title: Developing New Strategies for Treatment of Pseudomonas Aeruginosa Infection
Creators: name: Tran Thi, Minh Tam
Publisher: name: Griffith University
Year: 2022
Subjects: subject: Pseudomonas aeruginosa
subject: antibiotic-resistant biofilms
subject: biofilm dispersal assay
subject: Immunocompromised Populations

description: Pseudomonas aeruginosa (P. aeruginosa) is a pathogenic bacterium causing devastating acute and chronic infections among populations with compromised immune systems. Its highly notorious persistence in clinical settings is attributed to the ability to form antibiotic-resistant biofilms. Biofilm is an architecture built mostly by autogenic extracellular polymeric substances that functions as a scaffold to encase the bacteria together on surfaces, protect them from environmental stresses, evade host immune system, and thereby facilitate long-term persistence. Current antibiotics cannot penetrate the biofilm matrix as they were initially designed to modulate mainly the growth of planktonic bacteria. Therefore, there is a dire need to search for novel agents that can interfere with biofilm development and/or eradicate established biofilms. In this study, a biofilm dispersal assay in 384-well format was developed and optimized to identify anti-P. aeruginosa biofilm molecules from three compound collections. The first library, previously shown to inhibit planktonic P. aeruginosa, was screened against pre-existing biofilms of wild-type P. aeruginosa PAO1 and its isogenic mutant - mucoid variant PDO300, which were established in artificial sputum medium mimicking lung physiology of cystic fibrosis (CF) patients. Active fractions were subjected to bioassay-guided purification to confirm the activity and identify active compounds within the active fractions. The second library consists of NatureBank compounds which were evaluated against PAO1. The third library, David Open Access library, comprises known natural products and their analogues which have therapeutic effects on various diseases such as bacterial infections, viral infections, cancer, malaria, and neurodegenerative disorders. The library was assessed against established PAO1 biofilms. In addition, a set of three natural products, ianthelliformisamines A-C, previously identified as antibiotic adjuvants toward planktonic PAO1, was examined for the first time against PAO1 and its isogenic mutants including PAO1ΔpslA, PAO1ΔpelF, PDO300 and PDO300Δalg8, to determine their impacts on biofilms as well as biofilm matrix components (alginate, Psl and Pel). A cytotoxicity study against the HEK-293 cell line was carried out along with an investigation of the mechanism of action. Fifteen confirmed hits from the David Open Access library were identified as promising P. aeruginosa biofilm dispersers without adverse effects on the planktonic growth. These potential candidates will be subjected to further investigation. Ianthelliformisamines A-C inhibited biofilm formation of P. aeruginosa and interfered with different biofilm matrix components. Ianthelliformisamines A and B potentiated ciprofloxacin activity, corroborated by 3 - 4-fold reduction in MIC of antibiotic ciprofloxacin. Ianthelliformisamine C alone targeted the viability of suspended cells and biofilms of the studied strains at various levels. Interestingly, this molecule solely impeded both free-living and biofilm populations of mucoid variant PDO300 which was engineered to imitate clinical isolates from CF patient lungs. It was reported that PDO300 derived mucoid biofilms were greatly refractory to antibiotic tobramycin up to 1000 times compared to nonmucoid biofilms. These molecules were not toxic to the HEK-293 cell line. Although further research is required to elucidate the mechanisms of action of these compounds, the current data suggested that ianthelliformisamine C may be a potential efflux pump inhibitor. Overall, ianthelliformisamines A-C could be useful starting points for future anti- P. aeruginosa biofilm therapeutics.