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Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori.

Chiu HC, Lin TL, Yang JC, Wang JT - BMC Microbiol. (2009)

Bottom Line: The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant.Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability.Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei City 10051, Taiwan, Republic of China. gaki98@yahoo.com.tw

ABSTRACT

Background: Contamination of endoscopy equipment by Helicobacter pylori (H. pylori) frequently occurs after endoscopic examination of H. pylori-infected patients. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process to disinfect endoscopes. However, this might not be sufficient to remove H. pylori completely, and some glutaraldehyde-resistant bacteria might survive and be passed to the next patient undergoing endoscopic examination through unidentified mechanisms. We identified an Imp/OstA protein associated with glutaraldehyde resistance in a clinical strain, NTUH-C1, from our previous study. To better understand and manage the problem of glutaraldehyde resistance, we further investigated its mechanism.

Results: The minimal inhibitory concentrations (MICs) of glutaraldehyde andexpression of imp/ostA RNA in 11 clinical isolates from the National Taiwan University Hospital were determined. After glutaraldehyde treatment, RNA expression in the strains with the MICs of 4-10 microg/ml was higher than that in strains with the MICs of 1-3 microg/ml. We examined the full-genome expression of strain NTUH-S1 after glutaraldehyde treatment using a microarray and found that 40 genes were upregulated and 31 genes were downregulated. Among the upregulated genes, imp/ostA and msbA, two putative lipopolysaccharide biogenesis genes, were selected for further characterization. The sensitivity to glutaraldehyde or hydrophobic drugs increased in both of imp/ostA and msbA single mutants. The imp/ostA and msbA double mutant was also hypersensitive to these chemicals. The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant. Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability. Ethidium bromide accumulation assay demonstrated that MsbA was involved in efflux of hydrophobic drugs.

Conclusion: The expression levels of imp/ostA and msbA were correlated with glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment. Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.

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Permeability and efflux of ethidium bromide. (A) Determination of the outer membrane permeability in H. pylori wild-type and isogenic mutants. Each measurement was repeated three times. *, P < 0.05 vs. wild-type, and **, P < 0.001 vs. wild-type. (B) Ethidium bromide accumulation assay. Cells were preloaded with 10 μg/ml ethidium bromide. At the 12-min time point, 10 μM of CCCP was added to the cells suspensions to assess energy-dependent efflux. CCCP was not added to the cells serving as controls (dotted lines). Each measurement was repeated three times.
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Figure 8: Permeability and efflux of ethidium bromide. (A) Determination of the outer membrane permeability in H. pylori wild-type and isogenic mutants. Each measurement was repeated three times. *, P < 0.05 vs. wild-type, and **, P < 0.001 vs. wild-type. (B) Ethidium bromide accumulation assay. Cells were preloaded with 10 μg/ml ethidium bromide. At the 12-min time point, 10 μM of CCCP was added to the cells suspensions to assess energy-dependent efflux. CCCP was not added to the cells serving as controls (dotted lines). Each measurement was repeated three times.

Mentions: To investigate whether the permeability of the outer membrane was altered in the mutant strains, we measured the fluorescence intensity at a 40-min time point after addition of ethidium bromide and CCCP (Fig. 8A). The fluorescence intensity of the imp/ostA deletion mutant (1142.73 ± 12.38 relative fluorescence units [RFUs]) was higher than that of the wild-type (891.29 ± 20.62 RFUs, P = 0.0001). The fluorescence intensity of the msbA deletion mutant was also significantly higher than the wild-type (P = 0.00164). These results might due to the increase of outer membrane permeability when imp/ostA or msbA was mutated. Furthermore, the fluorescence intensity of the imp/ostA and msbA double mutant was also significantly higher than that of wild-type (P = 5.83 × 10-5). Therefore, the increased sensitivity to hydrophobic compounds conferred by imp/ostA and msbA mutations can be explained by the enhanced membrane permeability for the toxic substances moving in.


Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori.

Chiu HC, Lin TL, Yang JC, Wang JT - BMC Microbiol. (2009)

Permeability and efflux of ethidium bromide. (A) Determination of the outer membrane permeability in H. pylori wild-type and isogenic mutants. Each measurement was repeated three times. *, P < 0.05 vs. wild-type, and **, P < 0.001 vs. wild-type. (B) Ethidium bromide accumulation assay. Cells were preloaded with 10 μg/ml ethidium bromide. At the 12-min time point, 10 μM of CCCP was added to the cells suspensions to assess energy-dependent efflux. CCCP was not added to the cells serving as controls (dotted lines). Each measurement was repeated three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719649&req=5

Figure 8: Permeability and efflux of ethidium bromide. (A) Determination of the outer membrane permeability in H. pylori wild-type and isogenic mutants. Each measurement was repeated three times. *, P < 0.05 vs. wild-type, and **, P < 0.001 vs. wild-type. (B) Ethidium bromide accumulation assay. Cells were preloaded with 10 μg/ml ethidium bromide. At the 12-min time point, 10 μM of CCCP was added to the cells suspensions to assess energy-dependent efflux. CCCP was not added to the cells serving as controls (dotted lines). Each measurement was repeated three times.
Mentions: To investigate whether the permeability of the outer membrane was altered in the mutant strains, we measured the fluorescence intensity at a 40-min time point after addition of ethidium bromide and CCCP (Fig. 8A). The fluorescence intensity of the imp/ostA deletion mutant (1142.73 ± 12.38 relative fluorescence units [RFUs]) was higher than that of the wild-type (891.29 ± 20.62 RFUs, P = 0.0001). The fluorescence intensity of the msbA deletion mutant was also significantly higher than the wild-type (P = 0.00164). These results might due to the increase of outer membrane permeability when imp/ostA or msbA was mutated. Furthermore, the fluorescence intensity of the imp/ostA and msbA double mutant was also significantly higher than that of wild-type (P = 5.83 × 10-5). Therefore, the increased sensitivity to hydrophobic compounds conferred by imp/ostA and msbA mutations can be explained by the enhanced membrane permeability for the toxic substances moving in.

Bottom Line: The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant.Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability.Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei City 10051, Taiwan, Republic of China. gaki98@yahoo.com.tw

ABSTRACT

Background: Contamination of endoscopy equipment by Helicobacter pylori (H. pylori) frequently occurs after endoscopic examination of H. pylori-infected patients. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process to disinfect endoscopes. However, this might not be sufficient to remove H. pylori completely, and some glutaraldehyde-resistant bacteria might survive and be passed to the next patient undergoing endoscopic examination through unidentified mechanisms. We identified an Imp/OstA protein associated with glutaraldehyde resistance in a clinical strain, NTUH-C1, from our previous study. To better understand and manage the problem of glutaraldehyde resistance, we further investigated its mechanism.

Results: The minimal inhibitory concentrations (MICs) of glutaraldehyde andexpression of imp/ostA RNA in 11 clinical isolates from the National Taiwan University Hospital were determined. After glutaraldehyde treatment, RNA expression in the strains with the MICs of 4-10 microg/ml was higher than that in strains with the MICs of 1-3 microg/ml. We examined the full-genome expression of strain NTUH-S1 after glutaraldehyde treatment using a microarray and found that 40 genes were upregulated and 31 genes were downregulated. Among the upregulated genes, imp/ostA and msbA, two putative lipopolysaccharide biogenesis genes, were selected for further characterization. The sensitivity to glutaraldehyde or hydrophobic drugs increased in both of imp/ostA and msbA single mutants. The imp/ostA and msbA double mutant was also hypersensitive to these chemicals. The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant. Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability. Ethidium bromide accumulation assay demonstrated that MsbA was involved in efflux of hydrophobic drugs.

Conclusion: The expression levels of imp/ostA and msbA were correlated with glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment. Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.

Show MeSH
Related in: MedlinePlus