Development of an innovative method of Salmonella Typhi biofilm quantification using tetrahydrofuran and response surface methodology.

Salmonella Typhi (S. Typhi), the causative agent of typhoid fever, poses a severe global health threat, particularly due to its ability to form robust biofilms that confer high levels of antibiotic resistance. Biofilms act as a physical and chemical barrier, protecting the bacterial community from hostile environments and therapeutic interventions, thereby complicating infection management. Accurate and reproducible quantification of S. Typhi biofilms is essential for understanding pathogenesis, evaluating anti-biofilm agents, and improving clinical outcomes. However, conventional biofilm quantification methods, such as crystal violet staining, suffer from limitations, including biofilm loss during washing steps, variability in adherence to surfaces, and inconsistent readings due to biofilm accumulation at the air-liquid interface. In this study, we systematically investigated the efficiency of six chemicals (five solvents and one dye), dimethyl sulfoxide (DMSO), methanol, ethanol, acetone, tetrahydrofuran (THF), and crystal violet, for their interaction with pre-formed S. Typhi biofilms in 96-microtiter well plates. Our goal was to identify an optimal solvent that can effectively solubilize biofilm while preserving structural integrity, thus enhancing measurement precision. We developed a new solvent-assisted method that significantly reduces washing steps to minimize biofilm detachment and sample variability. The process was statistically optimized and validated using response surface methodology (RSM), enabling the identification of THF as a highly effective solvent that maximizes optical density signal without compromising reproducibility. Our findings demonstrate that THF-based biofilm quantification provides superior accuracy, consistency, and ease of use compared to traditional approaches. Notably, THF was found to be the most effective biofilm-dissolving solvent among the six chemicals. This optimized method not only streamlines biofilm measurement workflows but also holds great potential for high-throughput screening of anti-biofilm compounds.