Biofilm Formation and Disinfectant Susceptibility of Salmonella from Layer Hen Houses: Practical Implications for Poultry House Sanitation

Non-typhoidal Salmonella (NTS) remains a persistent challenge in poultry production due to its strong biofilm-forming capacity and reduced susceptibility to commonly used disinfectants. This study provides a combined evaluation of biofilm formation and disinfectant susceptibility among Salmonella serotypes isolated from commercial layer hen houses in Türkiye, offering practical insight for poultry house sanitation, including several serotypes that are detected in layer hens worldwide for the first time. A total of 56 Salmonella enterica isolates representing 16 serotypes, recovered during the sampling period, were assessed for biofilm formation using a crystal violet microtiter assay at 20°C and 37°C over incubation periods of 24, 48, and 72 h. All isolates developed biofilms by 72 h, with OD 570 values increasing significantly over time (Friedman test, p < 0.001), while no significant differences were observed among serotypes represented by at least five isolates (Kruskal–Wallis test, p > 0.29). The efficacy of five disinfectant formulations with different active ingredient compositions was evaluated using a qualitative suspension method. Disinfectants were initially tested at their original concentrations, and subsequently diluted stepwise to determine the minimum effective concentration (MEC) following a 1-min contact time. Hydrogen peroxide (26%)–peracetic acid (6%) at 0.04% showed the highest bactericidal activity against all serotypes, followed by potassium peroxymonosulfate (21.41%)–sodium chlorite (1.5%) at 0.05%. Glutaraldehyde (50%) was effective only at higher concentrations (4%), whereas quaternary ammonium compounds (16%) alone required higher doses and longer exposure times; however, a glutaraldehyde (15%)–QAC (10%) formulation demonstrated enhanced efficacy at 0.5%. These findings highlight the widespread biofilm-forming capacity of poultry-associated Salmonella and support the practical application of oxidizing disinfectants for improving sanitation strategies in commercial layer hen houses.