BACKGROUND: Pre-cleaning and soaking in glutaraldehyde is the necessary procedure to disinfect endoscopes. However, some chemical-solvent-tolerant bacteria may survive after incomplete endoscopic disinfection. The goal of this study was to identify glutaraldehyde resistance-related genes in Helicobacter pylori. MATERIALS AND METHODS: Lambda-Zap phagemid expression library of H. pylori strain NTUH-C1 was selected with 0.1% glutaraldehyde. The minimal inhibitory concentration (MIC) of glutaraldehyde-resistant DNA fragments of H. pylori NTUH-C1 strain were determined. Imp/OstA recombinant protein was expressed, purified, and used to generate anti-Imp/OstA polyclonal antibody. Imp/ostA knockout, deletion, and complementation strains were constructed. The function of Imp/OstA was monitored by organic solvent tolerance assay, antibiotics susceptibility test, and N-phenylnapthylamine assay. RESULTS: Using Imp/ostA polyclonal antibody against cell lysate of wild-type and imp/ostA mutant showed that it is not essential in H. pylori. Organic solvent tolerance assay demonstrated the role of Imp/ostA in n-hexane tolerance. MIC test showed that the mutation of imp/ostA was susceptible to hydrophobic and beta-lactam antibiotics. NPN assay demonstrated that the level of outer membrane permeability was increased by 50% in mutant strain comparing to wild-type strain (p < .001). CONCLUSIONS: We have identified an Imp/OstA protein that was associated with glutaraldehyde resistance in our clinical strain H. pylori NTUH-C1 by screening of lambda-Zap expression library. Disruption of this protein results in altering membrane permeability, sensitivity to organic solvent, and susceptibility to antibiotics.
BACKGROUND: Pre-cleaning and soaking in glutaraldehyde is the necessary procedure to disinfect endoscopes. However, some chemical-solvent-tolerant bacteria may survive after incomplete endoscopic disinfection. The goal of this study was to identify glutaraldehyde resistance-related genes in Helicobacter pylori. MATERIALS AND METHODS: Lambda-Zap phagemid expression library of H. pylori strain NTUH-C1 was selected with 0.1% glutaraldehyde. The minimal inhibitory concentration (MIC) of glutaraldehyde-resistant DNA fragments of H. pylori NTUH-C1 strain were determined. Imp/OstA recombinant protein was expressed, purified, and used to generate anti-Imp/OstA polyclonal antibody. Imp/ostA knockout, deletion, and complementation strains were constructed. The function of Imp/OstA was monitored by organic solvent tolerance assay, antibiotics susceptibility test, and N-phenylnapthylamine assay. RESULTS: Using Imp/ostA polyclonal antibody against cell lysate of wild-type and imp/ostA mutant showed that it is not essential in H. pylori. Organic solvent tolerance assay demonstrated the role of Imp/ostA in n-hexane tolerance. MIC test showed that the mutation of imp/ostA was susceptible to hydrophobic and beta-lactam antibiotics. NPN assay demonstrated that the level of outer membrane permeability was increased by 50% in mutant strain comparing to wild-type strain (p < .001). CONCLUSIONS: We have identified an Imp/OstA protein that was associated with glutaraldehyde resistance in our clinical strain H. pylori NTUH-C1 by screening of lambda-Zap expression library. Disruption of this protein results in altering membrane permeability, sensitivity to organic solvent, and susceptibility to antibiotics.
Authors: Eleanor Watson; Aileen Sherry; Neil F Inglis; Alex Lainson; Dushyanth Jyothi; Raja Yaga; Erin Manson; Lisa Imrie; Paul Everest; David G E Smith Journal: EuPA Open Proteom Date: 2014-06-26