# Resisting the Final Line: Phenotypic Detection of Resistance to Last-Resort Antimicrobials in Gram-Negative Bacteria Isolated from Wild Birds in Northern Italy

## TL;DR

Wild birds in northern Italy carry antibiotic-resistant bacteria, including those resistant to last-resort drugs, suggesting their role in spreading antimicrobial resistance.

## Contribution

This study identifies wild birds as carriers of Gram-negative bacteria with resistance to critical antimicrobials, emphasizing their role in environmental AMR dissemination.

## Key findings

- Gram-negative bacteria isolated from wild birds showed resistance to first-line and last-resort antimicrobials.
- 40% of Klebsiella pneumoniae isolates exhibited a phenotype compatible with ESBL production.
- 12.7% of isolates showed resistance profiles indicative of AmpC beta-lactamase production.

## Abstract

Antimicrobial resistance represents a critical global health challenge, affecting human, animal, and environmental health. Wild birds, due to their mobility and ecological diversity, are increasingly recognized as potential carriers and spreaders of resistant bacteria. In this study, we examined wild birds that died at a wildlife rescue center in northern Italy to investigate the presence of Gram-negative bacteria in their intestinal tract and assess their antimicrobial resistance profiles. Cloacal samples were collected from 112 birds. A total of 157 Gram-negative bacterial isolates were identified, including clinically significant species. Notably, many isolates exhibited resistance to first-line and critically important antimicrobials, including those reserved for human medicine. Additionally, several isolates showed resistance patterns suggestive of mechanisms that further enhance their ability to withstand treatment. Resistance was more pronounced in carnivorous, scavenging, and migratory birds. These findings highlight the important role wild birds may play in the environmental dissemination of antimicrobial resistance and underscore the need for their inclusion in surveillance programs, supporting a One Health approach.

Antimicrobial resistance (AMR) is a growing global health threat, with wild birds increasingly recognized as potential reservoirs of resistant pathogens and as sentinels of environmental AMR. This study investigated the occurrence and AMR profiles of Gram-negative bacteria isolated from wild birds that died at the Wildlife Rescue Center in Vanzago, Lombardy, in 2024. Cloacal swabs were collected from 112 birds representing various ecological categories. A total of 157 Gram-negative bacteria were isolated and identified, including clinically relevant genera and species, such as Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Salmonella spp., Pseudomonas aeruginosa, and Acinetobacter baumannii. Antimicrobial susceptibility testing revealed resistance to first-line and critically important antimicrobials, including those exclusively authorized for human use. Notably, a phenotype compatible with Extended-Spectrum Beta-Lactamase (ESBL) production was detected in four out of ten (40%) K. pneumoniae isolates. In addition, 20 out of the 157 (12.7%) isolated bacteria phenotypically exhibited a resistance profile indicative of AmpC beta-lactamase (AmpC) production, including Enterobacter spp. and P. aeruginosa. Resistance patterns were particularly interesting in birds with carnivorous, scavenging, or migratory-associated behaviors. These findings highlight the role of wild birds in the ecology and dissemination of antimicrobial-resistant bacteria (ARB) and highlight the need for wildlife-based AMR monitoring programs as part of a One Health approach.