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The quantification of cellular metabolic activity via MTT assay has become a widespread practice in eukaryotic cell studies and is progressively extending to bacterial cell investigations. This study pioneers the application of MTT assay to evaluate the metabolic activity of biofilm-forming cells within bacterial biofilms on nanofibrous materials. The biofilm formation of Staphylococcus aureus and Escherichia coli on nanomaterials electrospun from polycaprolactone (PCL), polylactic acid (PLA), and polyamide (PA) was examined. Various parameters of the MTT assay were systematically investigated, including (i) the dissolution time of the formed formazan, (ii) the addition of glucose, and (iii) the optimal wavelength for spectrophotometric determination. Based on interim findings, a refined protocol suitable for application to nanofibrous materials was devised. We recommend 2 h of the dissolution, the application of glucose, and spectrophotometric measurement at 595 nm to obtain reliable data. Comparative analysis with the reference CFU counting protocol revealed similar trends for both tested bacteria and all tested nanomaterials. The proposed MTT protocol emerges as a suitable method for assessing the metabolic activity of bacterial biofilms on PCL, PLA, and PA nanofibrous materials., Competing Interests: Declaration of competing interest The authors of the manuscript Application of MTT Assay for Probing Metabolic Activity in Bacterial Biofilm-Forming Cells on Nanofibrous Materials, namely Marta Stindlova, Vaclav Peroutka, Vera Jencova, Kristyna Havlickova, and Simona Lencova, declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024. Published by Elsevier B.V.)

To develop microbiologically safe nanofibrous materials, it is crucial to understand their interactions with microbial cells. Current research indicates that the morphology of nanofibers, particularly the diameter of the fibers, may play a significant role in biofilm formation and retention. However, it has not yet been determined how the fiber diameter of poly-ε-caprolactone (PCL), one of the most widely used biopolymers, affects these microbial interactions. In this study, two nanofibrous materials electrospun from PCL (PCL45 and PCL80) with different fiber diameter and characteristic distance δ between fibers were compared in terms of their ability to support or inhibit bacterial biofilm formation and retain bacterial cells. Strains of Escherichia coli (ATCC 25922 and ATCC 8739) and Staphylococcus aureus (ATCC 25923 and ATCC 6538) were used as model bacteria. Biofilm formation rate and retention varied significantly between the E. coli and S. aureus strains ( p < 0.05) for the tested nanomaterials. In general, PCL showed a lower tendency to be colonized by the tested bacteria compared to the control material (polystyrene). Fiber diameter did not influence the biofilm formation rate of S. aureus strains and E. coli 25922 ( p > 0.05), but it did significantly impact the biofilm formation rate of E. coli 8739 and biofilm morphology formed by all of the tested bacterial strains. In PCL45, thick uniform biofilm layers were formed preferably on the surface, while in PCL80 smaller clusters formed preferably inside the structure. Further, fiber diameter significantly influenced the retention of bacterial cells of all the tested strains ( p < 0.001). PCL45, with thin fibers (average fiber diameter of 376 nm), retained up to 7 log (CFU mL -1 ) of staphylococcal cells (100% retention). The overall results indicate PCL45's potential for further research and highlight the nanofibers' morphology influence on bacterial interactions and differences in bacterial strains' behavior in the presence of nanomaterials.

Electrospun polyamide (PA) nanofibers have great potential for medical applications (in dermatology as antimicrobial compound carriers or surgical sutures). However, little is known about microbial colonization on these materials. Suitable methods need to be chosen and optimized for the analysis of biofilms formed on nanofibers and the influence of their morphology on biofilm formation. We analyzed 11 PA nanomaterials, both nonfunctionalized and functionalized with AgNO 3 , and tested the formation of a biofilm by clinically relevant bacteria ( Escherichia coli CCM 4517, Staphylococcus aureus CCM 3953, and Staphylococcus epidermidis CCM 4418). By four different methods, it was confirmed that all of these bacteria attached to the PAs and formed biofilms; however, it was found that the selected method can influence the outcomes. For studying biofilms formed by the selected bacteria, scanning electron microscopy, resazurin staining, and colony-forming unit enumeration provided appropriate and comparable results. The values obtained by crystal violet (CV) staining were misleading due to the binding of the CV dye to the PA structure. In addition, the effect of nanofiber morphology parameters (fiber diameter and air permeability) and AgNO 3 functionalization significantly influenced biofilm maturation. Furthermore, the correlations between air permeability and surface density and fiber diameter were revealed. Based on the statistical analysis, fiber diameter was confirmed as a crucial factor influencing biofilm formation ( p ≤ 0.01). The functionalization of PAs with AgNO 3 (from 0.1 wt %) effectively suppressed biofilm formation. The PA functionalized with a concentration of 0.1 wt % AgNO 3 influenced the biofilm equally as nonfunctionalized PA 8% 2 g/m 2 . Therefore, biofilm formation could be affected by the above-mentioned morphology parameters, and ultimately, the risk of infections from contaminated medical devices could be reduced.