Acinetobacter baumannii biofilm was inhibited by tryptanthrin through disrupting its different stages and genes expression

Abstract

Biofilm formation plays a significant role in antibiotic resistance, necessitating the search for alternative therapies against biofilm-associated infections. This study demonstrates that 20 μg/mL tryptanthrin can hinder biofilm formation above 50% in various A. baumannii strains. Tryptanthrin impacts various stages of biofilm formation, including the inhibition of surface motility and eDNA release in A. baumannii, as well as an increase in its sensitivity to H₂0₂. RT-qPCR analysis reveals that tryptanthrin significantly decreases the expression of the following genes: abaI (19.07%), abaR (33.47%), bfmR (43.41%), csuA/B (64.16%), csuE (50.20%), ompA (67.93%), and katE (72.53%), which are related to biofilm formation and quorum sensing. Furthermore, tryptanthrin is relatively safe and can reduce the virulence of A. baumannii in a Galleria mellonella infection model. Overall, our study demonstrates the potential of tryptanthrin in controlling biofilm formation and virulence of A. baumannii by disrupting different stages of biofilm formation and intercellular signaling communication.

Keywords: Genetics; Microbiology; Molecular genetics.

Graphical abstract

Figure 1

Effects of tryptanthrin on A. baumannii biofilm formation (A) Biofilm formation ability of A. baumannii ATCC strain and clinical strains. (B) Gallium nitrate decreased A. baumannii ATCC 17978 biofilm formation; Tryptanthrin decreased A. baumannii ATCC 17978 (C) AB108 (D) and AB102 (E) biofilm formation. Data were shown as mean ± SD, and nonparametric one-way ANOVA was used to assess the statistical significance (∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 2

Effects of tryptanthrin on the growth of A. baumannii (A) (ATCC 17978), (C) (AB108), and (E) (AB102) were growth curves plotted by OD₆₀₀ nm measurement; (B) (ATCC 17978), (D) (AB108), and (F) (AB102) were growth curves plotted by colony counting. Data were presented as mean ± SD from three biological replicates.

Figure 3

Biofilm formation (A) Light microscopy showing the antibiofilm efficacy of 20 μg/mL tryptanthrin against ATCC17978, AB108, and AB102, scale bar is 400 μm. (B) CLSM images showing the antibiofilm efficacy of 20 μg/mL tryptanthrin against ATCC17978, AB108, and AB102, different colors in the picture represent the scale bar; Tryptanthrin inhibited the ring biofilm formation of ATCC17978, AB108, and AB102 on a polystyrene surface (C) and on glass (D).

Figure 4

Effect of tryptanthrin (5 μg/mL) on the swarming motility of ATCC17978, AB108 and AB102 (A) Plate images of swarming motility on 0.25% agar plates plus 0.1% DMSO, 0.25% agar plates and 0.25% agar plates plus tryptanthrin (5 μg/mL). (B) Measurement and analysis of the size of the swarming diameters. Data were presented as mean ± SD, and statistical significance was determined by Student’s t tests (∗∗∗p < 0.001).

Figure 5

Effect of tryptanthrin (20 μg/mL) on the synthesis of eDNA from AB102, AB108, and ATCC17978 (A) Agarose gel electropherogram showing the eDNA content, Lane M: Molecular weight marker. (B) ImageJ analysis of agarose gel electrophoresis bands. Data were presented as mean ± SD, and statistical significance was determined by Student’s t tests (∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 6

Effect of tryptanthrin (20 μg/mL) on the survival of AB102, AB108, and ATCC17978 in the presence of H₂O₂ (A) Colony count plots were generated with a PBS dilution. (B) Statistical analysis of the data were performed according to the colony count. Data were presented as mean ± SD, and statistical significance was determined by Student’s t tests (∗∗∗p < 0.001), and “ns” indicates no significant difference.

Figure 7

RT-qPCR analysis of the expression of candidate genes associated with biofilm formation and virulence production in A. baumannii in response to tryptanthrin (20 μg/mL) Data were presented as mean ± SD, and statistical significance was determined by Student’s t tests (∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 8

Effect of tryptanthrin on the survival of Galleria mellonella infected with A. baumannii ATCC17978 Survival analyses were performed using Kaplan-Meier survival curves.