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

Kazeem Adekunle Alayande; Olayinka Ayobami Aiyegoro; Thizwilondi Michael Nengwekhulu; Lebogang Katata-Seru; Collins Njie Ateba

doi:10.1371/journal.pone.0235873

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

PLoS One. 2020 Jul 20;15(7):e0235873. doi: 10.1371/journal.pone.0235873. eCollection 2020.

Authors

Kazeem Adekunle Alayande^{1

2}, Olayinka Ayobami Aiyegoro³, Thizwilondi Michael Nengwekhulu⁴, Lebogang Katata-Seru⁴, Collins Njie Ateba^{1

2}

Affiliations

¹ Antibiotic Resistance and Phage Biocontrol Research Group, Department of Microbiology, Faculty of Natural and Agricultural Sciences, North-West University, Mmabatho, South Africa.
² Food Security and Safety Niche Area, Faculty of Natural and Agricultural Sciences, North-West University, Mmabatho, South Africa.
³ Gastrointestinal Microbiology and Biotechnology Division, Agricultural Research Council, Animal Production Institute, Irene, South Africa.
⁴ Department of Chemistry, Faculty of Natural and Agricultural Sciences, North West University, Mmabatho, South Africa.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacterial Proteins / genetics
Bacteriocins / genetics
Bacteriocins / metabolism
Bacteriocins / pharmacology
Escherichia coli / drug effects
Genome, Bacterial*
Hydrolases / genetics
L-Lactate Dehydrogenase / genetics
Limosilactobacillus reuteri / genetics*
Limosilactobacillus reuteri / pathogenicity
Molecular Sequence Annotation
Probiotics / adverse effects*
Virulence Factors / genetics

Substances

Bacterial Proteins
Bacteriocins
Virulence Factors
L-Lactate Dehydrogenase
Hydrolases
arginine deiminase

Grants and funding

This work was supported in part by an Incentive Funding for Rated Researcher grant from the National Research Foundation, South Africa, that was awarded to O.A.A. and a research grant from the FNAS Research Committee of the North-West University awarded to C.N.A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.