Feasibility of Genome-Wide Screening for Biosafety Assessment of Probiotics: A Case Study of Lactobacillus helveticus MTCC 5463

Abstract

Recent years have witnessed an explosion in genome sequencing of probiotic strains for accurate identification and characterization. Regulatory bodies are emphasizing on the need for performing phase I safety studies for probiotics. The main hypothesis of this study was to explore the feasibility of using genome databases for safety screening of strains. In this study, we attempted to develop a framework for the safety assessment of a potential probiotic strain, Lactobacillus helveticus MTCC 5463 based on genome mining for genes associated with antibiotic resistance, production of harmful metabolites, and virulence. The sequencing of MTCC 5463 was performed using GS-FLX Titanium reagents. Genes coding for antibiotic resistance and virulence were identified using Antibiotic Resistance Genes Database and Virulence Factors Database. Results indicated that MTCC 5463 carried antibiotic resistance genes associated with beta-lactam and fluoroquinolone. There is no threat of transfer of these genes to host gut commensals because the genes are not plasmid encoded. The presence of genes for adhesion, biofilm, surface proteins, and stress-related proteins provides robustness to the strain. The presence of hemolysin gene in the genome revealed a theoretical risk of virulence. The results of in silico analysis complemented the in vitro studies and human clinical trials, confirming the safety of the probiotic strain. We propose that the safety assessment of probiotic strains administered live at high doses using a genome-wide screening could be an effective and time-saving tool for identifying prognostic biomarkers of biosafety.

In silico analysis of protein toxin and bacteriocins from Lactobacillus paracasei SD1 genome and available online databases

“…saniviri . Unsurprisingly, there are many reports about Hemolysin and its safety in Lactobacilli [74, 75]. These reports suggested that Lactobacilli can be included in probiotic products without any danger to the host organism [1, 74–77].…”

Regulatory and Safety Requirements for Food Cultures

“…For the probiotic strain Lactobacillus helveticus MTCC5463, safety related genes were categorised into resistome, heavy metals, adverse metabolic genes, virulence related genes, and stress related genes [27], and the presence or absence of genes in these categories were used to aid the safety assessment of this strain. A similar approach was used for another gram-positive probiotic strain, Bacillus coagulans GBI-30, 6086 [28].…”

Addressing the Antibiotic Resistance Problem with Probiotics: Reducing the Risk of Its Double-Edged Sword Effect

“…Given the separate approaches in screening probiotic bacterial strains, multiple independent studies have demonstrated several methods in screening different probiotic strains for antibiotic resistance genes (Tompkins et al, 2008; Vankerckhoven et al, 2008; Chang et al, 2009; Galopin et al, 2009; Xiao et al, 2010; Klein, 2011; Nueno-Palop and Narbad, 2011; Cebrián et al, 2012; Songisepp et al, 2012; Wei et al, 2012; Tan et al, 2013; Devi et al, 2015; Senan et al, 2015). In places where it is not bound to either US FDA or EFSA, screening for antibiotic resistance genes in probiotic strains becomes even more important as rigid guidelines and regulations for probiotic use are lacking (Chang et al, 2009).…”

“…Included in this group are Lactobacillus and Enterococcus. Both genera are currently extensively screened for species that can be used as probiotic bacteria (Tompkins et al, 2008; Vankerckhoven et al, 2008; Chang et al, 2009; Klein, 2011; Nueno-Palop and Narbad, 2011; Cebrián et al, 2012; Songisepp et al, 2012; Tan et al, 2013; Devi et al, 2015; Senan et al, 2015). …”

“…In addition, it has also been demonstrated that maintaining what is considered “normal” microbiota for certain host microbial environments may prevent diseased conditions that are not necessarily of infectious etiology and may improve general health outcome (Franz et al, 2011; Nueno-Palop and Narbad, 2011; Wei et al, 2012; Téllez et al, 2015; Varankovich et al, 2015). As a result, probiotic use, defined as the application of actual live beneficial microbes, has been increasingly practiced for both human and veterinary applications (Tompkins et al, 2008; Vankerckhoven et al, 2008; Sanders et al, 2010; Xiao et al, 2010; Nueno-Palop and Narbad, 2011; Songisepp et al, 2012; Devi et al, 2015; D’Orazio et al, 2015; Fuochi et al, 2015; Senan et al, 2015; Varankovich et al, 2015). Among the modes of probiotic use, the consumption of probiotics through the gastrointestinal route may be considered the most common application in both human and veterinary uses.…”