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Xiao-Ping Zhang, Wei-Hong Wang, Yu Tian, Wen Gao, Jiang Li, Department of Gastroenterology, Peking University First Hospital, Beijing 100034, China Author contributions: Zhang XP performed most parts of the experiment and wrote the manuscript; Wang WH designed the study and wrote the manuscript; Tian Y, Gao W, Li J offered the technical assistance. Supported by The National Natural Science Foundation of China, No. 30470777 Correspondence to: Wei-Hong Wang, Department of Gastroenterology, Peking University First Hospital, Beijing 100034, China. wangweihong@medmail.com.cn Telephone: +86-10-66551122 Fax: +86-10-66518105 Received: October 10, 2008 Revised: January 17, 2009 Accepted: January 24, 2009 Published online: February 28, 2009
Abstract AIM: To investigate the mechanisms of aspirin increasing the susceptibility of Helicobacter pylori (H pylori) to metronidazole.
METHODS: H pylori reference strain 26 695 and two metronidazole-resistant isolates of H pylori were included in this study. Strains were incubated in Brucella broth with or without aspirin (1 mmol/L). The rdxA gene of H pylori was amplified by PCR and sequenced. The permeability of H pylori to antimicrobials was determined by analyzing the endocellular radioactivity of the cells after incubated with [7-3H]-tetracycline. The outer membrane proteins (OMPs) of H pylori 26 695 were depurated and analyzed by SDS-PAGE. The expression of 5 porins (hopA, hopB, hopC, hopD and hopE) and the putative RND efflux system (hefABC) of H pylori were analyzed using real-time quantitative PCR.
RESULTS: The mutations in rdxA gene did not change in metronidazole resistant isolates treated with aspirin. The radioactivity of H pylori increased when treated with aspirin, indicating that aspirin improved the permeability of the outer membrane of H pylori. However, the expression of two OMP bands between 55 kDa and 72 kDa altered in the presence of aspirin. The expression of the mRNA of hopA, hopB, hopC, hopD, hopE and hefA, hefB, hefC of H pylori did not change when treated with aspirin.
CONCLUSION: Although aspirin increases the susceptibility of H pylori to metronidazole, it has no effect on the mutations of rdxA gene of H pylori. Aspirin increases endocellular concentrations of antimicrobials probably by altering the OMP expression. The expression of other porins and efflux systems of H pylori need to be determined.
© 2009 The WJG Press and Baishideng. All rights reserved.
Key words: Helicobacter pylori; Aspirin; Metronidazole; Resistance; Minimum inhibitory concentrations
Peer reviewer: Yoshio Yamaoka, MD, PhD, Associate Professor, Department of Medicine/Gastroenterology, Baylor College of Medicine and VA Medical Center (111D), 2002 Holcombe Blvd, Houston, Texas 77030, United States
Zhang XP, Wang WH, Tian Y, Gao W, Li J. Aspirin increases susceptibility of Helicobacter pylori to metronidazole by augmenting endocellular concentrations of antimicrobials. World J Gastroenterol 2009; 15(8): 919-926 Available from: URL: http://www.wjgnet.com/1007-9327/15/919.asp DOI: http://dx.doi.org/10.3748/wjg.15.919
INTRODUCTION Aspirin, referred to as non-steroidal anti-inflammatory drugs (NSAIDs), is one of the most widely used drugs worldwide. It inhibits cyclooxygenases (COX), thereby irreversibly blocking the conversion of arachidonic acid to prostanoids. In addition, aspirin is also considered to offer some protection against coronary heart disease[1], due in part to inhibition of the thromboxane A2, a potent platelet aggregator. It has been reported that aspirin demonstrates chemopreventative activity against cancers in the esophagus, stomach and colon by inducing apoptosis in epithelial cells and regulating angiogenesis[2-4]. Aspirin also has numerous effects in different bacterial species. Previous studies reported that aspirin could inhibit the growth of some bacteria, affect the production of virulence factors of some bacteria, and alter the susceptibility of bacteria to some antibiotics by influencing the gene expression and inducing a number of morphological and physiological alterations in bacteria[5]. We previously reported that NSAIDs, including sodium salicylate, aspirin, indomethacin and celecoxib, inhibited the growth of H pylori in a dose-dependent manner when incubated in brucella broth in vitro[6-9]. These drugs also significantly affected the activity of virulence factors of H pylori, for example, urease and vaculating cytotoxin[8,9]. In addition, the minimum inhibitory concentrations (MICs) of clarithromycin, metronidazole and amoxicillin to H pylori decreased when treated with a low concentration of aspirin[6-8], indicating that aspirin increased the susceptibility of H pylori to these antimicrobials. The aim of the present study was to investigate the mechanisms of aspirin increasing the susceptibility of H pylori to metronidazole. The rdxA gene of H pylori treated with and without aspirin was analyzed by PCR amplification and sequencing. The effect of aspirin on the permeability of the outer membrane of H pylori was determined using [7-3H]-tetracycline. The effects of aspirin on the expression of outer membrane proteins (OMPs) of H pylori were also determined.
MATERIALS AND METHODS Chemicals Aspirin (Sigma Chemical Co, St Louis, MO, USA) and proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma Chemical Co.) were dissolved in DMSO (Sigma Chemical Co.) in advance. [7-3H]-tetracycline (0.6 Ci/mmol; 22.2 GBq/mmol; Dupont/NEN Research Products, Boston, Mass.) was freshly dissolved in thin hydrochloric acid.
Strains and culture conditions
H pylori
reference strain 26 695
(susceptible to metronidazole) and two clinical isolates of H pylori
(metronidazole resistant, R1 and R2) were included in this study.
Strains were cultured on Columbia agar plates containing 8% sheep blood
in a microaerobic atmosphere (10% CO2 and 5% O2)
at 37℃
for 2-3 d. H pylori of 108 CFU/mL were then inoculated
in 20 mL
Determination of MICs Bacteria were prepared in Brain Heart Infusion broth to yield a viable count of 3×108 CFU/mL (equivalent to 1 McFarland turbidity standard unit) and used as the inocula for susceptibility testing. Bacterial suspension (100 mL) was spread, in duplicate, on Columbia agar plates with or without aspirin (1 mmol/L). A single E-test strip of metronidazole (OXOID Ltd, England) was applied to each plate. The MIC of metronidazole and the possible effect of aspirin on the MIC of metronidazole were determined after 72 h of incubation at 37℃ under microaerobic conditions.
Extraction of genomic DNA H pylori genomic DNA was extracted using silicon dioxicide method. Cells were harvested and washed twice in phosphate-buffered saline (PBS) (0.01 mol/L, pH 7.2). The cell precipitation was suspended in 100 mL TE. Then 5 mL SiO2 Liq. and 400 mL binding buffer (containing 4 mol/L guanidinium isothiocyanate, 50 mmol/L Tris-HCl, 20 mmol/L EDTA) was added and incubated at 55℃ for 5 min with shaking once every minute. The suspension was centrifuged at 8000 r/min for 30 s at room temperature and the precipitate was washed thrice in cleaning buffer (containing 20 mmol/L Tris-HCl, 1 mmol/L EDTA, 100 mmol/L NaCl and dehydrated alcohol). The resulting suspension was dried at 55℃ and stored at -20℃.
Amplification of H pylori rdxA gene and sequencing
The fragments (886 bp) containing the
complete rdxA gene was amplified by PCR. Forward primer:
5'-AGGGATTTTATTGTATGCTACAA-3'; Reverse primer:
5'-AGGAGCATCAGATAGTTCTGA-3'. The PCR amplification was carried out in 25
mL
reaction solution containing 2
mL of H pylori genomic DNA, 4.5
mL of 10 ×
PCR buffer (with 15 mmol/L
MgCl2), 2 mL
of dNTPs (each 2.5 mmol/L), 2 mL
of forward and reverse primers (each 5
mmol/L),
0.5 mL
of TaqDNA polymerase
Uptake studies using [7-3H] tetracycline Strain 26 695 was grown to mid-logarithmic phase (approximately from 3 × 109 to 5 × 109 CFU/mL) in Brucella broth and then 1 mmol/L of aspirin or DMSO (< 1%, vehicle control) were added for 6 h at 37℃ on a shaker at 60 r/min under microaerobic condition. Cell suspension was centrifuged at 8000 r/min for 10 min at room temperature and the precipitate washed and suspended in HEPES buffer (pH 7.2, containing 100 mmol/L MgCl2). At room temperature, 5 mCi [7-3H]-tetracycline (0.6 Ci/mmol; 22.2 GBq/mmol; Dupont/NEN Research Products, Boston, USA) was added to 10 mL cell suspension. After 20 min, each cell suspension was divided into two halves, and 100 mmol/L CCCP was added to one half. One milliliter aliquots were taken at 10 min intervals and washed three times in PBS. The resulting pellets were then diluted scintillation fluid and analyzed for radioactivity in an scintillation counter (TRI-CARB 2100TR).
Purification of OMPs H pylori 26 695 was incubated in Brucella broth for 48 h. The suspension was centrifuged at 8000 r/min for 10 min, washed, and suspended in ice-cold Tris-Mg buffer (10 mmol/L Tris-HCl containing 5 mmol/L MgCl2, pH 7.3) and sonicated (once 30 s at 3-5 s interval for 5 min) until most of the cells were disrupted as visualized microscopically. Unbroken cells were removed by centrifugation at 8000 r/min for 20 min at 4℃. The inner and outer membranes were concentrated by centrifugation at 50 000 r/min for 60 min at 4℃. The precipitate was suspended in 2% Triton Tris-Mg (pH 7.5) and incubated for 30 min at room temperature, and then centrifuged and incubated again under the same condition. The resulting pellets, OMPs, were washed twice in 10 mmol/L Tris-HCl and resolved in ddH2O. The final concentration of OMPs was determined by Coomassie brilliant blue R250 method.
SDS-PAGE gel electrophoresis Ten microgram OMPs were used for SDS-PAGE gel electrophoresis at permanent voltage (5% stacking gel at 60 V, 10% separating gel at 100 V). After incubation for 30 min in fixing liquid, the gel was dyed with Coomassie brilliant blue G250 for 30 min.
Isolation of total RNA and reverse transcription Total RNA was obtained by the TRIzol method as described by manufacturer (Invitrogen, Burlington, Ontario, Canada), and the contaminating DNA was removed by DNase Ⅰ treatment according to the manufacturer (Sigma). For cDNA synthesis, 4 mg RNA diluted with DEPC H2O was heated to 70℃ for 5 min and chilled quickly on ice for 15 min. The samples were then added to a 20 mL reaction mixture containing 2 mL random hexameric primers (1 mg/mL), 0.4 mL of RNasin, 1 mL of M-MLV, 4 mL of dNTPs (each 2.5 mmol/L) and 4 mL of 5 × RT buffer. The cDNA synthesis reaction was performed for 60 min at 37℃. The enzyme was subsequently inactivated at 95℃ for 5 min. Aliquots of cDNA were stored at -70℃.
Real-time quantitative PCR The mRNA levels of hopA, hopB, hopC, hopD, hopE and hefA, hefB, hefC were determined by real-time PCR using an ABI Prism 7700 sequence detection system (Perkin-Elmer Applied Biosystems, Foster City, CA.). Specific primers and TaqMan probes were designed with the aid of the Primer Express program 3.0 (Perkin-Elmer Applied Biosystems) (Table 1). A standard curve was constructed using 10-fold serial dilutions of each cDNA. Reaction mixtures for PCR (50 mL) were prepared by mixing 5 mL synthesized cDNA solution with 5 mL of 10 × PCR buffer (containing 15 mmol/L MgCl2), 3 mL forward and reverse primers (each 5 mmol/L), 4 mL dNTPs (each 2.5 mmol/L), 2 mL TagMan probe, 1 mL of ROX, 0.5 mL TaqDNA polymerase (1 U/mL). PCR was carried out at 95℃ for 10 min, followed by 40 cycles of 95℃ for 15 s and 60℃ for 60 s according to the manufacturers’ instructions. The levels of the protein mRNA were expressed as the ratio of the protein mRNA to 16S rRNA mRNA [protein mRNA (U/mL)/16S rRNA mRNA (U/mL) ratio × 100 000]. The PCR was carried out in quintuple using samples prepared at the same time.
Statistical analysis Statistical analysis was performed using SPSS, 13.0. Representative data of endocellular radioactivity and quantitative PCR were presented as mean ± SD. The Student’s t test was used to compare data. P < 0.05 was considered statistically significant.
RESULTS Effects of aspirin on MICs of metronidazole For strain R1, MIC of metronidazole decreased from 256 mg/mL to 0.25 mg/mL in the presence of aspirin (1 mmol/L), and for strain R2, MIC of metronidazole reduced from 64 mg/mL to below the readable value (0.016 mg/mL), indicating that aspirin increased the susceptibility of H pylori to metronidazole and converted these two resistant strains to susceptible strains.
Effects of aspirin on mutations of rdxA gene The 886 bp DNA fragments containing the complete rdxA gene were amplified by PCR for H pylori reference strain 26 695 and the two metronidazloe resistant strains (R1, R2) treated with or without aspirin. Sequence analysis revealed that there were point mutations, insertions and deletions in rdxA gene of strain R2 when compared with the reference strain 26 695, and the nucleotide homology between the two strains (26 695 and R2) was 94.4%. However, the nucleotide homology of rdxA gene between strain R2 and its corresponding isolate R2(A) (strain R2 treated with aspirin) was over 99.5% (Figures 1 and 2).
Effects of aspirin on permeability of outer membrane of H pylori Irrespective of the presence of CCCP, the radioactivity of H pylori treated with aspirin was higher when compared to that of the vehicle control (DMSO), indicating that aspirin enhanced the permeability of the outer membrane of H pylori to antimicrobial (Figure 3). Irrespective of the presence of aspirin, the radioactivity of H pylori cells decreased when the efflux pump inhibitor CCCP was added, indicating that aspirin did not interfere with the collapsion effect of CCCP on the proton gradient across the cytoplasmic membrane of H pylori (Figure 4).
Effects of aspirin on OMP profiles The OMP profiles of H pylori treated with aspirin were similar to that in control. However, the expression of two OMPs (Band 1 and 2) between 55 kDa and 72 kDa altered in the presence of aspirin (Figure 5).
Effects of aspirin on expression of mRNA of porins and efflux system Real-time quantitative PCR indicated that there was no significant difference in the levels of the cDNA of hopA, hopB, hopC, hopD, hopE and hefA, hefB, hefC for strains treated with aspirin when compared with that in control. This indicated that, irrespective of the presence of aspirin, the expression of the levels of mRNA of hopA, hopB, hopC, hopD, hopE and hefA, hefB, hefC did not change (Table 2).
DISCUSSION H pylori is a spiral-shaped gram-negative bacterium and an important human pathogen that colonizes in the stomach of 50% of the world human population[10,11]. Infection results in chronic inflammation of the gastric mucosa and peptic ulcer, and it has been proven that H pylori infection is strongly associated with adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. International Agency for Research on Cancer affiliated to World Health Organization (WHO) defined H pylori as one of the first class human carcinogens[12]. Recent Studies revealed that H pylori infection plays important roles in the invasion of heart and brain vascular disorders, autoimmune diseases, nutritional and metabolic diseases, hematopathy and dermatologic diseases. Eradication of H pylori infection is very important to prevent and cure these diseases. The most successful treatment regimens use combinations of two or more antibiotics, such as amoxicillin, clarithromycin, metronidazole, or tetracycline, along with a proton pump inhibitor or bismuth. However, with the wide use of antimicrobials in clinical practice, antibiotic resistance is more and more apparent and is considered one of the major causes of treatment failure[13]. Early studies suggested that salicylate inhibited the growth of some bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus epidermidis, and affected the activity of fimbriae, flagellum and the production of biofilm, slime, and thus might alter the pathogenicity of bacteria[14-21]. It has been reported that in vitro, salicylate could alter the susceptibilities of bacteria to some antimicrobials. Salicylate induced the intrinsic multiple antimicrobial resistance phenotype in many bacteria, such as Escherichia coli, Salmonella typhimurium and Staphylococcus aureus, and increased the susceptibilities of some bacteria to aminoglycosides[22-29]. Our previous studies also found that in vitro aspirin not only inhibited the growth of H pylori[6-9], but also decreased the MICs of metronidazole, clarithromycin and amoxicillin to H pylori, and even converted some resistant strains to susceptible ones[6-8]. Therefore, the present study investigated the mechanisms of aspirin increasing the susceptibility of H pylori to metronidazole. At least four distinctive mechanisms of antibiotic resistance have been described in bacteria: enzymatic inactivation, decreased permeability of bacterial membranes, active efflux of antimicrobial agents, and alteration of target sites of antimicrobials to bacteria[30]. Metronidazole, clarithromycin and amoxicillin are different kinds of antimicrobials, and each has its different effect on different target site in H pylori. Likewise, resistance of H pylori to these antimicrobial agents arises through various mechanisms. A reasonable explanation of aspirin increasing the susceptibility of H pylori to antimicrobials with different antibacterial mechanisms is that aspirin does not alter the target sites of bacteria, but increases the endocellular concentrations of antimicrobials. Metronidazole is a prodrug activated by nitrore-ductases in bacteria cells. Resistance of metronidazole is caused by either the absence or the inactivation of the nitroreductases[31]. It has been reported that the resistance of H pylori to metronidazole was mainly due to null mutations in the rdxA gene, which encoded an oxygen-insensitive NADPH nitroreductase[32]. However, studies also reported involvement of other reductases in the development of the resistant phenotype. In addition to oxygen-insensitive NADPH nitroreductases, several other nitroreductases in H pylori, such as NADPH flavin oxidoreductase, ferredoxin-like protein, flavodoxin, a-ketoglutarate oxidoreductase and pyruvate: flavodoxin oxidoreductase, have been found to reduce metronidazole and to generate active compounds[33,34]. In our study, mutations in rdxA gene might be involved for the resistance of the isolated strain (R2). However, in the presence of aspirin, the strain converted from metronidazole resistant to susceptible, while the mutations in rdxA gene did not change. By using isotope scintillation technique with [7-3H]-tetracycline, our study revealed that aspirin increased the endocellular concentration of antimicrobials in H pylori cells, indicating that aspirin increased the outer membrane permeability of H pylori to antimicrobials. With the higher endocellular concentration in the presence of aspirin, metronidazole might be reduced and activated by other nitroreductases in H pylori. Therefore, the MIC of H pylori to metronidazole decreased, and in some circumstances, resistant strains even converted to susceptible ones. Two pathways may be involved in the mechanisms for the increasing concentration of antimicrobials in bacteria cells. One is the augmentation of anti-microbials entering into the bacteria cells passively; the other is the impairment of antimicrobials pumping out of the bacteria actively. Previous studies on Escherichia coli revealed that salicylate increased resistance to multiple antibiotics, including quinolones, cephalosporins, ampicillin, nalidixic acid, tetracycline and chloramphenicol[22]. Aspirin could induce multiple antibiotic resistance (mar) gene, alter the expression of OMPs, and decrease the outer membrane permeability to antimicrobials or increase the efflux of antimicrobials[35-38]. There were three basic uptake systems across the outer membrane[39], namely, uptake of hydrophilic substances through the water-filled channels of porins, uptake of polycations via self-promoted uptake at divalent cation binding sites on lipopolysaccharide, and uptake of hydrophobic substances through the outer membrane bilayer. Bacteria could produce many porins, and were able to regulate the relative number of different porins in response to the osmolarity of the surrounding media. At least five porins named HopA, HopB, HopC, HopD and HopE (part of a 32-member family of outer membrane proteins) were present in a single cell of H pylori[40,41]. These porins were considered to be associated with antibiotic resistance[40]. On the other hand, some bacteria expressed a membrane transporter system that led to multidrug resistance by drug efflux. Three putative RND efflux systems, HefABC, HefDEF and HefGHI, identified in H pylori may be correlated with antibiotic resistance[42]. Of the three efflux systems, only HefABC was involved in multidrug resistance in vitro[42]. Therefore, we tested the five porin genes (hopA, hopB, hopC, hopD and hopE) and the efflux protein genes (hefA, hefB, hefC) using real-time quantitative PCR, and found that aspirin did not interfere with the expression of the above proteins at the levels of gene transcription. The alteration of the permeability of outer membrane of H pylori should be accompanied by the modification of some related OMPs. In the present study, the expression of two OMPs of H pylori between 55 kDa and 72 kDa altered in the presence of aspirin. However, the functions and identifications of these OMPs need to be determined by two-dimensional electrophoresis and protein mass-spectrum analysis. If these OMPs were associated with the increase of the permeability of outer membrane of H pylori, further studies should be performed to determine whether the functional and phenotypic alterations of these OMPs in the presence of aspirin occurred at the levels of protein translation or modification, or some other porins or efflux systems were involved. Park et al[43] conducted a pilot study aimed at comparing the efficacy of the standard omeprazole-amoxicillin-clarithromycin (OAC) regimen with a combined OAC regimen and aspirin (OACA). Follow-up endoscopic findings showed that the previous ulcers were completely healed in all subjects. Although the eradication rate for the OACA group (86.7%) was higher than that of the OAC group (80.3%), there was no statistically significant difference between the two groups. The overall adverse events were similar in the two groups. The OACA regimen was well tolerated in the group of patients with peptic ulcer disease. The potential of aspirin and other NSAIDs for clinical use to augment the efficacy of H pylori eradication may warrant further investigations. With the increasing attention paid to the detriment of H pylori and the resistance of antimicrobials to this microorganism, it is urgent to investigate new effective therapeutic regimens. Investigating the molecule mechanisms of aspirin increasing the susceptibility of H pylori to antimicrobials will help discover a more effective eradication regimen in clinical practice.
ACKNOWLEDGMENTS We thank Ding-Fang Bu for his technical assistance.
COMMENTS Background It was reported that aspirin inhibited the growth of Helicobacter pylori (H pylori) and the minimal inhibitory concentration (MICs) of clarithromycin, metronidazole and amoxicillin to H pylori decreased when treated with aspirin. This indicated that aspirin increased the susceptibility of H pylori to these antimicrobials, and even converted some resistant strains to susceptible ones. Research frontiers H pylori infection results in chronic inflammation of gastric mucosa, peptic ulcer and is strongly associated with adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Recent research revealed that H pylori infection played important roles in the invasion of heart and brain vascular disorders, autoimmune diseases, nutritional and metabolic diseases, hematopathy and dermatologic diseases. Eradication of H pylori infection is, therefore, very important to prevent and cure these diseases. However, with the wide use of antimicrobials in clinical practice, antibiotic resistance has become apparent and is considered one of the major causes of treatment failure. Innovations and breakthroughs In vitro, aspirin decreased the MICs of metronidazole, clarithromycin and amoxicillin to H pylori, and even converted some resistant strains to susceptible ones. This study investigated the mechanisms of aspirin increasing the susceptibility of H pylori to metronidazole. Applications Investigating the molecule mechanisms of aspirin increasing the susceptibility of H pylori to antimicrobials will help understand the mechanisms of the resistance of H pylori to antibiotics more intensively and discover a more effective eradication regimen in clinical practice. Terminology Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a kind of efflux pump inhibitor that is effective at a micromolar concentration, can alter the pH gradient across the cytoplasmic membrane, therefore, deprives the energy provision of the transport protein. Peer review The authors intensively reported that non-steroidal anti-inflammatory drugs (NSAIDs), including sodium salicylate, aspirin, indomethacin and celecoxib, inhibited the growth of H pylori in a dose-dependent manner and changed the susceptibility of H pylori to antibiotics. In this study, the authors demonstrated that although aspirin increased the susceptibility of H pylori to metronidazole, it had no effect on the mutations of rdxA gene of H pylori and that aspirin increased endocellular concentrations of antimicrobials probably by altering the outer membrane proteins (OMPs) expression of H pylori. This theme is interesting, and will give new insights of H pylori eradication for physicians.
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S- Editor Li LF L- Editor Ma JY E- Editor Zheng XM
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