- Email: [email protected]
Necrotizing Enterocolitis of Preterm Piglets is Characterized by a Unique Ileum Mucosa-Associated Microbiome
AGA Abstracts
both Gram-negative and Gram-positive bacteria. TLR4 activation by LPS rapidly and persistently alters biological and morphological SMC features via NFκB whereas that of TLR2 induces similar cellular effects after short exposure but of lower intensity than those of LPS. Nevertheless the effects of a prolonged TLR2 activation have still not been evaluated. AIMS of the study were to analyze direct effects of LGG on colonic human SMC and to evaluate its possible protective role in LPS-induced motor alterations. METHODS: The direct effect of LGG (ATCC 53103 strain) was tested on a highly pure human SMC culture either in the presence or absence of the TLR4 agonist LPS (1μg/ml). LGG effects, evaluated on muscular morphofunctional properties and NFκB activation, were compared to those induced by the synthetic agonists of TLR2/1 and TLR2/6, PAM2- and PAM3-CSKA respectively. NFκB activation was examined by ELISA determination of the phosphorylation of the p65 NFκB subunits Ser468 and Ser536. Data are expressed as mean±SD, p<0.05 considered significant. RESULTS: After a 30 min-exposure LGG induced a dose-dependent cell length shortening and an inhibition of contractile response to the muscarinic agonist acetylcholine, respectively of 15.1%±7.3 and 59.7%±15.1 at a dose of 120x106CFU/ml (p<0.05), confirming the direct effect of LGG on SMC. These cellular effects disappeared by prolonging exposure time. Indeed after 24h LGG exposure, cellular alterations were no more observed and similar time-dependent effects were observed with PAM2- and PAM3-CSKA. LGG influences then muscular activity in a reversible manner, a trend strongly in contrast to that observed with LPS. These differences resulted to be related to relative NFκB activation. Whereas LPS induced a rapid significant (p<0.05) increase in phosphorylation NFκB subunits (1h: Ser468 75.4%±22.1; Ser536 89.6%±33.8) that persisted over time (24h: Ser468 89.9%±12.6; Ser536 117.0%±15.9), LGG induced only a slight increase at 1h (Ser468 7.9%±7.5; Ser536 3.8%±2.7) with reversion at 24h. Furthermore, LGG inhibited LPS-induced NFκB activation in that LPS effects in its presence were blocked after 1 h exposure by up to 74.8%±12.1 for Ser468 and 90.3%±3.0 for Ser536 and similar effects were observed after 24. CONCLUSION: LGG has a direct anti-inflammatory effect on colonic muscle and might be an appropriate candidate for probiotic intervention in bacterial-related intestinal motor disorders.
16S rRNA gene specific for total Bacteria, Bacteroidetes, and Firmicutes. Two microbial genes involved in the production of butyrate and acetate, butyryl coA transferase (BCoAT) and formyl tetrahydrofolate synthetase (FTHFS), were also quantified. Gene counts for BCoAT and FTHFS were normalized to total 16S rRNA counts. Results: There were no significant differences between the PPI subjects and controls for total Bacteria and Firmicutes based on 16S rRNA counts. There was a significant decrease in Bacteroidetes in the PPI group (2.79 x 10^5 ± 8.78 x 10^4 copies/μl) when compared to controls (8.32 x 10^5 ± 3.03 x 10^5 copies/μl, p<0.05). There was also a significant decrease in BCoAT gene copies in the PPI group (1.78 x 10^3 ± 3.40 x 10^2 copies/μl) compared to controls (1.05 x 10^5 ± 3.91 x 10^3 copies/μl, p<0.05). There was no significant difference in FTHFS gene copies between PPI subjects and controls. Discussion: There is widespread use of PPIs in the United States and their effects on the colonic microbiome are not well known. Our data show that PPIs do not affect the overall census of the colonic microbiome but do alter the taxonomic composition, significantly decreasing Bacteroidetes. Furthermore, microbial genes involved in butyrate production appear to be decreased secondary to chronic PPI therapy, which may have implications on colonic epithelial health.
Mo1859 Necrotizing Enterocolitis of Preterm Piglets is Characterized by a Unique Ileum Mucosa-Associated Microbiome M. Andrea Azcarate-Peril, Derek Foster, Maria B. Cadenas, Maria Stone, Sheila Jacobi, Stephen Stauffer, Anthony Pease, Jody Gookin Necrotizing enterocolitis (NEC) is a tragic complication of prematurity affecting ~10% of verylow birth weight infants. Intestinal prematurity, formula feeding and bacterial colonization of the gut are recognized as key predisposing factors. Progress in characterizing GI bacteria and their role in NEC pathogenesis are limited by patient fragility, analysis restricted to fecal samples, use of culture-based methods, and lack of clinically-relevant animal models. The present studies used a unique piglet model of spontaneous NEC to explore a Central Hypothesis that infants developing NEC have a distinguishing pattern of microbial diversity within intestinal lesions that are absent in infants not developing NEC. Preterm piglets (n= 23; 720-1540gBW) were delivered by C-section, tube-fed formula (10ml/kg q3hr) and nurtured akin to human infants for 30hrs. Twelve piglets (52%) developed NEC and were euthanized an average of 24.2 ± 1.2 hrs after onset of feeding based on clinical (92%), radiographic (67%), gross pathologic (92%) and light microscopic (100%) diagnostic criteria. Piglets (48%) not developing NEC all survived to study endpoint. 16S rDNA was PCR amplified from terminal ileum mucosa, proximal colon mucosa and ileum content total DNA, digested using 3 restriction enzymes, and analyzed by restriction fragment length polymorphism (TRFLP). Based on TRFLP analysis selected bacterial groups were quantified by qPCR. A relatively low diversity was observed in the ileum mucosa of piglets without NEC where a mean of only 5.1 ± 3.2 unique bacterial TRFLP profiles were identified. The majority of the detected profiles were tentatively assigned to the phyla Proteobacteria, Firmicutes, Cyanobacteria and Chloroflexi. A greater diversity was observed in the colonic mucosa and ileum content where means of 10 ± 6.3 and 11.8 ± 5.8 unique TRFLP profiles were identified respectively. Again the majority of profiles were assigned to Proteobacteria, Firmicutes, Cyanobacteria and Chloroflexi with additional representation of Actinobacteria and uncharacterized phylotypes. Significant findings in piglets with NEC was an increase in diversity represented by increased mean number of profiles 9.5 ± 4.4 (p<0.01) identified in association with ileum mucosa but not colonic mucosa 11.6 ± 4.0 or ileum content 9.9 ± 6.5. In piglets with NEC, ileum mucosa had greater numbers of Firmicutes, acquired Actinobacteria and unidentified phylotypes. Five unique patterns were significantly more prevalent, detected at higher numbers, or only observed among samples from piglets with NEC including Streptomyces spp., Leptolyngbya, Clostridium butyricum, C.proteolyticum, and C.neonatale. Increased diversity and presence of atypical bacterial phyla directly associated with ileum mucosa of piglets with NEC suggest that unique attributes of these bacteria may play a key role in NEC pathogenesis.
The effect of chronic PPI use on the taxonomic composition of the colonic microbiome
Mo1860 Proton Pump Inhibitors Significantly Alter the Taxonomic Composition and Butyrate Production of the Colonic Microbiome Josephine Ni, Adam Snyder, Scott M. Smukalla, Hyunsoo Park, Animesh Jain, Abraham Khan, Ilseung Cho
Effect of chronic PPI use on microbial genes involved in short chain fatty acid synthesis
Background: Proton pump inhibitors (PPI) are among the most frequently prescribed medications in the United States. They have been shown to alter the gastric microbiome by increasing the pH and have also been associated with an increased risk of gastrointestinal infections, such as C. difficile infections and bacterial overgrowth. However, the effect of PPIs on the colonic microbiome and its ability to metabolize carbohydrates into short chain fatty acids has not been well studied. Objective: To study the effect of proton pump inhibitors on the colonic microbiome and its ability to metabolize carbohydrates. Methods: Subjects were recruited from patients presenting for screening colonoscopies. Stool samples were collected from patients at the time of study enrollment, prior to bowel preparation. 9 patients on PPI therapy for >6 months were age-, gender-, and ethnicity-matched in a 1:2 ratio to 18 controls. Microbial DNA was extracted from stool specimens using the MoBio PowerSoil DNA isolation kit. Quantitative PCR was performed using degenerate primers targeting the
AGA Abstracts
Mo1876 A Systematic Approach to Therapeutic Target Selection in Gastro-Esophageal Adenocarcinoma Anna L. Paterson, Nicholas B. Shannon, Pierre Lao-Sirieix, Christopher J. Peters, Maria O'Donovan, Rebecca Fitzgerald Introduction: The future success of individualised, molecular targeted therapy will depend on identification and thence inhibition of the oncogene(s) on which that tumour is dependent for survival. Gastro-esophageal tumours are common, lethal and molecularly heterogeneous. Currently chemotherapy relies on generic regimens and neo-adjuvant treatment only affords
S-666
both Gram-negative and Gram-positive bacteria. TLR4 activation by LPS rapidly and persistently alters biological and morphological SMC features via NFκB whereas that of TLR2 induces similar cellular effects after short exposure but of lower intensity than those of LPS. Nevertheless the effects of a prolonged TLR2 activation have still not been evaluated. AIMS of the study were to analyze direct effects of LGG on colonic human SMC and to evaluate its possible protective role in LPS-induced motor alterations. METHODS: The direct effect of LGG (ATCC 53103 strain) was tested on a highly pure human SMC culture either in the presence or absence of the TLR4 agonist LPS (1μg/ml). LGG effects, evaluated on muscular morphofunctional properties and NFκB activation, were compared to those induced by the synthetic agonists of TLR2/1 and TLR2/6, PAM2- and PAM3-CSKA respectively. NFκB activation was examined by ELISA determination of the phosphorylation of the p65 NFκB subunits Ser468 and Ser536. Data are expressed as mean±SD, p<0.05 considered significant. RESULTS: After a 30 min-exposure LGG induced a dose-dependent cell length shortening and an inhibition of contractile response to the muscarinic agonist acetylcholine, respectively of 15.1%±7.3 and 59.7%±15.1 at a dose of 120x106CFU/ml (p<0.05), confirming the direct effect of LGG on SMC. These cellular effects disappeared by prolonging exposure time. Indeed after 24h LGG exposure, cellular alterations were no more observed and similar time-dependent effects were observed with PAM2- and PAM3-CSKA. LGG influences then muscular activity in a reversible manner, a trend strongly in contrast to that observed with LPS. These differences resulted to be related to relative NFκB activation. Whereas LPS induced a rapid significant (p<0.05) increase in phosphorylation NFκB subunits (1h: Ser468 75.4%±22.1; Ser536 89.6%±33.8) that persisted over time (24h: Ser468 89.9%±12.6; Ser536 117.0%±15.9), LGG induced only a slight increase at 1h (Ser468 7.9%±7.5; Ser536 3.8%±2.7) with reversion at 24h. Furthermore, LGG inhibited LPS-induced NFκB activation in that LPS effects in its presence were blocked after 1 h exposure by up to 74.8%±12.1 for Ser468 and 90.3%±3.0 for Ser536 and similar effects were observed after 24. CONCLUSION: LGG has a direct anti-inflammatory effect on colonic muscle and might be an appropriate candidate for probiotic intervention in bacterial-related intestinal motor disorders.
16S rRNA gene specific for total Bacteria, Bacteroidetes, and Firmicutes. Two microbial genes involved in the production of butyrate and acetate, butyryl coA transferase (BCoAT) and formyl tetrahydrofolate synthetase (FTHFS), were also quantified. Gene counts for BCoAT and FTHFS were normalized to total 16S rRNA counts. Results: There were no significant differences between the PPI subjects and controls for total Bacteria and Firmicutes based on 16S rRNA counts. There was a significant decrease in Bacteroidetes in the PPI group (2.79 x 10^5 ± 8.78 x 10^4 copies/μl) when compared to controls (8.32 x 10^5 ± 3.03 x 10^5 copies/μl, p<0.05). There was also a significant decrease in BCoAT gene copies in the PPI group (1.78 x 10^3 ± 3.40 x 10^2 copies/μl) compared to controls (1.05 x 10^5 ± 3.91 x 10^3 copies/μl, p<0.05). There was no significant difference in FTHFS gene copies between PPI subjects and controls. Discussion: There is widespread use of PPIs in the United States and their effects on the colonic microbiome are not well known. Our data show that PPIs do not affect the overall census of the colonic microbiome but do alter the taxonomic composition, significantly decreasing Bacteroidetes. Furthermore, microbial genes involved in butyrate production appear to be decreased secondary to chronic PPI therapy, which may have implications on colonic epithelial health.
Mo1859 Necrotizing Enterocolitis of Preterm Piglets is Characterized by a Unique Ileum Mucosa-Associated Microbiome M. Andrea Azcarate-Peril, Derek Foster, Maria B. Cadenas, Maria Stone, Sheila Jacobi, Stephen Stauffer, Anthony Pease, Jody Gookin Necrotizing enterocolitis (NEC) is a tragic complication of prematurity affecting ~10% of verylow birth weight infants. Intestinal prematurity, formula feeding and bacterial colonization of the gut are recognized as key predisposing factors. Progress in characterizing GI bacteria and their role in NEC pathogenesis are limited by patient fragility, analysis restricted to fecal samples, use of culture-based methods, and lack of clinically-relevant animal models. The present studies used a unique piglet model of spontaneous NEC to explore a Central Hypothesis that infants developing NEC have a distinguishing pattern of microbial diversity within intestinal lesions that are absent in infants not developing NEC. Preterm piglets (n= 23; 720-1540gBW) were delivered by C-section, tube-fed formula (10ml/kg q3hr) and nurtured akin to human infants for 30hrs. Twelve piglets (52%) developed NEC and were euthanized an average of 24.2 ± 1.2 hrs after onset of feeding based on clinical (92%), radiographic (67%), gross pathologic (92%) and light microscopic (100%) diagnostic criteria. Piglets (48%) not developing NEC all survived to study endpoint. 16S rDNA was PCR amplified from terminal ileum mucosa, proximal colon mucosa and ileum content total DNA, digested using 3 restriction enzymes, and analyzed by restriction fragment length polymorphism (TRFLP). Based on TRFLP analysis selected bacterial groups were quantified by qPCR. A relatively low diversity was observed in the ileum mucosa of piglets without NEC where a mean of only 5.1 ± 3.2 unique bacterial TRFLP profiles were identified. The majority of the detected profiles were tentatively assigned to the phyla Proteobacteria, Firmicutes, Cyanobacteria and Chloroflexi. A greater diversity was observed in the colonic mucosa and ileum content where means of 10 ± 6.3 and 11.8 ± 5.8 unique TRFLP profiles were identified respectively. Again the majority of profiles were assigned to Proteobacteria, Firmicutes, Cyanobacteria and Chloroflexi with additional representation of Actinobacteria and uncharacterized phylotypes. Significant findings in piglets with NEC was an increase in diversity represented by increased mean number of profiles 9.5 ± 4.4 (p<0.01) identified in association with ileum mucosa but not colonic mucosa 11.6 ± 4.0 or ileum content 9.9 ± 6.5. In piglets with NEC, ileum mucosa had greater numbers of Firmicutes, acquired Actinobacteria and unidentified phylotypes. Five unique patterns were significantly more prevalent, detected at higher numbers, or only observed among samples from piglets with NEC including Streptomyces spp., Leptolyngbya, Clostridium butyricum, C.proteolyticum, and C.neonatale. Increased diversity and presence of atypical bacterial phyla directly associated with ileum mucosa of piglets with NEC suggest that unique attributes of these bacteria may play a key role in NEC pathogenesis.
The effect of chronic PPI use on the taxonomic composition of the colonic microbiome
Mo1860 Proton Pump Inhibitors Significantly Alter the Taxonomic Composition and Butyrate Production of the Colonic Microbiome Josephine Ni, Adam Snyder, Scott M. Smukalla, Hyunsoo Park, Animesh Jain, Abraham Khan, Ilseung Cho
Effect of chronic PPI use on microbial genes involved in short chain fatty acid synthesis
Background: Proton pump inhibitors (PPI) are among the most frequently prescribed medications in the United States. They have been shown to alter the gastric microbiome by increasing the pH and have also been associated with an increased risk of gastrointestinal infections, such as C. difficile infections and bacterial overgrowth. However, the effect of PPIs on the colonic microbiome and its ability to metabolize carbohydrates into short chain fatty acids has not been well studied. Objective: To study the effect of proton pump inhibitors on the colonic microbiome and its ability to metabolize carbohydrates. Methods: Subjects were recruited from patients presenting for screening colonoscopies. Stool samples were collected from patients at the time of study enrollment, prior to bowel preparation. 9 patients on PPI therapy for >6 months were age-, gender-, and ethnicity-matched in a 1:2 ratio to 18 controls. Microbial DNA was extracted from stool specimens using the MoBio PowerSoil DNA isolation kit. Quantitative PCR was performed using degenerate primers targeting the
AGA Abstracts
Mo1876 A Systematic Approach to Therapeutic Target Selection in Gastro-Esophageal Adenocarcinoma Anna L. Paterson, Nicholas B. Shannon, Pierre Lao-Sirieix, Christopher J. Peters, Maria O'Donovan, Rebecca Fitzgerald Introduction: The future success of individualised, molecular targeted therapy will depend on identification and thence inhibition of the oncogene(s) on which that tumour is dependent for survival. Gastro-esophageal tumours are common, lethal and molecularly heterogeneous. Currently chemotherapy relies on generic regimens and neo-adjuvant treatment only affords
S-666