Evolution of extraintestinal pathogenic Eescherichia coli within the gastrointestinal tract over time

Within the United States alone, Extraintestinal Pathogenic Ecsherichia coli (ExPEC) is responsible for adverse health outcomes that affect millions of people annually. ExPEC is the primary cause of urinary tract infections, which afflict about 50% of women at least once in their lifetime. It is thought that one of the main reservoirs for these infections are within the gut, where ExPEC does not typically cause disease. The mammalian gut utilizes a variety of defensive mechanisms to prevent infection from outside pathogens. Despite effective and complex host responses, ExPEC can colonize the intestinal tract and remain there for extended periods of time. The primary goal of this research is to identify factors that allow for not only successful ExPEC colonization of the gut, but to also note adaptational changes that may enhance ExPEC survival within this complex environment. Two reference ExPEC strains, the cystitis isolate F11 and the pyelonephritis isolate CFT073, were evaluated for their ability to colonize and adapt to the gastrointestinal (GI) tract of mice. Colonization of the gut was quantified by titering bacteria recovered from the feces over the course of 18 days following oral gavage of the indicated strains. Both CFT073 and F11 displayed varying degrees of colonization within the gut. It appears that the selective pressures and stochastic events within the intestinal tract likely contribute to this trend. This is being further investigated by using sequencing and imaging approaches to assess changes in ExPEC genomes and location within the gut over time.

EVOLUTION OF EXTRAINTESTINAL PATHOGENIC ESCHERICHIA COLI WITHIN THE GASTROINTESTINAL TRACT OVER TIME by Alejandra A. Mendez A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Kinesiology Approved: ___________________________________ _________________________________ Matthew Mulvey, Ph.D. Timothy Brusseau, Ph.D. Thesis Faculty Supervisor Director, Health and Kinesiology Program Janet Shaw, Ph.D. Honors Faculty Advisor Sylvia D. Torti, Ph.D. Dean, Honors College May 2020 Copyright © 2020 All Rights Reserved ABSTRACT Within the United States alone, Extraintestinal Pathogenic Ecsherichia coli (ExPEC) is responsible for adverse health outcomes that affect millions of people annually. ExPEC is the primary cause of urinary tract infections, which afflict about 50% of women at least once in their lifetime. It is thought that one of the main reservoirs for these infections are within the gut, where ExPEC does not typically cause disease. The mammalian gut utilizes a variety of defensive mechanisms to prevent infection from outside pathogens. Despite effective and complex host responses, ExPEC can colonize the intestinal tract and remain there for extended periods of time. The primary goal of this research is to identify factors that allow for not only successful ExPEC colonization of the gut, but to also note adaptational changes that may enhance ExPEC survival within this complex environment. Two reference ExPEC strains, the cystitis isolate F11 and the pyelonephritis isolate CFT073, were evaluated for their ability to colonize and adapt to the gastrointestinal (GI) tract of mice. Colonization of the gut was quantified by titering bacteria recovered from the feces over the course of 18 days following oral gavage of the indicated strains. Both CFT073 and F11 displayed varying degrees of colonization within the gut. It appears that the selective pressures and stochastic events within the intestinal tract likely contribute to this trend. This is being further investigated by using sequencing and imaging approaches to assess changes in ExPEC genomes and location within the gut over time. ii TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 METHODS 6 RESULTS 9 DISCUSSION 14 ACKNOWLEDGMENTS 17 REFERENCES 18 iii INTRODUCTION The leading cause of urinary tract infections and bacteremia is Extraintestinal Pathogenic Escherichia coli (ExPEC). It is estimated that urinary tract infections due to ExPEC sicken well over 11 million people annually within the United States alone and cost more than $6 billion a year [4]. Urinary tract infections can also lead to bacteremia, accounting for about 25% of sepsis cases [5]. Sepsis occurs as the result of bacteria entering the bloodstream (bacteremia) and causing an aberrant immune response that can lead to organ failure, tissue damage, and even death [5]. Uropathogenic Escherichia coli (UPEC), is a sub-category of ExPEC that is responsible for 70-95% of all communityacquired urinary tract infections [14]. In addition to urinary and bloodstream infections, ExPEC can cause a wide array of other common infections, including surgical sites and soft tissue infections, pneumonia, and meningitis [2]. Within the gut, ExPEC is usually considered innocuous, but some ExPEC isolates have been linked to incidences of Crohn’s disease and colorectal cancer [3]. The ability of ExPEC to colonize a niche and subsequently cause disease in a variety of diverse environments reflects the genetic repertoire available to these pathogens. The virulence potential of ExPEC arises not only from virulence factors that aid in colonization and disease, but also factors that enable ExPEC to resist stress and other challenges like antibiotic treatments. A major problem arising is the increased occurrence of the multidrug-resistant sequence type (ST) 131 clonal group as well as other ExPEC strains that are not treatable with frontline antibiotics [6]. Pathogens like ExPEC often develop antibiotic resistance through horizontal gene transfer. One such example is the passage of integrons, which usually encode antibiotic resistance cassettes, and are next to a recombination site to facilitate incorporation into the bacterial genome. This can further spread the occurrence of antibiotic resistant pathogens. ST131 is becoming the most populous ExPEC lineage and contributes significantly to the overall increase in antibiotic resistance, especially against fluoroquinolones, one of the frontline drugs currently used to combat UPEC infections [7]. The rise of antibiotic resistance further drives the need to better understand the pathways that ExPEC utilizes to cause disease, as this can identify novel drug targets and treatments for the common and diverse infections caused by ExPEC. However, due to the many different niches that ExPEC is able to survive in, both as a commensal organism and as a pathogen, determining factors that are necessary for ExPEC survival within and transmission between host environments can be difficult. ExPEC employs many modes of transmission, some of which include taking advantage of lapses in hygiene, fecal-oral routes, ingestion of contaminated food, and even sexual contact [8]. A vital part of ExPEC persistence is its establishment within the intestinal tract. Unfortunately, due to the within the host, the mechanisms of ExPEC colonization and survival within this complex and diverse environment are not well-established [9]. The gastrointestinal tract of mammals is a hostile and highly competitive environment. The intestinal tract has various host defenses such as the utilization of highly acidic digestive enzymes within the stomach, bile salts to emulsify solid matter, and assorted antimicrobial peptides [10]. Within the gut, there are massive populations of bacteria which create a competitive environment for space and resources that microbes like ExPEC need for colonization and survival. Collectively, the defense systems within the gut provide ‘colonization resistance’, with the native intestinal microbiota and the host working together to limit colonization by incoming, potentially pathogenic microbes, 2 plays a contributing role [20]. The mammalian intestinal tract is also lined by a mucus layer, which provides refuge for commensal bacteria, helping to modulate host immune responses and providing protection from invasive pathogens [11]. Much like humans, the GI tract of mice is a highly competitive and diverse environment [16], making it a useful model for our research. Despite the many defense mechanisms deployed by the host and the resident microbiota to prevent infections, ExPEC strains can still successfully colonize the gut. To examine this process, adult female Balb/c mice were infected via oral gavage with either the ExPEC isolate F11 or a non-pathogenic K12 commensal strain MG1655 (Fig. 1a). By following bacterial titers within the feces (which reflect bacterial numbers within the colon), we found that ExPEC strains like F11 are able to effectively colonize and persist within the gut (Fig. 1)*adapted from [21]. Typically, in these sorts of experiments, antibiotics are used to first clear out the resident microbes, reducing colonization resistance and clearing the way for new bacteria coming into the system. The ability of ExPEC, but not MG1655, to effectively colonize the GI tract without the having to pre-treat the mice with antibiotics was unexpected. Of note, these mice lack members of the Enterobacteriaceae family, including E. coli, which may facilitate ExPEC colonization of the gut in these animals. F11 is able to maintain a stable bacterial population for over 7 weeks in the GI tract (Fig. 1b). However, MG1655 was 3 outcompeted within a two-week period by the microbiota. In competition assays where F11 and MG1655 were inoculated into mice at a 1:1 ratio, F11 was only briefly below the limits of detection from one mouse before reestablishing itself, whereas MG1655 is lost from all mice within the first three days post-inoculation (Fig. 1c). Follow up studies showed that other ExPEC isolates, including UTI89 and CFT073, are also able to colonize and reside with the GI tract for a longer duration when compared to commensal E. coli strains [12]. Persistent reservoirs of ExPEC within the gut may help explain the fact that around 25% of patients who acquire a urinary tract infection will experience a second infection within 6 months, often due to identical or closely related strains [1]. Among ExPEC isolates, there is significant genetic variability that can impact host responses as well as the progression of disease [13]. This variability is one factor that makes studying these strains complicated and difficult. Due to the fast replication of bacteria, adaptations and mutations can arise quickly. In the K-12 MG1655 isolate, phenotypes generated from adaptational pressures can be seen as early as two days post inoculation [15]. The evolutionary process begins with large effect adaptations and mutational soft sweeps [15]. The ability of E. coli to rapidly change is likely instrumental to its ability to colonize and survive in stressful host environments like the GI tract. The primary aims of this research were to not only monitor ExPEC colonization of the gut, but to also evaluate adaptations that increase ExPEC fitness within this complex environment. To do this we used adult female SPF Balb/c mice aged ~8 weeks to evaluate the ability of differently tagged ExPEC strains to colonize the mouse gut. We also set out to monitor the emergence of mutations that may confer a fitness advantages to these strains. The mouse model utilized in these experiments is novel due to the fact 4 that pre-treatment of mice with antibiotics was not performed, and therefore the intestinal microbiota remained intact. Our data suggest that the success of an ExPEC strain within the gut is modulated by a combination of bacterial adaptive responses to selective pressures and stochastic effects. These lead to varying levels of specific ExPEC strains over time within the gut. We did not discern any distinct patterns of colonization and persistence when comparing different ExPEC isolates or isogenic, differently tagged versions of the same ExPEC strain. Furthermore, ExPEC strains that came to dominate within the mouse gut over the course of 18 days had no obvious advantage when collected and placed in competition with the parent strain within the gut or in in vitro assays. In total, these results suggest that the tested ExPEC strains are already well adapted to colonize and survive within the gut, and that strains that come to dominate may not differ too much phenotypically from their fellow ExPEC competitors. 5 METHODS In vitro Competitive Assay Chloramphenicol or kanamycin resistant cassettes were inserted onto the chromosomes of CFT073 and F11 using Lambda Red-mediated recombination [17]. These cassettes were inserted into an attTn7 site to avoid gene disruption. Static overnight cultures for the KanR and ClmR versions of CFT073 and F11 were incubated in both minimal M9 media and nutrient rich LB media. A 1:1000 dilution of KanR and ClmR were added to fresh M9 and LB media for both isolates and incubated shaking at 225 rpm. Bacterial titers were enumerated at 0, 24, 48, and 72-hours post-mixing and plated onto LB agar containing 50 mg/mL or 20 mg/mL of either kanamycin or chloramphenicol. 200µl of fresh media was added to each culture after tittering was performed. Growth Curves To ensure that the growth rates of each strain were not impacted by the insertion of chromosomal cassettes, growth curves were acquired using a Bioscreen C instrument (Oy Growth Curves Ab Ltd). The Bioscreen measures the optical density at 600 nm of each sample every 30 minutes over a 24-hour period. These data were analyzed and graphed using Prism (Graphpad). Data shown in this thesis are representative plots of at least two independent experiments, each performed with three technical replicates. 6 Biofilm Assay For these assays, 10 l of either CFT073 or F11 were spotted onto YESCA medium plates. Plates were kept at room temperature for the duration of the experiment. Biofilm formation by the input parent strains was compared with biofilms formed by isolates collected from mouse feces throughout the in vivo competitive assays (see below). Biofilm formation was assessed by appearance as well as diameter. In vivo Competitive Assay KanR and ClmR isogenic strains of both F11 and CFT073 were grown from frozen stocks in M9 minimal media at 37° C for 24 hours. After adjusting the OD600 of each culture to 0.5, 1:1 mixtures of the KanR and ClmR F11 or CFT073 strains were prepared. Single-housed female ~8-week-old SPF Balb/c mice were inoculated via oral gavage with approximately 109 colony forming units of either the KanR and ClmR CFT073 strains or KanR and ClmR F11 strains suspended in 50 µl sterile Phosphate Buffered Saline (PBS). Input inoculum was titered on selective media to ensure a 1:1 ratio was achieved. The mice were not treated with antibiotics prior to inoculation and did not have any endogenous E. coli strains based on plating of fecal samples on McConkey agar. Every three days, for 18 days, feces were collected from the mice and numbers of KanR and ClmR were enumerated by plating onto LB agar plates containing either kanamycin or chloramphenicol. Data were normalized to the percentage of ClmR bacteria present and graphed using Prism (Graphpad). 7 Isolating DNA for Sequencing Individual colonies recovered from feces collected at every time point were grown shaking for 4 hours in LB media and then frozen as 30% glycerol stocks at -80°C. DNA was isolated from strains grown from these frozen stocks using the DNeasy Blood and Tissue Kit from Qiagen. DNA samples were then sent to the Microbial Genome Sequencing Center at the University of Pittsburgh for whole genome sequencing using the NextSeq 550 platform with paired 2X150 bp reads (~150X coverage). Statistical analysis Except where indicated, P values were calculated by two-tailed Student’s t tests or MannWhitney U tests using Prism 6.0e software (GraphPad). P values of less than 0.05 were defined as significant for all experiments. Ethics Statement Animals used in this study were handled in accordance University of Utah approved IACUC protocols (protocol number 19-01001), according to U.S. federal guidelines indicated by the Office of Laboratory Animal Welfare (OLAW) and described in the Guide for the Care and Use of Laboratory Animals, 8th ed. 8 RESULTS In vitro Competitive Assay To determine if the ClmR or KanR strains had a competitive advantage in vitro, we incubated a (presumptive) 1:1 mixture of the paired F11 or CFT073 strains in either M9 or LB media shaking at 37°C. Results from these in vitro assays showed variations in Fig. 2 KanR and ClmR isolates display equal levels of competition in vitro. Isolates were competed against one another in M9 or LB media for 72 hours. (A) F11 in LB, (B) F11 in M9, (C) CFT073 in LB, and (D) CFT073 in M9. ratios of ClmR vs KanR strains over time, but in all but one case (Fig. 2A) fairly equal numbers of the ClmR vs KanR strains were recovered at the 72- hour timepoint (Fig. 2B-C). The loss of the KanR F11 strain after 72 hours of growth in LB (Fig. 2A) is likely an outlier, but additional experiments are required to assess this possibility. Of note, while the ClmR vs KanR variants of F11 grown in M9 media and CFT073 in LB did not begin at a true 1:1 ratio, the differently tagged strains reached nearly equal levels after 24 or 48 hours (Fig. 2B-C). These data indicate that, overall, the KanR- and ClmR-tagged strains are similarly competitive in vitro. 9 Growth Curves We measured the OD600 over the course of 24 hours to determine whether the parent (input) CFT073 ClmR and KanR strains grew similarly in vitro using the Bioscreen Fig. 3. Growth curves of CFT073 isolates in vitro are similar regardless of passage through the mouse gut. The strains labelled “Input Kan” and “Input Clm” refer to the original CFT073 KanR and ClmR strains that were used to gavage mice. The other “Kan” and “Clm” labels represent various isolates obtained from feces during the in vivo competitive assays. Each line represents the mean of technical triplicates. The y-axis indicates OD600 measurements against the x-axis time. C instrument. We also wanted to examine if passage through the murine intestinal tract impacted in vitro growth. Data presented in Fig. 3 indicate that all strains, both parent input strains and isolates recovered after passage through the mouse GI tract in competitive assays (see below), grew at similar rates in vitro (Fig. 3). 10 Biofilm Assays To assess if passage through the mouse gut in competitive assays affected the ability of either F11 or CFT073 to form biofilms was assessed using YESCA plates [19]. Fig. 4. Biofilm formation remains unchanged after passage through the murine gut. 10 µl of bacteria were plated on YESCA plates and kept at room temperature. Diameter measurement s were taken for (A) CFT073 and (B) F11. “Clm Mouse 1” indicates a ClmR isolate collected from mouse #1. Isolates collected from the day 18 of the in vivo competitive assays were used (see below). 10 µl of each bacterial culture were spotted in the center of a YESCA plate and stored at room temperature. Over the course of seven days, biofilm diameter measurements were taken. Overall, the CFT073 (Fig. 4A) and F11 (Fig. 4B) strains recovered from the mouse gut formed biofilms similarly, regardless of having Clm- and Kan-resistance cassettes. additional experiments are needed to determine if these isolates recovered from the mouse gut differ from their parent strains in terms of biofilm development. 11 In vivo Competitive Assay ClmR and KanR versions of the CFT073 or F11 isolates were inoculated 1:1 into mice via oral gavage. Fecal titers were determined by plating fecal homogenates on Fig. 5. There is no clear competitive “winner” between ClmR and KanR WT strains. Mice were gavaged with ~109 CFU. Fecal titers were performed at the indicated timepoints. Each line indicates a single mouse from two replicates followed over the course of 18 days. (A) F11 and (B) CFT073. selective media every 3 days over the course of 18 days. This was done to determine if the KanR or ClmR strain had a competitive advantage within the murine gut. F11 displayed a more divergent pattern of bacterial populations when compared to CFT073 (Fig. 5). For mice inoculated with F11, the majority diverged by day 6 to predominantly have either ClmR or KanR within the gut. However, one mouse did not display this trend and was equally colonized at day 18. The CFT073 assay displayed a less Fig. 6. Passage through the mouse gut did not confer a competitive advantage upon rechallenge in the gut. Mice were gavaged with ~109 CFUs. Competitive indices of the input F11 KanR and a collected ClmR isolate from day 18 were used. CI was calculated as log10 function. Bars represent median values. divergent trend. At day 18, five mice have exclusively ClmR bacteria whereas only one mouse had primarily KanR bacteria. There were also two mice with relatively equal amounts of KanR and ClmR bacteria at day 18. 12 After noticing that there did not appear to be a distinct advantage for either ClmR or KanR isolate, we wanted to determine if strains that had survived within the intestinal tract would be more competitive when compared to the respective input strains. As an initial test, a phenotypically successful F11 ClmR isolate (meaning one with a high quantity of CFUs on day 18) was competed against an input F11 KanR strain in mice. The competitive indices were graphed as log10 (ClmR/KanR). Despite already showing high F11 ClmR concentrations after being exposed to the GI environment for 18 days, this isolate did not display a superior competitive advantage when compared to the input strain (Fig. 6). 13 DISCUSSION ExPEC strains possess a variety of genetic characteristics that allow them to not only colonize a hostile environment such as the GI tract, but also stimulate an array of host responses [13]. We demonstrated that within the murine intestinal tract, the numbers of both KanR and ClmR isogenic variants of the same ExPEC reference strain (either F11 or CFT073) seem to vary randomly over time (Fig. 5). One potential explanation for the high variation in colonization populations are due to selective pressures within the gut. Some of these pressures include interactions with other resident microbiota, competition for nutrients, and limited space within the gut for colonization. After noticing these trends, we next determined whether the growth variation was observable outside of the gut. Using in vitro competitive assays, we found little evidence that the KanR and ClmR variants had any real advantage over the other, though occasionally one variant would come to dominate over the other in broth culture (Fig. 3). Furthermore, KanR and ClmR variants recovered from the mouse gut did not differ from one another in their ability to form biofilms in vitro (Fig. 4). Biofilm formation may aid the ability of ExPEC strains to cause prolonged infections by providing protection from host defenses as well as antibiotics [18]. When considering the results of our in vitro and in vivo assays, it appears that the pressures present within the intestinal tract environment likely impact the levels of ExPEC colonization over time by selecting for specific mutations within individual microbes. This would be in line with previous work showing that specific mutants “sweep” through a population of the K12 strain MG1655 following inoculation of antibiotic-treated mice [15]. Mutations that provide a selective advantage come to 14 dominate within the gut but may be replaced as other more fit mutants arise. Such a scenario would help explain our data, and especially results showing variable levels of the KanR and ClmR F11 variants over time (Fig. 5). However, if F11 and CFT073 acquires mutations that enhance their fitness within the gut, these changes are plastic and do not appear to provide a long-lasting benefit based on experiments in which we rechallenged a parent F11 strain with a “gut-adapted” isolate (Fig. 6). To better understand the importance if mutations in the adaption and persistence of ExPEC within the gut, we need to have genomic sequencing data. To accomplish this, we plan to sequence both input strains and strains recovered from mouse feces over time in competitive assays for comparative genomics analyses. To date, we have isolated and sequenced DNA collected from the input strains and dominant variants collected on the final day (18) of our competitive assays. Due to the high volume of sequencing data, we are still in processing these data. However, preliminary analysis of our sequencing data indicate that the bacteria recovered from the mouse gut 18 days post-inoculation look very similar to the input parent strains. In total, our preliminary sequencing data and rechallenge assays (Fig. 6) suggest that ExPEC strains like F11 and CFT073 are already optimized for survival within the intestinal tract environment. This could also mean that the specific KanR or ClmR variant come to dominate within the gut not solely because they have a clear selective advantage over their counterparts due to acquisition of random beneficial mutation(s). Rather, one variant may outcompete another due to other stochastic effects, such as where the microbe happened to end up within the gut and the composition of its particular micro-niche, including the presence or absence of competitors and nutrient availability. To test these ideas, in addition to comparative 15 genomics, we could in future competitive assays use different fluorescently tagged versions of CFT073 and F11 to examine localization of distinct bacteria within the intestinal tract. For example, would a 1:1 mix of red and green fluorescent F11 intermingle within the gut, or would entirely red or entirely green clonal colonies emerge dependent on their localization within the gut and with respect to other microbes? These and other approaches promise to shed light on the factors that impact ExPEC persistence within the gut and may eventually suggest strategies to combat these ubiquitous pathogens before they have the chance to disseminate from the GI tract. 16 ACKNOWLEDGMENTS I would like to thank The Mulvey Lab, especially Dr. Mulvey, Amanda Richards, and Brittany Fleming for their constant support and help. I have learned so much over these last two years and it is because of their mentorship that my research experience has been invaluable to my university experience. I would also like to thank the Undergraduate Research Opportunities Program (UROP) and the Chevron Foundation for providing funding for this research. 17 REFERENCES [1] Foxman B. Urinary Tract Infection Syndromes Occurrence, Recurrence, Bacteriology, Risk Factors, and Disease Burden. Infect Dis Clin N Am. 2014;28(1):1+.doi:10.1016/j.idc.2013.09.003. PubMed PMID: WOS:000331917500002. [2] Ewers, C., Li, G., Wilking, H., Kiessling, S., Alt, K., Antao, E. M., Laturnus, C., Diehl, I., Glodde, S., Homeier, T., Bohnke, U., Steinruck, H., Philipp, H. C., and Wieler, L. H. (2007). Avian pathogenic, uropathogenic, and newborn meningitis-causing Escherichia coli: how closely related are they? Int J Med Microbiol 297, 163-176. [3] Buc, E., Dubois, D., Sauvanet, P., Raisch, J., Delmas, J., Darfeuille-Michaud, A., Pezet, D., and Bonnet, R. (2013). High prevalence of mucosa-associated E. coli producing cyclomodulin and genotoxin in colon cancer. PLoS One 8, e56964. PMC3572998 [4] Mann R, Mediati DG, Duggin IG, Harry EJ, Bottomley AL. Metabolic Adaptations of Uropathogenic E. coli in the Urinary Tract. Front Cell Infect Microbiol. 2017;7:241. Published 2017 Jun 8. doi:10.3389/fcimb.2017.00241 [5] Making Health Care Safer: Think sepsis. Time matters.: CDC National Center for Emerging and Zoonotic Infectious Diseases; 2016 [updated August 23, 2016]. [6] Mathers, A. J., Peirano, G., and Pitout, J. D. (2015). Escherichia coli ST131: The quintessential example of an international multiresistant high-risk clone. Adv Appl Microbiol 90, 109-154. [7] Huang, J., Lan, F., Lu, Y., & Li, B. (2020). Characterization of Integrons and Antimicrobial Resistance in Escherichia coli Sequence Type 131 Isolates. The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies 18 infectieuses et de la microbiologie medicale, 2020, 3826186. https://doi.org/10.1155/2020/3826186 [8] Manges, A. R., Smith, S. P., Lau, B. J., Nuval, C. J., Eisenberg, J. N., Dietrich, P. S., and Riley, L. W. (2007). Retail meat consumption and the acquisition of antimicrobial resistant Escherichia coli causing urinary tract infections: a case-control study. Foodborne Pathog Dis 4, 419-431. [9] Chen, S. L., Wu, M., Henderson, J. P., Hooton, T. M., Hibbing, M. E., Hultgren, S. J., and Gordon, J. I. (2013). Genomic diversity and fitness of E. coli strains recovered from the intestinal and urinary tracts of women with recurrent urinary tract infection. Sci Transl Med 5, 184ra160. PMC3695744 [10] Stecher, B., and Hardt, W. D. (2011). Mechanisms controlling pathogen colonization of the gut. Curr Opin Microbiol 14, 82-91. [11] Round, J. L., and Mazmanian, S. K. (2009). The gut microbiota shapes intestinal immune responses during health and disease. Nature Rev Immunology 9, 313-323. [12] Nowrouzian, F. L., Adlerberth, I., and Wold, A. E. (2006). Enhanced persistence in the colonic microbiota of Escherichia coli strains belonging to phylogenetic group B2: role of virulence factors and adherence to colonic cells. Microbes Infect 8, 834-840. [13] Barber, A. E., Fleming, B. A., and Mulvey, M. A. (2016). Similarly Lethal Strains of Extraintestinal Pathogenic Escherichia coli Trigger Markedly Diverse Host Responses in a Zebrafish Model of Sepsis. mSphere 1, pii: e00062-16. PMC4894679 [14] Foxman, B. (2010). The epidemiology of urinary tract infection. Nature reviews. Urology 7, 653-660. 19 [15] Barroso-Batista J, Sousa A, Lourenc ̧o M, Bergman M-L, Sobral D, et al. (2014) The First Steps of Adaptation of Escherichia coli to the Gut Are Dominated by Soft Sweeps. PLoS Genet 10(3): e1004182. doi:10.1371/journal.pgen.1004182 [16] Hildebrand, F., Nguyen, T. L., Brinkman, B., Yunta, R. G., Cauwe, B., Vandenabeele, P., Liston, A., and Raes, J. (2013). Inflammation-associated enterotypes, host genotype, cage and inter-individual effects drive gut microbiota variation in common laboratory mice. Genome Biol 14, R4. [17] Murphy, K. C., and Campellone, K. G. (2003). Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol Biol 4, 11. [18] Yang, X., Sha, K., Xu, G., Tian, H., Wang, X., Chen, S., Wang, Y., Li, J., Chen, J., & Huang, N. (2016). Subinhibitory Concentrations of Allicin Decrease Uropathogenic Escherichia coli (UPEC) Biofilm Formation, Adhesion Ability, and Swimming Motility. International journal of molecular sciences, 17(7), 979. https://doi.org/10.3390/ijms17070979 [19] Zhou, Y., Blanco, L. P., Smith, D. R., & Chapman, M. R. (2012). Bacterial amyloids. Methods in molecular biology (Clifton, N.J.), 849, 303–320. https://doi.org/10.1007/978-1-61779-551-0_21 [20] Stecher, B., and Hardt, W. D. (2011). Mechanisms controlling pathogen colonization of the gut. Curr Opin Microbiol 14, 82-91. [21] Russell CW, Fleming BA, Jost CA, Tran A, Stenquist AT, Wambaugh MA, Bronner MP, Mulvey MA. 2018. Context-dependent requirements for FimH and other 20 canonical virulence factors in gut colonization by extraintestinal pathogenic Escherichia coli. Infect Immun 86:e00746-17. https://doi.org/10.1128/IAI.00746-17. 21