Studying bacterial population dynamics during biofilm dispersal and efficacy of Juglone as antibiofilm agent in a microfluidics device

Abstract

Pseudomonas aeruginosa (P. aeruginosa), a Gram-negative pathogen, often causes infection in patients with impaired host immune systems. P. aeruginosa can adhere to surfaces and form biofilms to survive as a community. P. aeruginosa infections are difficult to treat not only because their ability to develop resistance to antibiotics but also because its biofilm blocks the effective treatment of infections. Over 80% of chronic infections are caused by biofilms. Biofilms are composed of aggregates of bacteria and their secretion of extracellular polymers (EPS). Biofilm infection is challenging to treat because biofilm matrix protects the bacteria from being killed by antibiotics. It is the primary contributor to most persistent infections and can help bacteria to develop antibiotic resistance. The life cycle of a biofilm is divided into four steps, bacterial attachment, colonization and secretion of extracellular matrix, biofilm maturation and biofilm dispersal, the bacteria cells resume the planktonic phenotype that can reattach to a fresh surface and begin a new life cycle. There are two important systems that mediate the life cycle of biofilms, the c-di-GMP second messenger system and the Quorum sensing (QS) system. QS system enables bacteria to communicate, which is important for forming biofilms. And the upregulation and decrease of intracellular c-di-GMP levels will regulate the formation and dispersal of biofilms. Since the biofilm life cycle ends with biofilm dispersal, biofilm dispersal is believed to be an effective method of eliminating biofilm. There are two biofilm dispersal methods proposed for clinical treatment, namely chemically-dominated biofilm dispersal (CID), and enzymatic-dominated disassembly (EDA). CID induces active biofilm dispersal using chemical stimuli, such as nitric oxide (NO), while EDA targets the biofilm matrix for enzymatic degradation of biofilm matrix and direct destruction of the biofilm, such as glycoside hydrolases. However, there is currently limited knowledge about the differences between CID biofilm dispersal or EDA disassembly. Moreover, there is a lack of 3D biofilm models that are appropriate for studying and observing biofilm dispersal and recolonization in fresh areas. Therefore, to explore the mechanisms and population dynamics of biofilm dispersal, it is necessary to design a new model to study the dispersal and recolonization of biofilms, which can also be used to evaluate the differences between CID-dominated biofilm dispersal and EDA-dominated biofilm disassembly. Microfluidics is used to develop models for studying biofilms as a low-cost, high-throughput research tool that allows direct real-time observation of biofilms. And because the liquid environment in microfluidics can be precisely controlled, it is possible to study the effects of environmental changes on biofilms in the microfluidic model under well-defined conditions. Therefore, there are three aims of this study. [1] A biofilm-dispersal-then-recolonization (BDR) microfluidic model needs to be developed that can be used to study biofilm dispersal and recolonization. [2] The difference between biofilm dispersal and recolonization between CID biofilm dispersal and EDA biofilm degradation will be analyzed and evaluated in the BDR model. [3] The synergistic use of a new QS inhibitor Juglone with antibiotics to eliminate biofilms will be evaluated in the BDR model. The BDR device developed in this study was divided into two chambers, a primary chamber for observing biofilm dispersal and a secondary chamber for observing enbacteria recolonization, which is controlled by a valve between the two chambers. Then the CID-dominated biofilm dispersal experiments and EDA-dominated biofilm disassembly experiments were performed in the BDR device. The results showed that CID dispersed the biofilm more rapidly than EDA. In addition, there is also a difference in the ability of bacteria released by CID and EDA to recolonize. The released bacteria from CID could not immediately recolonize, in contrast, the bacteria aggregates released by EDA are able to recolonize within 2 hrs. Furthermore, human lung fibroblasts (HLF) were used as an in vitro model in this study. It is a cell that plays a key role in the lung's response to infection. The Caenorhabditis elegans (C. elegans) was also used as a model of infection in this study. It is particularly useful in infection research as it shares many conserved genes and signaling pathways with humans. Both HLF and C. elegans are useful infection model systems, providing a platform to explore host-pathogen interactions and allowing treatment of various therapeutics to be evaluated. In this study, it was found that the bacteria released by CID were less cytotoxic to the 3D-HLF spheroids and C. elegans infection models compared to EDA because they were unable to recolonize within 2 hrs. Juglone is a natural compound that binds to the residues of PqsR receptor to interfere with the pqs QS system. The efficacy of Juglone and colistin in the synergistic treatment of P. aeruginosa was evaluated. The results showed that synergistic treatment with Juglone and colistin significantly reduced biofilm and prevented biofilm cell recolonization. Moreover, through the combination of Juglone with colistin, biofilms could be effectively eliminated, and inflammatory responses reduced in a Medaka fish in vivo model. This project contributes to the understanding of biofilm dispersal processes and microbial responses to changes in the environment. The BDR microfluidic device was developed in this project that allows for the study of biofilm dispersal and recolonization in a controlled and real-time manner. The device offers advantages such as rapid biofilm formation, evaluation of drug concentrations and responses, and the inclusion of cell cultures and C. elegans within the device for studying host responses. The findings of this project have significant implications for the development of more effective therapeutic strategies against biofilm-mediated infections in both clinical and environmental settings. Additionally, the project also identifies Juglone as a QS inhibitor that can inhibit biofilm formation and reduce bacteria recolonization. The study shows that a combinatorial therapy of Juglone and colistin can effectively eliminate biofilms and prevent the recolonization of bacteria in both in vitro and in vivo infection models.