Integrated genome-based probiotic relevance and safety evaluation of Lactobacillus reuteri PNW1.

Abstract

This study evaluates whole-genome sequence of Lactobacillus reuteri PNW1 and identifies its safety genes that may qualify it as a putative probiotic. It further extracted the bacteriocin produced by the strain and tested its effectiveness against pathogenic STEC E. coli O177. The genomic DNA was sequenced on illuminal Miseq instrument and the sequenced data was assessed for quality reads before assembled with SPAdes. The draft assembly was annotated with Prokaryotic Genome Annotation Pipeline (PGAP) and Rapid Annotations using Subsystems Technology (RAST). Further downstream analyses were carried out using appropriate bioinformatic tools. Production of biogenic amines was biochemically confirmed through HPLC analysis. The assembled genome was 2,430,215 bp long in 420 contigs with 39% G+C content. Among all known genes, putatively responsible for the production of toxic biochemicals, only arginine deiminase (EC3.5.3.6) was spotted. Coding sequences (CDS) putative for D-lactate dehydrogenase (EC1.1.1.28), L-lactate dehydrogenase (EC1.1.1.27) and bacteriocin helveticin J were found within the genome together with plethora of other probiotic important genes. The strain harbours only resistant genes putative for Lincosamide (lnuC) and Tetracycline resistant genes (tetW). There was no hit found for virulence factors and probability of the strain being a human pathogen was zero. Two intact prophage regions were detected within the genome of L. reuteri PNW1 and nine CDS were identified for insertion sequence by OASIS which are belong to seven different families. Five putative CDS were identified for the CRISPR, each associated with Cas genes. Maximum zone of inhibition exhibited by the bacteriocin produced L. reuteri PNW1 is 20.0±1.00 mm (crude) and 23.3±1.15 mm (at 0.25 mg/ml) after being partially purified. With the strain predicted as non-human pathogen, coupled with many other identified desired features, L. reuteri PNW1 stands a chance of making good and safe candidates for probiotic, though further in-vivo investigations are still necessary.