Intern Emerg Med DOI 10.1007/s11739-013-1013-z

IM - ORIGINAL

Lactobacillus reuteri in the treatment of Helicobacter pylori infection Maria Pina Dore • Marianna Cuccu • Gianni Mario Pes • Alessandra Manca David Yates Graham

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Received: 29 September 2013 / Accepted: 21 October 2013 Ó SIMI 2013

Abstract Probiotics have proven to be useful in the treatment of a number of gastrointestinal diseases. Probiotics may compete directly with Helicobacter pylori, possibly by interference with adherence or by the production of antimicrobial molecules. Lactobacillus reuteri has been shown to inhibit H. pylori in vitro and in vivo, and theoretically may play a role in eradication therapy. The aim of this study was to examine the efficacy of L. reuteri in H. pylori eradication therapy. This was an open label single center study. H. pylori infection was defined as positive gastric histopathology and 13C-UBT. Intervention consisted of L. reuteri (DSM 17938) 108 cfu plus pantoprazole 20 mg twice a day for 8 weeks. Eradication was defined as a negative 13C-UBT, 4–6 weeks post therapy. Compliance was considered good if at least 90 % of the total number of the pills were taken. 21 of 22 subjects completed the study without protocol violation (mean age 52 years; 36 % men). L. reuteri plus pantoprazole twice a day cured 13.6 % (3/ 22; 95 % CI 2.9–34.9 %) of patients with H. pylori infection by ITT analysis and 14.2 % (3/21; 95 % CI 3.0–36 %) by PP analysis. Overall urease activity assessed before and 4–6 weeks post therapy showed a significant reduction with a difference of mean of 38.8 vs. 25.4 by one-tailed test (P = 0.002). In conclusion, L. reuteri may have a potential role in H. pylori eradication therapy if the cure rate can be improved by changes in dose, dosing interval, or duration of therapy. M. P. Dore (&)  M. Cuccu  G. M. Pes  A. Manca Dipartimento di Medicina Clinica e Sperimentale, Clinica Medica, University of Sassari, Viale San Pietro 8, 07100 Sassari, Italy e-mail: [email protected] M. P. Dore  D. Y. Graham Baylor College of Medicine, Houston, TX, USA

Keywords Probiotics  Lactobacillus reuteri  H. pylori  Treatment

Introduction Helicobacter pylori infection is etiologically involved in the gastritis-associated diseases, gastric ulcer, duodenal ulcer, gastric adenocarcinoma, and primary gastric B cell lymphoma and may play a role in some cases of non-ulcer dyspepsia. Cure of H. pylori infection dramatically alters the natural history of gastritis and its sequelae. Currently, antibiotics in combination with antisecretory therapy are the standard of care for patients with H. pylori infections. However, increasing antimicrobial resistance has made successful treatment of H. pylori infections a challenge as the effectiveness of many commonly recommended treatments has declined to unacceptably low levels [1, 2]. Newer regimens have improved treatment success, however, the widespread occurrence of the infection in many countries and the need of two or three antimicrobials suggest that antimicrobial resistance may soon undermine these currently more successful combinations [3]. The clinical manifestations of infectious diseases typically represent an interaction between the infecting microbe and the host, both of which may influence the outcome [4]. Normally, the local microbiome provides a stable environment that resists colonization by pathogens. To understand, and to enhance the ability to manipulate, the normal microbiome with therapeutic intention is an area of active research. Probiotics are live bacteria that may confer a health benefit to the host [5]. In the early 20th century, the Russian Nobel laureate E´lie Metchnikoff [6] suggested the possibility of colonizing the gut with beneficial flora. Currently Lactobacillus and Bifidus spp. are the

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most common microbes used as probiotics in the gastrointestinal tract [7, 8]. Probiotics have been used in the therapy of H. pylori infection, and have proven useful in reducing side effects of traditional antimicrobial therapy and for enhancing patients’ compliance [9]. Some strains of L. reuteri have previously been shown to prevent the early stage of H. pylori colonization in the human gastrointestinal tract, possibly by inhibition of H. pylori binding to putative glycolipid receptor molecules [10]. Colonization has been associated with alterations of the immune response of the gastrointestinal mucosa [11]. Importantly, L. reuteri has also been shown to produce an antimicrobial compound, 3-hydroxy propionaldehyde (reuterin), which is a broad spectrum antibiotic against Gram negative and positive bacteria including H. pylori [12, 13]. This was a pilot study to investigate the efficacy of L. reuteri for the treatment of human H. pylori infection.

Methods Patients This study was a prospective, single center, open label pilot study. Patients scheduled for upper endoscopy for any reason and found to be positive for H. pylori infection were invited to enter. Exclusion criteria Exclusion included: severe gastritis (mucosa intensively hyperemic, granular and fragile, easily bleeding by the scope touch), peptic ulcer, pregnancy or lactation; malignancy, severe liver, heart, kidney or endocrine diseases or any other clinically significant medical condition, alcohol abuse, drug addiction, or a history of allergy to pantoprazole or L. reuteri. Patients who received bismuth compounds, antisecretory drugs, or antibiotics during the 4 weeks before endoscopy were also excluded. Written informed consent was obtained from all participants, and the study was approved by the local ethics committee. There was no pharmaceutical company participation in any phase of the study. Definition of H. pylori infection Two biopsy specimens were taken from the antrum, one from the angulus, and two from the gastric corpus for histology to assess the presence of H. pylori. H. pylori infection was defined as the presence of typical histology and bacteria on histologic examination of the gastric biopsies stained by H&E and GIEMSA and a positive 13C-

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UBT. Post-treatment success was defined by a negative 13 C-UBT 30–40 days after completing therapy. 13

C-Urea breath test

The 13C-UBT was performed according to a standardized protocol, the sensitivity and specificity of which have been reported to be of 95 % [14]. All breath tests were analyzed at the same laboratory using a single gas isotope ratio mass spectrometer (ABCA, Europe Scientific, Crewe, UK). Medication The intervention consisted of L. reuteri (DSM 17938, ReuflorÒ, BioGaia AB, Sweden) 108 cfu/tablet plus pantoprazole 20 mg twice a day. The cfu declared contained per tablet was confirmed in two different labs (Sassari and Houston). The choice of the probiotic was based on the previous anecdotal experience. No treatment was administered thereafter. The L. reuteri was administered fasting at least 2–3 h before the mid-day and evening meals. Pantoprazole 20 mg was given just before breakfast and dinner. All drugs were given for 60 days. The duration of treatment chosen for the study was arbitrary. The cost of the drugs used for treatment (pantoprazole plus L. reuteri tablet) was € 141.66. Patient compliance The patients were evaluated by a physician for compliance and side effects after completing treatment and at followup by direct questioning. Side effects including diarrhea, nausea, vomiting, stomach ache, taste disturbance, dizziness, headache, skin rash, constipation and lack of appetite; were graded as mild (did not limit daily activities), moderate (limited daily activities to some extent), or severe (made daily activities all but impossible), and recorded. Study design and statistical analysis A two-stage design was applied. In the two-stage design the patients are divided into two groups. At the completion of the first stage, an interim analysis is made to determine if the second stage should be conducted (e.g., a stopping rule is utilized). If the number of patients responding is greater than a certain amount, the second stage will be conducted—otherwise, not. The optimal two-stage design to test the null hypothesis that P B 0.200 versus the alternative P C 0.350 has an expected sample size of 36 and a probability of early termination of 0.630. After testing the therapy on 20 patients in the first stage, the trial would be terminated if four or fewer responded and the details would be reconsidered (e.g., dose, dosing interval etc.). If more

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therapy showed a significant reduction with a difference of means of 38.8 vs. 25.4 by one-tailed test (P = 0.002) (Fig. 1). Treatment failures by L. reuteri-based therapy were, subsequently, cured with bismuth-based quadruple therapy twice a day [15].

than four were cured, a total of 62 patients would be studied. If the total number cured was less than or equal to 17, the drug would be rejected. Analysis of H. pylori eradication efficacy was performed on an intention-to-treat (ITT) basis including all eligible patients enrolled in the study, and on a per-protocol (PP) basis excluding patients lost to the follow-up and protocol violations.

Patient compliance and side effects Overall tolerability was good. Excellent compliance (100 %) was achieved in all cases. Data on side effects were available for all 21 treated patients. No patient complained of side effects.

Results A total of 22 consecutive patients fulfilling the inclusion criteria were enrolled in stage 1 (19 women, median age 51 years, range 21–68 years). All subjects had experienced dyspeptic symptoms. The enrollment stopped after the first stage according to the study design (four or fewer patients responded to the treatment). No patient had peptic ulcer disease, gastric or duodenal erosions. Twenty-two patients took the medications as prescribed and H. pylori eradication status was assessed after treatment. One patient was dropped from the study because he received antibiotics for pneumonia. None were lost to the follow-up (Table 1).

Discussion Lactobacillus species are rod-shaped Gram positive bacteria able to produce lactic acid leading to the microenvironment acidification [16]. Lactobacillus spp. have the capacity to colonize the gastric mucosa [17] and the lactic Table 1 Intervention status of patients enrolled in the study Status

Patients

Received intervention

22

H. pylori eradication 21 patients completed the study with one protocol violation. Twelve patients were treatment naive for H. pylori infection and nine patients had previous H. pylori treatments that failed to cure the infection. L. reuteri plus pantoprazole twice a day cured 13.6 % (3/22; 95 % CI 2.9–34.9 %) of patients with H. pylori infection by ITT analysis and 14.2 % (3/21; 95 % CI 3.0–36 %) by PP analysis (Table 1). The cured patients included one patient with a history of several previous unsuccessfully therapies. Overall urease activity assessed before and 4–6 weeks post Fig. 1 Difference in urease activity assessed before and 4–6 weeks post therapy by 13Curea breath test (UBT)

Drop-out

1

Lost to follow-up

0

Completed trial

21

Intervention ineffective

18

Cure rate ITT

14 % (3/22)

95 % CI

2.9–34.9

Cure rate PP

14 % (3/21)

95 % CI

3.0–36

ITT intention-to-treat analysis, PP per-protocol analysis, CI confidence interval

120

I 13C-UBT

II 13C-UBT

Delta 13C- UBT Value

100

80

60

40

20

0 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

Patient number

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acid produced by Lactobacilli has been shown to inhibit the growth of H. pylori at concentrations of 50–156 mmol/L [18]. Pre-treatment of rats with L. rhamnosus GG at 109 cfu/mL twice daily for three consecutive days markedly reduced the ethanol-induced mucosal lesion area by 45 %, and significantly increased the basal mucosal prostaglandin E(2) and PGE(2) levels suggesting that a local gastroprotective action may also be present [19]. L. gasseri in rat models also enhanced gastric ulcer healing of acetic acid-induced gastric ulcer [20]. These studies show a potential beneficial effect of Lactobacilli in the stomach. In a study from Japan, administration of L. reuteri strain ATCC 55730, four times daily for 8 weeks significantly decreases 13C-UBT delta levels in H. pylori positive subjects, demonstrating a suppression of H. pylori urease activity and H. pylori density. H. pylori density was investigated on the base of cfu obtained from the biopsy gastric cultured mucosa. Patients were divided in a low density (\1 9 105 cfu/mL), moderate density (between 1 9 105 and 5 9 105 cfu/mL), and high-density group (5 9 105 cfu/mL). The individual UBTs were then correlated to the established H. pylori quantity [21]. In a study from Italy, patients were assigned to receive standard 7-day triple without or with L. reuteri supplementation; or a 7-day triple therapy with a probiotic mixture; or a 14-day standard triple therapy plus a probiotic mixture. Overall, H. pylori eradication was achieved in up to 80 % of patients. A statistically significant difference among the different regimens (range 53–71 %) was not reported [22]. Despite this result other studies have demonstrated that Lactobacilli can produce beneficial effects [23–25]. The beneficial effects described for Lactobacillus strains include being able to stabilize the mucosal barrier by increasing mucin expression, reducing bacterial overgrowth, stimulating mucosal immunity and synthesizing antioxidant substances [25]. Lactic acid producing bacteria also generate a variety of metabolic products that are capable of interfering with the growth of other microbes [26]. However, none of these observations alone or together have prompted the use of Lactobacillus species to cure H. pylori infection. There have been a number of studies evaluating the probiotic effects of Lactobacillus strains in reducing antimicrobial side effect, enhancing compliance and improving the cure rate with traditional H. pylori therapies in humans [27–30]. Following a 3-week intake of 180 mL of LC-1 acidified milk twice a day in a placebo-controlled study a decrease in gastric inflammation and H. pylori density is reported. LC-1 acidified milk is fermented milk containing 107 cfu of L. johnsonii La1 per mL (Hirz, Production Plant and Nestle Research Centre, Orbe, Switzerland). During the last 2 weeks of therapy, volunteers in both groups were treated with clarithromycin 500 mg twice a day. Clarithromycin eradicates H. pylori in 26 % of the subjects and

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LC-1 does not improve the antibiotic effect [27]. In an uncontrolled study, Michetti et al. [28] report a decrease in urease activity as assessed by UBT in 80 % of patients positive for H. pylori infection treated for 2 weeks with 50 mL of L. johnsonii (La1) supernatant combined with either omeprazole 20 mg four times a day or with placebo. In accordance with these results a decrease in urease activity of 76 % was also observed in our patients. However, our study did not assess H. pylori urease activity or H. pylori density and our assessment was done at least 4 weeks after stopping therapy, a time lapse that should have allowed any direct effect to have dissipated. Subsequent studies are needed to assess the mechanism and whether the effect was L. reuteri related. In addition, we cannot exclude that such a phenomenon depends on the 60-day pantoprazole therapy itself. Because this was an open label study, the absence of a control group with pantoprazole without L. reuteri prevents any definite conclusion. An open label crossover study in 31 H. pylori positive Japanese subjects reports a slight, but significant decrease (Excess 13CO2 (%0); 26.6 ± 13.7 vs. 20.9 ± 11.8; P \ 0.05), in urease activity following consumption of 90 g of yogurt containing L. gasseri (LG 21) twice a day for 8 weeks [29]. Other studies have reported beneficial effects of Lactobacillus strains when added to the triple therapies against H. pylori and a meta-analysis by Wang et al. [30] on the efficacy and safety of Lactobacillus and Bifidobacterium-containing probiotic compound preparation in H. pylori eradication therapy, report an overall beneficial effects of probiotics. Eradication odds ratio (OR), was available for 1469 patients (10 trials; 708 in the probiotics supplementation group and 761 in the control group). The pooled OR by ITT analysis and by PP analysis in the probiotics supplementation over without probiotics was 2.066 (95 % CI 1.398–3.055) and 2.321 (95 % CI 1.715–3.142) in each group except in patients accepting rescue therapy, and in children. The incidence of total side effects is significantly decreased in the probiotics supplementation group (OR = 0.305; 95 % CI 0.117–0.793) [30]. Subgroup analysis for Lactobacillus was not performed. In another study, 120 H. pylori positive patients were randomized to receive rabeprazole, clarithromycin and amoxicillin, either with or without a lyophilized and inactivated culture of L. acidophilus twice daily. A significantly higher eradication rate was observed in the L. acidophilus-supplemented group (88 %), with respect to the control group (72 %) [31]. Diarrhea, nausea and taste disturbance, assessed by questionnaire, were significantly reduced in the Lactobacillus strain GG-supplemented group. Saggioro et al. [32] in a randomized, placebo-controlled pilot study published as an abstract reported 30 dyspeptic patients, positive for H. pylori infection by histology, rapid urease test (CLO-test) and 13C-UBT who

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were randomly and blindly assigned on either omeprazole 20 mg plus L. reuteri 89105 cfu twice a day before breakfast and dinner or omeprazole 20 mg plus placebo twice a day for 30 days. Patients were examined for H. pylori status by all the three tests 4 weeks after the end of therapy. L. reuteri plus omeprazole eradicated H. pylori infection in 9 of 15 treated patients (60 %) vs. no eradication occurred in the control population (omeprazole alone) (P \ 0.0001) [31]. Differences between our study and the Saggioro study (14 vs. 60 %) included different geographic areas, different H. pylori strains (Venetian vs. Sardinian), possibly differences in L. reuteri strains, doses, timing and duration of administrations. We believe the results are most consistent with L. reuteri having cured some patients particularly as the cure rate with PPI alone averages zero. Nevertheless, these results will need to be confirmed and to be useful clinically the effectiveness will need to be improved possibly by alterations in the duration, amount of cfu, or frequency of administration, etc.

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12. 13.

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16. Acknowledgments

This work was not financially supported.

Conflict of interest

None.

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18.

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