Abstract
BackgroundBurkholderia cenocepacia is a Gram-negative opportunistic pathogen displaying high resistance to antimicrobial peptides and polymyxins. We identified mechanisms of resistance by analyzing transcriptional changes to polymyxin B treatment in three isogenic B. cenocepacia strains with diverse polymyxin B resistance phenotypes: the polymyxin B-resistant parental strain K56-2, a polymyxin B-sensitive K56-2 mutant strain with heptoseless lipopolysaccharide (LPS) (RSF34), and a derivative of RSF34 (RSF34 4000B) isolated through multiple rounds of selection in polymyxin B that despite having a heptoseless LPS is highly polymyxin B-resistant.ResultsA heptoseless LPS mutant of B. cenocepacia was passaged through multiple rounds of selection to regain high levels of polymyxin B-resistance. This process resulted in various phenotypic changes in the isolate that could contribute to polymyxin B resistance and are consistent with LPS-independent changes in the outer membrane. The transcriptional response of three B. cenocepacia strains to subinhibitory concentrations of polymyxin B was analyzed using microarray analysis and validated by quantitative Real Time-PCR. There were numerous baseline changes in expression between the three strains in the absence of polymyxin B. In both K56-2 and RSF34, similar transcriptional changes upon treatment with polymyxin B were found and included upregulation of various genes that may be involved in polymyxin B resistance and downregulation of genes required for the synthesis and operation of flagella. This last result was validated phenotypically as both swimming and swarming motility were impaired in the presence of polymyxin B. RSF34 4000B had altered the expression in a larger number of genes upon treatment with polymyxin B than either K56-2 or RSF34, but the relative fold-changes in expression were lower.ConclusionsIt is possible to generate polymyxin B-resistant isolates from polymyxin B-sensitive mutant strains of B. cenocepacia, likely due to the multifactorial nature of polymyxin B resistance of this bacterium. Microarray analysis showed that B. cenocepacia mounts multiple transcriptional responses following exposure to polymyxin B. Polymyxin B-regulated genes identified in this study may be required for polymyxin B resistance, which must be tested experimentally. Exposure to polymyxin B also decreases expression of flagellar genes resulting in reduced swimming and swarming motility.
Highlights
Burkholderia cenocepacia is a Gram-negative opportunistic pathogen displaying high resistance to antimicrobial peptides and polymyxins
Strain RSF34 200E was isolated from a colony of RSF34 25A that grew on Luria Broth (LB) with 200 μg/ml polymyxin B, strain RSF34 1000D was isolated as a colony of RSF34 200E that grew on LB containing l000 μg/ml polymyxin B, and strain RSF34 4000B was isolated as a colony of RSF34 1000D that grew on LB containing 4000 μg/ml polymyxin B (Table 1)
Our data demonstrate that major transcriptional changes made by B. cenocepacia upon treatment with polymxyin B include downregulation of genes required for the synthesis and operation of the flagella and upregulation of a set of genes encoding proteins with diverse predicted functions
Summary
Burkholderia cenocepacia is a Gram-negative opportunistic pathogen displaying high resistance to antimicrobial peptides and polymyxins. We have recently proposed a two-tier model of antimicrobial peptide resistance in B. cenocepacia [14] with the first and most significant tier consisting of the complete lipopolysaccharide (LPS) core oligosaccharide (OS) [9,15] and the lipid A and core OS aminoarabinose residues that are essential for the viability of B. cenocepacia [16,17]. We cultured a polymyxin B-sensitive B. cenocepacia heptoseless LPS mutant, RSF34 [18], in a way that allowed for the isolation of clones with increased resistance to polymyxin B to identify other mechanisms of antimicrobial peptide resistance in this highly resistant organism. We established the baseline differences in transcription between the strains in the absence of polymyxin B challenge and identified genes transcriptionally regulated by the presence of polymyxin B in the three strains
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