The progress of the study.
MP
-11, DISEASE, ADVERSE EVENT, LYSIS
Initially, 65 mothers with their 79 newborn children were randomized into five intervention groups. Eventually, 68 mother-child pairs were eligible for the study. The abbreviations of the intervention groups are described in figure.The intervention strains were -Placebo: both the mother and the child received placebo-C-LGG: The mothers received placebo, the children were administered LGG.-C-LGG + Bb12: mother: placebo, child: LGG + Bb12-M-LGG: mother: LGG, child: placebo-M-LGG + Bb12: mother: LGG + Bb12, child: placeboRandom permuted block randomisation was performed by an independent statistician and the randomisation code was generated by SAS, version 9.3 for Windows. The allocation concealment was ensured using sequentially numbered containers. The University laboratory and the Hospital pharmacy held the emergency envelope to be opened in case of serious adverse event or disease in a study subject. Clinical research nurses recruited the patients and gave the intervention products in numerical order.The children received the intervention substances as probiotic capsules the contents of which were dispersed with a small amount of breastmilk and administered with their morning feed throughout their stay in the NICU. The mothers received the probiotics as capsules. The daily doses of the probiotics were 10The age of the children (days) when the administration of the intervention product was begun (median and range) by intervention group.The gut microbiota compositions of the study subjects (68 children) were assessed using faecal samples obtained 7 days after birth. The samples were stored in RNAlater solution at −80 °C. The gut microbiota compositions were assessed by analyzing the DNA isolated from faecal samples and utilizing 16S rRNA sequencing. The DNA was extracted by homogenizing 100–125 mg of faeces in the lysis buffer via bead beating with FastPrep-24 (MP Biomedicals, Irvine, CA). Then, commercial kit InviMag Stool DNA Kit (Stratec Molecular, Berlin, Germany) was used with the automated KingFisher DNA System (Thermo Fisher Scientific Oy, Vantaa, Finland). The manufacturer’s protocol was followed, and it included nucleic acid binding on magnetic beads, five-step washing, and elution. A Qubit® 2.0 Fluorometer (Life Technology, Carlsbad, CA) was used to measure the total DNA consentration. V3–V4 region of the 16S rRNA gene was amplified following the 16S rDNA gene Metagenomic Sequencing Library Preparation Illumina protocol (Cod. 15044223 Rev. A). The multiplexing was completed by using Nextera XT Index Kit (FC-131–2001). One microliter of the PCR product was run on a Bioanalyzer DNA 1000 chip to verify the size; the expected size being ca. 550 bp on a Bioanalyzer trace. The sequencing was made by utilising a 2 × 300 bp paired-end run (MiSeq Reagent Kit v3) on a MiSeq-Illumina platform following the manufacturer’s protocol. Both positive and negative sequencing controls were used to exclude contamination and batch effects. These sequencing results are available from the corresponding author as Fastq files upon reasonable request. In 16S rRNA sequencing method, the 16S rRNA gene, which codes the small subunit of procaryotic ribosome, is copied. Subsequently, these copies are grouped into coherent Amplicon Sequence Variants (ASVs) and mapped to a reference database for downstream compositional data analysis at the higher taxonomic levels. In our study, the QIIME2 (version 2019-07 and 2020-11) pipeline was used to analyze the raw sequences. Phred33-importing tool for paired-end data was used to import the data and DADA2 software package to correct Illumina-sequenced amplicon errors.CONSORT 2010 Checklist is included in the supplementary files (Supplementary File 