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Identification of commensal Escherichia coli genes involved in biofilm resistance to pathogen colonization.

Da Re S, Valle J, Charbonnel N, Beloin C, Latour-Lambert P, Faure P, Turlin E, Le Bouguénec C, Renauld-Mongénie G, Forestier C, Ghigo JM - PLoS ONE (2013)

Bottom Line: We showed that pathogens trigger specific responses in commensal bacteria and we identified genes involved in limiting colonization of incoming pathogens within commensal biofilm.We demonstrated that the absence of yiaF and bssS (yceP) differentially alters pathogen colonization in the mouse gut.This study therefore identifies previously uncharacterized colonization resistance genes and provides new approaches to unravelling molecular aspects of commensal/pathogen competitive interactions.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité de Génétique des Biofilms, Département de Microbiologie, Paris, France.

ABSTRACT
Protection provided by host bacterial microbiota against microbial pathogens is a well known but ill-understood property referred to as the barrier effect, or colonization resistance. Despite recent genome-wide analyses of host microbiota and increasing therapeutic interest, molecular analysis of colonization resistance is hampered by the complexity of direct in vivo experiments. Here we developed an in vitro-to-in vivo approach to identification of genes involved in resistance of commensal bacteria to exogenous pathogens. We analyzed genetic responses induced in commensal Escherichia coli upon entry of a diarrheagenic enteroaggregative E. coli or an unrelated Klebsiella pneumoniae pathogen into a biofilm community. We showed that pathogens trigger specific responses in commensal bacteria and we identified genes involved in limiting colonization of incoming pathogens within commensal biofilm. We tested the in vivo relevance of our findings by comparing the extent of intestinal colonization by enteroaggregative E. coli and K. pneumoniae pathogens in mice pre-colonized with E. coli wild type commensal strain, or mutants corresponding to identified colonization resistance genes. We demonstrated that the absence of yiaF and bssS (yceP) differentially alters pathogen colonization in the mouse gut. This study therefore identifies previously uncharacterized colonization resistance genes and provides new approaches to unravelling molecular aspects of commensal/pathogen competitive interactions.

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In vivo colonization of E. coli commensal biofilm by K. pneumoniae KpLM21 pathogen.A Schematic representation of the experimental procedure. B Streptomycin-treated mice were first challenged intragastrically with commensal wild-type MG1655-s F′ (C) or its mutant ΔyceP, ΔyliE, and ΔyiaF derivatives (C*), followed on day 11 by administration of the K. pneumoniae KpLM21-s pathogen. The numbers of commensal and pathogen cfus recovered per gram of feces were determined every other day from day 3 to day 20. The lower limit of detection for bacteria was 102 cfu/g of feces. Box-and-whiskers plots indicate high and low values, median and interquartile ranges; each group contained 8 to 12 mice. Pearson analysis of the bacterial count in faeces (impact of the initial colonization by the wild-type MG1655-s F′ or its derivatives on the capacity of the pathogen (K. pneumoniae KpLM21-s) to colonize the mice intestine) and Mann-Whitney analysis of the number of the pathogen CFUs recovered (comparison of pathogen colonization level in mice precolonized with either MG1655-s F′ (control) or its derivatives (yliE, yceP or yiaF)) were performed. Statistically different results (P<0.05), are indicated by an asterisk.
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pone-0061628-g005: In vivo colonization of E. coli commensal biofilm by K. pneumoniae KpLM21 pathogen.A Schematic representation of the experimental procedure. B Streptomycin-treated mice were first challenged intragastrically with commensal wild-type MG1655-s F′ (C) or its mutant ΔyceP, ΔyliE, and ΔyiaF derivatives (C*), followed on day 11 by administration of the K. pneumoniae KpLM21-s pathogen. The numbers of commensal and pathogen cfus recovered per gram of feces were determined every other day from day 3 to day 20. The lower limit of detection for bacteria was 102 cfu/g of feces. Box-and-whiskers plots indicate high and low values, median and interquartile ranges; each group contained 8 to 12 mice. Pearson analysis of the bacterial count in faeces (impact of the initial colonization by the wild-type MG1655-s F′ or its derivatives on the capacity of the pathogen (K. pneumoniae KpLM21-s) to colonize the mice intestine) and Mann-Whitney analysis of the number of the pathogen CFUs recovered (comparison of pathogen colonization level in mice precolonized with either MG1655-s F′ (control) or its derivatives (yliE, yceP or yiaF)) were performed. Statistically different results (P<0.05), are indicated by an asterisk.

Mentions: To test the in vivo role of colonization resistance genes identified via our approach, we used a mouse model of intragastric infection to compare the extent of intestinal colonization by 55989a and KpLM21 pathogens in streptomycin-treated mice, in which enterobacteria such as E. coli and Klebsiella were shown to colonize the same niches [45], [46]. Streptomycin-treated mice were pre-colonized with wild-type or mutant commensal E. coli MG1655 F′ and all three strains efficiently colonized the mouse intestine [47]–[49] (Fig. 4A). We chose to test the role of: i) yiaF, which affects in vitro colonization resistance to 55989a and was also induced upon KpLM21 colonization (Fig. 1B, and Table 4); ii) yliE, which was similarly involved in in vitro colonization resistance to 55989a, but was not induced upon KpLM21 colonization (Fig. 1B and Table 4 and Fig. S1); and iii) yceP, a gene induced in response to both pathogens, though without detectable in vitro effects on 55989a colonization (Fig. 1B and Table 4 and Fig. S1). To perform colonization experiments in streptomycin-treated mice, we first made E. coli 55989a and KpLM21 streptomycin-resistant derivatives (respectively, 55989a-s and KpLM21-s). We also introduced yiaF, yliE and yceP mutants into the same streptomycin-resistant derivative of MG1655 F′, (MG1655-s F′) and checked that these strains were not significantly affected in terms of growth and in vitro biofilm ability against this background (data not shown). We then determined bacterial counts in feces recovered from individually inoculated mice (n = at least 8 for each strain) and observed that wild-type and MG1655-s F′ yiaF, yliE and yceP mutants reached similar intestinal colonization capacity at day 10 (between 107 to 108 cfu/g of feces) prior to pathogen inoculation (Fig. 4B and 5B). At day 11 post-inoculation, streptomycin-treated mice colonized with wild-type MG1655-s F′ or corresponding yiaF, yliE and yceP mutants were inoculated intragastrically with either 55989a-s (Fig. 4) or KpLM21-s (Fig. 5). Determination of Pearson correlation coefficients indicated that there was no (−0.5<P<0.5) or only a moderate (0.5<P<0.8) correlation between colonization levels of wild-type MG1655-sF′ or its mutant derivatives and pathogens (KpLM21-s and 55989a-s) at days 10 and 12 post-inoculation by the commensal (wild-type or mutant) strains. Using the non-parametric Mann-Whitney test, comparison from day 12 to day 20 of the numbers of pathogen cfus in the feces of mice previously inoculated with wild-type MG1655-s or yliE, yceP or yiaF mutants indicated that pre-colonization of mice with MG1655-s F′ yceP, but not yliE, led to statistically significantly increased intestinal colonization by both pathogens (P = 2.3E10−7 and P = 0.19, respectively) (Fig. 4B and 5B). In addition, while mice pre-inoculated with MG1655-s F′ yiaF displayed lower level (P = 0.01) of KpLM21-s colonization, they showed higher levels (P = 0.01) of E. coli 55989a-s colonization compared to mice precolonized with wild-type MG1655-s control (Fig. 5B and 4B respectively)


Identification of commensal Escherichia coli genes involved in biofilm resistance to pathogen colonization.

Da Re S, Valle J, Charbonnel N, Beloin C, Latour-Lambert P, Faure P, Turlin E, Le Bouguénec C, Renauld-Mongénie G, Forestier C, Ghigo JM - PLoS ONE (2013)

In vivo colonization of E. coli commensal biofilm by K. pneumoniae KpLM21 pathogen.A Schematic representation of the experimental procedure. B Streptomycin-treated mice were first challenged intragastrically with commensal wild-type MG1655-s F′ (C) or its mutant ΔyceP, ΔyliE, and ΔyiaF derivatives (C*), followed on day 11 by administration of the K. pneumoniae KpLM21-s pathogen. The numbers of commensal and pathogen cfus recovered per gram of feces were determined every other day from day 3 to day 20. The lower limit of detection for bacteria was 102 cfu/g of feces. Box-and-whiskers plots indicate high and low values, median and interquartile ranges; each group contained 8 to 12 mice. Pearson analysis of the bacterial count in faeces (impact of the initial colonization by the wild-type MG1655-s F′ or its derivatives on the capacity of the pathogen (K. pneumoniae KpLM21-s) to colonize the mice intestine) and Mann-Whitney analysis of the number of the pathogen CFUs recovered (comparison of pathogen colonization level in mice precolonized with either MG1655-s F′ (control) or its derivatives (yliE, yceP or yiaF)) were performed. Statistically different results (P<0.05), are indicated by an asterisk.
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Related In: Results  -  Collection

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pone-0061628-g005: In vivo colonization of E. coli commensal biofilm by K. pneumoniae KpLM21 pathogen.A Schematic representation of the experimental procedure. B Streptomycin-treated mice were first challenged intragastrically with commensal wild-type MG1655-s F′ (C) or its mutant ΔyceP, ΔyliE, and ΔyiaF derivatives (C*), followed on day 11 by administration of the K. pneumoniae KpLM21-s pathogen. The numbers of commensal and pathogen cfus recovered per gram of feces were determined every other day from day 3 to day 20. The lower limit of detection for bacteria was 102 cfu/g of feces. Box-and-whiskers plots indicate high and low values, median and interquartile ranges; each group contained 8 to 12 mice. Pearson analysis of the bacterial count in faeces (impact of the initial colonization by the wild-type MG1655-s F′ or its derivatives on the capacity of the pathogen (K. pneumoniae KpLM21-s) to colonize the mice intestine) and Mann-Whitney analysis of the number of the pathogen CFUs recovered (comparison of pathogen colonization level in mice precolonized with either MG1655-s F′ (control) or its derivatives (yliE, yceP or yiaF)) were performed. Statistically different results (P<0.05), are indicated by an asterisk.
Mentions: To test the in vivo role of colonization resistance genes identified via our approach, we used a mouse model of intragastric infection to compare the extent of intestinal colonization by 55989a and KpLM21 pathogens in streptomycin-treated mice, in which enterobacteria such as E. coli and Klebsiella were shown to colonize the same niches [45], [46]. Streptomycin-treated mice were pre-colonized with wild-type or mutant commensal E. coli MG1655 F′ and all three strains efficiently colonized the mouse intestine [47]–[49] (Fig. 4A). We chose to test the role of: i) yiaF, which affects in vitro colonization resistance to 55989a and was also induced upon KpLM21 colonization (Fig. 1B, and Table 4); ii) yliE, which was similarly involved in in vitro colonization resistance to 55989a, but was not induced upon KpLM21 colonization (Fig. 1B and Table 4 and Fig. S1); and iii) yceP, a gene induced in response to both pathogens, though without detectable in vitro effects on 55989a colonization (Fig. 1B and Table 4 and Fig. S1). To perform colonization experiments in streptomycin-treated mice, we first made E. coli 55989a and KpLM21 streptomycin-resistant derivatives (respectively, 55989a-s and KpLM21-s). We also introduced yiaF, yliE and yceP mutants into the same streptomycin-resistant derivative of MG1655 F′, (MG1655-s F′) and checked that these strains were not significantly affected in terms of growth and in vitro biofilm ability against this background (data not shown). We then determined bacterial counts in feces recovered from individually inoculated mice (n = at least 8 for each strain) and observed that wild-type and MG1655-s F′ yiaF, yliE and yceP mutants reached similar intestinal colonization capacity at day 10 (between 107 to 108 cfu/g of feces) prior to pathogen inoculation (Fig. 4B and 5B). At day 11 post-inoculation, streptomycin-treated mice colonized with wild-type MG1655-s F′ or corresponding yiaF, yliE and yceP mutants were inoculated intragastrically with either 55989a-s (Fig. 4) or KpLM21-s (Fig. 5). Determination of Pearson correlation coefficients indicated that there was no (−0.5<P<0.5) or only a moderate (0.5<P<0.8) correlation between colonization levels of wild-type MG1655-sF′ or its mutant derivatives and pathogens (KpLM21-s and 55989a-s) at days 10 and 12 post-inoculation by the commensal (wild-type or mutant) strains. Using the non-parametric Mann-Whitney test, comparison from day 12 to day 20 of the numbers of pathogen cfus in the feces of mice previously inoculated with wild-type MG1655-s or yliE, yceP or yiaF mutants indicated that pre-colonization of mice with MG1655-s F′ yceP, but not yliE, led to statistically significantly increased intestinal colonization by both pathogens (P = 2.3E10−7 and P = 0.19, respectively) (Fig. 4B and 5B). In addition, while mice pre-inoculated with MG1655-s F′ yiaF displayed lower level (P = 0.01) of KpLM21-s colonization, they showed higher levels (P = 0.01) of E. coli 55989a-s colonization compared to mice precolonized with wild-type MG1655-s control (Fig. 5B and 4B respectively)

Bottom Line: We showed that pathogens trigger specific responses in commensal bacteria and we identified genes involved in limiting colonization of incoming pathogens within commensal biofilm.We demonstrated that the absence of yiaF and bssS (yceP) differentially alters pathogen colonization in the mouse gut.This study therefore identifies previously uncharacterized colonization resistance genes and provides new approaches to unravelling molecular aspects of commensal/pathogen competitive interactions.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité de Génétique des Biofilms, Département de Microbiologie, Paris, France.

ABSTRACT
Protection provided by host bacterial microbiota against microbial pathogens is a well known but ill-understood property referred to as the barrier effect, or colonization resistance. Despite recent genome-wide analyses of host microbiota and increasing therapeutic interest, molecular analysis of colonization resistance is hampered by the complexity of direct in vivo experiments. Here we developed an in vitro-to-in vivo approach to identification of genes involved in resistance of commensal bacteria to exogenous pathogens. We analyzed genetic responses induced in commensal Escherichia coli upon entry of a diarrheagenic enteroaggregative E. coli or an unrelated Klebsiella pneumoniae pathogen into a biofilm community. We showed that pathogens trigger specific responses in commensal bacteria and we identified genes involved in limiting colonization of incoming pathogens within commensal biofilm. We tested the in vivo relevance of our findings by comparing the extent of intestinal colonization by enteroaggregative E. coli and K. pneumoniae pathogens in mice pre-colonized with E. coli wild type commensal strain, or mutants corresponding to identified colonization resistance genes. We demonstrated that the absence of yiaF and bssS (yceP) differentially alters pathogen colonization in the mouse gut. This study therefore identifies previously uncharacterized colonization resistance genes and provides new approaches to unravelling molecular aspects of commensal/pathogen competitive interactions.

Show MeSH
Related in: MedlinePlus