Take a (statistical) look at microbial motility: analytical assessment of the early-stage interaction between B. Subtilis and ZnO-based antimicrobial surfaces

Understanding the bioactivity mechanisms and surface properties of antimicrobial coatings is crucial for the development of safer and more environmentally friendly materials, limiting the development of antimicrobial resistance through a correct control of dose-effect relations. In this work, the early stages of interaction between inorganic antimicrobial agents and microorganisms were investigated. Specifically, the short-time motility of bacteria after 30 minutes of contact with ZnO-based bioactive surfaces was inspected by laser scanning confocal microscopy (LSCM), proposing an innovative statistical method to study the bacteriostatic/bactericidal activity of antimicrobials. ZnO nanostructures (NSs) were synthesized using a scalable electrochemical synthesis in aqueous phase. Two stabilizers, Sodium Dodecyl Sulfate (SDS) and Poly-Diallyl-Dimethyl-Ammonium chloride (PDDA), were employed to produce elongated and flower-like ZnO NSs morphologies. Inorganic antimicrobials were embedded into three polymeric matrices (polyethylene oxide, polylactic acid and poly-vinyl-methyl-ketone) to produce nanocomposite coatings providing different release of Zn2+ ions, ranging from 20 to 120 ppm overtime (i.e. tunable bioactivity). The antimicrobial surfaces were characterized using UV-Vis, FTIR and X-ray photoelectron spectroscopies, and scanning and transmission electron microscopies. To establish an analytical dose-effect correlation, the influence of Zn2+ release on the motility of Bacillus subtilis was evaluated using particle tracking analysis of LSCM images to access the dynamical behavior of the bacteria at short times after direct contact with the bioactive surfaces. The average mean squared displacements (MSDs) revealed that the run-and-tumble dynamics of native B. Subtilis turns into sub-diffusive motion under the effect of Zn2+ ions release indicating a strong biostatic effect already in the first minutes of contact. For the highest concentration of Zn2+ released from the PEO and PVMK matrices the time-dependent diffusion coefficient D(τ) approaches zero, indicating an almost complete motility suppression. Direct LSCM imaging of live-dead bacteria was additionally used to assess viability as a function of bioactive ion release revealing that even for the larger Zn2+ ions release less than 20% of the cells were damaged after 30 minutes exposure, i.e. the bioactive surfaces have a biostatic rather than bactericidal effect. This direct approach offers a new paradigm in the development of bacteriostatic vs bactericidal strategies, allowing for a precise engineering of antimicrobial coatings to achieve a desired spatio-temporal effect.