Evaluation of the Antimicrobial Effects of Several Isothiocyanates on Helicobacter pylori

• Abstract
• Introduction
• Materials and Methods
• Bacterial strains
• Chemicals
• Determination of minimal inhibitory concentrations
• Determination of bactericidal activity
• Results
• Discussion
• References

Abstract

The antibacterial activity of sulforaphane [4-(methylsulfinyl)butyl isothiocyanate] has been previously described. We have analysed the activities of 12 isothiocyanates (ITC) including sulphoraphane on 25 strains of Helicobacter pylori using an agar dilution assay. In addition, bactericidal effects against H. pylori were determined for the 6 most active ITCs, both directly and against intracellular bacteria in cultured human epithelial (HEp-2) cells. The MIC₉₀ values for these ITCs ranged between 4 and 32 μg/mL and four of the most potent compounds exhibited bactericidal activity against both extra- and intracellular bacteria. Overall, our data indicate that ITCs have a potent antibacterial effect against H. pylori and these naturally occurring phytochemicals might have potential as novel therapeutic agents for H. pylori eradication.

Introduction

Helicobacter pylori, which causes chronic gastritis as well as gastric and duodenal ulcers, is also an important risk factor for development of gastric cancer and mucosa-associated lymphoid tissue lymphoma. About half of the world’s population is supposed to be infected, particularly in developing countries where the prevalence of the infection may reach 90 % [1]. H. pylori is difficult to eradicate in 15 - 20 % of patients even if multidrug therapies consisting of two or more antibiotics (e. g., amoxicillin, clarithromycin or metronidazole) with an inhibitor of acid secretion are used. This level of treatment failure appears to be due to the development of resistance to these antibiotics and poor patient compliance [1]. It may also be related to the persistence of organisms within gastric epithelial cells [2]. Therefore, there is a need for new antimicrobial agents that can be used to treat H. pylori infection.

The isothiocyanate sulforaphane [4-(methylsulfinyl)butyl isothiocyanate; (-)-1-isothiocyanato-(4R)-(methylsulfinyl)butane], a naturally occurring inducer of chemoprotective and antioxidant enzymes and a potent inhibitor of experimental carcinogenesis [3], [4], [5], [6], [7] was recently shown to be active against H. pylori [3], [8].

Broccoli sprouts, young plants of the family Cruciferae, are a rich source of glucoraphanin, the naturally occurring glucosinolate precursor of sulforaphane [4], [7]. Others plants, especially Cruciferae, contain glucosinolates that are converted into isothiocyanates by the action of myrosinase (E.C 3.2.3.1). Glucosinolates may act locally [9], or may be absorbed after conversion into isothiocyanates [4], [10]. It has been reported than isothiocyanates other than sulforaphane have antibacterial or chemoprotective activities [11], [12], [13]. They, too, may be of interest for the prevention and/or the treatment of H. pylori infection. However, the activities of these compounds against H. pylori are unknown at the present time.

In this study, we have evaluated in vitro the activity of the naturally occurring L-sulforaphane, its synthetic enantiomer (D-sulforaphane) and a synthetic chiral mixture (D,L-sulforaphane) against H. pylori both in pure culture and in cultured mammalian epithelial (HEp-2) cells, compared to 11 other isothiocyanates (Fig. [1]).

Materials and Methods

Bacterial strains

Two reference strains (26 695 and ATCC 43 504) and 23 clinical strains (LBN 202 to LBN 224) of H. pylori were used in this study. The clinical isolates were obtained in 2000 and 2001 at the University Hospital Center of Nancy, France, from individual patients with gastritis and gastric or duodenal ulcers. Identity as H. pylori was established by Gram stain, and oxidase, catalase and urease production. All strains were stored in Brucella broth (Oxoid, Basingstoke, England) containing 15 % (wt/vol) glycerol at -80 °C until use. Experiments were all performed at 37 °C in a microaerophilic (5 % O₂, 15 % CO₂, 80 % N₂) atmosphere and the culture medium was Columbia agar containing 10 % horse blood.

Chemicals

Iberin (1), cheirolin (2), erucin (3), D,L-sulforaphane (4), D-sulforaphane (4), L-sulforaphane (4), L-sulforaphene (5), erysolin (6), berteroin (7), alyssin (8), hirsutin (9), phenylethyl isothiocyanate (PEITC) (10) and benzyl isothiocyanate (BITC) (11) were obtained from Coger (Paris, France). 4-(α-L-rhamnopyranosyloxy)benzyl isothiocyanate (4RBITC) (12) was produced by myrosinase hydrolysis of the cognate glucosinolate isolated from seeds of the horseradish tree, Moringa oleifera as described previously [14]. The structures of all isothiocyanates used in this study are shown in Fig. [1]. Purity, common and chemical names, and botanical sources are included in Table [1]. Amoxicillin was supplied by GlaxoSmithKline and clarithromycin by Sanofi-Synthelabo (Paris). Metronidazole was purchased from Sigma-Aldrich (St Quentin, France). Stock solutions of isothiocyanates were prepared in acetonitrile, and the other antibiotics were prepared as recommended by their manufacturers. Subsequent dilution was made into sterile water.

Determination of minimal inhibitory concentrations

Minimal inhibitory concentrations (MICs) of isothiocyanates and conventional antibiotics were determined by using the agar dilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) [15]. Two-fold dilutions of each drug ranging from 64 to 0.03 μg/mL were tested. The MIC was defined as the lowest concentration of each compound that resulted in no visible growth after 3 days of incubation. Clarithromycin resistance was defined by an MIC ≥ 1 μg/mL, as recommended by NCCLS [15]. Resistance breakpoints for metronidazole and amoxicillin were defined as > 8 μg/mL and > 0.5 μg/mL, respectively, as described in [3].

Determination of bactericidal activity

The bactericidal activities of six isothiocyanates (2, 5, 6, 7, 9, and 12) showing the lowest modal MICs (Table [2]) were determined against H. pylori strains 26 695 and LBN 211. This evaluation was performed by monitoring, as previously described [3], the viability of H. pylori in extracellular (broth) or intracellular (human epithelial cells) locations after variable incubation times with each isothiocyanate at 0.5 × , 1 × , and 5 × MIC. To evaluate the extracellular bactericidal activity, time-to-kill studies were performed in Brucella broth supplemented with 10 % fetal calf serum with a final inoculum of approximately 5 × 10⁶ CFU/mL. After 0, 8, 16, and 24 h of incubation at 37 °C under microaerophilic conditions and gentle shaking, samples were serially diluted and plated onto Columbia blood agar. After 5 days of incubation under microaerophilic conditions at 37 °C, colonies were counted. The determination of intracellular bactericidal activity was performed in a human epithelial cell line (HEp-2; ATCC CCL 23). Briefly, 10⁶ HEp-2 cells were exposed to 10⁸ CFU of H. pylori strains in EMEM with FCS for 12 h at 37 °C in 5 % CO₂. Cells were gently washed with Hank’s balanced salt solution (HBSS) to remove any non-adherent bacteria, and then incubated with a solution containing 100 μg/mL of gentamicin for 1.5 h to kill extracellular bacteria. Monolayers were then washed 6 times with HBSS, and incubated in culture medium containing one of the compounds tested at 37 °C in 5 % CO₂. After 0, 4, 8, 12, and 24 h, wells were washed 6 times with HBSS, and cells were lysed with distilled water. At each time, samples were serially diluted and plated onto Columbia blood agar. After 5 days of incubation under microaerophilic conditions at 37 °C, colonies were counted. For all assays, tests were performed in duplicate and the limit of detection was 20 CFU/mL. Results are expressed as mean log₁₀ CFU/mL. Bactericidal activity was defined as a greater than 1,000-fold reduction in viable CFU compared to the inoculum.

Results

D,L-Sulforaphane, D-sulforaphane, and L-sulforaphane (compound 4) exhibited similar and high antibacterial activities (overall MIC range: 0.06 - 8 μg/mL) against all strains tested. For these three compounds, MICs varied by less than one single serial dilution step (Table [2]). The activities of 4 and 12 were greater (4- to 8-fold lower MICs) than those of the other compounds tested. Among the latter ITCs, 9 exhibited the highest activity (MIC₅₀ : 4 μg/mL - MIC₉₀ : 8 μg/mL). For all isothiocyanates tested, no differences in MIC₉₀ were found between clarithromycin- and metronidazole-susceptible strains and either clarithromycin-resistant strains (4 strains), metronidazole-resistant strains (4 strains) or clarithromycin- and metronidazole-resistant strains (4 strains) (data not shown).

At a concentration of 5 × MIC, all ITCs tested except 5 exhibited a significant bactericidal activity against the two strains tested after 8 h (2, 9 and 12) or 16 h (6 and 7) of incubation (Table [3]). Compound 5 only exhibited a bactericidal effect against H. pylori LBN 211 at supra-inhibitory concentrations after 16 h of incubation. At a concentration of 1 × MIC, 12 was bactericidal only against H. pylori LBN 211 after 24 h of incubation, whereas 9 was only bactericidal against H. pylori 26 695 after 16 h of incubation (Fig. [2]). The other ITCs studied did not exhibit any significant bactericidal activities at a concentration of 1 × MIC.

Against intracellular bacteria, 2, 6, 9 and 12 exhibited a significant bactericidal effect after 24 h at a concentration of 5 × MIC (Table [3]). For these compounds, no significant bactericidal effect was observed at the lower concentrations tested while 5 and 7 did not exhibit any intracellular bactericidal effect at any concentration tested.

Discussion

In the present work, we showed for the first time that ITCs other than sulforaphane also exhibit a potent effect against H. pylori. It is noteworthy that, as for sulforaphane, this inhibitory potency was not related to the resistance of strains to clarithromycin and/or metronidazole. Among the compounds tested in the present study, 4RBITC (12) and sulforaphane (4) exhibited the highest inhibitory activity against H. pylori. However, the less active molecules (1, 3, 6 and 10) were as potent as other plant-derived or phytochemicals such as epigallocatechin gallate from green tea, MIC₉₀ = 32 μg/mL [16]; resveratrol from red wine, MIC₉₀ = 25 μg/mL [17]; or allixin from garlic bulb, MIC₉₀ = 25 μg/mL [18]. The fact that all ITCs tested were active against H. pylori suggests that this potency may be related to the presence of the ITC group. This activity was not dependent upon chirality as similar antibacterial effects were observed with optical enantiomers of 4. This compound and its homologues (1, 8 and 9) exhibited variable antibacterial activity while the desaturation of the side chain, in the case of 5, was associated with a slight decrease of the inhibitory activity. These findings indicate that the number of carbon atoms in the side chain may be important for activity. Our results also suggest that the presence of an aromatic group in the side chain reduces antibacterial activity, since 10 and 11 were less active than 4. However 12, with a rhamnose moiety esterified to its benzyl side chain, was as active as 4 against H. pylori. The quinone reductase induction potential of 12 was recently shown to be considerably higher than that of 4 in cultured murine hepatoma cells, but lower in cultured mammalian cells [19]. The differential activities may be related to the facility with which these ITCs are taken up into cells, an effect that would be dramatically modified by the introduction of a side chain sugar substitution (e. g., in 12). Clearly, this effect has been known to be important in the quinone reductase inducer potency of ITCs on mammalian cells [6]. Whether bacterial uptake is related to antibacterial potency is currently under study in our laboratories. In the present study, we also found that, in addition to sulforaphane (4), four of the most inhibitory ITCs tested (2, 6, 9, and 12) were highly bactericidal against extra- and intracellular forms of H. pylori at concentrations of 5 × MIC. These results are similar to those previously obtained with sulforaphane [3]. Concentrations which are necessary to achieve such an effect could easily be achieved in vivo. For example, a person consuming a small portion (30 g) of broccoli sprouts consumes in the order of 180 μmoles of glucoraphanin, which when converted to sulforaphane accounts for ca. 32 mg of intake. Distributed across one litre of fluid in the stomach, this is equivalent to 32 μg/mL, for a short period of direct exposure. Consumption of a small portion of edible plant seeds in many cases provides an even richer source of glucosinolates than older plants or sprouts grown from these seeds [4], and transient levels in the stomach could reach as high as a few hundreds μg/mL. Exposure of H. pylori to these compounds may be equally, or even more, important via absorption and redistribution through the enterohepatic circulation. It has also been shown that the consumption of vegetables, especially cruciferous vegetables, and fruits reduces the incidence of cancer [20]. Some of the chemopreventive effects of fruits and vegetable are linked to their content of components such as glucosinolates/isothiocyanates. Among ITCs tested in this study, 4 (highest content in broccoli sprouts, Brassica oleracea L. var. italica, Cruciferae), 5 (highest content in radish seeds, Raphanus sativus L., Cruciferae), 9 (high content in cress, Lepidium sativum, and other species), 10 (high content in watercress and radish), 11 (high content in nasturtium, Tropaeolum majus L., Tropaeolacae, and watercress, Nasturtium officinale R.Br, Cruciferae), and 12 (highest content in Moringa oleifera Lam, Moringacae) (see Table [1]) have all been shown to exhibit cancer-preventive effects [11], [13], [21], [22].

Thus, the parallel findings that 4, 9 and 12 exhibit strong direct antibacterial activity against H. pylori, and are also chemoprotective by indirect measures, offers suggestive evidence that these effects might operate synergistically to treat H. pylori infection and to prevent gastric cancer. It has been recently shown in animal models that sulforaphane contributes in vivo to the eradication of H. pylori and to the prevention of chemically induced stomach tumors [3]. Similar evidence should be sought for the other two isothiocyanates (9 and 12) highlighted herein before investigating the activity of these compounds in humans.

References

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  PubMedSearch in Google Scholar
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• 12 Lin C M, Kim J, Du W X, Wei C I. Bactericidal activity of isothiocyanate against pathogens on fresh produce.  J Food Prot. 2000;  63 25-30
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• 13 Shishu Singla A K, Kaur I P. Inhibition of mutagenicity of food-derived heterocyclic amines by sulforaphene - an isothiocyanate isolated from radish.  Planta Med. 2003;  69 184-6
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• 15 National Committee for Clinical Laboratory S tandards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, Approved Standard M7-A5. NCCLS Wayne; 2003: 1-50
  Search in Google Scholar
• 16 Mabe K, Yamada M, Oguni I, Takahashi T. In vitro and in vivo activities of tea catechins against Helicobacter pylori .  Antimicrob Agents Chemother. 1999;  43 1788-91
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  CrossrefPubMedSearch in Google Scholar
• 19 Fahey J W, Dinkova-Kostova A T, Talalay P. The ”Prochaska” microtiter plate bioassay for inducers of NQO1. In: Sies H, Packer L, editors Methods in enzymology. Vol 382, part B San Diego; Elsevier Science 2004: pp 243-58
  Search in Google Scholar
• 20 Serafini M, Bellocco R, Wolk A, Ekstrom A M. Total antioxidant potential of fruit and vegetables and risk of gastric cancer.  Gastroenterology. 2002;  123 985-91
  CrossrefPubMedSearch in Google Scholar
• 21 Rose P, Faulkner K, Williamson G, Mithen R. 7-Methylsulfinylheptyl and 8-methylsulfinyloctyl isothiocyanates from watercress are potent inducers of phase II enzymes.  Carcinogenesis. 2000;  21 1983-8
  CrossrefPubMedSearch in Google Scholar
• 22 Guevara A P, Vargas C, Sakurai H, Fujiwara Y, Hashimoto K, Maoka T. et al . An antitumor promoter from Moringa oleifera Lam.  Mut Res. 1999;  440 181-8
  PubMedSearch in Google Scholar

Prof. Alain Lozniewski

Laboratoire de Bactériologie

Hôpital Central

29 Avenue du Maréchal de Lattre de Tassigny

54035 Nancy Cedex

France

Phone: +33-3-83-85-18-14

Fax: +33-3-83-85-26-73

Email: a.lozniewski@chu-nancy.fr

References

• 1 Ruggiero P, Peppoloni S, Berti D, Rappuoli R, Del Giudice G. New strategies for the prevention and treatment of Helicobacter pylori infection.  Expert Opin Investig Drugs. 2002;  11 1127-38
  CrossrefPubMedSearch in Google Scholar
• 2 Engstrand L, Graham D, Scheynius A, Genta R M, El-Zaatari F. Is the sanctuary where Helicobacter pylori avoids antibacterial treatment intracellular?.  Am J Clin Pathol. 1997;  108 504-9
  PubMedSearch in Google Scholar
• 3 Fahey J W, Haristoy X, Dolan P M, Kensler T W, Scholtus I, Stephenson K K. et al . Sulforaphane inhibits extracellular, intracellular and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumours.  Proc Natl Acad Sci USA. 2002;  99 7610-5
  CrossrefPubMedSearch in Google Scholar
• 4 Fahey J W, Zalcmann A T, Talalay P. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants.  Phytochemistry 2001; 56 : 5 - 51, and correction. 2002;  59 237
  PubMedSearch in Google Scholar
• 5 Talalay P, Fahey J W. Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism.  J Nutr. 2001;  131 S3027-33
  PubMedSearch in Google Scholar
• 6 Zhang Y, Talalay P. Mechanism of differential potencies of isothiocyanates as inducers of anticarcinogenic phase 2 enzymes.  Cancer Res. 1998;  58 4632-9
  PubMedSearch in Google Scholar
• 7 Fahey J W, Zhang Y, Talalay P. Broccoli sprouts: an exceptionally rich source of inducers of enzyme that protect against chemical carcinogens.  Proc Natl Acad Sci USA. 1997;  94 10 367-72
  PubMedSearch in Google Scholar
• 8 Haristoy X, Angioi-Duprez K, Duprez A, Lozniewski A. Efficacy of sulforaphane in eradicating Helicobacter pylori in human gastric xenografts implanted in nude mice.  Antimicrob Agents Chemother. 2003;  47 3982-4
  CrossrefPubMedSearch in Google Scholar
• 9 Halliwell B, Zhao K, Whiteman K. The gastrointestinal tract: a major site of antioxidant action?.  Free Radical Res. 2000;  33 819-30
  PubMedSearch in Google Scholar
• 10 Shapiro T A, Fahey J W, Wade K L, Stephenson K K, Talalay P. Chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in human.  Cancer Epidemiol Biomarkers Prev. 2001;  10 501-8
  PubMedSearch in Google Scholar
• 11 Hecht S S, Kenney P MJ, Wang M, Upadhyaya P. Benzyl isothiocyanate: an effective inhibitor of polycyclic aromatic hydrocarbon tumorigenesis in A/J mouse lung.  Cancer Lett. 2002;  187 87-94
  CrossrefPubMedSearch in Google Scholar
• 12 Lin C M, Kim J, Du W X, Wei C I. Bactericidal activity of isothiocyanate against pathogens on fresh produce.  J Food Prot. 2000;  63 25-30
  PubMedSearch in Google Scholar
• 13 Shishu Singla A K, Kaur I P. Inhibition of mutagenicity of food-derived heterocyclic amines by sulforaphene - an isothiocyanate isolated from radish.  Planta Med. 2003;  69 184-6
  Thieme ConnectPubMedSearch in Google Scholar
• 14 Fahey J W, Wade K L, Stephenson K K, Chou F E. Separation and purification of glucosinolates from crude plant homogenates by high-speed counter-current chromatography.  J Chromatogr A. 2003;  996 85-93
  CrossrefPubMedSearch in Google Scholar
• 15 National Committee for Clinical Laboratory S tandards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, Approved Standard M7-A5. NCCLS Wayne; 2003: 1-50
  Search in Google Scholar
• 16 Mabe K, Yamada M, Oguni I, Takahashi T. In vitro and in vivo activities of tea catechins against Helicobacter pylori .  Antimicrob Agents Chemother. 1999;  43 1788-91
  PubMedSearch in Google Scholar
• 17 Mahady G B, Pendland S L. Resveratrol inhibits the growth of Helicobacter pylori in vitro .  Am J Gastroenterol. 2000;  95 1849
  PubMedSearch in Google Scholar
• 18 Mahady G B, Matsuura H, Pendland S L. Allixin, a phytoalexin from garlic, inhibits the growth of Helicobacter pylori in vitro .  Am J Gastroenterol. 2001;  96 3454-5
  CrossrefPubMedSearch in Google Scholar
• 19 Fahey J W, Dinkova-Kostova A T, Talalay P. The ”Prochaska” microtiter plate bioassay for inducers of NQO1. In: Sies H, Packer L, editors Methods in enzymology. Vol 382, part B San Diego; Elsevier Science 2004: pp 243-58
  Search in Google Scholar
• 20 Serafini M, Bellocco R, Wolk A, Ekstrom A M. Total antioxidant potential of fruit and vegetables and risk of gastric cancer.  Gastroenterology. 2002;  123 985-91
  CrossrefPubMedSearch in Google Scholar
• 21 Rose P, Faulkner K, Williamson G, Mithen R. 7-Methylsulfinylheptyl and 8-methylsulfinyloctyl isothiocyanates from watercress are potent inducers of phase II enzymes.  Carcinogenesis. 2000;  21 1983-8
  CrossrefPubMedSearch in Google Scholar
• 22 Guevara A P, Vargas C, Sakurai H, Fujiwara Y, Hashimoto K, Maoka T. et al . An antitumor promoter from Moringa oleifera Lam.  Mut Res. 1999;  440 181-8
  PubMedSearch in Google Scholar

Prof. Alain Lozniewski

Laboratoire de Bactériologie

Hôpital Central

29 Avenue du Maréchal de Lattre de Tassigny

54035 Nancy Cedex

France

Phone: +33-3-83-85-18-14

Fax: +33-3-83-85-26-73

Email: a.lozniewski@chu-nancy.fr