Genetic analysis of colistin resistance in Salmonella enterica serovar Typhimurium

Abstract

Colistin is a cyclic cationic peptide that kills gram-negative bacteria by interacting with and disrupting the outer membrane. We isolated 44 independent mutants in Salmonella enterica serovar Typhimurium with reduced susceptibility to colistin and identified 27 different missense mutations located in the pmrA and pmrB genes (encoding the regulator and sensor of a two-component regulatory system) that conferred increased resistance. By comparison of the two homologous sensor kinases, PmrB and EnvZ, the 22 missense mutations identified in pmrB were shown to be located in four different structural domains of the protein. All five pmrA mutations were located in the phosphate receiver domain of the regulator protein. The mutants appeared at a mutation rate of 0.6 x 10(-6) per cell per generation. The MICs of colistin for the mutants increased 2- to 35-fold, and the extent of killing was reduced several orders of magnitude compared to the susceptible strain. The growth rates of the mutants were slightly reduced in both rich medium and M9-glycerol minimal medium, whereas growth in mice appeared unaffected by the pmrA and pmrB mutations. The low fitness costs and the high mutation rate suggest that mutants with reduced susceptibility to colistin could emerge in clinical settings.

FIG. 1.

Killing curves for the wild type and four mutants with reduced susceptibility to colistin. CFU are plotted as a function of time for different concentrations of colistin (0.25, 1, 4, and 16 mg/liter). Amino acid substitutions in the mutants (using the one-letter code) are indicated as follows: open circles, DA6192 (wild type); filled diamonds, DA10829 (PmrB [S29R]); open diamonds, DA10845 (PmrB [M186I]); filled triangles, DA10826 (PmrA [G53E]); open triangles, DA10840 (PmrA [R81H]).

FIG. 2.

Relative growth rates of mutants with reduced susceptibility to colistin measured in LB broth and M9-glycerol medium. The growth rate for each strain was determined from four to eight independent cultures. The standard deviation of the relative growth rates is ±2%.

FIG. 3.

Stationary-phase survival of four strains with reduced susceptibility to colistin compared to that of the fully susceptible strain. Four independent cultures of DA6192 (wild type), DA10826 (PmrA [G53E]), DA10840 (PmrA [R81H]), DA10833 (PmrB [E166K]), and DA10853 (PmrB [N130Y]) were incubated at 37°C for 21 days. The survival percentage was calculated as the average number of viable cells at each time point divided by the number of viable cells at the start of the experiment. Amino acid substitutions in the mutants (using the one-letter code) are indicated as follows: open circles, DA6192 (wild type); filled triangles, DA10826 (PmrA [G53E]); open triangles, DA10840 (PmrA [R81H]); open diamonds, DA10833 (PmrB [E166K]); filled diamonds, DA10853 (PmrB [N130Y]).

FIG. 4.

Relative expression levels of the pmrH gene in mutants with reduced susceptibility to colistin expressed as mutant/wild-type (wt) levels. Mutations in the pmrA (gray) and pmrB (black) genes are indicated. Amino acid substitutions in the mutants (using the one-letter code) are indicated.

FIG. 5.

Sequence locations of all mutations conferring colistin resistance and the MICs of colistin for the corresponding mutants. Mutations in pmrA (gray) and pmrB (black) and amino acid substitutions in the mutants (using the one-letter code) are indicated.