Draft Genome Sequence of Citrobacter rodentium DBS100 (ATCC 51459), a Primary Model of Enterohemorrhagic Escherichia coli Virulence.

Citrobacter rodentium is a Gram-negative bacterium which causes transmissible murine colonic hyperplasia and models the virulence of enterohemorrhagic Escherichia coli in vivo. Thus, C. rodentium is used to study human gastrointestinal disease. We present the draft genome sequence of C. rodentium strain ATCC 51459, also known as DBS100.

Genotype and phenotypes of an intestine-adapted Escherichia coli K-12 mutant selected by animal passage for superior colonization.

We previously isolated a spontaneous mutant of Escherichia coli K-12, strain MG1655, following passage through the streptomycin-treated mouse intestine, that has colonization traits superior to the wild-type parent strain (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). This intestine-adapted strain (E. coli MG1655*) grew faster on several different carbon sources than the wild type and was nonmotile due to deletion of the flhD gene. We now report the results of several high-throughput genomic analysis approaches to further characterize E. coli MG1655*. Whole-genome pyrosequencing did not reveal any changes on its genome, aside from the deletion at the flhDC locus, that could explain the colonization advantage of E. coli MG1655*. Microarray analysis revealed modest yet significant induction of catabolic gene systems across the genome in both E. coli MG1655* and an isogenic flhD mutant constructed in the laboratory. Catabolome analysis with Biolog GN2 microplates revealed an enhanced ability of both E. coli MG1655* and the isogenic flhD mutant to oxidize a variety of carbon sources. The results show that intestine-adapted E. coli MG1655* is more fit than the wild type for intestinal colonization, because loss of FlhD results in elevated expression of genes involved in carbon and energy metabolism, resulting in more efficient carbon source utilization and a higher intestinal population. Hence, mutations that enhance metabolic efficiency confer a colonization advantage.

E. coli O157:H7 catabolism of intestinal mucin-derived carbohydrates and colonization.

E. coli O157:H7 colonizes the bovine intestine, can contaminate food through fecal shedding, and causes human diarrheal and systemic illnesses. Catabolism of particular carbohydrates by E. coli has been found to be important for intestinal colonization of mice. In this study, we assessed whether catabolism of two mucin-derived carbohydrates are important for E. coli O157:H7 colonization of adult cattle. This was accomplished by competitively co-colonizing streptomycin-treated adult cattle with a wild-type strain of E. coli O157:H7 and isogenic mutants in catabolic pathways for mucin-derived carbohydrates N-acetylgalactosamine or l-fucose. Both mutants colonized poorly compared to the wild-type during the initiation stage of colonization (days 0-6). During the maintenance stage of colonization (days 7-15), the mutant unable to use N-acetylgalactosamine did not show a colonization defect, whereas the strain unable to use fucose had a significant colonization defect. These results support the concept that growth and colonization of E. coli O157:H7 in the bovine rectum has a nutritional basis, with a nutrient preference for l-fucose over N-acetylgalactosamine.

Comparison of carbon nutrition for pathogenic and commensal Escherichia coli strains in the mouse intestine.

The carbon sources that support the growth of pathogenic Escherichia coli O157:H7 in the mammalian intestine have not previously been investigated. In vivo, the pathogenic E. coli EDL933 grows primarily as single cells dispersed within the mucus layer that overlies the mouse cecal epithelium. We therefore compared the pathogenic strain and the commensal E. coli strain MG1655 modes of metabolism in vitro, using a mixture of the sugars known to be present in cecal mucus, and found that the two strains used the 13 sugars in a similar order and cometabolized as many as 9 sugars at a time. We conducted systematic mutation analyses of E. coli EDL933 and E. coli MG1655 by using lesions in the pathways used for catabolism of 13 mucus-derived sugars and five other compounds for which the corresponding bacterial gene system was induced in the transcriptome of cells grown on cecal mucus. Each of 18 catabolic mutants in both bacterial genetic backgrounds was fed to streptomycin-treated mice, together with the respective wild-type parent strain, and their colonization was monitored by fecal plate counts. None of the mutations corresponding to the five compounds not found in mucosal polysaccharides resulted in colonization defects. Based on the mutations that caused colonization defects, we determined that both E. coli EDL933 and E. coli MG1655 used arabinose, fucose, and N-acetylglucosamine in the intestine. In addition, E. coli EDL933 used galactose, hexuronates, mannose, and ribose, whereas E. coli MG1655 used gluconate and N-acetylneuraminic acid. The colonization defects of six catabolic lesions were found to be additive with E. coli EDL933 but not with E. coli MG1655. The data indicate that pathogenic E. coli EDL933 uses sugars that are not used by commensal E. coli MG1655 to colonize the mouse intestine. The results suggest a strategy whereby invading pathogens gain advantage by simultaneously consuming several sugars that may be available because they are not consumed by the commensal intestinal microbiota.

Respiration of Escherichia coli in the mouse intestine.

Mammals are aerobes that harbor an intestinal ecosystem dominated by large numbers of anaerobic microorganisms. However, the role of oxygen in the intestinal ecosystem is largely unexplored. We used systematic mutational analysis to determine the role of respiratory metabolism in the streptomycin-treated mouse model of intestinal colonization. Here we provide evidence that aerobic respiration is required for commensal and pathogenic Escherichia coli to colonize mice. Our results showed that mutants lacking ATP synthase, which is required for all respiratory energy-conserving metabolism, were eliminated by competition with respiratory-competent wild-type strains. Mutants lacking the high-affinity cytochrome bd oxidase, which is used when oxygen tensions are low, also failed to colonize. However, the low-affinity cytochrome bo(3) oxidase, which is used when oxygen tension is high, was found not to be necessary for colonization. Mutants lacking either nitrate reductase or fumarate reductase also had major colonization defects. The results showed that the entire E. coli population was dependent on both microaerobic and anaerobic respiration, consistent with the hypothesis that the E. coli niche is alternately microaerobic and anaerobic, rather than static. The results indicate that success of the facultative anaerobes in the intestine depends on their respiratory flexibility. Despite competition for relatively scarce carbon sources, the energy efficiency provided by respiration may contribute to the widespread distribution (i.e., success) of E. coli strains as commensal inhabitants of the mammalian intestine.

Evaluation of a model for Escherichia coli O157:H7 colonization in streptomycin-treated adult cattle.

OBJECTIVE: To develop a repeatable model for studying colonization with streptomycin-resistant Escherichia coli O157:H7 in adult cattle. ANIMALS: 5 adult mixed-breed beef cattle. PROCEDURES: Cattle were surgically cannulated in the duodenum, treated daily with streptomycin (33 mg/kg) via the duodenal cannula prior to and during experimental colonizations, and colonized with 10(10) CFUs of streptomycin-resistant E coli O157:H7 via the duodenal cannula. Colonization of rectal mucus and shedding in feces were monitored. Antimicrobials were administered to eliminate the colonizing strain so that 5 repeated colonization experiments could be performed. A comprehensive analysis of colonization was performed at necropsy. RESULTS: Streptomycin treatment resulted in improved experimental colonization variables, compared with untreated controls, during initiation (days 2 to 6) and early maintenance (days 7 to 12) of colonization. Elimination of the colonizing strain followed by 5 repeated colonizations in the same animals indicated the repeatability of the protocol. Positive results of bacteriologic culture of feces 7 and 12 days after colonization were obtained in 100% and 84% of samples, respectively, across all animals and trials. At necropsy, highest magnitude recovery was in terminal rectal mucus. CONCLUSIONS AND CLINICAL RELEVANCE: The model was highly repeatable and novel with respect to streptomycin treatment, use of duodenal cannulas, and repeated colonizations of the same animals. Its use in adult cattle, from which most bovine-derived food originates, is critical to the study of preharvest food safety. The findings have implications for understanding intermittency of shedding in the field and for proposed vaccine-based interventions.