Modular 3D-Printed Peg Biofilm Device for Flexible Setup of Surface-Related Biofilm Studies

Abstract

Medical device-related biofilms are a major cause of hospital-acquired infections, especially chronic infections. Numerous diverse models to study surface-associated biofilms have been developed; however, their usability varies. Often, a simple method is desired without sacrificing throughput and biological relevance. Here, we present an in-house developed 3D-printed device (FlexiPeg) for biofilm growth, conceptually similar to the Calgary Biofilm device but aimed at increasing ease of use and versatility. Our device is modular with the lid and pegs as separate units, enabling flexible assembly with up- or down-scaling depending on the aims of the study. It also allows easy handling of individual pegs, especially when disruption of biofilm populations is needed for downstream analysis. The pegs can be printed in, or coated with, different materials to create surfaces relevant to the study of interest. We experimentally validated the use of the device by exploring the biofilms formed by clinical strains of Escherichia coli and Klebsiella pneumoniae, commonly associated with device-related infections. The biofilms were characterized by viable cell counts, biomass staining, and scanning electron microscopy (SEM) imaging. We evaluated the effects of different additive manufacturing technologies, 3D printing resins, and coatings with, for example, silicone, to mimic a medical device surface. The biofilms formed on our custom-made pegs could be clearly distinguished based on species or strain across all performed assays, and they corresponded well with observations made in other models and clinical settings, for example, on urinary catheters. Overall, our biofilm device is a robust, easy-to-use, and relevant assay, suitable for a wide range of applications in surface-associated biofilm studies, including materials testing, screening for biofilm formation capacity, and antibiotic susceptibility testing.

Antibiotic Minimal Selective Concentrations and Fitness Costs during Biofilm and Planktonic Growth

“…During biofilm growth ( Fig. 1A ), ratios were obtained after 8 generations of growth on FlexiPegs, an in-house-modified Calgary device for biofilm growth ( 37 ). The mixes of susceptible and resistant bacteria were allowed to attach to the FlexiPegs for 3 h in the absence of antibiotics, and antibiotics were then added.…”

“…In this study, we used the biofilm-forming E. coli CFT073 strain ( 37 – 39 ) to study the selection of resistant mutants at subinhibitory concentrations of antibiotics under planktonic and biofilm growth conditions. The antibiotics included were streptomycin and rifampicin together with antibiotics that are clinically relevant for the treatment of E. coli infections, such as trimethoprim, nitrofurantoin, and fosfomycin.…”

“…To grow the biofilm, an in-house-developed biofilm growth system, FlexiPeg, was used ( 37 ). The FlexiPegs were printed using high-temperature resin (High Temp; Formlabs) at U-PRINT, Uppsala University’s three-dimensional (3D) printing facility at the Disciplinary Domain of Medicine and Pharmacy, using Formlabs form 3 (low-force stereolithography) 3D printers.…”

A Microfluidic Chip for Studies of the Dynamics of Antibiotic Resistance Selection in Bacterial Biofilms

“…Closed-systems are commonly used owing their simplicity and ease of use. Single- or mixed-species bacteria are incubated together in microtiter well plates and biofilm formation occur for a certain time under static conditions ( Zaborskytė et al., 2022 ). The system does not have constant supply of fresh nutrients essential for bacterial growth, leading to consumption of nutrients and accumulation of bacterial metabolic waste products.…”

“…Typically a flow of culture medium, with or without bacteria, is generated using a peristaltic or syringe pump to for example simulate the conditions during urinary tract infections ( Hong et al., 2012 ; Hol and Dekker, 2014 ; Azeredo et al., 2017 ; Yan et al., 2017 ; Pousti et al., 2019 ; Heyman et al., 2020 ). Mixed-systems combine the ease-of-use of close-systems and address the limitation of nutrient supply by a nutrient replenishment and waste discarding step at specific intervals during biofilm cultivation and propagation ( Poltak and Cooper, 2011 ; Zaborskytė et al., 2022 ).…”