Natural whey starter (NWS) is an undefined and variable microbial community of starter lactic acid bacteria (SLAB), traditionally produced by back-slopping and used in the manufacture of several hard-cooked Italian cheeses, including Parmigiano Reggiano PDO. Despite its importance, the parameters influencing the microbial composition and technological performance of NWS remain poorly investigated, largely due to the lack of controlled studies that do not interfere with actual cheese production. Although the Parmigiano Reggiano PDO regulations strictly forbid the use of selected starters and require NWS to be obtained exclusively through spontaneous whey fermentation following curd extraction, we developed a freeze-dried prototypical natural starter culture (NSC) system that simulates, under laboratory conditions, the back-slopping process used in Parmigiano Reggiano dairies to ferment sweet whey into NWS. This system (referred to as NSC₀) effectively replicated fresh NWS and was successfully propagated through three consecutive rounds of laboratory-scale cheese-making. Using this validated system, we demonstrated that raw cow milk spiked with penicillin G at or below the maximum residue limits (MRL) negatively impacted NSC fermentative performance during the first round of caseification and significantly reduced microbial counts during the second round, suggesting a cumulative inhibitory effect of penicillin G, non-observable in a single back-slopping cycle. As SLAB declined, spoilage yeasts increased. qPCR analysis revealed that 2 and 4 ppb penicillin G impaired NWS biodiversity by inhibiting Lactobacillus delbrueckii and streptococci, whereas Lactobacillus helveticus remained unaffected. The validated NSC system developed in this study offers a robust platform for future investigations into the abiotic and biotic factors affecting NWS, advancing our ability to predict the behavior of undefined starter cultures under realistic cheese-making conditions.

Background/Objectives: In food-producing animal (FPA) environments, healthy animals can act as reservoirs of potentially pathogenic Escherichia coli, which can be transmitted through the food chain to humans. This study aimed to evaluate cloacal E. coli in healthy Sicilian lambs subjected to an experimental feeding regimen by assessing bacterial levels, antimicrobial resistance, virulence traits, and the clonal relationships, as well as the impact of a pistachio skin as an agro-industrial by-product supplement during a 58-day feeding trial. Methods: A total of 295 E. coli isolates from the control (CTRL) and treatment (Treated) groups at initial time (T0) and final time (T1) were phenotypically and genotypically characterized using Kirby–Bauer antimicrobial testing, multiplex PCR for virulence genes, and PFGE for clonal analysis. Results: The feeding regimen did not significantly influence the prevalence, abundance, or virulence of the E. coli isolates. Shiga toxin-producing E. coli (STEC) were the most common pathotype, mainly carrying the stx1 gene, while the Enteroinvasive (EIEC) type was detected only sporadically. Enteropathogenic E. coli (EPEC) predominated at T0, while enteroaggregative E. coli (EAEC) at T1, and enterotoxigenic E. coli (ETEC), initially prevalent in Treated samples, disappeared by T1. Antimicrobial resistance profiles varied among isolates, with the highest resistance observed in the CTRL group. However, both groups exhibited high resistance to streptomycin, and 9% of CTRL isolates were multidrug resistant. A notable reduction in overall resistance rates, especially in the Treated group, was observed, indicating a dietary effect on the E. coli resistome. PFGE genotyping showed high genetic diversity, with resistance traits more frequently detected than virulence factors. Conclusions: This study highlights that healthy lambs serve as reservoirs for potentially human-pathogenic E. coli and suggests that dietary regimes could effectively reduce antibiotic resistance.