Sub-minimum inhibitory concentrations of colistin and polymyxin B promote Acinetobacter baumannii biofilm formation

Abstract

We investigated the numbers of planktonic and biofilm cells and the expression levels of genes encoding efflux pumps and biofilm-related proteins in 10 clinical isolates of multi-drug resistant Acinetobacter baumannii (MDRA) as well as in its standard strain ATCC 19606 in the presence of colistin (CST), polymyxin B (PMB), minomycin (MIN), and tigecycline (TGC) at their respective sub-MICs. The number of planktonic and biofilm cells of ATCC 19606 decreased in the presence of all aforementioned antibiotics in a dose-dependent manner. Cell number also decreased in two representative MDRA strains, R2 and R3, in the presence of MIN and TGC in a dose-dependent manner. In contrast, the number of biofilm cells in these two strains increased in the presence of CST, while they increased significantly in the presence of PMB in R2 only. Pearson correlation analysis revealed that the number of biofilm cells was positively and significantly correlated with the mRNA levels of genes encoding efflux pumps (adeB and adeG) and autoinducer synthase (abaI) in strain R2 and adeB, adeG, adeJ, poly-acetyl-glucosamine-porin (pgaA), and abaI in strain R3 in the presence of CST. It was positively and significantly correlated with the mRNA levels of genes encoding adeB in strain R2 and an outer membrane protein A (ompA) and biofilm-associated protein (bap) in strain R3 in the presence of PMB. These results provide valuable insights into the biofilm formation potency of clinical isolates of MDRA that depends on efflux pumps and biofilm-related genes and its regulation by antibiotics.

Fig 1. Effect of four antibiotics at sub-MICs on bacterial growth and biofilm formation of A. baumannii.

Summarized results showing the ratio of planktonic and biofilm cells in strain ATCC 19606 cultured in LB broth with (A) CST, (B) PMB, (C) MIN and (D) TGC at sub-MICs. Summarized results showing the ratio of planktonic and biofilm cells in strain R2 cultured in LB broth with (E) CST, (F) PMB, (G) MIN and (H) TGC at sub-MICs. Summarized results showing the ratio of planktonic and biofilm cells in strain R3 cultured in LB broth with (I) CST, (J) PMB, (K) MIN and (L) TGC at sub-MICs. Dark gray and gray bars indicate the ratio of planktonic and biofilm cells in A. baumannii, respectively. Bar graph data are shown as the mean ± SEM (n = 6) of 3 independent experiments. Asterisks indicate statistically significant differences in the number of planktonic cells (**P< 0.01; *P< 0.05, non-treated bacteria vs. antibiotics-treated bacteria; One-way ANOVA). Crosses indicate statistically significant differences in the number of biofilm cells (^††P<0.01; ^†P<0.05, non-treated bacteria vs. antibiotics-treated bacteria; One-way ANOVA).

Fig 2. Effect of CST at sub-MICs on the expression levels of efflux pumps and biofilm-related genes in A. baumannii.

Summarized results showing the mRNA levels of efflux pumps and biofilm-related genes in strains ATCC 19606, R2 and R3 cultured in LB-broth with CST at sub-MICs. The mRNA levels of (A) adeB, (B) adeG, (C) adeJ, (D) ompA, (E) bap, (F) pgaA and (G) abaI were analyzed by real-time PCR. Bar graph data are shown as the means ± SEM (n = 6) of 3 independent experiments. Asterisks indicate statistically significant differences (**P<0.01; *P<0.05, non-treated bacteria vs. antibiotics-treated bacteria; One-way ANOVA).

Fig 3. Effect of PMB at sub-MICs on the expression levels of efflux pumps and biofilm-related genes in A. baumannii.

Summarized results showing the mRNA levels of efflux pumps and biofilm-related genes in strains ATCC 19606, R2 and R3 cultured in LB-broth with sub-MICs of PMB. The mRNA levels of (A) adeB, (B) adeG, (C) adeJ, (D) ompA, (E) bap, (F) pgaA and (G) abaI were analyzed by real-time PCR. Bar graph data are shown as the means ± SEM (n = 6) of 3 independent experiments. Asterisks indicate statistically significant differences (**P< 0.01; *P<0.05, non-treated bacteria vs. antibiotics-treated bacteria; One-way ANOVA).