Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori
© Chiu et al; licensee BioMed Central Ltd. 2009
Received: 17 February 2009
Accepted: 13 July 2009
Published: 13 July 2009
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.
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 μg/ml was higher than that in strains with the MICs of 1–3 μg/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.
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.
Helicobacter pylori was first isolated from the gastric mucosa of a patient with gastritis and peptic ulceration by Marshall and Warren in 1982 . It is an important human pathogen, responsible for type B gastritis and peptic ulcers. Furthermore, infection by H. pylori is a risk factor for gastric adenocarcinoma and for lymphoma in the mucosa-associated lymphoid tissue of the stomach in humans [2–5].
H. pylori is believed to be transmitted from person to person by oral-oral or oral-fecal routes . However, another possible route involves transmission during endoscopic examination of patients because contamination of endoscopy equipment by H. pylori frequently occurs after endoscopic examination of H. pylori-infected patients [7–9]. Because H. pylori is prevalent in the population , it is important to prevent its transmission. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process used to disinfect endoscopes [7, 11]. However, endoscopic disinfection might not be sufficient to remove H. pylori completely [12, 13]. Some glutaraldehyde-resistant bacteria might survive and be passed to the next person undergoing endoscopic examination through unidentified mechanisms. Therefore, it is an important issue to clarify the mechanism of glutaraldehyde resistance.
In our previous study, we demonstrated that the Imp/OstA protein was associated with glutaraldehyde resistance in a clinical strain of H. pylori . OstA (organic solvent tolerance)  has also been called imp (increased membrane permeability) , and was recently named lptD in Escherichia coli . Imp/OstA exists widely in Gram-negative bacteria and participates in biogenesis of the cell envelope. It is an essential outer membrane protein in E. coli, depletion mutation of imp/ostA results in the formation of aberrant membranes . Furthermore, Imp/OstA forms a complex with the RlpB lipoprotein and is responsible for lipopolysaccharide (LPS) assembly at the surface of the cell [17, 19]. In addition, it mediates the transport of LPS to the surface in Neisseria meningitidis .
To further investigate the mechanism of glutaraldehyde resistance, we monitored the minimum inhibitory concentrations (MICs) and the expression of imp/ostA and Imp/OstA protein after glutaraldehyde treatment in 11 clinical isolates. Full-genome expression was also studied by microarray analysis; 40 genes were upregulated and 31 genes were downregulated in NTUH-S1 after glutaraldehyde treatment. Among the upregulated genes, msbA, was selected for further study. MsbA is an essential inner membrane protein in E. coli and a member of the ABC transporter superfamily of proteins . MsbA produced in the Gram-positive organism Lactococcus lactis is capable of conferring drug resistance to the organism . In addition, msbA is not essential in N. meningitidis and this organism can survive without LPS . In E. coli, msbA was implicated in lipid A-core moiety flipping from the inner leaflet to outer leaflet of the inner membrane [24, 25], and then Imp/RlpB protein complex was responsible for transport of LPS from the periplasm to the outer leaflet of the outer membrane . Here we showed that imp/ostA and msbA might be synergistic in hydrophobic drugs resistance and LPS transport in H. pylori.
Glutaraldehyde was purchased from Electron Microscopy Sciences (Hatfield, PA). Chloramphenicol, erythromycin, kanamycin, novobiocin, rifampicin, ethidium bromide, and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were purchased from Sigma Chemical Co (St Louis, MO).
Bacterial strains and culture conditions
Clinical isolates were collected from National Taiwan University Hospital (NTUH) as previously described . H. pylori strains were grown on Columbia agar plates containing 5% sheep blood under microaerophilic conditions (5% O2, 10% CO2, and 85% N2) at 37°C. For microarray analysis, we selected a rapidly growing strain NTUH-S1 with a higher MIC (MIC = 6 μg/ml) to glutaraldehyde from a patient with gastritis to study gene expression. To screen for mutant strains, blood agar plates were supplemented with 4 μg/ml chloramphenicol or 10 μg/ml kanamycin. To screen for imp/ostA and msbA double deletion mutant or complementation strains, blood agar plates were supplemented with 4 μg/ml chloramphenicol and 10 μg/ml kanamycin.
Determination the MICs of glutaraldehyde and hydrophobic drugs in H. pylori
The MICs of glutaraldehyde and hydrophobic drugs (erythromycin, novobiocin, rifampicin, and ethidium bromide) were determined by the agar dilution method. Suspension of H. pylori was adjusted to 107 cells/ml. Five microliters of bacterial suspensions were spotted on blood agar plates supplemented with different concentrations of drugs. Results were observed after 72 h incubation under microaerophilic condition at 37°C.
RNA slot blot hybridization
Four strains with the MICs of 7–10 μg/ml glutaraldehyde (designed numbers 1~4), four with the MICs of 4–6 μg/ml glutaraldehyde (numbers 5~8), and three with the MICs of 1–3 μg/ml glutaraldehyde (numbers 9~11) were grown on Columbia blood agar plates for 48 h, and further passaged on Columbia blood agar plates or 0.5 μg/ml glutaraldehyde-containing blood agar plates for 48 h. Since 0.5 μg/ml was the half concentration of the minimum MIC for the 11 strains, we defined this as the induction concentration. Subsequently, RNA was extracted from the bacteria with or without glutaraldehyde treatment. Total RNA from each H. pylori clinical isolate was extracted as described previously . Ten micrograms of total RNA was transferred onto a nylon membrane using a slot-blot system (Hoefer, Holliston, MA). The membrane was hybridized with DNA probes specific for 23S rRNA (0.9 kb PCR-amplified 23S rRNA-specific fragment using the forward primer: 5'-ATTGGAGGGAAGGCAAATCC-3' and the reverse primer: 5'-ATCACTATGACCGACTTTCG-3'), imp/ostA (0.8 kb PCR-amplified imp/ostA-specific fragment using the forward primer: 5'-CATTGATAACCCCATTTGGC-3' and the reverse primer: 5'-GCACATTCAAAGCGTTTTGC-3'), and msbA (0.8 kb PCR-amplified msbA-specific fragment using the forward primer: 5'-TAGCGTTAGTGGGGTTAGTC-3' and the reverse primer: 5'-ACACCCTTTGAGTGACAACG-3') labeled with DIG by PCR. Detection was performed with the DIG Luminescent Detection kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions.
RNA isolation and quantitative real-time PCR
It takes 48 to 72 h to recover colonies when H. pylori were grown on blood agar plates. A previous report also detected consistent RNA expression levels changes of H. pylori after 48 h of growth on acidified blood agar plates . H. pylori NTUH-S1 was grown on Columbia blood agar plates for 48 h, and further passaged on Columbia blood agar plates or 0.5 μg/ml glutaraldehyde-containing blood agar plates for 48 h. RNA was extracted by the QIAGEN RNeasy column purification kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Total RNA was quantified with a spectrophotometer and visualized on an ethidium bromide stained agarose gel. Total RNA served as a template for cDNA synthesis using the SuperScript II Reverse transcriptase (Invitrogen, Carlsbad, CA). Synthesis reactions were started with 1.5 μg total RNA per 20 μl reaction mixture. All reactions were normalized to the level of the 16S rRNA gene . In real-time RT-PCR, amplification and detection of the cDNAs were monitored using the KAPA SYBR FAST qPCR kit (Kapabiosystems, Boston, MA) in an ABI 7900 thermocycler (Applied Biosystems, Carlsbad, CA). Gene-specific primers imp/ostA RT (F): 5'-TTTGTCTTTAGGGCTTTGGAATG-3', imp/ostA RT (R): 5'-GCACGAAGGAATTTTTAGATTGC-3' and 16S rRNA RT (F):5'-TGCGAAGTGGAGCCAATCTT-3', 16S rRNA RT (R): 5'-GGAACGTATTCACCGCAACA-3' were used for amplification of cDNAs in this experiment. For the imp/ostA gene, the calculated threshold cycle (Ct) was normalized to the Ct of the 16S rRNA gene from the same cDNA sample before the fold change was calculated using the ΔΔCt method as described previously .
Western blots analysis of cell extracts
Eleven strains (numbers 1~11, the same isolates as previously described in RNA slot blot hybridization experiments) were selected and grown on Columbia blood agar plates for 48 h, and further passaged on Columbia blood agar plates or 0.5 μg/ml glutaraldehyde-containing blood agar plates for 48 h. Bacteria were harvested by centrifugation. Cells were washed in phosphate-buffered saline (PBS), resuspended in lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 0.1% SDS, 10% glycerol), and lysed by sonication. Total protein concentration was determined by using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Samples were loaded at identical protein concentrations from total cell extracts for Western blot analysis. SDS-PAGE was transferred to nitrocellulose for immunological detection. Membrane was blocked with 5% skimmed milk in TBS overnight at 4°C. Subsequently, membrane was incubated with anti-OstA polyclonal antibody  diluted 1:500 with 5% skimmed milk in TTBS (0.5% Tween-20) for 1 h at room temperature. Horseradish peroxidase-conjugated anti-rat IgG diluted 1:3000 with 5% skimmed milk in TTBS (0.5% Tween-20) was added and membrane was incubated for 1 h at room temperature. The membrane was washed three times with TTBS (0.5% Tween-20) between the incubation steps. Electrochemiluminescence (Amersham Biosciences, Fairfield, CT) was used for detection.
RNA isolation and microarray analysis of H. pyloriNTUH-S1
H. pylori NTUH-S1 was grown on Columbia blood agar plates for 48 h and further passaged on Columbia blood agar plates or 3 μg/ml glutaraldehyde-containing blood agar plates for 48 h. RNA was extracted using the QIAGEN RNeasy column purification kit (Qiagen) according to the manufacturer's instructions.
cDNA was synthesized according to the SuperScript™ indirect cDNA Labeling System (Invitrogen). cDNA was then purified using the S.N.A.P column purification (Invitrogen) according to the manufacturer's instructions.
Aminoallyl dUTP-labeled cDNA was resuspended in 2 × coupling buffer and labeled with either Alexa Fluor 555 or 647 according to the manufacturer's protocol (Molecular Probes, Eugene, OR). Labeled cDNA was mixed together and purified by S.N.A.P column purification. Then, the labeled cDNA was concentrated with a Microcon YM-30 column (Millipore, Billerica, MA).
The Institute for Genomic Research (TIGR) provided a H. pylori whole-genome microarray. It consisted of 2,572 70-mer oligonucleotides, printed in quadruplicate and representing open reading frames from H. pylori 26695 and strain J99. Labeled cDNA was resuspended in filtered hybridization buffer (50% formamide, 5 × SSC, 0.1% sodium dodecyl sulfate, 0.1 M DTT, and 0.6 μg/ml salmon sperm DNA), denatured at 95°C for 5 min, and flicked for an additional minute. It was then denatured for another 5 min. The labeled probe was applied to the pre-hybridized microarray and placed in a hybridization chamber at 42°C for 16~20 h. Microarray scanning and analysis were performed on a scanner (GenePix 4000B with GenePix Pro 5.0 software; Axon, Foster City, CA).
Processed microarray data files have been deposited in the Center for Information Biology Gene Expression Database (CIBEX; http://cibex.nig.ac.jp) under accession number CBX86.
Construction of imp/ostA and msbAdeletion mutants
The gene encoding Imp/OstA with the upstream and downstream 500 bp flanking region was amplified with the genomic DNA of wild-type NTUH-S1 by PCR. The forward primer was 5'-ATGCACTCTCCAAATTTAGA-3', and the reverse primer was 5'-GGGGCTAGGATAGGTTCTAA-3'. It was then cloned into a pGEM-T easy vector (Promega, Madison, WI).
The gene encoding MsbA with the upstream 458-bp and downstream 474-bp flanking regions was amplified with the genomic DNA of wild-type NTUH-S1. PCR was performed using the forward primer, 5'-ACGACAGGAAACCCTTTAGG-3' and the reverse primer was 5'-AGCGTAATAAACAGGCACGC-3'. It was also cloned into a pGEM-T easy vector (Promega).
The imp/ostA and msbA genes were deleted by inverse PCR, and a chloramphenicol-resistant cassette (a gift from Dr. D. E. Taylor, University of Alberta) with its coding region (from the 1-bp start codon to the 624-bp stop codon) was then cloned into the flanking regions to replace the full-length imp/ostA or msbA gene. This plasmid was natural transformed into the wild-type NTUH-S1 strain to generate deletion mutants. Chromosomal DNA of the transformants was checked by PCR with primers external and internal to the replacement site to verify the double-crossover event.
Complementation of imp/ostA and msbA
An imp/ostA complementation strain of NTUH-S1 was constructed as described previously . The promoter site of msbA gene was predicted by using a tool available at the following website: http://www.fruitfly.org/seq_tools/promoter.html. The msbA gene containing the predicted promoter region (upstream 73 bp) was obtained by PCR using the forward primer: 5'-CCAATCGCTTTAAGCTG-3', and the reverse primer: 5'-TTAGCATTCTGTCAAACGCC-3'. Then the DNA fragment was cloned into the pGEM-T easy vector (Promega). The msbA gene with its promoter region was cut from the constructed pGEM-T easy vector and ligated into the NruI site of the shuttle vector pHel3 (plasmid pHel3 was a gift from Dr. R. Haas, Max-Planck-Institute für Biologie, Tübingen, Germany). The constructed shuttle vector was natural transformed into an msbA deletion mutant strain to generate the msbA complementation strain.
Construction of the imp/ostA and msbAdouble deletion mutant
The gene encoding MsbA with its upstream 458-bp and downstream 474-bp flanking region was cloned into the pGEM-T easy vector as described above. A kanamycin-resistant gene aphA-3 from Campylobacter jejuni was then cloned between the flanking regions to replace the full length msbA gene. This plasmid was natural transformed into the wild-type NTUH-S1 strain to generate the deletion mutant. Chromosomal DNA of the transformants was checked by PCR with primers external and internal to the replacement site to verify the double-crossover event. Then, chromosomal DNA from msbA deletion mutant strain (Kmr) was natural transformed into the imp/ostA deletion mutant to obtain a double deletion mutant strain. It was also confirmed by PCR with primers external and internal to the msbA gene replacement site.
Approximately 5 μg of genomic DNA from H. pylori NTUH-S1 and the mutants was digested by Hind III and incubated at 37°C overnight for complete digestion. The digoxigenin-labeled imp/ostA and msbA probes (primers were the same as those described for slot blot) was generated by PCR. Hybridization and detection were performed with the DIG Luminescent Detection kit (Roche) according to the manufacturer's instructions.
Proteinase-K digested H. pylori
The procedure was followed as described previously . H. pylori cells were collected and adjusted to a concentration of 2.5 × 109 cells/ml in PBS. Bacteria were boiled with 150 μl sample dye for 10 min at 100°C to disrupt the whole cells. Subsequently, the whole cell lysates were treated with proteinase K (Sigma) for 60 min at 60°C in a water bath. Then, 2.5 × 108 cells/ml were analyzed by 12% SDS-PAGE and stained with silver. The protein concentration of the 2.5 × 108 cells/ml was also determined by using the Bio-Rad protein assay (Bio-Rad) to serve as a loading control.
Immunoblots of LPS from H. pyloriwith anti-Lewis (Le) monoclonal antibody
H. pylori strains that have been screened serologically [31–33], and a previous study suggested that Asian isolates express predominantly type 1 (Lea, Leb) antigens compared to Western strains (predominantly expressing type 2 Lex and Ley determinants) . We also primarily detected the Lewis antigen of NTUH-S1 with anti-Lea and anti-Leb antibody. Equivalent amounts of protein were loaded in each well and transferred to nitrocellulose for immunological detection with anti-Lea or anti-Leb monoclonal antibody (Seikagaku Corporation, Tokyo). For detection of Lewis antigen in proteinase K-digested whole cell lysates, nitrocellulose membrane was blocked with 5% skimmed milk in PBS for 1 h at room temperature. Subsequently, membrane was incubated with anti-Lea or anti-Leb antibody diluted 1:3000 with 5% skimmed milk in PBS overnight at 4°C. Horseradish peroxidase-conjugated anti-mouse IgG diluted 1:5000 with 5% skimmed milk in PBS was added and membrane was incubated for 1 h at room temperature. The membrane was washed three times with PBST (0.05% Tween-20) between the incubation steps. Electrochemiluminescence (Amersham Biosciences) was used for detection. Whole cells of the Lex and Ley antigen-expressing H. pylori 26695 strain  were used as a negative control in Western blots to ensure the specificity of the anti-Lea or anti-Leb antibody.
Measurement of outer membrane permeability by ethidium bromide
Outer membrane permeability can be measured by the fluorescence of the ethidium-polynucleotide complex in the cell because ethidium bromide displays approximately a 10-fold increase in fluorescence quantum yield upon binding to DNA . The assay was modified as described previously . Briefly,H. pylori were grown on Columbia blood agar plate for 48 h. Then, bacteria were pelleted and washed twice with ice-cold 50 mM potassium phosphate (pH 7.0) containing 5 mM MgSO4. Cells were resuspended in 1 ml of potassium phosphate buffer (pH 7.0) at an optical density (OD600) of 0.5 and incubated for 30 min at 37°C in the presence of 10 μM of CCCP to deplete cells of metabolic energy. Subsequently, cells were washed three times in ice-cold potassium phosphate (pH 7.0) containing 5 mM MgSO4 and loaded with 10 μg/ml ethidium bromide. At the 40-min time point, the increase in ethidium bromide fluorescence intensity was measured in Gemini XPS spectrofluorimeter (Molecular Devices, Sunnyvale, CA) at 30°C with the excitation wavelength set at 500 nm and the emission wavelength at 580 nm. Each measurement was repeated three times.
Ethidium bromide accumulation assay
The assay was modified as described previously . Briefly,H. pylori were grown on Columbia blood agar plate for 48 h. Then, bacteria were pelleted and washed twice with ice-cold 50 mM potassium phosphate (pH 7.0) containing 5 mM MgSO4. Cells were resuspended in 1 ml of potassium phosphate buffer (pH 7.0) at an optical density (OD600) of 0.5. 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 served as a control. The increase in ethidium bromide fluorescence intensity was measured in a Gemini XPS spectrofluorimeter at 30°C with excitation at 500 nm and emission at 580 nm. Each measurement was repeated three times.
For all experiments, a P value of < 0.05 was considered indicative of statistical significance, and all statistical analyses were determined with Student's t test.
The MICs for glutaraldehyde in clinical isolates
The MICs of glutaraldehyde in clinical isolates during 1991–2000.
Number of isolates
The MICs of glutaraldehyde in isolates (numbers)
7 μg/ml (n = 2), 6 μg/ml (n = 1)
5 μg/ml (n = 3), 4 μg/ml (n = 4)
3 μg/ml (n = 5)
10 μg/ml (n = 1), 7 μg/ml (n = 1)
6 μg/ml (n = 2), 5 μg/ml (n = 3)
4 μg/ml (n = 5), 2 μg/ml (n = 2)
1.5 μg/ml (n = 1), 1 μg/ml (n = 1)
10 μg/ml (n = 1), 7 μg/ml (n = 1)
6 μg/ml (n = 3), 5 μg/ml (n = 1)
3 μg/ml (n = 1), 1.5 μg/ml (n = 1)
1 μg/ml (n = 1)
7 μg/ml (n = 1), 5 μg/ml (n = 2)
6 μg/ml (n = 3), 5 μg/ml (n = 1)
3 μg/ml (n = 2)
The RNA and protein expression levels of imp/ostAin clinical isolates after glutaraldehyde treatment
Full genome expression after glutaraldehyde treatment
We next examined the alterations in RNA expression in H. pylori NTUH-S1 induced by glutaraldehyde. After treatment with glutaraldehyde for 48 h, 40 genes were upregulated at least 2.5-fold, and 31 genes were downregulated at least 2.5-fold (see Additional File 1), compared to the untreated bacteria. The upregulated genes included imp/ostA, which was upregulated 9.218-fold. These results are in agreement with the quantitative real-time PCR data, showing that this gene was notably expressed after glutaraldehyde treatment. Another interesting finding was that glutaraldehyde upregulated another lipid transport gene msbA (HP1082) by 2.661-fold. Previous reports indicated that this subfamily of ABC transporters is involved in transport of many different substrates, such as peptides, lipids, hydrophobic drugs, polysaccharides, and proteins . MsbA is a lipid flippase that transports the lipid A-core moiety from the inner to the outer leaflet of the inner membrane in E. coli [17, 41]. Imp/OstA also participates in transport of LPS to the cell surface in E. coli  and N. meningitidis . We proposed that MsbA might be correlated with LPS transport in H. pylori. The deficiency in a LPS biosynthesis gene could result in antibiotic susceptibility, especially for hydrophobic antibiotics [42–44]. Therefore, weregarded msbA as a suitable candidate for investigating glutaraldehyde or other hydrophobic drug transport in bacteria.
Reconfirmation of msbAexpression in the clinical isolates by slot blots hybridization
Effect of imp/ostAon the transcription of msbA after glutaraldehyde treatment
The MICs of glutaraldehyde in isogenic mutants
The MICs of hydrophobic antibiotics in isogenic mutants
Previous reports demonstrated that in Gram-negative bacteria, a deficiency of the LPS biosynthesis gene would result in antibiotic susceptibility, especially for hydrophobic antibiotics [42–44]. Therefore, we determined the MICs of two hydrophobic antibiotics, novobiocin and rifampicin, to investigate whether imp/ostA and msbA participated in resistance to hydrophobic antibiotics. Both imp/ostA and msbA single mutants were more sensitive to novobiocin and rifampicin than wild-type (Fig. 6C and 6D). These results indicated that imp/ostA and msbA are important for H. pylori resistence to hydrophobic antibiotics. The MIC of rifampicin for the imp/ostA and msbA double mutant (0.00037 ± 0.00013 μg/ml) decreased significantly.
In order to determine the transport route of hydrophobic drugs in bacteria, the hydrophobic compound ethidium bromide was used. In this way, the MIC of ethidium bromide for H. pylori was also examined. The result showed that the msbA mutant was more susceptible to ethidium bromide than the wild-type strain. This result suggests that MsbA might be involved in active efflux by H. pylori, as evidenced by an approximately 36-fold reduction in the MIC of the msbA mutant compared to the wild-type strain (Fig. 6E).
LPS production in H. pyloriwild-type and isogenic mutants
Outer membrane permeability to ethidium bromide
The role of msbAin ethidium bromide efflux
As ethidium bromide is a hydrophobic aromatic compound, we used this compound to mimic glutaraldehyde or hydrophobic antibiotics moving in and efflux. The Ethidium bromide accumulation assay was performed to determine whether the msbA deletion mutant was more susceptible to glutaraldehyde or hydrophobic antibiotics due to the loss of an active efflux mechanism. The result showed that the msbA deletion mutant accumulated more amounts of ethidium bromide than wild-type (Fig. 8B). When CCCP was added to the cells containing ethidium bromide at 12 min, the accumulation of ethidium bromide increased in wild-type and reached to the level almost equal to that of msbA deletion mutant. This indicated that ethidium bromide was retained in the cells when efflux was blocked after the collapse of the cells' metabolic energy by CCCP. In contrast, CCCP had no significant effect on the level of ethidium bromide accumulated in the msbA deletion mutant. In addition, ethidium bromide accumulation in the msbA complementation strain reached a level almost equal to that of wild-type. CCCP was not added to wild-type or complementation strain containing ethidium bromide at 12 min served as a control. These data indicated that MsbA was involved in hydrophobic drug efflux and that it pumped out ethidium bromide in an energy-dependent process. We concluded that MsbA might pump out glutaraldehyde or hydrophobic antibiotics through an active efflux mechanism in H. pylori.
We previously identified that imp/ostA was associated with glutaraldehyde resistance in a clinical H. pylori strain . In order to further investigate the mechanism of glutaraldehyde resistance, the MICs and the levels of imp/ostA expression in clinical isolates were monitored. The result indicated that RNA and protein 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. According to these results, we suggested that imp/ostA expression was correlated with glutaraldehyde resistance in H. pylori clinical isolates.
After treating NTUH-S1 with glutaraldehyde, 40 genes were found to be upregulated at least 2.5-fold by microarray analysis. For 14 of these genes, DNA or protein sequence alignment yielded no information about their function. The other genes could be divided into three groups: transporters, biosynthesis and metabolism genes, and motility and chemotaxis genes. Two genes were related to iron transport; nonheme iron-containing ferritin (HP0653, pfr), which participates in iron metabolism and in gastric colonization by H. pylori ; and an iron dicitrate ABC transporter (HP0889, fecD). Genes including aimF, bioC, ispB, NADH-flavin oxidoreductase (HP0642), and cytochrome c551 peroxidase (HP1461) were involved in biosynthesis and metabolism. Lastly, flagellar hook-associated protein 1 (HP1119, flgK)  and flagellar hook-associated protein 2 (HP0752, fliD) [49, 50] were related to motility and chemotaxis. However, these genes might not be directly involved in resistance to glutaraldehyde, and their association with glutaraldehyde resistance needs further investigation. In addition, 31 genes were downregulated at least 2.5-fold after glutaraldehyde treatment. Several adjacent genes seemed to be co-regulated, which is indicative of operon structures. For example, HP0690-HP0693  participated in fatty acid metabolism in the TCA cycle. HP0695-HP0696  participated in hydantoin utilization. In addition, some genes are transcribed at different loci but are involved in outer-membrane composition, which included hopG, hofH, and homA. Lastly, two subunits of the 2-oxoglutarate oxidoreductase, oorB and oorD , are also involved in the TCA cycle for energy metabolism. The correlation between TCA cycle-related genes and glutaraldehyde resistance also needs to be investigated further.
Silver staining revealed that both imp/ostA and msbA participated in the biogenesis of LPS in H. pylori. Similarly mutation of the E. coli LPS biosynthesis gene, lpxA2, resulted in extreme susceptibility to antibiotics, especially hydrophobic antibiotics [42–44]. Therefore, mutation of the LPS biosynthesis genes, imp/ostA and msbA, might account for the reduction of the MICs for hydrophobic antibiotics.
In the beginning, we observed that the MICs of two glutaraldehyde-resistant strains were 10 μg/ml glutaraldehyde. In fact, this is the half concentration used in our hospital for disinfection during endoscopy. We proposed that some bacteria could survive at the low concentrations in the glutaraldehyde-treated endoscopic environment. According to the MICs tests, LPS analysis, outer membrane permeability assay, and ethidium bromide accumulation assay, the increased sensitivity to hydrophobic compounds conferred by mutations of imp/ostA and msbA can be explained by the defect in LPS production and increased outer membrane permeability. In addition, the increased sensitivity to hydrophobic compounds conferred by mutation of msbA might to the result of accumulation of chemicals that are not pumped out by the MsbA efflux pump. The combination of these effects of the imp/ostA and msbA would reduce the MICs of cells toward glutaraldehyde and hydrophobic antibiotics. These findings might help us to understand the mechanism of bacterial tolerance to chemical disinfectant and hydrophobic drugs.
The expression levels of imp/ostA and msbA were correlated with glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment. Imp/OstA and MsbA play an important role in hydrophobic drugs resistance and LPS biogenesis in H. pylori.
This work was supported by grants from the National Science Council, Taiwan.
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