Standardization of an Adaptative Methodology for In Vitro Biofilm Evaluation of Staphylococcus hominis

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Abstract
Biofilm formation by Staphylococcus hominis in pharmaceutical environments can promote microbial persistence on surfaces and hinder cleaning and disinfection, posing a risk to product quality and safety. This study aimed to standardize an in vitro adaptive methodology to evaluate biofilm for-mation on surfaces of interest to the pharmaceutical industry and analyze the impact of different interpretation criteria. Fifty-four S. hominis strains isolated between 2014 and 2022 from a pharma-ceutical production chain were evaluated. Biofilm formation was analyzed on stainless steel, acrylic, and vinyl coating using the Cell Counting Kit-8 (CCK-8) assay to quantify bacterial cell viability, as well as on polystyrene using the crystal violet method. Qualitative and quantitative interpretations applied different equations to define the negative control cutoff. Vinyl coating showed the highest formation, with 31 (57.41%) strains classified as moderate or strong producers, followed by stainless steel (n=20; 37.01%), acrylic (n=16; 29.63%), and polystyrene (n=2; 3.70%). Quantitative analysis identified surface-dependent hyper-producing strains, reaching maximum values from 49.75 to 61.60%. Applying three standard deviations to the negative control generated reclassifications without statistically significant differences between criteria. The proposed standardized method supports the CCK-8 assay as an adaptive methodology to evaluate biofilm formation by different bacterial strains and surfaces.

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