Study approval

This study was conducted according to the principles of the Declaration of Helsinki and with approval from the Ethical Committee of Comac Medical, Sofia, Bulgaria (Reference: #127/20.06.2017). The study design has been deposited in the ISRCTN registry (ISRCTN12562026 Registration date: 12.03.2019).

Study design

This was a single-centre, double-blind, randomised and placebo-controlled superiority study with equal allocation of participants between two parallel study groups. As an exploratory study, there was no formal sample size calculation.

Recruitment and randomisation

The study was performed by the trials company Comac Medical. Adults aged 30–65 were recruited at the trials facility (Sofia, Bulgaria) between 17/07/17 and 26/07/17. The included participants had a waist circumference > 89 cm (women) or > 100 cm (men); a body mass index (BMI, kg/m2) between 25 and 34.9; receiving no statins or on stabilised statin therapy for at least 3 months and were willing to provide blood samples. Participants were not considered if they were undergoing immunodeficiency/immunosuppressive therapy; had diagnosed diabetes; pregnant or planning pregnancy; had history of ischemic heart disease, heart failure, prolonged QTc interval, rhythm, conduction disorders or any other cardiovascular disease deemed by the investigator as a risk for the participation in the study; had severe systemic disease (cancer, dementia, advanced organ failure); or had experienced significant unexplained weight loss in the previous 3 months.

All participants entering the study provided written informed consent and received a financial incentive as stipulated by the ethics committee. Eligible participants were sequentially assigned an order number and allocated to one of 2 arms of the study in a 1:1 ratio according to a computer-generated random sequence using permuted block randomisation with a block-size of four. The randomisation scheme was generated by an independent statistician using SAS PROC PLAN (SAS v9.4) and the study product was randomised before arrival at the trial site. The allocation sequence was not available to any member of the research team until databases had been completed and locked but was held at the trial site in tamper-proof sealed envelopes in case of emergency.

Study product

The active product (Lab4P) comprised Lactobacillus acidophilus CUL60 (NCIMB 30157), Lactobacillus acidophilus CUL21 (NCIMB 30156), Lactobacillus plantarum CUL66 (NCIMB 30280) Bifidobacterium bifidum CUL20 (NCIMB 30153) and Bifidobacterium animalis subsp. lactis CUL34 (NCIMB 30172) on a base of microcrystalline cellulose at a total of 5 × 1010 colony forming units (cfu) per capsule. The placebo product was capsules of microcrystalline cellulose and was identical in appearance to the active product. All products were prepared by Cultech Ltd, Port Talbot, UK and packed into induction-sealed high-density polyethylene pots and stored at 4–8 °C at the trial site; participants were instructed to refrigerate the supplement throughout the study.

Intervention

One capsule was taken daily for 6 months (180 days). Participants were asked to consume the supplement with food (with or without a cool drink) at any time of the day and to avoid consumption within 2 hours of any antibiotic intake. Participants were asked to maintain their normal diet and lifestyle throughout the study while avoiding the consumption of other probiotic supplements. Participants were provided with pots containing 93 capsules at baseline and 3 months and unused capsules were collected at 3 months and 6 months for compliance monitoring and enumeration of viable bacteria; no deterioration in the product occurred during the intervention period (data not shown).

Outcomes

Primary outcomes were changes from baseline in body weight, waist circumference (WC), blood pressure (BP) and plasma lipid profile (total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG)) in the total population. Secondary outcomes were changes from baseline in plasma CRP level, diversity of the faecal microbiota of volunteers, QoL (as measured by QoL questionnaire (QoLQ), Supplementary Fig. S1) and the incidence of URTI in the total population. Changes in body weight and plasma lipids in a stratified study population were also assessed.

Data and sample collection

The schedule of data and sample collection is shown in Fig. 1a. Physiological measurements were taken at each visit. Overnight fasted blood samples were taken at baseline and 6 months. Participants were asked to complete daily diaries monitoring URTI symptoms14 throughout the duration of the intervention period and QoLQ were completed (Supplementary Fig. S1) at each visit. Participants volunteering to provide faecal samples used faecal collection kits for sample collection/transport.

Figure 1 (a) Scheme of sample/data collection and (b) Flow diagram of the study. QoLQ, quality of life questionnaire; URTI, upper respiratory tract infection. Full size image

Physiological measurements

Body weights were recorded using a calibrated column scale (Seca 709, Hamburg, Germany) after the removal of shoes and jackets. Waist circumference was measured 2 fingers below the umbilicus and seated blood pressure was measured after 5 minutes respite using a calibrated blood pressure monitor (Omron, Kyoto, Japan). Height was measured after the removal of shoes. Efforts were made to ensure the time of day when measurements were taken was standardised for each participant.

Processing and analysis of blood plasma

Overnight fasted bloods were collected into heparinised tubes and plasma separated by centrifugation (2,000 × g, 10 mins) and aliquoted within 1.5 hours of collection (stored at 4–8 °C). One aliquot was used to measure TC, HDL-C, LDL-C, TG and CRP immediately (Architect System clinical analyser®, Abbot Laboratories, Illinois, USA) and remaining plasma was stored at −80 °C until required. Levels of small dense (sd)LDL-C were determined using the sLDL-C-EX “SEIKEN” assay (Randox Laboratories Ltd, UK).

Processing and analysis of faecal samples

Eighty volunteers provided faecal samples at baseline but at 6 months only 64/80 returned samples. Samples were stored refrigerated in anaerobic containers for no more than 48 h prior to storage at −80 °C pending analysis. Genomic DNA was extracted using the QIAamp® Fast DNA Stool Mini Kit (Qiagen, UK) in accordance with manufacturer’s instructions in conjunction with a cell lysis step using Matrix Lysing B tubes (MP Biomedicals, USA) and a FastPrep®−24 bead beater (3 × 30 s cycles (5 m/s) with 5 minute intervals). Sample libraries were prepared using the Illumina 16S Metagenomic Sequencing Library Preparation Protocol with slight modifications; primers targeting Bifidobacteria species were included during amplification of the V1-V2 regions of the 16S rRNA as described elsewhere15 and PCR reactions were cleaned and normalised using the SequalPrep Normalization Plate Kit (Life Technologies, Paisley, UK). Sample libraries were then quantified using the NEBNext Library Quant Kit for Illumina (New England Biolabs, Hitchin, UK) and sequencing was performed on an Illumina MiSeq platform (Illumina Inc., Saffron Walden, UK) using the MiSeq Reagent Kit v3 (Illumina Inc) and paired-end 300 base pair (bp) chemistry. Negative and positive control reactions were included during sequencing that contained no template DNA or DNA from a reference bacterial community respectively.

SeqPrep C++ software was used to join the paired-end R1 and R2 reads. QIIME 1.916 was used to process joined reads for quality-filtering with the following parameters: i) maximum of three consecutive base calls with Phred <19 (1 error in base calling in 100 bp), ii) a minimum read length including consecutive high-quality base calls (Phred > 19) and iii) no “n”-bases allowed. Quality-filtered reads were then aligned against the SILVA17 closed-reference database 123 release, with 97% cluster identity. To reduce the effect of uneven sampling and sequencing, each sample library was rarefied to the smallest library size.

Data management and statistical analysis

Analysis of study outcomes was performed on an intention-to-treat basis. Outcome variables with measurements at 3 and 6 months were analysed using a linear mixed model (LMM) that included treatment, time, interaction between treatment and time as fixed effects, baseline measurement as a covariate, and subject as random effect. The treatment difference at each time point with 95% confidence intervals (CI) from t-test was calculated from the LMM. Outcome variables with only measurement at 6 months were analysed using a generalized linear model (GLM) that included treatment as the only predictor, and baseline measurement as a covariate, from which the treatment difference at 6 months with 95% CI was calculated.

Incidence rates were calculated from daily diaries by dividing the number of episodes of each symptom (coughing, runny nose, blocked nose, sore throat, headache, earache, muscle pain, chest wheeze and itchy eyes) or antibiotic usage by the number of days in the study and are expressed per 100 person days. Each episode comprised a continuous sequence of symptoms or antibiotic usage and was separated from another episode by a minimum of 24 h. Incidence rate ratios (IRR) were calculated using a GLM with Poisson distribution and log link function.

Covariate adjusted analyses within the LMM/GLM framework as described above were performed on all outcomes with age, gender and BMI as covariates. Where appropriate, subgroup analysis was performed by gender, age, BMI and TC level at baseline. Values of p were considered statistically significant when less than 0.05. Continuous variables were summarised using mean ± standard deviation (SD). Data analyses were performed using SAS® version 9.4 (SAS Institute Inc., Cary, NC, USA).

Statistical analysis of faecal microbiota

Statistical analysis of faecal next generation sequencing data was performed by calculating the unique number of operational taxonomic units (OTU) and indices of alpha-diversity (Chao1 and Shannon) and beta-diversity (weighted Unifrac) using QIIME 1.9. OTUs with less than 15 counts in at least two samples were removed from analysis. Differences in the alpha-diversity indices were tested using a mixed-effects linear model, implemented in the lmer function of the lme4 R package, with randomisation (active or placebo), time-point (baseline or 6 months), age, gender and BMI as fixed effects and each participant as a random effect. Two-sample comparisons were performed using either the t-test or the non-parametric Wilcoxon-Mann-Whitney test according to the normality distribution of the data. Between-group differences in beta-diversity were tested with the permutational analysis of variance (PERMANOVA), using the Adonis function of the Vegan R package. The assumption of homogeneity of dispersion amongst groups, required by PERMANOVA, was tested using the betadisper function in the Vegan R package.