UBC Theses and Dissertations
Aminoglycoside susceptibility and acquired resistance in Burkholderia vietnamiensis Jassem, Agatha Natalie
The Burkholderia cepacia complex (BCC) group of Gram-negative bacteria are highly virulent, opportunistic pathogens in cystic fibrosis (CF) patients and other immunocompromised individuals. It is the current dogma that all species of the complex are highly and intrinsically resistant to polycationic antimicrobials, including aminoglycosides, and that this resistance is due to unusual characteristics of the lipopolysaccharide (LPS). Cationic agents enter Gram-negative bacteria through LPS-mediated uptake, relying on anionic lipid A binding sites. Here we observed that environmental and clinical isolates of B. vietnamiensis were more often susceptible to aminoglycosides than those of other BCC species, but were not inhibited by other cationic agents (natural and synthetic cationic antimicrobial peptides, polymyxin B). Furthermore, B. vietnamiensis strains acquired aminoglycoside resistance during chronic CF infection, and in vitro under tobramycin, azithromycin, and hydrogen peroxide pressure. B. vietnamiensis strains also displayed enhanced catalase activity and became less mucoid. Gentamicin and tobramycin time-kill assays revealed drug concentrations up to 8 × the minimum inhibitory concentration were unable to kill a susceptible B. vietnamiensis CF isolate. Aminoglycoside resistant B. vietnamiensis CF isolates accumulated significantly less [³H]gentamicin than susceptible isolates. Aminoglycoside resistance, however, was not correlated with LPS chemotype, and mass spectrometry revealed the presence of lipid A-associated 4-amino-4-deoxy-L-arabinose moieties, residues that neutralize anionic lipid A binding sites, in aminoglycoside-susceptible and -resistant B. vietnamiensis isolates. Furthermore, permeability to the fluorescent hydrophobic probe 1-N-phenylnapthylamine was not enhanced following incubation with gentamicin or tobramycin in any B. vietnamiensis isolates. Aminoglycoside-resistant B. vietnamiensis isolates overexpressed a putative resistance-nodulation-division (RND) efflux system transporter gene, amrB. After serial exposure to tobramycin and azithromycin, but not hydrogen peroxide, amrB expression was induced in an aminoglycoside-susceptible B. vietnamiensis CF isolate. Moreover, inhibition of the putative efflux system enhanced B. vietnamiensis susceptibility to aminoglycosides. These data suggest that active efflux via a RND efflux system, not LPS modification, impairs aminoglycoside accumulation in clinical B. vietnamiensis strains that have acquired aminoglycoside resistance, and in those exposed to tobramycin and azithromycin, but not hydrogen peroxide, in vitro. These new insights may help in the design of improved therapeutic regimens against Burkholderia species.
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