Transition of colistin dependence into colistin resistance in Acinetobacter baumannii

Abstract

We recently demonstrated a high rate of colistin dependence in Acinetobacter baumannii isolates exposed to colistin in vitro. In the present study, we obtained a colistin-resistant (H08-391R) and colistin-dependent mutant (H08-391D) from a colistin-susceptible parental strain (H08-391). We found that the colistin-dependent mutant converted into a stable colistin-resistant mutant (H08-391D-R) in vitro after four serial passages without colistin. H08-391D and H08-391D-R were both found to harbor defective lipid A, as indicated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis. Additionally, both contained an ISAba1 insertion in lpxC, which encodes a lipid A biosynthetic enzyme. Further, membrane potential measurements using the fluorescent dye 3,3'-diethyloxacarbocyanine iodide (DiOC₂[3]) showed that the membrane potential of H08-391D and H08-391D-R was significantly decreased as compared to that of the parental strain, H08-391. Moreover, these mutant strains exhibited increased susceptibilities to antibiotics other than colistin, which may be attributed to their outer membrane fragility. Such phenomena were identified in other A. baumannii strains (H06-855 and its derivatives). Taken together, our study reveals that the colistin-dependent phenotype is a transient phenotype that allows A. baumannii to survive under colistin pressure, and can transition to the extremely resistant phenotype, even in an antibiotic-free environment.

Figure 1

Colistin disc diffusion assays showing the development of colistin-dependent and -resistant mutants from the colistin-susceptible wild-type (WT) strain, H08-391. From a colistin-susceptible strain, H08-391, a colistin-resistant (H08-391R) and -dependent mutant (H08-391D) were selected in vitro using culture media containing 10 mg/L colistin. H08-391D-R was derived from the colistin-dependent mutant, H08-391D, through subsequent passages in the absence of colistin selection pressure, and exhibited the colistin-resistant phenotype.

Figure 2

Expression levels of the pmrC, lpxA, and lpxC genes in the strains in A. baumannii H08-391 (A) and H06-855 (B) lineages. The three genes were found to be overexpressed only in the colistin-resistant mutants, H08-391R and/or H06-855R. Error bars represent the standard deviation of three biological repeats, each performed in duplicate. Statistical significance between each strain was determined using Student’s unpaired t-test. Data were analyzed using GraphPad Prism 5.

Figure 3

Genes showing high expression levels in colistin-dependent and its converted colistin-resistant strains. The expression levels were evaluated by mRNA sequencing. (A) H08-391 lineage; (B) H06-855 lineage. Error bars represent the standard deviation of three biological repeats, each performed in duplicate.

Figure 4

Negative-ion mode matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry spectra of the lipid A moieties of lipopolysaccharide isolated from the four strains: H08-391, H08-391R, H08-391D, and H08-391D-R.

Figure 5

LPS profile of the strains H08-391, H08-391R, H08-391D, and H08-391D-R.

Figure 6

Comparison of the membrane potential in the strains H08-391, H08-391R, H08-391D, and H08-391D-R. Error bars represent the standard deviation of three biological repeats, each performed in duplicate. Statistically significant differences in membrane potential between the strains were analyzed using Student’s unpaired t-test using GraphPad Prism 5.