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159 Modulation of TLR and Cytokine Gene Expression in Commensal Enterococcus Faecalis Dominated Infant Mouse Intestine
AGA Abstracts
on gene expression in intestinal tissues of infant mice in vivo. E. faecalis strains colonizing MAT infant mice are not vancomycin resistant, thus allowing characterization of nonpathogenic E. faecalis in vivo. Methods: We used the Mouse Innate and Adaptive Immune Responses RT2 Profiler PCR Array (Qiagen) to assess the expression of 84 genes involved in the host response to bacterial infection and sepsis. To distinguish tissue- and age-specific effects, distal ileum and proximal colon total tissue RNA from MAT infant littermates at day of life (DOL) 15 and 21 was compared gene expression relative to age matched controls (CTRL). To assess cell type specific gene and protein expression of candidate genes we further isolated intestinal epithelial cells and intestinal and splenic lymphocytes . Results: In MAT infant mice at DOL 15, 60 genes were modulated in the colon and 30 were modulated in the ileum, whereas at DOL 21, 59 genes were modulated in the colon versus 64 genes in the ileum. Based on the array data we found that IL10, TLR3, TLR9, and Traf6 were significantly regulated in MAT infant mouse intestines. We confirmed differential expression of TLRs, cytokines and signaling molecule genes in MAT and CTRL intestines by qPCR from 3 separate litters of mice. IL10, IL1a, TGFbeta, and TLR3 were down-regulated in colonic tissue from MAT infants, while Tyk2 and TLR2 were up-regulated. Conclusions: Infant mice born of mothers treated with antibiotics during the perinatal period can acquire an intestinal microbiota dominated by non-pathogenic bacteria. We show that an E. faecalis dominated microbiota in infant mice modulates TLR and cytokine expression. This result is similar to those in which the effect of E. faecalis strains from newborn infants cultured in vitro with human intestinal cell lines was examined. Our mouse model provides the opportunity for targeted experiments that further characterize how the limited diversity intestinal microbiota of infancy regulates immune homeostasis during this period.
160 Alterations in the Gut Microbiome and Correlation With Bloodstream Infection in Infants With Necrotizing Enterocolitis-Induced Short Bowel Syndrome Conrad R. Cole, Ethan A. Mezoff, Charles E. Robertson, Daniel N. Frank, Thomas Ziegler Introduction: Necrotizing enterocolitis is a major predisposing factor for surgical short bowel syndrome (SBS) in infants. Central line-associated blood stream infection (CLA-BSI) is a significant contributor to the morbidity and mortality of these children. Gut permeability defects leading to translocation of gut-associated bacteria may contribute to CLA-BSI. The aim of this study was to gain insight into the microbial diversity of stool of these infants in relationship to CLA-BSI. Methods: Ten infants with SBS caused by necrotizing enterocolitis who were dependent on parenteral nutrition for > 6 weeks were evaluated, along with 9 age-matched control subjects without intestinal failure or SBS. Stool was collected at baseline and if the child was hospitalized for CLA-BSI. Gut microbiome profiling of collected stool was accomplished by pan-bacterial 16S rRNA gene PCR and pyrosequencing. Results: An average of 53.9 genus-level bacterial groups were identified per subject. Klebsiella spp, were the most abundant microorganisms (mean = 17% of sequences) in SBS infants, but were notably less abundant in control specimens (mean = 2% of sequences; p = 0.053). Other genera, such as Enterococcus and Lactobacillus also were enriched in the stool of infants with SBS relative to controls, but the results were not statistically significant. Klebsiella pneumonia (35%) was the most common bacteria cultured in CLA-BSI. Conclusion: These results suggest the potential utility for use of culture-independent analysis to characterize microbial diversity in SBS. The preliminary data support the hypothesis that alterations in the gut luminal microbiome in pediatric SBS patients, versus non-SBS controls, is a predisposing factor in SBS-associated CLA-BSI.
158 Novel Insight Into Salmonella Typhi Pathogenesis From Ex Vivo Human Tissue Models Kourtney Nickerson, Stefania Senger, Marcelo B. Sztein, Alessio Fasano, Maria R. Fiorentino Introduction. Salmonella enterica serovar Typhi is the causative agent of Typhoid fever, from which an estimated 22 million cases occur annually resulting in 200,000 deaths. In some areas of the globe, the incidence of Typhoid fever is as high as 500 cases out of every 100,000 children. At present, little is understood about host response to Typhi infection. As such, no long-term preventive vaccine therapy is available. Current therapeutics include antibiotic treatment, however antibiotic resistant serovars are increasing worldwide. Furthermore, chronic Typhi colonization of the gall bladder is sufficient to cause gall bladder cancer therefore demonstrating significant health risks upon both long and short-term infection. To design alternative therapeutic strategies, there is immediate need to understand Typhi infection and pathogenesis. Materials and Methods. Terminal ileum biopsies were collected from donors for direct infection or generation of organoids. Whole biopsy infections were conducted using micro-snapwell mounting of tissue followed by addition of Salmonella enterica serovar Typhi 2a to the apical surface. Organoids were differentiated into epithelial compartment monolayers followed by infection with Typhi. Prior to infection, Typhi was grown in Luria Burtoni broth under static or shaking conditions. Upon infection, changes in trans-epithelial electrical resistance (TEER), cytokine release, gene expression, and cellular localization were assessed. Results and Conclusions. Terminal ileum derived organoids give rise to a diversity of epithelial cells, including goblet, paneth and M cells, which are grown as a monolayer in vitro. Use of the epithelial monolayer (EM) and whole biopsy (WB) model identified specific contributions of the epithelium in response to Typhi infection as assessed by RNA-sequencing, qPCR, and cytokine secretion. Consideration for bacterial inoculum preparation revealed that Typhi grown under static conditions express the Vi antigen, as well as, SPI-1 and SPI-2 effector proteins. Therefore, static or shaking inoculums were applied to both models. Differences in cellular association of bacteria were assessed using IF and TEM. To identify how the gut mucosa responds to infection, apical and basolateral culture supernatants were collected for ELISA analysis. ELISA analysis demonstrated differences in IL-8, IL-1b and IL-12p70 cytokine production dependent on infection model and secretion location. Interestingly, infected monolayers showed the highest levels of basolateral cytokine release. Finally, infection decreased TEER under both inoculum conditions in the EM model, but only the static inoculum decreased TEER in the WB model. All together, our data characterizes key aspects of terminal ileum response to Typhi infection addressing a critical gap in our current understanding of Typhoid fever pathogenesis.
161 Impact of Early Life Intervention With Bifidobacteria on the Structure and Function of Infant Fecal Microbiota Monika Bazanella, Tanja Maier, Thomas Clavel, Ilias Lagkouvardos, Lars Bode, Philippe Schmitt-Kopplin, Dirk Haller Background: Development of the intestinal microbiota in infants is a dynamic process and probiotic intervention was shown to be effective in preventing newborn pathologies like necrotizing enterocolitis and infantile colic. However, the impact of early-life bacterial intervention on the natural assembly of fecal communities and metabolic functions is unknown. We designed a randomized, double-blinded, placebo controlled intervention trial with healthy neonates receiving infant formula supplemented with or without a mixture of four different bifidobacteria during the first year of life. Methods: 106 infants were randomized to receive bacteria-supplemented (Bifidobacterium longum, B. infantis, B. breve, B. bifidum ) infant formula (n=48) or non-supplemented formula (n=49) after weaning. 9 infants were exclusively breast-fed. Fecal samples were collected monthly over a period of one year. High-throughput 16S rRNA amplicon sequencing by Illumina, high resolution metabolite analysis (UPLC-MS), and human milk oligosaccharide (HMO) analysis were applied. Results: Formula- and breast-fed infants harbored distinct bacterial communities, even after 1 year of age, and breast feeding was characterized by significantly reduced diversity. Metabolite profiling identified distinct clusters according to the feeding regiment and age of the infants. Supplementation of infant formula with bifidobacteria did not affect community structures or metabolite profiles in infants exclusively fed with formula (N=22). However in combination with breast feeding, bifidobacteria supplementation modulated the microbiota up to 7 months of age. All feeding groups were dominated by bifidobacteria, representing 69% (breastfed), 35% (bacteria-supplemented formula), and 32% (control formula) of total sequences. Bacteroidaceae, with Bacteroides fragilis as prevalent member, showed increased relative abundances during the first weeks of colonization in the control formula group. A core microbiota including molecular species classified as two Bifidobacterium spp., Escherichia-Shigella sp., Streptococcus sp. and Enterococcus sp., was detected in all infants at least once in the first year of life. Caesarean section was associated with increased relative abundance of Firmicutes and decrease in Actinobacteria during the first three months. Breast milk HMO analysis pointed towards a link between maternal secretor status (presence or absence of fucosyltransferase 2) and infantile microbiota composition. Conclusion: Infant formula significantly influenced assembly and metabolite profiles of the early life microbiota, particularly associated
159 Modulation of TLR and Cytokine Gene Expression in Commensal Enterococcus Faecalis Dominated Infant Mouse Intestine Tessa M. Tekieli, Esi Lamouse-Smith Background: Enterococcus faecalis is normal member of the intestinal microbiota and a founding member of commensal bacteria colonizing the intestine of humans and mice at birth. While well known as a human pathogen its role and function as a commensal symbiont in the intestine of infants is less well characterized. In our mouse model of maternal antibiotic treatment (MAT), infant MAT mice maintain an intestinal flora almost entirely dominated by E. faecalis. These mice gain weight and grow appropriately, but are unable to control a systemic vaccinia virus infection. We hypothesized that the E. faecalis dominated microbiota modulates the expression of genes that can regulate mucosal and systemic immunity. Our aim in this study was to characterize the selective impact of commensal E. faecalis colonization
AGA Abstracts
S-40
on gene expression in intestinal tissues of infant mice in vivo. E. faecalis strains colonizing MAT infant mice are not vancomycin resistant, thus allowing characterization of nonpathogenic E. faecalis in vivo. Methods: We used the Mouse Innate and Adaptive Immune Responses RT2 Profiler PCR Array (Qiagen) to assess the expression of 84 genes involved in the host response to bacterial infection and sepsis. To distinguish tissue- and age-specific effects, distal ileum and proximal colon total tissue RNA from MAT infant littermates at day of life (DOL) 15 and 21 was compared gene expression relative to age matched controls (CTRL). To assess cell type specific gene and protein expression of candidate genes we further isolated intestinal epithelial cells and intestinal and splenic lymphocytes . Results: In MAT infant mice at DOL 15, 60 genes were modulated in the colon and 30 were modulated in the ileum, whereas at DOL 21, 59 genes were modulated in the colon versus 64 genes in the ileum. Based on the array data we found that IL10, TLR3, TLR9, and Traf6 were significantly regulated in MAT infant mouse intestines. We confirmed differential expression of TLRs, cytokines and signaling molecule genes in MAT and CTRL intestines by qPCR from 3 separate litters of mice. IL10, IL1a, TGFbeta, and TLR3 were down-regulated in colonic tissue from MAT infants, while Tyk2 and TLR2 were up-regulated. Conclusions: Infant mice born of mothers treated with antibiotics during the perinatal period can acquire an intestinal microbiota dominated by non-pathogenic bacteria. We show that an E. faecalis dominated microbiota in infant mice modulates TLR and cytokine expression. This result is similar to those in which the effect of E. faecalis strains from newborn infants cultured in vitro with human intestinal cell lines was examined. Our mouse model provides the opportunity for targeted experiments that further characterize how the limited diversity intestinal microbiota of infancy regulates immune homeostasis during this period.
160 Alterations in the Gut Microbiome and Correlation With Bloodstream Infection in Infants With Necrotizing Enterocolitis-Induced Short Bowel Syndrome Conrad R. Cole, Ethan A. Mezoff, Charles E. Robertson, Daniel N. Frank, Thomas Ziegler Introduction: Necrotizing enterocolitis is a major predisposing factor for surgical short bowel syndrome (SBS) in infants. Central line-associated blood stream infection (CLA-BSI) is a significant contributor to the morbidity and mortality of these children. Gut permeability defects leading to translocation of gut-associated bacteria may contribute to CLA-BSI. The aim of this study was to gain insight into the microbial diversity of stool of these infants in relationship to CLA-BSI. Methods: Ten infants with SBS caused by necrotizing enterocolitis who were dependent on parenteral nutrition for > 6 weeks were evaluated, along with 9 age-matched control subjects without intestinal failure or SBS. Stool was collected at baseline and if the child was hospitalized for CLA-BSI. Gut microbiome profiling of collected stool was accomplished by pan-bacterial 16S rRNA gene PCR and pyrosequencing. Results: An average of 53.9 genus-level bacterial groups were identified per subject. Klebsiella spp, were the most abundant microorganisms (mean = 17% of sequences) in SBS infants, but were notably less abundant in control specimens (mean = 2% of sequences; p = 0.053). Other genera, such as Enterococcus and Lactobacillus also were enriched in the stool of infants with SBS relative to controls, but the results were not statistically significant. Klebsiella pneumonia (35%) was the most common bacteria cultured in CLA-BSI. Conclusion: These results suggest the potential utility for use of culture-independent analysis to characterize microbial diversity in SBS. The preliminary data support the hypothesis that alterations in the gut luminal microbiome in pediatric SBS patients, versus non-SBS controls, is a predisposing factor in SBS-associated CLA-BSI.
158 Novel Insight Into Salmonella Typhi Pathogenesis From Ex Vivo Human Tissue Models Kourtney Nickerson, Stefania Senger, Marcelo B. Sztein, Alessio Fasano, Maria R. Fiorentino Introduction. Salmonella enterica serovar Typhi is the causative agent of Typhoid fever, from which an estimated 22 million cases occur annually resulting in 200,000 deaths. In some areas of the globe, the incidence of Typhoid fever is as high as 500 cases out of every 100,000 children. At present, little is understood about host response to Typhi infection. As such, no long-term preventive vaccine therapy is available. Current therapeutics include antibiotic treatment, however antibiotic resistant serovars are increasing worldwide. Furthermore, chronic Typhi colonization of the gall bladder is sufficient to cause gall bladder cancer therefore demonstrating significant health risks upon both long and short-term infection. To design alternative therapeutic strategies, there is immediate need to understand Typhi infection and pathogenesis. Materials and Methods. Terminal ileum biopsies were collected from donors for direct infection or generation of organoids. Whole biopsy infections were conducted using micro-snapwell mounting of tissue followed by addition of Salmonella enterica serovar Typhi 2a to the apical surface. Organoids were differentiated into epithelial compartment monolayers followed by infection with Typhi. Prior to infection, Typhi was grown in Luria Burtoni broth under static or shaking conditions. Upon infection, changes in trans-epithelial electrical resistance (TEER), cytokine release, gene expression, and cellular localization were assessed. Results and Conclusions. Terminal ileum derived organoids give rise to a diversity of epithelial cells, including goblet, paneth and M cells, which are grown as a monolayer in vitro. Use of the epithelial monolayer (EM) and whole biopsy (WB) model identified specific contributions of the epithelium in response to Typhi infection as assessed by RNA-sequencing, qPCR, and cytokine secretion. Consideration for bacterial inoculum preparation revealed that Typhi grown under static conditions express the Vi antigen, as well as, SPI-1 and SPI-2 effector proteins. Therefore, static or shaking inoculums were applied to both models. Differences in cellular association of bacteria were assessed using IF and TEM. To identify how the gut mucosa responds to infection, apical and basolateral culture supernatants were collected for ELISA analysis. ELISA analysis demonstrated differences in IL-8, IL-1b and IL-12p70 cytokine production dependent on infection model and secretion location. Interestingly, infected monolayers showed the highest levels of basolateral cytokine release. Finally, infection decreased TEER under both inoculum conditions in the EM model, but only the static inoculum decreased TEER in the WB model. All together, our data characterizes key aspects of terminal ileum response to Typhi infection addressing a critical gap in our current understanding of Typhoid fever pathogenesis.
161 Impact of Early Life Intervention With Bifidobacteria on the Structure and Function of Infant Fecal Microbiota Monika Bazanella, Tanja Maier, Thomas Clavel, Ilias Lagkouvardos, Lars Bode, Philippe Schmitt-Kopplin, Dirk Haller Background: Development of the intestinal microbiota in infants is a dynamic process and probiotic intervention was shown to be effective in preventing newborn pathologies like necrotizing enterocolitis and infantile colic. However, the impact of early-life bacterial intervention on the natural assembly of fecal communities and metabolic functions is unknown. We designed a randomized, double-blinded, placebo controlled intervention trial with healthy neonates receiving infant formula supplemented with or without a mixture of four different bifidobacteria during the first year of life. Methods: 106 infants were randomized to receive bacteria-supplemented (Bifidobacterium longum, B. infantis, B. breve, B. bifidum ) infant formula (n=48) or non-supplemented formula (n=49) after weaning. 9 infants were exclusively breast-fed. Fecal samples were collected monthly over a period of one year. High-throughput 16S rRNA amplicon sequencing by Illumina, high resolution metabolite analysis (UPLC-MS), and human milk oligosaccharide (HMO) analysis were applied. Results: Formula- and breast-fed infants harbored distinct bacterial communities, even after 1 year of age, and breast feeding was characterized by significantly reduced diversity. Metabolite profiling identified distinct clusters according to the feeding regiment and age of the infants. Supplementation of infant formula with bifidobacteria did not affect community structures or metabolite profiles in infants exclusively fed with formula (N=22). However in combination with breast feeding, bifidobacteria supplementation modulated the microbiota up to 7 months of age. All feeding groups were dominated by bifidobacteria, representing 69% (breastfed), 35% (bacteria-supplemented formula), and 32% (control formula) of total sequences. Bacteroidaceae, with Bacteroides fragilis as prevalent member, showed increased relative abundances during the first weeks of colonization in the control formula group. A core microbiota including molecular species classified as two Bifidobacterium spp., Escherichia-Shigella sp., Streptococcus sp. and Enterococcus sp., was detected in all infants at least once in the first year of life. Caesarean section was associated with increased relative abundance of Firmicutes and decrease in Actinobacteria during the first three months. Breast milk HMO analysis pointed towards a link between maternal secretor status (presence or absence of fucosyltransferase 2) and infantile microbiota composition. Conclusion: Infant formula significantly influenced assembly and metabolite profiles of the early life microbiota, particularly associated
159 Modulation of TLR and Cytokine Gene Expression in Commensal Enterococcus Faecalis Dominated Infant Mouse Intestine Tessa M. Tekieli, Esi Lamouse-Smith Background: Enterococcus faecalis is normal member of the intestinal microbiota and a founding member of commensal bacteria colonizing the intestine of humans and mice at birth. While well known as a human pathogen its role and function as a commensal symbiont in the intestine of infants is less well characterized. In our mouse model of maternal antibiotic treatment (MAT), infant MAT mice maintain an intestinal flora almost entirely dominated by E. faecalis. These mice gain weight and grow appropriately, but are unable to control a systemic vaccinia virus infection. We hypothesized that the E. faecalis dominated microbiota modulates the expression of genes that can regulate mucosal and systemic immunity. Our aim in this study was to characterize the selective impact of commensal E. faecalis colonization
AGA Abstracts
S-40