Antimicrobial activity of ellagic acid against Helicobacter pylori isolates from India and during infections in mice

Antimicrobial activity of ellagic acid against Helicobacter pylori isolates from India and during... Abstract Objectives Because of the rise in antimicrobial resistance, an inexpensive, diet-based treatment against Helicobacter pylori infection would be of great interest. The present study was performed to assess the in vitro effects of ellagic acid against clinical H. pylori strains that were resistant to antibiotics used for therapy and also to observe the morphological structure following treatment with ellagic acid. The effectiveness of ellagic acid in eradicating H. pylori infection in a murine (C57BL/6) infection model, one of the standard inbred mouse lines often used for experimental infection, was also assessed. Methods A total of 55 strains were screened. The agar dilution method was used to determine the susceptibility of isolates to test compounds. Transmission electron microscopy was used to observe the morphology following treatment with ellagic acid. The antibacterial activity of ellagic acid in an H. pylori SS1-infected mouse model and its effect on gastric mucosal injury were determined by histology and PCR. Results Ellagic acid inhibited the growth of all 55 of the H. pylori strains tested. The MIC of ellagic acid ranged from 5 to 30 mg/L, showing its bactericidal properties in vitro. Ellagic acid also demonstrated anti-H. pylori efficacy in eradication of this organism in an in vivo model, as well as restitution and repair of H. pylori-induced gastric mucosal damage. Conclusions The present study paves the way for the preventive and therapeutic use of ellagic acid against H. pylori infection and, thus, ellagic acid can be considered a promising antibacterial agent against H. pylori-associated gastroduodenal diseases in humans. Introduction Helicobacter pylori is a Gram-negative, motile, spiral pathogen that infects >50% of the global population. Infection is associated with chronic active gastritis, peptic ulcer and ultimately gastric carcinoma.1 The clinical manifestations of microbial infection depend on complex interactions established between bacterial virulence factors and the host, contributing to this complex interplay are the host’s genetic background and environmental factors, mainly diet.2 Treatment of H. pylori infection has not significantly changed over the last decade, and drug-resistant strains of H. pylori and poor patient compliance are the major causes of therapy failure.3 The consensus treatment regimen is a triple therapy consisting of a proton pump inhibitor and two antibiotics. However, the efficacy of conventional antibiotic therapy is not always satisfactory and therefore the acquisition by H. pylori isolates of high-level resistance to antibiotics, including metronidazole (antiprotozoal), clarithromycin (antibacterial) and tetracycline, could signify a serious dilemma that may reduce treatment efficacy.4,5 In the context of incomplete cure accomplished with standard triple therapy because of the increasing number of drug-resistant strains, adverse side effects such as nausea and diarrhoea,6 poor patient compliance,7 the escalating cost of the multiple antibiotics8 and some unknown factors for their ineffectiveness, there is a strong need to explore new non-antimicrobial agents that are cost-effective, safe and applicable to all H. pylori infections. Several strategies have been developed to overcome the issue of the increasing failure of H. pylori eradication therapies, namely, adoption of novel, more effective, empirical treatment, the generation of a vaccine to stimulate the host’s immune defences, adjunct use of probiotics and the development of new and more potent substances that could inhibit bacterial growth. Using a mouse-adapted H. pylori strain (Sydney strain 1, SS1), Lee et al.9 established a robust model of long-term and high bacterial load colonization in C57BL/6 mice. Although extensive studies in this H. pylori mouse model have established the feasibility of both therapeutic and prophylactic immunizations, the mechanism of immunogen-evoked protection is still inadequately understood, meaning that vaccination as a tool to eradicate H pylori is still very contentious. Numerous studies have shown that plant polyphenols are an important class of antimicrobial agents against organisms that include bacteria, viruses and protozoa.10 Ellagic acid (C14H6O8), also known as 2,3,7,8-tetrahydroxy[1]benzopyrano-[5,4,3-cde][1]benzopyran-5,10-dione, 4′,5,5′,6,6′-hexahydroxy-diphenic acid 2,6,2′,6′-dilactone (Figure 1) and benzoaric acid, is a naturally occurring dietary polyphenol, present in free form or in the form of ellagitannins or glucosides. It is generally found in certain rich dietary sources such as walnuts, pomegranates, strawberries, blackberries, cloudberries and raspberries.11 It is known to possess an array of pharmacological and biological activities including antioxidant activity,12 free radical-scavenging capacity, chemopreventive properties13 and anti-apoptotic properties,14 and has also been reported to possess considerable in vitro antibacterial activity. Numerous studies demonstrated in vitro antimicrobial efficacy of ellagic acid against H. pylori,15–18 but in vivo efficacy has not been demonstrated. Figure 1. View largeDownload slide Chemical structure of ellagic acid (2,3,7,8-tetrahydroxy[1]benzopyrano-[5,4,3-cde][1]benzopyran-5,10-dione). Figure 1. View largeDownload slide Chemical structure of ellagic acid (2,3,7,8-tetrahydroxy[1]benzopyrano-[5,4,3-cde][1]benzopyran-5,10-dione). Accordingly, the present study has been conducted: (i) to assess the efficacy of ellagic acid as an antibacterial agent against H. pylori strains isolated from patients in India; (ii) to observe alteration of H. pylori morphology upon ellagic acid treatment; and (iii) to understand the effectiveness of ellagic acid in eradicating H. pylori infection in a murine (C57BL/6) infection model. Methods Strains and culture conditions Fifty-five archived strains of H. pylori, which were isolated from antral mucosal biopsy specimens of patients aged between 18 and 80 years with gastrointestinal disease, were used for this study. H. pylori colonies were identified on the basis of typical translucent water droplet colony morphology, Gram staining and positive reactions in biochemical tests (catalase, urease and oxidase). H. pylori strains were streaked onto Petri plates containing brain heart infusion (BHI) agar (Difco Laboratories, Detroit, MI, USA) supplemented with 7% heat-inactivated horse serum (Invitrogen, NY, USA), 0.4% IsoVitaleX (Becton Dickinson, MD, USA), trimethoprim (5 mg/L), vancomycin (8 mg/L) and polymyxin B (10 mg/L). Nalidixic acid (10 mg/L) and bacitracin (200 mg/L) were added to this medium when culturing H. pylori from mouse stomachs. All plates were incubated inverted at 37°C in a microaerobic atmosphere (5% O2/10% CO2/85% N2) (double gas incubator; Heraeus, Langenselbold, Germany) for 3–6 days. Stock cultures were maintained at −70°C as suspensions of fresh exponentially growing cells suspended in BHI broth with 20% glycerol. Determination of MICs Briefly, frozen stock cultures were subcultured on BHI agar supplemented with 7% horse serum (Invitrogen, NY, USA) and incubated for 72 h under microaerophilic conditions. H. pylori cultures in the exponential phase of growth were suspended in sterile physiological saline (PBS) solution and adjusted to an optical density of 0.1 at 600 nm using a UV spectrophotometer. Briefly, the final test concentrations were 5, 10, 15, 20 and 30 mg/L for each sample. Anti-H. pylori activity of the compounds was determined by the agar dilution method. A total of 55 clinical samples and the mouse-colonizing strain SS1 were used in the susceptibility testing. Samples were inverted and incubated under humid microaerobic conditions at 37°C for 5 days. The MIC was the lowest concentration at which the compound inhibited visible bacterial growth. For quality control and comparative analyses, the antibiotics amoxicillin (Sigma Chemical Co., St Louis, MO, USA) and clarithromycin (Abbott Laboratories, Abbott Park, IL, USA) were also tested with each batch of ellagic acid. MICs of metronidazole, amoxicillin and clarithromycin were determined by the agar dilution method, as described in Mendonça et al.19 Transmission electron microscopy (TEM) analysis In order to observe the effect of ellagic acid treatment on H. pylori structure and morphology, the mouse-colonizing strain SS1 was grown for 12 h under microaerophilic conditions (10% CO2/5% O2/85% N2) in the presence or absence of various concentrations of ellagic acid (5, 10 and 15 μM) while shaking the bacteria on a shaker at 125 rpm. Then, control and ellagic acid-treated H. pylori cultures were observed using TEM. Briefly, samples were washed in PBS at 3000 g for 5 min and bacterial pellets fixed by re-suspending in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (Ted Pella, Redding, CA, USA) at 4°C overnight. Dehydration and embedding in Agar 100 resin (Agar Scientific, UK) were performed according to a standard protocol. After embedding in resin, blocks were kept at 60°C for 24 h. Ultra-thin sections were prepared using a Leica Ultracut UCT microtome and stained with uranyl acetate and lead citrate, and bacterial cells were observed with a Tecnai 12 Biotwin transmission electron microscope (Fei, the Netherlands) operating at 80 kV.20 Mouse challenge study followed by treatment with ellagic acid Six-week-old, pathogen-free C57BL/6 male mice weighing 20 ± 5 g bred in-house were used in all in vivo experiments. Animals were housed in standard conditions with fresh sterile bedding, fed a conventional pellet diet and provided with sterile water ad libitum unless otherwise stated for the duration of the experiment. The animal facility had a regulated room temperature (21–24°C) and humidity (55% ± 10%) and an artificial standard 12 h light/12 h dark cycle. All animal studies were conducted in compliance with protocols approved by the Animal Ethics Committee of NICED (registration number 68/GO/ReBi/S/99/CPCSEA; permit number NICED/CPCSEA/AW/(225)/2013-IAEC/AM-1). Six animals per group were challenged with the bacterial strain SS1, which is known to colonize the mouse gastric mucosa efficiently. The strain was revived on Petri plates containing brain heart infusion agar as described earlier for mouse infection.21 Bacterial cultures were harvested by centrifugation at 8000 g for 5 min and resuspended in sterile PBS for the animal challenge. Each mouse received 0.2 mL (108 cfu/mL)21 of bacterial suspension by oral gavage three times at 2 day intervals using a feeding needle, excluding the no-H. pylori controls, which received sterile PBS. At 2 weeks following the initial bacterial challenge, the mice were divided into three groups: control group (n = 6); infected group (n = 6); and infected group treated with ellagic acid (n = 6). The experimental group of mice was orogastrically fed with ellagic acid (10 mg/kg) once daily for 1 week consecutively, while the non-infected control mice received sterile PBS. At 6 weeks post-infection, mice were euthanized by cervical dislocation. The stomach was removed and dissected longitudinally along the greater curvature, and the gastric content was removed. A small section of the stomach was analysed for a urease test, H. pylori enumeration, PCR and a histological evaluation study as described previously.22–24 For quantitative culture, the tissues were homogenized in PBS using a disposable pellet pestle (Tarson), homogenates of each suspension were spread on BHI agar medium and H. pylori colonies were enumerated by the procedure described above. Data are presented as cfu/g of stomach. Histological evaluation Stomachs were aseptically removed and dissected along the greater curvature in the different groups of mice, washed vigorously in PBS and preserved in 10% neutral buffered formalin for histological evaluation. The tissue samples were dehydrated with alcohol and xylene prior to embedding in paraffin wax and sectioning. The sections (5 μm) were cut with a microtome, mounted on glass slides with mounting medium, stained with haematoxylin and eosin and observed under a microscope. Inflammation was observed and graded from 0 to 3 (where 0 means no inflammation, 1 is mild inflammation, 2 is moderate inflammation and 3 is high inflammation) by a pathologist in a blinded fashion as described elsewhere.25 PCR from H. pylori-infected stomach tissue Genomic DNA from mouse gastric tissues was extracted with a DNeasy tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA content and purity were determined by measuring the absorbance at 260–280 nm. The presence or absence of the vacA gene and the specificity for the mouse genome were confirmed using the gene-specific PCR primers listed in Table 1 and DNA from the respective gastric tissues. PCR amplification was carried out in a final volume of 20 μL containing 40 ng of template DNA, 2 μL of 10× PCR buffer (Roche), 2.5 mM dNTPs (Roche), 10 pmol of appropriate primers and 1 U of Taq polymerase (Roche). The cycling programme was: initial denaturation at 94°C for 3 min; 35 cycles of denaturation at 94°C for 30 s; primer–template annealing at 57°C for 30 s; and DNA synthesis at 72°C for 1 min; and final extension at 72°C for 7 min. The amplified products were electrophoresed in 2% agarose gels (containing 0.5 μg/mL ethidium bromide) in 1× TAE buffer. Amplicon sizes were estimated with 100 bp standards (New England Biolabs, MA, USA). Table 1. Primers used in this study Locus  Primer  Sequence  Amplicon size (bp)  Reference  vacA m1/m2  VAG-F  5′-CAATCTGTCCAATCAAGCGAG-3′  642  21  VAG-R  5′-GCGTCAAAATAATTCCAAGG-3′  GAPDH (mouse)  GAPDH F  5′-GCAGTGGCAAAGTGGAGATT-3′  249  21  GAPDH R  5′-TCTCCATGGTGGTGAAGACA-3′  Locus  Primer  Sequence  Amplicon size (bp)  Reference  vacA m1/m2  VAG-F  5′-CAATCTGTCCAATCAAGCGAG-3′  642  21  VAG-R  5′-GCGTCAAAATAATTCCAAGG-3′  GAPDH (mouse)  GAPDH F  5′-GCAGTGGCAAAGTGGAGATT-3′  249  21  GAPDH R  5′-TCTCCATGGTGGTGAAGACA-3′  Table 1. Primers used in this study Locus  Primer  Sequence  Amplicon size (bp)  Reference  vacA m1/m2  VAG-F  5′-CAATCTGTCCAATCAAGCGAG-3′  642  21  VAG-R  5′-GCGTCAAAATAATTCCAAGG-3′  GAPDH (mouse)  GAPDH F  5′-GCAGTGGCAAAGTGGAGATT-3′  249  21  GAPDH R  5′-TCTCCATGGTGGTGAAGACA-3′  Locus  Primer  Sequence  Amplicon size (bp)  Reference  vacA m1/m2  VAG-F  5′-CAATCTGTCCAATCAAGCGAG-3′  642  21  VAG-R  5′-GCGTCAAAATAATTCCAAGG-3′  GAPDH (mouse)  GAPDH F  5′-GCAGTGGCAAAGTGGAGATT-3′  249  21  GAPDH R  5′-TCTCCATGGTGGTGAAGACA-3′  Statistical analysis Differences in the histology score were tested with the Mann–Whitney U-test. Student’s t-test was used for the remaining statistics. Both tests were considered as statistically significant when P < 0.05. Results are expressed as average ± SEM of n observations. Results In vitro susceptibility of H. pylori to ellagic acid The antimicrobial efficacy of ellagic acid against 55 H. pylori isolates was evaluated by the agar dilution method. Among 55 H. pylori clinical strains examined against ellagic acid, 39 were isolated from duodenal ulcer patients, whereas 10 were isolated from antral gastritis patients and 6 from non-ulcer dyspepsia patients. Ellagic acid inhibited the growth of all 55 clinical isolates and SS1, a mouse-adapted strain. The MIC of ellagic acid varied between 5 and 30 mg/L and for the majority (89.08%) of the strains the MIC was either 10 mg/L (14.54%) or 15 mg/L (74.54%) (Figure 2). A few strains (3.63%) showed a higher MIC of 30 mg/L (Table 2). The mouse-colonizing strain SS1 showed an MIC of 15 mg/L. These data suggest that ellagic acid has an anti-H. pylori effect in Indian strains. The MICs of amoxicillin, clarithromycin and metronidazole ranged from 0.03 to 2 mg/L, 0.02 to 4 mg/L and 3 to 64 mg/L, respectively (Table 2). Of the 55 H. pylori isolates, resistance to metronidazole (MIC > 8 mg/L) was observed in 52 isolates (94.5%, Tables 2 and 3). Resistance to clarithromycin (MIC > 0.5 mg/L) was found in 5 (9.1%) of the 55 isolates. The remaining 50 susceptible isolates showed MICs varying from 0.02 to 0.125 mg/L (Table 2 and 3). All the strains included in this study were susceptible to amoxicillin with MICs ranging from 0.03 to 2 mg/L. Table 2. MICs of ellagic acid, clarithromycin, amoxicillin and metronidazole for H. pylori strains Number  Strain  MIC (mg/L)   ellagic acid  clarithromycin  amoxicillin  metronidazole  1  186  5  0.125  0.125  16  2  144_1A  5  0.125  0.125  16  3  185_1A  5  0.125  0.125  3  4  156_1A  5  0.125  0.25  16  5  139(1)  10  0.125  0.25  16  6  213(4)  10  0.125  0.25  16  7  233  10  0.125  0.25  3  8  129(1)  10  0.125  0.03  16  9  155_10B  10  0.125  0.25  16  10  L7  10  0.125  0.25  32  11  34  10  2  0.25  16  12  39  10  2  0.25  16  13  I-77  15  0.125  0.25  16  14  162  15  0.125  0.25  32  15  I-90  15  0.125  0.25  32  16  218_1A  15  0.125  0.25  32  17  I-87  15  0.125  0.25  32  18  I-119  15  0.125  0.25  32  19  217  15  0.125  0.25  64  20  I-80  15  0.125  0.25  32  21  I-27  15  0.125  0.25  3  22  127  15  0.125  0.25  32  23  157_6B  15  0.125  0.125  16  24  227_1A  15  0.125  0.5  16  25  I-91  15  0.125  0.5  32  26  138  15  0.125  0.5  32  27  130  15  0.125  0.5  16  28  L10  15  0.125  1  16  29  197  15  0.125  0.125  16  30  157(1A)  15  0.125  0.25  32  31  137(1)  15  0.125  0.25  16  32  135(2)  15  0.125  0.25  16  33  171  15  0.125  0.25  32  34  170(1A)  15  0.125  0.25  32  35  152_1A  15  0.125  0.25  32  36  216_1A  15  0.125  0.25  32  37  169  15  0.02  2  16  38  L1660  15  0.125  0.25  16  39  191  15  0.02  0.25  16  40  152(7B)  15  0.125  0.25  16  41  M27  15  0.125  0.125  16  42  M28  15  0.125  0.125  16  43  M29  15  0.125  0.125  16  44  M12B  15  0.125  0.125  16  45  179  15  0.125  0.125  16  46  148(1)  15  0.125  0.25  64  47  163  15  0.125  0.25  16  48  136  15  4  0.25  32  49  195  15  4  0.25  32  50  481  15  4  0.25  32  51  168  15  0.125  0.25  16  52  209(1)  15  0.125  0.25  16  53  205(1)  15  0.125  0.25  16  54  PCR24  30  0.125  0.25  16  55  225_6B  30  0.125  0.25  16  56  SS1  15  0.015  0.5  0.25  Number  Strain  MIC (mg/L)   ellagic acid  clarithromycin  amoxicillin  metronidazole  1  186  5  0.125  0.125  16  2  144_1A  5  0.125  0.125  16  3  185_1A  5  0.125  0.125  3  4  156_1A  5  0.125  0.25  16  5  139(1)  10  0.125  0.25  16  6  213(4)  10  0.125  0.25  16  7  233  10  0.125  0.25  3  8  129(1)  10  0.125  0.03  16  9  155_10B  10  0.125  0.25  16  10  L7  10  0.125  0.25  32  11  34  10  2  0.25  16  12  39  10  2  0.25  16  13  I-77  15  0.125  0.25  16  14  162  15  0.125  0.25  32  15  I-90  15  0.125  0.25  32  16  218_1A  15  0.125  0.25  32  17  I-87  15  0.125  0.25  32  18  I-119  15  0.125  0.25  32  19  217  15  0.125  0.25  64  20  I-80  15  0.125  0.25  32  21  I-27  15  0.125  0.25  3  22  127  15  0.125  0.25  32  23  157_6B  15  0.125  0.125  16  24  227_1A  15  0.125  0.5  16  25  I-91  15  0.125  0.5  32  26  138  15  0.125  0.5  32  27  130  15  0.125  0.5  16  28  L10  15  0.125  1  16  29  197  15  0.125  0.125  16  30  157(1A)  15  0.125  0.25  32  31  137(1)  15  0.125  0.25  16  32  135(2)  15  0.125  0.25  16  33  171  15  0.125  0.25  32  34  170(1A)  15  0.125  0.25  32  35  152_1A  15  0.125  0.25  32  36  216_1A  15  0.125  0.25  32  37  169  15  0.02  2  16  38  L1660  15  0.125  0.25  16  39  191  15  0.02  0.25  16  40  152(7B)  15  0.125  0.25  16  41  M27  15  0.125  0.125  16  42  M28  15  0.125  0.125  16  43  M29  15  0.125  0.125  16  44  M12B  15  0.125  0.125  16  45  179  15  0.125  0.125  16  46  148(1)  15  0.125  0.25  64  47  163  15  0.125  0.25  16  48  136  15  4  0.25  32  49  195  15  4  0.25  32  50  481  15  4  0.25  32  51  168  15  0.125  0.25  16  52  209(1)  15  0.125  0.25  16  53  205(1)  15  0.125  0.25  16  54  PCR24  30  0.125  0.25  16  55  225_6B  30  0.125  0.25  16  56  SS1  15  0.015  0.5  0.25  Table 2. MICs of ellagic acid, clarithromycin, amoxicillin and metronidazole for H. pylori strains Number  Strain  MIC (mg/L)   ellagic acid  clarithromycin  amoxicillin  metronidazole  1  186  5  0.125  0.125  16  2  144_1A  5  0.125  0.125  16  3  185_1A  5  0.125  0.125  3  4  156_1A  5  0.125  0.25  16  5  139(1)  10  0.125  0.25  16  6  213(4)  10  0.125  0.25  16  7  233  10  0.125  0.25  3  8  129(1)  10  0.125  0.03  16  9  155_10B  10  0.125  0.25  16  10  L7  10  0.125  0.25  32  11  34  10  2  0.25  16  12  39  10  2  0.25  16  13  I-77  15  0.125  0.25  16  14  162  15  0.125  0.25  32  15  I-90  15  0.125  0.25  32  16  218_1A  15  0.125  0.25  32  17  I-87  15  0.125  0.25  32  18  I-119  15  0.125  0.25  32  19  217  15  0.125  0.25  64  20  I-80  15  0.125  0.25  32  21  I-27  15  0.125  0.25  3  22  127  15  0.125  0.25  32  23  157_6B  15  0.125  0.125  16  24  227_1A  15  0.125  0.5  16  25  I-91  15  0.125  0.5  32  26  138  15  0.125  0.5  32  27  130  15  0.125  0.5  16  28  L10  15  0.125  1  16  29  197  15  0.125  0.125  16  30  157(1A)  15  0.125  0.25  32  31  137(1)  15  0.125  0.25  16  32  135(2)  15  0.125  0.25  16  33  171  15  0.125  0.25  32  34  170(1A)  15  0.125  0.25  32  35  152_1A  15  0.125  0.25  32  36  216_1A  15  0.125  0.25  32  37  169  15  0.02  2  16  38  L1660  15  0.125  0.25  16  39  191  15  0.02  0.25  16  40  152(7B)  15  0.125  0.25  16  41  M27  15  0.125  0.125  16  42  M28  15  0.125  0.125  16  43  M29  15  0.125  0.125  16  44  M12B  15  0.125  0.125  16  45  179  15  0.125  0.125  16  46  148(1)  15  0.125  0.25  64  47  163  15  0.125  0.25  16  48  136  15  4  0.25  32  49  195  15  4  0.25  32  50  481  15  4  0.25  32  51  168  15  0.125  0.25  16  52  209(1)  15  0.125  0.25  16  53  205(1)  15  0.125  0.25  16  54  PCR24  30  0.125  0.25  16  55  225_6B  30  0.125  0.25  16  56  SS1  15  0.015  0.5  0.25  Number  Strain  MIC (mg/L)   ellagic acid  clarithromycin  amoxicillin  metronidazole  1  186  5  0.125  0.125  16  2  144_1A  5  0.125  0.125  16  3  185_1A  5  0.125  0.125  3  4  156_1A  5  0.125  0.25  16  5  139(1)  10  0.125  0.25  16  6  213(4)  10  0.125  0.25  16  7  233  10  0.125  0.25  3  8  129(1)  10  0.125  0.03  16  9  155_10B  10  0.125  0.25  16  10  L7  10  0.125  0.25  32  11  34  10  2  0.25  16  12  39  10  2  0.25  16  13  I-77  15  0.125  0.25  16  14  162  15  0.125  0.25  32  15  I-90  15  0.125  0.25  32  16  218_1A  15  0.125  0.25  32  17  I-87  15  0.125  0.25  32  18  I-119  15  0.125  0.25  32  19  217  15  0.125  0.25  64  20  I-80  15  0.125  0.25  32  21  I-27  15  0.125  0.25  3  22  127  15  0.125  0.25  32  23  157_6B  15  0.125  0.125  16  24  227_1A  15  0.125  0.5  16  25  I-91  15  0.125  0.5  32  26  138  15  0.125  0.5  32  27  130  15  0.125  0.5  16  28  L10  15  0.125  1  16  29  197  15  0.125  0.125  16  30  157(1A)  15  0.125  0.25  32  31  137(1)  15  0.125  0.25  16  32  135(2)  15  0.125  0.25  16  33  171  15  0.125  0.25  32  34  170(1A)  15  0.125  0.25  32  35  152_1A  15  0.125  0.25  32  36  216_1A  15  0.125  0.25  32  37  169  15  0.02  2  16  38  L1660  15  0.125  0.25  16  39  191  15  0.02  0.25  16  40  152(7B)  15  0.125  0.25  16  41  M27  15  0.125  0.125  16  42  M28  15  0.125  0.125  16  43  M29  15  0.125  0.125  16  44  M12B  15  0.125  0.125  16  45  179  15  0.125  0.125  16  46  148(1)  15  0.125  0.25  64  47  163  15  0.125  0.25  16  48  136  15  4  0.25  32  49  195  15  4  0.25  32  50  481  15  4  0.25  32  51  168  15  0.125  0.25  16  52  209(1)  15  0.125  0.25  16  53  205(1)  15  0.125  0.25  16  54  PCR24  30  0.125  0.25  16  55  225_6B  30  0.125  0.25  16  56  SS1  15  0.015  0.5  0.25  Table 3. Antibacterial activities of clarithromycin, amoxicillin and metronidazole against H. pylori strains Number  Strain  Clarithromycin  Amoxicillin  Metronidazole  1  186  S  S  R  2  144_1A  S  S  R  3  185_1A  S  S  S  4  156_1A  S  S  R  5  139(1)  S  S  R  6  213(4)  S  S  R  7  233  S  S  S  8  129(1)  S  S  R  9  155_10B  S  S  R  10  L7  S  S  R  11  34  R  S  R  12  39  R  S  R  13  I-77  S  S  R  14  162  S  S  R  15  I-90  S  S  R  16  218_1A  S  S  R  17  I-87  S  S  R  18  I-119  S  S  R  19  217  S  S  R  20  I-80  S  S  R  21  I-27  S  S  S  22  127  S  S  R  23  157_6B  S  S  R  24  227_1A  S  S  R  25  I-91  S  S  R  26  138  S  S  R  27  130  S  S  R  28  L10  S  S  R  29  197  S  S  R  30  157(1A)  S  S  R  31  137(1)  S  S  R  32  135(2)  S  S  R  33  171  S  S  R  34  170(1A)  S  S  R  35  152_1A  S  S  R  36  216_1A  S  S  R  37  169  S  S  R  38  L1660  S  S  R  39  191  S  S  R  40  152(7B)  S  S  R  41  M27  S  S  R  42  M28  S  S  R  43  M29  S  S  R  44  M12B  S  S  R  45  179  S  S  R  46  148(1)  S  S  R  47  163  S  S  R  48  136  R  S  R  49  195  R  S  R  50  481  R  S  R  51  168  S  S  R  52  209(1)  S  S  R  53  205(1)  S  S  R  54  PCR24  S  S  R  55  225_6B  S  S  R  56  SS1  S  S  S  Number  Strain  Clarithromycin  Amoxicillin  Metronidazole  1  186  S  S  R  2  144_1A  S  S  R  3  185_1A  S  S  S  4  156_1A  S  S  R  5  139(1)  S  S  R  6  213(4)  S  S  R  7  233  S  S  S  8  129(1)  S  S  R  9  155_10B  S  S  R  10  L7  S  S  R  11  34  R  S  R  12  39  R  S  R  13  I-77  S  S  R  14  162  S  S  R  15  I-90  S  S  R  16  218_1A  S  S  R  17  I-87  S  S  R  18  I-119  S  S  R  19  217  S  S  R  20  I-80  S  S  R  21  I-27  S  S  S  22  127  S  S  R  23  157_6B  S  S  R  24  227_1A  S  S  R  25  I-91  S  S  R  26  138  S  S  R  27  130  S  S  R  28  L10  S  S  R  29  197  S  S  R  30  157(1A)  S  S  R  31  137(1)  S  S  R  32  135(2)  S  S  R  33  171  S  S  R  34  170(1A)  S  S  R  35  152_1A  S  S  R  36  216_1A  S  S  R  37  169  S  S  R  38  L1660  S  S  R  39  191  S  S  R  40  152(7B)  S  S  R  41  M27  S  S  R  42  M28  S  S  R  43  M29  S  S  R  44  M12B  S  S  R  45  179  S  S  R  46  148(1)  S  S  R  47  163  S  S  R  48  136  R  S  R  49  195  R  S  R  50  481  R  S  R  51  168  S  S  R  52  209(1)  S  S  R  53  205(1)  S  S  R  54  PCR24  S  S  R  55  225_6B  S  S  R  56  SS1  S  S  S  S, susceptible; R, resistant. Table 3. Antibacterial activities of clarithromycin, amoxicillin and metronidazole against H. pylori strains Number  Strain  Clarithromycin  Amoxicillin  Metronidazole  1  186  S  S  R  2  144_1A  S  S  R  3  185_1A  S  S  S  4  156_1A  S  S  R  5  139(1)  S  S  R  6  213(4)  S  S  R  7  233  S  S  S  8  129(1)  S  S  R  9  155_10B  S  S  R  10  L7  S  S  R  11  34  R  S  R  12  39  R  S  R  13  I-77  S  S  R  14  162  S  S  R  15  I-90  S  S  R  16  218_1A  S  S  R  17  I-87  S  S  R  18  I-119  S  S  R  19  217  S  S  R  20  I-80  S  S  R  21  I-27  S  S  S  22  127  S  S  R  23  157_6B  S  S  R  24  227_1A  S  S  R  25  I-91  S  S  R  26  138  S  S  R  27  130  S  S  R  28  L10  S  S  R  29  197  S  S  R  30  157(1A)  S  S  R  31  137(1)  S  S  R  32  135(2)  S  S  R  33  171  S  S  R  34  170(1A)  S  S  R  35  152_1A  S  S  R  36  216_1A  S  S  R  37  169  S  S  R  38  L1660  S  S  R  39  191  S  S  R  40  152(7B)  S  S  R  41  M27  S  S  R  42  M28  S  S  R  43  M29  S  S  R  44  M12B  S  S  R  45  179  S  S  R  46  148(1)  S  S  R  47  163  S  S  R  48  136  R  S  R  49  195  R  S  R  50  481  R  S  R  51  168  S  S  R  52  209(1)  S  S  R  53  205(1)  S  S  R  54  PCR24  S  S  R  55  225_6B  S  S  R  56  SS1  S  S  S  Number  Strain  Clarithromycin  Amoxicillin  Metronidazole  1  186  S  S  R  2  144_1A  S  S  R  3  185_1A  S  S  S  4  156_1A  S  S  R  5  139(1)  S  S  R  6  213(4)  S  S  R  7  233  S  S  S  8  129(1)  S  S  R  9  155_10B  S  S  R  10  L7  S  S  R  11  34  R  S  R  12  39  R  S  R  13  I-77  S  S  R  14  162  S  S  R  15  I-90  S  S  R  16  218_1A  S  S  R  17  I-87  S  S  R  18  I-119  S  S  R  19  217  S  S  R  20  I-80  S  S  R  21  I-27  S  S  S  22  127  S  S  R  23  157_6B  S  S  R  24  227_1A  S  S  R  25  I-91  S  S  R  26  138  S  S  R  27  130  S  S  R  28  L10  S  S  R  29  197  S  S  R  30  157(1A)  S  S  R  31  137(1)  S  S  R  32  135(2)  S  S  R  33  171  S  S  R  34  170(1A)  S  S  R  35  152_1A  S  S  R  36  216_1A  S  S  R  37  169  S  S  R  38  L1660  S  S  R  39  191  S  S  R  40  152(7B)  S  S  R  41  M27  S  S  R  42  M28  S  S  R  43  M29  S  S  R  44  M12B  S  S  R  45  179  S  S  R  46  148(1)  S  S  R  47  163  S  S  R  48  136  R  S  R  49  195  R  S  R  50  481  R  S  R  51  168  S  S  R  52  209(1)  S  S  R  53  205(1)  S  S  R  54  PCR24  S  S  R  55  225_6B  S  S  R  56  SS1  S  S  S  S, susceptible; R, resistant. Figure 2. View largeDownload slide Distribution of ellagic acid MICs for the H. pylori strains isolated from gastroduodenal patients in India. Figure 2. View largeDownload slide Distribution of ellagic acid MICs for the H. pylori strains isolated from gastroduodenal patients in India. Morphological changes induced by ellagic acid TEM, a powerful imaging technique, was used to examine the morphological changes of H. pylori strain SS1 following ellagic acid treatment (Figure 3). Briefly, H. pylori growth under control conditions was exponential until ∼12 h, after which it reached stationary phase. After treatment with 15 μM ellagic acid, the bacterial (Figure 3a and b) morphology changed from bacillary (untreated, control) to spherical/coccoid forms, but no changes were observed with lower concentrations of ellagic acid. In strains that have MICs of 5 and 10 mg/L, we observed a change in morphology (data not shown). The logic behind the selection of SS1 is its well-known property of infecting animals and its worldwide use. Thus, ellagic acid is an effective anti-H. pylori molecule, promoting coccoid bacterial morphology, which is known to be a non-cultivable form. Figure 3. View largeDownload slide Transmission electron micrographs of H. pylori control (a) and after 12 h of treatment with ellagic acid (b). Figure 3. View largeDownload slide Transmission electron micrographs of H. pylori control (a) and after 12 h of treatment with ellagic acid (b). Effect of ellagic acid on bacterial colonization Initially all the mice were challenged with H. pylori strain SS1. Two weeks following the initial bacterial challenge, the experimental group of mice was orogastrically fed with ellagic acid (10 mg/kg) and non-infected control mice received sterile PBS as described in the Methods section. At 6 weeks post-infection a small tissue section from the stomach was used for H. pylori enumeration. Tissue homogenates were spread on BHI agar medium. H. pylori colonies were enumerated and results are presented as cfu/g of stomach tissue. In infected tissue the average number of cfu was estimated as 5.2 × 106/g. Here, maximum, minimum and median colony counts of infected mouse samples were 61, 42 and 56, respectively. No colonies were found in the control group. The group treated with ellagic acid also did not show any colonies (P < 0.05 compared with the infected group; Figure 4). Figure 4. View largeDownload slide Effect of ellagic acid on the viability of H. pylori in H. pylori-infected mice. Histographic representation of colonization efficiency of the SS1 strain of H. pylori in mice and the effect of ellagic acid thereon, obtained by quantitative culture. EA10, ellagic acid 10 mg/kg. Figure 4. View largeDownload slide Effect of ellagic acid on the viability of H. pylori in H. pylori-infected mice. Histographic representation of colonization efficiency of the SS1 strain of H. pylori in mice and the effect of ellagic acid thereon, obtained by quantitative culture. EA10, ellagic acid 10 mg/kg. Anti-H. pylori activity in vivo In view of the fact that the in vitro study confirmed that ellagic acid had anti-H. pylori activity, we investigated the effect of ellagic acid on H. pylori colonization in an invivo model. Briefly, positive control and experimental groups of C57BL/6 mice were infected with 1 × 108 cfu/mL of H. pylori strain SS1 via orogastric inoculation. Six weeks post-infection, animals were deprived of feed, but allowed free access to water for 24 h and then sacrificed. Stomach tissues were collected and rapid urease test (RUT) confirmed H. pylori colonization. Samples were also screened for the presence of the vacA gene using DNA extracted from H. pylori SS1-infected mouse gastric tissues by PCR. Two weeks after bacterial inoculation, a group of mice was administered 10 mg/kg body weight ellagic acid daily for 1 week. The efficacy of ellagic acid was evaluated by RUT with the respective mouse gastric tissues. Mouse gastric tissues from the infected and ellagic acid-treated groups were all positive and negative for RUT, respectively. To further substantiate ellagic acid antibacterial potential, the bacterial-specific gene primer vacA was amplified by PCR using genomic DNA extracted from the gastric tissues of H. pylori-infected and ellagic acid-fed C57BL/6 mice (10 mg/kg), while the mouse-specific GAPDH gene served as an internal reference. Figure 5 shows the therapeutic efficacy of ellagic acid treatment, which completely abolished H. pylori from mouse stomach tissues. Thus, this in vivo information is in accordance with the in vitro results, confirming the anti-H. pylori potential of ellagic acid. Figure 5. View largeDownload slide Effect of ellagic acid on H. pylori viability in H. pylori-infected C57BL/6 mice. Lane 1 (second from left) shows amplification of the vacA middle region of 642 bp using DNA from the mouse-colonizing strain SS1 with primers VAG-F and VAG-R (Table 1). Lane 2 shows amplification of H. pylori-specific vacA and the mouse-specific GAPDH gene using DNA isolated from the stomach of uninfected C57BL/6 mice. Lane 3 shows the presence of H. pylori-specific gene vacA and GAPDH (housekeeping gene) using DNA isolated from the gastric sample of H. pylori SS1-infected mice 4 weeks post-infection. Lane 4 shows the amplification of vacA and GAPDH using DNA isolated from the gastric tissue of ellagic acid-fed (10 mg/kg body weight) H. pylori SS1-infected C57BL/6 mice. +ve, positive. Figure 5. View largeDownload slide Effect of ellagic acid on H. pylori viability in H. pylori-infected C57BL/6 mice. Lane 1 (second from left) shows amplification of the vacA middle region of 642 bp using DNA from the mouse-colonizing strain SS1 with primers VAG-F and VAG-R (Table 1). Lane 2 shows amplification of H. pylori-specific vacA and the mouse-specific GAPDH gene using DNA isolated from the stomach of uninfected C57BL/6 mice. Lane 3 shows the presence of H. pylori-specific gene vacA and GAPDH (housekeeping gene) using DNA isolated from the gastric sample of H. pylori SS1-infected mice 4 weeks post-infection. Lane 4 shows the amplification of vacA and GAPDH using DNA isolated from the gastric tissue of ellagic acid-fed (10 mg/kg body weight) H. pylori SS1-infected C57BL/6 mice. +ve, positive. Histology of mouse gastric tissues during H. pylori infection and the effect of ellagic acid Briefly, freshly prepared aliquots (200 μL; 108 cfu/mL) of H. pylori strain SS1 were administered to a group of C57BL/6 mice, while the uninfected group received sterile PBS. At 2 weeks post-inoculation a group of H. pylori-infected mice was orally fed with 10 mg/kg body weight ellagic acid (daily for 1 week). All groups of mice were sacrificed at 6 weeks from the start of the experiment. Collected stomach tissues were stained and subjected to histological analysis. As compared with the untreated group (control; Figure 6a and d) in infected (colonizing strain SS1 for 6 weeks) mouse gastric tissue (Figure 6b and e) considerable damage was observed by histological analysis of longitudinal sections. Denudation of the surface epithelial layer was visible in the SS1-infected mouse gastric tissues; this layer was restored to almost normal after ellagic acid treatment. Furthermore, inflammation in the gastric pit cells, as observed in the infected tissues (Figure 6b and e), was checked noticeably by ellagic acid treatment (Figure 6c and f). The inflammatory gradation or score was analysed blindly by a pathologist. For the control and ellagic acid-treated mice no inflammation of gastric mucosa was observed (score 0). Among the infected mice mild (score 1, n = 2) and moderate (score 2, n = 4) inflammation was observed after 6 weeks of infection, which was significant compared with the control and treated groups. No adverse effect or death was observed in any group of mice in our experiments. Figure 6. View largeDownload slide Histopathology of mouse gastric tissues after H. pylori infection and eradication. Representative images of haematoxylin and eosin-stained sections of mouse gastric tissues taken at ×40 magnification. Histological analysis of gastric tissues from (a and d) negative control (i.e. no H. pylori) and (b and e) positive control (i.e. H. pylori with no treatment). There was significant damage of gastric tissues of mice infected with the mouse-colonizing strain SS1 for 3 weeks, like loss of normal mucosal architecture. (c and f) This layer was restored to almost normal after treating with ellagic acid (EA) at 10 mg/kg body weight. The gastric mucosal epithelium, gastric glands, parietal cells (top)/chief cells (bottom) and inflammatory cell infiltration are shown as a black arrow, orange stars, green stars and white stars, respectively. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 6. View largeDownload slide Histopathology of mouse gastric tissues after H. pylori infection and eradication. Representative images of haematoxylin and eosin-stained sections of mouse gastric tissues taken at ×40 magnification. Histological analysis of gastric tissues from (a and d) negative control (i.e. no H. pylori) and (b and e) positive control (i.e. H. pylori with no treatment). There was significant damage of gastric tissues of mice infected with the mouse-colonizing strain SS1 for 3 weeks, like loss of normal mucosal architecture. (c and f) This layer was restored to almost normal after treating with ellagic acid (EA) at 10 mg/kg body weight. The gastric mucosal epithelium, gastric glands, parietal cells (top)/chief cells (bottom) and inflammatory cell infiltration are shown as a black arrow, orange stars, green stars and white stars, respectively. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Discussion Standard triple therapy consisting of a proton pump inhibitor-based regimen is currently used to ameliorate gastric inflammation caused by H. pylori infection.26 Though considerable progress in therapeutic modalities has been achieved, obstacles such as therapeutic failure, poor patient compliance, resistance to conventional antibiotics and escalating cost are frequent. Moreover, the increasing emergence of H. pylori strains resistant to conventional therapy is a major public health concern.27,28 Recently, H. pylori has been registered by the WHO among 16 antibiotic-resistant bacteria that pose the greatest menace to human health.29 Metronidazole and clarithromycin resistance rates are alarming, although they vary among populations.30,31 Clarithromycin resistance has been rapidly increasing in many countries over the past decade and it is ∼30% in Italy and 40% in Turkey.32 One study from Japan demonstrated that the prevalence of clarithromycin resistance is around 31.1%.33 Similarly, China has experienced an increase in clarithromycin resistance from 14.8% in 2000 to 52.6% in 2014.34,35 A study from Kolkata, India, during 2005 reported that 85% of the H. pylori strains were resistant to metronidazole but none of the strains was found to be resistant to clarithromycin.36 Another study, during 2016, reported clarithromycin resistance in 11.8% of the H. pylori isolates in North India.37 Increasing resistance to H. pylori infection has heightened trepidation about a decline in the efficacy of the same antibiotics in treatment of other diseases. Because of the emergence of antibiotic-resistant H. pylori clinical strains, there is an imperative need for alternative, cost-effective antimicrobial agents that are non-antibiotic and suitable for the next generation of eradication therapy. The evolving role of natural products, especially plant-derived active principles, have an evolving role and have been an important source of cancer treatments since ancient times.38–41 Much of the world’s population (∼70%–80%) still depends on plant-derived medicines for healthcare. This is especially true in developing countries like India, where traditional herbal medicines have played a prominent role in the strategy to treat acute diseases, including dyspepsia, gastritis and peptic ulcer.42 Previous literature has shown that ellagic acid, a major polyphenolic component of fruits, vegetables and nuts, has anti-H. pylori activity.15–18 Therefore, the purpose of the present study was to explore the antimicrobial potential of ellagic acid against Indian H. pylori clinical strains, which are phylogeographically distinct from strains of East Asian and Western origin.43 Furthermore, although data are scanty, around 64% of the Indian population harbours H. pylori and, overall, infected populations suffer from H. pylori-associated gastrointestinal disorders having a varying type of antibiotic resistance profile.37,44–46 In the present study, the antibacterial activity of ellagic acid against 55 H. pylori strains from clinical isolates from patients suffering from various gastroduodenal pathologies was examined. Significantly, the majority of these strains were resistant to metronidazole, with MICs ranging from 16 to 64 mg/L, and 9.1% strains were resistant to clarithomycin.36,46 These results suggest that ellagic acid acts through pathways markedly different from the mechanism of action of the antibiotics used for elimination of H. pylori from patients. The mechanism underlying the inhibitory effect of ellagic acid on H. pylori growth is still unknown. However, our TEM observations revealed H. pylori morphological changes upon ellagic acid treatment, which might be the mechanism responsible for bactericidal activity. H. pylori that had been treated with ellagic acid showed coccoid forms47 known to be associated with loss of viability. The morphological transformation of H. pylori from a helical bacillary to a non-culturable coccoid shape has previously been reported to be related to alteration of the bacterial cell wall peptidoglycan. Flagellar structures were also either disrupted or absent. These morphological changes might completely inhibit the motility of the bacteria, key for their migration in the gastric niche and colonization of the host gastric mucosa.48 However, further exploration is needed to clearly explain the antibacterial action of this compound. The murine infection model has been widely used in explorations of host responses to H. pylori infection in addition to eradication studies. In vivo models suggest various parameters, such as the bio-distribution of compounds and their chemical stability, biodegradability and resistance to the excessively acidic conditions of the gastric environment, can influence their bioactivity. In the present study, we utilized an established H. pylori infection model to evaluate the potential therapeutic effect of ellagic acid. Despite the low number of tested mice, the histological analysis showed that ellagic acid at a dose of 10 mg/kg body weight completely eliminated H. pylori from infected mice. This information is of clinical significance for the development of alternative therapy against this ulcer-causing organism. Histological study demonstrated that ellagic acid is very effective in the elimination of the pathogen from infected mice and also repairing damaged gastric tissue as compared with mice infected with SS1. Numerous clinical trials suggest a therapeutic efficacy of ellagic acid in diseases such as cardiovascular disease, diabetes and prostate cancer.49 It has been claimed in a recent patent application50 that ellagic acid has a beneficial effect in reducing the damaging effects of gastric acid and H. pylori infection of the upper digestive tract in the prevention of gastrointestinal disorders. In conclusion, the present study clearly indicates the antimicrobial activity of ellagic acid against H. pylori in vitro, and that the gastric epithelial damage induced by H. pylori infection was almost completely restored by ellagic acid, thus highlighting its promise as a potential therapeutic candidate against H. pylori-related gastrointestinal diseases. Our results suggest that ellagic acid looks very promising as a future alternative for treatment of H. pylori infections, although research in advance of clinical application is necessary to determine its effects in humans. Acknowledgements We thank Mr Bivash Ranjan Mallick of the Electron Microscopy Facility at the National Institute of Cholera and Enteric Diseases for his help in processing samples for histology. Funding This study was supported in part by: (i) the Indian Council of Medical Research (ICMR), Government of India; (ii) the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) of the Japan Agency for Medical Research and Development (AMED); and (iii) the Council of Scientific and Industrial Research (CSIR) [ref. no. 37(1640)/14/EMR-II]. R. D. acknowledges the Indian Council of Medical Research, Government of India for providing a postdoctoral fellowship [grant ref. no. 3/1/3/PDF (3)/2011-MPD]. Transparency declarations None to declare. References 1 Covacci A, Telford JL, Del Giudice G et al.   Helicobacter pylori virulence and genetic geography. Science  1999; 284: 1328– 33. Google Scholar CrossRef Search ADS PubMed  2 Peek M, Balser M. Helicobacter pylori and gastrointestinal tract adenocarcinoma. Nat Rev Cancer  2002; 2: 28– 37. 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Google Scholar CrossRef Search ADS PubMed  49 Johanningsmeier SD, Harris GK. Pomegranate as a functional food and nutraceutical source. Annu Rev Food Sci Technol  2011; 2: 181– 201. Google Scholar CrossRef Search ADS PubMed  50 Holt S. Composition and Method for Treating the Effects of Diseases and Maladies of the Upper Digestive Tract. US Patent, US 2008/0038370 A1, 2008. © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Antimicrobial activity of ellagic acid against Helicobacter pylori isolates from India and during infections in mice

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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0305-7453
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10.1093/jac/dky079
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Abstract

Abstract Objectives Because of the rise in antimicrobial resistance, an inexpensive, diet-based treatment against Helicobacter pylori infection would be of great interest. The present study was performed to assess the in vitro effects of ellagic acid against clinical H. pylori strains that were resistant to antibiotics used for therapy and also to observe the morphological structure following treatment with ellagic acid. The effectiveness of ellagic acid in eradicating H. pylori infection in a murine (C57BL/6) infection model, one of the standard inbred mouse lines often used for experimental infection, was also assessed. Methods A total of 55 strains were screened. The agar dilution method was used to determine the susceptibility of isolates to test compounds. Transmission electron microscopy was used to observe the morphology following treatment with ellagic acid. The antibacterial activity of ellagic acid in an H. pylori SS1-infected mouse model and its effect on gastric mucosal injury were determined by histology and PCR. Results Ellagic acid inhibited the growth of all 55 of the H. pylori strains tested. The MIC of ellagic acid ranged from 5 to 30 mg/L, showing its bactericidal properties in vitro. Ellagic acid also demonstrated anti-H. pylori efficacy in eradication of this organism in an in vivo model, as well as restitution and repair of H. pylori-induced gastric mucosal damage. Conclusions The present study paves the way for the preventive and therapeutic use of ellagic acid against H. pylori infection and, thus, ellagic acid can be considered a promising antibacterial agent against H. pylori-associated gastroduodenal diseases in humans. Introduction Helicobacter pylori is a Gram-negative, motile, spiral pathogen that infects >50% of the global population. Infection is associated with chronic active gastritis, peptic ulcer and ultimately gastric carcinoma.1 The clinical manifestations of microbial infection depend on complex interactions established between bacterial virulence factors and the host, contributing to this complex interplay are the host’s genetic background and environmental factors, mainly diet.2 Treatment of H. pylori infection has not significantly changed over the last decade, and drug-resistant strains of H. pylori and poor patient compliance are the major causes of therapy failure.3 The consensus treatment regimen is a triple therapy consisting of a proton pump inhibitor and two antibiotics. However, the efficacy of conventional antibiotic therapy is not always satisfactory and therefore the acquisition by H. pylori isolates of high-level resistance to antibiotics, including metronidazole (antiprotozoal), clarithromycin (antibacterial) and tetracycline, could signify a serious dilemma that may reduce treatment efficacy.4,5 In the context of incomplete cure accomplished with standard triple therapy because of the increasing number of drug-resistant strains, adverse side effects such as nausea and diarrhoea,6 poor patient compliance,7 the escalating cost of the multiple antibiotics8 and some unknown factors for their ineffectiveness, there is a strong need to explore new non-antimicrobial agents that are cost-effective, safe and applicable to all H. pylori infections. Several strategies have been developed to overcome the issue of the increasing failure of H. pylori eradication therapies, namely, adoption of novel, more effective, empirical treatment, the generation of a vaccine to stimulate the host’s immune defences, adjunct use of probiotics and the development of new and more potent substances that could inhibit bacterial growth. Using a mouse-adapted H. pylori strain (Sydney strain 1, SS1), Lee et al.9 established a robust model of long-term and high bacterial load colonization in C57BL/6 mice. Although extensive studies in this H. pylori mouse model have established the feasibility of both therapeutic and prophylactic immunizations, the mechanism of immunogen-evoked protection is still inadequately understood, meaning that vaccination as a tool to eradicate H pylori is still very contentious. Numerous studies have shown that plant polyphenols are an important class of antimicrobial agents against organisms that include bacteria, viruses and protozoa.10 Ellagic acid (C14H6O8), also known as 2,3,7,8-tetrahydroxy[1]benzopyrano-[5,4,3-cde][1]benzopyran-5,10-dione, 4′,5,5′,6,6′-hexahydroxy-diphenic acid 2,6,2′,6′-dilactone (Figure 1) and benzoaric acid, is a naturally occurring dietary polyphenol, present in free form or in the form of ellagitannins or glucosides. It is generally found in certain rich dietary sources such as walnuts, pomegranates, strawberries, blackberries, cloudberries and raspberries.11 It is known to possess an array of pharmacological and biological activities including antioxidant activity,12 free radical-scavenging capacity, chemopreventive properties13 and anti-apoptotic properties,14 and has also been reported to possess considerable in vitro antibacterial activity. Numerous studies demonstrated in vitro antimicrobial efficacy of ellagic acid against H. pylori,15–18 but in vivo efficacy has not been demonstrated. Figure 1. View largeDownload slide Chemical structure of ellagic acid (2,3,7,8-tetrahydroxy[1]benzopyrano-[5,4,3-cde][1]benzopyran-5,10-dione). Figure 1. View largeDownload slide Chemical structure of ellagic acid (2,3,7,8-tetrahydroxy[1]benzopyrano-[5,4,3-cde][1]benzopyran-5,10-dione). Accordingly, the present study has been conducted: (i) to assess the efficacy of ellagic acid as an antibacterial agent against H. pylori strains isolated from patients in India; (ii) to observe alteration of H. pylori morphology upon ellagic acid treatment; and (iii) to understand the effectiveness of ellagic acid in eradicating H. pylori infection in a murine (C57BL/6) infection model. Methods Strains and culture conditions Fifty-five archived strains of H. pylori, which were isolated from antral mucosal biopsy specimens of patients aged between 18 and 80 years with gastrointestinal disease, were used for this study. H. pylori colonies were identified on the basis of typical translucent water droplet colony morphology, Gram staining and positive reactions in biochemical tests (catalase, urease and oxidase). H. pylori strains were streaked onto Petri plates containing brain heart infusion (BHI) agar (Difco Laboratories, Detroit, MI, USA) supplemented with 7% heat-inactivated horse serum (Invitrogen, NY, USA), 0.4% IsoVitaleX (Becton Dickinson, MD, USA), trimethoprim (5 mg/L), vancomycin (8 mg/L) and polymyxin B (10 mg/L). Nalidixic acid (10 mg/L) and bacitracin (200 mg/L) were added to this medium when culturing H. pylori from mouse stomachs. All plates were incubated inverted at 37°C in a microaerobic atmosphere (5% O2/10% CO2/85% N2) (double gas incubator; Heraeus, Langenselbold, Germany) for 3–6 days. Stock cultures were maintained at −70°C as suspensions of fresh exponentially growing cells suspended in BHI broth with 20% glycerol. Determination of MICs Briefly, frozen stock cultures were subcultured on BHI agar supplemented with 7% horse serum (Invitrogen, NY, USA) and incubated for 72 h under microaerophilic conditions. H. pylori cultures in the exponential phase of growth were suspended in sterile physiological saline (PBS) solution and adjusted to an optical density of 0.1 at 600 nm using a UV spectrophotometer. Briefly, the final test concentrations were 5, 10, 15, 20 and 30 mg/L for each sample. Anti-H. pylori activity of the compounds was determined by the agar dilution method. A total of 55 clinical samples and the mouse-colonizing strain SS1 were used in the susceptibility testing. Samples were inverted and incubated under humid microaerobic conditions at 37°C for 5 days. The MIC was the lowest concentration at which the compound inhibited visible bacterial growth. For quality control and comparative analyses, the antibiotics amoxicillin (Sigma Chemical Co., St Louis, MO, USA) and clarithromycin (Abbott Laboratories, Abbott Park, IL, USA) were also tested with each batch of ellagic acid. MICs of metronidazole, amoxicillin and clarithromycin were determined by the agar dilution method, as described in Mendonça et al.19 Transmission electron microscopy (TEM) analysis In order to observe the effect of ellagic acid treatment on H. pylori structure and morphology, the mouse-colonizing strain SS1 was grown for 12 h under microaerophilic conditions (10% CO2/5% O2/85% N2) in the presence or absence of various concentrations of ellagic acid (5, 10 and 15 μM) while shaking the bacteria on a shaker at 125 rpm. Then, control and ellagic acid-treated H. pylori cultures were observed using TEM. Briefly, samples were washed in PBS at 3000 g for 5 min and bacterial pellets fixed by re-suspending in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (Ted Pella, Redding, CA, USA) at 4°C overnight. Dehydration and embedding in Agar 100 resin (Agar Scientific, UK) were performed according to a standard protocol. After embedding in resin, blocks were kept at 60°C for 24 h. Ultra-thin sections were prepared using a Leica Ultracut UCT microtome and stained with uranyl acetate and lead citrate, and bacterial cells were observed with a Tecnai 12 Biotwin transmission electron microscope (Fei, the Netherlands) operating at 80 kV.20 Mouse challenge study followed by treatment with ellagic acid Six-week-old, pathogen-free C57BL/6 male mice weighing 20 ± 5 g bred in-house were used in all in vivo experiments. Animals were housed in standard conditions with fresh sterile bedding, fed a conventional pellet diet and provided with sterile water ad libitum unless otherwise stated for the duration of the experiment. The animal facility had a regulated room temperature (21–24°C) and humidity (55% ± 10%) and an artificial standard 12 h light/12 h dark cycle. All animal studies were conducted in compliance with protocols approved by the Animal Ethics Committee of NICED (registration number 68/GO/ReBi/S/99/CPCSEA; permit number NICED/CPCSEA/AW/(225)/2013-IAEC/AM-1). Six animals per group were challenged with the bacterial strain SS1, which is known to colonize the mouse gastric mucosa efficiently. The strain was revived on Petri plates containing brain heart infusion agar as described earlier for mouse infection.21 Bacterial cultures were harvested by centrifugation at 8000 g for 5 min and resuspended in sterile PBS for the animal challenge. Each mouse received 0.2 mL (108 cfu/mL)21 of bacterial suspension by oral gavage three times at 2 day intervals using a feeding needle, excluding the no-H. pylori controls, which received sterile PBS. At 2 weeks following the initial bacterial challenge, the mice were divided into three groups: control group (n = 6); infected group (n = 6); and infected group treated with ellagic acid (n = 6). The experimental group of mice was orogastrically fed with ellagic acid (10 mg/kg) once daily for 1 week consecutively, while the non-infected control mice received sterile PBS. At 6 weeks post-infection, mice were euthanized by cervical dislocation. The stomach was removed and dissected longitudinally along the greater curvature, and the gastric content was removed. A small section of the stomach was analysed for a urease test, H. pylori enumeration, PCR and a histological evaluation study as described previously.22–24 For quantitative culture, the tissues were homogenized in PBS using a disposable pellet pestle (Tarson), homogenates of each suspension were spread on BHI agar medium and H. pylori colonies were enumerated by the procedure described above. Data are presented as cfu/g of stomach. Histological evaluation Stomachs were aseptically removed and dissected along the greater curvature in the different groups of mice, washed vigorously in PBS and preserved in 10% neutral buffered formalin for histological evaluation. The tissue samples were dehydrated with alcohol and xylene prior to embedding in paraffin wax and sectioning. The sections (5 μm) were cut with a microtome, mounted on glass slides with mounting medium, stained with haematoxylin and eosin and observed under a microscope. Inflammation was observed and graded from 0 to 3 (where 0 means no inflammation, 1 is mild inflammation, 2 is moderate inflammation and 3 is high inflammation) by a pathologist in a blinded fashion as described elsewhere.25 PCR from H. pylori-infected stomach tissue Genomic DNA from mouse gastric tissues was extracted with a DNeasy tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA content and purity were determined by measuring the absorbance at 260–280 nm. The presence or absence of the vacA gene and the specificity for the mouse genome were confirmed using the gene-specific PCR primers listed in Table 1 and DNA from the respective gastric tissues. PCR amplification was carried out in a final volume of 20 μL containing 40 ng of template DNA, 2 μL of 10× PCR buffer (Roche), 2.5 mM dNTPs (Roche), 10 pmol of appropriate primers and 1 U of Taq polymerase (Roche). The cycling programme was: initial denaturation at 94°C for 3 min; 35 cycles of denaturation at 94°C for 30 s; primer–template annealing at 57°C for 30 s; and DNA synthesis at 72°C for 1 min; and final extension at 72°C for 7 min. The amplified products were electrophoresed in 2% agarose gels (containing 0.5 μg/mL ethidium bromide) in 1× TAE buffer. Amplicon sizes were estimated with 100 bp standards (New England Biolabs, MA, USA). Table 1. Primers used in this study Locus  Primer  Sequence  Amplicon size (bp)  Reference  vacA m1/m2  VAG-F  5′-CAATCTGTCCAATCAAGCGAG-3′  642  21  VAG-R  5′-GCGTCAAAATAATTCCAAGG-3′  GAPDH (mouse)  GAPDH F  5′-GCAGTGGCAAAGTGGAGATT-3′  249  21  GAPDH R  5′-TCTCCATGGTGGTGAAGACA-3′  Locus  Primer  Sequence  Amplicon size (bp)  Reference  vacA m1/m2  VAG-F  5′-CAATCTGTCCAATCAAGCGAG-3′  642  21  VAG-R  5′-GCGTCAAAATAATTCCAAGG-3′  GAPDH (mouse)  GAPDH F  5′-GCAGTGGCAAAGTGGAGATT-3′  249  21  GAPDH R  5′-TCTCCATGGTGGTGAAGACA-3′  Table 1. Primers used in this study Locus  Primer  Sequence  Amplicon size (bp)  Reference  vacA m1/m2  VAG-F  5′-CAATCTGTCCAATCAAGCGAG-3′  642  21  VAG-R  5′-GCGTCAAAATAATTCCAAGG-3′  GAPDH (mouse)  GAPDH F  5′-GCAGTGGCAAAGTGGAGATT-3′  249  21  GAPDH R  5′-TCTCCATGGTGGTGAAGACA-3′  Locus  Primer  Sequence  Amplicon size (bp)  Reference  vacA m1/m2  VAG-F  5′-CAATCTGTCCAATCAAGCGAG-3′  642  21  VAG-R  5′-GCGTCAAAATAATTCCAAGG-3′  GAPDH (mouse)  GAPDH F  5′-GCAGTGGCAAAGTGGAGATT-3′  249  21  GAPDH R  5′-TCTCCATGGTGGTGAAGACA-3′  Statistical analysis Differences in the histology score were tested with the Mann–Whitney U-test. Student’s t-test was used for the remaining statistics. Both tests were considered as statistically significant when P < 0.05. Results are expressed as average ± SEM of n observations. Results In vitro susceptibility of H. pylori to ellagic acid The antimicrobial efficacy of ellagic acid against 55 H. pylori isolates was evaluated by the agar dilution method. Among 55 H. pylori clinical strains examined against ellagic acid, 39 were isolated from duodenal ulcer patients, whereas 10 were isolated from antral gastritis patients and 6 from non-ulcer dyspepsia patients. Ellagic acid inhibited the growth of all 55 clinical isolates and SS1, a mouse-adapted strain. The MIC of ellagic acid varied between 5 and 30 mg/L and for the majority (89.08%) of the strains the MIC was either 10 mg/L (14.54%) or 15 mg/L (74.54%) (Figure 2). A few strains (3.63%) showed a higher MIC of 30 mg/L (Table 2). The mouse-colonizing strain SS1 showed an MIC of 15 mg/L. These data suggest that ellagic acid has an anti-H. pylori effect in Indian strains. The MICs of amoxicillin, clarithromycin and metronidazole ranged from 0.03 to 2 mg/L, 0.02 to 4 mg/L and 3 to 64 mg/L, respectively (Table 2). Of the 55 H. pylori isolates, resistance to metronidazole (MIC > 8 mg/L) was observed in 52 isolates (94.5%, Tables 2 and 3). Resistance to clarithromycin (MIC > 0.5 mg/L) was found in 5 (9.1%) of the 55 isolates. The remaining 50 susceptible isolates showed MICs varying from 0.02 to 0.125 mg/L (Table 2 and 3). All the strains included in this study were susceptible to amoxicillin with MICs ranging from 0.03 to 2 mg/L. Table 2. MICs of ellagic acid, clarithromycin, amoxicillin and metronidazole for H. pylori strains Number  Strain  MIC (mg/L)   ellagic acid  clarithromycin  amoxicillin  metronidazole  1  186  5  0.125  0.125  16  2  144_1A  5  0.125  0.125  16  3  185_1A  5  0.125  0.125  3  4  156_1A  5  0.125  0.25  16  5  139(1)  10  0.125  0.25  16  6  213(4)  10  0.125  0.25  16  7  233  10  0.125  0.25  3  8  129(1)  10  0.125  0.03  16  9  155_10B  10  0.125  0.25  16  10  L7  10  0.125  0.25  32  11  34  10  2  0.25  16  12  39  10  2  0.25  16  13  I-77  15  0.125  0.25  16  14  162  15  0.125  0.25  32  15  I-90  15  0.125  0.25  32  16  218_1A  15  0.125  0.25  32  17  I-87  15  0.125  0.25  32  18  I-119  15  0.125  0.25  32  19  217  15  0.125  0.25  64  20  I-80  15  0.125  0.25  32  21  I-27  15  0.125  0.25  3  22  127  15  0.125  0.25  32  23  157_6B  15  0.125  0.125  16  24  227_1A  15  0.125  0.5  16  25  I-91  15  0.125  0.5  32  26  138  15  0.125  0.5  32  27  130  15  0.125  0.5  16  28  L10  15  0.125  1  16  29  197  15  0.125  0.125  16  30  157(1A)  15  0.125  0.25  32  31  137(1)  15  0.125  0.25  16  32  135(2)  15  0.125  0.25  16  33  171  15  0.125  0.25  32  34  170(1A)  15  0.125  0.25  32  35  152_1A  15  0.125  0.25  32  36  216_1A  15  0.125  0.25  32  37  169  15  0.02  2  16  38  L1660  15  0.125  0.25  16  39  191  15  0.02  0.25  16  40  152(7B)  15  0.125  0.25  16  41  M27  15  0.125  0.125  16  42  M28  15  0.125  0.125  16  43  M29  15  0.125  0.125  16  44  M12B  15  0.125  0.125  16  45  179  15  0.125  0.125  16  46  148(1)  15  0.125  0.25  64  47  163  15  0.125  0.25  16  48  136  15  4  0.25  32  49  195  15  4  0.25  32  50  481  15  4  0.25  32  51  168  15  0.125  0.25  16  52  209(1)  15  0.125  0.25  16  53  205(1)  15  0.125  0.25  16  54  PCR24  30  0.125  0.25  16  55  225_6B  30  0.125  0.25  16  56  SS1  15  0.015  0.5  0.25  Number  Strain  MIC (mg/L)   ellagic acid  clarithromycin  amoxicillin  metronidazole  1  186  5  0.125  0.125  16  2  144_1A  5  0.125  0.125  16  3  185_1A  5  0.125  0.125  3  4  156_1A  5  0.125  0.25  16  5  139(1)  10  0.125  0.25  16  6  213(4)  10  0.125  0.25  16  7  233  10  0.125  0.25  3  8  129(1)  10  0.125  0.03  16  9  155_10B  10  0.125  0.25  16  10  L7  10  0.125  0.25  32  11  34  10  2  0.25  16  12  39  10  2  0.25  16  13  I-77  15  0.125  0.25  16  14  162  15  0.125  0.25  32  15  I-90  15  0.125  0.25  32  16  218_1A  15  0.125  0.25  32  17  I-87  15  0.125  0.25  32  18  I-119  15  0.125  0.25  32  19  217  15  0.125  0.25  64  20  I-80  15  0.125  0.25  32  21  I-27  15  0.125  0.25  3  22  127  15  0.125  0.25  32  23  157_6B  15  0.125  0.125  16  24  227_1A  15  0.125  0.5  16  25  I-91  15  0.125  0.5  32  26  138  15  0.125  0.5  32  27  130  15  0.125  0.5  16  28  L10  15  0.125  1  16  29  197  15  0.125  0.125  16  30  157(1A)  15  0.125  0.25  32  31  137(1)  15  0.125  0.25  16  32  135(2)  15  0.125  0.25  16  33  171  15  0.125  0.25  32  34  170(1A)  15  0.125  0.25  32  35  152_1A  15  0.125  0.25  32  36  216_1A  15  0.125  0.25  32  37  169  15  0.02  2  16  38  L1660  15  0.125  0.25  16  39  191  15  0.02  0.25  16  40  152(7B)  15  0.125  0.25  16  41  M27  15  0.125  0.125  16  42  M28  15  0.125  0.125  16  43  M29  15  0.125  0.125  16  44  M12B  15  0.125  0.125  16  45  179  15  0.125  0.125  16  46  148(1)  15  0.125  0.25  64  47  163  15  0.125  0.25  16  48  136  15  4  0.25  32  49  195  15  4  0.25  32  50  481  15  4  0.25  32  51  168  15  0.125  0.25  16  52  209(1)  15  0.125  0.25  16  53  205(1)  15  0.125  0.25  16  54  PCR24  30  0.125  0.25  16  55  225_6B  30  0.125  0.25  16  56  SS1  15  0.015  0.5  0.25  Table 2. MICs of ellagic acid, clarithromycin, amoxicillin and metronidazole for H. pylori strains Number  Strain  MIC (mg/L)   ellagic acid  clarithromycin  amoxicillin  metronidazole  1  186  5  0.125  0.125  16  2  144_1A  5  0.125  0.125  16  3  185_1A  5  0.125  0.125  3  4  156_1A  5  0.125  0.25  16  5  139(1)  10  0.125  0.25  16  6  213(4)  10  0.125  0.25  16  7  233  10  0.125  0.25  3  8  129(1)  10  0.125  0.03  16  9  155_10B  10  0.125  0.25  16  10  L7  10  0.125  0.25  32  11  34  10  2  0.25  16  12  39  10  2  0.25  16  13  I-77  15  0.125  0.25  16  14  162  15  0.125  0.25  32  15  I-90  15  0.125  0.25  32  16  218_1A  15  0.125  0.25  32  17  I-87  15  0.125  0.25  32  18  I-119  15  0.125  0.25  32  19  217  15  0.125  0.25  64  20  I-80  15  0.125  0.25  32  21  I-27  15  0.125  0.25  3  22  127  15  0.125  0.25  32  23  157_6B  15  0.125  0.125  16  24  227_1A  15  0.125  0.5  16  25  I-91  15  0.125  0.5  32  26  138  15  0.125  0.5  32  27  130  15  0.125  0.5  16  28  L10  15  0.125  1  16  29  197  15  0.125  0.125  16  30  157(1A)  15  0.125  0.25  32  31  137(1)  15  0.125  0.25  16  32  135(2)  15  0.125  0.25  16  33  171  15  0.125  0.25  32  34  170(1A)  15  0.125  0.25  32  35  152_1A  15  0.125  0.25  32  36  216_1A  15  0.125  0.25  32  37  169  15  0.02  2  16  38  L1660  15  0.125  0.25  16  39  191  15  0.02  0.25  16  40  152(7B)  15  0.125  0.25  16  41  M27  15  0.125  0.125  16  42  M28  15  0.125  0.125  16  43  M29  15  0.125  0.125  16  44  M12B  15  0.125  0.125  16  45  179  15  0.125  0.125  16  46  148(1)  15  0.125  0.25  64  47  163  15  0.125  0.25  16  48  136  15  4  0.25  32  49  195  15  4  0.25  32  50  481  15  4  0.25  32  51  168  15  0.125  0.25  16  52  209(1)  15  0.125  0.25  16  53  205(1)  15  0.125  0.25  16  54  PCR24  30  0.125  0.25  16  55  225_6B  30  0.125  0.25  16  56  SS1  15  0.015  0.5  0.25  Number  Strain  MIC (mg/L)   ellagic acid  clarithromycin  amoxicillin  metronidazole  1  186  5  0.125  0.125  16  2  144_1A  5  0.125  0.125  16  3  185_1A  5  0.125  0.125  3  4  156_1A  5  0.125  0.25  16  5  139(1)  10  0.125  0.25  16  6  213(4)  10  0.125  0.25  16  7  233  10  0.125  0.25  3  8  129(1)  10  0.125  0.03  16  9  155_10B  10  0.125  0.25  16  10  L7  10  0.125  0.25  32  11  34  10  2  0.25  16  12  39  10  2  0.25  16  13  I-77  15  0.125  0.25  16  14  162  15  0.125  0.25  32  15  I-90  15  0.125  0.25  32  16  218_1A  15  0.125  0.25  32  17  I-87  15  0.125  0.25  32  18  I-119  15  0.125  0.25  32  19  217  15  0.125  0.25  64  20  I-80  15  0.125  0.25  32  21  I-27  15  0.125  0.25  3  22  127  15  0.125  0.25  32  23  157_6B  15  0.125  0.125  16  24  227_1A  15  0.125  0.5  16  25  I-91  15  0.125  0.5  32  26  138  15  0.125  0.5  32  27  130  15  0.125  0.5  16  28  L10  15  0.125  1  16  29  197  15  0.125  0.125  16  30  157(1A)  15  0.125  0.25  32  31  137(1)  15  0.125  0.25  16  32  135(2)  15  0.125  0.25  16  33  171  15  0.125  0.25  32  34  170(1A)  15  0.125  0.25  32  35  152_1A  15  0.125  0.25  32  36  216_1A  15  0.125  0.25  32  37  169  15  0.02  2  16  38  L1660  15  0.125  0.25  16  39  191  15  0.02  0.25  16  40  152(7B)  15  0.125  0.25  16  41  M27  15  0.125  0.125  16  42  M28  15  0.125  0.125  16  43  M29  15  0.125  0.125  16  44  M12B  15  0.125  0.125  16  45  179  15  0.125  0.125  16  46  148(1)  15  0.125  0.25  64  47  163  15  0.125  0.25  16  48  136  15  4  0.25  32  49  195  15  4  0.25  32  50  481  15  4  0.25  32  51  168  15  0.125  0.25  16  52  209(1)  15  0.125  0.25  16  53  205(1)  15  0.125  0.25  16  54  PCR24  30  0.125  0.25  16  55  225_6B  30  0.125  0.25  16  56  SS1  15  0.015  0.5  0.25  Table 3. Antibacterial activities of clarithromycin, amoxicillin and metronidazole against H. pylori strains Number  Strain  Clarithromycin  Amoxicillin  Metronidazole  1  186  S  S  R  2  144_1A  S  S  R  3  185_1A  S  S  S  4  156_1A  S  S  R  5  139(1)  S  S  R  6  213(4)  S  S  R  7  233  S  S  S  8  129(1)  S  S  R  9  155_10B  S  S  R  10  L7  S  S  R  11  34  R  S  R  12  39  R  S  R  13  I-77  S  S  R  14  162  S  S  R  15  I-90  S  S  R  16  218_1A  S  S  R  17  I-87  S  S  R  18  I-119  S  S  R  19  217  S  S  R  20  I-80  S  S  R  21  I-27  S  S  S  22  127  S  S  R  23  157_6B  S  S  R  24  227_1A  S  S  R  25  I-91  S  S  R  26  138  S  S  R  27  130  S  S  R  28  L10  S  S  R  29  197  S  S  R  30  157(1A)  S  S  R  31  137(1)  S  S  R  32  135(2)  S  S  R  33  171  S  S  R  34  170(1A)  S  S  R  35  152_1A  S  S  R  36  216_1A  S  S  R  37  169  S  S  R  38  L1660  S  S  R  39  191  S  S  R  40  152(7B)  S  S  R  41  M27  S  S  R  42  M28  S  S  R  43  M29  S  S  R  44  M12B  S  S  R  45  179  S  S  R  46  148(1)  S  S  R  47  163  S  S  R  48  136  R  S  R  49  195  R  S  R  50  481  R  S  R  51  168  S  S  R  52  209(1)  S  S  R  53  205(1)  S  S  R  54  PCR24  S  S  R  55  225_6B  S  S  R  56  SS1  S  S  S  Number  Strain  Clarithromycin  Amoxicillin  Metronidazole  1  186  S  S  R  2  144_1A  S  S  R  3  185_1A  S  S  S  4  156_1A  S  S  R  5  139(1)  S  S  R  6  213(4)  S  S  R  7  233  S  S  S  8  129(1)  S  S  R  9  155_10B  S  S  R  10  L7  S  S  R  11  34  R  S  R  12  39  R  S  R  13  I-77  S  S  R  14  162  S  S  R  15  I-90  S  S  R  16  218_1A  S  S  R  17  I-87  S  S  R  18  I-119  S  S  R  19  217  S  S  R  20  I-80  S  S  R  21  I-27  S  S  S  22  127  S  S  R  23  157_6B  S  S  R  24  227_1A  S  S  R  25  I-91  S  S  R  26  138  S  S  R  27  130  S  S  R  28  L10  S  S  R  29  197  S  S  R  30  157(1A)  S  S  R  31  137(1)  S  S  R  32  135(2)  S  S  R  33  171  S  S  R  34  170(1A)  S  S  R  35  152_1A  S  S  R  36  216_1A  S  S  R  37  169  S  S  R  38  L1660  S  S  R  39  191  S  S  R  40  152(7B)  S  S  R  41  M27  S  S  R  42  M28  S  S  R  43  M29  S  S  R  44  M12B  S  S  R  45  179  S  S  R  46  148(1)  S  S  R  47  163  S  S  R  48  136  R  S  R  49  195  R  S  R  50  481  R  S  R  51  168  S  S  R  52  209(1)  S  S  R  53  205(1)  S  S  R  54  PCR24  S  S  R  55  225_6B  S  S  R  56  SS1  S  S  S  S, susceptible; R, resistant. Table 3. Antibacterial activities of clarithromycin, amoxicillin and metronidazole against H. pylori strains Number  Strain  Clarithromycin  Amoxicillin  Metronidazole  1  186  S  S  R  2  144_1A  S  S  R  3  185_1A  S  S  S  4  156_1A  S  S  R  5  139(1)  S  S  R  6  213(4)  S  S  R  7  233  S  S  S  8  129(1)  S  S  R  9  155_10B  S  S  R  10  L7  S  S  R  11  34  R  S  R  12  39  R  S  R  13  I-77  S  S  R  14  162  S  S  R  15  I-90  S  S  R  16  218_1A  S  S  R  17  I-87  S  S  R  18  I-119  S  S  R  19  217  S  S  R  20  I-80  S  S  R  21  I-27  S  S  S  22  127  S  S  R  23  157_6B  S  S  R  24  227_1A  S  S  R  25  I-91  S  S  R  26  138  S  S  R  27  130  S  S  R  28  L10  S  S  R  29  197  S  S  R  30  157(1A)  S  S  R  31  137(1)  S  S  R  32  135(2)  S  S  R  33  171  S  S  R  34  170(1A)  S  S  R  35  152_1A  S  S  R  36  216_1A  S  S  R  37  169  S  S  R  38  L1660  S  S  R  39  191  S  S  R  40  152(7B)  S  S  R  41  M27  S  S  R  42  M28  S  S  R  43  M29  S  S  R  44  M12B  S  S  R  45  179  S  S  R  46  148(1)  S  S  R  47  163  S  S  R  48  136  R  S  R  49  195  R  S  R  50  481  R  S  R  51  168  S  S  R  52  209(1)  S  S  R  53  205(1)  S  S  R  54  PCR24  S  S  R  55  225_6B  S  S  R  56  SS1  S  S  S  Number  Strain  Clarithromycin  Amoxicillin  Metronidazole  1  186  S  S  R  2  144_1A  S  S  R  3  185_1A  S  S  S  4  156_1A  S  S  R  5  139(1)  S  S  R  6  213(4)  S  S  R  7  233  S  S  S  8  129(1)  S  S  R  9  155_10B  S  S  R  10  L7  S  S  R  11  34  R  S  R  12  39  R  S  R  13  I-77  S  S  R  14  162  S  S  R  15  I-90  S  S  R  16  218_1A  S  S  R  17  I-87  S  S  R  18  I-119  S  S  R  19  217  S  S  R  20  I-80  S  S  R  21  I-27  S  S  S  22  127  S  S  R  23  157_6B  S  S  R  24  227_1A  S  S  R  25  I-91  S  S  R  26  138  S  S  R  27  130  S  S  R  28  L10  S  S  R  29  197  S  S  R  30  157(1A)  S  S  R  31  137(1)  S  S  R  32  135(2)  S  S  R  33  171  S  S  R  34  170(1A)  S  S  R  35  152_1A  S  S  R  36  216_1A  S  S  R  37  169  S  S  R  38  L1660  S  S  R  39  191  S  S  R  40  152(7B)  S  S  R  41  M27  S  S  R  42  M28  S  S  R  43  M29  S  S  R  44  M12B  S  S  R  45  179  S  S  R  46  148(1)  S  S  R  47  163  S  S  R  48  136  R  S  R  49  195  R  S  R  50  481  R  S  R  51  168  S  S  R  52  209(1)  S  S  R  53  205(1)  S  S  R  54  PCR24  S  S  R  55  225_6B  S  S  R  56  SS1  S  S  S  S, susceptible; R, resistant. Figure 2. View largeDownload slide Distribution of ellagic acid MICs for the H. pylori strains isolated from gastroduodenal patients in India. Figure 2. View largeDownload slide Distribution of ellagic acid MICs for the H. pylori strains isolated from gastroduodenal patients in India. Morphological changes induced by ellagic acid TEM, a powerful imaging technique, was used to examine the morphological changes of H. pylori strain SS1 following ellagic acid treatment (Figure 3). Briefly, H. pylori growth under control conditions was exponential until ∼12 h, after which it reached stationary phase. After treatment with 15 μM ellagic acid, the bacterial (Figure 3a and b) morphology changed from bacillary (untreated, control) to spherical/coccoid forms, but no changes were observed with lower concentrations of ellagic acid. In strains that have MICs of 5 and 10 mg/L, we observed a change in morphology (data not shown). The logic behind the selection of SS1 is its well-known property of infecting animals and its worldwide use. Thus, ellagic acid is an effective anti-H. pylori molecule, promoting coccoid bacterial morphology, which is known to be a non-cultivable form. Figure 3. View largeDownload slide Transmission electron micrographs of H. pylori control (a) and after 12 h of treatment with ellagic acid (b). Figure 3. View largeDownload slide Transmission electron micrographs of H. pylori control (a) and after 12 h of treatment with ellagic acid (b). Effect of ellagic acid on bacterial colonization Initially all the mice were challenged with H. pylori strain SS1. Two weeks following the initial bacterial challenge, the experimental group of mice was orogastrically fed with ellagic acid (10 mg/kg) and non-infected control mice received sterile PBS as described in the Methods section. At 6 weeks post-infection a small tissue section from the stomach was used for H. pylori enumeration. Tissue homogenates were spread on BHI agar medium. H. pylori colonies were enumerated and results are presented as cfu/g of stomach tissue. In infected tissue the average number of cfu was estimated as 5.2 × 106/g. Here, maximum, minimum and median colony counts of infected mouse samples were 61, 42 and 56, respectively. No colonies were found in the control group. The group treated with ellagic acid also did not show any colonies (P < 0.05 compared with the infected group; Figure 4). Figure 4. View largeDownload slide Effect of ellagic acid on the viability of H. pylori in H. pylori-infected mice. Histographic representation of colonization efficiency of the SS1 strain of H. pylori in mice and the effect of ellagic acid thereon, obtained by quantitative culture. EA10, ellagic acid 10 mg/kg. Figure 4. View largeDownload slide Effect of ellagic acid on the viability of H. pylori in H. pylori-infected mice. Histographic representation of colonization efficiency of the SS1 strain of H. pylori in mice and the effect of ellagic acid thereon, obtained by quantitative culture. EA10, ellagic acid 10 mg/kg. Anti-H. pylori activity in vivo In view of the fact that the in vitro study confirmed that ellagic acid had anti-H. pylori activity, we investigated the effect of ellagic acid on H. pylori colonization in an invivo model. Briefly, positive control and experimental groups of C57BL/6 mice were infected with 1 × 108 cfu/mL of H. pylori strain SS1 via orogastric inoculation. Six weeks post-infection, animals were deprived of feed, but allowed free access to water for 24 h and then sacrificed. Stomach tissues were collected and rapid urease test (RUT) confirmed H. pylori colonization. Samples were also screened for the presence of the vacA gene using DNA extracted from H. pylori SS1-infected mouse gastric tissues by PCR. Two weeks after bacterial inoculation, a group of mice was administered 10 mg/kg body weight ellagic acid daily for 1 week. The efficacy of ellagic acid was evaluated by RUT with the respective mouse gastric tissues. Mouse gastric tissues from the infected and ellagic acid-treated groups were all positive and negative for RUT, respectively. To further substantiate ellagic acid antibacterial potential, the bacterial-specific gene primer vacA was amplified by PCR using genomic DNA extracted from the gastric tissues of H. pylori-infected and ellagic acid-fed C57BL/6 mice (10 mg/kg), while the mouse-specific GAPDH gene served as an internal reference. Figure 5 shows the therapeutic efficacy of ellagic acid treatment, which completely abolished H. pylori from mouse stomach tissues. Thus, this in vivo information is in accordance with the in vitro results, confirming the anti-H. pylori potential of ellagic acid. Figure 5. View largeDownload slide Effect of ellagic acid on H. pylori viability in H. pylori-infected C57BL/6 mice. Lane 1 (second from left) shows amplification of the vacA middle region of 642 bp using DNA from the mouse-colonizing strain SS1 with primers VAG-F and VAG-R (Table 1). Lane 2 shows amplification of H. pylori-specific vacA and the mouse-specific GAPDH gene using DNA isolated from the stomach of uninfected C57BL/6 mice. Lane 3 shows the presence of H. pylori-specific gene vacA and GAPDH (housekeeping gene) using DNA isolated from the gastric sample of H. pylori SS1-infected mice 4 weeks post-infection. Lane 4 shows the amplification of vacA and GAPDH using DNA isolated from the gastric tissue of ellagic acid-fed (10 mg/kg body weight) H. pylori SS1-infected C57BL/6 mice. +ve, positive. Figure 5. View largeDownload slide Effect of ellagic acid on H. pylori viability in H. pylori-infected C57BL/6 mice. Lane 1 (second from left) shows amplification of the vacA middle region of 642 bp using DNA from the mouse-colonizing strain SS1 with primers VAG-F and VAG-R (Table 1). Lane 2 shows amplification of H. pylori-specific vacA and the mouse-specific GAPDH gene using DNA isolated from the stomach of uninfected C57BL/6 mice. Lane 3 shows the presence of H. pylori-specific gene vacA and GAPDH (housekeeping gene) using DNA isolated from the gastric sample of H. pylori SS1-infected mice 4 weeks post-infection. Lane 4 shows the amplification of vacA and GAPDH using DNA isolated from the gastric tissue of ellagic acid-fed (10 mg/kg body weight) H. pylori SS1-infected C57BL/6 mice. +ve, positive. Histology of mouse gastric tissues during H. pylori infection and the effect of ellagic acid Briefly, freshly prepared aliquots (200 μL; 108 cfu/mL) of H. pylori strain SS1 were administered to a group of C57BL/6 mice, while the uninfected group received sterile PBS. At 2 weeks post-inoculation a group of H. pylori-infected mice was orally fed with 10 mg/kg body weight ellagic acid (daily for 1 week). All groups of mice were sacrificed at 6 weeks from the start of the experiment. Collected stomach tissues were stained and subjected to histological analysis. As compared with the untreated group (control; Figure 6a and d) in infected (colonizing strain SS1 for 6 weeks) mouse gastric tissue (Figure 6b and e) considerable damage was observed by histological analysis of longitudinal sections. Denudation of the surface epithelial layer was visible in the SS1-infected mouse gastric tissues; this layer was restored to almost normal after ellagic acid treatment. Furthermore, inflammation in the gastric pit cells, as observed in the infected tissues (Figure 6b and e), was checked noticeably by ellagic acid treatment (Figure 6c and f). The inflammatory gradation or score was analysed blindly by a pathologist. For the control and ellagic acid-treated mice no inflammation of gastric mucosa was observed (score 0). Among the infected mice mild (score 1, n = 2) and moderate (score 2, n = 4) inflammation was observed after 6 weeks of infection, which was significant compared with the control and treated groups. No adverse effect or death was observed in any group of mice in our experiments. Figure 6. View largeDownload slide Histopathology of mouse gastric tissues after H. pylori infection and eradication. Representative images of haematoxylin and eosin-stained sections of mouse gastric tissues taken at ×40 magnification. Histological analysis of gastric tissues from (a and d) negative control (i.e. no H. pylori) and (b and e) positive control (i.e. H. pylori with no treatment). There was significant damage of gastric tissues of mice infected with the mouse-colonizing strain SS1 for 3 weeks, like loss of normal mucosal architecture. (c and f) This layer was restored to almost normal after treating with ellagic acid (EA) at 10 mg/kg body weight. The gastric mucosal epithelium, gastric glands, parietal cells (top)/chief cells (bottom) and inflammatory cell infiltration are shown as a black arrow, orange stars, green stars and white stars, respectively. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 6. View largeDownload slide Histopathology of mouse gastric tissues after H. pylori infection and eradication. Representative images of haematoxylin and eosin-stained sections of mouse gastric tissues taken at ×40 magnification. Histological analysis of gastric tissues from (a and d) negative control (i.e. no H. pylori) and (b and e) positive control (i.e. H. pylori with no treatment). There was significant damage of gastric tissues of mice infected with the mouse-colonizing strain SS1 for 3 weeks, like loss of normal mucosal architecture. (c and f) This layer was restored to almost normal after treating with ellagic acid (EA) at 10 mg/kg body weight. The gastric mucosal epithelium, gastric glands, parietal cells (top)/chief cells (bottom) and inflammatory cell infiltration are shown as a black arrow, orange stars, green stars and white stars, respectively. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Discussion Standard triple therapy consisting of a proton pump inhibitor-based regimen is currently used to ameliorate gastric inflammation caused by H. pylori infection.26 Though considerable progress in therapeutic modalities has been achieved, obstacles such as therapeutic failure, poor patient compliance, resistance to conventional antibiotics and escalating cost are frequent. Moreover, the increasing emergence of H. pylori strains resistant to conventional therapy is a major public health concern.27,28 Recently, H. pylori has been registered by the WHO among 16 antibiotic-resistant bacteria that pose the greatest menace to human health.29 Metronidazole and clarithromycin resistance rates are alarming, although they vary among populations.30,31 Clarithromycin resistance has been rapidly increasing in many countries over the past decade and it is ∼30% in Italy and 40% in Turkey.32 One study from Japan demonstrated that the prevalence of clarithromycin resistance is around 31.1%.33 Similarly, China has experienced an increase in clarithromycin resistance from 14.8% in 2000 to 52.6% in 2014.34,35 A study from Kolkata, India, during 2005 reported that 85% of the H. pylori strains were resistant to metronidazole but none of the strains was found to be resistant to clarithromycin.36 Another study, during 2016, reported clarithromycin resistance in 11.8% of the H. pylori isolates in North India.37 Increasing resistance to H. pylori infection has heightened trepidation about a decline in the efficacy of the same antibiotics in treatment of other diseases. Because of the emergence of antibiotic-resistant H. pylori clinical strains, there is an imperative need for alternative, cost-effective antimicrobial agents that are non-antibiotic and suitable for the next generation of eradication therapy. The evolving role of natural products, especially plant-derived active principles, have an evolving role and have been an important source of cancer treatments since ancient times.38–41 Much of the world’s population (∼70%–80%) still depends on plant-derived medicines for healthcare. This is especially true in developing countries like India, where traditional herbal medicines have played a prominent role in the strategy to treat acute diseases, including dyspepsia, gastritis and peptic ulcer.42 Previous literature has shown that ellagic acid, a major polyphenolic component of fruits, vegetables and nuts, has anti-H. pylori activity.15–18 Therefore, the purpose of the present study was to explore the antimicrobial potential of ellagic acid against Indian H. pylori clinical strains, which are phylogeographically distinct from strains of East Asian and Western origin.43 Furthermore, although data are scanty, around 64% of the Indian population harbours H. pylori and, overall, infected populations suffer from H. pylori-associated gastrointestinal disorders having a varying type of antibiotic resistance profile.37,44–46 In the present study, the antibacterial activity of ellagic acid against 55 H. pylori strains from clinical isolates from patients suffering from various gastroduodenal pathologies was examined. Significantly, the majority of these strains were resistant to metronidazole, with MICs ranging from 16 to 64 mg/L, and 9.1% strains were resistant to clarithomycin.36,46 These results suggest that ellagic acid acts through pathways markedly different from the mechanism of action of the antibiotics used for elimination of H. pylori from patients. The mechanism underlying the inhibitory effect of ellagic acid on H. pylori growth is still unknown. However, our TEM observations revealed H. pylori morphological changes upon ellagic acid treatment, which might be the mechanism responsible for bactericidal activity. H. pylori that had been treated with ellagic acid showed coccoid forms47 known to be associated with loss of viability. The morphological transformation of H. pylori from a helical bacillary to a non-culturable coccoid shape has previously been reported to be related to alteration of the bacterial cell wall peptidoglycan. Flagellar structures were also either disrupted or absent. These morphological changes might completely inhibit the motility of the bacteria, key for their migration in the gastric niche and colonization of the host gastric mucosa.48 However, further exploration is needed to clearly explain the antibacterial action of this compound. The murine infection model has been widely used in explorations of host responses to H. pylori infection in addition to eradication studies. In vivo models suggest various parameters, such as the bio-distribution of compounds and their chemical stability, biodegradability and resistance to the excessively acidic conditions of the gastric environment, can influence their bioactivity. In the present study, we utilized an established H. pylori infection model to evaluate the potential therapeutic effect of ellagic acid. Despite the low number of tested mice, the histological analysis showed that ellagic acid at a dose of 10 mg/kg body weight completely eliminated H. pylori from infected mice. This information is of clinical significance for the development of alternative therapy against this ulcer-causing organism. Histological study demonstrated that ellagic acid is very effective in the elimination of the pathogen from infected mice and also repairing damaged gastric tissue as compared with mice infected with SS1. Numerous clinical trials suggest a therapeutic efficacy of ellagic acid in diseases such as cardiovascular disease, diabetes and prostate cancer.49 It has been claimed in a recent patent application50 that ellagic acid has a beneficial effect in reducing the damaging effects of gastric acid and H. pylori infection of the upper digestive tract in the prevention of gastrointestinal disorders. In conclusion, the present study clearly indicates the antimicrobial activity of ellagic acid against H. pylori in vitro, and that the gastric epithelial damage induced by H. pylori infection was almost completely restored by ellagic acid, thus highlighting its promise as a potential therapeutic candidate against H. pylori-related gastrointestinal diseases. Our results suggest that ellagic acid looks very promising as a future alternative for treatment of H. pylori infections, although research in advance of clinical application is necessary to determine its effects in humans. Acknowledgements We thank Mr Bivash Ranjan Mallick of the Electron Microscopy Facility at the National Institute of Cholera and Enteric Diseases for his help in processing samples for histology. Funding This study was supported in part by: (i) the Indian Council of Medical Research (ICMR), Government of India; (ii) the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) of the Japan Agency for Medical Research and Development (AMED); and (iii) the Council of Scientific and Industrial Research (CSIR) [ref. no. 37(1640)/14/EMR-II]. R. D. acknowledges the Indian Council of Medical Research, Government of India for providing a postdoctoral fellowship [grant ref. no. 3/1/3/PDF (3)/2011-MPD]. Transparency declarations None to declare. References 1 Covacci A, Telford JL, Del Giudice G et al.   Helicobacter pylori virulence and genetic geography. Science  1999; 284: 1328– 33. Google Scholar CrossRef Search ADS PubMed  2 Peek M, Balser M. Helicobacter pylori and gastrointestinal tract adenocarcinoma. Nat Rev Cancer  2002; 2: 28– 37. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Mar 16, 2018

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