An adaptable microreactor to investigate the influence of interfaces on Pseudomonas aeruginosa biofilm growth

Abstract

Biofilms are ubiquitous and notoriously difficult to eradicate and control, complicating human infections and industrial and agricultural biofouling. However, most of the study had used the biofilm model that attached to solid surface and developed in liquid submerged environments which generally have neglected the impact of interfaces. In our study, a reusable dual-chamber microreactor with interchangeable porous membranes was developed to establish multiple growth interfaces for biofilm culture and test. Protocol for culturing Pseudomonas aeruginosa (PAO1) on the air–liquid interface (ALI) and liquid–liquid interface (LLI) under static environmental conditions for 48 h was optimized using this novel device. This study shows that LLI model biofilms are more susceptible to physical disruption compared to ALI model biofilm. SEM images revealed a unique “dome-shaped” microcolonies morphological feature, which is more distinct on ALI biofilms than LLI. Furthermore, the study showed that ALI and LLI biofilms produced a similar amount of extracellular polymeric substances (EPS). As differences in biofilm structure and properties may lead to different outcomes when using the same eradication approaches, the antimicrobial effect of an antibiotic, ciprofloxacin (CIP), was chosen to test the susceptibility of a 48-h-old P. aeruginosa biofilms grown on ALI and LLI. Our results show that the minimum biofilm eradication concentration (MBEC) of 6-h CIP exposure for ALI and LLI biofilms is significantly different, which are 400 μg/mL and 200 μg/mL, respectively. These results highlight the importance of growth interface when developing more targeted biofilm management strategies, and our novel device provides a promising tool that enables manipulation of realistic biofilm growth. Key points • A novel dual-chamber microreactor device that enables the establishment of different interfaces for biofilm culture has been developed. • ALI model biofilms and LLI model biofilms show differences in resistance to physical disruption and antibiotic susceptibility.

Understanding the effects of aerodynamic and hydrodynamic shear forces on Pseudomonas aeruginosa biofilm growth

“…The total viable cell number determined by the sum of the bacteria detached from the membrane during rinsing (rinsed) and sonication (sonicated) is represented in Figure 4. Compared to the biofilms cultured under static flow (nutrient-limited) conditions (Ye et al, 2022), the CFUs number with continuous nutrient supply was higher by at least 10-fold in both biofilms formed on ALI or LLI.…”

“…Using a dual‐channel microfluidic device, biofilms were cultured under static flow or dynamic flow conditions and replicated a nutrient limiting environment (Ye et al, 2022) or an environment where nutrients are supplied continuously. As demonstrated in this study, the distinct difference in biofilm's physical properties obtained from these two different culture methods (static vs. dynamic flow) shows that nutrient supply plays an important role in biofilm growth.…”

“…All the statistical analysis was performed using Graphpad Prism 7.0. Data of static culture are obtained from Ye et al (2022).…”

“…The top channel serves as the biofilm culture channel and has two inlets, one for bacteria inoculation and the other for air or liquid flow to study the effects of dynamic flow conditions on biofilm growth. The PDMS chips were cast using 3-D printing microfluidic techniques, and fabrication details have been illustrated in previous work (Ye et al, 2022).…”

“…This study aimed to understand the effects of environmental factors specifically, nutrient availability, mechanical shear stress and interface type on the development of biofilm. A dynamic platform was set up in the ALI and LLI configuration using a dual-channel microfluidic device (Ye et al, 2022) for this purpose, with the dynamic system providing a controllable air or liquid flow rate for biofilm culture. We hypothesized that biofilm properties, as determined by the viable cell number, spatial distribution, resistance to physical disruption, permeability, and antibiotic susceptibility to a model antibiotic (ciprofloxacin-CIP) was significantly different between biofilms cultured in ALI and LLI.…”