JAC Advance Access published online on October 26, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm404
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Inhibitory effect of Gram-negative and Gram-positive microorganisms against Helicobacter pylori clinical isolates
Department of Microbiology, Hospital Universitario de La Princesa, Diego de León 62, 28006 Madrid, Spain
* Corresponding author. Tel/Fax: +34-915202317; E-mail: talarcon{at}helicobacterspain.com
Received 29 July 2007; returned 24 August 2007; revised 27 September 2007; accepted 1 October 2007
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Abstract |
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Objectives: To determine the in vitro inhibitory effect of several Gram-negative and Gram-positive microorganisms against Helicobacter pylori clinical isolates.
Methods: The in vitro effect of 32 microorganisms against H. pylori clinical isolates was determined by a diffusion method. Time–kill assay was performed with two Staphylococcus spp. strains.
Results: Anti-H. pylori activity was detected with Saccharomyces cerevisiae, Bacillus spp., 1 Enterococcus faecium and 2 Lactobacillus spp. against 7, 11, 1, 5 and 6 H. pylori strains tested. All Staphylococcus spp. showed an anti-H. pylori effect: one Staphylococcus auricularis and two Staphylococcus epidermidis against all H. pylori tested; Staphylococcus aureus, Staphylococcus hominis and S. auricularis against six, five and seven H. pylori strains; and two other coagulase-negative Staphylococcus against one H. pylori strain. An inhibitory effect was detected with one Escherichia coli against one H. pylori. Klebsiella pneumoniae, Salmonella spp. and Acinetobacter baumannii showed activity against four H. pylori strains, and Enterobacter cloacae and Stenotrophomonas maltophilia showed activity against 14 H. pylori isolates. No anti-H. pylori activity was detected with one Lactobacillus spp., two Lactococcus lactis, four Streptococcus spp., one Bacillus cereus, one E. faecium, one Enterococcus faecalis, one E. coli, Pseudomonas aeruginosa and Klebsiella oxytoca. Time–kill assay showed bactericidal activity at 24 h with the two Staphylococcus spp. strains tested.
Conclusions: Several strains of human pathogens or commensal bacteria are able to inhibit H. pylori growth in vitro and it is a strain-dependent phenomenon.
Key Words: probiotics , time–killing , Staphylococcus spp. , Lactobacillus spp.
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Introduction |
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Helicobacter pylori is associated with different digestive diseases such as peptic ulcer, gastritis and mucosa-associated lymphoid tissue lymphoma, and it is considered a risk factor in the development of gastric cancer.1 Although several therapeutic regimens have been proposed for H. pylori treatment, including antibiotics and antacids, there is a failure rate of up to 20% because of bad compliance, side effects or antibiotic resistance. This leads to a significant number of patients remaining infected after a first eradication attempt, and so the constant need for new strategies in the treatment of H. pylori, alternative or complementary to antibiotic therapy.2
The in vitro inhibitory effect of probiotics (live microorganisms that, when applied to animal or man, beneficially affect the host by improving the properties of the indigenous flora3) against H. pylori has been described previously. The effect of several microorganisms not considered probiotics such as Clostridium butyricum or commensal flora has been described too.4,5
The purpose of this study was to determine the in vitro inhibitory effect of several microorganisms (both Gram-negative and Gram-positive bacteria) against H. pylori clinical isolates.
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Materials and methods |
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Microorganisms
H. pylori isolates were obtained from 35 gastric biopsies from patients after endoscopy. They were plated on selective and non-selective agar and incubated under a micro-aerobic atmosphere at 37°C for up to 15 days. Identification was made by Gram stain and the presence of oxidase, catalase and urease.
Several microorganisms were obtained from commercial products (milk and yoghurt) and clinical specimens such as blood cultures and vaginal secretions. They were processed depending on their requirements and identification was made by Gram stain and MicroScan WalkAway (Dade), API (bioMérieux) or Auxacolor (Bio-Rad). The microorganisms studied were three Lactobacillus spp., two Lactococcus spp., four Streptococcus spp., two Bacillus spp., three Enterococcus spp., one Saccharomyces cerevisiae, eight Staphylococcus spp., two Escherichia coli, two Klebsiella spp., one Enterobacter cloacae, one Salmonella spp., one Pseudomonas aeruginosa, one Acinetobacter baumannii and one Stenotrophomonas maltophilia.
In vitro activity of microorganisms by the diffusion method
To determine the effect of the different bacteria against H. pylori strains, a diffusion method was used. An antibiotic-free blood agar plate was inoculated with an H. pylori fresh culture (turbidity equivalent to that of a 2 McFarland standard). Two 10 µL drops of each bacterium were deposited on the blood agar containing different concentrations of the microorganism. Plates were incubated at 37°C under micro-aerobic atmosphere for 2–5 days, and the diameters of inhibition zones around the drop were measured.
Microorganisms with an inhibitory effect against all H. pylori strains were chosen for further characterization. (i) Culture supernatants were obtained by centrifugation (10 min at 13 000 rpm) and filter-sterilized (0.45 µm pore size filter) to eliminate the presence of viable cells. (ii) Cultures were sonicated for 10 min, centrifuged and filter-sterilized to obtain the components from inside the cell. (iii) Cell-free culture supernatant was concentrated to 1000 mg/mL by freeze-drying. The agar diffusion method was performed with all the products obtained.
In vitro activity of microorganisms by a time–killing assay
Bacterial viability was estimated by a time–kill assay for the microorganisms that showed in vitro activity against all the H. pylori tested (Staphylococcus spp.). Tubes containing brain heart infusion (BHI) supplemented with 10% bovine fetal serum were inoculated with H. pylori (turbidity equivalent to that of a 3 McFarland standard) and Staphylococcus spp., both as whole viable cells (turbidity equivalent to that of a 0.5 McFarland standard) and the freeze-dried concentrated product. A tube with H. pylori alone was used as a growth control and a tube with H. pylori and amoxicillin was used as a killing control. Aliquots were taken at time 0 and after 1, 2, 6 and 24 h of incubation at 37°C under a micro-aerobic atmosphere and colony counting was performed. The number of viable cfu was determined by 10-fold serial dilutions and plating 10 µL of each one on Mueller–Hinton agar with 5% horse blood, 10 mg/L vancomycin and 10 mg/L amphotericin B; colonies were counted after 5 days of incubation at 37°C under a micro-aerobic atmosphere.
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Results |
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The agar diffusion method was able to detect the inhibitory effect of the different microorganisms tested against H. pylori strains. An inhibitory zone was observed surrounding the drop with some bacteria from 27 to 36 mm.
The inhibitory effect was observed in two of the three Lactobacillus spp., one of the two Bacillus spp., one of the two Enterococcus faecium, the S. cerevisiae, one of the two E. coli, the Klebsiella pneumoniae, E. cloacae, Salmonella spp., A. baumannii and S. maltophilia. All the Staphylococcus spp. tested showed an inhibitory effect against some H. pylori isolates tested, including one Staphylococcus aureus studied. In fact, three Staphylococcus (one Staphylococcus auricularis and two Staphylococcus epidermidis) showed an inhibitory effect against all H. pylori tested (Table 1).
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One H. pylori isolate was inhibited by more microorganisms than any other isolate (the eight Staphylococcus spp., one Bacillus sp., S. cerevisiae and E. faecium), showing more susceptibility than the other H. pylori isolates.
The three Staphylococcus spp. that showed activity against all the H. pylori tested were selected for further studies to characterize the product involved in the production of the inhibitory effect. However, the inhibitory effect was detected when testing whole cells but not when testing: (i) the filtered supernatant; (ii) the supernatant obtained after whole cell sonication and centrifugation; or (iii) the freeze-dried concentrated supernatant.
Killing curves were performed with two Staphylococcus spp. against six H. pylori strains, both with whole cells and with freeze-dried concentrated supernatant. After 24 h of incubation, a bactericidal effect (at least 1000 cfu/mL reduction of initial inoculum) was observed with whole cells of S. epidermidis against five of the six strains tested and with whole cells of S. auricularis against four of the six strains tested. The freeze-dried concentrated supernatant of both Staphylococcus spp. showed no effect against the six H. pylori strains tested. Amoxicillin showed bactericidal activity against all the strains tested (Figure 1).
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Discussion |
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H. pylori treatment, combination of antacids and antibiotics, has good cure rates, but has the disadvantages of being expensive, risking poor compliance, causing side effects and, in particular, encouraging resistance development.2 There is considerable interest in measures capable of reducing those factors as alternative or complementary to the conventional treatment.6 The description of microorganisms with anti-H. pylori activity, at least in vitro, has resulted in new expectations for the treatment of H. pylori infections.
In this study, the inhibitory effect of several microorganisms (Lactobacillus spp., Bacillus spp., E. faecium and S. cerevisiae) against H. pylori clinical isolates was observed. Several previous studies showed that various lactobacilli, or their metabolites, can inhibit or kill H. pylori in vitro or in vivo, such as Lactobacillus salivarius, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri or Lactobacillus johnsonii.4 Other microorganisms with a proven inhibition effect against H. pylori are Weissella confusa, E. faecium, Bacillus subtilis and C. butyricum.7
The inhibitory effect of three Staphylococcus strains against all H. pylori strains in vitro was evident by agar diffusion and also with a time–kill assay after 24 h of incubation. However, the effect was detected with whole cell viable Staphylococcus, but no anti-H. pylori activity was obtained with a cell-free culture supernatant. Our results are in accordance with other studies, in which authors were unable to produce evidence of inhibitory activity in cell-free supernatants.5,8
Krausse et al.5 also described an anti-H. pylori effect of S. epidermidis and S. aureus. S. epidermidis ATCC 14990 produced inhibition in 94% of H. pylori clinical isolates, and S. aureus ATCC 25923 and S. aureus ATCC 29213 inhibited 90% and 61% of H. pylori strains, respectively. The same authors described the inhibitory effect of some Gram-negative bacilli such as S. maltophilia, P. aeruginosa, Serratia marcescens and Morganella morganii, whereas an Enterobacter aerogenes showed no anti-H. pylori activity. In our study, S. maltophilia inhibited 14 H. pylori strains, but P. aeruginosa did not produce inhibition in any of the 25 H. pylori strains tested.
H. pylori occupies a narrow ecological niche because it has the capability to adhere to gastric epithelium and can survive in the hostile acidic environment of the stomach. Apart from H. pylori, the stomach is generally thought to have few other microorganisms and, when they are isolated, reflect swallowed oral and nasopharyngeal biota. Contamination of gastric mucosa with non-Helicobacter bacteria is produced primarily with 
-haemolytic Streptococcus, Micrococcus, Staphylococcus, Enterobacteriaceae and yeasts.9 However, acid secretion has an important effect on bacterial flora. An increase in the quantity and quality of non-H. pylori bacteria was described in the scientific literature, both in experimental models and in humans associated with hypochlorydia.9 The colonization by H. pylori decreases at the endstage of atrophic gastritis, and colonization of the gastric mucosa by Staphylococcus, Klebsiella, Streptococcus and Pseudomonas spp. was described.
In contrast, endoscope-transmitted infections with bacteria such as Salmonella spp., S. epidermidis, -haemolytic Streptococcus, E. coli, S. aureus, K. pneumoniae, Enterococcus faecalis and P. aeruginosa have been described.10 Moreover, the presence of contamination in H. pylori culture plates can reduce the sensitivity of the method. Some of the contaminant bacteria, such as Lactobacillus, Bacillus, Enterococcus, Staphylococcus or Gram-negative bacilli, could inhibit H. pylori growth in the culture media.
In conclusion, several microorganisms (human pathogens and commensal bacteria) are able to inhibit H. pylori growth in vitro and it is a strain-dependent phenomenon. Further research into these H. pylori-inhibiting bacteria could lead to alternatives for H. pylori eradication.
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This work was supported by Fondo de Investigaciones Sanitarias de la Seguridad Social grant FIS PI052442.
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None to declare.
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References |
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9 . Kato S, Fujimura S, Kimura K, et al. Non-Helicobacter bacterial flora rarely develops in the gastric mucosal layer of children. Dig Dis Sci (2006) 51:641–6.[CrossRef][Web of Science][Medline]
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