Table 1.
Bacterial strains, plasmids and primers used in this study.
Fig 1.
The influence of the BaeSR TCS on AdeIJK and MacAB-TolC pump gene expression.
baeR deletion in the ATCC 17978 strain significantly reduced adeI, adeJ, adeK, tolC, macA and macB expression. The reduced expression could be restored through trans-complementation with pWH1266-kanr-baeR. Introducing pWH1266-kanr-baeR into the ATCC 17978 strain mildly increased expression of these genes. The results are shown as the means ± SD from three independent experiments. *, P < 0.05 and **, P < 0.01 and ***, P < 0.001 between ATCC 17978 and AB1026; #, P < 0.05 and ##, P < 0.01 and ###, and P < 0.001 between AB1026 and AB1027.
Fig 2.
Expression analyses of the AdeIJK and MacAB-TolC pump genes in the laboratory-induced and clinical tigecycline-resistant A. baumannii and their baeR mutant strains.
(A) Gene expression of the AdeIJK pump in ABtc increased compared with the wild-type strain, whereas gene expression of MacAB-TolC in ABtc exhibited a more marked increase after tigecycline induction. The expression levels of adeI, adeJ, and adeK in the ABtcm strain were 44%, 47%, and 59% lower, respectively, than in ABtc, and the expression levels of tolC, macB, and macA in ABtcm were 80%, 81%, and 85% lower, respectively, than in ABtc. (B) The expression levels of adeI, adeJ, and adeK in ABhl1 exhibited a statistically significant decrease compared with the wild-type strain, whereas the expression levels of tolC and macB increased 1.7 and 2.3-fold, respectively. (C) The gene expression of adeI, adeJ, adeK, tolC, macB and macA in the ABhl1tc strain increased 9.4-, 27-, 37-, 15-, 4.6-, 3.1-fold respectively compared with the ABhl1 strain. The results are displayed as the means ± SD from three independent experiments. *, P < 0.05 and, **, P < 0.01 and ***, P < 0.001 between ATCC 17978 and ABtc or ABhl1. #, P < 0.05 and ##, P < 0.01 between ABtc and ABtcm.
Fig 3.
Phenotype microarray using the baeR mutant.
Differential susceptibility to some of the 72 compounds studied was observed between the ABwt and AB∆baeR strains. Four different concentrations of each compound were placed in individual wells next to each other in a row with increasing concentrations from the left to the right. The growth of A. baumannii was determined by measuring the optical density at 595 nm (OD595). Compared to ABwt, growth of AB∆baeR was much sensitive with increasing concentrations of several compounds, including procaine, alexidine, and puromycin (in PM 15B); aminopyridine, oxycarboxin, caffeine, ethionamide, tannic acid (in PM17A); and difulphiram, iodonitro tetrazolium violet, and thioglycerol (in PM 19). All these compounds inhibited AB∆baeR cell growth were marked with ↓. Among them, tannic acid sensitivity was the most markedly compromised after deleting baeR. In contrast, AB∆baeR exhibited better tolerance to oleandomycin, methyl viologen, niaproof, compound 48/80, josamycin and FCCP compared to ABwt (All these compounds were marked with ↑). *: the color of the chemical compounds somehow interferes with the observation of bacterial growth.
Fig 4.
Spot assays and gene expression analyses after tannic acid exposure.
(A) Spot assay. The wild-type strain exhibited better tolerance to tannic acid than the baeR mutant strain. (B) The gene expression levels of the baeR and pump genes in the wild-type strain. At 50 μg/mL tannic acid, only macB showed a statistically significant increase in the wild-type strain. Through increasing medium tannic acid up to 100 μg/mL, baeR, adeJ and macB exhibited increased gene expression. If the medium contained tannic acid 500 μg/mL, the expression of each gene investigated in the wild-type strain increased dramatically. (C) The gene expression levels of the pump genes in the baeR mutant strain. The gene expression of adeB, adeJ, and macB in the baeR mutant strain increased significantly at the tannic acid concentration 50 μg/mL. Through increasing the tannic acid concentration to 100 μg/mL, expression of each gene decreased to levels without tannic acid exposure. (D) The gene expression levels of the baeR and pump genes in the clinical strain ABhl1. The gene expression of baeR, adeB, and macB demonstrated a statistically significant increase upon being exposed to 100 and 500 μg/mL tannic acid. The results are shown as the means ± SD from three independent experiments. *, P < 0.05 and, **, P < 0.01 and ***, P < 0.001.