Nutritional Basis for Colonization Resistance by Human Commensal \u3cem\u3eEscherichia coli\u3c/em\u3e Strains HS and Nissle 1917 Against \u3cem\u3eE. coli\u3c/em\u3e O157:H7 in the Mouse Intestine

Escherichia coli is a single species consisting of many biotypes, some of which are commensal colonizers of mammals and others that cause disease. Humans are colonized on average with five commensal biotypes, and it is widely thought that the commensals serve as a barrier to infection by pathogens. Previous studies showed that a combination of three pre-colonized commensal E. coli strains prevents colonization of E. coli O157:H7 in a mouse model (Leatham, et al., 2010, Infect Immun 77: 2876–7886). The commensal biotypes included E. coli HS, which is known to successfully colonize humans at high doses with no adverse effects, and E. coli Nissle 1917, a human commensal strain that is used in Europe as a preventative of traveler\u27s diarrhea. We hypothesized that commensal biotypes could exert colonization resistance by consuming nutrients needed by E. coli O157:H7 to colonize, thus preventing this first step in infection. Here we report that to colonize streptomycin-treated mice E. coli HS consumes six of the twelve sugars tested and E. coli Nissle 1917 uses a complementary yet divergent set of seven sugars to colonize, thus establishing a nutritional basis for the ability of E. coli HS and Nissle 1917 to occupy distinct niches in the mouse intestine. Together these two commensals use the five sugars previously determined to be most important for colonization of E. coli EDL933, an O157:H7 strain. As predicted, the two commensals prevented E. coli EDL933 colonization. The results support a model in which invading pathogenic E. coli must compete with the gut microbiota to obtain the nutrients needed to colonize and establish infection; accordingly, the outcome of the challenge is determined by the aggregate capacity of the native microbiota to consume the nutrients required by the pathogen

Nutritional Basis for Colonization Resistance by Human Commensal Escherichia coli Strains HS and Nissle 1917 against E. coli O157:H7 in the Mouse Intestine

The authors wish to thank Sal Rizzuto for assistance with the animal colonization experiment shown in Figure 4.Conceived and designed the experiments: RM MPL PSC TC. Performed the experiments: RM MPL TG. Analyzed the data: RM MPL PSC TC. Contributed reagents/materials/analysis tools: RM MPL TG PSC TC. Wrote the paper: RM PSC TC.Escherichia coli is a single species consisting of many biotypes, some of which are commensal colonizers of mammals and others that cause disease. Humans are colonized on average with five commensal biotypes, and it is widely thought that the commensals serve as a barrier to infection by pathogens. Previous studies showed that a combination of three pre-colonized commensal E. coli strains prevents colonization of E. coli O157:H7 in a mouse model (Leatham, et al., 2010, Infect Immun 77: 2876–7886). The commensal biotypes included E. coli HS, which is known to successfully colonize humans at high doses with no adverse effects, and E. coli Nissle 1917, a human commensal strain that is used in Europe as a preventative of traveler's diarrhea. We hypothesized that commensal biotypes could exert colonization resistance by consuming nutrients needed by E. coli O157:H7 to colonize, thus preventing this first step in infection. Here we report that to colonize streptomycin-treated mice E. coli HS consumes six of the twelve sugars tested and E. coli Nissle 1917 uses a complementary yet divergent set of seven sugars to colonize, thus establishing a nutritional basis for the ability of E. coli HS and Nissle 1917 to occupy distinct niches in the mouse intestine. Together these two commensals use the five sugars previously determined to be most important for colonization of E. coli EDL933, an O157:H7 strain. As predicted, the two commensals prevented E. coli EDL933 colonization. The results support a model in which invading pathogenic E. coli must compete with the gut microbiota to obtain the nutrients needed to colonize and establish infection; accordingly, the outcome of the challenge is determined by the aggregate capacity of the native microbiota to consume the nutrients required by the pathogen.Yeshttp://www.plosone.org/static/editorial#pee

An \u3cem\u3eEscherichia coli\u3c/em\u3e Nissle 1917 Missense Mutant Colonizes the Streptomycin-Treated Mouse Intestine Better than the Wild Type but Is Not a Better Probiotic

Previously we reported that the streptomycin-treated mouse intestine selected for two different Escherichia coli MG1655 mutants with improved colonizing ability: nonmotile E. coli MG1655 flhDC deletion mutants that grew 15% faster in vitro in mouse cecal mucus and motile E. coli MG1655 envZ missense mutants that grew slower in vitro in mouse cecal mucus yet were able to cocolonize with the faster-growing flhDC mutants. The E. coli MG1655 envZ gene encodes a histidine kinase that is a member of the envZ-ompR two-component signal transduction system, which regulates outer membrane protein profiles. In the present investigation, the envZ P41L gene was transferred from the intestinally selected E. coli MG1655 mutant to E. coli Nissle 1917, a human probiotic strain used to treat gastrointestinal infections. Both the E. coli MG1655 and E. coli Nissle 1917 strains containing envZ P41L produced more phosphorylated OmpR than their parents. The E. coli Nissle 1917 strain containing envZ P41L also became more resistant to bile salts and colicin V and grew 50% slower in vitro in mucus and 15% to 30% slower on several sugars present in mucus, yet it was a 10-fold better colonizer than E. coli Nissle 1917. However, E. coli Nissle 1917 envZ P41L was not better at preventing colonization by enterohemorrhagic E. coli EDL933. The data can be explained according to our “restaurant” hypothesis for commensal E. coli strains, i.e., that they colonize the intestine as sessile members of mixed biofilms, obtaining the sugars they need for growth locally, but compete for sugars with invading E. coli pathogens planktonically

The Streptomycin-Treated Mouse Intestine Selects \u3cem\u3eEscherichia coli envZ\u3c/em\u3e Missense Mutants That Interact with Dense and Diverse Intestinal Microbiota

Previously, we reported that the streptomycin-treated mouse intestine selected nonmotile Escherichia coli MG1655 flhDC deletion mutants of E. coli MG1655 with improved colonizing ability that grow 15% faster in vitro in mouse cecal mucus and 15 to 30% faster on sugars present in mucus (M. P. Leatham et al., Infect. Immun. 73:8039–8049, 2005). Here, we report that the 10 to 20% remaining motile E. coli MG1655 are envZ missense mutants that are also better colonizers of the mouse intestine than E. coli MG1655. One of the flhDC mutants, E. coli MG1655 ΔflhD, and one of the envZ missense mutants, E. coli MG1655 mot-1, were studied further. E. coli MG1655 mot-1 is more resistant to bile salts and colicin V than E. coli MG1655 ΔflhD and grows ca. 15% slower in vitro in mouse cecal mucus and on several sugars present in mucus compared to E. coli MG1655 ΔflhD but grows 30% faster on galactose. Moreover, E. coli MG1655 mot-1 and E. coli MG1655 ΔflhD appear to colonize equally well in one intestinal niche, but E. coli MG1655 mot-1 appears to use galactose to colonize a second, smaller intestinal niche either not colonized or colonized poorly by E. coli MG1655 ΔflhD. Evidence is also presented that E. coli MG1655 is a minority member of mixed bacterial biofilms in the mucus layer of the streptomycin-treated mouse intestine. We offer a hypothesis, which we call the “Restaurant” hypothesis, that explains how nutrient acquisition in different biofilms comprised of different anaerobes can account for our results

Uropathogenic <i>Escherichia coli</i> metabolite-dependent quiescence and persistence may explain antibiotic tolerance during urinary tract infection

ABSTRACT In the present study, it is shown that although Escherichia coli CFT073, a human uropathogenic (UPEC) strain, grows in liquid glucose M9 minimal medium, it fails to grow on glucose M9 minimal medium agar plates seeded with ≤106 CFU. The cells on glucose plates appear to be in a “quiescent” state that can be prevented by various combinations of lysine, methionine, and tyrosine. Moreover, the quiescent state is characteristic of ~80% of E. coli phylogenetic group B2 multilocus sequence type 73 strains, as well as 22.5% of randomly selected UPEC strains isolated from community-acquired urinary tract infections in Denmark. In addition, E. coli CFT073 quiescence is not limited to glucose but occurs on agar plates containing a number of other sugars and acetate as sole carbon sources. It is also shown that a number of E. coli CFT073 mini-Tn5 metabolic mutants (gnd, gdhA, pykF, sdhA, and zwf) are nonquiescent on glucose M9 minimal agar plates and that quiescence requires a complete oxidative tricarboxylic acid (TCA) cycle. In addition, evidence is presented that, although E. coli CFT073 quiescence and persistence in the presence of ampicillin are alike in that both require a complete oxidative TCA cycle and each can be prevented by amino acids, E. coli CFT073 quiescence occurs in the presence or absence of a functional rpoS gene, whereas maximal persistence requires a nonfunctional rpoS. Our results suggest that interventions targeting specific central metabolic pathways may mitigate UPEC infections by interfering with quiescence and persistence. IMPORTANCE Recurrent urinary tract infections (UTIs) affect 10 to 40% of women. In up to 77% of those cases, the recurrent infections are caused by the same uropathogenic E. coli (UPEC) strain that caused the initial infection. Upon infection of urothelial transitional cells in the bladder, UPEC appear to enter a nongrowing quiescent intracellular state that is thought to serve as a reservoir responsible for recurrent UTIs. Here, we report that many UPEC strains enter a quiescent state when ≤106 CFU are seeded on glucose M9 minimal medium agar plates and show that mutations in several genes involved in central carbon metabolism prevent quiescence, as well as persistence, possibly identifying metabolic pathways involved in UPEC quiescence and persistence in vivo

Uropathogenic Escherichia coli Metabolite-Dependent Quiescence and Persistence May Explain Antibiotic Tolerance during Urinary Tract Infection

In the present study, it is shown that although Escherichia coli CFT073, a human uropathogenic (UPEC) strain, grows in liquid glucose M9 minimal medium, it fails to grow on glucose M9 minimal medium agar plates seeded with ≤106 CFU. The cells on glucose plates appear to be in a “quiescent” state that can be prevented by various combinations of lysine, methionine, and tyrosine. Moreover, the quiescent state is characteristic of ~80% of E. coli phylogenetic group B2 multilocus sequence type 73 strains, as well as 22.5% of randomly selected UPEC strains isolated from community-acquired urinary tract infections in Denmark. In addition, E. coli CFT073 quiescence is not limited to glucose but occurs on agar plates containing a number of other sugars and acetate as sole carbon sources. It is also shown that a number of E. coliCFT073 mini-Tn5 metabolic mutants (gnd, gdhA, pykF, sdhA, and zwf) are nonquiescent on glucose M9 minimal agar plates and that quiescence requires a complete oxidative tricarboxylic acid (TCA) cycle. In addition, evidence is presented that, although E. coli CFT073 quiescence and persistence in the presence of ampicillin are alike in that both require a complete oxidative TCA cycle and each can be prevented by amino acids, E. coli CFT073 quiescence occurs in the presence or absence of a functional rpoS gene, whereas maximal persistence requires a nonfunctional rpoS. Our results suggest that interventions targeting specific central metabolic pathways may mitigate UPEC infections by interfering with quiescence and persistence

Competitive colonization of <i>E. coli</i> HS mutants vs. wild type.

<p>Sets of three mice were fed 10⁵ CFU of both the <i>E. coli</i> HS wild type strain and mutant strain. At the times indicated, fecal samples were homogenized, diluted, and plated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053957#s4" target="_blank">Materials and Methods</a>. Compiled data from at least two distinct experiments (6 mice) are shown. Standard errors of the log₁₀ means of CFU/gram feces are indicated with error bars. (<b>A</b>) Δ<i>uxaC</i>, which does not have a colonization defect; (<b>B</b>) Δ<i>rbsK</i>, which has a moderate colonization defect; (<b>C</b>) Δ<i>galK</i>, which has a severe colonization defect.</p

Phenotypic assay results for <i>E. coli</i> HS mutants.

<p><b>A–L.</b> Biolog GN2 (Gram negative carbon nutrition) plates were utilized to assess the overall metabolic capacity of the mutants constructed in <i>E. coli</i> HS to consume 95 individual carbon sources (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053957#s4" target="_blank">Materials and Methods</a> for details). The kinetic curves plot substrate oxidation vs. time. Green represents carbon source utilization by the <i>E. coli</i> HS wild type strain, red represents the mutant strain, and yellow represents utilization by both strains. Mutant strains of <i>E. coli</i> HS: (<b>A</b>) Δ<i>araBAD</i>, (<b>B</b>) <i>araBAD+</i>, (<b>C</b>) Δ<i>galK</i>, (<b>D</b>) <i>galK+</i>, (<b>E</b>) Δ<i>fucK</i>, (<b>F</b>) Δ<i>gntK</i> Δ<i>idnK</i>, (<b>G</b>) Δ<i>lacZ</i>, (<b>H</b>) Δ<i>manA</i>, (<b>I</b>) Δ<i>nagE</i>, (<b>J</b>) Δ<i>nanAT</i>, (<b>K</b>) Δ<i>rbsK</i>, and (<b>L</b>) Δ<i>uxaC</i>. <b>M.</b> Sugar utilization was assessed in minimal media containing 0.2% carbon source. The final O.D.₆₀₀'s following 12 hour incubations are shown. Note: the generation time of <i>E. coli HS</i> Δ<i>nagE</i> on N-acetylglucosamine is 2.6 h compared to 1 h for the wild type (see text for details). The sugars N-acetylneuraminate and ribose are not represented on the Biolog plate (NA).</p

Bacterial strains and plasmids used in this study.

1<p>Str^R, streptomycin resistance; Nal^R, nalidixic acid resistance; Rif^R, rifampicin resistance; Amp^R, ampicillin resistance; T^S, temperature sensitive replication.</p

<i>E. coli</i> HS sugar utilization in the mouse intestine.

<p>The difference between population density of the wild type and each mutant strain is shown at Day 1 and Day 9, plus or minus the standard error of the mean. Significant values (bold) indicate log difference >0.8 and student's <i>t</i> test value P<0.05 at Day 9.</p