Figure 8: Representative microphotographs of 48h-old biofilms exposed to CIP for 6h: A) untreated biofilm grown in ALI; B) and C) biofilms grown in ALI and treated with 50µg/mL and 1600µg/mL of CIP, respectively; D) untreated biofilm was grown in LLI; E) and F) biofilms grown in LLI and treated with 50µg/mL and 1600µg/mL of CIP, respectively.
Conventional methods to determine antibiotic efficacy are routinely performed under the standard liquid culture condition using the broth microdilution technique, and 96-well microtiter plates [28-31] and the standard protocol for measuring minimal inhibitory concentration (MIC), which required to inhibit growth or kill planktonic bacteria [32] and the minimal biofilm eradication concentration (MBEC) [33] were proposed. Several novel methods, such as the Calgary Biofilm Device [34] and microfluidic devices [35], were developed to improve the measurement of MBEC as well. However, the biofilms were mostly cultured by submerging in the liquid, and antibiotics can only be delivered through one approach (biofilm submerged in antibiotics). In our study, injecting the antibiotic solution into the basal chamber and interacting with biofilm from the substrate attaching side illustrates an alternative way to mimic the antimicrobial mechanism that the drug being administrated through oral or parenteral routes and taken to the biofilm site through the systemic circulation. The MBEC of 3-day PAO1 biofilm using CIP reported by Wu et. al. using the microtiter plate method was 64μg/mL [31], while our results show that significantly higher CIP concentration is needed to achieve a 50% decrease in the biofilm. The differences across models highlight the importance of having a model that can closely mimic the environmental conditions where these communities persist. Thus, recognizing the differences in biofilms thrived on various interfaces could shed light on designing more effective and efficient control methods.
Conclusions
This paper introduces a novel reusable dual-chamber microreactor to provide multiple interfaces for biofilm culture and test. The reusable assembling technique provides greater flexibility to grow biofilms and to screen the effects of different interfaces on biofilms, both quantitatively and qualitatively. Protocols for culturing biofilm on ALI and LLI in static condition and following test methods are feasible and repeatable. PAO1 biofilms grow on ALI and LLI show differences in morphology and resistance to physical interruptions. Our dual chamber device also enables efficacy testing of antibiofilm strategies in an environment that closely mimics in vivo biofilm growth conditions, producing more accurate test results. 48h-old PAO1 biofilms cultured on ALI and LLI show high resistance when antibiotic delivered from the substrate side.