In vitro visualization and quantitative characterization of Pseudomonas aeruginosa biofilm growth dynamics on polyether ether ketone

Abstract

Prevention and treatment of orthopedic device‐related infection (ODRI) is complicated by the formation of bacterial biofilms. Biofilm formation involves dynamic production of macromolecules that contribute to the structure of the biofilm over time. Limitations to clinically relevant and translational biofilm visualization and measurement hamper advances in this area of research. In this paper, we present a multimodal methodology for improved characterization of Pseudomonas aeruginosa grown on polyether ether ketone (PEEK) as a model for ODRI. PEEK discs were inoculated with P. aeruginosa, incubated for 4−48 h time intervals, and fixed with 10% neutral‐buffered formalin. Samples were stained with fluorescent dyes to measure biofilm components, imaged with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), and quantified. We were able to visualize and quantify P. aeruginosa biofilm growth on PEEK implants over 48 h. Based on imaging data, we propose a generalized growth cycle that can inform orthopedic diagnostic and treatment for this pathogen on PEEK. These results demonstrate the potential of using a combined CLSM and SEM approach for determining biofilm structure, composition, post‐adherence development on orthopedic materials. This model may be used for quantitative biofilm analysis for other pathogens and other materials of orthopedic relevance for translational study of ODRI.

Virtual High‐Throughput Screening of Vapor‐Deposited Amphiphilic Polymers for Inhibiting Biofilm Formation

“…This was followed by static incubation for 8 h at 37 °C based on P. aeruginosa biofilm lifecycle and previous work. [12,57,58] At this point in time (8 h) when culture transitions from stage II to III, quantification is most reliable due to the presence of linear and vertical biofilm development; further growth would lead to biofilm maturation, giving rise to a 3D community (stage IV, 14-24 h) where biomass accumulation would reflect more of multiplication and quorum sensing, [59] rather than bacteriamaterial interactions. For the biofilm assay at solid-liquid interfaces, the hydrophobic pHFBA-coated substrates (i.e., "0% HEMA"), along with the uncoated controls, were placed horizontally with the coated side facing upward on the well bottom of six-well culture plates.…”

Amphiphilic Copolymer Thin Films with Short Fluoroalkyl Side Chains for Antibiofilm Properties at the Solid–Liquid–Air Interface

“…This 8-h incubation was selected because it allows P. aeruginosa biofilm to reach stage III of its lifecycle, leading to the formation of vertical and linear biofilms, the qualification of which is known to be reliable. , Similar conditions have been used in the past to assess the biofilm formation on amphiphilic copolymers . After the incubation, the cover slips were washed with Milli-Q water and then submerged into 0.5% (w/v) CV solution for 15 min.…”

What’s New in Musculoskeletal Infection

“…In an effort to understand the precise timing and biology of Pseudomonas aeruginosa biofilm production, Spake et al reported on a polyetheretherketone (PEEK) disc model for in vitro biofilm creation 57 . Their model allowed for consistent imaging and quantification of biofilm production and has implications for understanding the variables associated with biofilm production across multiple species.…”