An in-vitro evaluation of C-di-GMP mediated biofilm dispersal as a potential strategy to restore antimicrobial efficacy

• Main Author: Roizman, Dan
• Other Authors: Yang Liang
• Format: Theses and Dissertations
• Language: English
• Published: 2018
• Subjects: DRNTU::Science::Biological sciences::Microbiology::Drug Resistance
• Online Access: https://hdl.handle.net/10356/87655; http://hdl.handle.net/10220/46811
• Institution: Nanyang Technological University

The World Health Organization (WHO) has deemed antimicrobial resistance (AMR) in bacteria to be a critical threat to society (World Health Organization 2016, World Health Organization 2017). AMR and specifically multi-drug resistant (MDR) bacteria represent an increasingly serious public health concern worldwide. The need for novel therapeutic approaches to prevent and/or eradicate microbial infections is urgent. AMR can evolve due to mutations that reduce antibiotic susceptibility or due to activation of various innate mechanisms of tolerance, for example, biofilm formation. Bacteria exhibit a complex life-cycle, whereby they can switch from a planktonic, free-living form, to a densely packed biofilm form. Biofilms are an aggregated, sessile, often surface attached form of bacterial mode of growth. Biofilms provide complex 3D structures to bacterial communities, allowing them to interact with each other and respond to changes in their environment differently than their planktonic counterparts (Costerton, Geesey et al. 1978). 60-80% of infections are biofilm-related, once biofilms attach and mature bacteria can exhibit 10 to 1000 times less susceptibility to antimicrobials (Høiby, Bjarnsholt et al. 2010). The use of dispersal agents has been suggested as a potential treatment strategy against microbial biofilms (Banin, Brady et al. 2006, Frederiksen, Pressler et al. 2006, Landini, Antoniani et al. 2010, Barraud, J. Kelso et al. 2015). This strategy consists of manipulating biofilm dispersal to induce changes in biofilm matrix and metabolic states of biofilm cells. These changes are believed to enhance antimicrobial efficacy, increasing antimicrobial killing efficiency, shortening duration of treatment and reducing the risk of emergence of resistance. Investigating the effectiveness of biofilm dispersal as part of an antimicrobial treatment strategy is challenging, and the consequences and the underlying mechanisms of such strategy remain unclear. So far, no dispersal agents have yet been used extensively in a clinical setting (Fleming and Rumbaugh 2017). The present research aims at providing knowledge about how c-di-GMP mediated biofilm dispersal will impact antimicrobial synergy treatment against bacterial biofilms and whether it will result in the emergence of resistant/tolerant variant populations. To achieve these objectives, a strain of Pseudomonas aeruginosa, PAO1/pBAD-yhjH which carries an inducible phosphodiesterase (PDE) enzyme allowing for controlled biofilm dispersal, was utilized (Chua, Tan et al. 2013). A strain PAO1/pJN105 carrying an empty vector plasmid was used as a control. Using antimicrobial agents, alone or in combination, under static in-vitro conditions, c-di-GMP mediated biofilm dispersal was shown to enhance drug synergy against P. aeruginosa biofilms (Chapter 2), without increasing the risk of releasing resistant/tolerant variants (Chapter 3). The potential of biofilm dispersal strategy was also assessed against an evolved AMR strain (Chapter 4). A colistin-resistant derivative of PAO1/pBAD-yhjH was generated and characterized: Significant differences in morphology, motility, and virulence factor production were identified between the parent PAO1/pBAD-yhjH strain and its colistin resistant derivative. Whole genome sequencing and analysis revealed point mutations in phoQ and lpxC, two genes involved in the biosynthesis of lipid-A located in the bacterial membranes and the biological target of colistin. These mutations resulted in upregulated expression of the arn operon which is known to lead to the modification of Lipid-A. The colistin-resistant derivative also showed enhanced susceptibility to carbapenems and glycopeptides, suggesting that the development of colistin resistance resulted in a trade-off in sensitivity to other classes of antimicrobials. This was further explored by testing biofilms for sensitivity to a range of antimicrobial combinations. It was observed that colistin plus vancomycin demonstrated a synergistic effect only against the dispersed biofilms of the colistin resistant strain. The results of this work indicate the potential of c-di-GMP mediated biofilm dispersal as a potential strategy for efficient treatment of biofilm-associated infections.