Effect of single versus multistrain probiotic in extremely preterm infants: a randomised trial

Abstract

Objective: Evidence indicates that multistrain probiotics benefit preterm infants more than single-strain (SS) probiotics. We assessed the effects of SS versus triple-strain (TS) probiotic supplementation (PS) in extremely preterm (EP) infants.

Design: EP infants (gestational age (GA) <28 weeks) were randomly allocated to TS or SS probiotic, assuring blinding. Reference (REF) group was EP infants in the placebo arm of our previous probiotic trial. PS was commenced with feeds and continued until 37 weeks' corrected GA. Primary outcome was time to full feed (TFF: 150 mL/kg/day). Secondary outcomes included short-chain fatty acids and faecal microbiota collected at T1 (first week) and T2 (after 3 weeks of PS) using 16S ribosomal RNA gene sequencing.

Results: 173 EP (SS: 86, TS: 87) neonates with similar GA and birth weight (BW) were randomised. Median TFF was comparable (11 (IQR 8-16) vs 10 (IQR 8-16) days, p=0.92). Faecal propionate (SS, p<0.001, and TS, p=0.0009) and butyrate levels (TS, p=0.029) were significantly raised in T2 versus T1 samples. Secondary clinical outcomes were comparable. At T2, alpha diversity was comparable (p>0.05) between groups, whereas beta-diversity analysis revealed significant differences between PS and REF groups (both p=0.001). Actinobacteria were higher (both p<0.01), and Proteobacteria, Firmicutes and Bacteroidetes were lower in PS versus REF. Gammaproteobacteria, Clostridia and Negativicutes were lower in both PS versus REF.

Conclusion: TFF in EP infants was similar between SS and TS probiotics. Both probiotics were effective in reducing dysbiosis (higher bifidobacteria and lower Gammaproteobacteria). Long-term significance of increased propionate and butyrate needs further studies.

Trial registration number: ACTRN 12615000940572.

Keywords: infant/neonatal nutrition; intestinal microbiology; neonatal gut; probiotics.

Figure 1

Consolidated Standards of Reporting Trials flow diagram showing details of the study recruitment. TGA, Transposition of Great Arteries; TOF, Tracheo-oesophageal fistula; ITT: Intention To Treat.

Figure 2

Combined SCFA (butyric acid, propionic acid and acetic acid) and BCFA (isobutyric and isovaleric acid) for all groups. SCFA and BCFA levels. SCFA (acetic acid (B), butyric acid (C) and propionic acid (D)) and BCFA (isobutyric (E) and isovaleric acid (F)) levels in SimPro groups (SS and TS). Box shows IQR; the line, median and the dots represent an individual sample. Differences between groups and time were calculated using linear mixed effects test with Tukey correction to adjust for multiple testing. Significant differences are indicated by *p<0.05, ***p<0.001. BCFA, branched-chain fatty acid; SCFA, short-chain fatty acid; SS, single strain; TS, triple strain.

Figure 3

(A–I) Alpha and beta diversity at T2. Box plots of bacterial richness measures; Ace (A) and Chao1 (B) and alpha evenness measures; Shannon (C) and Simpson index (D). Box shows IQR; the line, median and the error bars; the range and the dots are outliers. Differences between groups were calculated using Wilcoxon rank-sum test with Benjamini-Hochberg correction to adjust for multiple testing. Significant differences are indicated by *p<0.05, ** <0.01. (E) Principal coordinate analysis based on weighted Unifrac distances. Each sample is depicted as a dot. (F) Permutational Analysis of Variance (PERMANOVA) was used to identify if there were differences in the community structures by group and time followed by pairwise Adonis test for comparisons between the groups. REF, reference; SS, single strain; TS, triple strain.

Figure 4

(A–E) Infant gut microbiota by taxa at T2. Box plots showing RA of class (A), genus (C) and subspecies (E). Box shows IQR; the line, median and the dots represent an individual sample. (B) Shows phylum level composition. (D) Bubble plot of significantly different Species with RA >0.01%. Differences between groups were calculated using Wilcoxon rank-sum test with Benjamini-Hochberg correction to adjust for multiple testing. Significant differences are indicated by **p<0.01, ***p<0.001, ****p<0.0001. RA, relative abundance; REF, reference; SS, single-strain; TS, triple-strain.