Evaluation of Biofilm Cultivation Models for Efficacy Testing of Disinfectants against Salmonella Typhimurium Biofilms

Abstract

Within the European Union, Salmonella is frequently reported in food and feed products. A major route of transmission is upon contact with contaminated surfaces. In nature, bacteria such as Salmonella are often encountered in biofilms, where they are protected against antibiotics and disinfectants. Therefore, the removal and inactivation of biofilms is essential to ensure hygienic conditions. Currently, recommendations for disinfectant usage are based on results of efficacy testing against planktonic bacteria. There are no biofilm-specific standards for the efficacy testing of disinfectants against Salmonella. Here, we assessed three models for disinfectant efficacy testing on Salmonella Typhimurium biofilms. Achievable bacterial counts per biofilm, repeatability, and intra-laboratory reproducibility were analyzed. Biofilms of two Salmonella strains were grown on different surfaces and treated with glutaraldehyde or peracetic acid. Disinfectant efficacy was compared with results for planktonic Salmonella. All methods resulted in highly repeatable cell numbers per biofilm, with one assay showing variations of less than 1 log10 CFU in all experiments for both strains tested. Disinfectant concentrations required to inactivate biofilms were higher compared to planktonic cells. Differences were found between the biofilm methods regarding maximal achievable cell numbers, repeatability, and intra-laboratory reproducibility of results, which may be used to identify the most appropriate method in relation to application context. Developing a standardized protocol for testing disinfectant efficacy on biofilms will help identify conditions that are effective against biofilms.

Evaluation of Disinfectant Efficacy against Biofilm-Residing Wild-Type Salmonella from the Porcine Industry

“…Moreover, our recently published study demonstrated that disinfectant concentrations, which were evaluated using standardized protocols for the disinfection of cell suspensions (EN 1656), are not high enough to achieve a ≥4 log 10 CFU reduction when tested against biofilms. In this study, on the S. Typhimurium reference strain ATCC 14028, the concentrations of the same disinfectants needed to give a ≥5 log 10 reduction, according to EN 1656, were 0.03% with glutaraldehyde and 0.002% with peracetic acid [24,29]. This shows that there is a necessity for significantly higher concentrations of disinfectants when biofilms are a concern and emphasizes the importance of also testing bacteria in biofilms when evaluating disinfectants on wild-type strains [30].…”

“…Both disinfectants were neutralized for at least five minutes. Neutralizer toxicity and disinfectant neutralization validation checks were performed, as described in Richter/Konrat et al 2023 [24]. The method was performed according to Richter/Konrat et al 2023 [24] with some modifications.…”

“…Neutralizer toxicity and disinfectant neutralization validation checks were performed, as described in Richter/Konrat et al 2023 [24]. The method was performed according to Richter/Konrat et al 2023 [24] with some modifications. To create a working culture in broth, bacterial cultures on blood agar were transferred to 5 mL LB, and the OD was measured and adjusted to 1 McFarland.…”

“…Level of detection (LOD) was set to 1.7 log 10 when no viable bacteria were observed. See reference [24] for more detail on calculation of LOD.…”

“…Bacteria in biofilms are more tolerant to disinfectants than when in their free-living state. The required ≥4 log 10 colony forming units (CFUs) reduction in bacteria stated in the European disinfection standards (EN 13697:2015+A1:2019) is more difficult to reach when bacteria are associated with a biofilm [20][21][22][23], and it has also been shown that higher concentrations of disinfectant are required to achieve a ≥5 log 10 CFU reduction in Salmonella reference strains in biofilms [24].…”