Acinetobacter baumannii increases tolerance to antibiotics in response to monovalent cations

Abstract

Acinetobacter baumannii is well adapted to the hospital environment, where infections caused by this organism are associated with significant morbidity and mortality. Genetic determinants of antimicrobial resistance have been described extensively, yet the mechanisms by which A. baumannii regulates antibiotic resistance have not been defined. We sought to identify signals encountered within the hospital setting or human host that alter the resistance phenotype of A. baumannii. In this regard, we have identified NaCl as being an important signal that induces significant tolerance to aminoglycosides, carbapenems, quinolones, and colistin upon the culturing of A. baumannii cells in physiological NaCl concentrations. Proteomic analyses of A. baumannii culture supernatants revealed the release of outer membrane proteins in high NaCl, including two porins (CarO and a 33- to 36-kDa protein) whose loss or inactivation is associated with antibiotic resistance. To determine if NaCl affected expression at the transcriptional level, the transcriptional response to NaCl was determined by microarray analyses. These analyses highlighted 18 genes encoding putative efflux transporters that are significantly upregulated in response to NaCl. Consistent with this, the effect of NaCl on the tolerance to levofloxacin and amikacin was significantly reduced upon the treatment of A. baumannii with an efflux pump inhibitor. The effect of physiological concentrations of NaCl on colistin resistance was conserved in a panel of multidrug-resistant isolates of A. baumannii, underscoring the clinical significance of these observations. Taken together, these data demonstrate that A. baumannii sets in motion a global regulatory cascade in response to physiological NaCl concentrations, resulting in broad-spectrum tolerance to antibiotics.

FIG. 1.

NaCl induces increased release of proteins into culture supernatants. Total protein was precipitated with trichloroacetic acid from filtered supernatants of A. baumannii cells grown to stationary phase in LB (−) or LB supplemented with 200 mM NaCl (+) and resolved by SDS-PAGE in 15% polyacrylamide gels. *, bands that increased in high NaCl.

FIG. 2.

A. baumannii upregulates putative efflux transporters upon culture in high NaCl medium. (A) RNA was extracted from A. baumannii cells grown to stationary phase in LB or LB supplemented with 200 mM NaCl. The fold changes in transcript levels determined by microarray analyses are shown for putative transporters with levels that increased significantly in A. baumannii cells upon culture in LB supplemented with 200 mM NaCl relative to A. baumannii cells cultured without NaCl supplementation. RND, resistance-nodulation-division; DMT, drug/metabolite transporter; MFS, major facilitator superfamily; MATE, multidrug and toxic compound extrusion; ABC, ATP-binding cassette. (B) Fold changes in transcript levels of selected transporters and transcriptional regulatory genes in 200 mM and 260 mM NaCl compared to medium alone as determined by real-time PCR. The fold change in expression was determined by using the ΔΔC_T method. Error bars represent 1 standard deviation (SD) from the mean and in some cases are too small to be seen. The data are representative of at least three independent biological replicates. (C) Fold change in transcript levels of selected genes in MHB with 150 mM NaCl relative to MHB without NaCl as determined by real-time PCR. Error bars represent the means ± SD. Each bar represents the average of data for three independent biological replicates. Expression changes comparing MHB with NaCl to MHB alone were statistically significant (P < 0.05 by a Student's t test) for each gene tested. (D) SDS-PAGE analyses of proteins released into culture supernatants. Total protein was precipitated with trichloroacetic acid from filtered supernatants of A. baumannii cells grown to stationary phase in MHB (−) or MHB supplemented with NaCl to final concentrations of 50 mM, 90 mM, 150 mM, and 300 mM and resolved by SDS-PAGE in 15% polyacrylamide gels. M, molecular mass marker (in kDa); *, bands that increased with increasing NaCl concentrations.

FIG. 3.

NaCl induces increased tolerance to distinct classes of antibiotics in A. baumannii. A. baumannii strain ATCC 17978 cells were challenged with amikacin (4.5 mg/liter), colistin (0.75 mg/liter), gentamicin (1.125 mg/liter), imipenem (0.0625 mg/liter), or levofloxacin (0.09 mg/liter) with (dashed lines) or without (solid lines) NaCl supplementation of the culture medium to a final concentration of 150 mM. Bacterial growth was monitored by measuring the optical density of the cultures at 600 nm, with each point representing the mean ± SD for at least three cultures (error bars may be too small to be seen). Asterisks indicate statistically significant changes in growth upon antibiotic challenge in medium containing NaCl compared to medium lacking NaCl as determined by a Student's t test (*, P < 0.05; **, P < 0.005; ***, P < 0.0005).

FIG. 4.

Effects of KCl on resistance to antibiotics and on release of proteins into culture medium. (A) Growth curve analyses of A. baumannii cells challenged with antibiotics in MHB (solid lines) or MHB supplemented with 150 mM KCl (dashed lines) at the indicated concentrations. Error bars represent the mean ± 1 SD and may be obscured by the symbol in some cases. Asterisks indicate statistically significant changes in growth upon antibiotic challenge in medium containing KCl compared to medium lacking KCl as determined by a Student's t test (*, P < 0.05; **, P < 0.005; ***, P < 0.0005). (B) SDS-PAGE of TCA-precipitated proteins from A. baumannii culture supernatants. A. baumannii cells were grown to stationary phase in MHB supplemented with 50 mM, 86 mM, 154 mM, or 308 mM KCl. *, bands that increased with increasing concentrations of KCl.

FIG. 5.

NaCl-induced resistance to levofloxacin and amikacin is due in part to increased antibiotic efflux. A. baumannii cells were treated with amikacin, levofloxacin, or colistin in MHB (filled bars) or MHB with 150 mM NaCl (white bars) in the presence or absence of 30 mg/liter of the efflux pump inhibitor PAβN. Bacterial growth was monitored for 12 h, and the optical densities of the cultures were normalized to the respective untreated (i.e., antibiotic-free) controls and expressed as the percent bacterial growth. Statistical analysis was performed by a Student's t test comparing PAβN-treated with the respective PAβN-untreated controls. Error bars are ±1 SD from the mean. *, P < 0.05; **, P < 0.005.

FIG. 6.

NaCl-induced resistance to colistin is conserved among drug-susceptible and multidrug-resistant A. baumannii strains. A. baumannii ATCC 17978 and nine clinical isolates were treated with colistin (0.75 mg/liter) with (solid bars) or without (open bars) NaCl supplementation of the culture medium to a final concentration of 150 mM. Bacterial growth was monitored for 12 h, and the optical densities of the cultures were normalized to growth in the absence of colistin in the respective media (i.e., with or without NaCl). Statistically significant differences comparing medium alone to medium supplemented with NaCl were determined by a Student's t test. Error bars are ±1 SD from the mean. *, P < 0.005.