A Bead Biofilm Reactor for High-Throughput Growth and Translational Applications

Abstract

Bacteria in natural ecosystems such as soil, dirt, or debris preferentially reside in the biofilm phenotype. When a traumatic injury, such as an open fracture, occurs, these naturally dwelling biofilms and accompanying foreign material can contaminate the injury site. Given their high tolerance of systemic levels of antibiotics that may be administered prophylactically, biofilms may contribute to difficult-to-treat infections. In most animal models, planktonic bacteria are used as initial inocula to cause infection, and this might not accurately mimic clinically relevant contamination and infection scenarios. Further, few approaches and systems utilize the same biofilm and accompanying substrate throughout the experimental continuum. In this study, we designed a unique reactor to grow bacterial biofilms on up to 50 silica beads that modeled environmental wound contaminants. The data obtained indicated that the reactor system repeatably produced mature Staphylococcus aureus and Pseudomonas aeruginosa biofilms on the silica beads, with an average of 5.53 and 6.21 log₁₀ colony-forming units per mm², respectively. The bead substrates are easily manipulable for in vitro or in vivo applications, thus improving translatability. Taken together, the bead biofilm reactor presented herein may be a useful system for repeatably growing established biofilms on silica beads that could be used for susceptibility testing and as initial inocula in future animal models of trauma-related injuries.

Keywords: P. aeruginosa; S. aureus; SEM; antibiofilm; biofilm; in vitro; reactor; repeatability; surface coverage.

Figure 1

Reactor design from original prototype to final dish, viewed top-down and from the side. (a) Initial glass dish prototype with fifty divots etched with a Dremel tool. We found that this approach could adequately hold 4 mm glass beads in place when exposed to 40 rpm shear force. However, the manufacturing process was laborious and somewhat inconsistent. Biofilm growth could be seen adhering to Dremel surface defects along the dish. (b) A SolidWorks insert that contained fifty divots and could be exchanged between crystallizing dishes or manufactured with different materials. (c) The final reactor system with an aluminum foil lid. The metal insert prevented bead substrates from dislodging in the assembled reactor, even when broth was added in a biosafety cabinet.

Figure 2

SEM images of a 4 mm glass bead surface before (a) and after (b) hand sanding it between two sheets of 60-grit sandpaper.

Figure 3

Images of S. aureus and P. aeruginosa biofilm growth on glass bead substrates at different SEM magnifications and live/dead-stained. (a) Representative SEM images of S. aureus and P. aeruginosa biofilm growth on the surface of a sanded glass bead taken at 500× magnification. S. aureus growth appeared to increase in thickness around textured areas, whereas P. aeruginosa dwelled in a multilayer sheet evenly covering the bead surface. (b) Representative SEM images of S. aureus and P. aeruginosa biofilm on the surface of a sanded glass bead at 1000× magnification. S. aureus growth correlated with substrate texture: biofilm plumes appeared to increase where sanded defects were most prominent. Comparatively, P. aeruginosa continued to grow in a sheet across the bead surface. (c) A representative z-slice image of live/dead-stained biofilms taken with an inverted light microscope showed predominately living (green) and fewer dead (red) stained bacteria cells. These images were taken at 60× magnification.

Figure 4

Log₁₀ CFU per mm² of S. aureus biofilm on beads grown in eight reactor runs. Gray bars represent the average CFU per eight trials, and black circles denote the specific CFU of each bead quantified (n = 6 per trial). There was greater variability in bioburden between beads of the same reactor than averages of each trial.

Figure 5

Log₁₀ CFU per mm² of P. aeruginosa biofilm on beads grown in eight reactor runs. Gray bars represent the average CFU per eight trials, and black circles the specific CFU of each bead (n = 6 per trial). Variability of P. aeruginosa bioburden occurred both between trials and within substrates of the same trial.