Giovanni Milani

Bifidobacterium animalis subsp. lactis is widely used as a probiotic; however, the presence of the tetracycline resistance gene tetW raises safety and regulatory concerns due to its potential mobility within the gut microbiome. Here, we applied a Safe‐by‐Design strategy using the endogenous CRISPR‐Cas system of B. animalis subsp. lactis BLC01 to inactivate tetW through the introduction of premature stop codons. Whole‐genome sequencing confirmed the intended editing and excluded relevant off‐target effects. tetW inactivation markedly reduced the tetracycline minimum inhibitory concentration, restoring susceptibility below the tetracycline cut‐off value for bifidobacteria (8 μg/mL). Comparative phenotypic analyses demonstrated that the edited strain (BLC01‐2F3G10) retained key probiotic traits, including tolerance to acid, bile, and osmotic stress, exopolysaccharide production, aggregation capacity, survival during simulated gastrointestinal digestion and adhesion to intestinal epithelial cells. Importantly, no reversion to tetracycline resistance was observed after prolonged exposure to sub‐inhibitory minimal selective antimicrobial concentration, indicating genetic stability of the edited phenotype. Collectively, these findings demonstrate that endogenous CRISPR‐based genome editing can be leveraged to selectively remove antimicrobial resistance determinants from probiotic strains while preserving functionality, supporting the development of next‐generation probiotics with an improved safety profile and reduced potential for antimicrobial resistance dissemination in the human gut.

The global threat of antimicrobial resistance (AMR) is exacerbated by the mobilization of antimicrobial resistance genes (ARGs) occurring in different environmental niches, including seawater. Marine environments serve as reservoirs for resistant bacteria and ARGs, further complicated by the ubiquity of microplastics (MPs). MPs can adsorb pollutants and promote bacterial biofilm formation, creating conditions favorable to the dissemination of ARGs. This study explores the dynamics of ARG transfer in the marine bivalve Mytilus galloprovincialis within a seawater model, focusing on the influence of polyethylene MPs on the mobilization of the Tn916-carrying tetM gene and plasmid-encoded ermB. Experiments revealed that biofilm formation on MPs by Enterococcus faecium and Listeria monocytogenes facilitated the transfer of the tetM resistance gene, but not the ermB gene. Furthermore, the presence of MPs significantly increased the conjugation frequency of tetM within mussels, indicating that MPs enhance the potential for ARG mobilization in marine environments. These findings highlight the role of MPs and marine organisms in ARG spread, underscoring the ecological and public health implications.

Fermented meat products represent an important industrial sector in Europe, particularly in the Mediterranean Countries (MC), where the presence of numerous local productions, still obtained through spontaneous fermentation, is recognized as a formidable treasure chest of unexplored microbial biodiversity. Lactobacillaceae naturally occurring in fifteen spontaneously fermented sausages from MC (Italy, Spain, Croatia, and Slovenia) were isolated and taxonomically characterized using molecular techniques. Additionally, a safety assessment for the presence of antibiotic resistances and biogenic amine (BA) production was performed to determine their suitability as autochthonous starter cultures. Molecular typing, performed using REP-PCR, discriminated 151 strains belonging to Latilactobacillus sakei (59.6%), Latilactobacillus curvatus (26.5%) and Companilactobacillus alimentarius (13.9%). The minimum inhibitory concentrations (MICs) of eight different antibiotics revealed a high resistance to streptomycin (27%), tetracycline (16%), followed by gentamycin (14%) and kanamycin (13%). Interestingly, the results showed a geographical distribution of resistant biotypes. tetM/tetS or ermB genes were identified in only six strains. The amino-biogenic potential of the strains was assessed, confirming the absence of this trait among L. sakei, while a high number of producer strains was found among L. curvatus. On the 151 analyzed strains, 45 demonstrated safety traits for their future use as starter food cultures. These results open the way to further studies on the technological properties of these promising autochthonous strains, strongly linked to the Mediterranean environment.