This study examined the biofilm-forming capacity and antibiotic resistance of 93 L. monocytogenes isolates from poultry to better understand how this pathogen persists and how it can be more effectively controlled throughout the poultry production process. Biofilms were evaluated on polystyrene microtiter plates at 12 and 30 °C in a nutrient-rich laboratory medium (Brain Heart Infusion, BHI). Susceptibility to eight clinically and food-relevant antibiotics was tested using disk diffusion and interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints when available. Most isolates produced detectable but generally weak biofilms at both temperatures, with a subset shifting to moderate biofilm formation after prolonged incubation at 12 °C; no strong biofilm producers were identified. This highlights the significant influence of incubation time and temperature on surface colonization. Overall, the isolates remained largely susceptible to ampicillin, penicillin G, vancomycin, tetracycline, and chloramphenicol, although resistant or low-susceptibility subpopulations were noted for trimethoprim–sulphamethoxazole (TMP-SMX) and, particularly, erythromycin and streptomycin. No consistent correlation was found between biofilm-forming ability and antibiotic susceptibility, indicating that these phenotypic traits are largely independent in this collection. These findings reveal that poultry-derived L. monocytogenes isolates can form weak to moderate biofilms under the tested monoculture conditions while generally maintaining susceptibility to first-line antibiotics. However, the development of macrolide- and aminoglycoside-resistant subpopulations, along with the potential for increased colonization within complex multispecies biofilms in real processing environments, emphasizes the importance of ongoing integrated surveillance across animal food systems.

Background/Objectives: Listeria monocytogenes is a critical foodborne pathogen, with poultry products serving as a potential reservoir. Its ability to form biofilms may aid in its persistence on processing equipment and food-contact surfaces, while antibiotic resistance complicates efforts to control and treat infections. This study aimed to characterize, in parallel, the biofilm-forming capacity and antibiotic susceptibility of a large collection of poultry-derived L. monocytogenes isolates (n = 93) to better understand their potential for persistence and to clarify how the biofilm phenotype may relate to the bacterial antibiotic response and to inform risk assessment and targeted control strategies along poultry processing and supply chains. Methods: Biofilms were evaluated on polystyrene microtiter plates at 12 and 30 °C in a nutrient-rich laboratory medium. Susceptibility to eight clinically and food-relevant antibiotics was tested using disk diffusion and interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints when available. Results: At 30 °C for 48 h, 69.9% of isolates were classified as weak biofilm formers and 30.1% as non-biofilm formers, whereas at 12 °C for 120 h, 55.9% were weak, 16.1% moderate, and 28.0% non-biofilm formers, with no strong biofilm producers identified under either condition. Overall, the isolates remained largely susceptible to ampicillin, penicillin G, vancomycin, tetracycline, and chloramphenicol, with 87.3% of inhibition zones across all drugs falling within the 20–29 mm and 30–39 mm categories, while small subpopulations showed reduced susceptibility or resistance to trimethoprim–sulphamethoxazole (TMP-SMX) and, particularly, erythromycin and streptomycin. No consistent correlation was found between biofilm-forming ability and antibiotic susceptibility, indicating that these phenotypic traits are largely independent in this collection. Conclusions: These findings reveal that poultry-derived L. monocytogenes isolates can form weak to moderate biofilms under the tested monoculture conditions while generally maintaining susceptibility to first-line antibiotics. However, the development of macrolide- and aminoglycoside-resistant subpopulations, along with the potential for increased colonization within complex multispecies biofilms in real processing environments, emphasizes the importance of ongoing integrated surveillance across animal food systems.