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1074 Lactobacillus GG Secretes Small Peptides That Inhibit the Growth of Antibiotic-Resistant Bacteria
AGA Abstracts
platform. Differentially expressed miRs were identified by Significance Analysis of Microarrays (SAM). Results; Five primary CCAs and 5 normal bile duct specimens (NBDs) were analyzed. Twenty miRs were over- and 112 were underexpressed in CCAs vs. NBDs. The top 5 miRs in each category are listed in the Table. MiR expression was verified by quantitative RTPCR (qRT-PCR). To evaluate the ability of miR-21 to diagnose CCA, qRT-PCR was performed on 15 additional primary CCAs and 9 normal liver (NL) specimens. NLs displayed uniformly low levels of miR-21, with a standard deviation (SD) of 1.05. In contrast, CCAs displayed more variable expression levels (SD 8.64). The fold difference between CCA and NL was 6.21. Receiver-operating characteristic (ROC) curve analyses showed that miR-21 was 95% sensitive and 100% specific in distinguishing between CCA and normal tissues, with an area under the ROC curve (AUC) of 0.992. Conclusions: MiR-21 was found overexpressed in a variety of solid cancers, but this is the first report of its expression in human CCAs. MiR-21 was found to have a consistently higher expression in CCAs compared to normal hepatobiliary tissues and shows promise as a biomarker of this cancer. Furthermore, a panel combining miR-21 and other differentially expressed miRs may further refine our ability to correctly diagnose CCAs. Moreover, miR-21 along with other differentially expressed miRs in CCAs found in this study, deserve further consideration to elucidate their role in the CCA carcinogenesis. MicroRNA species differentially expressed between CCA and NBD
in the number of Bifidobacterium spp. This was associated with an increase in TLR2 (0.6±0.14) and a reduction in CB2 expression (0.64±0.12), when compared to that observed in the conventional group. Conclusions: Expression of TLR2 and CB2 receptors in the rat cecum followed changes in the microbiota, particularly in the content of Bifidobacterium spp. Bifidobacterium spp have been identified as an active component in probiotic treatments with clinical efficacy in IBS. These observations suggest that changes in the expression of TLR2, implicated in epithelial function and immune responses, and CB2 receptors, implicated in nociceptive responses, might be part of the mechanisms underlying the beneficial effects of probiotics in IBS. 1076 Clinical and Physiological Effects of Yogurt Drink Containing the Probiotic Bacteria Bifidobacterium lactis Bb12 and Prebiotic Inulin in Subjects with Functional Bowel Symptoms. Tamar Ringel-Kulka, Olafur S. Palsson, Joseph Galanko, Danielle M. Maier, Yehuda . Ringel Evidence suggests that intestinal bacteria play a role in the pathophysiology of functional GI disorders and that modulation of intestinal microbiota may have beneficial clinical effect in these conditions. Several studies looked at clinical effects of probiotics on functional GI symptoms but only a few have correlated the clinical outcome with physiological effects. Aim: To investigate the clinical and physiological effects of a yogurt drink containing the probiotic bacteria Bifidobacterium animalis ssp. lactis Bb12 (Bb12) and the prebiotic inulin in subjects with functional bowel symptoms. Methods: Subjects who met the Rome II criteria for chronic functional bowel symptoms were enrolled in a prospective double-blind, placebo-control trial. Subjects received a yogurt drink containing 5x109 cfu total Bb12 and 1.0g inulin (probiotic arm) or acidified dairy drink (placebo arm) for 6-weeks. Clinical variables included overall well being, pain, bloating and ease of defecation. Physiological variables included stool frequency and consistency, and colonic transit time (CTT). CTT was measured at baseline and during intervention using radio-opaque markers. Mean changes in variables from baseline to mid- (3 weeks) and end-of- intervention (6 weeks) were calculated and compared between the groups. Results: A total of 60 (probiotic n=30; placebo n=30) subjects were enrolled. The probiotic group reported significant improvement in overall well being (p=0.02) while no change was observed in the placebo group (p=0.08, for between group comparison). No significant between-group differences were found in reported changes in bloating, pain, and ease of defecation. CTT was significantly shorter in the active (n=17) group (30.5h vs. 21.3h, p=0.02), while no significant change was observed in the placebo (n=19) group (23.0h vs. 25.0h, p=NS). This effect on CTT strongly correlated with change in stool consistency (r=0.4, p=0.02) and showed a trend for correlation with improvement in ease of defecation (r=-0.4 p=0.088). The intervention group reported softer and more frequent stools; these differences did not reach statistical significance. Conclusions: Yogurt containing probiotic bacteria Bb12 and inulin have beneficial effects on intestinal physiology. The shorter CTT with the positive effect on stool consistency can explain the observed improvement in the overall well being. In view of the current lack of effective intestinal promotility agents, this dietary intervention may be a useful addition to the management of subjects with slow transit-related functional bowel symptoms. (Supported by grants K23 DK075621 and General Mills, Inc.)
1074 Lactobacillus GG Secretes Small Peptides That Inhibit the Growth of Antibiotic-Resistant Bacteria Ruiliang Lu, Alessio Fasano Background and Objective: We have previously reported that several small peptides isolated from lactobacillus GG conditional media exert both gram-negative and gram-positive bactericidal activity. Among them, peptide NPSRQERR, VHTAPK and PDENK have more activities than others. This research was aimed at establishing whether these peptides exert their bactericidal effect also on microorganisms that have developed multiple antibiotic resistance. Method: Peptides were synthesized, purified, identified and dissolved in different media. Lactobacillus GG Conditional Media (LGGCM) (19.7 X 1012 CFU/ML) was used as positive control, while media only was used as negative control. Antibacterial activity was monitored by measuring A600 of the culture after 3 h incubation. Results: Peptides NPSRQERR(2.76mM), VHTAPK(3.25mM) and PDENK(3.31mM) all inhibit Kanamycineresistant E. coli(2.6 X 1013 CFU/ML) growth by 43.75%, 29.45% and 68.54%, respectively. The same peptides tested at the same concentrations were also able to inhibit tetracyclineresistant E. coli (8.7 X1013CFU/ML) growth by 69.08%, 48.53% and 81.4% respectively. Finally, methicillin-resistant Staphylococcus aureus (MRSA)(2.89 X 1014 CFU/ML) growth can be inhibited by NPSEQERR(6.9mM), VHTAPK(9.0mM) and PDENK(9.0mM) by 20.4%, 14.76% and 34.93% respectively. Interestingly, peptide NPSRQERR has 2 positive charges that dictate its anti-bacterial activity. Indeed, modification of the peptide by decreasing its positive charge from 2 to 0 caused a drop in its anti-bacterial activity by 12%, while the addition of an extra positive charge caused an increase of the anti-bacterial activity by16.5%. Conclusions: Lactobacillus GG peptides NPSRQERR, VHTAPK and PDENK exert an antibacterial activity not only on gram-negative and gram-positive bacteria growth, but also on strains with multi-antibiotic resistance. These results suggest that probiotics or their derivative peptides can be effective alternative treatments for infections caused by multi-drug resistant bacteria.
1077 Bifidobacterium bifidum in a Rat Model of Necrotizing Enterocolitis: Changes in Toll-Like Receptors and Inflammatory Cytokines Ludmila Khailova, Kelly M. Arganbright, Andrew Maynard, Amber Johnson, Melissa Halpern, Toshi Kinouchi, Masako Yajima, Bohuslav Dvorak Background: Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Oral administration of probiotics has been suggested as a promising strategy for prevention of NEC. However, little is known about the mechanism(s) of probiotic-mediated protection against NEC. The host response to bacteria is mediated by innate immune receptors called Toll-like receptors (TLRs), which are involved in many inflammatory signaling pathways. TLR signaling may play a critical role in intestinal inflammation development. We have previously shown that the production of pro-inflammatory cytokines is upregulated in NEC animals. Objective: The aim of this study was to evaluate Toll-like receptors and inflammatory cytokines in ileum after oral administration of Bifidobacterium bifidum in a rat model of NEC. Methods: Premature rats were divided into three groups: dam fed (DF), hand-fed with formula (NEC), or hand-fed with formula supplemented with 5 x 106 CFU Bifidobacterium bifidum OLB6378 per day (BB). All groups were exposed to asphyxia/cold stress to develop NEC. The incidence of NEC, pro-inflammatory cytokines and TLRs were evaluated at the site of injury - the terminal ileum. Results: Bifidobacterium bifidum OLB6378 significantly lowered the incidence of NEC in neonatal rat model. TLR4 was significantly elevated in animals that received BB compare to DF and NEC animals. TLR-2 showed increased levels in both NEC and BB groups when compared to healthy DF controls. TNF-α and IL-6 levels were significantly upregulated in animals with NEC and downregulated in the BB group to levels seen in DF animals. In contrast, IFN-γ showed dramatic increase in BB group compare to DF and NEC groups. Conclusions: Oral administration of B. bifidum OLB6378 protects against NEC in neonatal rat model. Regulation of the pro-inflammatory pathway can be one of the mechanisms by which Bifidobacterium bifidum improves NEC. Supported by the NIH Grant HD-39657 (to B.D.) and a gift from Meiji Dairies Corporation.
1075 Gut Microbiota Affects Toll-Like Receptors (TLR) and Cannabinoid 2 Receptor (CB2) Expression in the Intestine of Rats Evangelina Teran, Maite Martin, Ferran Jardi, Patri Vergara, Vicente Martinez Background: Gut microbiota has been implicated in the pathophysiology of functional gastrointestinal disorders, particularly IBS. Gut flora is recognized mainly through toll-like receptors 2 and 4 (TLR2 and TLR4), presenting a flora-modulated expression. Moreover, specific bacterial strains have been demonstrated to improve IBS symptoms and to modulate visceral pain responses and the colonic content of cannabinoid 2 receptors (CB2) (Nat. Med. 13:25, 2007). Aim: To determine how changes in gut microbiota modulate the expression of TLR2, TLR4 and CB2 receptors in the cecum of rats. Methods: Cecal microbiota was characterized using fluorescent in situ hybridization in SD rats bred under controlled microbiological conditions (barrier group, n=12) or under conventional housing conditions (conventional group, n=5). In the same animals, the expression of TLR2, TLR4 and CB2 receptors was determined by RT-PCR. In addition, spontaneous changes in the same parameters were assessed in rats bred under controlled microbiological conditions and adapted for 3 weeks to conventional conditions (adapted group, n=5). Results: Total number of cecal bacteria was similar in the barrier and conventional groups. Compared with the conventional group, the barrier group had a higher number of strict anaerobic bacteria (Bacteroides spp and Clostridium spp; P<0.05), while the number of Bifidobacterium spp was reduced (P<0.01). This was associated to a lower expression of Bifidobacterium spp-recognition TLR2 in the barrier group (0.24±0.08) vs. the conventional group (0.47±0.11; P=0.07), while TLR4 expression was similar in both groups (barrier: 0.5±0.09; conventional: 0.49±0.14). CB2 expression was 3-fold higher in the barrier (1.17±0.09) than in the conventional group (0.41±0.12, P=0.015). In the adapted group, cecal flora showed a spontaneous transition towards that of the conventional group, with a reduction in strict anaerobics and an increase
AGA Abstracts
A-166
platform. Differentially expressed miRs were identified by Significance Analysis of Microarrays (SAM). Results; Five primary CCAs and 5 normal bile duct specimens (NBDs) were analyzed. Twenty miRs were over- and 112 were underexpressed in CCAs vs. NBDs. The top 5 miRs in each category are listed in the Table. MiR expression was verified by quantitative RTPCR (qRT-PCR). To evaluate the ability of miR-21 to diagnose CCA, qRT-PCR was performed on 15 additional primary CCAs and 9 normal liver (NL) specimens. NLs displayed uniformly low levels of miR-21, with a standard deviation (SD) of 1.05. In contrast, CCAs displayed more variable expression levels (SD 8.64). The fold difference between CCA and NL was 6.21. Receiver-operating characteristic (ROC) curve analyses showed that miR-21 was 95% sensitive and 100% specific in distinguishing between CCA and normal tissues, with an area under the ROC curve (AUC) of 0.992. Conclusions: MiR-21 was found overexpressed in a variety of solid cancers, but this is the first report of its expression in human CCAs. MiR-21 was found to have a consistently higher expression in CCAs compared to normal hepatobiliary tissues and shows promise as a biomarker of this cancer. Furthermore, a panel combining miR-21 and other differentially expressed miRs may further refine our ability to correctly diagnose CCAs. Moreover, miR-21 along with other differentially expressed miRs in CCAs found in this study, deserve further consideration to elucidate their role in the CCA carcinogenesis. MicroRNA species differentially expressed between CCA and NBD
in the number of Bifidobacterium spp. This was associated with an increase in TLR2 (0.6±0.14) and a reduction in CB2 expression (0.64±0.12), when compared to that observed in the conventional group. Conclusions: Expression of TLR2 and CB2 receptors in the rat cecum followed changes in the microbiota, particularly in the content of Bifidobacterium spp. Bifidobacterium spp have been identified as an active component in probiotic treatments with clinical efficacy in IBS. These observations suggest that changes in the expression of TLR2, implicated in epithelial function and immune responses, and CB2 receptors, implicated in nociceptive responses, might be part of the mechanisms underlying the beneficial effects of probiotics in IBS. 1076 Clinical and Physiological Effects of Yogurt Drink Containing the Probiotic Bacteria Bifidobacterium lactis Bb12 and Prebiotic Inulin in Subjects with Functional Bowel Symptoms. Tamar Ringel-Kulka, Olafur S. Palsson, Joseph Galanko, Danielle M. Maier, Yehuda . Ringel Evidence suggests that intestinal bacteria play a role in the pathophysiology of functional GI disorders and that modulation of intestinal microbiota may have beneficial clinical effect in these conditions. Several studies looked at clinical effects of probiotics on functional GI symptoms but only a few have correlated the clinical outcome with physiological effects. Aim: To investigate the clinical and physiological effects of a yogurt drink containing the probiotic bacteria Bifidobacterium animalis ssp. lactis Bb12 (Bb12) and the prebiotic inulin in subjects with functional bowel symptoms. Methods: Subjects who met the Rome II criteria for chronic functional bowel symptoms were enrolled in a prospective double-blind, placebo-control trial. Subjects received a yogurt drink containing 5x109 cfu total Bb12 and 1.0g inulin (probiotic arm) or acidified dairy drink (placebo arm) for 6-weeks. Clinical variables included overall well being, pain, bloating and ease of defecation. Physiological variables included stool frequency and consistency, and colonic transit time (CTT). CTT was measured at baseline and during intervention using radio-opaque markers. Mean changes in variables from baseline to mid- (3 weeks) and end-of- intervention (6 weeks) were calculated and compared between the groups. Results: A total of 60 (probiotic n=30; placebo n=30) subjects were enrolled. The probiotic group reported significant improvement in overall well being (p=0.02) while no change was observed in the placebo group (p=0.08, for between group comparison). No significant between-group differences were found in reported changes in bloating, pain, and ease of defecation. CTT was significantly shorter in the active (n=17) group (30.5h vs. 21.3h, p=0.02), while no significant change was observed in the placebo (n=19) group (23.0h vs. 25.0h, p=NS). This effect on CTT strongly correlated with change in stool consistency (r=0.4, p=0.02) and showed a trend for correlation with improvement in ease of defecation (r=-0.4 p=0.088). The intervention group reported softer and more frequent stools; these differences did not reach statistical significance. Conclusions: Yogurt containing probiotic bacteria Bb12 and inulin have beneficial effects on intestinal physiology. The shorter CTT with the positive effect on stool consistency can explain the observed improvement in the overall well being. In view of the current lack of effective intestinal promotility agents, this dietary intervention may be a useful addition to the management of subjects with slow transit-related functional bowel symptoms. (Supported by grants K23 DK075621 and General Mills, Inc.)
1074 Lactobacillus GG Secretes Small Peptides That Inhibit the Growth of Antibiotic-Resistant Bacteria Ruiliang Lu, Alessio Fasano Background and Objective: We have previously reported that several small peptides isolated from lactobacillus GG conditional media exert both gram-negative and gram-positive bactericidal activity. Among them, peptide NPSRQERR, VHTAPK and PDENK have more activities than others. This research was aimed at establishing whether these peptides exert their bactericidal effect also on microorganisms that have developed multiple antibiotic resistance. Method: Peptides were synthesized, purified, identified and dissolved in different media. Lactobacillus GG Conditional Media (LGGCM) (19.7 X 1012 CFU/ML) was used as positive control, while media only was used as negative control. Antibacterial activity was monitored by measuring A600 of the culture after 3 h incubation. Results: Peptides NPSRQERR(2.76mM), VHTAPK(3.25mM) and PDENK(3.31mM) all inhibit Kanamycineresistant E. coli(2.6 X 1013 CFU/ML) growth by 43.75%, 29.45% and 68.54%, respectively. The same peptides tested at the same concentrations were also able to inhibit tetracyclineresistant E. coli (8.7 X1013CFU/ML) growth by 69.08%, 48.53% and 81.4% respectively. Finally, methicillin-resistant Staphylococcus aureus (MRSA)(2.89 X 1014 CFU/ML) growth can be inhibited by NPSEQERR(6.9mM), VHTAPK(9.0mM) and PDENK(9.0mM) by 20.4%, 14.76% and 34.93% respectively. Interestingly, peptide NPSRQERR has 2 positive charges that dictate its anti-bacterial activity. Indeed, modification of the peptide by decreasing its positive charge from 2 to 0 caused a drop in its anti-bacterial activity by 12%, while the addition of an extra positive charge caused an increase of the anti-bacterial activity by16.5%. Conclusions: Lactobacillus GG peptides NPSRQERR, VHTAPK and PDENK exert an antibacterial activity not only on gram-negative and gram-positive bacteria growth, but also on strains with multi-antibiotic resistance. These results suggest that probiotics or their derivative peptides can be effective alternative treatments for infections caused by multi-drug resistant bacteria.
1077 Bifidobacterium bifidum in a Rat Model of Necrotizing Enterocolitis: Changes in Toll-Like Receptors and Inflammatory Cytokines Ludmila Khailova, Kelly M. Arganbright, Andrew Maynard, Amber Johnson, Melissa Halpern, Toshi Kinouchi, Masako Yajima, Bohuslav Dvorak Background: Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Oral administration of probiotics has been suggested as a promising strategy for prevention of NEC. However, little is known about the mechanism(s) of probiotic-mediated protection against NEC. The host response to bacteria is mediated by innate immune receptors called Toll-like receptors (TLRs), which are involved in many inflammatory signaling pathways. TLR signaling may play a critical role in intestinal inflammation development. We have previously shown that the production of pro-inflammatory cytokines is upregulated in NEC animals. Objective: The aim of this study was to evaluate Toll-like receptors and inflammatory cytokines in ileum after oral administration of Bifidobacterium bifidum in a rat model of NEC. Methods: Premature rats were divided into three groups: dam fed (DF), hand-fed with formula (NEC), or hand-fed with formula supplemented with 5 x 106 CFU Bifidobacterium bifidum OLB6378 per day (BB). All groups were exposed to asphyxia/cold stress to develop NEC. The incidence of NEC, pro-inflammatory cytokines and TLRs were evaluated at the site of injury - the terminal ileum. Results: Bifidobacterium bifidum OLB6378 significantly lowered the incidence of NEC in neonatal rat model. TLR4 was significantly elevated in animals that received BB compare to DF and NEC animals. TLR-2 showed increased levels in both NEC and BB groups when compared to healthy DF controls. TNF-α and IL-6 levels were significantly upregulated in animals with NEC and downregulated in the BB group to levels seen in DF animals. In contrast, IFN-γ showed dramatic increase in BB group compare to DF and NEC groups. Conclusions: Oral administration of B. bifidum OLB6378 protects against NEC in neonatal rat model. Regulation of the pro-inflammatory pathway can be one of the mechanisms by which Bifidobacterium bifidum improves NEC. Supported by the NIH Grant HD-39657 (to B.D.) and a gift from Meiji Dairies Corporation.
1075 Gut Microbiota Affects Toll-Like Receptors (TLR) and Cannabinoid 2 Receptor (CB2) Expression in the Intestine of Rats Evangelina Teran, Maite Martin, Ferran Jardi, Patri Vergara, Vicente Martinez Background: Gut microbiota has been implicated in the pathophysiology of functional gastrointestinal disorders, particularly IBS. Gut flora is recognized mainly through toll-like receptors 2 and 4 (TLR2 and TLR4), presenting a flora-modulated expression. Moreover, specific bacterial strains have been demonstrated to improve IBS symptoms and to modulate visceral pain responses and the colonic content of cannabinoid 2 receptors (CB2) (Nat. Med. 13:25, 2007). Aim: To determine how changes in gut microbiota modulate the expression of TLR2, TLR4 and CB2 receptors in the cecum of rats. Methods: Cecal microbiota was characterized using fluorescent in situ hybridization in SD rats bred under controlled microbiological conditions (barrier group, n=12) or under conventional housing conditions (conventional group, n=5). In the same animals, the expression of TLR2, TLR4 and CB2 receptors was determined by RT-PCR. In addition, spontaneous changes in the same parameters were assessed in rats bred under controlled microbiological conditions and adapted for 3 weeks to conventional conditions (adapted group, n=5). Results: Total number of cecal bacteria was similar in the barrier and conventional groups. Compared with the conventional group, the barrier group had a higher number of strict anaerobic bacteria (Bacteroides spp and Clostridium spp; P<0.05), while the number of Bifidobacterium spp was reduced (P<0.01). This was associated to a lower expression of Bifidobacterium spp-recognition TLR2 in the barrier group (0.24±0.08) vs. the conventional group (0.47±0.11; P=0.07), while TLR4 expression was similar in both groups (barrier: 0.5±0.09; conventional: 0.49±0.14). CB2 expression was 3-fold higher in the barrier (1.17±0.09) than in the conventional group (0.41±0.12, P=0.015). In the adapted group, cecal flora showed a spontaneous transition towards that of the conventional group, with a reduction in strict anaerobics and an increase
AGA Abstracts
A-166