Metabolism of L-arabinose converges with virulence regulation to promote enteric pathogen fitness.

Virulence and metabolism are often interlinked to control the expression of essential colonisation factors in response to host-associated signals. Here, we identified an uncharacterised transporter of the dietary monosaccharide ʟ-arabinose that is widely encoded by the zoonotic pathogen enterohaemorrhagic Escherichia coli (EHEC), required for full competitive fitness in the mouse gut and highly expressed during human infection. Discovery of this transporter suggested that EHEC strains have an enhanced ability to scavenge ʟ-arabinose and therefore prompted us to investigate the impact of this nutrient on pathogenesis. Accordingly, we discovered that ʟ-arabinose enhances expression of the EHEC type 3 secretion system, increasing its ability to colonise host cells, and that the underlying mechanism is dependent on products of its catabolism rather than the sensing of ʟ-arabinose as a signal. Furthermore, using the murine pathogen Citrobacter rodentium, we show that ʟ-arabinose metabolism provides a fitness benefit during infection via virulence factor regulation, as opposed to supporting pathogen growth. Finally, we show that this mechanism is not restricted to ʟ-arabinose and extends to other pentose sugars with a similar metabolic fate. This work highlights the importance integrating central metabolism with virulence regulation in order to maximise competitive fitness of enteric pathogens within the host-niche.

Citrobacter rodentium possesses a functional type II secretion system necessary for successful host infection.

Infectious diarrheal diseases are the third leading cause of mortality in young children, many of which are driven by Gram-negative bacterial pathogens. To establish successful host infections these pathogens employ a plethora of virulence factors necessary to compete with the resident microbiota, and evade and subvert the host defenses. The type II secretion system (T2SS) is one such conserved molecular machine that allows for the delivery of effector proteins into the extracellular milieu. To explore the role of the T2SS during natural host infection, we used Citrobacter rodentium, a murine enteric pathogen, as a model of human intestinal disease caused by pathogenic Escherichia coli such as Enteropathogenic and Enterohemorrhagic E. coli (EPEC and EHEC). In this study, we determined that the C. rodentium genome encodes one T2SS and 22 potential T2SS-secreted protein effectors, as predicted via sequence homology. We demonstrated that this system was functional in vitro, identifying a role in intestinal mucin degradation allowing for its utilization as a carbon source, and promoting C. rodentium attachment to a mucus-producing colon cell line. During host infection, loss of the T2SS or associated effectors led to a significant colonization defect and lack of systemic spread. In mice susceptible to lethal infection, T2SS-deficient C. rodentium was strongly attenuated, resulting in reduced morbidity and mortality in infected hosts. Together these data highlight the important role of the T2SS and its effector repertoire during C. rodentium pathogenesis, aiding in successful host mucosal colonization.

Turicibacterales protect mice from severe Citrobacter rodentium infection.

One of the major contributors to child mortality in the world is diarrheal diseases, with an estimated 800,000 deaths per year. Many pathogens are causative agents of these illnesses, including the enteropathogenic or enterohemorrhagic forms of Escherichia coli. These bacteria are characterized by their ability to cause attaching and effacing lesions in the gut mucosa. Although much has been learned about the pathogenicity of these organisms and the immune response against them, the role of the intestinal microbiota during these infections is not well characterized. Infection of mice with E. coli requires pre-treatment with antibiotics in most mouse models, which hinders the study of the microbiota in an undisturbed environment. Using Citrobacter rodentium as a murine model for attaching and effacing bacteria, we show that C57BL/6 mice deficient in granzyme B expression are highly susceptible to severe disease caused by C. rodentium infection. Although a previous publication from our group shows that granzyme B-deficient CD4+ T cells are partially responsible for this phenotype, in this report, we present data demonstrating that the microbiota, in particular members of the order Turicibacterales, have an important role in conferring resistance. Mice deficient in Turicibacter sanguinis have increased susceptibility to severe disease. However, when these mice are co-housed with resistant mice or colonized with T. sanguinis, susceptibility to severe infection is reduced. These results clearly suggest a critical role for this commensal in the protection against enteropathogens.

Quantitative dose-response analysis untangles host bottlenecks to enteric infection.

Host bottlenecks prevent many infections before the onset of disease by eliminating invading pathogens. By monitoring the diversity of a barcoded population of the diarrhea causing bacterium Citrobacter rodentium during colonization of its natural host, mice, we determine the number of cells that found the infection by establishing a replicative niche. In female mice the size of the pathogen's founding population scales with dose and is controlled by a severe yet slow-acting bottleneck. Reducing stomach acid or changing host genotype modestly relaxes the bottleneck without breaking the fractional relationship between dose and founders. In contrast, disrupting the microbiota causes the founding population to no longer scale with the size of the inoculum and allows the pathogen to infect at almost any dose, indicating that the microbiota creates the dominant bottleneck. Further, in the absence of competition with the microbiota, the diversity of the pathogen population slowly contracts as the population is overtaken by bacteria having lost the critical virulence island, the locus of enterocyte effacement (LEE). Collectively, our findings reveal that the mechanisms of protection by colonization bottlenecks are reflected in and can be generally defined by the impact of dose on the pathogen's founding population.

Distinct Molecular Features of NleG Type 3 Secreted Effectors Allow for Different Roles during Citrobacter rodentium Infection in Mice.

The NleGs are the largest family of type 3 secreted effectors in attaching and effacing (A/E) pathogens, such as enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and Citrobacter rodentium. NleG effectors contain a conserved C-terminal U-box domain acting as a ubiquitin protein ligase and target host proteins via a variable N-terminal portion. The specific roles of these effectors during infection remain uncertain. Here, we demonstrate that the three NleG effectors-NleG1Cr, NleG7Cr, and NleG8Cr-encoded by C. rodentium DBS100 play distinct roles during infection in mice. Using individual nleGCr knockout strains, we show that NleG7Cr contributes to bacterial survival during enteric infection while NleG1Cr promotes the expression of diarrheal symptoms and NleG8Cr contributes to accelerated lethality in susceptible mice. Furthermore, the NleG8Cr effector contains a C-terminal PDZ domain binding motif that enables interaction with the host protein GOPC. Both the PDZ domain binding motif and the ability to engage with host ubiquitination machinery via the intact U-box domain proved to be necessary for NleG8Cr function, contributing to the observed phenotype during infection. We also establish that the PTZ binding motif in the EHEC NleG8 (NleG8Ec) effector, which shares 60% identity with NleG8Cr, is engaged in interactions with human GOPC. The crystal structure of the NleG8Ec C-terminal peptide in complex with the GOPC PDZ domain, determined to 1.85 Å, revealed a conserved interaction mode similar to that observed between GOPC and eukaryotic PDZ domain binding motifs. Despite these common features, nleG8Ec does not complement the ΔnleG8Cr phenotype during infection, revealing functional diversification between these NleG effectors.

Gastric acid and escape to systemic circulation represent major bottlenecks to host infection by Citrobacter rodentium.

The gastrointestinal (GI) environment plays a critical role in shaping enteric infections. Host environmental factors create bottlenecks, restrictive events that reduce the genetic diversity of invading bacterial populations. However, the identity and impact of bottleneck events on bacterial infection are largely unknown. We used Citrobacter rodentium infection of mice, a model of human pathogenic Escherichia coli infections, to examine bacterial population dynamics and quantify bottlenecks to host colonization. Using Sequence Tag-based Analysis of Microbial Populations (STAMP) we characterized the founding population size (Nb') and relatedness of C. rodentium populations at relevant tissue sites during early- and peak-infection. We demonstrate that the GI environment severely restricts the colonizing population, with an average Nb' of only 12-43 lineages (of 2,000+ inoculated) identified regardless of time or biogeographic location. Passage through gastric acid and escape to the systemic circulation were identified as major bottlenecks during C. rodentium colonization. Manipulating such events by increasing gastric pH dramatically increased intestinal Nb'. Importantly, removal of the stomach acid barrier had downstream consequences on host systemic colonization, morbidity, and mortality. These findings highlight the capability of the host GI environment to limit early pathogen colonization, controlling the population of initial founders with consequences for downstream infection outcomes.

Simulated Colonic Fluid Replicates the In Vivo Growth Capabilities of Citrobacter rodentium cpxRA Mutants and Uncovers Additive Effects of Cpx-Regulated Genes on Fitness.

Citrobacter rodentium is an attaching and effacing (A/E) pathogen used to model enteropathogenic and enterohemorrhagic Escherichia coli infections in mice. During colonization, C. rodentium must adapt to stresses in the gastrointestinal tract, such as antimicrobial peptides, pH changes, and bile salts. The Cpx envelope stress response (ESR) is a two-component system used by some bacteria to remediate stress by modulating gene expression, and it is necessary for C. rodentium pathogenesis in mice. Here, we utilized simulated colonic fluid (SCF) to mimic the gastrointestinal environment, which we show strongly induces the Cpx ESR and highlights a fitness defect specific to the ΔcpxRA mutant. While investigating genes in the Cpx regulon that may contribute to C. rodentium pathogenesis, we found that the absence of the Cpx ESR resulted in higher expression of the locus of enterocyte effacement (LEE) master regulator, ler, and that the genes yebE, ygiB, bssR, and htpX relied on CpxRA for proper expression. We then determined that CpxRA and select gene mutants were essential for proper growth in SCF when in the presence of extraneous stressors and in competition. Although none of the Cpx-regulated gene mutants exhibited marked virulence phenotypes in vivo, the ΔcpxRA mutant had reduced colonization and attenuated virulence, as previously determined, which replicated the in vitro growth phenotypes specific to SCF. Overall, these results indicate that the ΔcpxRA virulence defect is not due to any single Cpx regulon gene examined. Instead, attenuation may be the result of defective growth in the colonic environment resulting from the collective impact of multiple Cpx-regulated genes.

The type III secretion system effector network hypothesis.

Type III secretion system (T3SS) effectors are key virulence factors that underpin the infection strategy of many clinically important Gram-negative pathogens, including Salmonella enterica, Shigella spp., enteropathogenic and enterohemorrhagic Escherichia coli and their murine equivalent, Citrobacter rodentium. The cellular processes or proteins targeted by the effectors can be common to multiple pathogens or pathogen-specific. The main approach to understanding T3SS-mediated pathogenesis has been to determine the contribution of one effector at a time, with the aim of piecing together individual functions and unveiling infection mechanisms. However, in contrast to this prevailing approach, simultaneous deletion of multiple effectors revealed that they function as an interconnected network in vivo, uncovering effector codependency and context-dependent effector essentiality. This paradigm shift in T3SS biology is at the heart of this opinion article.

Citrobacter rodentium(ϕStx2dact), a murine infection model for enterohemorrhagic Escherichia coli.

The formation of attaching and effacing (A/E) lesions on intestinal epithelium, combined with Shiga toxin production, are hallmarks of enterohemorrhagic Escherichia coli (EHEC) infection that can lead to lethal hemolytic uremic syndrome. Although an animal infection model that fully recapitulates human disease remains elusive, mice orally infected with Citrobacter rodentium(ϕStx2dact), a natural murine pathogen lysogenized with an EHEC-derived Shiga toxin 2-producing bacteriophage, develop intestinal A/E lesions and toxin-dependent systemic disease. This model has facilitated investigation of how: (A) phage gene expression and prophage induction contribute to disease and are potentially triggered by antibiotic treatment; (B) virulence gene expression is altered by microbiota and the colonic metabolomic milieu; and (C) innate immune signaling is affected by Stx. Thus, the model provides a unique tool for accessing diverse aspects of EHEC pathogenesis.

Intimate Adhesion Is Essential for the Pathogen-Specific Inflammatory and Immune Responses in the Gut of Mice Infected with Citrobacter rodentium.

Citrobacter rodentium is a murine pathogenic bacterium that adheres to intestinal epithelial cells, resulting in loss of microvilli and pedestal formation, and alters multiple cellular processes, including actin dynamics. Translocated intimin receptor (Tir), one of its virulence factors, functions as receptor for intimin, a bacterial adhesin, thereby mediating bacterial adhesion to epithelial cells. Although robust immune responses are induced to eliminate pathogenic bacteria in the host, they are suppressed against harmless commensal bacteria. The mechanism(s) underlying such a differentiation remains unclear. This study sought to determine the roles of intimate adhesion in the induction of specific immune responses upon C. rodentium infection. To this end, microbiota-depleted mice were infected with the Tir-F strain expressing full-length Tir or mutant strains expressing the C-terminal truncated Tir that is defective in intimin binding and host cell actin polymerization. There were no differences in the colonization kinetics and Abs responses against C. rodentium LPS among the strains, whereas Abs against the virulence factors were only produced on Tir-F infection. Although there were no differences in the virulence factors mRNA expression levels, colonic hyperplasia, and bacterial translocation to the systemic organs irrespective of the strain, adhesion to colonic epithelial cells was reduced in the mutant strain-infected mice. Furthermore, transcriptomic analysis indicated that robust inflammatory and immune responses were only induced in the Tir-F-infected group and were suppressed in the mutant-infected groups. Taken together, these findings suggest that Tir-mediated intimate adhesion induces inflammatory and immune responses, resulting in the induction of virulence factor-specific Abs.

Citrobacter amalonaticus Inhibits the Growth of Citrobacter rodentium in the Gut Lumen.

The gut microbiota plays a crucial role in susceptibility to enteric pathogens, including Citrobacter rodentium, a model extracellular mouse pathogen that colonizes the colonic mucosa. C. rodentium infection outcomes vary between mouse strains, with C57BL/6 and C3H/HeN mice clearing and succumbing to the infection, respectively. Kanamycin (Kan) treatment at the peak of C57BL/6 mouse infection with Kan-resistant C. rodentium resulted in relocalization of the pathogen from the colonic mucosa and cecum to solely the cecal luminal contents; cessation of the Kan treatment resulted in rapid clearance of the pathogen. We now show that in C3H/HeN mice, following Kan-induced displacement of C. rodentium to the cecum, the pathogen stably colonizes the cecal lumens of 65% of the mice in the absence of continued antibiotic treatment, a phenomenon that we term antibiotic-induced bacterial commensalization (AIBC). AIBC C. rodentium was well tolerated by the host, which showed few signs of inflammation; passaged AIBC C. rodentium robustly infected naive C3H/HeN mice, suggesting that the AIBC state is transient and did not select for genetically avirulent C. rodentium mutants. Following withdrawal of antibiotic treatment, 35% of C3H/HeN mice were able to prevent C. rodentium commensalization in the gut lumen. These mice presented a bloom of a commensal species, Citrobacter amalonaticus, which inhibited the growth of C. rodentium in vitro in a contact-dependent manner and the luminal growth of AIBC C. rodentium in vivo. Overall, our data suggest that commensal species can confer colonization resistance to closely related pathogenic species. IMPORTANCE Gut bacterial infections involve three-way interactions between virulence factors, the host immune responses, and the microbiome. While the microbiome erects colonization resistance barriers, pathogens employ virulence factors to overcome them. Treating mice infected with kanamycin-resistant Citrobacter rodentium with kanamycin caused displacement of the pathogen from the colonic mucosa to the cecal lumen. Following withdrawal of the kanamycin treatment, 65% of the mice were persistently colonized by C. rodentium, which seemed to downregulate virulence factor expression. In this model of luminal gut colonization, 35% of mice were refractory to stable C. rodentium colonization, suggesting that their microbiotas were able to confer colonization resistance. We identify a commensal bacterium of the Citrobacter genus, C. amalonaticus, which inhibits C. rodentium growth in vitro and in vivo. These results show that the line separating commensal and pathogenic lifestyles is thin and multifactorial and that commensals may play a major role in combating enteric infection.

Scalable Reporter Assays to Analyze the Regulation of stx2 Expression in Shiga Toxin-Producing Enteropathogens.

Stx2 is the major virulence factor of EHEC and is associated with an increased risk for HUS in infected patients. The conditions influencing its expression in the intestinal tract are largely unknown. For optimal management and treatment of infected patients, the identification of environmental conditions modulating Stx2 levels in the human gut is of central importance. In this study, we established a set of chromosomal stx2 reporter assays. One system is based on superfolder GFP (sfGFP) using a T7 polymerase/T7 promoter-based amplification loop. This reporter can be used to analyze stx2 expression at the single-cell level using FACSs and fluorescence microscopy. The other system is based on the cytosolic release of the Gaussia princeps luciferase (gluc). This latter reporter proves to be a highly sensitive and scalable reporter assay that can be used to quantify reporter protein in the culture supernatant. We envision that this new set of reporter tools will be highly useful to comprehensively analyze the influence of environmental and host factors, including drugs, small metabolites and the microbiota, on Stx2 release and thereby serve the identification of risk factors and new therapies in Stx-mediated pathologies.

Post-transcriptional regulation in attaching and effacing pathogens: integration of environmental cues and the impact on gene expression and host interactions.

To establish infection, enteric pathogens integrate environmental cues to navigate the gastrointestinal tract and precisely control expression of virulence determinants. Emerging data indicate that post-transcriptional and post-translational gene regulation plays a key role in virulence regulation and pathogen adaptation to the host environment. Here, we highlight recent studies that reveal how physiologically relevant signals initiate post-transcriptional and post-translational regulatory circuits and the impact on virulence gene expression in the attaching and effacing pathogens, enteropathogenic Escherichia coli, enterohemorrhagic E. coli O157:H7, and Citrobacter rodentium.

Type III secretion system effectors form robust and flexible intracellular virulence networks.

Infections with many Gram-negative pathogens, including Escherichia coli, Salmonella, Shigella, and Yersinia, rely on type III secretion system (T3SS) effectors. We hypothesized that while hijacking processes within mammalian cells, the effectors operate as a robust network that can tolerate substantial contractions. This was tested in vivo using the mouse pathogen Citrobacter rodentium (encoding 31 effectors). Sequential gene deletions showed that effector essentiality for infection was context dependent and that the network could tolerate 60% contraction while maintaining pathogenicity. Despite inducing very different colonic cytokine profiles (e.g., interleukin-22, interleukin-17, interferon-γ, or granulocyte-macrophage colony-stimulating factor), different networks induced protective immunity. Using data from >100 distinct mutant combinations, we built and trained a machine learning model able to predict colonization outcomes, which were confirmed experimentally. Furthermore, reproducing the human-restricted enteropathogenic E. coli effector repertoire in C. rodentium was not sufficient for efficient colonization, which implicates effector networks in host adaptation. These results unveil the extreme robustness of both T3SS effector networks and host responses.

Citrobacter rodentium Lysogenized with a Shiga Toxin-Producing Phage: A Murine Model for Shiga Toxin-Producing E. coli Infection.

Shiga toxin-producing E. coli (STEC) is a common foodborne pathogen in developed countries. STEC generates "attaching and effacing" (AE) lesions on colonic epithelium, characterized by effacement of microvilli and the formation of actin "pedestals" beneath intimately attached bacteria. In addition, STEC are lysogenized with a phage that, upon induction, can produce potent Shiga toxins (Stx), potentially leading to both hemorrhagic colitis and hemolytic uremic syndrome. Investigation of the pathogenesis of this disease has been challenging because STEC does not readily colonize conventional mice.Citrobacter rodentium (CR) is a related mouse pathogen that also generates AE lesions. Whereas CR does not produce Stx, a murine model for STEC utilizes CR lysogenized with an E. coli-derived Stx phage, generating CR(Φstx), which both colonizes conventional mice and readily gives rise to systemic disease. We present here key methods for the use of CR(Φstx) infection as a highly predictable murine model for infection and disease by STEC. Importantly, we detail CR(Φstx) inoculation by feeding, determination of pathogen colonization, production of phage and toxin, and assessment of intestinal and renal pathology. These methods provide a framework for studying STEC-mediated systemic disease that may aid in the development of efficacious therapeutics.

Roles of the Tol-Pal system in the Type III secretion system and flagella-mediated virulence in enterohemorrhagic Escherichia coli.

The Tol-Pal system is a protein complex that is highly conserved in many gram-negative bacteria. We show here that the Tol-Pal system is associated with the enteric pathogenesis of enterohemorrhagic E. coli (EHEC). Deletion of tolB, which is required for the Tol-Pal system decreased motility, secretion of the Type III secretion system proteins EspA/B, and the ability of bacteria to adhere to and to form attaching and effacing (A/E) lesions in host cells, but the expression level of LEE genes, including espA/B that encode Type III secretion system proteins were not affected. The Citrobacter rodentium, tolB mutant, that is traditionally used to estimate Type III secretion system associated virulence in mice did not cause lethality in mice while it induced anti-bacterial immunity. We also found that the pal mutant, which lacks activity of the Tol-Pal system, exhibited lower motility and EspA/B secretion than the wild-type parent. These combined results indicate that the Tol-Pal system contributes to the virulence of EHEC associated with the Type III secretion system and flagellar activity for infection at enteric sites. This finding provides evidence that the Tol-Pal system may be an effective target for the treatment of infectious diseases caused by pathogenic E. coli.

The Ethanolamine-Sensing Transcription Factor EutR Promotes Virulence and Transmission during Citrobacter rodentium Intestinal Infection.

Enteric pathogens exploit chemical and nutrient signaling to gauge their location within a host and control expression of traits important for infection. Ethanolamine-containing molecules are essential in host physiology and play important roles in intestinal processes. The transcription factor EutR is conserved in the Enterobacteriaceae and is required for ethanolamine sensing and metabolism. In enterohemorrhagic Escherichia coli (EHEC) O157:H7, EutR responds to ethanolamine to activate expression of traits required for host colonization and disease; however, the importance of EutR to EHEC intestinal infection has not been examined. Because EHEC does not naturally colonize or cause disease in mice, we employed the natural murine pathogen Citrobacter rodentium as a model of EHEC virulence to investigate the importance of EutR in vivo EHEC and C. rodentium possess the locus of enterocyte effacement (LEE), which is the canonical virulence trait of attaching and effacing pathogens. Our findings demonstrate that ethanolamine sensing and EutR-dependent regulation of the LEE are conserved in C. rodentium Moreover, during infection, EutR is required for maximal LEE expression, colonization, and transmission efficiency. These findings reveal that EutR not only is important for persistence during the primary host infection cycle but also is required for maintenance in a host population.

The Serotonin Neurotransmitter Modulates Virulence of Enteric Pathogens.

The gut-brain axis is crucial to microbial-host interactions. The neurotransmitter serotonin is primarily synthesized in the gastrointestinal (GI) tract, where it is secreted into the lumen and subsequently removed by the serotonin transporter, SERT. Here, we show that serotonin decreases virulence gene expression by enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium, a murine model for EHEC. The membrane-bound histidine sensor kinase, CpxA, is a bacterial serotonin receptor. Serotonin induces dephosphorylation of CpxA, which inactivates the transcriptional factor CpxR controlling expression of virulence genes, notably those within the locus of enterocyte effacement (LEE). Increasing intestinal serotonin by genetically or pharmacologically inhibiting SERT decreases LEE expression and reduces C. rodentium loads. Conversely, inhibiting serotonin synthesis increases pathogenesis and decreases host survival. As other enteric bacteria contain CpxA, this signal exploitation may be engaged by other pathogens. Additionally, repurposing serotonin agonists to inhibit CpxA may represent a potential therapeutic intervention for enteric bacteria.

l-Arginine sensing regulates virulence gene expression and disease progression in enteric pathogens.

Microbiota, host and dietary metabolites/signals compose the rich gut chemical environment, which profoundly impacts virulence of enteric pathogens. Enterohemorrhagic Escherichia coli (EHEC) engages a syringe-like machinery named type-III secretion system (T3SS) to inject effectors within host cells that lead to intestinal colonization and disease. We previously conducted a high-throughput screen to identify metabolic pathways that affect T3SS expression. Here we show that in the presence of arginine, the arginine sensor ArgR, identified through this screen, directly activates expression of the genes encoding the T3SS. Exogenously added arginine induces EHEC virulence gene expression in vitro. Congruently, a mutant deficient in arginine transport (ΔartP) had decreased virulence gene expression. ArgR also augments murine disease caused by Citrobacter rodentium, which is a murine pathogen extensively employed as a surrogate animal model for EHEC. The source of arginine sensed by C. rodentium is not dietary. At the peak of C. rodentium infection, increased arginine concentration in the colon correlated with down-regulation of the host SLC7A2 transporter. This increase in the concentration of colonic arginine promotes virulence gene expression in C. rodentium Arginine is an important modulator of the host immune response to pathogens. Here we add that arginine also directly impacts bacterial virulence. These findings suggest that a delicate balance between host and pathogen responses to arginine occur during disease progression.

Identification of Antibiotics That Diminish Disease in a Murine Model of Enterohemorrhagic Escherichia coli Infection.

Infections with enterohemorrhagic Escherichia coli (EHEC) cause disease ranging from mild diarrhea to hemolytic-uremic syndrome (HUS) and are the most common cause of renal failure in children in high-income countries. The severity of the disease derives from the release of Shiga toxins (Stx). The use of antibiotics to treat EHEC infections is generally avoided, as it can result in increased stx expression. Here, we systematically tested different classes of antibiotics and found that their influence on stx expression and release varies significantly. We assessed a selection of these antibiotics in vivo using the Citrobacter rodentium ϕstx2dact mouse model and show that stx2d-inducing antibiotics resulted in weight loss and kidney damage despite clearance of the infection. However, several non-Stx-inducing antibiotics cleared bacterial infection without causing Stx-mediated pathology. Our results suggest that these antibiotics might be useful in the treatment of EHEC-infected human patients and decrease the risk of HUS development.