Reason: Access restricted by the author. A copy can be requested for private research and study by contacting your institution's library service. This copy cannot be republished
Investigation of the surface of colistin susceptible and resistant Acinetobacter baumannii
thesisposted on 09.02.2017, 05:04 authored by Soon, Rachel Li-Xia
Colistin, a cationic amphipathic polymyxin antibiotic, has been revived as a last-line therapy for Gram-negative multidrug-resistant infections. Colistin heteroresistant and -resistant Acinetobacter baumannii have prompted fears that these infections may become untreatable. The proposed ‘self-promoted uptake’ mechanism of colistin action suggests that electrostatic and hydrophobic interactions with lipopolysaccharide (LPS) facilitate permeation through the complex Gram-negative outer membrane. Proteomic and genomic characterization of colistin-resistant A. baumannii has revealed structural outer-membrane alterations, including the absence of LPS arising from mutations in lipid A biosynthesis genes (lpxA, lpxC or lpxD). To broaden our understanding of colistin action and resistance, this thesis examined the surface properties of paired colistin-susceptible and resistant A. baumannii. These properties include morphology, topography, surface charge, surface hydrophobicity, mechanical stiffness and adhesive properties, which are potentially associated with the interaction with colistin. Atomic force microscopy (AFM) studies were conducted on dried cells and live hydrated cells. Rod-shaped colistin-susceptible cells were differentiated from spherical colistin-resistant cells; the latter frequently appeared in chains or clumps, and extracellular appendages were reduced in number and length compared to susceptible cells. Cellular elongation was revealed at stationary phase for both phenotypes. Protrusions observed on colistin-susceptible cells were suggested to represent LPS bundles. The rough featureless topography of colistin-resistant cells corresponded with the loss of LPS. Surface disruption of both phenotypes illustrated in AFM images captured in air following colistin treatment (4 µg/mL) was likely accentuated by dehydration, as disruption of colistin-treated cells in liquid was not evident. High colistin concentrations (32 µg/mL) resulted in cellular aggregation, and imparted a smoothening effect to colistin-susceptible and resistant cells. Electrostatic forces mediate the initial interaction between cationic colistin and anionic lipid-A phosphoresters. Zeta potentials of bacterial cells were thus determined as a measure of surface charge. The less electronegative charge detected for colistin-resistant versus susceptible cells at mid-logarithmic phase may theoretically impede the electrostatic binding component of colistin activity. Opposing growth-phase trends were detected at stationary phase whereby colistin-susceptible cells exhibited a lower electronegative charge, while colistin-resistant cells exhibited a higher electronegative charge, in comparison to mid-logarithmic phase cells. Neutralization of the surface charge of both phenotypes by colistin and PBN (a derivative of polymyxin B (PmB) lacking the terminal N-fatty-acyl chain) occurred in a concentration-dependent manner, emphasizing the importance of the cationic polymyxin charge for antimicrobial activity. Hydrophobic interactions between colistin and LPS are proposed to mediate outer-membrane disruption. Consequently, the cell surface hydrophobicity (CSH) of A. baumannii was determined using contact angles, which describe the tendency of a water droplet to spread across a bacterial lawn on a filter. AFM images illustrated that highly porous colistin-resistant lawns were formed compared to lawns of colistin-susceptible cells. For both phenotypes, a reduction in contact angle over time paralleled a reduction in droplet volume, suggesting that results were influenced by droplet leakage through the porous bacterial lawn. Contact angles captured 0.66 sec after droplet deposition revealed significantly lower CSH for colistin-resistant versus susceptible cells at both growth phases. At stationary phase and after colistin treatment, the CSH of both phenotypes increased, which is consistent with the surface charge alterations determined by zeta potential measurements. Cellular surface modifications following colistin treatment highlighted the ability of colistin to bind to both phenotypes, creating the impetus to evaluate the interaction between polymyxins and colistin-susceptible versus -resistant A. baumannii. Investigations were also conducted using LPS from various Gram-negative strains. Structural and mechanistic deficiencies of the fluorescent dansyl-polymyxin B (DPmB) assay to determine binding affinity were identified. Polymyxin-LPS affinity was thus quantified using an improved mono-substituted fluorescent probe, [dansyl-Lys]1polymyxinB3 (DPmB3), which exhibited a comparable affinity to colistin and PmB for LPS. Thermodynamic characterization of the polymyxin-LPS interaction using isothermal titration calorimetry (ITC) revealed enthalpically driven binding of PmB and DPmB3 to LPS, attributed to electrostatic interactions. Hydrophobic association of the [dansyl-Lys]1 substituent with LPS contributed an unfavourable entropic response to the DPmB3-LPS interaction. Attempts to characterize the polymyxin interaction with colistin-susceptible versus resistant A. baumannii whole cells were unsuccessful using both the DPmB3 fluorescence assay and ITC. Finally, the force-sensing ability of the AFM was utilized to determine bacterial mechanical and adhesive properties. Measurement of bacterial spring constants indicated that colistin-susceptible cells were stiffer than LPS-deficient colistin-resistant cells at both growth phases. Multiple large adhesive peaks were noted from force curves captured on colistin-susceptible cells; these were not observed for colistin-resistant cells, which corresponds with the reduced expression of LPS and surface appendages. Colistin treatment increased cellular rigidity and caused a marked reduction in adhesion events for both phenotypes. This thesis was the first to highlight considerable alterations to the surface properties of colistin-susceptible and resistant A. baumannii as a function of growth phase and colistin treatment. These properties reflect changes to the outer-membrane structure that potentially influence the crucial binding interaction of colistin, and may contribute to the development of colistin resistance. Important insights into the mechanisms of colistin action and resistance in this problematic pathogen have been provided.