Evaluation of Helicobacter pylori eradication by triple therapy plus Lactobacillus acidophilus compared to triple therapy alone
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- da Silva Medeiros, J.A., F. O. Gonçalves, T.M., Boyanova, L. et al. Eur J Clin Microbiol Infect Dis (2011) 30: 555. doi:10.1007/s10096-010-1119-4
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The purpose of this study was to evaluate the influence of adding Lactobacillus acidophilus to a triple regimen for Helicobacter pylori eradication in untreated patients with peptic ulcers or ulcer-scars. This was a pre-randomized, single-blind, interventional, treatment-efficacy study with active controls and parallel-assignment, set in Coimbra, Portugal, on 62 consecutive H. pylori-positive untreated adults with peptic ulcers or ulcer-scars, diagnosed by gastroduodenoscopy, with pre-treatment direct Gram-staining and culture of gastric biopsies. The first 31 patients received esomeprazole 20 mg, amoxicillin 1000 mg and clarithromycin 500 mg (EAC), all b.i.d., for 8 days. The remaining 31 added L. acidophilus, 5 × 109 organisms per capsule, 3 + 2 i.d. for 8 days (EACL). The main outcome measure was 13C urea breath test (UBT), ≥6 weeks after completion of therapy. Successful eradication (UBT-negativity after treatment), was similar in both groups (EAC = 80.6%; EACL = 83.9%, p = 0.740) by both intention-to-treat and per-protocol analysis. The non-eradicated strains were susceptible in vitro to both antibiotics. Adding L. acidophilus to EAC triple therapy did not increase H. pylori eradication rates. Considering the cost and the burden of ingesting five extra capsules daily, supplementing the EAC therapy with L. acidophilus, at this dose, shows no benefit. Further studies with different dosages and duration of treatment, and other probiotics or probiotic combinations are required to improve eradication.
Helicobacter pylori infection is associated with chronic gastritis and peptic ulcer disease, and is a risk factor for the development of gastric cancer and MALT lymphoma . Successful eradication of the infection by triple associations of antibiotics and proton-pump inhibitors is curative. However, the treatment may fail in 10–35% of all cases due to H. pylori resistance, antibiotic side effects and other reasons . Searching for new or additional therapeutic agents is necessary to overcome treatment failures.
There is an ongoing controversy in the literature regarding the usefulness of the addition of probiotics, including Lactobacillus acidophilus, to the standard triple therapy for H. pylori eradication [2–7]. While the activity of L. acidophilus , L. johnsonii and L. casei subsp. rhamnosus strains against H. pylori has been demonstrated in vitro, in vivo successful H. pylori eradication by lactobacilli has been uncommon [2, 8, 9].
There are relatively few studies using triple regimens supplemented with L. acidophilus in humans [5–7]. The aim of our study was to evaluate the influence of adding L. acidophilus to a triple regimen for H. pylori eradication in untreated patients with peptic ulcers or ulcer scars.
Patients and methods
Eligibility criteria included previously untreated H. pylori-positive adults aged 20 years or over who had endoscopically confirmed peptic ulcers or peptic ulcer scars. Exclusion criteria were previous or current treatment with proton pump inhibitors or triple therapy, and neoplastic ulcers.
Patients involved in the study
Patients in the EACL group
Patients in the EAC group
Duodenal ulcer scar
Culture and antibacterial susceptibility testing
The biopsies were frozen in Brucella broth with 25% glycerol and kept at –80 °C before being cultured, as previously described . Immediately prior to culture, the biopsies were chopped and spread evenly onto H. pylori selective medium (PYL agar, Bio Mérieux, France). The plates were incubated for 3–10 days at 37°C under microaerophilic conditions (CampyGen system, Oxoid, UK). The suspected colonies were tested with a home-made rapid urease test (personal communication, L Monteiro, Lisboa, Portugal) and subcultured onto H. pylori selective medium (Wilkins-Chalgren agar with 10% horse blood, vancomycin 10 mg/L, cefsulodin 5 mg/L, trimethoprim 5 mg/L, and cycloheximide 100 mg/L; Biogerm, Porto, Portugal). The plates were incubated at 37°C for 48 h in a microaerophilic atmosphere (CampyGen). H. pylori identification was made by Gram staining and testing for the presence of oxidase, catalase, and urease activity.
The isolated H. pylori strains were subcultured. The minimal inhibitory concentrations (MICs) of clarithromycin and amoxicillin were evaluated against 31 strains from the patients in the EACL group and 28 strains from those in the EAC group. Three strains from the EAC group were lost while being subcultured. The strain susceptibility to the antibiotics used in this study was determined by E test, according to a previously described method [11, 12]. The plates were incubated for 48 h in a microaerophilic atmosphere and were read according to the supplier’s recommendations.
Helicobacter pylori eradication by adding Lactobacillus acidophilus to the eradication regimens of patients with gastroduodenal diseases
Number of patients treated by standard regimen plus probiotic
Number of patients treated by standard regimen
Duration of treatment (days)
Eradication rate (%) by classic regimen
Eradication rate (%) by probiotic- supplemented regimen
Significant increase in eradication rate
L. acidophilus lyophilized and inactivated culture (t.i.d.)
70 (ITT), 72 (PP)
87 (ITT), 88 (PP)
L. acidophilus LB spent culture supernatant
Lacidofil containing L. acidophilus and L. rhamnosus (two capsules b.i.d.)
L. acidophilus 5 × 109 organisms per capsule, (b.i.d. 3 capsules in the morning and 2 capsules at night)
80.6 (ITT and PP)
83.9 (ITT and PP)
Urea breath test
To detect possible differences in H. pylori eradication rates between the patients of the EACL group and those of the EAC group, a 13C urea breath test (UBT, Helico-Test 13C-Ureia, Isomed S.L. Pharmaceutical Laboratory, Madrid, Spain) was performed six to seven weeks after the end of therapy. The specimen collection was performed immediately before (basal sample) and 30 min after (post-test sample) the ingestion of the marked urea; the 13C/12C ratio was measured in both specimens, and the result was expressed as the difference between the basal and post-test measurements. The UBT was considered positive when this value was >5 delta over baseline (DOB) per 1,000. This study had a single-blind design, with the technicians administering the UBT and evaluating the outcomes being blinded to patient group assignments.
The chi-square test and Fisher's exact test of independence (when appropriate) were used for the statistical analysis. The outcome variable was the presence of H. pylori eradication (yes vs. no) and the exposure variable was Lactobacillus supplementation of the triple therapy (yes vs. no). A p value of <0.05 was considered statistically significant.
This study was approved by the Ethics Committee of the Social Services of the University of Coimbra (Coimbra, Portugal) with the registration number 36/07. It was registered at ClinicalTrials.gov, of the National Institutes of Health (USA), with the protocol ID “SASUC-36/07”. Informed written consent was obtained from all patients.
The MICs of the 31 strains in the EACL group and the 28 strains in the EAC group for amoxicillin were MIC50, <0.016 and <0.016 mg/L, MIC90, 0.023 and 0.023 mg/L, and ranges of <0.016–0.047 and <0.016–0.19 mg/L, respectively. The MICs of the 31 strains in the EACL group and the 28 strains in the EAC group for clarithromycin were MIC50, <0.016 and <0.016 mg/L, MIC90, 0.032 and 0.064 mg/L, and ranges of <0.016 to >256 and <0.016–48 mg/L, respectively.
Successful eradication rates, as determined by a negative UBT after treatment, were similar in the patients of the EAC group (80.6%, 25 of 31) and the EACL group (83.9%, 26 of 31; p = 0.740, OR 0.80; 95% CI 0.22–2.96) by both intention to treat and per protocol analysis. The strains of the 11 non-eradicated patients were in vitro susceptible to both clarithromycin and amoxicillin. The MIC ranges against the five strains of the EACL group and the six strains of the EAC group were <0.016–0.016 and <0.016–0.19 mg/L for clarithromycin, and <0.016–0.047 mg/L and <0.016–0.19 for amoxicillin, respectively.
There were no reported dropouts from the study due to non-compliance of the patients or side effects from the medications.
Probiotics are live, nonpathogenic microbial feeds or food supplements that have been used to improve the balance of the microflora, primarily in the gastrointestinal tract . Lactobacilli are acid resistant and can persist in the stomach longer than most other bacteria ; therefore, they have the potential to inhibit H. pylori, both for prophylaxis and for treatment of the infection.
Previous studies have found that the supplementation of standard anti-H. pylori regimens with probiotics can reduce the side effects of triple therapy and, therefore, can improve patients' compliance to therapy .
Other authors have identified the in vitro activity of L. acidophilus against H. pylori, which has been associated with the secretion of lactic acid, CRL639 autolysins and competition for adhesion . However, most of these properties have been observed only in animal models or in vitro data. In addition, the activity of several probiotic species against H. pylori has been reported to be strain-dependant .
In vivo, the administration of probiotics alone usually does not lead to H. pylori eradication . However, in several studies, adding Lactobacillus species (e.g., L. johnsonii La1 lyophilized + inactivated culture or L. casei DN 114001) to a triple therapy of amoxicillin, clarithromycin and a proton pump inhibitor has increased the eradication rate compared to the standard triple regimen alone .
In our study, the addition of 1.5 × 1010L. acidophilus organisms in the morning and 1 × 1010 organisms at night, failed to improve H. pylori eradication. Although 13C UBT was performed at least six weeks after the end of therapy, the annual recurrence of H. pylori infection has been reported to be low (<1–2.7%) in most developed countries ; therefore, UBT-positivity in our sample is most likely due to persistent infection than to reinfection, the latter being unlikely to influence the study outcome.
There are several possible reasons for the absence of effect observed in this study. First, the choice of probiotic to be added to the standard therapy seems to be important. It is still unclear which Lactobacillus species and strains are most active against H. pylori. So far, the most effective (both in vitro and in vivo) Lactobacillus species have been L. casei and L. johnsonii La1 . In the relatively few studies on the addition of L. acidophilus to the standard eradication regimens (Table 2), only two sets of data have reported an increase in H. pylori eradication rate. However, in one of the successful studies, a probiotic preparation containing both L. acidophilus and L. rhamnosus had been used [5, 7].
In addition, the length (14 days in the study by De Francesco et al. , three weeks in the study by Cremonini et al.  and 8 days in our study) and frequency of administration (t.i.d. in the study of Canducci et al.  and b.i.d. in our study) could also be of importance.
Finally, a combination of probiotics could provide a better effect on H. pylori eradication than the use of single species. Several authors [7, 20] have obtained an increase in H. pylori eradication, compared to the standard therapy with probiotic combinations such as Lactobacillus- and Bifidobacterium-containing yogurt (AB-Yogurt) or L. acidophilus and L. rhamnosus. A recent study has demonstrated a significantly higher H. pylori eradication rate by a quadruple therapy following pre-treatment with AB-yogurt containing a mixture of L. acidophilus La5, Bifidobacterium lactis Bb12, Lactobacillus bulgaricus, and Streptococcus thermophilus, than by the quadruple therapy alone .
H. pylori eradication rates in the EAC and EACL groups were comparable; adding L. acidophilus to the triple therapy used in this study did not increase the eradication rate. Considering both the patients’ expenses and the need for the daily ingestion of five extra capsules, the supplementation of the amoxicillin + clarithromycin + esomeprazole therapy with L. acidophilus, at a daily dose of 2.5 × 1010 cells, does not benefit the ulcer patient.
Further studies with different posologies or duration of treatment, and with alternative probiotics or probiotic combinations are required to improve the eradication of H. pylori infection.
The authors would like to thank the “Dr. Ricardo Jorge” National Institute of Health, Portugal, for its support in the execution of the microbiology tests.
Conflict of interest statement
The authors declare that they have no conflicts of interest, including financial or personal relationships with other people or organisations, that could influence their work.