The goal was to investigate the mechanisms of colistin resistance and heteroresistance in Pseudomonas aeruginosa clinical isolates. Colistin resistance was determined by the broth microdilution method. Colistin heteroresistance was evaluated by population analysis profiling. Time-kill assays were also conducted. PCR sequencing was performed to detect the resistance genes among (hetero)resistant isolates, and quantitative real-time PCR assays were performed to determine their expression levels. Pulsed-field gel electrophoresis and multilocus sequence typing were performed. Lipid A characteristics were determined via matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS). Two resistant isolates and 9 heteroresistant isolates were selected in this study. Substitutions in PmrB were detected in 2 resistant isolates. Among heteroresistant isolates, 8 of 9 heteroresistant isolates had nonsynonymous PmrB substitutions, and 2 isolates, including 1 with a PmrB substitution, had PhoQ alterations. Correspondingly, the expression levels of pmrA or phoP were upregulated in PmrB- or PhoQ-substituted isolates. One isolate also found alterations in ParRS and CprRS. The transcript levels of the pmrH gene were observed to increase across all investigated isolates. MALDI-TOF MS showed additional 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties in lipid A profiles in (hetero)resistant isolates. In conclusion, both colistin resistance and heteroresistance in P. aeruginosa in this study mainly involved alterations of the PmrAB regulatory system. There were strong associations between mutations in specific genetic loci for lipid A synthesis and regulation of modifications to lipid A. The transition of colistin heteroresistance to resistance should be addressed in future clinical surveillance.
Keywords: Pseudomonas aeruginosa; colistin; heteroresistance; lipid A modification; molecular mechanisms; resistance.
Copyright © 2019 American Society for Microbiology.