Influence of Fiber Diameter of Polycaprolactone Nanofibrous Materials on Biofilm Formation and Retention of Bacterial Cells

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.

. The average pores size has here also a meaning of a characteristic distance  =  4    between fibres creating a pore in planar fibre process.Parameter is the fibre diameter an denotes

𝑑 𝑉 𝑉
the volume fraction of fibres.This approach motivates for characteristic distance estimation using the formula , where is the area density of the fibre process, i.e., the total fibre length in a unitary  = 1     area.The density is estimated here using the Buffon's needle problem (Stoyan 1995) as where is a length density of crossings between a testing line with a fibrous system, i.e., the number

𝐼 𝑁
of crossings per a unitary length of the testing line.Therefore, we estimate here the characteristic intra-fiber distance as  Wettability of the materials Figure S1: Water wicking into PCL45 and PCL80 nanofibrous materials over time.These are the average values of five individual measurements for each sample.The results represent the dynamic of the liquid wicking into the samples.The graphs presented are the average values for measurements of all samples from both types of materials.The graph can be divided into the first part where capillary forces are dominant and the second part where gravitational forces are already involved in the process against capillary forces.The break between the first and the second part occurs for PCL80 at a lower mass of the wicked liquid.This is probably due to the larger fibre diameters and hence the smaller specific surface area that the liquid gradually occupies in this type of test.Otherwise, it is not possible to see significant differences between the two materials with respect to their water wetting.

PCLs retention of bacterial cells
Table S8: Statistical evaluation of data -testing of a significant difference in the retention of S. aureus (SA) and E. coli (EC) by PCL45 and PCL80; the one-factor ANOVA method was used (p < 0.001 = retention differs significantly within the selected groups at the level of significance α = 0.001, marked as ***; p > 0.05 = retention does not differ significantly within the selected groups at the level of significance α = 0.05, marked as -), the condition of normal data distribution was met and verified using the Shapiro-Wilk test.

Table S1 :
The results of biofilm growth measurements of all bacterial strains on the plate, PCL45 and PCL80, the Shapiro-Wilk test confirmed the normality of the distributions of the measured data (pvalue > 0.05).

Table S2 :
The results of filtration of all bacterial strains through PCL45 and PCL80, the Shapiro-Wilk test confirmed the normality of the distributions of the measured data (p-value > 0.05).

Table S3 :
Biofilm formation by E. coli 8739, E. coli 25922, S. aureus 6538 and S. aureus 25923 on PCL nanomaterials PCL45 and PCL80 and control (PS) expressed as determination of CFUml -1 and biofilm formation rate compared to control (%).

Table S4 :
Statistical evaluation of data -testing whether biofilm growth on PCL is significantly different from the control on the plate.The paired t-test method was used, the condition of normal data distribution was met and verified using the Shapiro-Wilk test (at α = 0.05).

Table S5 :
Statistical evaluation of data -testing of difference in biofilm growth on PCL45 versus PCL80; the unpaired t-test method was used, the condition of normal data distribution was met and verified using the Shapiro-Wilk test (at α = 0.05).

Table S6 :
Statistical evaluation of data -testing of a significant difference in the growth of biofilm of S. aureus and E. coli on individual materials; the one-factor ANOVA method was used (p < 0.05 = biofilm formation differs significantly within the selected groups at the level of significance α = 0.05), the condition of normal data distribution was met and verified using the Shapiro-Wilk test.

Table S7 :
Statistical evaluation of data -testing for which particular strains does the biofilm formation differ.Multiple pairwise comparisons was performed to determine if the mean difference between specific pairs of a group is statistically significant.Since the ANOVA test is significant, we can calculate Tukey HSD (Tukey Honest Significant Differences) to perform multiple pairwise comparisons (group means) -a) for PCL45 and b) for PCL80.For both PCL45 and PCL80, the results of the TukeyHSD multiple comparison test show that there is a significant difference between the biofilm formation of EC8739 and EC25922, between SA25923 and EC8739, and between SA6538 and EC8739 at the given adjusted p-values (p adj).In general: on both PCL45 and PCL80 there is a significantly difference in EC8739 biofilm compared to the other strains.The differences in biofilm formation of the other tested isolates are not statistically different.