Precolonized human commensal Escherichia coli strains serve as a barrier to E. coli O157:H7 growth in the streptomycin-treated mouse intestine.

Different Escherichia coli strains generally have the same metabolic capacity for growth on sugars in vitro, but they appear to use different sugars in the streptomycin-treated mouse intestine (Fabich et al., Infect. Immun. 76:1143-1152, 2008). Here, mice were precolonized with any of three human commensal strains (E. coli MG1655, E. coli HS, or E. coli Nissle 1917) and 10 days later were fed 10(5) CFU of the same strains. While each precolonized strain nearly eliminated its isogenic strain, confirming that colonization resistance can be modeled in mice, each allowed growth of the other commensal strains to higher numbers, consistent with different commensal E. coli strains using different nutrients in the intestine. Mice were also precolonized with any of five commensal E. coli strains for 10 days and then were fed 10(5) CFU of E. coli EDL933, an O157:H7 pathogen. E. coli Nissle 1917 and E. coli EFC1 limited growth of E. coli EDL933 in the intestine (10(3) to 10(4) CFU/gram of feces), whereas E. coli MG1655, E. coli HS, and E. coli EFC2 allowed growth to higher numbers (10(6) to 10(7) CFU/gram of feces). Importantly, when E. coli EDL933 was fed to mice previously co-colonized with three E. coli strains (MG1655, HS, and Nissle 1917), it was eliminated from the intestine (<10 CFU/gram of feces). These results confirm that commensal E. coli strains can provide a barrier to infection and suggest that it may be possible to construct E. coli probiotic strains that prevent growth of pathogenic E. coli strains in the intestine.

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.

L-fucose stimulates utilization of D-ribose by Escherichia coli MG1655 DeltafucAO and E. coli Nissle 1917 DeltafucAO mutants in the mouse intestine and in M9 minimal medium.

Escherichia coli MG1655 uses several sugars for growth in the mouse intestine. To determine the roles of L-fucose and D-ribose, an E. coli MG1655 DeltafucAO mutant and an E. coli MG1655 DeltarbsK mutant were fed separately to mice along with wild-type E. coli MG1655. The E. coli MG1655 DeltafucAO mutant colonized the intestine at a level 2 orders of magnitude lower than that of the wild type, but the E. coli MG1655 DeltarbsK mutant and the wild type colonized at nearly identical levels. Surprisingly, an E. coli MG1655 DeltafucAO DeltarbsK mutant was eliminated from the intestine by either wild-type E. coli MG1655 or E. coli MG1655 DeltafucAO, suggesting that the DeltafucAO mutant switches to ribose in vivo. Indeed, in vitro growth experiments showed that L-fucose stimulated utilization of D-ribose by the E. coli MG1655 DeltafucAO mutant but not by an E. coli MG1655 DeltafucK mutant. Since the DeltafucK mutant cannot convert L-fuculose to L-fuculose-1-phosphate, whereas the DeltafucAO mutant accumulates L-fuculose-1-phosphate, the data suggest that L-fuculose-1-phosphate stimulates growth on ribose both in the intestine and in vitro. An E. coli Nissle 1917 DeltafucAO mutant, derived from a human probiotic commensal strain, acted in a manner identical to that of E. coli MG1655 DeltafucAO in vivo and in vitro. Furthermore, L-fucose at a concentration too low to support growth stimulated the utilization of ribose by the wild-type E. coli strains in vitro. Collectively, the data suggest that L-fuculose-1-phosphate plays a role in the regulation of ribose usage as a carbon source by E. coli MG1655 and E. coli Nissle 1917 in the mouse intestine.

Mouse intestine selects nonmotile flhDC mutants of Escherichia coli MG1655 with increased colonizing ability and better utilization of carbon sources.

D-gluconate which is primarily catabolized via the Entner-Doudoroff (ED) pathway, has been implicated as being important for colonization of the streptomycin-treated mouse large intestine by Escherichia coli MG1655, a human commensal strain. In the present study, we report that an MG1655 Deltaedd mutant defective in the ED pathway grows poorly not only on gluconate as a sole carbon source but on a number of other sugars previously implicated as being important for colonization, including L-fucose, D-gluconate, D-glucuronate, N-acetyl-D-glucosamine, D-mannose, and D-ribose. Furthermore, we show that the mouse intestine selects mutants of MG1655 Deltaedd and wild-type MG1655 that have improved mouse intestine-colonizing ability and grow 15 to 30% faster on the aforementioned sugars. The mutants of MG1655 Deltaedd and wild-type MG1655 selected by the intestine are shown to be nonmotile and to have deletions in the flhDC operon, which encodes the master regulator of flagellar biosynthesis. Finally, we show that DeltaflhDC mutants of wild-type MG1655 and MG1655 Deltaedd constructed in the laboratory act identically to those selected by the intestine; i.e., they grow better than their respective parents on sugars as sole carbon sources and are better colonizers of the mouse intestine.