Limits...
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.

Show MeSH

Related in: MedlinePlus

Identification of colonization resistance factors interfering with establishment of mixed pathogen/commensal biofilm.A Experimental set-up: continuous flow biofilm growth in microfermentor. After initial inoculation of the microfermentor with E. coli MG1655 F′ commensal (C), biofilm develops for 6 h before re-inoculation (colonization) with exogenous pathogen E. coli 55989a (P). At 24 h post-colonization, mixed biofilm developing on the glass slide was resuspended and used for gene expression analysis and determination of colonization phenotype (% of pathogens in the mixed biofilm). B Microfermentors were inoculated with wild-type or mutant commensal (MG1655 F′ is abbreviated as MG) as indicated in the x-axis. After 6 h of growth, commensal biofilm was re-inoculated (colonized) with the 55989a (P). Colonization phenotype of each mixed biofilm was estimated and results are represented as ratio of colonization level in Cmutant+P mixed biofilms compared to wild-type C+P mixed biofilms. Black bar represents wild-type colonization level in C+P mixed biofilms arbitrarily set to one. White bars represent colonization level of Cmutant+P mixed biofilms. Results are averages of at least 6 replicates ± standard deviation of the mean. Stars indicate mutant mixed biofilm with a colonization level significantly different from that of wild-type C+P mixed biofilm, p<0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3646849&req=5

pone-0061628-g001: Identification of colonization resistance factors interfering with establishment of mixed pathogen/commensal biofilm.A Experimental set-up: continuous flow biofilm growth in microfermentor. After initial inoculation of the microfermentor with E. coli MG1655 F′ commensal (C), biofilm develops for 6 h before re-inoculation (colonization) with exogenous pathogen E. coli 55989a (P). At 24 h post-colonization, mixed biofilm developing on the glass slide was resuspended and used for gene expression analysis and determination of colonization phenotype (% of pathogens in the mixed biofilm). B Microfermentors were inoculated with wild-type or mutant commensal (MG1655 F′ is abbreviated as MG) as indicated in the x-axis. After 6 h of growth, commensal biofilm was re-inoculated (colonized) with the 55989a (P). Colonization phenotype of each mixed biofilm was estimated and results are represented as ratio of colonization level in Cmutant+P mixed biofilms compared to wild-type C+P mixed biofilms. Black bar represents wild-type colonization level in C+P mixed biofilms arbitrarily set to one. White bars represent colonization level of Cmutant+P mixed biofilms. Results are averages of at least 6 replicates ± standard deviation of the mean. Stars indicate mutant mixed biofilm with a colonization level significantly different from that of wild-type C+P mixed biofilm, p<0.01.

Mentions: To establish conditions of MG1655 F′ colonization upon exogenous introduction of 55989a, we first produced MG1655 F′ biofilms formed for 6 to 24 h in continuous flow microfermentors. We then inoculated them with various titers of E. coli 55989a and allowed the resulting mixed biofilm to grow an additional 24 h. We defined E. coli 55989a colonization efficiency as the percentage of pathogens present in the resulting 24 h C+P mixed biofilm, as determined using the 55989a ampicillin antibiotic resistance marker (Table 1). At 24 h, a commensal colony-forming unit (cfu) had increased by a 2-log factor and the presence of the pathogen did not significantly alter development of the commensal biofilm, since C and C+P biofilm displayed similar biomass (data not shown). We found that the proportion of 55989a in C+P biofilm depended on both the 55989a initial inoculation titer and the age of MG1655 F′ biofilm. When MG1655 F′ 6 h biofilms were inoculated with a titer of 109 bacteria/ml of 55989a, we reproducibly obtained 25+/−5% of 55989a in 24 h C+P mixed biofilm; we used these experimental conditions throughout the rest of the study (Fig. 1A).


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)

Identification of colonization resistance factors interfering with establishment of mixed pathogen/commensal biofilm.A Experimental set-up: continuous flow biofilm growth in microfermentor. After initial inoculation of the microfermentor with E. coli MG1655 F′ commensal (C), biofilm develops for 6 h before re-inoculation (colonization) with exogenous pathogen E. coli 55989a (P). At 24 h post-colonization, mixed biofilm developing on the glass slide was resuspended and used for gene expression analysis and determination of colonization phenotype (% of pathogens in the mixed biofilm). B Microfermentors were inoculated with wild-type or mutant commensal (MG1655 F′ is abbreviated as MG) as indicated in the x-axis. After 6 h of growth, commensal biofilm was re-inoculated (colonized) with the 55989a (P). Colonization phenotype of each mixed biofilm was estimated and results are represented as ratio of colonization level in Cmutant+P mixed biofilms compared to wild-type C+P mixed biofilms. Black bar represents wild-type colonization level in C+P mixed biofilms arbitrarily set to one. White bars represent colonization level of Cmutant+P mixed biofilms. Results are averages of at least 6 replicates ± standard deviation of the mean. Stars indicate mutant mixed biofilm with a colonization level significantly different from that of wild-type C+P mixed biofilm, p<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646849&req=5

pone-0061628-g001: Identification of colonization resistance factors interfering with establishment of mixed pathogen/commensal biofilm.A Experimental set-up: continuous flow biofilm growth in microfermentor. After initial inoculation of the microfermentor with E. coli MG1655 F′ commensal (C), biofilm develops for 6 h before re-inoculation (colonization) with exogenous pathogen E. coli 55989a (P). At 24 h post-colonization, mixed biofilm developing on the glass slide was resuspended and used for gene expression analysis and determination of colonization phenotype (% of pathogens in the mixed biofilm). B Microfermentors were inoculated with wild-type or mutant commensal (MG1655 F′ is abbreviated as MG) as indicated in the x-axis. After 6 h of growth, commensal biofilm was re-inoculated (colonized) with the 55989a (P). Colonization phenotype of each mixed biofilm was estimated and results are represented as ratio of colonization level in Cmutant+P mixed biofilms compared to wild-type C+P mixed biofilms. Black bar represents wild-type colonization level in C+P mixed biofilms arbitrarily set to one. White bars represent colonization level of Cmutant+P mixed biofilms. Results are averages of at least 6 replicates ± standard deviation of the mean. Stars indicate mutant mixed biofilm with a colonization level significantly different from that of wild-type C+P mixed biofilm, p<0.01.
Mentions: To establish conditions of MG1655 F′ colonization upon exogenous introduction of 55989a, we first produced MG1655 F′ biofilms formed for 6 to 24 h in continuous flow microfermentors. We then inoculated them with various titers of E. coli 55989a and allowed the resulting mixed biofilm to grow an additional 24 h. We defined E. coli 55989a colonization efficiency as the percentage of pathogens present in the resulting 24 h C+P mixed biofilm, as determined using the 55989a ampicillin antibiotic resistance marker (Table 1). At 24 h, a commensal colony-forming unit (cfu) had increased by a 2-log factor and the presence of the pathogen did not significantly alter development of the commensal biofilm, since C and C+P biofilm displayed similar biomass (data not shown). We found that the proportion of 55989a in C+P biofilm depended on both the 55989a initial inoculation titer and the age of MG1655 F′ biofilm. When MG1655 F′ 6 h biofilms were inoculated with a titer of 109 bacteria/ml of 55989a, we reproducibly obtained 25+/−5% of 55989a in 24 h C+P mixed biofilm; we used these experimental conditions throughout the rest of the study (Fig. 1A).

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