Identification and characterization of an organic solvent tolerance gene in Helicobacter pylori.
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.


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.
Chiu
Hung-Chuan
HC
Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan.
Lin
Tzu-Lung
TL
Wang
Jin-Town
JT
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D004202:Disinfectants / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics; D004724:Endoscopy / Q000009:adverse effects; D017353:Gene Deletion; D005798:Genes, Bacterial / Q000502:physiology; D005976:Glutaral / Q000494:pharmacology*; D016481:Helicobacter Infections / Q000209:etiology; D016480:Helicobacter pylori / Q000187:drug effects / Q000235:genetics*; D006586:Hexanes / Q000494:pharmacology; D007291:Inosine Monophosphate; D008826:Microbial Sensitivity Tests
D001425:Bacterial Outer Membrane Proteins; D004202:Disinfectants; D006586:Hexanes; D007291:Inosine Monophosphate; C026385:n-hexane; D005976:Glutaral