Safety Assessment of a Nham Starter Culture Lactobacillus plantarum BCC9546 via Whole-genome Analysis

Abstract

The safety of microbial cultures utilized for consumption is vital for public health and should be thoroughly assessed. Although general aspects on the safety assessment of microbial cultures have been suggested, no methodological detail nor procedural guideline have been published. Herein, we propose a detailed protocol on microbial strain safety assessment via whole-genome sequence analysis. A starter culture employed in traditional fermented pork production, nham, namely Lactobacillus plantarum BCC9546, was used as an example. The strain’s whole-genome was sequenced through several next-generation sequencing techniques. Incomplete plasmid information from the PacBio sequencing platform and shorter chromosome size from the hybrid Oxford Nanopore-Illumina platform were noted. The methods for 1) unambiguous species identification using 16S rRNA gene and average nucleotide identity, 2) determination of virulence factors and undesirable genes, 3) determination of antimicrobial resistance properties and their possibility of transfer, and 4) determination of antimicrobial drug production capability of the strain were provided in detail. Applicability of the search tools and limitations of databases were discussed. Finally, a procedural guideline for the safety assessment of microbial strains via whole-genome analysis was proposed.

Genomic Insight into Pediococcus acidilactici HN9, a Potential Probiotic Strain Isolated from the Traditional Thai-Style Fermented Beef Nhang

“…PacBio reads are long and may exceed the lengths of small plasmid sequences with a size smaller than 10 kb [ 29 ]. During assembly, a parameter setting on the seed read length cut-off that is greater than actual plasmid size in the hierarchical genome assembly process (HGAP) software [ 30 ] may miss small plasmids [ 31 ].…”

Characterization of the first vaginal Lactobacillus crispatus genomes isolated in Brazil

“…In the first four L. crispatus sequenced genomes from Brazil, we identified genes involved in the protective properties of vaginal lactobacilli (Smith & Ravel, 2017) such as the production of lactic acid, bacteriocins, hydrogen peroxide and adhesion (Table 3). For safety reasons, a probiotic strain should not present features associated with virulence and antimicrobial resistance, as well as mobile elements that could transfer those features to other microorganisms from the host microbiome (Pariza et al, 2015;Chokesajjawatee et al, 2020). No antimicrobial resistance gene was found, and only three hemolysins were predicted as virulence factors, which have also been previously identified in other Lactobacillus probiotic strains (Zafar & Saier, 2020) (Table 4).…”

“…Protein IDs were identified using BLASTp (Camacho et al, 2009) using the GenBank non-redundant (nr) database, selecting hits with 100% identity and coverage. Pathways involved in the biosynthesis of antimicrobial drugs with clinical importance (Chokesajjawatee et al, 2020) were predicted using KEGG Mapper.…”

“…The screening using these databases was performed using ABRicate (https://github.com/tseemann/abricate) with default parameters. The presence of the toxins hemolysins, enzymes involved in the synthesis of biogenic amines and other undesirable genes, as listed by Chokesajjawatee et al (2020), were manually screened using KEGG Mapper (Kanehisa & Sato, 2020) implemented in BlastKOALA v2.2 (Kanehisa, Sato & Morishima, 2016).…”

KITSUNE: A Tool for Identifying Empirically Optimal K-mer Length for Alignment-Free Phylogenomic Analysis

“…Lactobacillus plantarum BCC9546 (BIOTEC culture collection, Thailand) (Chokesajjawatee et al, 2020) was grown in (Man, Rogosa, and Sharpe) MRS broth at 30 • C for 16 h. The genomic DNA was extracted with the Wizard Genomic DNA Purification Kit (Promega, United States).…”