In vitro and In vivo characterization of anti-biofilm activity of LpxC inhibitor alone, and in combination with different classes of antimicrobials

• Main Author: Tan, Jun Hou
• Other Authors: Michael Givskov
• Format: Theses and Dissertations
• Language: English
• Published: 2018
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/73866
• Institution: Nanyang Technological University

Biofilms are multicellular bacterial communities that are formed on a surface and encased in extracellular polymeric substance (EPS) made up of eDNA, proteins and exopolysacchrides. Due to their distinct - instrinic, acquired and adaptive - antimicrobial resistance, biofilm formation is one of the main common causes of chronic infections and is becoming increasingly difficult to treat with existing antibiotics. Without anti-biofilm therapy that targets biofilm specifically, combination antimicrobial therapy remains one of the most effective strategies to eradicate biofilms. CHIR-090 is a new class of antipseudomonial agent that inhibits LpxC enzyme, which in turn disrupts the formation of the lipopolysaccharides (LPS) anchoring protein, Lipid A. While CHIR-090 has been extensively characterised against planktonic bacteria, its anti-biofilm activity has yet been studied. Understanding the stress responses and possible resistance mechanisms that arise during CHIR-090 treatment can provide useful information to help prolong the therapeutic longevity of the LpxC inhibiting compounds and the data could be used for the selection criteria for antimicrobial combinations. In this study, we applied RNA-sequencing (RNA-seq) analysis and experimental adaptive evolution assays to study the stress responses and development of resistance mechanisms by bacterial pathogen during CHIR-090 treatment. We also evaluated the anti-biofilm activity of CHIR-090 alone, and in combination of other classes of antimicrobials using in vitro and in vivo methods such as antibiotic susceptibility testing, flow cell biofilms model and mouse implant biofilm model. Our transcriptomic analyses showed upregulation of three gene clusters associated with CHIR-090 treatment: (i) faoAB operon with its psrA transcription regulator involved in fatty acid synthesis, (ii) mexCD-OrpJ that encodes for RND efflux pump and (iii) phoPQ-oprH, a two component transcription regulator. Meanwhile, experimental adaptive evolution assays identified six mutated genes from CHIR-090 resistant variants after 21 days of cultivation of P. aeruginosa strain in sub-inhibitory concentrations of CHIR-090. These mutated genes are classified into three categories based on their functions: (i) efflux pump (mexR), (ii) cell membrane and fatty acid synthesis (murB, pyrG and fabF1) and (iii) motility (morA and fliF). In vitro and in vivo evaluation of anti-biofilm activity of CHIR-090 demonstrated that the compound targeted the metabolically active subpopulation of P. aeruginosa biofilms, but administrated alone was ineffective in eradicating the entire biofilms. The combination of CHIR-090 and colistin displayed synergistic efficacy against colistin-susceptible and non-susceptible P. aeruginosa biofilms, while that combination of CHIR-090 and tobramycin was also synergistic against P. aeruginosa biofilms, albeit less effective than the CHIR-090-colistin combination. In conclusion, this study provided evidence for the stress response and resistance mechanisms that P. aeruginosa developed during CHIR-090 treatment; it also demonstrated the therapeutic potential of CHIR-090 in combination with other antimicrobial agents against P. aeruginosa biofilms.