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

Abstract

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.

Keywords: AmpC; CIAs; ESBL; HCIAs; HPCIAs; One Health; antimicrobial resistance; last-line antimicrobials; wild birds.

Figure 1

Network graph showing associations between wild bird species and bacterial genera isolated from cloacal samples. The circular representation displays connections between wild bird species (black nodes with stylized illustrations on the left) and the bacterial genera isolated (colored nodes on the right). The size of the bacterial nodes is proportional to the total number of isolates per genus. Similarly, the size of black nodes is proportional to the number of sampled birds belonging to the indicated species. Node color represents bacterial taxonomic order: Enterobacterales (dark blue), Pseudomonadales (pink), and Burkholderiales (yellow). Grey lines indicate the presence of at least one bacterial isolate of the corresponding genus isolated from the linked bird species. For some species, no Gram-negative bacteria were isolated from fecal samples.

Figure 2

Acquired antimicrobial resistance profiles among Enterobacterales isolates. Bar chart illustrating the number of Enterobacterales isolates exhibiting acquired resistance to selected antimicrobial agents. Intrinsic resistance traits were excluded from the analysis. Antimicrobial molecules are reported along the x-axis, while the y-axis indicates the number of resistant isolates. Bar colors correspond to individual bacterial species, as detailed in the legend on the right. CN: gentamicin; CTX: cefotaxime; CAZ: ceftazidime; FEP: cefepime; CZA: ceftazidime/avibactam; FOX: cefoxitin; TE: tetracycline; DO: doxycycline; TGC: tigecycline; LEV: levofloxacin; CIP: ciprofloxacin; MEM: meropenem; IPM: imipenem; AMP: ampicillin; AMC: amoxicillin/clavulanic acid; PRL: piperacillin; TZP: piperacillin/tazobactam; SXT: sulfamethoxazole/trimethoprim; C: chloramphenicol; ATM: aztreonam; CS: colistin.