Resistance to colistin in Acinetobacter baumannii associated with mutations in the PmrAB two-component system

Abstract

The mechanism of colistin resistance (Col(r)) in Acinetobacter baumannii was studied by selecting in vitro Col(r) derivatives of the multidrug-resistant A. baumannii isolate AB0057 and the drug-susceptible strain ATCC 17978, using escalating concentrations of colistin in liquid culture. DNA sequencing identified mutations in genes encoding the two-component system proteins PmrA and/or PmrB in each strain and in a Col(r) clinical isolate. A colistin-susceptible revertant of one Col(r) mutant strain, obtained following serial passage in the absence of colistin selection, carried a partial deletion of pmrB. Growth of AB0057 and ATCC 17978 at pH 5.5 increased the colistin MIC and conferred protection from killing by colistin in a 1-hour survival assay. Growth in ferric chloride [Fe(III)] conferred a small protective effect. Expression of pmrA was increased in Col(r) mutants, but not at a low pH, suggesting that additional regulatory factors remain to be discovered.

FIG. 1.

Selection of Col^r and Col^s mutants. The scheme for selection or isolation of Col^r mutants and revertants of each mutant is shown. Col^r strains are indicated by bold circles.

FIG. 2.

Survival of strain AB0057 in colistin. (A) Mid-log-phase cultures of AB0057 in LB were treated with the indicated concentration of colistin for 1 h at 37°C, and then quantitative counts of live cells were determined by plating 0.1-ml samples of serial 10-fold dilutions on LB agar plates. (B) Cells were grown in LB for 4 hours, and then colistin was added at the indicated concentration. Aliquots were removed at the given intervals, and quantitative counts were performed as described above. “Percent survival” corresponds to 100 × the ratio of the number of CFU in the presence of colistin to the number of CFU in the absence of colistin at each time/concentration point.

FIG. 3.

Reversion of colistin resistance. Each clone was passaged in successive overnight and daily cultures. Each daily culture was plated on LB agar with and without 10 μg/ml colistin. The ratio of CFU from the two plates is plotted.

FIG. 4.

Mutational analysis of the pmr operon. Mutations in bold affect conserved residues in functional domains. All strains are Col^r, except for AB060 and MAC203, which are Col^s.

FIG. 5.

Gene expression analysis of pmrA. The level of expression of pmrA was measured by real-time quantitative RT-PCR. Expression levels were normalized to 16S rRNA. (A) Expression analysis of wild-type and mutant strains. Changes with respect to wild-type expression levels are shown. (B) Expression analysis at pH 5.5 and 7.7. Changes are expressed with respect to expression levels measured at pH 7.7. (C) Expression analysis in the presence of 1 mM FeCl₃. Changes are expressed with respect to expression levels of the wild-type strain grown without iron. Each value represents the mean ± standard deviation for at least three independent cultures. An asterisk indicates that the difference is significant (P < 0.01) by the t test.