Phenotypic changes associated with Colistin resistance due to Lipopolysaccharide loss in Acinetobacter baumannii

Figures & data

Table 1. Colistin MIC values and endotoxin levels of strains used in this study.

Strain	Mutation/Description	Colistin MIC (mg/l)	EU/ 10 [6] cells
ATCC 19606	Antibiotic susceptible reference strain	≤ 0.25	1060 ± 814
IB002	ISAba11 insertion at nucleotide 372 of lpxC gene	> 128	< 1
IB003	ISAba11 insertion at nucleotide 394 of lpxC gene	> 128	< 1
IB004	Deletion of nucleotide 461 of lpxA gene generating frameshift after S153	> 128	< 1
IB006	ISAba11 insertion at nucleotide 393 of lpxC gene	> 128	< 1
IB007	ISAba11 insertion at nucleotide 393 of lpxC gene	> 128	< 1
IB004 + pWH1266-lpxA	IB004 complemented with the pWH1266 plasmid expressing the wild type lpxA gene	≤ 0.25	421 ± 67.0
Ab-84	Multidrug resistant clinical isolate	≤ 0.25	17.78 ± 3.5
Ab-108	Multidrug resistant clinical isolate	≤ 0.25	18.53 ± 4.80
Ab-167	Multidrug resistant clinical isolate	≤ 0.25	20.02 ±2.36
Ab-176	Multidrug resistant clinical isolate	≤ 0.25	42.97 ±21.52
Ab-208	Multidrug resistant clinical isolate	≤ 0.25	25.02±5.04
Ab-84R	Strain Ab-84 containing a 40 nucleotide insertion at nucleotide 321 of lpxC generating a premature stop codon	> 128	< 1
Ab-108R	Strain Ab-108 with a T614A mutation in lpxA producing an I205N amino acid substitution	> 128	< 1
Ab-167R	Strain Ab-167 containing an ISAba1 insertion at nucleotide 321 of lpxC	> 128	< 1
Ab-176R	Strain Ab-176 with a G739T substitution at nucleotide 739 of the lpxD gene producing a premature stop codon	> 128	< 1
Ab-208R	Strain Ab-208with a C593A substitution at nucleotide 198 of the lpxD gene producing A198E	>256	< 1
CS01	Colistin susceptible A. baumannii clinical isolate	≤ 0.25	ND
CR17	Colistin resistant derivative of CS01 containing a M12K mutation in pmrA	64	ND

^a Data represent the mean ± the standard error of the mean of three independent assays.

EU; Endotoxin Units.

Figure 1. In vitro growth of LPS-deficient A. baumannii. Graphs represent the growth of ATCC 19606, IB004 and IB004 + pWH1266-lpxA in Mueller Hinton broth (A) and human serum (B). Data points represent the average of three independent cultures with error bars representing the standard deviation.

Figure 2. Growth and dissemination of LPS-deficient A. baumannii in an experimental mouse model. Tissue bacterial loads of ATCC 19606, IB004 and IB004 + pWH1266-lpxA in spleen, lungs and kidneys of mice infected with 2.5 × 10⁵ CFU of each strain (n = 4 mice/timepoint). Data points represent the average independent mice with error bars representing the standard deviation.

Figure 3. Fitness of LPS-deficient A. baumannii with its wild type counterpart in laboratory medium, human serum and mouse tissue. Competition indices comparing ATCC 19606 and IB004, and ATCC 19606 and IB004 + pWH1266-lpxA in Muller Hinton broth, human serum and mouse spleen. Bars represent the average of three independent assays with error bars representing the standard deviation.

Figure 4. Serum cytokine levels after infection with LPS-deficient A. baumannii in a mouse model of experimental infection. Serum TNF-α (A) and IL-6 (B) levels in uninfected, ATCC 19606 infected, IB004-infected and IB004 + pWH1266-lpxA-infected mice (n = 6 mice/group) 12 hours after infection with 2.5 × 10⁵ CFU of the indicated strain. Bars represent average values and error bars represent the standard deviation. #P < 0.01 compared to uninfected mice, † P < 0.01 compared to IB004-infected mice; Tukey post-hoc test.

Figure 5. Virulence of LPS-deficient A. baumannii in a mouse model of disseminated infection. Survival of mice infected with 2.5 × 10⁵ CFU the indicated strains in a disseminated sepsis model during 7 days post-infection. #P < 0.005, log-rank test compared to ATCC 19606 and IB004 + pWH1266-lpxA infected mice.

Figure 6. Effect of LPS loss on growth rate and biofilm production. Growth rate (A) and biofilm production (B) were determined for ATCC 19606, IB004, and IB004 + pWH1266-lpxA (IB004 C), and 5 pairs of multidrug resistant clinical isolates (Ab-84, Ab-108, Ab-167, Ab-178 and Ab-208) and their LPS-deficient derivatives (Ab-84R, Ab-108R, Ab-167R, Ab-178R and Ab-208R) and CS01/CR17. Bars represent the average of three separate assays, with error bars representing the standard deviation. #P < 0.005 compared to parental, LPS-replete strain, Student's t test. # P < 0.005 compared to complemented strain, Student's t test.

Figure 7. Effect of LPS loss on surface motility. Surface motility on semisolid media of ATCC 19606, IB004, and IB004 + pWH1266-lpxA, and 5 pairs of multidrug resistant clinical isolates (Ab-84, Ab-108, Ab-167, Ab-178 and Ab-208) and their LPS-deficient derivatives (Ab-84R, Ab-108R, Ab-167R, Ab-178R and Ab-208R) and CS01/CR17 after 72 hours of incubation. Bars represent the average of three separate assays, with error bars representing the standard deviation. #P < 0.005 compared to parental, LPS-replete strain, Student's t test. # P < 0.005 compared to complemented strain, Student's t test (A). Surface motility of the Ab-176 strain and its LPS-deficient counterpart after 72 hours of incubation (B).

Figure 8. Effect of LPS loss on growth under iron limitation. Growth in iron limiting conditions of ATCC 19606, IB004, and IB004 + pWH1266-lpxA, and 5 pairs of multidrug resistant clinical isolates (Ab-84, Ab-108, Ab-167, Ab-178 and Ab-208) and their LPS-deficient derivatives (Ab-84R, Ab-108R, Ab-167R, Ab-178R and Ab-208R) and CS01/CR17 over 24 hours. Bars represent the average of three separate cultures, with error bars representing the standard deviation.

Table 2. Susceptibility (MIC) of A. baumannii strains to disinfectants.

Strain	Chlorhexidine (mg/l)	Deoxycholate (mM)	SDS (g/l)	Ethanol (%)	Povidone iodine (g/l)
ATCC 19606	0.487	> 10	1.56	25	6.25
IB004	0.122	0.3125	0.1	12.5	6.25
IB004 + pWH1266-lpxA	0.487	> 10	1.56	50	6.25
84S	0.975	> 10	1.56	6.25	6.25
84R	0.061	0.3125	0.1	12.5	6.25
108S	0.487	> 10	6.25	6.25	6.25
108R	0.244	0.625	0.2	6.25	6.25
167S	0.975	> 10	1.56	6.25	6.25
167R	0.122	0.3125	0.1	6.25	6.25
176S	0.975	> 10	3.125	12.5	6.25
176R	0.122	0.15	0.2	3.125	6.25
208S	0.975	> 10	3.125	12.5	6.25
208R	0.122	0.15	0.2	12.5	6.25
CS01	0.487	>10	0.4	12.5	6.25
CR17	0.487	>10	0.4	12.5	6.25

Adams MD, Nickel GC, Bajaksouzian S, et al. Resistance to colistin in Acinetobacter baumannii associated with mutations in the PmrAB two-component system. Antimicrob Agents Chemother. 2009;53:3628–34.

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