Assessment of pathogenic potential in non-pathogenic bacterial species for industrial production of live bacteria

Assessment of the pathogenic potential (virulence and toxicity) in non-pathogenic bacterial species is a challenge as it relies on methods developed for assessment of species known to be pathogenic. Here we have applied and evaluated some of these methods on industrially relevant bacteria to differentiate between “true” virulence factors applying only to pathogens and niche factors being defined as promoting colonization and survival rather than pathogenicity and as being present also in non-pathogenic bacteria.   We examined the pathogenicity of 49 strains from nine industrially relevant bacterial species (Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus delbrueckii, Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, Latilactobacillus curvatus, Ligilactobacillus salivarius, Staphylococcus carnosus, and Staphylococcus xylosus), including 14 clinical isolates of the same species, through genomic screening and phenotypically through assays established for pathogenic bacteria. The genomes were screened against the Virulence Factor Database (VFDB) and thresholds (>80% nucleotide or protein identity, >70% coverage) provided by the European Food Safety Authority (EFSA) were adopted to differentiate between genes of potential concern and genes of no concern. Core genome analysis was performed to determine whether the clinical isolates were phylogenetically related to the industrial isolates. The genotypic assessment did not reveal the presence of true virulence factors in the examined strains, and in the core genome analysis the clinical isolates could not be distinguished from the industrial strains. Furthermore, cytotoxicity toward Vero cells, negative impact on Caco-2 cell viability, and hemolytic activity on blood agar plates were examined, and none of the tested strains exhibited any activity in these assays. Overall, the results suggest that VFDB screening with the EFSA thresholds can be used to differentiate between true virulence factors and niche factors. Furthermore, the use of phenotypic assays supports the genotypic assessments and Caco-2 cell viability and hemolysis assays are useful to determine the presence of acquired virulence genes, albeit expert knowledge is required to interpret the results.