Safety evaluation of Lactobacillus reuteri PNW1 and Lactobacillus acidophilus PNW3 as probiotics and purification of their bacteriocins

Abstract

Background: Probiotics are live microorganisms that confer health benefit on the host when administered in adequate dose. Strains with probiotic potential are to be carefully selected based on their functionality, safety and genome stability. Objectives: This study enumerates important probiotic features harboured by two identified lactic acid bacteria as probiotic candidates and evaluate possible undesirable traits in both organisms. It further purifies and assesses the antimicrobial efficacy of the bioactive peptides produced by these isolates. Methodology: Identification of the isolates was confirmed via PCR amplification of the 16S rRNA region while the genomic DNA of the isolate was extracted and the entire genome was sequenced using illuminal Miseq instrument. The draft assemblies for both Lactobacillus reuteri PNW1 and Lactobacillus acidophilus PNW3 were annotated with Prokaryotic Genome Annotation Pipeline (PGAP) and Rapid Annotations using Subsystems Technology (RAST). Further genome-based down stream analyses were carried out using a number of bioinformatic tools which includes antiSMASH, PathogenFinder, ResFinder, Comprehensive Antibiotic Resistance Database (CARD), Phage Search Tool Enhanced Release (PHASTER), ISfinder search tool, Insertion Sequence Semi-Automatic Genome Annotation (ISsaga), Optimized Annotation System for Insertion Sequences (OASIS) and CRISPRCasFinder among others. Efficacy of the bioactive peptides produced by the isolates against pathogenic Escherichia coli O177 was assessed using agar well diffusion method. The bioactive peptides were thereafter precipitated with 80% saturated ammonium sulphate and further purified using HPLC. Results: Among all known genes which may be responsible for production of toxic biochemicals, arginine deiminase (EC3.5.3.6) and Ornithine decarboxylase (EC 4.1.1.17) were spotted harbouring within the genome of L. reuteri PNW1 and L. acidophilus PNW3, respectively. Resistance genes against lincosamide (lnuC) and tetracycline (tetW) were found present in both isolates; only the lnuC is flanked by a passenger gene found within the genome of L. reuteri PNW1. Other mobile genetic elements found within the genome are not in association with the indentified resistance genes. There are plethora of probiotic important genes found within the genome of both isolates and no hit was found for the virulent determinants. Five putative coding sequences were also identified for the CRISPR in L. reuteri PNW1 genome and only one was found in the L. acidophilus PNW3 genome; each of CRISPR is associated with Cas genes. This trait, thus denotes genome stability for both isolates. The maximum zone of inhibition exhibited by the bacteriocin produced by L. reuteri PNW1 is 20.0±1.00 mm (crude) and 23.3±1.15 mm (at 0.25 mg/ml) after partial purification. While on the other hand the maximum for the L. acidophilus PNW3 is 21.7±0.58 mm (crude) and 24.3±1.15 mm after partially purified and tested at a concentration of 0.25 mg/ml. Conclussion: Both isolates possess desired trait for a typical viable and safe probiotic, though further in vivo assessments are required before developed into functional products for application in animal husbandry within the nearest future.