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A small number of C. rodentium founders initiates enteric infectionTo enable monitoring of the pathogen population’s diversity during infection, we introduced short, random, ~20 nucleotide DNA tags (barcodes) at a neutral location in the C. rodentium genome. As previously described5, monitoring barcode diversity using high-throughput DNA sequencing and the STAMP (Sequence Tag-based Analysis of Microbial Populations) computational framework can quantify the constriction of the pathogen population that often occurs during establishment of infection (schematized in Fig. 1a). We created two independent STAMP libraries of barcoded bacteria. Library “STAMP-CR253” contains 253 unique barcodes integrated in the intergenic region between genes ROD_05521 and selU. The neutrality of the barcode insertions was confirmed by measuring growth in lysogeny broth (LB; Supplemental Fig. 1a). Library “STAMP-CR69K” contains approximately 69,000 unique barcodes inserted into the genome on a Tn7 vector, which integrates at a neutral site downstream of the glmS gene6,7. While the libraries were not directly compared, both yielded similar results in our studies.To validate that these barcoded STAMP libraries can quantify the population effects of a bottleneck, we created in vitro bottlenecks by plating serial dilutions of the libraries grown in culture. The number of colony forming units (CFU) per plate provides a true measure of the number of founders, i.e., the number of cells from the initial population (culture) that gave rise to the observed population (plated colonies). Bacteria were harvested from the plates, the barcodes were amplified and sequenced, and barcode frequencies were analyzed using the recently updated STAMP analysis pipeline “STAMPR”8. The size of the founding population (founders) was calculated by comparing the diversity and frequency of barcodes recovered from plated samples to those in the initial cultures. There was a strong correlation between the counted founders (CFU) and the calculated founders (Nr for STAMP-CR253 and Ns for STAMP-CR69K) up to 104 founders (Supplemental Fig. 1b). These data were also used as standard curves to increase the resolution of the experiments described below to approximately 106 founders.Contraction of a barcoded population during colonization changes the frequency and number of barcodes relative to the inoculum. To determine when a C. rodentium infection is founded, C57BL/6 J (B6) mice were orally gavaged with 4 × 108 CFUs (enumerated by serial dilution and plating). Remarkably, despite this relatively large dose, within 24 hours (h) there was an average of only 9 founders (geometric mean), and as few as one founder per mouse (a single barcode; Fig. 1b). Thus, only ~1 of every 4 × 107 cells in the inoculum establishes infection, revealing that host bottlenecks result in a massive constriction of the pathogen population. Beyond 24 h, the founding population remained stable at ~10 founders. The diminutive C. rodentium founding population indicates that the vast majority of the inoculum does not survive to give rise to detectable offspring and is thus either killed by the host or passes through the intestine and is excreted in feces. Consistent with the latter possibility, 5 h after inoculation there were 1 × 107 CFU C. rodentium per gram of feces with 9×104 founders, suggesting that at this early point a numerous and diverse population has already reached the colon and cecum but failed to become founders. Surprisingly, the contraction of the pathogen population continued beyond 5 h, when the pathogen had already reached the principal sites of colonization. Despite the profound bottleneck to infection, the ~10 founders were capable of replication, and by 5 days post inoculation the C. rodentium burden in the feces was on average 9 × 108 CFU/gram. Together, these observations reveal that there is a severe bottleneck to infection with this natural, mouse enteric pathogen; however, even though the restrictive bottleneck leaves a founding population that is a miniscule fraction of the inoculum, the founders robustly replicate, creating a total pathogen burden that ultimately exceeds the inoculum (Fig. 1b).The size of the founding population increases with doseWe reasoned that determining how dose impacts the number of founders could provide insight into the mechanisms underlying the bottleneck9,10. For example, one explanation proposed for the C. rodentium bottleneck is that it is created by finite niches or resources (e.g., sugar or amino acids) whose scarcity limits the size of the population11,12. At doses where the pathogen saturates this limited resource, the ‘finite resource’ hypothesis predicts that increasing dose will not increase the number of founders (schematized in Fig. 2a). An alternate possibility is that the bottleneck eliminates potential founders through a mechanism such as acid killing in the stomach13,14, which is expected to result in a founding population that increases with dose.Fig. 2: The C. rodentium bottleneck is defined by a fractional relationship between dose and founders.a Models for the relationship between dose and founding population. In the absence of a bottleneck, all bacteria from the inoculum become founders. If the inoculum contracts due to the limited availability of finite nutrients or niches (e.g. iron, sugar, binding sites), the diversity of the population and thus the size of the founding population will remain fixed once those nutrients are saturated. If increasing dose increases the number of founders, then the underlying mechanism is not due to a limited resource; instead, the bottleneck acts proportionally on the inoculum by eliminating potential founders. b, c C57BL/6 J mice were inoculated with doses ranging from 107 to 1010 CFU of STAMP-CR253 and the C. rodentium population was monitored in the feces (geometric means and standard deviations; ND not detected counted as 0.5). Additional shedding analysis in Supplemental Fig. 2. c The bottleneck impeding B6 colonization is described 5 days post inoculation by comparing dose and founders with a linear regression of the log10-transformed data (regression line with 95% confidence intervals; not detected counted as 0.8; x-intercept “ID50” 107.2–107.9 CFU). 4–8 animals per dose. Source data are provided as a Source Data file.Full size imageTo characterize the bottleneck, we orally inoculated B6 mice with C. rodentium doses ranging 1000-fold from 107 to 1010 CFUs. Doses (ge)108 CFUs led to infection, with the founding population decreasing for ~2 days before reaching a steady value that persisted until the infection began clearing, as indicated by a simultaneous decrease in the total population (burden) and founding population (Fig. 2b). Lower doses of C. rodentium resulted in fewer founders and a longer period to reach peak shedding, with a correspondingly longer time from inoculation to pathogen elimination. As the delay in shedding at lower doses correlated with the delay in clearance, all mice were infected for a similar number of days and had similar total fecal burdens, regardless of dose (Supplemental Fig. 2).The founding population was small in number. Even at the maximum inoculum of 1010 CFUs relatively few founders were detected (83, geometric mean; Fig. 2b). While founders were never numerous, increasing the size of the inoculum always increased the size of the founding population. The bottleneck eliminated a proportion of the C. rodentium population, resulting in a founding population that scaled with dose (increasing dose 100-fold also increased founders ~100-fold). These observations indicate that the number of founders is likely not dictated by limited space or resources, contradicting the finite resource hypothesis (Fig. 2a).As an increase in dose resulted in a proportional increase in the number of founders, we represented their relationship as a line by plotting log10-transformed dose and founding population data from 5 days post inoculation (Fig. 2c). This line indicates that the bottleneck is not fixed, but rather functions by eliminating a fraction of potential founders, as schematized in Fig. 2a (‘elimination bottleneck’). Since our findings conform to a simple fractional relationship between dose and founding population, we will use this relationship to define the bottleneck: in B6 mice 1 of every ~108 inoculated C. rodentium establish a replicative niche.The x-intercept of the log-linear relationship between dose and founders can be used to calculate the dose at which we expect 1 founder. This dose corresponds to the ID50 – the dose that leads to infection of ~50% of animals. Thus, for C. rodentium infection, the ID50, a critical parameter describing a pathogen’s infectivity, is a property biologically defined by the infection bottleneck. For B6 mice, the x-intercept of this line is between 107.2 and 107.9 CFU (95% confidence-intervals) and explains why infection did not result from an inoculum of 107 CFU (Fig. 2b). Surprisingly, even though C. rodentium is a natural mouse pathogen, at least ~100-million organisms are required to routinely establish infection.Stomach acid contributes a 10- to 100-fold bottleneck to C. rodentium colonizationWe next probed the contribution of stomach acid to the highly restrictive B6 enteric colonization bottleneck. The acidity of the stomach is thought to be a potent barrier against ingested bacteria; human studies find that taking stomach acid reducing drugs increases the risk of contracting multiple enteric pathogens15. Notably, it has been observed that eliminating stomach acid decreases the minimum infectious dose for C. rodentium and increases the size of the founding population13,14. Further, acid is mechanistically consistent with the fractional relationship which we observe between dose and founding population (Fig. 2). To test the role of stomach acid in restricting C. rodentium enteric colonization, we treated mice with the fast-acting, irreversible H2-antagonist Loxtidine (aka Lavoltidine)16. 3–5 h after Loxtidine treatment, the pH of the stomach rose from 2.5 to 4.7 (Fig. 3a). Importantly, a pH of 2.5 sterilized 1010 CFUs of C. rodentium in under 15 minutes (min), whereas pH 4.7 did not kill C. rodentium even after a 1 h exposure (Fig. 3b).Fig. 3: Stomach acid constricts the C. rodentium population by 10- to 100-fold.a The effect of Loxtidine on stomach acid in C57BL/6 J mice 3–5 h after intraperitoneal administration of 1 mg in 0.1 ml PBS. pH determined post-mortem in aspirated stomach fluid. Boxes arithmetic mean (2.5 for mock and 4.7 for Loxtidine). Two-tailed t test with p-value 0.0002. Animals are 7 (vehicle) and 5 (Loxtidine). b The acid tolerance of STAMP-CR69K in culture measured by diluting cells in LB at pH 2.5 or 4.7 and incubating at 37 °C with shaking. pH 2.5 sterilized 1010 CFU in 15 min. c, d 3–5 h after intraperitoneal administration of PBS (vehicle) or Loxtidine (1 mg), C57BL/6 J mice were orally gavaged with doses ranging from 107 to 1010 CFU of STAMP-CR69K. c Bacterial burden monitored in the feces for 5 days following inoculation (geometric means and standard deviations; ND not detected counted as 0.5). d Bottleneck to colonization measured 5 days post inoculation by comparing dose and founders with linear regression of the log10-transformed data (regression line and 95% confidence intervals; significance compares elevation with p-value 5.5 × 10−7; not detected counted as 0.8). 4 animals per dose per group. Source data are provided as a Source Data file.Full size imageLoxtidine treatment prior to inoculating B6 mice resulted in infection at a lower dose, a higher pathogen burden in the feces 1 day post inoculation, and more founders on day 5 (Fig. 3c, d). The fractional relationship between dose and founding population was also observed in the absence of stomach acid, but the line depicting this relationship was shifted upward. Loxtidine treatment increased the number of C. rodentium founders approximately 10-fold at every dose, reducing the ID50 computed from the founding population from 107.3 to 105.4 CFUs. Thus, stomach acid significantly contributes to the bottleneck restricting C. rodentium colonization. However, the magnitude of stomach acid’s contribution is relatively small, between 10- and 100-fold of the observed ~108-fold B6 bottleneck to C. rodentium colonization. In the absence of stomach acid, the C. rodentium population constricts >106-fold prior to establishing a replicative niche, indicating that other factors must more potently contribute to the bottleneck.Constriction of the C. rodentium inoculum occurs distal to the stomach, at the sites of infectionTo further define the C. rodentium population dynamics and host barriers that accompany establishment of infection, we probed where and when the bottleneck occurs. Five days post-inoculation, the largest pathogen burdens were detected in the cecum and distal colon, with less numerous populations in the small intestine (SI) (Fig. 4a), consistent with previous observations17. Within individual mice (intra-mouse) the cecum, colon, and feces contained related populations of C. rodentium, with approximately the same number of founders and similar barcodes (Fig. 4b–e). Importantly, the near identity of the barcodes found in the fecal population to those in the cecum and colon indicates that fecal samples can be used to report on the pathogen population at these primary infection sites, facilitating longitudinal monitoring. While intra-mouse populations were related, comparisons of barcodes between cohoused mice (inter-mouse) inoculated with the same inoculum revealed that each mouse contained a distinct C. rodentium population (Fig. 4c–e). The distinct identities of the founding populations in each of five cohoused, co-inoculated mice was apparent when comparing pathogen barcode frequencies with principal component analysis (PCA), where intra-mouse samples formed their own tight clusters (Fig. 4c). Similarly, analysis of barcode genetic distances showed that the intra-mouse pathogen populations were highly similar (low genetic distance), whereas they were dissimilar to the populations in cohoused, co-inoculated mice (Fig. 4d, e). A notable exception were the C. rodentium populations from some SI samples that were more closely related to cage-mates than other intra-mouse samples, likely reflecting recent inter-mouse exchange via coprophagy (Supplemental Fig. 3). These data suggest that despite the consumption of C. rodentium-laden feces, C. rodentium infection leads to super-colonization resistance at the primary infection sites in the cecum and colon, preventing transmission to cohoused, co-infected mice.Fig. 4: Infection is initiated by related populations of C. rodentium in the cecum and colon.a–e C. rodentium populations in whole organ homogenates from 5 cohoused (intra-cage) C57BL/6 J mice 5 days post inoculation with 4 × 108 CFU of STAMP-CR253. Within a mouse (intra-mouse) the C. rodentium populations at the primary sites of colonization (cecum, proximal colon, distal colon, feces) share founders (number, identity, and frequency of barcodes). a, b Lines connect intra-mouse samples. c Clustering of barcode populations by principal component analysis (PCA). d, e Relatedness determined by comparing the barcode frequencies by genetic distance (arithmetic means) with zero indicating no difference between populations (identical). e Two-tailed t test with p-value 5.8 × 10−48. p proximal, m mid, d distal, SI small intestine. Heatmap depicting genetic distance relationships of all intra-cage populations in Supplemental Fig. 3. Source data are provided as a Source Data file. f, g To determine when/where C. rodentium establishes a replicative niche, C57BL/6 J mice were orally gavaged with between 3 × 109 and 6 × 109 CFU STAMP-CR253. Following dissection, the cecum and colon were flushed to separate organ adherent (f) and luminal (g) bacteria. Burden and founders display geometric means and standard deviations. Bacteria not detected (ND) counted as 0.5. Relatedness of populations was determined by comparing the barcode frequencies of colon and cecal populations from within the same animal (intra-mouse) by genetic distance (arithmetic mean and standard deviation). 22 animals. Source data are provided as a Source Data file. h Model depicting how related C. rodentium populations could initiate infection in both the cecum and colon: (1) the inoculum minorly constricts passing through the stomach and SI to deposit diverse populations in the cecum and colon, (2) the populations in the cecum and colon contract separately over the first 24–48 h becoming dissimilar, and then (3) expansion occurs in either the cecum or colon moving to both locations. We depict the movement from cecum to colon as we judge this to be more likely, but the opposite is possible.Full size imageTo test this super-colonization resistance hypothesis, we separately infected two groups of ‘seed’ mice with different sets (A and B) of barcoded C. rodentium (Supplemental Fig. 4a). At the peak of colonization in the seed mice, 7 days post-inoculation, they were cohoused for 16 h along with an uninfected ‘contact’ mouse, three mice per cage. After 16 h, the mice were separated back into 3 cages containing mice originally inoculated with the A barcodes, inoculated with the B barcodes, or uninoculated. No transmission of barcodes was detected between the animals originally inoculated with the A and B barcodes (Supplemental Fig. 4b), confirming that C. rodentium infection prevents super-colonization. In marked contrast, the contact mice became infected with founders from seed A and/or B, demonstrating the ready transmission of C. rodentium from infected to uninfected mice. Furthermore, the co-infection of contact mice with barcodes from A and B confirms a previous report from super-infection experiments in mice lacking a microbiota18 that immunity to super-colonization takes time, providing a window for co-colonization. Importantly, super-colonization resistance indicates that founders are more likely to originate from the inoculum than other cohoused, infected animals.Based on the high burdens of C. rodentium in the cecum but not the colon during the first 3 days following inoculation, prior studies proposed that infection begins with pathogen expansion in the cecum, followed by subsequent spread to the colon17; a hypothesis that is consistent with the closely related intra-mouse C. rodentium populations that we observe in the cecum and colon 5 days after inoculation (Fig. 4a–e). To determine when and where C. rodentium initiates infection, we monitored the luminal and adherent C. rodentium populations in the cecum and colon. Within the first 5 h a large burden ( >107 CFU) and numerous founders ( >105 Nr’) were detected in both locations (Fig. 4f, g). Since a large founding population was observed in the cecum and colon early after inoculation, we can discount the model that the primary bottleneck occurs proximal to these locations (e.g., stomach acid or bile). The number of founders and the total burden contracted over the first 24 h, resulting in small (0.4) populations in the cecum and colon one day post inoculation. Expansion was detected first in the cecum, on day 2. Concomitant with cecal expansion, the populations in the cecum and colon became increasingly similar; i.e., the genetic distance between the populations became smaller. The most plausible model to fit these data (depicted in Fig. 4h) is that (1) within hours many bacteria pass through the stomach, reaching the cecum and colon, and then (2) these populations diverge as they separately constrict, and finally, (3) spread to both locations when a small number of founders begin to replicate. We propose that the initial population expansion begins in the cecum and then spreads to the colon, but we cannot rule out the opposite directionality because we were unable to serially sample the internal populations from a single mouse. However, displacement of the cecal population by bacteria from the colon seems unlikely because it would require non-flagellated C. rodentium to move against the bulk flow of the gut and thus we favor the model that infection initiates in the cecum.C3H/HeOuJ mice have a less restrictive bottleneck than C57BL/6 JWe next interrogated the host’s contribution to the bottleneck impeding C. rodentium colonization by quantifying the bottleneck in a more disease susceptible genotype of mice. While C. rodentium causes self-limited diarrhea in B6 mice, infection leads to a lethal diarrheal disease in C3H/HeOuJ (C3Ou) mice (Fig. 5a, b)19. We found that increased vulnerability to disease correlated with a less restrictive bottleneck. C. rodentium is 10- to 100-fold more infectious in C3Ou than B6 mice, infecting at a ~10-fold lower dose and producing ~10-times more founders at every dose (Fig. 5c). While the bottleneck was relaxed in C3Ou mice, a fractional relationship remained between dose and founding population, suggesting a similar underlying mechanism restricts colonization in both mouse genotypes. Also, as in B6 animals, higher doses and more founders accelerated the dynamics of pathogen shedding in C3Ou mice (Fig. 5a). These observations demonstrate that in addition to dose, the size of the founding population is determined in part by host genetics, which may impact the bottleneck through several mechanisms. Notably, changing host genotype caused a more lethal disease while only alleviating ~10-fold of the ~108-fold B6 bottleneck.Fig. 5: Host genotype impacts the bottleneck to C. rodentium colonization.C3H/HeOuJ (a) or C57BL/6 J (b) mice were inoculated with doses ranging from 106 to 1010 CFU of STAMP-CR253. The C. rodentium population was monitored in the feces (geometric means and standard deviations; ND not detected counted as 0.5) and animal health assessed by weight loss (percent compared to pre-inoculation; arithmetic means and standard deviations) and body condition. For survival, lines are percent of initial animals not moribund. c The bottleneck to C3Ou and B6 colonization is described 5 days post inoculation by comparing dose and founders with a linear regression of the log10-transformed data (regression line with 95% confidence intervals; significance compares elevation with p-value 5.5 × 10−7; not detected counted as 0.8). B6 bottleneck data is repeated from Fig. 2. 4 animals per dose. Source data are provided as a Source Data file.Full size imageThe bottleneck to C. rodentium enteric colonization is microbiota dependentAs shown above, a large portion of the restrictive, fractional, B6 bottleneck to C. rodentium colonization occurs distal to the stomach, at the chief sites of infection in the cecum and colon. These data strongly suggest that the principal step limiting colonization occurs during the pathogen’s establishment of a replicative niche in the cecum and/or colon. One factor present at these sites and previously linked to limiting C. rodentium colonization is the microbiota12,18. We therefore tested whether acute microbiota depletion eliminated the bottleneck to C. rodentium colonization. Treating mice with the antibiotic streptomycin for the 3 days prior to inoculation with streptomycin-resistant C. rodentium greatly accelerated pathogen population expansion, with mice shedding >109 CFUs per gram of feces within the first day (Fig. 6a). Further, streptomycin pretreatment almost completely ablated the bottleneck, with colonization at doses as low as ~100 CFUs; at this low dose, we measured an average of 25 founders 5 days post inoculation, indicating that C. rodentium experiences less than a 10-fold bottleneck following microbiota depletion (Fig. 6b). Significantly, streptomycin treatment does not alter the acidity of the animal’s stomach (Supplemental Fig. 5). Since an ~10-fold bottleneck remains after microbiota depletion and an ~10-fold bottleneck is stomach acid dependent (Fig. 3d), these data suggest that the combination of the microbiota and stomach acid can account for the majority of factors restricting C. rodentium colonization.Fig. 6: Streptomycin treatment ablates most of the bottleneck preventing C. rodentium colonization.The microbiota of conventional C57BL/6 J mice was reduced by treatment with the antibiotic streptomycin for the 3 days prior to inoculation with a streptomycin resistant library of STAMP-CR69K. a Founding population and bacterial burden monitored in the feces, and (b, c) the bottleneck to colonization measured by comparing dose and founders (geometric means and standard deviations; resolution limit is ~106 founders). (a) Animal health monitored by weight loss (percent compared to pre-inoculation; arithmetic means and standard deviations) and body condition. For survival, lines are percent of initial animals not moribund. Untreated B6 bottleneck data is repeated from Fig. 2 for comparison. 4 animals per dose. Streptomycin treatment does not impact stomach acidity (Supplemental Fig. 5). Source data are provided as a Source Data file.Full size imageTo confirm that streptomycin’s ablation of the bottleneck to C. rodentium colonization occurs because of microbiota depletion rather than an off-target effect, we also determined the bottleneck in B6 mice lacking a microbiota (germ-free). In germ-free mice, like streptomycin pretreated animals, there was almost no bottleneck to C. rodentium colonization (Fig. 7a, b). Animals lacking a microbiota were colonized at a dose of 150 CFU and shed numerous C. rodentium within 1 day of inoculation. Together, experiments with germ-free and streptomycin-pretreated mice reveal that the primary barrier to enteric colonization is linked to the microbiota.Fig. 7: The bottleneck to C. rodentium colonization is microbiota dependent.Germ-free C57BL/6 J mice were orally inoculated with doses ranging from 102 to 1010 CFU of STAMP-CR69K. 4 animals per dose, cohoused with animals receiving the same dose in sterile cages. Measurement of germ-free stomach acidity in Supplemental Fig. 5. Source data are provided as a Source Data file. a Bacterial burden and founding population monitored in the feces (geometric means and standard deviations; resolution limit is ~106 founders). Animal health monitored by weight loss (percent compared to pre-inoculation; arithmetic means and standard deviations) and body condition. No animals became moribund. b, c Bottleneck to colonization measured by comparing dose and founders (geometric means and standard deviations). SPF B6 bottleneck data is repeated from Fig. 2 for comparison. d To determine if colonization was accompanied by changes in the C. rodentium genome, whole genome sequencing was performed on 3 clones (colonies) from the STAMP-CR69K input library and compared to clones isolated from feces of infected mice (1 colony per mouse). Boxes represent the genome status of the LEE pathogenicity island. No LEE genomic changes wild-type (wt), deletion of the entire LEE region (del), insertion within the LEE (ins). Mice with a conventional microbiota SPF specific pathogen free. Other genomic changes listed in Supplemental table 1. e Depiction of a ~100,000 base-pair region of the C. rodentium genome containing the LEE pathogenicity island. Read depth from STAMP-CR69K inoculum and 5 clones isolated after 20 days passage in otherwise germ-free animals. Large deletions in 3 of 5 clones revealed by lack of specifically mapped reads in regions of up to 97,691 base-pairs. Non-specific reads map to multiple loci in the genome (primarily transposons).Full size imageMicrobiota disruption also impaired the capacity of mice to clear C. rodentium infection (Figs. 6a, 7a)12. Pathogen burden in the feces of germ-free mice did not decrease over time, in marked contrast to mice with an intact microbiota (specific pathogen free; SPF). Similarly, most cages of streptomycin-pretreated mice failed to clear the pathogen, with heterogeneity presumably caused by variation in the rebound of the microbiota after streptomycin treatment (Fig. 6a). Despite high fecal burdens, germ-free animals only exhibited mild diarrhea and did not lose weight for the 30 days of observation. These data indicate that the microbiota is the primary impediment to C. rodentium replication in the gastrointestinal tract, antagonizing the pathogen’s capacity to initiate a replicative niche and promoting its clearance.In germ-free and streptomycin pretreated animals the number of C. rodentium founders ceased to be fractionally related to dose; doses ranging 10,000-fold, from 106 to 1010 CFUs, all yielded a similar number of founders 5-days post-inoculation (Figs. 6b, 7b). These data suggest that there is an upper limit to the size of the C. rodentium founding population of ~105 on day 5 (i.e., a bottleneck caused by limited resources as illustrated in Fig. 2a). Furthermore, in the absence of a microbiota dependent bottleneck, the maximum size of the founding population continuously decreased for the 20 days of observation (Figs. 6c, 7c). Although there was no contraction in the C. rodentium burden following infection of germ-free animals, the maximum number of founders decreased from ~105 on day 5 to ~102 on day 20 (Fig. 7a, c). A decrease in diversity without a decrease in abundance suggests that C. rodentium adapts to the germ-free environment, introducing a new bottleneck caused by intra-pathogen competition.To test the hypothesis that C. rodentium evolved during colonization of germ-free animals, we sequenced the genomes of single C. rodentium colonies isolated from infected SPF or germ-free mice 5 or 20 days post inoculation. 5 days post-inoculation, the C. rodentium genomes isolated from SPF and germ-free mice were similar to the inoculum, with 6/10 colonies lacking detectable variations (Supplemental table 1). These data indicate that the initial contraction of the C. rodentium population observed during establishment of infection is not caused by selection of a genetically distinct subpopulation of the inoculum. By contrast, 20 days growth in the absence of a microbiota was always accompanied by changes in the C. rodentium genome. Notably, C. rodentium with structural variations in the LEE pathogenicity island became dominant in 4 out of 5 cages of infected germ-free animals (Fig. 7d, Supplemental table 1). These variations included large deletions of up to 97,691 bps (Fig. 7e, isolate from mouse F1) that eliminated the entire island, which is essential for colonization of SPF mice20. These genome alterations suggest that in the absence of a microbiota, a common mechanism for C. rodentium adaption to the host environment is to lose the LEE pathogenicity island. Thus, we conclude that competition among C. rodentium constricts the diversity of the population in the absence of a microbiota-dependent bottleneck, with organisms that lose the LEE virulence island outcompeting bacteria possessing the LEE. Additional mutations were also detected in the C. rodentium isolated on day 20 from germ-free animals, including in the galactonate operon, which have previously been observed in Escherichia coli colonizing microbiota depleted mice21 (Supplemental Table 1). Thus, there may be common evolutionary strategies for pathogenic and non-pathogenic bacteria to adapt to growth without competition in the host intestine.Collectively these experiments show that of the multiple host factors protecting against enteric infection, the microbiota is by far the most restrictive. Diminution of the microbiota markedly increases host susceptibility, permitting infection at almost any dose. In the absence of competition with the microbiota, a new slow-acting bottleneck constricted the C. rodentium population as the pathogen evolved increased fitness, notably through loss of the LEE pathogenicity island. More