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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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The RNA and protein expression levels of imp/ostA in clinical isolates after glutaraldehyde treatment. (A) Quantitative real-time PCR analysis of the relative expression of imp/ostA mRNA after glutaraldehyde treatment in 11 clinical isolates. The MICs of the corresponding strains are shown in the lower portion of the figure. Each bar represents the relative expression after glutaraldehyde treatment. (B) Western blot analysis of Imp/OstA protein expression. (+) represents glutaraldehyde treatment; (-) represents no glutaraldehyde treatment. (C) The ratio of Imp/OstA protein expression with and without glutaraldehyde treatment. The results were from three independent experiments.
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Figure 2: The RNA and protein expression levels of imp/ostA in clinical isolates after glutaraldehyde treatment. (A) Quantitative real-time PCR analysis of the relative expression of imp/ostA mRNA after glutaraldehyde treatment in 11 clinical isolates. The MICs of the corresponding strains are shown in the lower portion of the figure. Each bar represents the relative expression after glutaraldehyde treatment. (B) Western blot analysis of Imp/OstA protein expression. (+) represents glutaraldehyde treatment; (-) represents no glutaraldehyde treatment. (C) The ratio of Imp/OstA protein expression with and without glutaraldehyde treatment. The results were from three independent experiments.

Mentions: In our previous study [14], we found that the barrier function of the outer membrane against drugs might be decreased in the imp/ostA mutant strain, suggesting that glutaraldehyde may enter the mutant strain more rapidly than it enters the wild-type strain. Four strains with the MICs of 7–10 μg/ml (designed numbers 1~4), four with the MICs of 4–6 μg/ml (numbers 5~8), and three with the MICs of 1–3 μg/ml (numbers 9~11) were selected to clarify the correlation of imp/ostA expression with glutaraldehyde resistance. Subsequently, RNA was extracted from bacteria after 48 h with or without 0.5 μg/ml glutaraldehyde treatment. However, RNA expression of imp/ostA in strains without glutaraldehyde treatment was not detected by slot blot (data not shown). Therefore, we further examined RNA expression of imp/ostA by quantitative real-time PCR. The result indicated that RNA expression of imp/ostA induced by glutaraldehyde was higher in strains with the MICs of 4–10 μg/ml than that in strains with the MICs of 1–3 μg/ml (P= 0.001455) (Fig. 2A). Expression of Imp/OstA protein in these 11 strains after glutaraldehyde treatment was also examined (Fig. 2B). The intensity of protein expression in three independent experiments was analyzed by Image Quant 5.1, and the ratio of Imp/OstA protein expression in the 11 strains with and without glutaraldehyde treatment was calculated. The ratio of Imp/OstA expression induced by glutaraldehyde was higher for strains with the MICs of 4–10 μg/ml (numbers 1~8) than strains with the MICs of 1–3 μg/ml (numbers 9~11) (P = 6.1 × 10-5) (Fig. 2C). These results suggested that the expression of imp/ostA and Imp/OstA was involved in glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment.


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)

The RNA and protein expression levels of imp/ostA in clinical isolates after glutaraldehyde treatment. (A) Quantitative real-time PCR analysis of the relative expression of imp/ostA mRNA after glutaraldehyde treatment in 11 clinical isolates. The MICs of the corresponding strains are shown in the lower portion of the figure. Each bar represents the relative expression after glutaraldehyde treatment. (B) Western blot analysis of Imp/OstA protein expression. (+) represents glutaraldehyde treatment; (-) represents no glutaraldehyde treatment. (C) The ratio of Imp/OstA protein expression with and without glutaraldehyde treatment. The results were from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The RNA and protein expression levels of imp/ostA in clinical isolates after glutaraldehyde treatment. (A) Quantitative real-time PCR analysis of the relative expression of imp/ostA mRNA after glutaraldehyde treatment in 11 clinical isolates. The MICs of the corresponding strains are shown in the lower portion of the figure. Each bar represents the relative expression after glutaraldehyde treatment. (B) Western blot analysis of Imp/OstA protein expression. (+) represents glutaraldehyde treatment; (-) represents no glutaraldehyde treatment. (C) The ratio of Imp/OstA protein expression with and without glutaraldehyde treatment. The results were from three independent experiments.
Mentions: In our previous study [14], we found that the barrier function of the outer membrane against drugs might be decreased in the imp/ostA mutant strain, suggesting that glutaraldehyde may enter the mutant strain more rapidly than it enters the wild-type strain. Four strains with the MICs of 7–10 μg/ml (designed numbers 1~4), four with the MICs of 4–6 μg/ml (numbers 5~8), and three with the MICs of 1–3 μg/ml (numbers 9~11) were selected to clarify the correlation of imp/ostA expression with glutaraldehyde resistance. Subsequently, RNA was extracted from bacteria after 48 h with or without 0.5 μg/ml glutaraldehyde treatment. However, RNA expression of imp/ostA in strains without glutaraldehyde treatment was not detected by slot blot (data not shown). Therefore, we further examined RNA expression of imp/ostA by quantitative real-time PCR. The result indicated that RNA expression of imp/ostA induced by glutaraldehyde was higher in strains with the MICs of 4–10 μg/ml than that in strains with the MICs of 1–3 μg/ml (P= 0.001455) (Fig. 2A). Expression of Imp/OstA protein in these 11 strains after glutaraldehyde treatment was also examined (Fig. 2B). The intensity of protein expression in three independent experiments was analyzed by Image Quant 5.1, and the ratio of Imp/OstA protein expression in the 11 strains with and without glutaraldehyde treatment was calculated. The ratio of Imp/OstA expression induced by glutaraldehyde was higher for strains with the MICs of 4–10 μg/ml (numbers 1~8) than strains with the MICs of 1–3 μg/ml (numbers 9~11) (P = 6.1 × 10-5) (Fig. 2C). These results suggested that the expression of imp/ostA and Imp/OstA was involved in glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment.

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