Phenotypic resistance to life-saving antimicrobials in Gram-positive bacteria isolated from wild birds in Northern Italy: a One Health surveillance perspective

Antimicrobial resistance (AMR) is a global health challenge, and wild birds are recognized as sentinels of environmental AMR circulation at the human-animal-environment interface. This study investigated the phenotypic profiles of AMR in Gram-positive bacteria from wild avifauna in Lombardy, Northern Italy, focusing on resistance to Critically Important Antimicrobials (CIAs) for human medicine. In total, 205 wild birds admitted to a wildlife rescue center were sampled (2024–2025). Isolates were identified by MALDI-TOF MS and characterized by disk diffusion susceptibility testing following validated international breakpoints; supplementary phenotypic tests were applied for vancomycin susceptibility (MIC strips), methicillin-resistance (MR; cefoxitin disk), high-level aminoglycoside resistance (HLAR; gentamicin disk), and beta-lactamase production (penicillin G morphology and nitrocefin test). Among 469 total isolates, 188 (40.1%) were Gram-positive, predominantly Lactobacillales (95.2%), with Enterococcus spp. as the dominant genus. Multidrug-resistance (MDR) was detected in 17% of Gram-positive isolates. Among Lactobacillales, resistance to rifampicin (35.7%) and tetracycline (29.6%) was most prevalent. Resistance to linezolid (6.1%) and fluoroquinolones (2.8%), authorized exclusively for human medicine, was detected in multiple Enterococcus species, including isolates from non-synanthropic birds. No acquired resistance to vancomycin in Enterococcus isolates was detected. HLAR affected 5.6% of Enterococcus isolates. Among Bacillales, a MR Staphylococcus epidermidis and a nitrocefin-confirmed beta-lactamase-producing Staphylococcus saprophyticus were recovered. Since sampled birds were admitted to a wildlife rescue center following injury, illness, or death, results should not be considered representative of healthy free-ranging populations. Furthermore, as characterization was exclusively phenotypic, the genetic basis of the resistance profiles detected remains to be confirmed by molecular analysis. Nonetheless, this study provides original phenotypic data across a broad taxonomic diversity of avian hosts, contributing to the evidence base for the integration of wild birds as environmental sentinels within One Health AMR surveillance frameworks.