Article Figures & Data
Figures
- FIG 1
In vitro growth of ATCC 19606 and the colistin-resistant laboratory derivates. The numbers of CFU per milliliter were determined at 1, 2, 3, 5, 7, and 20 h. The means of four independent replicates (for each growth curve) are shown. The error bars represent the standard deviation (SD).
- FIG 2
Relative in vitro competition indexes of colistin-resistant mutants. The CI values were calculated as the number of Col-R mutants divided by the number of bacteria of the Col-S parental strain of A. baumannii, and the median CI values are shown in parentheses. Each circle represents the CI obtained in each in vitro replicate. Experiments were performed at 5 and 20 h. In all cases, the differences in bacterial counts were statistically significant (P < 0.01). The error bars represent the standard deviation.
- FIG 3
Relative in vivo competition indexes determined in a mouse systemic-infection model over 20 h. The CI values were calculated as the number of Col-R mutants divided by the number of bacteria of the Col-S parental strain of A. baumannii, and the median CI values are shown in parentheses. Circles represent the CI obtained in each in vivo replicate. The differences in bacterial counts were statistically significant (P < 0.01). The error bars represent the standard deviation.
- FIG 4
Abilities of different A. baumannii strains to cause cell death of A549 alveolar cells. (A) Fluorescence microscopy images of human alveolar A549 cells infected with each of the A. baumannii strains and stained with the LIVE/DEAD Cellstain double-staining kit. Healthy cells with intact membranes are stained green, and dead cells with permeabilized membranes are stained red. A549 cells were incubated with the A. baumannii strains ATCC 19606 WT, AL1851 ΔlpxA, Al1852 ΔlpxD, AL1842 ΔlpxC, and ATCC 19606 pmrB for 20 h or left uninfected. (B) Quantification of A549 cell death caused by A. baumannii ATCC 19606 WT and AL1851 ΔlpxA, AL1852 ΔlpxD, and AL1842 ΔlpxC mutants and the pmrB mutant. The results of 6 independent experiments are shown as means and SD. *, P < 0.01 between the parental strain and each of the designated mutants; **, not statistically different.
- FIG 5
Survival of BALB/c (n = 10 per group) mice following intraperitoneal infection with 3 × 108 CFU of the ATCC 19606, AL1842 ΔlpxC, or ATCC 19606 pmrB strain. No difference in survival was found between the mice infected with the pmrB mutant or the parental strain, but survival was significantly higher in mice infected with the AL1842 ΔlpxC mutant (P < 0.01).
- FIG 6
C. elegans fertility assays. The number of progeny from L4 stage worms was monitored for 5 days in the presence of the nonvirulent control E. coli OP50, the A. baumannii ATCC 19606 WT strain and its colistin-resistant mutants (A), or A. baumannii ABRIM and ABRIM pmrB (B). The error bars represent the standard deviation.
Tables
- TABLE 1
Bacterial strains used in this study
Strain Description Colistin MIC (μg/ml) Source or reference ATCC 19606 A. baumannii type strain; parent of AL1842 ΔlpxC, AL1851 ΔlpxA, and AL1852 ΔlpxD 1 American Type Culture Collection AL1851 ΔlpxA Derivate of ATCC 19606; colistin resistant; 445-bp deletion within lpxA 64 12 AL1852 ΔlpxD Derivate of ATCC 19606; colistin resistant; single base deletion at nucleotide [nt] 364 of lpxD and frame shift after K317 >128 12 AL1842 ΔlpxC Derivate of ATCC 19606; colistin resistant; 84-bp deletion within lpxC 128 12 ATCC 19606 pmrB Derivate of ATCC 19606; colistin resistant; single amino acid substitution (Ala227Val) in PmrB. Ala227Val is adjacent to the conserved histidine at the site of phosphorylation (His228). 64 15 ABRIM A. baumannii clinical strain carrying the carbapenemase OXA-24, isolated in a Spanish nosocomial outbreak in 1997 1 21 ABRIM pmrB Derivate of A. baumannii ABRIM; colistin resistant; single amino acid substitution (Asn353Tyr) in PmrB. This substitution is inside the ATP binding site and may have an effect on the phosphorylation of His228 and thereby on the phosphorylation levels of PmrA. 32 15 OP50 E. coli strain used for maintenance of C. elegans NAa 24 ↵a NA, not applicable.
- TABLE 2
A. baumannii doubling times and growth rates measured in the exponential phase of growth over the first 300 mina
Strain Doubling time (g) (min) Growth rate (μ) (h−1) ATCC 19606 27 ± 3 1.56 ± 0.27 AL1851 ΔlpxA 49 ± 6 0.85 ± 0.09 AL1842 ΔlpxC 40 ± 3 1.03 ± 0.09 AL1852 ΔlpxD 83 ± 8 0.49 ± 0.03 ATCC 19606 pmrB 32 ± 1 1.32 ± 0.03 ↵a Data were extracted from Fig. 1.
- TABLE 3
C. elegans fertility assay
Strain Quantification (per day) on daya: Total no. of progenya Difference from controlb (%) 1 2 3 4 A. baumannii ATCC 19606 45 99.3 47.7 2.33 194.2 ± 6.9 79.5 AL1851 ΔlpxA 70.2 158.7 87.3 0.7 316.9 ± 12 129.7 AL1852 ΔlpxD 73.7 202.7 61.3 0.7 338.3 ± 26 138.4 AL1842 ΔlpxC 62.5 147.2 72.3 2.3 284.3 ± 50 116.3 ATCC 19606 pmrB 46.33 92 37.2 9.7 180.2 ± 22 73.7 ABRIM 37.3 83.7 76.5 2.8 200.3 ± 13 81.9 ABRIM pmrB 38.8 75 75.3 13.5 202.7 ± 30.3 82.9 E. coli OP50 38.6 122.4 73.2 10.2 244.4 ± 28 100
