JAC Advance Access originally published online on February 8, 2008
Journal of Antimicrobial Chemotherapy 2008 61(4):831-834; doi:10.1093/jac/dkn040
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Original research |
Strain-specific inhibition of Helicobacter pylori by Lactobacillus salivarius and other lactobacilli
1 Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland; 2 Department of Microbiology, University College Cork, Cork, Ireland; 3 Department of Microbiology, Cork University Hospital, Cork, Ireland
* Corresponding author. Tel: +353-21-490-3997; Fax: +353-21-490-3101; E-mail: pwotoole{at}ucc.ie
Received 3 December 2007; returned 9 January 2008; revised 11 January 2008; accepted 15 January 2008
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Abstract |
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Objectives: To investigate the anti-Helicobacter pylori activity of 28 strains of Lactobacillus salivarius and 12 other lactobacilli, isolated from different sites and from different geographical regions.
Methods: An in vitro agar plate diffusion assay was employed to assess the Lactobacillus anti-H. pylori activity.
Results: Nine out of 28 L. salivarius strains and 3/12 other Lactobacillus species tested inhibited H. pylori growth. There was no correlation between ecological niche/geographical location of isolation of the lactobacilli and their inhibitory capability. Further studies on strain L. salivarius UCC119 showed that this strain could inhibit growth of 6/6 clinical isolates of H. pylori, five of which were antibiotic-resistant. This inhibition was not due to acid production and was not mediated by a protein, but did require the presence of live cells.
Conclusions: Growth inhibition of H. pylori by L. salivarius is strain-dependent and is not linked to any particular environmental niche or geographic location. Strains of L. salivarius showing highest anti-H. pylori activity may be useful as an adjunct in the treatment of strains that are resistant to conventional antibiotics.
Keywords: probiotics , antibiotic resistance , bacteriocins
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Introduction |
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Helicobacter pylori is a causative agent of a range of human diseases including gastritis, gastric and duodenal ulcer and gastric cancer.1 Although antibiotics are generally effective in the treatment of H. pylori, it remains a significant human health problem.2 In the developed world where antibiotics are freely available, failure rates of H. pylori therapy are rising steadily due to increasing antibiotic resistance rates.3
Organisms from the lactic acid group of bacteria have been extensively studied for their ability to protect against pathogens like H. pylori.4,5 The antimicrobial activity of a broad-range bacteriocin produced by a well-characterized probiotic strain of Lactobacillus salivarius, strain UCC118, was recently shown to be entirely responsible for the protective effect of this organism in a mouse model of listeriosis.4
The aim of the current study was to investigate if L. salivarius UCC118 or other strains of this organism could inhibit the growth of H. pylori and have potential for use as adjuncts to current therapy strategies.
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Materials and methods |
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Bacteria
Growth conditions and sources of all 40 Lactobacillus strains used are described in detail elsewhere.6,7 H. pylori type strain CCUG 17874 was obtained from the Culture Collection University of Gothenburg, Sweden, and cultured on Colombia base agar (CBA) (Oxoid, Basingstoke, UK) supplemented with 5% horse blood (Charles River Labs, Wilmington, MA, USA) at 37°C and 5% CO2. The clinical isolates of H. pylori have been described previously.8
A 48 h culture of H. pylori 17874 was harvested into 1 mL of brain heart infusion broth (Oxoid). Cells were washed in sterile phosphate-buffered saline (PBS; Sigma, Dorset, UK) and OD600 was adjusted to 0.3. A 600 µL volume of cells was then spread on a pre-warmed CBA plate and allowed to dry. Cultures of various Lactobacillus strains were grown in M17 broth (Oxoid) supplemented with 10% lactose (Oxoid). Cell-free supernatants (CFSs) were prepared by centrifuging the cultures and filtering the supernatant with a 0.2 µM filter (Sartorius, Goettingen, Germany). Whole-cell preparations were resuspended in fresh M17 medium and the OD600 was adjusted to 0.2. Finally, 5 µL of either suspension was placed directly on a 7 mm diameter paper disc on the CBA plate containing H. pylori. Plates were incubated for at least 72 h at 37°C and 5% CO2 before zones of inhibition were measured. Experiments were repeated on three different cultures and scored positive if inhibition was observed on at least two out of three plates. To investigate the nature of the inhibitory substance, either 20 µg of pronase (Roche, Basel, Switzerland) or up to 50 mM N-acetyl cysteine (NAC; Sigma) was spotted onto the agar plate adjacent to the paper disc.
Results are expressed as means ± SEM. Results were compared using a paired t-test and considered significant if P values were <0.05.
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Results and discussion |
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L. salivarius, and particularly strain UCC118, has been extensively studied as a probiotic organism.4,9 This strain has many beneficial properties making it an ideal probiotic candidate. Recently, it has been shown that a bacteriocin secreted by this strain is capable of protecting mice in a Listeria monocytogenes infection model.4 Administration to pigs of a probiotic strain mixture in which L. salivarius becomes dominant, resulted in reduction of Salmonella enterica shedding.10 We hypothesized that if strain UCC118 had a similar activity against H. pylori, it might be effective in treating H. pylori infection. We therefore investigated if L. salivarius strain UCC118, as well as 27 others isolated from diverse sources, could similarly inhibit the growth of H. pylori, another human gastrointestinal tract pathogen.
We had observed that MRS medium inhibits growth of H. pylori, so M17 broth supplemented with lactose was used to culture the various lactobacilli instead. Whole cells from 9/28 strains (32%) of L. salivarius were capable of inhibiting H. pylori growth but L. salivarius UCC118 was not one of these (Table 1). Only three from the 12 other Lactobacillus species tested (25%) inhibited growth of H. pylori. There was no statistical difference in the ability of L. salivarius strains to inhibit H. pylori when compared with the other lactobacilli. Interestingly, Lactobacillus kalixensis, which was isolated from the human stomach,7 did inhibit H. pylori, so this species may warrant further study. There was no correlation between the environmental niche from which the L. salivarius strain was isolated or the geographic region from which it came and the ability to inhibit H. pylori. We found a similar lack of correlation when we compared the other lactobacilli for their ability to inhibit H. pylori growth, indicating that in general L. salivarius strains are not unique within the lactobacilli in this respect.
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None of the CFS samples from any of the 40 strains tested inhibited H. pylori growth, indicating that the inhibitory factor was either not secreted or secreted in quantities too small to cause observable growth inhibition. This also suggested that growth inhibition was not due to lactic acid production by the lactobacilli. We measured the pH of the Lactobacillus culture supernatants, and there was no correlation between pH reduction achieved in the medium and the ability to inhibit growth of H. pylori (Table 1). To further investigate this, we tested M17 broth adjusted to pH 5 and 4 with lactic acid and found that this was similarly unable to inhibit H. pylori growth (data not shown).
Strain L. salivarius UCC119 was a very consistent inhibitor of H. pylori, so we used this strain to further investigate the nature of the inhibitory substance. This strain also had the advantage that it grew better than other L. salivarius strains at 37°C and under 5% CO2, conditions which are required for H. pylori growth. Although neat CFS from any of the strains tested did not inhibit H. pylori growth, we did observe some anti-H. pylori activity when we concentrated the CFS from strain L. salivarius UCC119 40-fold. This activity was absent in uninoculated, concentrated medium.
We have observed that UCC119 secretes a bacteriocin whose gene is identical to that of Abp118 from UCC1189 and it is effective against many Gram-positives such as Lactobacillus sakei and L. monocytogenes (data not shown). Despite this, using pronase (which is a cocktail of proteases), we observed that the anti-H. pylori agent from UCC119 was not proteinaceous in nature and thus is unlikely to be a bacteriocin. It is also unlikely that the production of H2O2 by UCC119 is responsible for its anti-H. pylori activity, as the use of NAC, a potent hydroxyl radical scavenging antioxidant, did not reduce the inhibitory effect.
When we tested dead cells of UCC119 (either boiled or UV-killed) or cells suspended in PBS instead of culture medium, inhibition of H. pylori was not observed, showing that viable metabolically active cells were required for inhibition to take place. We surmised that perhaps the inhibitor was only produced when the lactobacilli came in contact with H. pylori. However, the filtered culture supernatant from a UCC119/H. pylori co-culture was not able to inhibit subsequent growth of H. pylori in our plate diffusion assay (data not shown).
We tested the ability of L. salivarius UCC119 to inhibit the growth of six clinical isolates of H. pylori (Table 2). Although all six H. pylori isolates were inhibited by UCC119, the degree of inhibition varied between strains. There was a statistically significant difference in the degree of inhibition of isolate 3828 when compared with isolates 134 and 8235 (P < 0.05). Perhaps as expected, there was no clear correlation between the sensitivity of an isolate to L. salivarius UCC119 and its antibiotic resistance profile (Table 2).
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Conclusions
Together, these data suggest that H. pylori is not sensitive to bacteriocins or bacteriocin-like peptides produced by L. salivarius, and that live metabolizing cells are required for production of the inhibitory substance(s), which appear to be secreted in small quantities.
L. salivarius UCC119 is currently a better candidate than UCC118 for further studies as a therapy adjunct due to its potent anti-H. pylori activity especially against antibiotic-resistant clinical isolates.
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Funding |
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This work was supported by grants from the Irish Research Council for Science Engineering and Technology (to K. A. R.) and Science Foundation Ireland (to P. W. O'T.).
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None to declare.
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References |
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1 Uemura N, Okamoto S, Yamamoto S, et al. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med (2001) 345:784–9.
2 Hunt RH. WGO-OMGE practice guideline highlights: Helicobacter pylori in developing countries. World Gastroenterol News (2006) 11:22–9.
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Lopez-Brea M, Alarcon T, Domingo D, et al. Inhibitory effect of Gram-negative and Gram-positive microorganisms against Helicobacter pylori clinical isolates. J Antimicrob Chemother (2008) 61:139–42.
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Li Y, Canchaya C, Fang F, et al. Distribution of megaplasmids in Lactobacillus salivarius and other lactobacilli. J Bacteriol (2007) 189:6128–39.
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Roos S, Engstrand L, Jonsson H. Lactobacillus gastricus sp. nov. Lactobacillus antri sp. nov. Lactobacillus kalixensis sp. nov. and Lactobacillus ultunensis sp. nov. isolated from human stomach mucosa. Int J Syst Evol Microbiol (2005) 55:77–82.
8 Hooton C, Dempsey C, Keohane J, et al. Helicobacter pylori: prevalence of antimicrobial resistance in clinical isolates. Br J Biomed Sci (2006) 63:113–6.[CrossRef][Web of Science][Medline]
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Claesson MJ, Li Y, Leahy S, et al. Multireplicon genome architecture of Lactobacillus salivarius. Proc Natl Acad Sci USA (2006) 103:6718–23.
10
Casey PG, Gardiner GE, Casey G, et al. A five-strain probiotic combination reduces pathogen shedding and alleviates disease signs in pigs challenged with Salmonella enterica serovar Typhimurium. Appl Environ Microbiol (2007) 73:1858–63.
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