Phenotypic and Genome-based Approaches for Antibiotic Resistance Assessment in Lactic Acid Bacteria: a Focus on the Genera Leuconostoc and Lactobacillus

The emergence of antibiotic resistance (AR) among bacteria in foodstuff has become a huge issue to public healthcare systems in recent decades. Indeed, resistant foodborne bacteria can cause serious health effects directly or via the transmission of the AR genes to pathogens, causing illnesses that are difficult to treat. Lactic acid bacteria (LAB) represent a chief component within the composite microbiota of fermented foods. Recently, the crucial role of LAB as reservoir of potentially transmissible AR genes has been increasingly recognized, highlighting the need for expanding the current knowledge on LAB mobile resistome and providing further insight into the evolution and spread of AR in the food chain. In the present study, the antimicrobial susceptibility profiles and the genetic basis of the resistance were investigated for two relevant genera of LAB, i.e. Leuconostoc and Lactobacillus, through the application of standard methods, such as phenotypic testing, conjugation experiments and PCR assay, and whole-genome sequencing (WGS)-based approaches. In the first part of the dissection, the contribution of these two approaches in the characterization of AR features for the genus Leuconostoc was investigated, revealing that genome-based analysis was more informative than conventional molecular techniques. Indeed, WGS-based analysis revealed the presence of genes coding for aminoglycoside resistance, such as aad6, sat4 and aphA-3, for streptogramin A resistance, as vatE, and for tetracycline, such as tet(S), in the genome sequence of the multidrug resistant L. mesenteroides subsp. mesenteroides LbE16 strain. In addition, this approach highlighted for the first time the presence of a erm(B)-bearing Tn917 transposon in the genome of L. mesenteroides subsp. dextranicum LbE15. Antibiotic susceptibility testing and conjugation experiments allowed to confirm the resistance phenotype for those Leuconostoc strains and provided the first evidence of the erythromycin resistance transfer between L. mesenteroides and E. faecalis, supplying novel proof that AR LAB can act as a reservoir of acquired AR genes. Therefore, standard methods should be combined with the WGS-based approaches to validate the hypothesis emerged from the in silico prediction of AR features. In the last part of the dissection, the combination of phenotypic susceptibility testing and genome-based analysis was performed for the whole genus Lactobacillus. This analysis revealed a positive correlation between phenotype and genotype for the 67% of the cases examined, where the genes aac(3), lsa and cml(A) involved in the resistance towards aminoglycoside, clindamycin and chloramphenicol, respectively, were found for the first time in Lactobacillus strains. In addition, acquired determinants coding for tetracycline and erythromycin resistance were simultaneously detected in L. amylophilus DSM 20533T and L. amylotrophicus DSM 20534T. Whereas, L. ingluviei DSM 15946T harboured the Tn916-like transposon carrying the genes tet(M) and tet(L), highlighting the potential of these AR genes to be horizontally transferred to other microorganisms. The results reported in this study may be utilized as a starting point for the generation of new and more focused scientific protocols and regulatory procedures based on WGS approaches for the safety assessment of Leuconostoc and Lactobacillus strains employed as starter cultures, food preservatives or probiotic by food and probiotic stakeholders.