Patients who were eligible for this study were aged 19–75 years and were diagnosed with IBS according to the Rome III diagnostic criteria. A colonoscopy or barium enema study had been performed in all patients within the previous 5 years. Exclusion criteria included a history of organic bowel disease (e.g. colon cancer, intestinal tuberculosis and inflammatory bowel disease), acute or chronic liver/kidney disease, significant allergic disorders (e.g. asthma), previous major abdominal surgery other than appendectomy, uncontrolled thyroid disease, and acute illness within the previous 2 weeks. No alcoholics, pregnant or nursing women were included. Patients who were using probiotics, prebiotics, synbiotics, antibiotics, corticosteroids, antidepressants, antihistamines, non-steroidal anti-inflammatory drugs, and other drugs that affect intestinal motility (e.g. laxatives, antidiarrheals, prokinetics, and antispasmotics) were excluded.
All enrolled participants received comprehensive information about this study, and informed consent was obtained before any study-related processes began.
Study Design and Procedures
The study was conducted at Hanyang University Hospital in Korea between March 2011 and August 2011, and was approved by the Clinical Research Ethics Committee of the Hanyang University Hospital of Korea (2010-04-009). After a 2-week run-in period, enrolled patients were randomly assigned to receive either one capsule (500 mg) of LacClean Gold-S (Cell Biotech, Co. Ltd, Gimpo, Korea; a multispecies probiotics) or one capsule (500 mg) of a placebo twice daily (total dosage 1000 mg/day) for 4 weeks (Fig. 1). The patients were instructed to take the study product between meals because the increased gastric pH is more favorable for the ingested bacteria. LacClean Gold-S is a capsule-form probiotics containing six species of live bacteria. The six strains of probiotics were Bifidobacterium bifidum (KCTC 12 199BP), Bifidobacterium lactis (KCTC 11 904BP), Bifidobacterium longum (KCTC 12 200BP), Lactobacillus acidophilus (KCTC 11 906BP), Lactobacillus rhamnosus (KCTC 12 202BP), and Streptococcus thermophilus (KCTC 11 870BP). A total of 5 × 109 viable cells in a lyophilized powder form were included in each capsule and constituted 13.1% (w/w) of the total weight (500 mg/capsule). The amount of probiotics equally consisted in each of the six strains. The dose was determined based on previous studies where the daily doses were between 5 × 107 and 3.6 × 1011 colony forming units (CFUs)/day, and ≥ 5 × 109 CFUs/day has been suggested. The placebo powder contained the same "other ingredients" as the active medication and maltodextrin instead of bacteria. OY Lee and KN Lee enrolled the patients for this study. Patients were allocated to the probiotics or placebo group using a computer-generated randomization schedule with a 1 : 1 allocation ratio. Dr. Jun generated the random allocation sequence, and no one but him knew the allocation sequence. The practice nurse gave a questionnaire and explained the protocol to the patients. The nurse did not know the allocation sequence and met the patients in regular sequence. The patient received the medication from the clinical pharmacist. No one could differentiate the two drugs without the sequence information. Stool samples for fecal microflora analysis were obtained immediately before the start of treatment and at the end of the 4 weeks of treatment. Fecal microbiota was analyzed only from patients who agreed to the stool sample collection.
Consolidated Standards of Reporting Trials flowsheet. Probiotics: a probiotic mixture (total 5 × 109 colony-forming unit) containing Bifidobacterium bifidum, B. lactis, B. longum, Lactobacillus acidophilus, L. rhamnosus, and Streptococcus thermophiles. Participants were randomly assigned to receive either one capsule of probiotics or one capsule of placebo twice daily for 4 weeks. ITT, intention to treat.
IBS symptoms were assessed by examiners and patients at baseline and week 4 using a questionnaire. Global relief of IBS symptoms, drug compliance, and adverse events were evaluated by a questionnaire after the 4 weeks of treatment.
Efficacy Measurements The primary efficacy end-point was the proportion of patients who experienced global relief of IBS symptoms after the 4-week treatment. Efficacy was estimated by the response (yes or no) to the question: "Compared with your health before treatment started, have overall IBS symptoms improved during the past seven days?"
Secondary efficacy end-points were: (i) intensity of abdominal pain/discomfort and bloating, for which patients scored their worst symptoms over the previous 24 h on a numeric scale from 0 to 10; (ii) defecation frequency defined as the average number of episodes per week; (iii) stool consistency using the 7-point Bristol Stool Form Scale (BSFS); and (iv) alterations in the composition of fecal microflora as a result of treatment.
Compliance and Safety Assessments Drug compliance was defined as the ratio of number of drugs taken to the number of drugs prescribed. All adverse reactions were reported.
Fecal Samples Patients enrolled in the study provided fecal specimens at the beginning and end of the study. These were collected in sterile containers, brought to the laboratory in a frozen condition, and stored at −80°C until analysis.
Preparation of Genomic DNA From Reference Strains and Fecal Samples Bacterial genomic DNA from pure cultures or fecal samples was prepared using an AccuPrep Genomic DNA extraction kit (Bioneer, Daejeon, Korea). Genomic DNA was extracted from 1 mL of pure culture according to the manufacturer's instructions.
Real-time Quantitative Polymerase Chain Reaction Real-time quantitative polymerase chain reaction (PCR) was carried out using a LightCycler 480 (Roche, Germany), and the group and species-specific primers for PCR are listed in Table 1 . The primers were synthesized commercially by Bioneer, and their specificity was previously verified using DNA from closely or distantly related bacteria. Quantitative PCR was performed in 96-well plates in final volumes of 20 μL consisting of 1 μL of fecal DNA, 0.5 μL of primers (10 pmol each), 10 μL SYBR Green I master (Roche, Mannheim, Germany), and 8 μL of H2O. PCR amplification involved: pre-incubation at 94°C for 4 min followed by 55 cycles of amplification (denaturation at 94°C for 15 s, primer annealing at 55°C for 15 s, and elongation at 72°C for 20 s). Melting curves were obtained by heating samples from 50 to 90°C at a rate of 5°C/s.
The sample size for this study was calculated assuming a 40% difference in the primary end-point between two groups. From this assumption, we calculated that a total of 48 patients would have a statistical power of 80% and a two-sided α risk of 0.05. We planned to enroll 50 patients, as we expected some participants to dropout of the study.
Efficacy and safety were assessed by intention-to-treat (ITT) analysis. The ITT analysis included all participants who had taken any medication, and dropouts were regarded as non-responders. All significance tests were two-sided, and a P value of less than 0.05 was regarded as significant. All statistical analyses were performed using SPSS for Windows release 18.0 (SPSS, Inc., Chicago, IL, USA).
J Gastroenterol Hepatol. 2014;29(1):52-59. © 2014 Blackwell Publishing