- Email: [email protected]
558 Intestine-Specific Deletion of the Mouse KrüPpel-Like Factor 4 Gene Results in Altered Homeostasis of Intestinal Epithelial Cells
558
of AIEC reference strain LF82 was screened to search for putative virulence factors allowing bacteria to target PP. Wild-type LF82 bacteria and mutants were tested for their abilities to interact with murine and human isolated PP, and M cells. Results: Numerous AIEC LF82 bacteria were observed within PP as shown by ex vivo assays using murine and human PP. The AIEC LF82 bacteria translocated at a very high level through M cell monolayers, but not as the result of the loss of monolayer integrity. A functional operon encoding long polar fimbriae (LPF) is present in AIEC strain LF82. The LF82-delta lpfA mutant was impaired in its ability to interact with murine and human isolated PP and to translocate across M cell monolayers. In addition, increased numbers of E. coli are associated with PP from CD patients compared to those from controls. Increased numbers of AIEC LF82 bacteria associated with PP were also observed in Nod2-/- mice due to the increased numbers of M cells and confocal analysis confirmed that AIEC LF82 bacteria target M cells at the surface of PP dependently on the expression of LPF. The prevalence of subjects positive for ileum-associated AIEC strains harboring lpf operon was 21.8% in CD patients as against only 3.5 % in controls (P=0.027). Conclusions: As a key factor for AIEC to target M cells in PP, LPF encoding gene should be screened among ileal-associated E. coli strains to define patients at high risk of developing CD or a recurrence of the disease.
AGA Abstracts
Intestine-Specific Deletion of the Mouse KrüPpel-Like Factor 4 Gene Results in Altered Homeostasis of Intestinal Epithelial Cells Amr Ghaleb, Beth B. McConnell, Jonathan P. Katz, Klaus H. Kaestner, Vincent W. Yang BACKGROUND: The zinc finger transcription factor, Krüppel-like factor 4 (KLF4), is normally expressed in the differentiated epithelial cells lining the villus border and surface epithelium of the small and large intestine, respectively. In Vitro, KLF4 inhibits cell proliferation by functioning as a cell cycle checkpoint protein. In Vivo, KLF4 exhibits a tumor suppressive effect on intestinal tumorigenesis. Klf4-null mice die by postnatal day 1 and show changes in differentiation of epithelial tissues including the conjunctiva, epidermis and gastrointestinal tract. AIM: To characterize the intestinal phenotype in mice following conditional deletion of the Klf4 gene from the intestine. METHODS: Conditional gene ablation was used to generate mice lacking Klf4 in their intestinal epithelium by mating floxed Klf4 (Klf4fl/fl) mice with villin-Cre (Vil-Cre) mice. Klf4 mutant mice (Vil-Cre;Klf4fl/fl) and control (Klf4fl/fl) mice were sacrificed at 3 weeks of age for histological and immunuohistochemical characterization of the intestinal tract. Cell migration was measured by 5-bromo2-deoxyuridine (BrdU) pulse labeling experiments. RESULTS: Vil-Cre;Klf4fl/fl mice were born in a Mendelian ratio and remained viable at 4 months. At 3 weeks of age, the colon of the mutant mice displayed distorted architecture characterized by dilated crypt glands. Both the small and large intestine of the mutant mice exhibited reduced number and size of mature goblet cells compared to controls as demonstrated by Alcian blue staining. Brush-border alkaline phosphatase staining in the villus epithelial cells was also significantly reduced in the small intestine of mutant mice. In addition, Paneth cells were not restricted to the base of the crypts and were mispositioned in the upper crypt region in the small intestine of mutant mice. In contrast, the number of enteroendocrine cells was not affected by Klf4 deletion. Although the number of Ki67 or cyclin D1-positive cells per crypt in the small intestine was not significantly different between mutant and control mice, mutant mice had a statistically significant increase in both the number and migratory rate of BrdU-positive cells following a 24-hour pulse when compared to controls. CONCLUSION: The results of this study provide new insights into the function of KLF4 in postnatal maturation, proliferation, migration and positioning of intestinal epithelial cells, demonstrating an essential role for KLF4 in maintaining normal intestinal epithelial homeostasis.
561 The Stress Response Significantly Changes Microbial Populations in the Intestines and Increases Susceptibility to Enteric Infection Michael T. Bailey, Jeffrey D. Galley, Scot E. Dowd, Mark Lyte The ability of the stress response to influence susceptibility to enteric disease through the direct modulation of the commensal microbiota is unknown. The commensal microbiota have many beneficial effects on health and participate in host defense against enteric pathogens. Previous studies using culture-based methods have demonstrated that stressor exposure can change microbial populations in the intestines, but the extent, and biological importance, of these changes are not well known. We have been using next generation pyrosequencing to characterize the microbiota of mice exposed to an overnight restraint stressor. The diversity of the microbiota within the luminal contents, as well as adhered to colonic tissue, was significantly reduced by exposure to the stressor. Double clustering analysis of the microbiota indicated that although a single night of stressor exposure was not enough to significantly affect community structure, repeated nights of stressor exposure significantly changed community structure at the class, family, and genus level of analysis. This clustering was primarily due to a stressor-induced reduction in the relative abundance of bacteria in the genus Tannerella in the luminal contents, and a reduction of bacteria in the genus Lactobacillus associated with the colonic tissue. To begin testing whether stressor-induced changes in the microbiota impact susceptibility to enteric infection, mice were restrained overnight for 7 consecutive nights prior to oral challenge with the murine pathogen Citrobacter rodentium, which causes a disease that is similar to human infection with enteropathogenic Escherichia coli. Exposure to the restraint stressor increased C. rodentium colonization by approximately 4 log units. This increased colonization was associated with increased gene expression for TNF-α, iNOS, and IL-1β, and decreased gene expression for IL-10 in the colonic tissue. Gene expression for mouse β-defensin 1 and colonic levels of IgA were unaffected by stressor exposure. Thus, the data are consistent with the view that stressor-induced changes in the microbiota result in increased susceptibility to C. rodentium; higher levels of C. rodentium in turn lead to increased colonic inflammation. Thus, the ability of stressors to directly modulate the composition of the enteric microbiota may represent a new mechanism governing susceptibility to enteric infection. This model may also be a useful tool for studying post-infectious irritable bowel syndrome, where alterations of the microbiota, prolonged inflammation, and psychological distress are all known to be predictive of this clinically important disease.
559 The Human Variant D299G of the TLR4 Gene Links Inflammation and Cancer Progression in the Intestinal Epithelium Annette Eyking, Guido Gerken, Daniel K. Podolsky, Elke Cario Background: Chronic recurrent intestinal inflammation in inflammatory bowel diseases (IBD) may result from aberrant stimulation of the mucosal immune system by the resident microflora. TLR4 expression is significantly upregulated in intestinal epithelial cells (IEC) in IBD colitis. The variant D299G of the TLR4 gene has been associated with increased IBD susceptibility. However, the phenotypic consequences of this gene variant in IEC have not been resolved yet. The aim of the study was to investigate how the IBD-associated variant TLR4-D299G may affect IEC biology and molecular function. Methods: IEC lines (Caco2) stably overexpressing HA-tagged full-length (FL) TLR4 or mutant TLR4-D299G or TLR4T399I were generated. Gene expression profiling using DNA microarray analysis (Human Gene 1.0 ST; Affymetrix) was performed and network data were generated through Ingenuity Pathways Analysis. Results were confirmed by qRT-PCR and validated by western/ELISA. Mitosis was examined by confocal immunofluorescence. Functional studies included assessment of proliferation (BrdU), metabolic activity (MTT) and invasive potential (matrigel). InVivo tumorigenicity was tested using the CD-1 nu/nu mouse xenograft model. Results: Functional clustering of the microarray datasets revealed that the major TLR4-D299G targets were dominated by genes involved with promotion of inflammation and tumorigenesis, and these genes were markedly downregulated in IEC expressing TLR4-FL or TLR4-T399I. Three distinct pro-inflammatory systems (acute phase response, coagulation and complement) were the most significant canonical pathways modulated by the differentially regulated genes in TLR4-D299G. In contrast to TLR4-FL, TLR4-D299G IEC constitutively secreted large amounts of complement proteins (C3a; C5a) and coagulation factors (TFPI; A2M). Functionally, IEC expressing TLR4-D299G revealed central features of tumorigenicity - which were not evident in TLR4-FL: TLR4-D299G IEC 1) were multinucleated with multipolar mitotic spindles, 2) exhibited increased proliferation associated with elevated metabolic activity and repressed differentiation, and 3) demonstrated pronounced invasion via STAT3 activation. Compared with TLR4-FL-xenografts, In-Vivo tumor growth was significantly stimulated in TLR4-D299G-xenografts. Conclusions: Our results suggest that the TLR4-D299G variant represents a gain-of-function mutation with enhanced oncogenic potential. The TLR4-D299G variant establishes a pro-inflammatory microenvironment which may drive cancer progression in IEC. Thus, IBD patients with TLR4-D299G may be at increased risk to develop a more aggressive phenotype of colitis-associated neoplasia.
562 Microbiota-Induced Serum Amyloid A is an Important Mediator of Innate Immune Response in Zebrafish Ja Seol Koo, Michelle Kanther, John F. Rawls, Christian Jobin Background: The serum amyloid A family of acute phase protein (SAA1-4) have been traditionally viewed as markers of inflammation and their role in modulating innate host response is currently unknown. Since the zebrafish encodes only a single SAA homolog, this vertebrate animal represents an ideal system to investigate SAA function in host-microbe interaction. Methods: Germ-free (GF) transgenic NFKB:EGFP reporter zebrafish (Tg(NFKB:EGFP)) were conventionalized (CONVD) and pattern of EGFP expression was determined by fluorescent microscopy. SAA expression in GF and CONVD zebrafish was determined by wholemount in situ hybridization (WISH). Conventionally-raised Tg(NFKB:EGFP) zebrafish were injected with SAA morpholino (MO) at the 1-cell stage (0.9 pg/embryo) to block SAA expression. Impact of SAA on NF-κB signaling and gene expression In Vitro was determined using zebrafish PAC2 embryonic fibroblasts. NF-κB signaling was determined using immunofluorescent staining (RelA), western blot (phospho-IκBa), and transcriptional activity (luciferase reporter gene, NFKB:EGFP reporter plasmid). Transcription of NF-κB target genes In Vitro and In Vivo was determined using ABI Prism 7700HT detection system. Results: Recombinant human SAA (1μM) stimulated IκBα serine 32/36 phosphorylation, RelA nuclear translocation, and luciferase activity (relative fold change 3.5±0.07 vs ctl, p<0.01) in PAC2 cells. SAA induced EGFP expression in PAC2 cells transfected with pNFKB:EGFP. In addition, SAA induced NF-κB target genes IκBαa and MMP9 (relative fold change, 25.8±1.70 and 19.9±0.69 vs ctl respectively, p<0.01). Colonization of GF Tg(NFKB:EGFP) zebrafish with a normal microbiota resulted in elevated EGFP expression in the liver, dorsal root ganglia, the neuromasts of the lateral line, and a distinct population of intestinal cells. WISH and quantitative RT-PCR showed that CONVD Tg(NFKB:EGFP) zebrafish strongly up-regulated SAA gene expression in the liver and intestine compared to GF zebrafish. Importantly, although global cellular EGFP expression was not significantly inhibited in SAA-MO injected Tg(NFKB:EGFP) zebrafish, a strong decrease in endogenous mRNA accumulation of NF-κB targets IκBαa (40%) and MMP9 (90%) was observed in these fish compared to control MO injected zebrafish. Conclusion: Our data indicate that microbial colonization enhances SAA expression, which then impacts on NF-κB
560 Crohn's Disease-Associated Adherent-Invasive Escherichia coli Target Peyer's Patches via Long Polar Fimbriae Benoit Chassaing, Nathalie Rolhion, Amelie De Vallee, Sa'ad Salim, Maelle Prorok-Hamon, Christel Neut, Barry J. Campbell, Jean-Pierre Hugot, Jean-Frederic Colombel, Arlette Darfeuille-Michaud Background & Aims: Ileal lesions of patients with Crohn's disease (CD) are colonized by adherent-invasive Escherichia coli (AIEC) and the earliest observable lesions of CD are microscopic erosions of the follicle-associated epithelium (FAE) lining the Peyer's patches(PP). We aimed in the present study to investigate the ability of AIEC to interact with PP and M cells of the FAE, to quantify the PP-associated E. coli in CD patients compared to controls, and to search for the presence of a virulence factor in AIEC that could be involved in the targeting of PP by these invasive bacteria. Methods: PP from CD patients and controls were analyzed by q-PCR for the presence of E. coli 16S r-RNA encoding gene, and the genome
AGA Abstracts
S-78
of AIEC reference strain LF82 was screened to search for putative virulence factors allowing bacteria to target PP. Wild-type LF82 bacteria and mutants were tested for their abilities to interact with murine and human isolated PP, and M cells. Results: Numerous AIEC LF82 bacteria were observed within PP as shown by ex vivo assays using murine and human PP. The AIEC LF82 bacteria translocated at a very high level through M cell monolayers, but not as the result of the loss of monolayer integrity. A functional operon encoding long polar fimbriae (LPF) is present in AIEC strain LF82. The LF82-delta lpfA mutant was impaired in its ability to interact with murine and human isolated PP and to translocate across M cell monolayers. In addition, increased numbers of E. coli are associated with PP from CD patients compared to those from controls. Increased numbers of AIEC LF82 bacteria associated with PP were also observed in Nod2-/- mice due to the increased numbers of M cells and confocal analysis confirmed that AIEC LF82 bacteria target M cells at the surface of PP dependently on the expression of LPF. The prevalence of subjects positive for ileum-associated AIEC strains harboring lpf operon was 21.8% in CD patients as against only 3.5 % in controls (P=0.027). Conclusions: As a key factor for AIEC to target M cells in PP, LPF encoding gene should be screened among ileal-associated E. coli strains to define patients at high risk of developing CD or a recurrence of the disease.
AGA Abstracts
Intestine-Specific Deletion of the Mouse KrüPpel-Like Factor 4 Gene Results in Altered Homeostasis of Intestinal Epithelial Cells Amr Ghaleb, Beth B. McConnell, Jonathan P. Katz, Klaus H. Kaestner, Vincent W. Yang BACKGROUND: The zinc finger transcription factor, Krüppel-like factor 4 (KLF4), is normally expressed in the differentiated epithelial cells lining the villus border and surface epithelium of the small and large intestine, respectively. In Vitro, KLF4 inhibits cell proliferation by functioning as a cell cycle checkpoint protein. In Vivo, KLF4 exhibits a tumor suppressive effect on intestinal tumorigenesis. Klf4-null mice die by postnatal day 1 and show changes in differentiation of epithelial tissues including the conjunctiva, epidermis and gastrointestinal tract. AIM: To characterize the intestinal phenotype in mice following conditional deletion of the Klf4 gene from the intestine. METHODS: Conditional gene ablation was used to generate mice lacking Klf4 in their intestinal epithelium by mating floxed Klf4 (Klf4fl/fl) mice with villin-Cre (Vil-Cre) mice. Klf4 mutant mice (Vil-Cre;Klf4fl/fl) and control (Klf4fl/fl) mice were sacrificed at 3 weeks of age for histological and immunuohistochemical characterization of the intestinal tract. Cell migration was measured by 5-bromo2-deoxyuridine (BrdU) pulse labeling experiments. RESULTS: Vil-Cre;Klf4fl/fl mice were born in a Mendelian ratio and remained viable at 4 months. At 3 weeks of age, the colon of the mutant mice displayed distorted architecture characterized by dilated crypt glands. Both the small and large intestine of the mutant mice exhibited reduced number and size of mature goblet cells compared to controls as demonstrated by Alcian blue staining. Brush-border alkaline phosphatase staining in the villus epithelial cells was also significantly reduced in the small intestine of mutant mice. In addition, Paneth cells were not restricted to the base of the crypts and were mispositioned in the upper crypt region in the small intestine of mutant mice. In contrast, the number of enteroendocrine cells was not affected by Klf4 deletion. Although the number of Ki67 or cyclin D1-positive cells per crypt in the small intestine was not significantly different between mutant and control mice, mutant mice had a statistically significant increase in both the number and migratory rate of BrdU-positive cells following a 24-hour pulse when compared to controls. CONCLUSION: The results of this study provide new insights into the function of KLF4 in postnatal maturation, proliferation, migration and positioning of intestinal epithelial cells, demonstrating an essential role for KLF4 in maintaining normal intestinal epithelial homeostasis.
561 The Stress Response Significantly Changes Microbial Populations in the Intestines and Increases Susceptibility to Enteric Infection Michael T. Bailey, Jeffrey D. Galley, Scot E. Dowd, Mark Lyte The ability of the stress response to influence susceptibility to enteric disease through the direct modulation of the commensal microbiota is unknown. The commensal microbiota have many beneficial effects on health and participate in host defense against enteric pathogens. Previous studies using culture-based methods have demonstrated that stressor exposure can change microbial populations in the intestines, but the extent, and biological importance, of these changes are not well known. We have been using next generation pyrosequencing to characterize the microbiota of mice exposed to an overnight restraint stressor. The diversity of the microbiota within the luminal contents, as well as adhered to colonic tissue, was significantly reduced by exposure to the stressor. Double clustering analysis of the microbiota indicated that although a single night of stressor exposure was not enough to significantly affect community structure, repeated nights of stressor exposure significantly changed community structure at the class, family, and genus level of analysis. This clustering was primarily due to a stressor-induced reduction in the relative abundance of bacteria in the genus Tannerella in the luminal contents, and a reduction of bacteria in the genus Lactobacillus associated with the colonic tissue. To begin testing whether stressor-induced changes in the microbiota impact susceptibility to enteric infection, mice were restrained overnight for 7 consecutive nights prior to oral challenge with the murine pathogen Citrobacter rodentium, which causes a disease that is similar to human infection with enteropathogenic Escherichia coli. Exposure to the restraint stressor increased C. rodentium colonization by approximately 4 log units. This increased colonization was associated with increased gene expression for TNF-α, iNOS, and IL-1β, and decreased gene expression for IL-10 in the colonic tissue. Gene expression for mouse β-defensin 1 and colonic levels of IgA were unaffected by stressor exposure. Thus, the data are consistent with the view that stressor-induced changes in the microbiota result in increased susceptibility to C. rodentium; higher levels of C. rodentium in turn lead to increased colonic inflammation. Thus, the ability of stressors to directly modulate the composition of the enteric microbiota may represent a new mechanism governing susceptibility to enteric infection. This model may also be a useful tool for studying post-infectious irritable bowel syndrome, where alterations of the microbiota, prolonged inflammation, and psychological distress are all known to be predictive of this clinically important disease.
559 The Human Variant D299G of the TLR4 Gene Links Inflammation and Cancer Progression in the Intestinal Epithelium Annette Eyking, Guido Gerken, Daniel K. Podolsky, Elke Cario Background: Chronic recurrent intestinal inflammation in inflammatory bowel diseases (IBD) may result from aberrant stimulation of the mucosal immune system by the resident microflora. TLR4 expression is significantly upregulated in intestinal epithelial cells (IEC) in IBD colitis. The variant D299G of the TLR4 gene has been associated with increased IBD susceptibility. However, the phenotypic consequences of this gene variant in IEC have not been resolved yet. The aim of the study was to investigate how the IBD-associated variant TLR4-D299G may affect IEC biology and molecular function. Methods: IEC lines (Caco2) stably overexpressing HA-tagged full-length (FL) TLR4 or mutant TLR4-D299G or TLR4T399I were generated. Gene expression profiling using DNA microarray analysis (Human Gene 1.0 ST; Affymetrix) was performed and network data were generated through Ingenuity Pathways Analysis. Results were confirmed by qRT-PCR and validated by western/ELISA. Mitosis was examined by confocal immunofluorescence. Functional studies included assessment of proliferation (BrdU), metabolic activity (MTT) and invasive potential (matrigel). InVivo tumorigenicity was tested using the CD-1 nu/nu mouse xenograft model. Results: Functional clustering of the microarray datasets revealed that the major TLR4-D299G targets were dominated by genes involved with promotion of inflammation and tumorigenesis, and these genes were markedly downregulated in IEC expressing TLR4-FL or TLR4-T399I. Three distinct pro-inflammatory systems (acute phase response, coagulation and complement) were the most significant canonical pathways modulated by the differentially regulated genes in TLR4-D299G. In contrast to TLR4-FL, TLR4-D299G IEC constitutively secreted large amounts of complement proteins (C3a; C5a) and coagulation factors (TFPI; A2M). Functionally, IEC expressing TLR4-D299G revealed central features of tumorigenicity - which were not evident in TLR4-FL: TLR4-D299G IEC 1) were multinucleated with multipolar mitotic spindles, 2) exhibited increased proliferation associated with elevated metabolic activity and repressed differentiation, and 3) demonstrated pronounced invasion via STAT3 activation. Compared with TLR4-FL-xenografts, In-Vivo tumor growth was significantly stimulated in TLR4-D299G-xenografts. Conclusions: Our results suggest that the TLR4-D299G variant represents a gain-of-function mutation with enhanced oncogenic potential. The TLR4-D299G variant establishes a pro-inflammatory microenvironment which may drive cancer progression in IEC. Thus, IBD patients with TLR4-D299G may be at increased risk to develop a more aggressive phenotype of colitis-associated neoplasia.
562 Microbiota-Induced Serum Amyloid A is an Important Mediator of Innate Immune Response in Zebrafish Ja Seol Koo, Michelle Kanther, John F. Rawls, Christian Jobin Background: The serum amyloid A family of acute phase protein (SAA1-4) have been traditionally viewed as markers of inflammation and their role in modulating innate host response is currently unknown. Since the zebrafish encodes only a single SAA homolog, this vertebrate animal represents an ideal system to investigate SAA function in host-microbe interaction. Methods: Germ-free (GF) transgenic NFKB:EGFP reporter zebrafish (Tg(NFKB:EGFP)) were conventionalized (CONVD) and pattern of EGFP expression was determined by fluorescent microscopy. SAA expression in GF and CONVD zebrafish was determined by wholemount in situ hybridization (WISH). Conventionally-raised Tg(NFKB:EGFP) zebrafish were injected with SAA morpholino (MO) at the 1-cell stage (0.9 pg/embryo) to block SAA expression. Impact of SAA on NF-κB signaling and gene expression In Vitro was determined using zebrafish PAC2 embryonic fibroblasts. NF-κB signaling was determined using immunofluorescent staining (RelA), western blot (phospho-IκBa), and transcriptional activity (luciferase reporter gene, NFKB:EGFP reporter plasmid). Transcription of NF-κB target genes In Vitro and In Vivo was determined using ABI Prism 7700HT detection system. Results: Recombinant human SAA (1μM) stimulated IκBα serine 32/36 phosphorylation, RelA nuclear translocation, and luciferase activity (relative fold change 3.5±0.07 vs ctl, p<0.01) in PAC2 cells. SAA induced EGFP expression in PAC2 cells transfected with pNFKB:EGFP. In addition, SAA induced NF-κB target genes IκBαa and MMP9 (relative fold change, 25.8±1.70 and 19.9±0.69 vs ctl respectively, p<0.01). Colonization of GF Tg(NFKB:EGFP) zebrafish with a normal microbiota resulted in elevated EGFP expression in the liver, dorsal root ganglia, the neuromasts of the lateral line, and a distinct population of intestinal cells. WISH and quantitative RT-PCR showed that CONVD Tg(NFKB:EGFP) zebrafish strongly up-regulated SAA gene expression in the liver and intestine compared to GF zebrafish. Importantly, although global cellular EGFP expression was not significantly inhibited in SAA-MO injected Tg(NFKB:EGFP) zebrafish, a strong decrease in endogenous mRNA accumulation of NF-κB targets IκBαa (40%) and MMP9 (90%) was observed in these fish compared to control MO injected zebrafish. Conclusion: Our data indicate that microbial colonization enhances SAA expression, which then impacts on NF-κB
560 Crohn's Disease-Associated Adherent-Invasive Escherichia coli Target Peyer's Patches via Long Polar Fimbriae Benoit Chassaing, Nathalie Rolhion, Amelie De Vallee, Sa'ad Salim, Maelle Prorok-Hamon, Christel Neut, Barry J. Campbell, Jean-Pierre Hugot, Jean-Frederic Colombel, Arlette Darfeuille-Michaud Background & Aims: Ileal lesions of patients with Crohn's disease (CD) are colonized by adherent-invasive Escherichia coli (AIEC) and the earliest observable lesions of CD are microscopic erosions of the follicle-associated epithelium (FAE) lining the Peyer's patches(PP). We aimed in the present study to investigate the ability of AIEC to interact with PP and M cells of the FAE, to quantify the PP-associated E. coli in CD patients compared to controls, and to search for the presence of a virulence factor in AIEC that could be involved in the targeting of PP by these invasive bacteria. Methods: PP from CD patients and controls were analyzed by q-PCR for the presence of E. coli 16S r-RNA encoding gene, and the genome
AGA Abstracts
S-78