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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 rodentiuminfection of mice, a model of human pathogenic Escherichia coliinfections, 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. rodentiumpopulations 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. rodentiumcolonization. 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.

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 rodentiuminfection of mice, a model of human pathogenic Escherichia coliinfections, 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. rodentiumpopulations 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. rodentiumcolonization. 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.Graphical Abstract

ABSTRACTEnterohemorrhagic Escherichia coli(EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC’s ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites. In this study, we used a combined RNA-Seq and Tn-Seq approach to characterize EHEC’s response to metabolites from an in vitroculture of 33 human gut microbiota isolates (MET-1), previously demonstrated to effectively resolve recurrent Clostridioides difficileinfection in human patients. Collectively, the results revealed that EHEC adjusts to growth in the presence of microbiota metabolites in two major ways: by altering its metabolism and by activating stress responses. Metabolic adaptations to the presence of microbiota metabolites included increased expression of systems for maintaining redox balance and decreased expression of biotin biosynthesis genes, reflecting the high levels of biotin released by the microbiota into the culture medium. In addition, numerous genes related to envelope and oxidative stress responses (including cpxP, spy, soxS, yhcN, and bhsA) were upregulated during EHEC growth in a medium containing microbiota metabolites. Together, these results provide insight into the molecular mechanisms by which pathogens adapt to the presence of competing microbes in the host environment, which ultimately may enable the development of therapies to enhance colonization resistance and prevent infection.

ABSTRACTQuorum Sensing (QS) is a form of cell-to-cell communication that enables bacteria to modify behavior according to their population density. While QS has been proposed as a potential intervention against pathogen infection, QS-mediated communication within the mammalian digestive tract remains understudied. Using an LC-MS/MS approach, we discovered that Citrobacter rodentium, a natural murine pathogen used to model human infection by pathogenic Escherichia coli, utilizes the CroIR system to produce three QS-molecules. We then profiled their accumulation both in vitroand across different gastrointestinal sites over the course of infection. Importantly, we found that in the absence of QS capabilities the virulence of C. rodentiumis enhanced. This highlights the role of QS as an effective mechanism to regulate virulence according to the pathogen’s spatio-temporal context to optimize colonization and transmission success. These results also demonstrate that inhibiting QS may not always be an effective strategy for the control of virulence.

The type VI secretion system (T6SS) is a contractile nanomachine widely distributed among pathogenic and commensal Gram-negative bacteria. The T6SS is used for inter-bacterial competition to directly kill competing species; however, its importance during bacterial infection in vivoremains poorly understood. We report that the murine pathogen Citrobacter rodentium, used as a model for human pathogenic Escherichia coli,harbors two functional T6SSs. C. rodentiumemploys its T6SS-1 to colonize the murine gastrointestinal tract by targeting commensal Enterobacteriaceae. We identify VgrG1 as a C. rodentiumT6SS antibacterial effector, which exhibits toxicity in E. coli. Conversely, commensal prey species E. coliMt1B1 employs two T6SSs of its own to counter C. rodentiumcolonization. Collectively, these data demonstrate that the T6SS is a potent weapon during bacterial competition and is used by both invading pathogens and resident microbiota to fight for a niche in the hostile gut environment.