles of genes such as rpsL, rpoB, gyrA that confer resistance to three classes of broad-spectrum antibiotics, segregating at high frequency []. Of note, nearly 40% of host-associated bacteria carried quinolone-resistance genes, even in subjects never exposed clinically. The ‘resistome’ is defined as the pool of all ARGs present in a given microbiome, and appears early in life, being detectable in infants as early as 2 months after birth []. Interestingly, twins display remarkably greater resistome similarity to each other than to unrelated infants or their mothers, suggesting that vertical transmission does not play a crucial role in its establishment. Moreover, the resistome expands over time because the number of ARGs within the human microbiome positively correlates with age []. Together, these data provide evidence that antibiotic resistance is naturally present in bacterial communities, including the gut microbiota, where it can be induced by several driving forces. However, exposure to exogenous antibiotics via medical or industrial practices has a marked capacity to enhance the insurgence and spread of ARGs.

Antibiotic Resistance Generation: De Novo Mutations 107 Toprak E.

et al. Evolutionary paths to antibiotic resistance under dynamically sustained drug selection. 107 Toprak E.

et al. Evolutionary paths to antibiotic resistance under dynamically sustained drug selection. 107 Toprak E.

et al. Evolutionary paths to antibiotic resistance under dynamically sustained drug selection. As previously discussed, antibiotics exert a selective pressure that drives rapid development of resistant strains. This process generally requires multiple DNA mutations. To understand how such mutations are acquired, in one study E. coli cultures were challenged with increasing doses of three different antibiotics for 20 days in vitro []. Antibiotic resistance was found to arise following similar or identical mutational patterns in replicate experiments; mutations affecting the same, or functionally-analogous genes, consistently emerged in bacterial cultures treated with a given drug []. Mutations also occurred in multidrug-resistance genes. Furthermore, antibiotics with a common molecular target also induced resistance to one another. Thus, it appears that there is an evolutionary trajectory in the acquisition of mutations conferring antibiotic resistance []. 108 Lee H.H.

et al. Bacterial charity work leads to population-wide resistance. 108 Lee H.H.

et al. Bacterial charity work leads to population-wide resistance. 109 Kelsic E.D.

et al. Counteraction of antibiotic production and degradation stabilizes microbial communities. 109 Kelsic E.D.

et al. Counteraction of antibiotic production and degradation stabilizes microbial communities. Importantly, within a clonal bacterial population under antibiotic pressure, not all cells acquire resistance. ‘Bacterial charity work’ has been described as a mechanism to enhance the resistance of the whole community with respect to a single resistant clone in an initially homogeneous population []. This study documented that, within an E. coli culture exposed to norfloxacin or gentamicin, only a few cells rapidly acquired resistance-conferring mutations. Surprisingly, the antibiotic-resistant cells did not outcompete the antibiotic-sensitive members of the community, but instead kept them alive. In fact, cells acquiring resistance to the drug also gained the ability (normally inhibited by antibiotics) to secrete indole, which acted on neighboring cells by activating defense mechanisms such as drug efflux pumps and oxidative-stress responses, ultimately allowing the survival of drug-sensitive bacteria. This mechanism resulted in higher growth rates of the entire population when compared to those of isolated resistant clones, which displayed lower proliferative capacity owing to the metabolic cost of acquired mutations []. This study highlighted how the acquisition of antibiotic resistance modifies ecological interactions within a homogeneous bacterial community, contributing to its own well-being. In well-mixed environments where different microbial taxa coexist in close proximity, distinct strains generally have the capacity to produce and to inactivate specific sets of antibacterial molecules. In this scenario, it has been proposed that leaky protection conferred by an antibiotic-degrading species to other surrounding bacteria is of public benefit []. In fact, ecological modeling has shown that, in a bacterial consortium where every bacterial strain protects only itself from the action of a given antibiotic produced by a competing species (’rock, paper, scissors’ model), fluctuations in the composition of the consortium increase, giving rise to a very unstable community that is ultimately dominated by one strain. By contrast, if protection against a bactericidal molecule is provided in a leaky manner to surrounding species–by secretion of antibiotic-degrading enzymes in outer spaces, for instance–the overall community composition is stabilized over time, becoming less subject to fluctuations []. Thus, antibiotic resistance plays a strong role in regulating microbial interactions and shaping microbial communities, possibly representing a prominent factor in the ecological regulation of the gut microbiota.

Resistance Genes Are Spread via HGT 97 Modi S.R.

et al. Antibiotics and the gut microbiota. 110 Costello E.K.

et al. The application of ecological theory toward an understanding of the human microbiome. 97 Modi S.R.

et al. Antibiotics and the gut microbiota. 110 Costello E.K.

et al. The application of ecological theory toward an understanding of the human microbiome. Bacteria can exchange ARGs via HGT. Specifically, HGT includes: (i) transformation, the acquisition of DNA fragments from the environment; (ii) conjugation, the delivery of genetic material from one cell to another through a pilus; and (iii) transduction, an exchange mediated by bacteriophages []. All three mechanisms have been implicated in the transfer of ARGs ( Figure 3 ) []. 111 Hehemann J.H.

et al. Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota. Generally speaking, HGT can take place among commensals, environmental bacteria or, importantly, between the two. For instance, a porphyranase necessary to process specific seaweed carbohydrates originally carried by a marine Bacteroidetes, Zobellia galactanivorans, was also found in commensal bacteria from Japanese but not North American subjects []. Because Japanese diets are highly enriched in seaweed containing porphyran, this work strongly implicated HGT between environmental and gut commensal bacterial species. 112 Schloissnig S.

et al. Genomic variation landscape of the human gut microbiome. HGT of ARGs occurs at high rates among intestinal bacteria. In fact, clindamycin resistance transfer factor gene (btgA) has been identified as carrying the highest number of single-nucleotide polymorphisms (SNPs) within 200 human gut metagenomes, and other conjugal transfer factors have followed in this ranking []. Interestingly, all three mechanisms of HGT seem to be upregulated in bacteria upon antibiotic treatment ( Figure 3 ). Conjugation 113 Beaber J.W.

et al. SOS response promotes horizontal dissemination of antibiotic resistance genes. Ciprofloxacin induces DNA damage-related SOS stress responses in E. coli, leading to a 100-fold enhancement in conjugative transfer of SXT, a genetic element conferring resistance to several antibiotics. Notably, in recent decades the SXT element has emerged, commonly presenting itself in V. cholerae isolates from patients, potentially a result of antibiotic-induced resistance transfer []. Transduction 114 Modi S.R.

et al. Antibiotic treatment expands the resistance reservoir and ecological network of the phage metagenome. 114 Modi S.R.

et al. Antibiotic treatment expands the resistance reservoir and ecological network of the phage metagenome. The bacteriophage metagenome (or ‘phageome’) recovered from the intestinal content of mice treated with ciprofloxacin or ampicillin is highly enriched in ARGs []. Furthermore, bacteriophages recovered from antibiotic-treated mice have been shown to be significantly more capable of transferring resistance to cultured microbiota ex vivo []. Phage genes conferring bacterial survival advantages under stress conditions (e.g., DNA repair enzymes), or an increase in fitness (e.g., carbohydrate degradation) were also enriched upon antibiotic treatment, suggesting that the ‘phageome’ can represent a reservoir for bacterial adaptation genes to be spread in times of need. Transformation 115 Slager J.

et al. Antibiotic-induced replication stress triggers bacterial competence by increasing gene dosage near the origin. 115 Slager J.

et al. Antibiotic-induced replication stress triggers bacterial competence by increasing gene dosage near the origin. Antibiotics and drugs interfering with DNA replication induce competence in bacteria, allowing the acquisition of ARGs and other genetic material from the environment []. Treatment of S. pneumoniae with ciprofloxacin or HPUra (an inhibitor of DNA replication) has been shown to stall the progression of the DNA replication fork without inhibiting the initiation of DNA replication []. This has been reported to result in increased copy numbers and, consequently, enhanced transcription of genes neighboring the origin of replication. Genes encoding competence functions are located in this region and, consequently, they are upregulated in the presence of these drugs, promoting a state of competence. Thus, ARGs spread among bacteria through conjugation, transduction, and transformation, and this process is promoted by antibiotic exposure. Because the human intestine is densely colonized with an amazing variety of microbes, antibiotic therapies are likely to promote diffusion of resistance genes within the microbiota, an aspect of antibiotic therapy that should not be neglected.

Moving Beyond Antibiotics Figure 4 Novel Approaches to Substitute or Complement Antibiotic Therapies. Antibiotic treatment depletes commensal communities in the gut, decreases mucus layer thickness and the expression of antimicrobial peptides (AMPs), and predisposes to infection (upper panel). Transfer of microbiota by fecal transplantation (FMT, fecal microbiota transplantation) can restore a healthy microbiota, mucus production, AMP secretion, and provide colonization resistance against pathogens, that consequently can no longer expand or are cleared. Transfer of selected bacterial communities, as shown in mouse models, can achieve the same effect. Similarly, administration of microbial ligands, here depicted as fragments of bacteria, can restore basal production of mucus and AMPs following antibiotic treatment. (Lower panel) Strategies to selectively deplete pathogens without perturbing the microbiota. All the approaches illustrated have proved to be successful in mouse models, providing high levels of protection and leaving the composition of the surrounding communities unaltered. Consequently, colonization resistance mechanisms can be potentially preserved. Abbreviations, Ab, antibody; ABX, antibiotic resistance. Considering the important roles of the microbiota in regulating host physiology, and the multiple drawbacks of antibiotic use discussed above, finding alternative or complementary strategies to fight infections is imperative. Different promising approaches have been proposed to tackle this problem ( Figure 4 ). 116 Laxminarayan R. Antibiotic effectiveness: balancing conservation against innovation. First and foremost, reforming or establishing a set of complementary public health measures can greatly diminish the need for antibiotic use. As pointed out by Laxminarayan [], improving sanitation, expanding the use of vaccines, and strengthening hospital infection control have proved to be extremely effective tools in reducing the needs for antibiotic therapies. Moreover, reducing the unnecessary use of these molecules in farming practices must also be considered ( Box 4 ). 53 van Nood E.

et al. Duodenal infusion of donor feces for recurrent Clostridium difficile. 117 Kelly C.R.

et al. Fecal microbiota transplant for treatment of Clostridium difficile infection in immunocompromised patients. 54 Buffie C.G.

et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. 118 Lawley T.D.

et al. Targeted restoration of the intestinal microbiota with a simple, defined bacteriotherapy resolves relapsing Clostridium difficile disease in mice. 119 Reeves A.E.

et al. Suppression of Clostridium difficile in the gastrointestinal tracts of germfree mice inoculated with a murine isolate from the family Lachnospiraceae. 54 Buffie C.G.

et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. 83 Ubeda C.

et al. Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes bloodstream invasion in humans. 120 Ubeda C.

et al. Intestinal microbiota containing Barnesiella species cures vancomycin-resistant Enterococcus faecium colonization. 56 Kommineni S.

et al. Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract. Colonization resistance through microbiota transfer can substitute for antibiotics by restoring commensal communities ( Figure 4 ). Fecal microbiota transplantation (FMT) has been shown to be more effective than conventional antibiotic therapy in the treatment of patients with recurrent C. difficile infection []. Bacterial strains [] and metabolites [] associated with protection from C. difficile have now been identified, paving the way for eventual replacement of fecal material with selected probiotic strains or effector molecules. VRE colonizes the intestine following antibiotic treatment, before spreading to the bloodstream, and thus, represents a major threat for hospitalized patients []. In one study, transplantation of fecal microbiota containing Barnesiella species effectively cleared intestinal VRE from colonized mice, a promising finding that requires further investigation []. Enterococci carrying the conjugative plasmid pPD1, encoding bacteriocin bac-21, were shown to outcompete VRE from mice colonized with the pathogen []. However, in this model, the plasmid was acquired also by commensal bacteria, raising the possibility of pPD1 conjugative transfer to VRE itself, and therefore suggesting that caution should be taken with the clinical application of this approach. 18 Brandl K.

et al. Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits. 19 Kinnebrew M.A.

et al. Bacterial flagellin stimulates Toll-like receptor 5-dependent defense against vancomycin-resistant Enterococcus infection. 20 Kinnebrew M.A.

et al. Interleukin 23 production by intestinal CD103+CD11b+ dendritic cells in response to bacterial flagellin enhances mucosal innate immune defense. 121 Abt M.C.

et al. TLR-7 activation enhances IL-22-mediated colonization resistance against vancomycin-resistant enterococcus. Bacterial ligand administration following microbiota depletion can protect mice from infection. Systemic administration of the TLR5 agonist flagellin, or oral administration of the TLR4 agonist LPS, were shown to reinstate resistance to VRE or C. difficile in mice previously treated with antibiotics, through restoration of RegIII-γ production []. Moreover, R848, a synthetic agonist of TLR7/8, orally delivered to mice, was reported to protect from VRE colonization []. Notably, R848 is already used in the clinics to treat papillomavirus infections, although it is topically administered. Thus, controlled administration of microbial ligands represents an important potential means by which to restore basal innate immune status and protection in hospitalized subjects undergoing antibiotic treatment. 122 Donia M.S.

et al. A systematic analysis of biosynthetic gene clusters in the human microbiome reveals a common family of antibiotics. 122 Donia M.S.

et al. A systematic analysis of biosynthetic gene clusters in the human microbiome reveals a common family of antibiotics. 123 Howe J.A.

et al. Selective small-molecule inhibition of an RNA structural element. 123 Howe J.A.

et al. Selective small-molecule inhibition of an RNA structural element. Computational platforms and high-throughput screenings are currently being exploited in the quest for novel antimicrobial molecules. Screening of 2000 bacterial genomes from the human microbiome project have identified biosynthetic gene clusters (BGCs) encoding thiopeptides, carried by commensals present at multiple anatomical locations []. Lactocillin, a thiopeptide encoded by one such cluster (i.e., bgc66 from L. gasseri), has been produced and characterized, showing strong inhibitory activity against common pathogens such as S. aureus and G. vaginalis, but not against commensals []. Moreover, a high-throughput screening of small molecules recently identified an inhibitor of riboflavin synthesis, ‘ribocil’, that targets a regulatory non-coding region (i.e., riboswitch) in the mRNA for a synthase involved in the pathway, RibB []. Ribocil binding inhibits translation of the ribB transcript in E. coli, inducing cell death by riboflavin starvation. Accordingly, ribocil administration, although only at high concentrations, significantly reduced bacterial burden in a mouse model of systemic infection with E. coli, suggesting that novel molecular targets may have promising potential for drug development []. 124 Rea M.C.

et al. Effect of broad- and narrow-spectrum antimicrobials on Clostridium difficile and microbial diversity in a model of the distal colon. 125 Gebhart D.

et al. A modified R-type bacteriocin specifically targeting Clostridium difficile prevents colonization of mice without affecting gut microbiota diversity. Recognition of the marked effect of antibiotics on microbiota composition has led to the search for a set of more narrow-spectrum bactericidal compounds. For instance, thuricin-CD, a bacteriocin produced by Bacillus thuringiensis, has been shown to be as effective as vancomycin or metronidazole against C. difficile without impacting on microbiota composition in a fecal-culture system that models the human colon []. Along the same lines, the bacteriocin avidocin-CDs, engineered to selectively target the clinically-relevant C. difficile strain BI/NAP1/027, has been reported to promote pathogen clearance in mice upon oral administration. Furthermore, avidocin-CDs, unlike antibiotics, was not found to interfere with the ability of the microbiota to provide colonization resistance []. 126 Lehar S.M.

et al. Novel antibody-antibiotic conjugate eliminates intracellular S. aureus. 126 Lehar S.M.

et al. Novel antibody-antibiotic conjugate eliminates intracellular S. aureus. Exploiting the principle of selective targeting, a recent study cleverly conjugated an analog of rifampicin (i.e., rifalogue) and an S. aureus-specific antibody to target intracellular MRSA []. Indeed, intracellular MRSA represents an important pathogen reservoir that is shielded from antibiotic action. Mice challenged with infected cells rather than bacterial particles were shown to present higher bacterial burdens despite vancomycin treatment []. In this study, rifalogue was conjugated to an S. aureus-specific antibody via a cleavable bridge such that opsonized MRSA, once phagocytosed, could be effectively killed by the protease-liberated antibiotic. Importantly, the Ab–rifalogue complex was more effective than uncoupled antibiotics in combating infection in an in vivo mouse model. This approach may in principle be suitable for many intracellular pathogens. 127 Bikard D.

et al. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. 128 Citorik R.J.

et al. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. 127 Bikard D.

et al. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. 128 Citorik R.J.

et al. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. 127 Bikard D.

et al. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. 128 Citorik R.J.

et al. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Finally, a strategy to eliminate selected bacterial targets exploiting the CRISPR/Cas9 system was recently proposed []. CRISPR/Cas9 is a bacterial immune system that can be easily engineered to cleave DNA sequences of interest. Phagemids (i.e., plasmids carrying the information to package phage particles) bearing CRISPR/Cas9 components were generated to cleave ARGs on chromosomes or plasmids of specific pathogens such as E. coli and S. aureus []. Phage-mediated delivery of this genetic material resulted in efficient killing of the bacteria in both cases. Importantly, no resistance developed against the phagemids, and the sensitivity of the system allowed discrimination of single polymorphic nucleotides. Accordingly, within a consortium of 2–3 bacterial isolates of the same strain, the CRISPR/Cas9 phagemids killed only bacteria carrying the target gene, sparing surrounding microbes in in vivo models, such as infection of moth larvae with E. coli and murine skin with S. aureus []. Thus, this system has allowed selective killing of pathogens, even antibiotic-resistant ones, witho