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Difluoromethylornithine is a novel inhibitor of Helicobacter pylori growth, CagA translocation, and interleukin-8 induction.

Barry DP, Asim M, Leiman DA, de Sablet T, Singh K, Casero RA, Chaturvedi R, Wilson KT - PLoS ONE (2011)

Bottom Line: We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner.H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism.These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
Helicobacter pylori infects half the world's population, and carriage is lifelong without antibiotic therapy. Current regimens prescribed to prevent infection-associated diseases such as gastroduodenal ulcers and gastric cancer can be thwarted by antibiotic resistance. We reported that administration of 1% D,L-α-difluoromethylornithine (DFMO) to mice infected with H. pylori reduces gastritis and colonization, which we attributed to enhanced host immune response due to inhibition of macrophage ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Although no ODC has been identified in any H. pylori genome, we sought to determine if DFMO has direct effects on the bacterium. We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner. Two other gram-negative pathogens possessing ODC, Escherichia coli and Citrobacter rodentium, were resistant to the DFMO effect. The effect of DFMO on H. pylori required continuous exposure to the drug and was reversible when removed, with recovery of growth rate in vitro and the ability to colonize mice. H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism. DFMO inhibited expression of the H. pylori virulence factor cytotoxin associated gene A, and its translocation and phosphorylation in gastric epithelial cells, which was associated with a reduction in interleukin-8 expression. These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

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DFMO suppresses the growth of H. pylori.Brucella broth cultures, some containing DFMO, were inoculated with H. pylori SS1 at an OD600 of ∼0.1 and growth was monitored for 24 h. (A) Bacteria were grown in control broth or broth supplemented with 0.01%, 0.1%, or 1% (w/v) DFMO and growth was monitored by measuring OD600 at the indicated time points. Solid lines depict the growth curve obtained for each treatment and error bars represent the standard error (n = 3). (B) H. pylori were grown and monitored as described for panel A in control broth or broth with 1% DFMO. Solid lines are experimentally obtained growth curves, with standard errors (n = 9), while the dashed lines indicate the calculated exponential regression curves using the first 12 h of data. The generation time (g) and goodness of fit (R2) are indicated for each curve. (C) Bacteria were cultured as in panel B, and at the indicated time points samples were collected, diluted and plated on solid medium. Colonies were counted once visible to calculate concentrations of viable bacteria. Solid lines depict the growth curve obtained for each treatment and error bars represent the standard errors (n = 3). For all graphs, *, p<0.05; **, p<0.01; ***, p<0.001 versus the control condition.
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pone-0017510-g001: DFMO suppresses the growth of H. pylori.Brucella broth cultures, some containing DFMO, were inoculated with H. pylori SS1 at an OD600 of ∼0.1 and growth was monitored for 24 h. (A) Bacteria were grown in control broth or broth supplemented with 0.01%, 0.1%, or 1% (w/v) DFMO and growth was monitored by measuring OD600 at the indicated time points. Solid lines depict the growth curve obtained for each treatment and error bars represent the standard error (n = 3). (B) H. pylori were grown and monitored as described for panel A in control broth or broth with 1% DFMO. Solid lines are experimentally obtained growth curves, with standard errors (n = 9), while the dashed lines indicate the calculated exponential regression curves using the first 12 h of data. The generation time (g) and goodness of fit (R2) are indicated for each curve. (C) Bacteria were cultured as in panel B, and at the indicated time points samples were collected, diluted and plated on solid medium. Colonies were counted once visible to calculate concentrations of viable bacteria. Solid lines depict the growth curve obtained for each treatment and error bars represent the standard errors (n = 3). For all graphs, *, p<0.05; **, p<0.01; ***, p<0.001 versus the control condition.

Mentions: We recently reported that in mice infected with the mouse-adapted strain H. pylori SS1, administration of 1% DFMO suppressed both bacterial colonization and gastric inflammation [6]. Notably, this is the same dose of DFMO that has been shown to be efficacious in mouse models of colon tumorigenesis [35]. Although no sequenced H. pylori genome has been found to contain an ODC, we investigated whether DFMO had any direct effect on the bacterium. H. pylori are primarily found in the surface mucous layer of the stomach, and we began with the assumption that in our in vivo mouse model the 1% DFMO in the drinking water would reach the H. pylori bacteria at this concentration, and the DFMO is replenished continuously by ongoing intake of this agent in the drinking water. Therefore, we initiated our studies with 1% DFMO (55 mM) in liquid culture media. Flasks of broth, some supplemented with different percentages of DFMO, were inoculated with H. pylori SS1 at an OD600 of 0.1 and growth was monitored by optical density for 24 h (Figure 1A). Growth of H. pylori in broth containing 1% DFMO was restricted beginning 4 h post-inoculation, while lower concentrations had no effect. When we repeated these growth curves to quantify the inhibitory effects of the chemical, we observed that H. pylori SS1 grown in standard broth had a generation time of 4.1 h during the exponential growth phase from 0–12 h (Figure 1B). In contrast, 1% DFMO significantly increased the generation time to 7.3 h (p<0.001), an increase of 78% (Figure 1B). We also monitored growth of H. pylori by diluting and plating the bacteria at time points from 0 to 24 h. At 8 h there were 38±7% fewer viable, culturable bacteria and at 24 h there was an 86±4% decrease (Figure 1C). These findings indicated that there was a direct effect of DFMO on H. pylori that was observable as a decrease in growth rate.


Difluoromethylornithine is a novel inhibitor of Helicobacter pylori growth, CagA translocation, and interleukin-8 induction.

Barry DP, Asim M, Leiman DA, de Sablet T, Singh K, Casero RA, Chaturvedi R, Wilson KT - PLoS ONE (2011)

DFMO suppresses the growth of H. pylori.Brucella broth cultures, some containing DFMO, were inoculated with H. pylori SS1 at an OD600 of ∼0.1 and growth was monitored for 24 h. (A) Bacteria were grown in control broth or broth supplemented with 0.01%, 0.1%, or 1% (w/v) DFMO and growth was monitored by measuring OD600 at the indicated time points. Solid lines depict the growth curve obtained for each treatment and error bars represent the standard error (n = 3). (B) H. pylori were grown and monitored as described for panel A in control broth or broth with 1% DFMO. Solid lines are experimentally obtained growth curves, with standard errors (n = 9), while the dashed lines indicate the calculated exponential regression curves using the first 12 h of data. The generation time (g) and goodness of fit (R2) are indicated for each curve. (C) Bacteria were cultured as in panel B, and at the indicated time points samples were collected, diluted and plated on solid medium. Colonies were counted once visible to calculate concentrations of viable bacteria. Solid lines depict the growth curve obtained for each treatment and error bars represent the standard errors (n = 3). For all graphs, *, p<0.05; **, p<0.01; ***, p<0.001 versus the control condition.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046249&req=5

pone-0017510-g001: DFMO suppresses the growth of H. pylori.Brucella broth cultures, some containing DFMO, were inoculated with H. pylori SS1 at an OD600 of ∼0.1 and growth was monitored for 24 h. (A) Bacteria were grown in control broth or broth supplemented with 0.01%, 0.1%, or 1% (w/v) DFMO and growth was monitored by measuring OD600 at the indicated time points. Solid lines depict the growth curve obtained for each treatment and error bars represent the standard error (n = 3). (B) H. pylori were grown and monitored as described for panel A in control broth or broth with 1% DFMO. Solid lines are experimentally obtained growth curves, with standard errors (n = 9), while the dashed lines indicate the calculated exponential regression curves using the first 12 h of data. The generation time (g) and goodness of fit (R2) are indicated for each curve. (C) Bacteria were cultured as in panel B, and at the indicated time points samples were collected, diluted and plated on solid medium. Colonies were counted once visible to calculate concentrations of viable bacteria. Solid lines depict the growth curve obtained for each treatment and error bars represent the standard errors (n = 3). For all graphs, *, p<0.05; **, p<0.01; ***, p<0.001 versus the control condition.
Mentions: We recently reported that in mice infected with the mouse-adapted strain H. pylori SS1, administration of 1% DFMO suppressed both bacterial colonization and gastric inflammation [6]. Notably, this is the same dose of DFMO that has been shown to be efficacious in mouse models of colon tumorigenesis [35]. Although no sequenced H. pylori genome has been found to contain an ODC, we investigated whether DFMO had any direct effect on the bacterium. H. pylori are primarily found in the surface mucous layer of the stomach, and we began with the assumption that in our in vivo mouse model the 1% DFMO in the drinking water would reach the H. pylori bacteria at this concentration, and the DFMO is replenished continuously by ongoing intake of this agent in the drinking water. Therefore, we initiated our studies with 1% DFMO (55 mM) in liquid culture media. Flasks of broth, some supplemented with different percentages of DFMO, were inoculated with H. pylori SS1 at an OD600 of 0.1 and growth was monitored by optical density for 24 h (Figure 1A). Growth of H. pylori in broth containing 1% DFMO was restricted beginning 4 h post-inoculation, while lower concentrations had no effect. When we repeated these growth curves to quantify the inhibitory effects of the chemical, we observed that H. pylori SS1 grown in standard broth had a generation time of 4.1 h during the exponential growth phase from 0–12 h (Figure 1B). In contrast, 1% DFMO significantly increased the generation time to 7.3 h (p<0.001), an increase of 78% (Figure 1B). We also monitored growth of H. pylori by diluting and plating the bacteria at time points from 0 to 24 h. At 8 h there were 38±7% fewer viable, culturable bacteria and at 24 h there was an 86±4% decrease (Figure 1C). These findings indicated that there was a direct effect of DFMO on H. pylori that was observable as a decrease in growth rate.

Bottom Line: We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner.H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism.These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
Helicobacter pylori infects half the world's population, and carriage is lifelong without antibiotic therapy. Current regimens prescribed to prevent infection-associated diseases such as gastroduodenal ulcers and gastric cancer can be thwarted by antibiotic resistance. We reported that administration of 1% D,L-α-difluoromethylornithine (DFMO) to mice infected with H. pylori reduces gastritis and colonization, which we attributed to enhanced host immune response due to inhibition of macrophage ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Although no ODC has been identified in any H. pylori genome, we sought to determine if DFMO has direct effects on the bacterium. We found that DFMO significantly reduced the growth rate of H. pylori in a polyamine-independent manner. Two other gram-negative pathogens possessing ODC, Escherichia coli and Citrobacter rodentium, were resistant to the DFMO effect. The effect of DFMO on H. pylori required continuous exposure to the drug and was reversible when removed, with recovery of growth rate in vitro and the ability to colonize mice. H. pylori exposed to DFMO were significantly shorter in length than those untreated and they contained greater internal levels of ATP, suggesting severe effects on bacterial metabolism. DFMO inhibited expression of the H. pylori virulence factor cytotoxin associated gene A, and its translocation and phosphorylation in gastric epithelial cells, which was associated with a reduction in interleukin-8 expression. These findings suggest that DFMO has effects on H. pylori that may contribute to its effectiveness in reducing gastritis and colonization and may be a useful addition to anti-H. pylori therapies.

Show MeSH
Related in: MedlinePlus