Article Search
검색
검색 팝업 닫기

Metrics

Help

  • 1. Aims and Scope

    Gut and Liver is an international journal of gastroenterology, focusing on the gastrointestinal tract, liver, biliary tree, pancreas, motility, and neurogastroenterology. Gut atnd Liver delivers up-to-date, authoritative papers on both clinical and research-based topics in gastroenterology. The Journal publishes original articles, case reports, brief communications, letters to the editor and invited review articles in the field of gastroenterology. The Journal is operated by internationally renowned editorial boards and designed to provide a global opportunity to promote academic developments in the field of gastroenterology and hepatology. +MORE

  • 2. Editorial Board

    Editor-in-Chief + MORE

    Editor-in-Chief
    Yong Chan Lee Professor of Medicine
    Director, Gastrointestinal Research Laboratory
    Veterans Affairs Medical Center, Univ. California San Francisco
    San Francisco, USA

    Deputy Editor

    Deputy Editor
    Jong Pil Im Seoul National University College of Medicine, Seoul, Korea
    Robert S. Bresalier University of Texas M. D. Anderson Cancer Center, Houston, USA
    Steven H. Itzkowitz Mount Sinai Medical Center, NY, USA
  • 3. Editorial Office
  • 4. Articles
  • 5. Instructions for Authors
  • 6. File Download (PDF version)
  • 7. Ethical Standards
  • 8. Peer Review

    All papers submitted to Gut and Liver are reviewed by the editorial team before being sent out for an external peer review to rule out papers that have low priority, insufficient originality, scientific flaws, or the absence of a message of importance to the readers of the Journal. A decision about these papers will usually be made within two or three weeks.
    The remaining articles are usually sent to two reviewers. It would be very helpful if you could suggest a selection of reviewers and include their contact details. We may not always use the reviewers you recommend, but suggesting reviewers will make our reviewer database much richer; in the end, everyone will benefit. We reserve the right to return manuscripts in which no reviewers are suggested.

    The final responsibility for the decision to accept or reject lies with the editors. In many cases, papers may be rejected despite favorable reviews because of editorial policy or a lack of space. The editor retains the right to determine publication priorities, the style of the paper, and to request, if necessary, that the material submitted be shortened for publication.

Search

Search

Year

to

Article Type

Original Article

Split Viewer

In Vitro Activity of Diphenyleneiodonium toward Multidrug-Resistant Helicobacter pylori Strains

Jun-Won Chung1, Su Young Kim1, Hee Jung Park2, Chang Su Chung1, Hee Woo Lee1, Sun Mi Lee3, Inki Kim3, Jhang Ho Pak3, Gin Hyug Lee2, Jin-Yong Jeong3

1Division of Gastroenterology, Department of Internal Medicine, Gachon University Gil Medical Center, Incheon, Korea, 2Department of Gastroenterology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea, 3Department of Convergence Medicine and Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea

Correspondence to: Jin-Yong Jeonga and Gin Hyug Leeb. aDepartment of Convergence Medicine and Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea, Tel: +82-2-3010-4105, Fax: +82-2-3010-4182, E-mail: jyjeong@amc.seoul.kr. bDepartment of Gastroenterology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea, Tel: +82-2-3010-3197, Fax: +82-2-485-5782, E-mail: jhlee409@chollian.net

Received: October 13, 2016; Revised: January 6, 2017; Accepted: February 13, 2017

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Gut Liver 2017;11(5):648-654. https://doi.org/10.5009/gnl16503

Published online July 28, 2017, Published date September 15, 2017

Copyright © Gut and Liver.

Background/Aims

The increased resistance of Helicobacter pylori to antibiotics has increased the need to develop new treatments for this bacterium. The aim of our study was to identify new drugs with anti-H. pylori activity.

Methods

We screened a small molecule library—the library of pharmacologically active compounds (LOPAC), which includes 1,280 pharmacologically active compounds—to identify inhibitors of H. pylori growth. The minimal inhibitory concentrations (MICs) of antibiotics against multidrug-resistant H. pylori strains were determined using the agar dilution method.

Results

We identified diphenyleneiodonium (DPI) as a novel anti-H. pylori agent. The MIC values for DPI were <0.03 μg/mL against all tested H. pylori strains. DPI also exhibited strong antibacterial activity against common gram-negative and gram-positive pathogenic bacteria.

Conclusions

DPI may be a candidate anti-H. pylori drug for future development.

Keywords: Helicobacter pylori, Diphenyleneiodonium, Drug resistance, multiple, Anti-bacterial agents, Minimal inhibitory concentration

Helicobacter pylori infection is the main cause of peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. The eradication of H. pylori has been shown to dramatically decrease the recurrence of peptic ulcer disease, including gastric and duodenal ulcers.1 Currently, it is estimated that around 50% of the world’s population has this bacterium.2 Hence, eradicating this organism is of significant clinical importance. According to various guidelines published since 1993, the first-line choice of treatment for H. pylori eradication consists of conventional triple therapy, which includes a proton pump inhibitor (PPI), clarithromycin, and amoxicillin for 7 to 14 days. Over the past few years, however, the efficacy of conventional triple therapy has decreased and now demonstrates eradication rates of less than 80%.3,4 This decrease is mainly due to the emergence of clarithromycin-resistant H. pylori strains.

To improve first-line treatments for H. pylori, a four-drug treatment (including metronidazole), with sequential and concomitant bismuth-based quadruple therapy for 7 to 14 days, was introduced. In many previous studies, this regimen has demonstrated a better eradication rate than that of conventional triple treatment as the first-line treatment.57 However, all of these treatments include metronidazole, which has been conventionally used as a second-line treatment. Hence, there is concern that these four-drug regimens could worsen antibiotic resistance and decrease the second-line eradication rate. Another drawback to these complex regimens is that they increase both the cost of therapy and patient noncompliance. In addition, quinolone is popularly used as a second-line treatment after a four-drug treatment regimen.8 The quinolone resistance rate also has dramatically increased in recent years.9

Various methods have recently been introduced to overcome the drawbacks of four-drug treatment and fluoroquinolone-containing therapy. Rifabutin-containing therapy, probiotics, and tailored therapy are alternative methods.10,11 However, these methods involve some challenges. Rifabutin causes serious complications, such as myelosuppression, and is difficult to use in countries with a high incidence of Mycobacterium tuberculosis.12,13 Probiotics are relatively invulnerable agents, but the cost of eradication has increased and they have not been clinically effective in some randomized control trials.14,15 Most importantly, the ultimate disadvantage of the various methods presented so far is that they could increase antibiotic resistance. Antibiotic resistance has been increasing gradually since H. pylori eradication began, and H. pylori will eventually become resistant to several antibiotics.10,16 This will increase the incidence of multidrug-resistant H. pylori, and effective antibiotic regimens will gradually disappear. Therefore, resistance to diverse antibiotics indicates the need to develop new drugs that are effective against resistant strains.

The library of pharmacologically active compounds (LOPAC) is a collection of high-quality innovative molecules, such as antibiotics, enzyme inhibitors, cell-cycle regulators, and various other substances. It is designed for the identification of novel drug discovery assay and commonly used for screening of novel agents in drug discovery fields. LOPAC includes a large number of small molecules, and small molecules have certain benefits, such as high chemical stability and simple synthesis. Additionally, small molecules registered with LOPAC are commercially available and their effects on human cells are well known. Therefore, it is easier to apply these substances in clinical practice. Many studies have demonstrated that LOPAC is useful and effective for detecting new drugs against various pathogens, such as fungi, tuberculosis, malaria, and viruses.1720 Therefore, we utilized LOPAC as a way to identify anti-H. pylori drugs. The primary purposes of our present study were to (1) identify new chemical agents with potential anti-H. pylori activity among the 1,200 compounds included in the LOPAC Chemical Library; and (2) measure the minimal inhibitory concentrations (MICs) of these candidates against reference and resistant strains of H. pylori.

1. Bacterial strains and culture conditions

The well-characterized ATCC 43504 H. pylori strain (ATCC, Manassas, VA, USA) was used as the reference strain for the chemical library screening assay and the initial antimicrobial susceptibility test. Twenty clinical isolates (known to be resistant to antibiotics currently used to treat H. pylori) and three susceptible strains were used to test the anti-H. pylori activity of diphenyleneiodonium (DPI; Sigma D2926; Sigma-Aldrich Co., St Louis, MO, USA). Resistant strains included single drug- and multidrug-resistant strains. Single drug-resistant strains were resistant to clarithromycin (Sigma C9742), metronidazole (Sigma M1547), levofloxacin (Sigma 28266), or amoxicillin (Sigma A8523), all of which are frequently used to eradicate H. pylori. Multidrug-resistant strains were resistant to at least two of these four drugs. Antibiotic resistance was assessed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.21H. pylori strains were cultured using a selective medium that contained Brucella agar (BD 211088) and 7% defibrinated sheep blood. Incubation of the cultured isolates was performed at 37°C under microaerobic conditions (10% CO2) for 72 hours.

2. Screening for inhibitors of H. pylori growth

The LOPAC Chemical Library was purchased from Sigma-Aldrich Co. and is composed of 1,280 small pharmacologically active molecules. These chemical compounds (2 mM) were serially diluted in 96-well source plates to select the growth inhibition concentration in a 50-μL volume. Each well of the 96-well microplate contained 180 μL of culture medium to which 10 μL of the chemical compound and 10 μL of a stock solution of 106H. pylori ATCC 43504 bacteria/mL was added. The plates were then incubated under microaerobic conditions at 37°C for 72 hours to allow for bacterial growth. After 3 days, the chemical compounds that prevented the growth of H. pylori were regarded as candidates for anti-H. pylori activity and further analyzed in antimicrobial susceptibility testing.

3. Determination of MIC

The susceptibilities of the H. pylori isolates to antibiotics were examined using the serial 2-fold agar dilution method, as previously described.21 Briefly, the bacteria were subcultured on Mueller–Hinton agar supplemented with 5% defibrinated sheep blood for 48 hours. The bacterial suspension was adjusted to 107 colony-forming units and directly inoculated onto each antibiotic-containing agar dilution plate. After incubation for 72 hours, the MIC of each antibiotic was determined. The MIC range for amoxicillin, clarithromycin, and DPI is 0.03125 to 32 μg/mL. The MIC range for metronidazole is 0.5 to 128 μg/mL. The standard H. pylori ATCC 43504 strain was included in these susceptibility tests as a control. The resistance breakpoints for amoxicillin, clarithromycin, metronidazole, and levofloxacin were defined as ≥1.0, >1.0, ≥8 and >1.0 μg/mL, respectively. The MIC results were obtained from two experiments.

1. Anti-H. pylori activities of DPI

More than 50 chemical compounds from the small-molecule LOPAC library prevented any visible H. pylori growth, and were regarded as having an inhibitory effect on H. pylori. Based on currently known pharmacological applications, we excluded antibacterial and antifungal agents that are currently used in clinical practice. Anticancer drugs acting on the cell cycle, apoptosis, DNA metabolism, and phosphorylation were also excluded. We also excluded antipsychotics and antidepressants that could affect the central nervous system through unexpected mechanisms of action. Calcium channel blockers and calcium channel activators were also excluded due to the possibility of cardiac toxicity. Finally, we selected a promising candidate, DPI, for further analysis (Fig. 1). DPI is a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor that reduces the production of reactive oxygen species (ROS) and may reverse atherosclerosis. DPI is presently under investigation for its possible neuroprotective effects against focal cerebral ischemia, but there are no reports on its antimicrobial activity. The MIC value of DPI against the H. pylori ATCC 43504 strain was <0.03 μg/mL.

We further evaluated the possible anti-H. pylori activities of DPI using multidrug-resistant H. pylori strains. The MIC values of DPI for the ATCC 43504 strain and 23 clinical isolates are listed in Table 1. The antibiotic resistance of each strain is also stated in Table 1. The MIC value of DPI was <0.03 μg/mL against all of the tested H. pylori strains, indicating strong anti-H. pylori activity.

2. Antibacterial activity of DPI against common pathogenic bacteria

The activity of DPI against common gram-negative and -positive pathogenic bacteria was also assessed. The MIC results are indicated in Table 2 and ranged between 0.5 and 2 μg/mL against gram-negative bacteria. The MIC of DPI against all of the tested gram-positive bacteria was 1 μg/mL. These MIC values are comparable to those of vancomycin, which is a commonly used antibiotic against gram-positive bacteria including methicillin-resistant Staphylococcus aureus.

We demonstrate that the NADPH oxidase inhibitor DPI has inhibitory activities against reference and resistant H. pylori strains and could be a candidate for further studies and future drug development. Importantly, the MIC value of DPI is lower than previously reported chemical compounds.22 The direct mechanism by which DPI inhibits H. pylori has not been clearly evaluated, but our indirect in vitro evidence suggests that DPI noncompetitively inhibits NADPH oxidase via covalent binding to flavin adenine dinucleotide. Although DPI is a nonspecific inhibitor of flavoenzymes, a decrease in ROS production has been reported in DPI-treated cells.23 Based on this decrease in ROS production, it has been speculated that DPI may reverse atherosclerosis and this compound is now under investigation to determine its neuroprotective effects in focal cerebral ischemia, a neurodegenerative disease.24 Micromolar concentrations of DPI are highly toxic, but sub-picomolar concentrations of DPI inhibit NADPH oxidase activation with high specificity.25 In another study, DPI was reported to inhibit H. pylori-induced increases in ROS, NADPH oxidase activity, MCP-1 expression, and the activation of MAPKs (mitogen-activated protein kinases), including the extracellular signal-regulated kinases, p38, and Jun N-terminal kinases, in AGS cells.26

DPI is likely to be a novel agent in the future for two reasons. First, a NADPH oxidase inhibitor is a new substance that has never been used to treat H. pylori infection. Previous studies have shown that some chemicals inhibit activation of NADPH oxidase, suggesting that they may alleviate cell inflammation in H. pylori-infected gastric epithelial cells.27,28 DPI may also suppress unnatural apoptosis in gastric epithelial cells infected with H. pylori.29 However, whether DPI can directly eradicate H. pylori has not been reported. In our in vitro study, we demonstrated that DPI effectively eradicated H. pylori. This is the first study to investigate the efficacy of DPI for eradicating H. pylori. This novel agent may be free from current antibiotic resistance, so it may help to overcome antibiotic resistance. Second, NADPH oxidase exists in gastric mucosal tissue, and DPI is a nonselective inhibitor of NADPH oxidase.30,31H. pylori infections increase ROS in infected cells using NADPH oxidase. This increase in ROS causes oxidative DNA damage in infected cells and may provoke H. pylori-infected carcinogenesis.32 Therefore, a substance with a NADPH oxidase inhibitor mechanism, such as DPI, may inhibit carcinogenesis and eradicate H. pylori. Finally, in this study, DPI was effective against all H. pylori strains regardless of antibiotic resistance. Considering these findings, we expect that DPI may help to overcome multidrug-resistant H. pylori in the future.

As mentioned earlier, the rate of resistance to commonly used antibiotics such as clarithromycin, metronidazole, and fluoroquinolone is the main cause of the decrease in the eradication rate of certain bacteria. In addition, the number of multidrug-resistant bacterial strains has increased. This highlights a critical need to develop selective antibacterial agents with novel target sites and establish an effective drug-resistance management strategy. In contrast, the development of new antimicrobial agents is somewhat out of proportion. Rifabutin, furazolidone, sitafloxacin, and nitazoxanide have been introduced but are not always available in some countries. In addition, rifabutin demonstrates serious side effects, such as myelosuppression, and should be reserved to treat mycobacterial infections. Furazolidone is a nitrofuran antibiotic that has demonstrated efficacy in some trials. However, it has been recognized by the U.S. Food and Drug Administration as a carcinogenic agent and thus is no longer used, except in a few developing countries.33 Sitafloxacin seems to be effective, but clinical data are still limited in Japan.34,35 Nitazoxanide is an antiprotozoal agent that has demonstrated efficacy in a restricted study, but it is a somewhat expensive agent.36,37 Further studies of these antibiotics are needed.

In addition to these antimicrobial approaches, therapeutic alternatives beyond antibiotics have been investigated in recent years, including natural phytotherapy and probiotics.22,38 Representative agents include Lactobacillus reuteri, Korean red ginseng, and sulforaphane. Micro- and nano-technologies have also been used to develop gastric drug delivery systems.39,40

Acid stability is an important factor for antibiotics used to treat H. pylori. Some studies have reported that major antibiotics (clarithromycin, amoxicillin, metronidazole) for H. pylori treatment degrade in human gastric acid at different pH values. Metronidazole is stable in an acidic environment (half-life >800 hours); however, clarithromycin is unstable in an acidic environment (half-life <1 hour).41,42 The quick destruction of antibiotics in gastric acid makes them less effective at eradicating H. pylori. Therefore, it is important that this problem is solved. No study has reported on the influence of gastric juice on antibacterial efficacy and acid stability of DPI. Additionally, DPI has not been reported as an antibiotic in humans. However, DPI has excellent antibiotic effects in vitro, and it has an antimicrobial effect on H. pylori as well as other gram-negative and -positive pathogenic bacteria. DPI is also expected to maintain acid stability using these methods, but more studies are needed.

We demonstrated that DPI has the ability to strongly eradicate H. pylori. Although DPI is relatively highly toxic, some studies have reported that ultra-low dose DPI is not overtly toxic in mice and has potent neuroprotective efficacy.24,43 Therefore, we could produce a substance that strongly inhibits H. pylori infection without toxic side effects by identifying the proper dose of DPI to treat H. pylori. Additionally, DPI has excellent antibacterial effects even in multidrug-resistant H. pylori. These advantages could help DPI serve as a novel agent in the future. Although we identified a novel H. pylori chemical inhibitor, additional steps are needed to develop such a substance into a viable drug because of several limitations. First, DPI is a non-specific NADPH inhibitor, so drug development is somewhat limited. Second, the interactions between DPI and other medications (PPIs and other antibiotics) for treating H. pylori have not been elucidated. Finally, the effects of an acidic environment on the activities of anti-H. pylori agent have not yet been considered. The high environmental pH of the stomach may affect the susceptibility of these bacteria to antibiotics. Further research is needed to examine the effects of pH on anti-H. pylori activity in different agents. Animal experiments will also be needed to determine whether substances such as DPI are effective under in vivo conditions.

In conclusion, DPI shows a potent MIC value against H. pylori, suggesting that it might be a promising new agent for that could be used to significantly improve anti-H. pylori treatment success and eradicate this pathogen.

This work was supported by a grant from the Asan Institute for Life Sciences (number: 2013-348, Asan Medical Center, Seoul, Korea) and Il-Yang Pharmaceutical (GCU 2014–5115).

Antimicrobial Susceptibilities of the Tested Helicobacter pylori Strains

StrainResistanceMIC, μg/mL

CLAAMOMETLEVDPI
HP43504MET 0.03  <0.03 1280.25 <0.03 
CS_S1-0.030.12540.25<0.03
CS_S2-0.030.12540.25<0.03
CS_S3-0.03<0.0320.25<0.03
CS_C1CLA640.2540.5<0.03
CS_C2CLA320.12540.5<0.03
CS_C3CLA64<0.0320.25<0.03
CS_M1MET0.06<0.031280.5<0.03
CS_M2MET0.06<0.031280.5<0.03
CS_M3MET0.060.1251280.5<0.03
CS_L1LEV0.030.25216<0.03
CS_L2LEV0.060.125232<0.03
CS_L3LEV0.06<0.0348<0.03
CS_A1AMO0.06140.25<0.03
CS_A2AMO0.06240.25<0.03
CS_A3AMO0.03440.25<0.03
CS_CM1CLA/MET32<0.031280.5<0.03
CS_CM2CLA/MET64<0.03160.25<0.03
CS_CM3CLA/MET320.03640.25<0.03
CS_CA1CLA/AMO64120.25<0.03
CS_CA2CLA/AMO64140.25<0.03
CS_CA3CLA/AMO32210.25<0.03
CS_CMLCLA/MET/LEV1280.0612864<0.03
CS_CMACLA/MET/AMO 641640.5<0.03

Antimicrobial Susceptibility of Gram-Negative and Gram-Positive Pathogenic Bacteria to DPI and Vancomycin

StrainMIC, μg/mL

DPIVancomycin
Gram-positiveStaphylococcus aureus 29213 (MSSA) 11
Staphylococcus aureus 33591 (MRSA)11
Enterococcus faecalis 2921214
Staphylococcus epidermidis 1222812
Gram-negative Escherichia coli 259220.5>32
Pseudomonas aeruginosa 278532>32
Acinetobacter baumannii 196061>32
Salmonella enteritidis 130761>32
Salmonella typhimurium 133111>32
Enterobacter cloacae 130491>32

  1. Hentschel, E, Brandstätter, G, and Dragosics, B (1993). Effect of ranitidine and amoxicillin plus metronidazole on the eradication of Helicobacter pylori and the recurrence of duodenal ulcer. N Engl J Med. 328, 308-312.
    Pubmed CrossRef
  2. Suerbaum, S, and Josenhans, C (2007). Helicobacter pylori evolution and phenotypic diversification in a changing host. Nat Rev Microbiol. 5, 441-452.
    Pubmed CrossRef
  3. Chung, JW, Lee, GH, and Han, JH (2011). The trends of one-week first-line and second-line eradication therapy for Helicobacter pylori infection in Korea. Hepatogastroenterology. 58, 246-250.
    Pubmed
  4. Graham, DY, and Fischbach, L (2010). Helicobacter pylori treatment in the era of increasing antibiotic resistance. Gut. 59, 1143-1153.
    Pubmed CrossRef
  5. Gisbert, JP, and Calvet, X (2011). Review article: non-bismuth quadruple (concomitant) therapy for eradication of Helicobater pylori. Aliment Pharmacol Ther. 34, 604-617.
    Pubmed CrossRef
  6. Chung, JW, Jung, YK, and Kim, YJ (2012). Ten-day sequential versus triple therapy for Helicobacter-pylori eradication: a prospective, open-label, randomized trial. J Gastroenterol Hepatol. 27, 1675-1680.
    Pubmed CrossRef
  7. Chung, JW, Ha, M, and Yun, SC (2013). Meta-analysis: sequential therapy is superior to conventional therapy for Helicobacter pylori infection in Korea. Korean J Gastroenterol. 62, 267-271.
    Pubmed CrossRef
  8. Malfertheiner, P, Megraud, F, and O’Morain, CA (2012). Management of Helicobacter pylori infection: the Maastricht IV/Florence consensus report. Gut. 61, 646-664.
    Pubmed CrossRef
  9. Ierardi, E, Giorgio, F, Losurdo, G, Di Leo, A, and Principi, M (2013). How antibiotic resistances could change Helicobacter pylori treatment: a matter of geography?. World J Gastroenterol. 19, 8168-8180.
    Pubmed KoreaMed CrossRef
  10. Kim, SY, Choi, DJ, and Chung, JW (2015). Antibiotic treatment for Helicobacter pylori: is the end coming?. World J Gastrointest Pharmacol Ther. 6, 183-198.
    Pubmed KoreaMed CrossRef
  11. Molina-Infante, J, and Shiotani, A (2015). Practical aspects in choosing a Helicobacter pylori therapy. Gastroenterol Clin North Am. 44, 519-535.
    Pubmed CrossRef
  12. Jeong, MH, Chung, JW, and Lee, SJ (2012). Comparison of rifabutin- and levofloxacin-based third-line rescue therapies for Helicobacter pylori. Korean J Gastroenterol. 59, 401-406.
    Pubmed CrossRef
  13. Heep, M, Rieger, U, Beck, D, and Lehn, N (2000). Mutations in the beginning of the rpoB gene can induce resistance to rifamycins in both Helicobacter pylori and Mycobacterium tuberculosis. Antimicrob Agents Chemother. 44, 1075-1077.
    Pubmed KoreaMed CrossRef
  14. Navarro-Rodriguez, T, Silva, FM, and Barbuti, RC (2013). Association of a probiotic to a Helicobacter pylori eradication regimen does not increase efficacy or decreases the adverse effects of the treatment: a prospective, randomized, double-blind, placebo-controlled study. BMC Gastroenterol. 13, 56.
    Pubmed KoreaMed CrossRef
  15. Mirzaee, V, and Rezahosseini, O (2012). Randomized control trial: comparison of triple therapy plus probiotic yogurt vs. standard triple therapy on Helicobacter pylori eradication. Iran Red Crescent Med J. 14, 657-666.
  16. Lee, JW, Kim, N, and Kim, JM (2013). Prevalence of primary and secondary antimicrobial resistance of Helicobacter pylori in Korea from 2003 through 2012. Helicobacter. 18, 206-214.
    CrossRef
  17. Watamoto, T, Egusa, H, Sawase, T, and Yatani, H (2015). Screening of pharmacologically active small molecule compounds identifies antifungal agents against Candida biofilms. Front Microbiol. 6, 1453.
    CrossRef
  18. Altaf, M, Miller, CH, Bellows, DS, and O’Toole, R (2010). Evaluation of the Mycobacterium smegmatis and BCG models for the discovery of Mycobacterium tuberculosis inhibitors. Tuberculosis (Edinb). 90, 333-337.
    CrossRef
  19. Lucumi, E, Darling, C, and Jo, H (2010). Discovery of potent small-molecule inhibitors of multidrug-resistant Plasmodium falciparum using a novel miniaturized high-throughput luciferase-based assay. Antimicrob Agents Chemother. 54, 3597-3604.
    Pubmed KoreaMed CrossRef
  20. Che, P, Wang, L, and Li, Q (2009). The development, optimization and validation of an assay for high throughput antiviral drug screening against Dengue virus. Int J Clin Exp Med. 2, 363-373.
  21. Jean, B (2015). Performance standards for antimicrobial susceptibility testing: 25th informational supplement. Wayne: Clinical and Laboratory Standards Institute
  22. Makobongo, MO, Gilbreath, JJ, and Merrell, DS (2014). Nontraditional therapies to treat Helicobacter pylori infection. J Microbiol. 52, 259-272.
    Pubmed CrossRef
  23. Bhunia, AK, Han, H, Snowden, A, and Chatterjee, S (1997). Redox-regulated signaling by lactosylceramide in the proliferation of human aortic smooth muscle cells. J Biol Chem. 272, 15642-15649.
    Pubmed CrossRef
  24. Wang, Q, Chu, CH, and Oyarzabal, E (2014). Subpicomolar diphenyleneiodonium inhibits microglial NADPH oxidase with high specificity and shows great potential as a therapeutic agent for neurodegenerative diseases. Glia. 62, 2034-2043.
    Pubmed KoreaMed CrossRef
  25. Aldieri, E, Riganti, C, and Polimeni, M (2008). Classical inhibitors of NOX NAD(P)H oxidases are not specific. Curr Drug Metab. 9, 686-696.
    Pubmed CrossRef
  26. Cho, SO, Lim, JW, Kim, KH, and Kim, H (2011). Diphenyleneiodonium inhibits the activation of mitogen-activated protein kinases and the expression of monocyte chemoattractant protein-1 in Helicobacter pylori-infected gastric epithelial AGS cells. Inflamm Res. 60, 501-507.
    CrossRef
  27. Cha, B, Lim, JW, Kim, KH, and Kim, H (2011). 15-Deoxy-D12,14-prostaglandin J2 suppresses RANTES expression by inhibiting NADPH oxidase activation in Helicobacter pylori-infected gastric epithelial cells. J Physiol Pharmacol. 62, 167-174.
    Pubmed
  28. Cho, SO, Lim, JW, and Kim, H (2013). Red ginseng extract inhibits the expression of MCP-1 and iNOS in Helicobacter pylori-infected gastric epithelial cells by suppressing the activation of NADPH oxidase and Jak2/Stat3. J Ethnopharmacol. 150, 761-764.
    Pubmed CrossRef
  29. Cho, SO, Lim, JW, and Kim, H (2015). Diphenyleneiodonium inhibits apoptotic cell death of gastric epithelial cells infected with Helicobacter pylori in a Korean isolate. Yonsei Med J. 56, 1150-1154.
    Pubmed KoreaMed CrossRef
  30. Tominaga, K, Kawahara, T, and Sano, T (2007). Evidence for cancer-associated expression of NADPH oxidase 1 (Nox1)-based oxidase system in the human stomach. Free Radic Biol Med. 43, 1627-1638.
    Pubmed CrossRef
  31. Jones, RD, Hancock, JT, and Morice, AH (2000). NADPH oxidase: a universal oxygen sensor?. Free Radic Biol Med. 29, 416-424.
    Pubmed CrossRef
  32. Jang, SH, Lim, JW, Morio, T, and Kim, H (2012). Lycopene inhibits Helicobacter pylori-induced ATM/ATR-dependent DNA damage response in gastric epithelial AGS cells. Free Radic Biol Med. 52, 607-615.
    CrossRef
  33. World Gastroenterology Organisation (2011). World Gastroenterology Organisation global guideline: Helicobacter pylori in developing countries. J Clin Gastroenterol. 45, 383-388.
    Pubmed CrossRef
  34. Furuta, T, Sugimoto, M, and Kodaira, C (2014). Sitafloxacin-based third-line rescue regimens for Helicobacter pylori infection in Japan. J Gastroenterol Hepatol. 29, 487-493.
    CrossRef
  35. Murakami, K, Furuta, T, and Ando, T (2013). Multi-center randomized controlled study to establish the standard third-line regimen for Helicobacter pylori eradication in Japan. J Gastroenterol. 48, 1128-1135.
    Pubmed CrossRef
  36. Basu, PP, Rayapudi, K, Pacana, T, Shah, NJ, Krishnaswamy, N, and Flynn, M (2011). A randomized study comparing levofloxacin, omeprazole, nitazoxanide, and doxycycline versus triple therapy for the eradication of Helicobacter pylori. Am J Gastroenterol. 106, 1970-1975.
    Pubmed KoreaMed CrossRef
  37. Watson, JB, and Moss, SF (2011). Will H. pylori stagger under the weight of this LOAD? A novel but expensive eradication regimen. Am J Gastroenterol. 106, 1976-1977.
    Pubmed CrossRef
  38. Vale, FF, and Oleastro, M (2014). Overview of the phytomedicine approaches against Helicobacter pylori. World J Gastroenterol. 20, 5594-5609.
    Pubmed KoreaMed CrossRef
  39. Lopes, D, Nunes, C, Martins, MC, Sarmento, B, and Reis, S (2014). Eradication of Helicobacter pylori: past, present and future. J Control Release. 189, 169-186.
    Pubmed CrossRef
  40. Gao, W, Thamphiwatana, S, Angsantikul, P, and Zhang, L (2014). Nanoparticle approaches against bacterial infections. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 6, 532-547.
    Pubmed KoreaMed CrossRef
  41. Erah, PO, Goddard, AF, Barrett, DA, Shaw, PN, and Spiller, RC (1997). The stability of amoxycillin, clarithromycin and metronidazole in gastric juice: relevance to the treatment of Helicobacter pylori infection. J Antimicrob Chemother. 39, 5-12.
    Pubmed CrossRef
  42. Labenz, J (2001). Current role of acid suppressants in Helicobacter pylori eradication therapy. Best Pract Res Clin Gastroenterol. 15, 413-431.
    Pubmed CrossRef
  43. Wang, Q, Qian, L, and Chen, SH (2015). Post-treatment with an ultra-low dose of NADPH oxidase inhibitor diphenyleneiodonium attenuates disease progression in multiple Parkinson’s disease models. Brain. 138, 1247-1262.
    Pubmed KoreaMed CrossRef

Article

Original Article

Gut and Liver 2017; 11(5): 648-654

Published online September 15, 2017 https://doi.org/10.5009/gnl16503

Copyright © Gut and Liver.

In Vitro Activity of Diphenyleneiodonium toward Multidrug-Resistant Helicobacter pylori Strains

Jun-Won Chung1, Su Young Kim1, Hee Jung Park2, Chang Su Chung1, Hee Woo Lee1, Sun Mi Lee3, Inki Kim3, Jhang Ho Pak3, Gin Hyug Lee2, Jin-Yong Jeong3

1Division of Gastroenterology, Department of Internal Medicine, Gachon University Gil Medical Center, Incheon, Korea, 2Department of Gastroenterology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea, 3Department of Convergence Medicine and Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea

Correspondence to: Jin-Yong Jeonga and Gin Hyug Leeb. aDepartment of Convergence Medicine and Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea, Tel: +82-2-3010-4105, Fax: +82-2-3010-4182, E-mail: jyjeong@amc.seoul.kr. bDepartment of Gastroenterology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea, Tel: +82-2-3010-3197, Fax: +82-2-485-5782, E-mail: jhlee409@chollian.net

Received: October 13, 2016; Revised: January 6, 2017; Accepted: February 13, 2017

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background/Aims

The increased resistance of Helicobacter pylori to antibiotics has increased the need to develop new treatments for this bacterium. The aim of our study was to identify new drugs with anti-H. pylori activity.

Methods

We screened a small molecule library—the library of pharmacologically active compounds (LOPAC), which includes 1,280 pharmacologically active compounds—to identify inhibitors of H. pylori growth. The minimal inhibitory concentrations (MICs) of antibiotics against multidrug-resistant H. pylori strains were determined using the agar dilution method.

Results

We identified diphenyleneiodonium (DPI) as a novel anti-H. pylori agent. The MIC values for DPI were <0.03 μg/mL against all tested H. pylori strains. DPI also exhibited strong antibacterial activity against common gram-negative and gram-positive pathogenic bacteria.

Conclusions

DPI may be a candidate anti-H. pylori drug for future development.

Keywords: Helicobacter pylori, Diphenyleneiodonium, Drug resistance, multiple, Anti-bacterial agents, Minimal inhibitory concentration

INTRODUCTION

Helicobacter pylori infection is the main cause of peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. The eradication of H. pylori has been shown to dramatically decrease the recurrence of peptic ulcer disease, including gastric and duodenal ulcers.1 Currently, it is estimated that around 50% of the world’s population has this bacterium.2 Hence, eradicating this organism is of significant clinical importance. According to various guidelines published since 1993, the first-line choice of treatment for H. pylori eradication consists of conventional triple therapy, which includes a proton pump inhibitor (PPI), clarithromycin, and amoxicillin for 7 to 14 days. Over the past few years, however, the efficacy of conventional triple therapy has decreased and now demonstrates eradication rates of less than 80%.3,4 This decrease is mainly due to the emergence of clarithromycin-resistant H. pylori strains.

To improve first-line treatments for H. pylori, a four-drug treatment (including metronidazole), with sequential and concomitant bismuth-based quadruple therapy for 7 to 14 days, was introduced. In many previous studies, this regimen has demonstrated a better eradication rate than that of conventional triple treatment as the first-line treatment.57 However, all of these treatments include metronidazole, which has been conventionally used as a second-line treatment. Hence, there is concern that these four-drug regimens could worsen antibiotic resistance and decrease the second-line eradication rate. Another drawback to these complex regimens is that they increase both the cost of therapy and patient noncompliance. In addition, quinolone is popularly used as a second-line treatment after a four-drug treatment regimen.8 The quinolone resistance rate also has dramatically increased in recent years.9

Various methods have recently been introduced to overcome the drawbacks of four-drug treatment and fluoroquinolone-containing therapy. Rifabutin-containing therapy, probiotics, and tailored therapy are alternative methods.10,11 However, these methods involve some challenges. Rifabutin causes serious complications, such as myelosuppression, and is difficult to use in countries with a high incidence of Mycobacterium tuberculosis.12,13 Probiotics are relatively invulnerable agents, but the cost of eradication has increased and they have not been clinically effective in some randomized control trials.14,15 Most importantly, the ultimate disadvantage of the various methods presented so far is that they could increase antibiotic resistance. Antibiotic resistance has been increasing gradually since H. pylori eradication began, and H. pylori will eventually become resistant to several antibiotics.10,16 This will increase the incidence of multidrug-resistant H. pylori, and effective antibiotic regimens will gradually disappear. Therefore, resistance to diverse antibiotics indicates the need to develop new drugs that are effective against resistant strains.

The library of pharmacologically active compounds (LOPAC) is a collection of high-quality innovative molecules, such as antibiotics, enzyme inhibitors, cell-cycle regulators, and various other substances. It is designed for the identification of novel drug discovery assay and commonly used for screening of novel agents in drug discovery fields. LOPAC includes a large number of small molecules, and small molecules have certain benefits, such as high chemical stability and simple synthesis. Additionally, small molecules registered with LOPAC are commercially available and their effects on human cells are well known. Therefore, it is easier to apply these substances in clinical practice. Many studies have demonstrated that LOPAC is useful and effective for detecting new drugs against various pathogens, such as fungi, tuberculosis, malaria, and viruses.1720 Therefore, we utilized LOPAC as a way to identify anti-H. pylori drugs. The primary purposes of our present study were to (1) identify new chemical agents with potential anti-H. pylori activity among the 1,200 compounds included in the LOPAC Chemical Library; and (2) measure the minimal inhibitory concentrations (MICs) of these candidates against reference and resistant strains of H. pylori.

MATERIALS AND METHODS

1. Bacterial strains and culture conditions

The well-characterized ATCC 43504 H. pylori strain (ATCC, Manassas, VA, USA) was used as the reference strain for the chemical library screening assay and the initial antimicrobial susceptibility test. Twenty clinical isolates (known to be resistant to antibiotics currently used to treat H. pylori) and three susceptible strains were used to test the anti-H. pylori activity of diphenyleneiodonium (DPI; Sigma D2926; Sigma-Aldrich Co., St Louis, MO, USA). Resistant strains included single drug- and multidrug-resistant strains. Single drug-resistant strains were resistant to clarithromycin (Sigma C9742), metronidazole (Sigma M1547), levofloxacin (Sigma 28266), or amoxicillin (Sigma A8523), all of which are frequently used to eradicate H. pylori. Multidrug-resistant strains were resistant to at least two of these four drugs. Antibiotic resistance was assessed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.21H. pylori strains were cultured using a selective medium that contained Brucella agar (BD 211088) and 7% defibrinated sheep blood. Incubation of the cultured isolates was performed at 37°C under microaerobic conditions (10% CO2) for 72 hours.

2. Screening for inhibitors of H. pylori growth

The LOPAC Chemical Library was purchased from Sigma-Aldrich Co. and is composed of 1,280 small pharmacologically active molecules. These chemical compounds (2 mM) were serially diluted in 96-well source plates to select the growth inhibition concentration in a 50-μL volume. Each well of the 96-well microplate contained 180 μL of culture medium to which 10 μL of the chemical compound and 10 μL of a stock solution of 106H. pylori ATCC 43504 bacteria/mL was added. The plates were then incubated under microaerobic conditions at 37°C for 72 hours to allow for bacterial growth. After 3 days, the chemical compounds that prevented the growth of H. pylori were regarded as candidates for anti-H. pylori activity and further analyzed in antimicrobial susceptibility testing.

3. Determination of MIC

The susceptibilities of the H. pylori isolates to antibiotics were examined using the serial 2-fold agar dilution method, as previously described.21 Briefly, the bacteria were subcultured on Mueller–Hinton agar supplemented with 5% defibrinated sheep blood for 48 hours. The bacterial suspension was adjusted to 107 colony-forming units and directly inoculated onto each antibiotic-containing agar dilution plate. After incubation for 72 hours, the MIC of each antibiotic was determined. The MIC range for amoxicillin, clarithromycin, and DPI is 0.03125 to 32 μg/mL. The MIC range for metronidazole is 0.5 to 128 μg/mL. The standard H. pylori ATCC 43504 strain was included in these susceptibility tests as a control. The resistance breakpoints for amoxicillin, clarithromycin, metronidazole, and levofloxacin were defined as ≥1.0, >1.0, ≥8 and >1.0 μg/mL, respectively. The MIC results were obtained from two experiments.

RESULTS

1. Anti-H. pylori activities of DPI

More than 50 chemical compounds from the small-molecule LOPAC library prevented any visible H. pylori growth, and were regarded as having an inhibitory effect on H. pylori. Based on currently known pharmacological applications, we excluded antibacterial and antifungal agents that are currently used in clinical practice. Anticancer drugs acting on the cell cycle, apoptosis, DNA metabolism, and phosphorylation were also excluded. We also excluded antipsychotics and antidepressants that could affect the central nervous system through unexpected mechanisms of action. Calcium channel blockers and calcium channel activators were also excluded due to the possibility of cardiac toxicity. Finally, we selected a promising candidate, DPI, for further analysis (Fig. 1). DPI is a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor that reduces the production of reactive oxygen species (ROS) and may reverse atherosclerosis. DPI is presently under investigation for its possible neuroprotective effects against focal cerebral ischemia, but there are no reports on its antimicrobial activity. The MIC value of DPI against the H. pylori ATCC 43504 strain was <0.03 μg/mL.

We further evaluated the possible anti-H. pylori activities of DPI using multidrug-resistant H. pylori strains. The MIC values of DPI for the ATCC 43504 strain and 23 clinical isolates are listed in Table 1. The antibiotic resistance of each strain is also stated in Table 1. The MIC value of DPI was <0.03 μg/mL against all of the tested H. pylori strains, indicating strong anti-H. pylori activity.

2. Antibacterial activity of DPI against common pathogenic bacteria

The activity of DPI against common gram-negative and -positive pathogenic bacteria was also assessed. The MIC results are indicated in Table 2 and ranged between 0.5 and 2 μg/mL against gram-negative bacteria. The MIC of DPI against all of the tested gram-positive bacteria was 1 μg/mL. These MIC values are comparable to those of vancomycin, which is a commonly used antibiotic against gram-positive bacteria including methicillin-resistant Staphylococcus aureus.

DISCUSSION

We demonstrate that the NADPH oxidase inhibitor DPI has inhibitory activities against reference and resistant H. pylori strains and could be a candidate for further studies and future drug development. Importantly, the MIC value of DPI is lower than previously reported chemical compounds.22 The direct mechanism by which DPI inhibits H. pylori has not been clearly evaluated, but our indirect in vitro evidence suggests that DPI noncompetitively inhibits NADPH oxidase via covalent binding to flavin adenine dinucleotide. Although DPI is a nonspecific inhibitor of flavoenzymes, a decrease in ROS production has been reported in DPI-treated cells.23 Based on this decrease in ROS production, it has been speculated that DPI may reverse atherosclerosis and this compound is now under investigation to determine its neuroprotective effects in focal cerebral ischemia, a neurodegenerative disease.24 Micromolar concentrations of DPI are highly toxic, but sub-picomolar concentrations of DPI inhibit NADPH oxidase activation with high specificity.25 In another study, DPI was reported to inhibit H. pylori-induced increases in ROS, NADPH oxidase activity, MCP-1 expression, and the activation of MAPKs (mitogen-activated protein kinases), including the extracellular signal-regulated kinases, p38, and Jun N-terminal kinases, in AGS cells.26

DPI is likely to be a novel agent in the future for two reasons. First, a NADPH oxidase inhibitor is a new substance that has never been used to treat H. pylori infection. Previous studies have shown that some chemicals inhibit activation of NADPH oxidase, suggesting that they may alleviate cell inflammation in H. pylori-infected gastric epithelial cells.27,28 DPI may also suppress unnatural apoptosis in gastric epithelial cells infected with H. pylori.29 However, whether DPI can directly eradicate H. pylori has not been reported. In our in vitro study, we demonstrated that DPI effectively eradicated H. pylori. This is the first study to investigate the efficacy of DPI for eradicating H. pylori. This novel agent may be free from current antibiotic resistance, so it may help to overcome antibiotic resistance. Second, NADPH oxidase exists in gastric mucosal tissue, and DPI is a nonselective inhibitor of NADPH oxidase.30,31H. pylori infections increase ROS in infected cells using NADPH oxidase. This increase in ROS causes oxidative DNA damage in infected cells and may provoke H. pylori-infected carcinogenesis.32 Therefore, a substance with a NADPH oxidase inhibitor mechanism, such as DPI, may inhibit carcinogenesis and eradicate H. pylori. Finally, in this study, DPI was effective against all H. pylori strains regardless of antibiotic resistance. Considering these findings, we expect that DPI may help to overcome multidrug-resistant H. pylori in the future.

As mentioned earlier, the rate of resistance to commonly used antibiotics such as clarithromycin, metronidazole, and fluoroquinolone is the main cause of the decrease in the eradication rate of certain bacteria. In addition, the number of multidrug-resistant bacterial strains has increased. This highlights a critical need to develop selective antibacterial agents with novel target sites and establish an effective drug-resistance management strategy. In contrast, the development of new antimicrobial agents is somewhat out of proportion. Rifabutin, furazolidone, sitafloxacin, and nitazoxanide have been introduced but are not always available in some countries. In addition, rifabutin demonstrates serious side effects, such as myelosuppression, and should be reserved to treat mycobacterial infections. Furazolidone is a nitrofuran antibiotic that has demonstrated efficacy in some trials. However, it has been recognized by the U.S. Food and Drug Administration as a carcinogenic agent and thus is no longer used, except in a few developing countries.33 Sitafloxacin seems to be effective, but clinical data are still limited in Japan.34,35 Nitazoxanide is an antiprotozoal agent that has demonstrated efficacy in a restricted study, but it is a somewhat expensive agent.36,37 Further studies of these antibiotics are needed.

In addition to these antimicrobial approaches, therapeutic alternatives beyond antibiotics have been investigated in recent years, including natural phytotherapy and probiotics.22,38 Representative agents include Lactobacillus reuteri, Korean red ginseng, and sulforaphane. Micro- and nano-technologies have also been used to develop gastric drug delivery systems.39,40

Acid stability is an important factor for antibiotics used to treat H. pylori. Some studies have reported that major antibiotics (clarithromycin, amoxicillin, metronidazole) for H. pylori treatment degrade in human gastric acid at different pH values. Metronidazole is stable in an acidic environment (half-life >800 hours); however, clarithromycin is unstable in an acidic environment (half-life <1 hour).41,42 The quick destruction of antibiotics in gastric acid makes them less effective at eradicating H. pylori. Therefore, it is important that this problem is solved. No study has reported on the influence of gastric juice on antibacterial efficacy and acid stability of DPI. Additionally, DPI has not been reported as an antibiotic in humans. However, DPI has excellent antibiotic effects in vitro, and it has an antimicrobial effect on H. pylori as well as other gram-negative and -positive pathogenic bacteria. DPI is also expected to maintain acid stability using these methods, but more studies are needed.

We demonstrated that DPI has the ability to strongly eradicate H. pylori. Although DPI is relatively highly toxic, some studies have reported that ultra-low dose DPI is not overtly toxic in mice and has potent neuroprotective efficacy.24,43 Therefore, we could produce a substance that strongly inhibits H. pylori infection without toxic side effects by identifying the proper dose of DPI to treat H. pylori. Additionally, DPI has excellent antibacterial effects even in multidrug-resistant H. pylori. These advantages could help DPI serve as a novel agent in the future. Although we identified a novel H. pylori chemical inhibitor, additional steps are needed to develop such a substance into a viable drug because of several limitations. First, DPI is a non-specific NADPH inhibitor, so drug development is somewhat limited. Second, the interactions between DPI and other medications (PPIs and other antibiotics) for treating H. pylori have not been elucidated. Finally, the effects of an acidic environment on the activities of anti-H. pylori agent have not yet been considered. The high environmental pH of the stomach may affect the susceptibility of these bacteria to antibiotics. Further research is needed to examine the effects of pH on anti-H. pylori activity in different agents. Animal experiments will also be needed to determine whether substances such as DPI are effective under in vivo conditions.

In conclusion, DPI shows a potent MIC value against H. pylori, suggesting that it might be a promising new agent for that could be used to significantly improve anti-H. pylori treatment success and eradicate this pathogen.

ACKNOWLEDGEMENTS

This work was supported by a grant from the Asan Institute for Life Sciences (number: 2013-348, Asan Medical Center, Seoul, Korea) and Il-Yang Pharmaceutical (GCU 2014–5115).

Fig 1.

Figure 1.Array
Gut and Liver 2017; 11: 648-654https://doi.org/10.5009/gnl16503

Table 1 Antimicrobial Susceptibilities of the Tested Helicobacter pylori Strains

StrainResistanceMIC, μg/mL

CLAAMOMETLEVDPI
HP43504MET 0.03  <0.03 1280.25 <0.03 
CS_S1-0.030.12540.25<0.03
CS_S2-0.030.12540.25<0.03
CS_S3-0.03<0.0320.25<0.03
CS_C1CLA640.2540.5<0.03
CS_C2CLA320.12540.5<0.03
CS_C3CLA64<0.0320.25<0.03
CS_M1MET0.06<0.031280.5<0.03
CS_M2MET0.06<0.031280.5<0.03
CS_M3MET0.060.1251280.5<0.03
CS_L1LEV0.030.25216<0.03
CS_L2LEV0.060.125232<0.03
CS_L3LEV0.06<0.0348<0.03
CS_A1AMO0.06140.25<0.03
CS_A2AMO0.06240.25<0.03
CS_A3AMO0.03440.25<0.03
CS_CM1CLA/MET32<0.031280.5<0.03
CS_CM2CLA/MET64<0.03160.25<0.03
CS_CM3CLA/MET320.03640.25<0.03
CS_CA1CLA/AMO64120.25<0.03
CS_CA2CLA/AMO64140.25<0.03
CS_CA3CLA/AMO32210.25<0.03
CS_CMLCLA/MET/LEV1280.0612864<0.03
CS_CMACLA/MET/AMO 641640.5<0.03

MIC, minimal inhibitory concentration; CLA, clarithromycin; AMO, amoxicillin; MET, metronidazole; LEV, levofloxacin; DPI, diphenyleneiodonium; HP, Helicobacter pylori; CS, clinical strain.


Table 2 Antimicrobial Susceptibility of Gram-Negative and Gram-Positive Pathogenic Bacteria to DPI and Vancomycin

StrainMIC, μg/mL

DPIVancomycin
Gram-positiveStaphylococcus aureus 29213 (MSSA) 11
Staphylococcus aureus 33591 (MRSA)11
Enterococcus faecalis 2921214
Staphylococcus epidermidis 1222812
Gram-negative Escherichia coli 259220.5>32
Pseudomonas aeruginosa 278532>32
Acinetobacter baumannii 196061>32
Salmonella enteritidis 130761>32
Salmonella typhimurium 133111>32
Enterobacter cloacae 130491>32

DPI, diphenyleneiodonium; MIC, minimal inhibitory concentration; MSSA, methicillin-sensitive Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus.


References

  1. Hentschel, E, Brandstätter, G, and Dragosics, B (1993). Effect of ranitidine and amoxicillin plus metronidazole on the eradication of Helicobacter pylori and the recurrence of duodenal ulcer. N Engl J Med. 328, 308-312.
    Pubmed CrossRef
  2. Suerbaum, S, and Josenhans, C (2007). Helicobacter pylori evolution and phenotypic diversification in a changing host. Nat Rev Microbiol. 5, 441-452.
    Pubmed CrossRef
  3. Chung, JW, Lee, GH, and Han, JH (2011). The trends of one-week first-line and second-line eradication therapy for Helicobacter pylori infection in Korea. Hepatogastroenterology. 58, 246-250.
    Pubmed
  4. Graham, DY, and Fischbach, L (2010). Helicobacter pylori treatment in the era of increasing antibiotic resistance. Gut. 59, 1143-1153.
    Pubmed CrossRef
  5. Gisbert, JP, and Calvet, X (2011). Review article: non-bismuth quadruple (concomitant) therapy for eradication of Helicobater pylori. Aliment Pharmacol Ther. 34, 604-617.
    Pubmed CrossRef
  6. Chung, JW, Jung, YK, and Kim, YJ (2012). Ten-day sequential versus triple therapy for Helicobacter-pylori eradication: a prospective, open-label, randomized trial. J Gastroenterol Hepatol. 27, 1675-1680.
    Pubmed CrossRef
  7. Chung, JW, Ha, M, and Yun, SC (2013). Meta-analysis: sequential therapy is superior to conventional therapy for Helicobacter pylori infection in Korea. Korean J Gastroenterol. 62, 267-271.
    Pubmed CrossRef
  8. Malfertheiner, P, Megraud, F, and O’Morain, CA (2012). Management of Helicobacter pylori infection: the Maastricht IV/Florence consensus report. Gut. 61, 646-664.
    Pubmed CrossRef
  9. Ierardi, E, Giorgio, F, Losurdo, G, Di Leo, A, and Principi, M (2013). How antibiotic resistances could change Helicobacter pylori treatment: a matter of geography?. World J Gastroenterol. 19, 8168-8180.
    Pubmed KoreaMed CrossRef
  10. Kim, SY, Choi, DJ, and Chung, JW (2015). Antibiotic treatment for Helicobacter pylori: is the end coming?. World J Gastrointest Pharmacol Ther. 6, 183-198.
    Pubmed KoreaMed CrossRef
  11. Molina-Infante, J, and Shiotani, A (2015). Practical aspects in choosing a Helicobacter pylori therapy. Gastroenterol Clin North Am. 44, 519-535.
    Pubmed CrossRef
  12. Jeong, MH, Chung, JW, and Lee, SJ (2012). Comparison of rifabutin- and levofloxacin-based third-line rescue therapies for Helicobacter pylori. Korean J Gastroenterol. 59, 401-406.
    Pubmed CrossRef
  13. Heep, M, Rieger, U, Beck, D, and Lehn, N (2000). Mutations in the beginning of the rpoB gene can induce resistance to rifamycins in both Helicobacter pylori and Mycobacterium tuberculosis. Antimicrob Agents Chemother. 44, 1075-1077.
    Pubmed KoreaMed CrossRef
  14. Navarro-Rodriguez, T, Silva, FM, and Barbuti, RC (2013). Association of a probiotic to a Helicobacter pylori eradication regimen does not increase efficacy or decreases the adverse effects of the treatment: a prospective, randomized, double-blind, placebo-controlled study. BMC Gastroenterol. 13, 56.
    Pubmed KoreaMed CrossRef
  15. Mirzaee, V, and Rezahosseini, O (2012). Randomized control trial: comparison of triple therapy plus probiotic yogurt vs. standard triple therapy on Helicobacter pylori eradication. Iran Red Crescent Med J. 14, 657-666.
  16. Lee, JW, Kim, N, and Kim, JM (2013). Prevalence of primary and secondary antimicrobial resistance of Helicobacter pylori in Korea from 2003 through 2012. Helicobacter. 18, 206-214.
    CrossRef
  17. Watamoto, T, Egusa, H, Sawase, T, and Yatani, H (2015). Screening of pharmacologically active small molecule compounds identifies antifungal agents against Candida biofilms. Front Microbiol. 6, 1453.
    CrossRef
  18. Altaf, M, Miller, CH, Bellows, DS, and O’Toole, R (2010). Evaluation of the Mycobacterium smegmatis and BCG models for the discovery of Mycobacterium tuberculosis inhibitors. Tuberculosis (Edinb). 90, 333-337.
    CrossRef
  19. Lucumi, E, Darling, C, and Jo, H (2010). Discovery of potent small-molecule inhibitors of multidrug-resistant Plasmodium falciparum using a novel miniaturized high-throughput luciferase-based assay. Antimicrob Agents Chemother. 54, 3597-3604.
    Pubmed KoreaMed CrossRef
  20. Che, P, Wang, L, and Li, Q (2009). The development, optimization and validation of an assay for high throughput antiviral drug screening against Dengue virus. Int J Clin Exp Med. 2, 363-373.
  21. Jean, B (2015). Performance standards for antimicrobial susceptibility testing: 25th informational supplement. Wayne: Clinical and Laboratory Standards Institute
  22. Makobongo, MO, Gilbreath, JJ, and Merrell, DS (2014). Nontraditional therapies to treat Helicobacter pylori infection. J Microbiol. 52, 259-272.
    Pubmed CrossRef
  23. Bhunia, AK, Han, H, Snowden, A, and Chatterjee, S (1997). Redox-regulated signaling by lactosylceramide in the proliferation of human aortic smooth muscle cells. J Biol Chem. 272, 15642-15649.
    Pubmed CrossRef
  24. Wang, Q, Chu, CH, and Oyarzabal, E (2014). Subpicomolar diphenyleneiodonium inhibits microglial NADPH oxidase with high specificity and shows great potential as a therapeutic agent for neurodegenerative diseases. Glia. 62, 2034-2043.
    Pubmed KoreaMed CrossRef
  25. Aldieri, E, Riganti, C, and Polimeni, M (2008). Classical inhibitors of NOX NAD(P)H oxidases are not specific. Curr Drug Metab. 9, 686-696.
    Pubmed CrossRef
  26. Cho, SO, Lim, JW, Kim, KH, and Kim, H (2011). Diphenyleneiodonium inhibits the activation of mitogen-activated protein kinases and the expression of monocyte chemoattractant protein-1 in Helicobacter pylori-infected gastric epithelial AGS cells. Inflamm Res. 60, 501-507.
    CrossRef
  27. Cha, B, Lim, JW, Kim, KH, and Kim, H (2011). 15-Deoxy-D12,14-prostaglandin J2 suppresses RANTES expression by inhibiting NADPH oxidase activation in Helicobacter pylori-infected gastric epithelial cells. J Physiol Pharmacol. 62, 167-174.
    Pubmed
  28. Cho, SO, Lim, JW, and Kim, H (2013). Red ginseng extract inhibits the expression of MCP-1 and iNOS in Helicobacter pylori-infected gastric epithelial cells by suppressing the activation of NADPH oxidase and Jak2/Stat3. J Ethnopharmacol. 150, 761-764.
    Pubmed CrossRef
  29. Cho, SO, Lim, JW, and Kim, H (2015). Diphenyleneiodonium inhibits apoptotic cell death of gastric epithelial cells infected with Helicobacter pylori in a Korean isolate. Yonsei Med J. 56, 1150-1154.
    Pubmed KoreaMed CrossRef
  30. Tominaga, K, Kawahara, T, and Sano, T (2007). Evidence for cancer-associated expression of NADPH oxidase 1 (Nox1)-based oxidase system in the human stomach. Free Radic Biol Med. 43, 1627-1638.
    Pubmed CrossRef
  31. Jones, RD, Hancock, JT, and Morice, AH (2000). NADPH oxidase: a universal oxygen sensor?. Free Radic Biol Med. 29, 416-424.
    Pubmed CrossRef
  32. Jang, SH, Lim, JW, Morio, T, and Kim, H (2012). Lycopene inhibits Helicobacter pylori-induced ATM/ATR-dependent DNA damage response in gastric epithelial AGS cells. Free Radic Biol Med. 52, 607-615.
    CrossRef
  33. World Gastroenterology Organisation (2011). World Gastroenterology Organisation global guideline: Helicobacter pylori in developing countries. J Clin Gastroenterol. 45, 383-388.
    Pubmed CrossRef
  34. Furuta, T, Sugimoto, M, and Kodaira, C (2014). Sitafloxacin-based third-line rescue regimens for Helicobacter pylori infection in Japan. J Gastroenterol Hepatol. 29, 487-493.
    CrossRef
  35. Murakami, K, Furuta, T, and Ando, T (2013). Multi-center randomized controlled study to establish the standard third-line regimen for Helicobacter pylori eradication in Japan. J Gastroenterol. 48, 1128-1135.
    Pubmed CrossRef
  36. Basu, PP, Rayapudi, K, Pacana, T, Shah, NJ, Krishnaswamy, N, and Flynn, M (2011). A randomized study comparing levofloxacin, omeprazole, nitazoxanide, and doxycycline versus triple therapy for the eradication of Helicobacter pylori. Am J Gastroenterol. 106, 1970-1975.
    Pubmed KoreaMed CrossRef
  37. Watson, JB, and Moss, SF (2011). Will H. pylori stagger under the weight of this LOAD? A novel but expensive eradication regimen. Am J Gastroenterol. 106, 1976-1977.
    Pubmed CrossRef
  38. Vale, FF, and Oleastro, M (2014). Overview of the phytomedicine approaches against Helicobacter pylori. World J Gastroenterol. 20, 5594-5609.
    Pubmed KoreaMed CrossRef
  39. Lopes, D, Nunes, C, Martins, MC, Sarmento, B, and Reis, S (2014). Eradication of Helicobacter pylori: past, present and future. J Control Release. 189, 169-186.
    Pubmed CrossRef
  40. Gao, W, Thamphiwatana, S, Angsantikul, P, and Zhang, L (2014). Nanoparticle approaches against bacterial infections. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 6, 532-547.
    Pubmed KoreaMed CrossRef
  41. Erah, PO, Goddard, AF, Barrett, DA, Shaw, PN, and Spiller, RC (1997). The stability of amoxycillin, clarithromycin and metronidazole in gastric juice: relevance to the treatment of Helicobacter pylori infection. J Antimicrob Chemother. 39, 5-12.
    Pubmed CrossRef
  42. Labenz, J (2001). Current role of acid suppressants in Helicobacter pylori eradication therapy. Best Pract Res Clin Gastroenterol. 15, 413-431.
    Pubmed CrossRef
  43. Wang, Q, Qian, L, and Chen, SH (2015). Post-treatment with an ultra-low dose of NADPH oxidase inhibitor diphenyleneiodonium attenuates disease progression in multiple Parkinson’s disease models. Brain. 138, 1247-1262.
    Pubmed KoreaMed CrossRef
Gut and Liver

Vol.19 No.1
January, 2025

pISSN 1976-2283
eISSN 2005-1212

qrcode
qrcode

Share this article on :

  • line

Popular Keywords

Gut and LiverQR code Download
qr-code

Editorial Office