Bacterial persistence has been extensively studied as a possible explanation for strain survival under stress; however, in Enterococcus spp., this ability is still an understudied phenomenon. In this study, 40 Enterococcus spp. isolates of human clinical (n = 10), veterinary commensal (n = 10), veterinary clinical (n = 10) and veterinary environmental (hospital surfaces) (n = 10) origins, were exposed to a high concentration of ciprofloxacin. Time–kill curves were established, after which antimicrobial susceptibility profiles were reassessed. Subsequently, the only presumptive persister was selected for Whole-Genome Sequencing, together with one isolate showing no evidence of persister formation. Comparative genomic analyses were conducted to identify genetic variations between exposed and non-exposed isolates and to explore potential genetic determinants associated with persistence. Observed genetic features present in the persister isolate included toxin–antitoxin systems, a cold-shock protein and the tyrosine-type recombinase/integrase XerC, which may represent putative candidates for further investigation. Interestingly, the majority of toxin–antitoxin system-associated genes were found in plasmids. This study represents an important step towards a better understanding of persistence development in Enterococcus spp.; however, validation using other methodologies such as RNA-sequencing is an important next step.

Protected Designation of Origin (PDO) cheeses are strongly linked to specific geographical areas, where local raw materials, artisanal practices, and microbial communities collectively shape product identity. Metataxonomic-based approaches have been increasingly proposed as tools for food authentication; however, the robustness of taxonomic profiling alone for defining regional biomarkers remains insufficiently tested, particularly in spontaneously fermented cheeses. Here, we conducted a two-year longitudinal analysis of bacterial and fungal communities in Portuguese PDO ewe’s milk cheeses from two regions, Azeitão and Nisa, including four and two producers, respectively, using a metataxonomic analysis to 16S and ITS.Alpha diversity analyses revealed no regional differences in bacteria or fungi richness, however some differences at the producer’s level were found. Beta diversity highlighted patterns of regional variation in microbial community structures. While permutational analyses supported the idea that producer-specific effects explained a larger proportion of variance than geographical origin. The analysis of differentially abundant amplicon sequence variants (ASVs) identified 36 and 20 bacterial ASVs, and 26 and 4 fungi ASVs, capable of discriminating Azeitão and Nisa cheeses, respectively. However, further examination showed that these ASVs did not meet the criteria for true regional biomarkers, as their prevalence was uneven across producers within each region.Our findings suggest that regional identity is not primarily determined by the taxonomic composition of cheese microbiota, but rather by the functional and metabolic activities expressed during ripening. Overall, this study underscores the limitations of taxonomy-based profiling and supports the need for integrated multi-omic approaches to accurately capture PDO-specific signatures.

Portuguese Protected Designation of Origin (PDO) cheeses from Azeitão and Nisa harbor unique autochthonous microbial communities integral to their terroir, sensory identity, and authenticity. Rediscovering and preserving this microbial heritage is essential for safeguarding traditional cheesemaking practices and unlocking the biotechnological potential of indigenous microorganisms. This study used a polyphasic approach to isolate, preserve, and functionally characterize lactic acid bacteria (LAB) from these cheeses, aiming both to conserve autochthonous microbial resources and evaluate their suitability for food applications and preliminary probiotic-related traits. From 2304 bacterial isolates, 119 representative LAB were selected and screened for safety based on hemolytic and gelatinolytic activities and antimicrobial susceptibility. Technological potential was assessed through acidification, proteolytic and lipolytic activities, and antagonism against foodborne pathogens, while probiotic traits were evaluated via tolerance to acidic pH and bile salts, survival under simulated gastrointestinal conditions, and auto- and co-aggregation. Thirty-nine isolates exhibited desirable technological traits, including proteolytic and lipolytic activities and broad-spectrum pathogen antagonism. Among these, three isolates (two Leuconostoc mesenteroides and one Enterococcus hirae) combined desirable technological traits with survival under simulated gastrointestinal conditions, representing preliminary multifunctional candidates. These findings reveal Azeitão and Nisa PDO cheeses as living reservoirs of terroir, where indigenous LAB rediscovered through this approach represent both a key component of Portugal’s microbial heritage and a source of functional cultures for sustainable dairy innovation.