Isolation of polymyxin B-susceptible mutants of Burkholderia pseudomallei and molecular characterization of genetic loci involved in polymyxin B resistance

Burkholderia pseudomallei is a gram-negative bacterium that causes the dise ase known as melioidosis, This pathogen is endemic to Southeast Asia and no rthern Australia and is particularly problematic in northeastern Thailand, It has been previously reported that B. pseudomallei is resistant to the ki lling action of cationic antimicrobial peptides, including human neutrophil peptide, protamine sulfate, poly-L-lysine, magainins, and polymyxins. Rece ntly, we have also found that the virulent clinical isolate B. pseudomallei 1026b is capable of replicating in media containing polymyxin B at concent rations of >100 mg/ml. In order to identify genetic loci that are associate d with this particular resistance phenotype, we employed a Tn5-OT182 mutage nesis system in coordination with a replica plating screen to isolate polym yxin B-susceptible mutants. Of the 17,000 Tn5-OT182 mutants screened via th is approach, five polymyxin B-susceptible mutants were obtained. Three of t hese mutants harbored Tn5-OT182 insertions within a genetic locus demonstra ting strong homology to the lytB gene present in other gram-negative bacter ia. Of the remaining two mutants, one contained a transposon insertion in a locus involved in lipopolysaccharide core biosynthesis (waaF), while the o ther contained an insertion in an open reading frame homologous to UDP-gluc ose dehydrogenase genes. Isogenic mutants were also constructed via allelic exchange and used in complementation analysis studies to further character ize the relative importance of each of the various genetic loci with respec t to the polymyxin B resistance phenotype exhibited by B, pseudomallei 1026 b.