Effects of Lactobacillus reuteri supplementation on the gut microbiota in extremely preterm infants in a randomized placebo-controlled trial

Magalí Martí, Johanne E Spreckels, Purnika Damindi Ranasinghe, Erik Wejryd, Giovanna Marchini, Eva Sverremark-Ekström, Maria C Jenmalm, Thomas Abrahamsson, Magalí Martí, Johanne E Spreckels, Purnika Damindi Ranasinghe, Erik Wejryd, Giovanna Marchini, Eva Sverremark-Ekström, Maria C Jenmalm, Thomas Abrahamsson

Abstract

Extremely low birth weight (ELBW) infants often develop an altered gut microbiota composition, which is related to clinical complications, such as necrotizing enterocolitis and sepsis. Probiotic supplementation may reduce these complications, and modulation of the gut microbiome is a potential mechanism underlying the probiotic effectiveness. In a randomized, double-blind, placebo-controlled trial, we assessed the effect of Lactobacillus reuteri supplementation, from birth to post-menstrual week (PMW)36, on infant gut microbiota. We performed 16S amplicon sequencing in 558 stool samples from 132 ELBW preterm infants at 1 week, 2 weeks, 3 weeks, 4 weeks, PMW36, and 2 years. Probiotic supplementation results in increased bacterial diversity and increased L. reuteri abundance during the 1st month. At 1 week, probiotic supplementation also results in a lower abundance of Enterobacteriaceae and Staphylococcaceae. No effects were found at 2 years. In conclusion, probiotics may exert benefits by modulating the gut microbiota composition during the 1st month in ELBW infants.

Trial registration: ClinicalTrials.gov NCT01603368.

Keywords: Lactobacillus; Staphylococcus; extremely low birth weight infant; microbial diversity; microbiota; necrotizing enterocolitis; preterm infant; probiotic; randomized controlled trial; supplementation.

Conflict of interest statement

T.A. has received honoraria for lectures and a grant for the present trial from BioGaia AB. M.C.J. has received honoraria for lectures from BioGaia AB. E.S.-E. has received honoraria for lectures and a research grant from BioGaia AB.

© 2021 The Author(s).

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Flow chart of the study PMW36: post-menstrual week 36. aStudy product was discontinued by mistake after transfer to other hospital (n = 1). bStudy product was not administrated again by mistake after temporarily being withheld during nil oral (n = 3). cStudy product ran out temporarily at the study site (n = 3). dInsufficient amounts of DNA were recovered from extraction (n = 6). eLibrary preparation failed (n = 6). fSequencing failed (n = 12).
Figure 2
Figure 2
Gut microbiota α-diversity of ELBW preterm infants supplemented with L. reuteri or placebo Boxplots (median with 25% and 75% percentiles and 1.5× the interquartile range; diamond shape depicts the mean) showing the α-diversity (Shannon index), richness (observed ASVs), and evenness (Pielou’s evenness index) from 1 week to 2 years of life in ELBW preterm infants supplemented with L. reuteri (Lr) or placebo (Pl). PMW36, post-menstrual week 36; w, week; y, year. ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 with Mann-Whitney U test and p value adjustment for multiple comparisons with the method from Benjamini and Hochberg.
Figure 3
Figure 3
Clustering of the gut microbiota composition (β-diversity) of ELBW preterm infants supplemented with L. reuteri or placebo Non-metric multidimensional scaling (NMDS) of bacterial community composition from 1 week to 2 years of life across ELBW preterm infants supplemented with L. reuteri or placebo. (A–F) The ASVs that significantly contributed to the variance explained (envfit(); p < 0.01 and R2 > 0.3) were classified at genus level, and only one genus for all ASVs pointing toward the same direction was displayed (Table S6). At 1 week, (A) the ellipses (confidence level 0.95) show Linköping and Stockholm because inclusion site also affected the bacterial community composition and Lactobacillus had different effects depending on the site. (G–I) The abundance of L. reuteri DSM 17938 (qPCR data) significantly (envfit(); p < 0.01 and R2 > 0.3) correlated to bacterial community composition in the placebo group. Weight, length, and head circumference were adjusted for gestational age using the standard deviation score (Z score). ∗∗∗p < 0.001 with ANOSIM and p value adjustment for multiple comparisons with the method from Benjamini and Hochberg.
Figure 4
Figure 4
Taxonomic composition of the gut bacteria in ELBW preterm infants supplemented with L. reuteri or placebo Relative abundance of the dominant taxa is displayed at phylum (A), family (B), and genus (C) level. At family and genus levels, the taxa with a relative abundance of L. reuteri and placebo groups (LEfSE; p = 0.01; Table S3).
Figure 5
Figure 5
Prevalence and abundance of L. reuteri DSM 17938 Prevalence (A) and abundance (B) of supplemented L. reuteri DSM 17938 in L. reuteri and placebo groups at different time points. (A) Percentage of infants with a stool sample positive for the supplemented strain. (B) Boxplots (median with 25% and 75% percentiles and 1.5× the interquartile range) show the abundance as the number of L. reuteri DSM 17938 bacteria per 1 g wet feces. Colored dots indicate the L. reuteri DSM 17938 abundance in individual stool samples positive for the supplemented L. reuteri strain; the number of L. reuteri DSM 17938 bacteria per 1 g wet feces for infants with a L. reuteri negative stool sample was set to 1 for graphical display; (n) indicates the number of infants with a stool sample positive for supplemented L. reuteri DSM 17938; (N) indicates the total number of infants with a stool sample in the L. reuteri or placebo group at the indicated time point. Prevalence and abundance between groups were compared using Fisher’s exact tests and Mann-Whitney U tests, respectively, and adjusted for multiple comparisons with the method from Benjamini and Hochberg. Significant differences in L. reuteri DSM 17938 abundance in the L. reuteri group across neonatal time points (1 week to PMW36) were tested for with a Kruskal-Wallis test with Dunn post hoc test and p value adjustment for multiple comparisons with the method from Benjamini and Hochberg. ∗∗∗p < 0.001; ∗∗p < 0.01.
Figure 6
Figure 6
Correlation between growth parameters and ELBW preterm infant gut microbiota composition (A and B) Head growth until 4 weeks of life correlated to microbial diversity at 1 week (simple linear regression; p = 0.007; adjusted R2 = 0.06; A) and microbial richness at 2 weeks (simple linear regression; p = 0.035; adjusted R2 = 0.03; B). (C and D) Head (head) and weight (weight) growth rate significantly (envfit(); p < 0.05 and R2 0.1–0.2) correlated to the microbial community composition at 1 week (C) and 3 weeks (D) of life.

References

    1. Norman M., Hallberg B., Abrahamsson T., Björklund L.J., Domellöf M., Farooqi A., Foyn Bruun C., Gadsbøll C., Hellström-Westas L., Ingemansson F. Association between year of birth and 1-year survival among extremely preterm infants in Sweden during 2004-2007 and 2014-2016. JAMA. 2019;321:1188–1199.
    1. Pammi M., Cope J., Tarr P.I., Warner B.B., Morrow A.L., Mai V., Gregory K.E., Kroll J.S., McMurtry V., Ferris M.J. Intestinal dysbiosis in preterm infants preceding necrotizing enterocolitis: a systematic review and meta-analysis. Microbiome. 2017;5:31.
    1. Stewart C.J., Embleton N.D., Marrs E.C.L., Smith D.P., Fofanova T., Nelson A., Skeath T., Perry J.D., Petrosino J.F., Berrington J.E., Cummings S.P. Longitudinal development of the gut microbiome and metabolome in preterm neonates with late onset sepsis and healthy controls. Microbiome. 2017;5:75.
    1. Borre Y.E., O’Keeffe G.W., Clarke G., Stanton C., Dinan T.G., Cryan J.F. Microbiota and neurodevelopmental windows: implications for brain disorders. Trends Mol. Med. 2014;20:509–518.
    1. Jin Y.T., Duan Y., Deng X.K., Lin J. Prevention of necrotizing enterocolitis in premature infants - an updated review. World J. Clin. Pediatr. 2019;8:23–32.
    1. Dong Y., Speer C.P. Late-onset neonatal sepsis: recent developments. Arch. Dis. Child. Fetal Neonatal Ed. 2015;100:F257–F263.
    1. Levy M., Kolodziejczyk A.A., Thaiss C.A., Elinav E. Dysbiosis and the immune system. Nat. Rev. Immunol. 2017;17:219–232.
    1. Lindberg T.P., Caimano M.J., Hagadorn J.I., Bennett E.M., Maas K., Brownell E.A., Matson A.P. Preterm infant gut microbial patterns related to the development of necrotizing enterocolitis. J. Matern. Fetal Neonatal Med. 2020;33:349–358.
    1. Thomas J.P., Raine T., Reddy S., Belteki G. Probiotics for the prevention of necrotising enterocolitis in very low-birth-weight infants: a meta-analysis and systematic review. Acta Paediatr. 2017;106:1729–1741.
    1. Aceti A., Maggio L., Beghetti I., Gori D., Barone G., Callegari M.L., Fantini M.P., Indrio F., Meneghin F., Morelli L., Italian Society of Neonatology. Probiotics prevent late-onset sepsis in human milk-fed, very low birth weight preterm infants: systematic review and meta-analysis. Nutrients. 2017;9:E904.
    1. Cui X., Shi Y., Gao S., Xue X., Fu J. Effects of Lactobacillus reuteri DSM 17938 in preterm infants: a double-blinded randomized controlled study. Ital. J. Pediatr. 2019;45:140.
    1. Indrio F., Riezzo G., Tafuri S., Ficarella M., Carlucci B., Bisceglia M., Polimeno L., Francavilla R. Probiotic supplementation in preterm: feeding intolerance and hospital cost. Nutrients. 2017;9:E965.
    1. Wejryd E., Marchini G., Frimmel V., Jonsson B., Abrahamsson T. Probiotics promoted head growth in extremely low birthweight infants in a double-blind placebo-controlled trial. Acta Paediatr. 2019;108:62–69.
    1. Oncel M.Y., Sari F.N., Arayici S., Guzoglu N., Erdeve O., Uras N., Oguz S.S., Dilmen U. Lactobacillus reuteri for the prevention of necrotising enterocolitis in very low birthweight infants: a randomised controlled trial. Arch. Dis. Child. Fetal Neonatal Ed. 2014;99:F110–F115.
    1. van den Akker C.H.P., van Goudoever J.B., Szajewska H., Embleton N.D., Hojsak I., Reid D., Shamir R., ESPGHAN Working Group for Probiotics, Prebiotics & Committee on Nutrition Probiotics for preterm infants: a strain-specific systematic review and network meta-analysis. J. Pediatr. Gastroenterol. Nutr. 2018;67:103–122.
    1. Dermyshi E., Wang Y., Yan C., Hong W., Qiu G., Gong X., Zhang T. The “golden age” of probiotics: a systematic review and meta-analysis of randomized and observational studies in preterm infants. Neonatology. 2017;112:9–23.
    1. Hill C., Guarner F., Reid G., Gibson G.R., Merenstein D.J., Pot B., Morelli L., Canani R.B., Flint H.J., Salminen S. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat. Rev. Gastroenterol. Hepatol. 2014;11:506–514.
    1. Mu Q., Tavella V.J., Luo X.M. Role of Lactobacillus reuteri in human health and diseases. Front. Microbiol. 2018;9:757.
    1. Singh T.P., Kaur G., Kapila S., Malik R.K. Antagonistic activity of Lactobacillus reuteri strains on the adhesion characteristics of selected pathogens. Front. Microbiol. 2017;8:486.
    1. La Rosa P.S., Warner B.B., Zhou Y., Weinstock G.M., Sodergren E., Hall-Moore C.M., Stevens H.J., Bennett W.E., Jr., Shaikh N., Linneman L.A. Patterned progression of bacterial populations in the premature infant gut. Proc. Natl. Acad. Sci. USA. 2014;111:12522–12527.
    1. Arboleya S., Sánchez B., Milani C., Duranti S., Solís G., Fernández N., de los Reyes-Gavilán C.G., Ventura M., Margolles A., Gueimonde M. Intestinal microbiota development in preterm neonates and effect of perinatal antibiotics. J. Pediatr. 2015;166:538–544.
    1. Pereira F.C., Berry D. Microbial nutrient niches in the gut. Environ. Microbiol. 2017;19:1366–1378.
    1. Hayashi H., Takahashi R., Nishi T., Sakamoto M., Benno Y. Molecular analysis of jejunal, ileal, caecal and recto-sigmoidal human colonic microbiota using 16S rRNA gene libraries and terminal restriction fragment length polymorphism. J. Med. Microbiol. 2005;54:1093–1101.
    1. Rougé C., Piloquet H., Butel M.J., Berger B., Rochat F., Ferraris L., Des Robert C., Legrand A., de la Cochetière M.F., N’Guyen J.M. Oral supplementation with probiotics in very-low-birth-weight preterm infants: a randomized, double-blind, placebo-controlled trial. Am. J. Clin. Nutr. 2009;89:1828–1835.
    1. Neu J., Walker W.A. Necrotizing enterocolitis. N. Engl. J. Med. 2011;364:255–264.
    1. Plummer E.L., Bulach D.M., Murray G.L., Jacobs S.E., Tabrizi S.N., Garland S.M., ProPrems Study Group Gut microbiota of preterm infants supplemented with probiotics: sub-study of the ProPrems trial. BMC Microbiol. 2018;18:184.
    1. Millar M., Seale J., Greenland M., Hardy P., Juszczak E., Wilks M., Panton N., Costeloe K., Wade W.G. The microbiome of infants recruited to a randomised placebo-controlled probiotic trial (PiPS trial) EBioMedicine. 2017;20:255–262.
    1. Alcon-Giner C., Dalby M.J., Caim S., Ketskemety J., Shaw A., Sim K., Lawson M.A.E., Kiu R., Leclaire C., Chalklen L. Microbiota supplementation with Bifidobacterium and Lactobacillus modifies the preterm infant gut microbiota and metabolome: an observational study. Cell Rep. Med. 2020;1:100077.
    1. Costeloe K., Hardy P., Juszczak E., Wilks M., Millar M.R., Probiotics in Preterm Infants Study Collaborative Group Bifidobacterium breve BBG-001 in very preterm infants: a randomised controlled phase 3 trial. Lancet. 2016;387:649–660.
    1. Egervärn M., Danielsen M., Roos S., Lindmark H., Lindgren S. Antibiotic susceptibility profiles of Lactobacillus reuteri and Lactobacillus fermentum. J. Food Prot. 2007;70:412–418.
    1. Ridaura V.K., Faith J.J., Rey F.E., Cheng J., Duncan A.E., Kau A.L., Griffin N.W., Lombard V., Henrissat B., Bain J.R. Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science. 2013;341:1241214.
    1. Diaz Heijtz R., Wang S., Anuar F., Qian Y., Björkholm B., Samuelsson A., Hibberd M.L., Forssberg H., Pettersson S. Normal gut microbiota modulates brain development and behavior. Proc. Natl. Acad. Sci. USA. 2011;108:3047–3052.
    1. Goodrich J.K., Waters J.L., Poole A.C., Sutter J.L., Koren O., Blekhman R., Beaumont M., Van Treuren W., Knight R., Bell J.T. Human genetics shape the gut microbiome. Cell. 2014;159:789–799.
    1. Dao M.C., Everard A., Aron-Wisnewsky J., Sokolovska N., Prifti E., Verger E.O., Kayser B.D., Levenez F., Chilloux J., Hoyles L., MICRO-Obes Consortium Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology. Gut. 2016;65:426–436.
    1. White R.A., Bjørnholt J.V., Baird D.D., Midtvedt T., Harris J.R., Pagano M., Hide W., Rudi K., Moen B., Iszatt N. Novel developmental analyses identify longitudinal patterns of early gut microbiota that affect infant growth. PLoS Comput. Biol. 2013;9:e1003042.
    1. Younge N.E., Newgard C.B., Cotten C.M., Goldberg R.N., Muehlbauer M.J., Bain J.R., Stevens R.D., O’Connell T.M., Rawls J.F., Seed P.C., Ashley P.L. Disrupted maturation of the microbiota and metabolome among extremely preterm infants with postnatal growth failure. Sci. Rep. 2019;9:8167.
    1. Valeur N., Engel P., Carbajal N., Connolly E., Ladefoged K. Colonization and immunomodulation by Lactobacillus reuteri ATCC 55730 in the human gastrointestinal tract. Appl. Environ. Microbiol. 2004;70:1176–1181.
    1. Smits H.H., Engering A., van der Kleij D., de Jong E.C., Schipper K., van Capel T.M.M., Zaat B.A., Yazdanbakhsh M., Wierenga E.A., van Kooyk Y., Kapsenberg M.L. Selective probiotic bacteria induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin. J. Allergy Clin. Immunol. 2005;115:1260–1267.
    1. Klindworth A., Pruesse E., Schweer T., Peplies J., Quast C., Horn M., Glöckner F.O. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 2013;41:e1.
    1. Aloisio I., Quagliariello A., De Fanti S., Luiselli D., De Filippo C., Albanese D., Corvaglia L.T., Faldella G., Di Gioia D. Evaluation of the effects of intrapartum antibiotic prophylaxis on newborn intestinal microbiota using a sequencing approach targeted to multi hypervariable 16S rDNA regions. Appl. Microbiol. Biotechnol. 2016;100:5537–5546.
    1. Bushnell B. 2019. BBMap short read aligner, and other bioinformatic tools.
    1. Ewels P., Magnusson M., Lundin S., Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32:3047–3048.
    1. Callahan B.J., McMurdie P.J., Rosen M.J., Han A.W., Johnson A.J.A., Holmes S.P. DADA2: high-resolution sample inference from Illumina amplicon data. Nat. Methods. 2016;13:581–583.
    1. Love M.I., Huber W., Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.
    1. Guevara M.R., Hartmann D., Mendoza M. diverse: an R package to analyze diversity in complex systems. R J. 2016;8:60–78.
    1. McMurdie P.J., Holmes S. Waste not, want not: why rarefying microbiome data is inadmissible. PLoS Comput. Biol. 2014;10:e1003531.
    1. Niklasson A., Albertsson-Wikland K. Continuous growth reference from 24th week of gestation to 24 months by gender. BMC Pediatr. 2008;8:8.
    1. Bell M.J., Ternberg J.L., Feigin R.D., Keating J.P., Marshall R., Barton L., Brotherton T. Neonatal necrotizing enterocolitis. Therapeutic decisions based upon clinical staging. Ann. Surg. 1978;187:1–7.
    1. Koletzko B., Goulet O., Hunt J., Krohn K., Shamir R., Parenteral Nutrition Guidelines Working Group. European Society for Clinical Nutrition and Metabolism. European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) European Society of Paediatric Research (ESPR) 1. Guidelines on paediatric parenteral nutrition of the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and the European Society for Clinical Nutrition and Metabolism (ESPEN), supported by the European Society of Paediatric Research (ESPR) J. Pediatr. Gastroenterol. Nutr. 2005;41(Suppl 2):S1–S87.
    1. Rosander A., Connolly E., Roos S. Removal of antibiotic resistance gene-carrying plasmids from Lactobacillus reuteri ATCC 55730 and characterization of the resulting daughter strain, L. reuteri DSM 17938. Appl. Environ. Microbiol. 2008;74:6032–6040.
    1. Walker A.W., Martin J.C., Scott P., Parkhill J., Flint H.J., Scott K.P. 16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice. Microbiome. 2015;3:26.
    1. Romani Vestman N., Hasslöf P., Keller M.K., Granström E., Roos S., Twetman S., Stecksén-Blicks C. Lactobacillus reuteri influences regrowth of mutans streptococci after full-mouth disinfection: a double-blind, randomised controlled trial. Caries Res. 2013;47:338–345.
    1. Clarke K.R. Non-parametric multivariate analyses of changes in community structure. Aust. J. Ecol. 1993;18:117–143.
    1. Segata N., Izard J., Waldron L., Gevers D., Miropolsky L., Garrett W.S., Huttenhower C. Metagenomic biomarker discovery and explanation. Genome Biol. 2011;12:R60.
    1. Wang C., Hu J., Blaser M.J., Li H. Estimating and testing the microbial causal mediation effect with high-dimensional and compositional microbiome data. Bioinformatics. 2020;36:347–355.

Source: PubMed

스폰서 및 공동 작업자

건강 상태

약물 개입

3
구독하다