Lentinula edodes, commonly known as shiitake mushroom, is renowned for its potential health advantages. This research delves into the often-overlooked by-product of shiitake cultivation, namely spent mushroom substrate (SMS), to explore its nutraceutical properties. The SMS samples were collected and subjected to different extraction methods, namely short or long agitation, and ultrasound-assisted extractions using different temperatures and distilled water or a 50% (v/v) ethanol as solvents. The extracts were tested for phenolic content (total phenols, ortho-diphenols, and flavonoids), antioxidant capacity (DPPH, 2,2-diphenyl-1 picrylhydrazyl; ABTS, 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid; and FRAP, ferric reducing antioxidant power), and antibacterial activity. The different extraction methods revealed substantial variations (p < 0.05) in phenolic composition and antioxidant capacity. The highest phenolic content and antioxidant capacity were achieved using 24 h extraction, agitation, 50 °C, and ethanol as the solvent. Furthermore, the extracted compounds displayed antibacterial activity in specific tested bacterial strains. This study highlights the nutraceutical potential of L. edodes’ SMS, positioning it as a valuable dietary supplement for animal nutrition, with emphasis on its prebiotic properties. Hence, this research unveils the promising health benefits of SMS in both human and animal nutrition.

Horses are considered as reservoirs of multidrug resistant bacteria that can be spread through the environment and possibly to humans. The aim of this study was to characterize the oral Gram-negative microbiota of healthy horses and evaluate their antimicrobial susceptibility profile in a One Health approach. For this purpose, samples were collected from the gingival margin of healthy horses, free of antimicrobial therapy, cultured in selective mediums, identified, and tested for antimicrobial susceptibility. Fifty-five Gram-negative isolates were identified, with 89.5% being zoonotic and 62% affecting humans, which were also found commonly in the environment. Forty-eight isolates (96%) were MDR. The phenotypic resistance presented as higher to macrolides (81.8%), β-lactams (55.4%), and quinolones (50%), and lower to sulfonamides (27.3%), tetracyclines, and amphenicols (both with 30.9%). In total, 51.5% of the isolates presented resistance to carbapenems. In addition to being the first report on the commensal oral microbiota of horses and respective susceptibility profile, this study highlights the horse as a valuable sentinel that can control the evolution and transmission of multidrug-resistant bacteria between the “One Health triad” since it is in contact with humans, other animals, and the environment, in different geographic locations.

As in human medicine, in veterinary medicine, chronic wounds are often related to polymicrobial infections and the presence of a biofilm, which compromises the effectiveness of therapeutic approaches. In this study, a Lusitano mare presented a 21-day-old chronic wound that was only being treated with an antiseptic. A swab sample was collected, and three isolates of Staphylococcus aureus and one of Pseudomonas aeruginosa were isolated. S. aureus did not show resistance to a panel of antibiotics. However, the P. aeruginosa isolate showed a resistance profile to carbapenems and fluoroquinolones, which may suggest a cross-resistance between antiseptic and antibiotics, given that no antibiotic therapy was applied to the wound or the mare in the previous year. Further experiments were conducted to assess the ability of the isolates to form biofilms, and to ascertain their susceptibility to gentamicin. The results demonstrated that the isolates produced biofilms. Gentamicin at the minimum inhibitory concentration (MIC) and 10× MIC caused biofilm removal between 59.3% and 85.7%, with the highest removal percentage being obtained for the P. aeruginosa isolate (at 10× MIC concentration). This study reveals that an equine wound was colonized by antibiotic resistant bacteria, and that all the wound colonizers could form biofilms, demonstrating the relevance of an adequate diagnosis and treatment when there is a suspicion of a biofilm-infected wound. It also highlights the possibility of resistance transmission between animals, animals and humans, or animals and the environment.

The oral microbiota has a diversity of microorganisms that together maintain the homeostasis of the oral cavity. Disruptions in the balance of these microbial populations can lead to the development of oral and dental diseases. To characterize the normal oral microbiota of captive ophidians, samples were obtained from the oral cavity of eight Burmese pythons (Python bivittatus) and 11 royal pythons (Python regius), using oral swab, during routine evaluations. In the Laboratory-Antimicrobials, Biocides and Biofilms Unit at University of Trás-os-Montes e Alto Douro, the identification and antimicrobial susceptibility profile was determined using the Vitek^® 2 Compact automated device (bioMérieux, Marcy-l’Étoile, France). Of the 106 bacterial isolates obtained, 69% were Gram-negative species and 31% were Gram-positive bacteria. The genus Pseudomonas was the most frequently isolated. Chryseobacterium indologenes, Escherichia coli and Pseudomonas aeruginosa were the most isolated species. Antimicrobial susceptibility testing revealed that the phenotypic resistance was highest for nitrofurans (47.2%), beta-lactams (45.8%) and sulfonamides (30.6%). Twenty-one multidrug-resistant isolates (58.3%) were identified with Acinetobacter baumannii, Serratia plymuthica, Chryseobacterium indologenes, Providencia rettgeri and Pseudomonas aeruginosa showing the highest resistance frequencies.

Coastal environments have been recognized as key reservoirs for antibiotic-resistant bacteria. The present study evaluated marine gastropods, Phorcus lineatus, as potential bioindicators to assess the spread of antibiotic-resistant bacteria. P. lineatus was sampled in four sites, with different anthropogenic pressures, along the northwest Portuguese coastal area. From these specimens, bacteria were isolated and tested for their antimicrobial susceptibility, followed by their phylogenetic and pathotypic determination. All the Escherichia coli isolates showed resistance to at least one antimicrobial agent. The highest levels of multidrug resistance (25%) were observed in E. coli isolates obtained from SITE 2, which is impacted by the city of Porto and industrial settlements, while nearly 17% of these isolates showed a multiple antibiotic resistance (MAR) index higher than 0.2. Among the isolates, phylogroups A and B2 were the most prevalent, followed by phylogroup B1. The isolates of phylogroup A showed a higher prevalence of antimicrobial resistance. This study offers valuable insights into the antibiotic resistance risks posed to marine ecosystems and underscores the need for microbiological monitoring and the development of effective management strategies. The findings suggest P. lineatus as a potential bioindicator of antibiotic-resistant bacteria in marine environments.

Microplastics (MPs) and metals frequently co-occur in aquatic environments, yet their combined effects on gut health and antimicrobial resistance in fish remain poorly understood. This study investigated the chronic effects of polyethylene (PE) and polystyrene (PS) microplastics, alone or combined with copper (Cu), on intestinal integrity, the gut-associated Gram-negative cultivable fraction, and phenotypic antimicrobial resistance in adult zebrafish (Danio rerio). Fish were exposed for 21 days to MPs (1 mg/L), Cu (25 µg/L), or their combinations. Histopathological analysis revealed that Cu-containing treatments induced more severe intestinal alterations, including edema, villus degeneration, and necrosis, whereas MPs-only exposures produced milder and heterogeneous responses. The composition of the Gram-negative cultivable fraction varied among treatments, with Cu, particularly in combination with MPs, associated with a broader occurrence of opportunistic and potentially pathogenic taxa. Antimicrobial susceptibility testing showed a high prevalence of multidrug resistance across treatments, with broader resistance spectra observed in Cu-containing exposures, consistent with metal-driven co-selection. In contrast, MPs alone did not systematically increase resistance and, for some antibiotics, showed resistance levels comparable to or lower than controls. Integrated multivariate analyses indicated that intestinal pathology and antimicrobial resistance co-varied along gradients of overall stress severity and stressor type, with Cu acting as the dominant driver and MPs exerting a modulatory, context-dependent influence. Overall, these findings highlight the importance of integrated assessments of gut pathology, microbial composition, and antimicrobial resistance to better understand the ecological and One Health implications of combined microplastic–metal exposure in aquatic systems.