Dietary Pistachio Skin Effects on Antibiotic-Free Lamb: Virulence Traits, Antimicrobial Resistance, and Clonal Relatedness in Commensal Escherichia coli Strains

<b>Background/Objectives</b>: In food-producing animal (FPA) environments, healthy animals can act as reservoirs of potentially pathogenic <i>Escherichia coli</i>, which can be transmitted through the food chain to humans. This study aimed to evaluate cloacal <i>E. coli</i> 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. <b>Methods</b>: A total of 295 <i>E. coli</i> 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. <b>Results</b>: The feeding regimen did not significantly influence the prevalence, abundance, or virulence of the <i>E. coli</i> isolates. Shiga toxin-producing <i>E. coli</i> (STEC) were the most common pathotype, mainly carrying the <i>stx1</i> gene, while the Enteroinvasive (EIEC) type was detected only sporadically. Enteropathogenic <i>E. coli</i> (EPEC) predominated at T0, while enteroaggregative <i>E. coli</i> (EAEC) at T1, and enterotoxigenic <i>E. coli</i> (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 <i>E. coli</i> resistome. PFGE genotyping showed high genetic diversity, with resistance traits more frequently detected than virulence factors. <b>Conclusions</b>: This study highlights that healthy lambs serve as reservoirs for potentially human-pathogenic <i>E. coli</i> and suggests that dietary regimes could effectively reduce antibiotic resistance.