Fig 1.
Chemical structures of the membrane-active macromolecules.
Fig 2.
Antibacterial activity of antibiotics and QCybuAP against E. coli.
~106 CFU mL-1 of bacteria in M9 media were treated with compounds for 2 h. Stars represent no survival detected (limit of detection < 50 CFU/mL).
Fig 3.
Antibacterial activity of antibiotics and cationic polymer (Qn-prAP) against E. coli antibiotic-tolerant cells.
(A) Ampicillin (100 μg mL-1), kanamycin (100 μg mL-1) do not kill whereas Qn-prAP shows concentration dependent (10, 30 and 50 μg mL-1) elimination of E. coli antibiotic-tolerant cells. ~106 CFU mL-1 of bacteria in M9 media were treated with compounds for 2 h. Stars represent no survival detected (limit of detection < 50 CFU/mL).
Fig 4.
Antibacterial activity of antibiotics and cationic polymers (Qn-prAP) against A. baumannii.
(A) Actively growing and (B) stationary phase cells. ~106 CFU mL-1 of bacteria in BM2 media were treated with compounds for 2 h at a concentration of 10 μg mL-1. Stars represent no survival detected (limit of detection < 50 CFU/mL).
Fig 5.
Membrane-active properties of QCybuAP against E. coli.
(A) Concentration dependent effect of QCybuAP on membrane depolarization and (B) membrane permeabilization against antibiotic-tolerant cells; Membrane permeabilization of QCybuAP and colistin against actively growing (C) and antibiotic-tolerant (D) cells. The concentrations used were 20 μg mL-1.
Fig 6.
Disruption of E. coli biofilms.
(A) Confocal laser scanning microscopy (CLSM) images of biofilms either treated with QCybuAP (50 μg mL-1), erythromycin (Ery, 50 μg mL-1) and erythromycin + QCybuAP (50 μg mL-1 + 50 μg mL-1) or left untreated for 24 h. Biofilms were stained with SYTO 9 dye and each image is a 3D reconstruction of z-stack images; (B) Reduction in bacterial cell counts in biofilms with and without treatment of compounds; (C) Absorbance of the crystal violet (CV) staining of the biofilms.
Fig 7.
Disruption of A. baumannii biofilms.
(A) Absorbance of crystal violet staining of the A. baumannii biofilms grown on glass cover slips in presence of colistin (30 μg mL-1), Qn-prAP and QCybuAP (both at 30 μg mL-1), erythromycin (Ery, 30 μg mL-1), tobramycin (Tobra, 30 μg mL-1), erythromycin + Qn-prAP/QCybuAP (30 μg mL-1 + 30 μg mL-1); (B) Reduction in bacterial cell counts in biofilms with the treatment of colistin (50 μg mL-1), Qn-prAP and QCybuAP (both at 50 μg mL-1), erythromycin (Ery, 50 μg mL-1), tobramycin (Tobra, 50 μg mL-1), erythromycin + Qn-prAP/QCybuAP (50 μg mL-1 + 50 μg mL-1) compared to untreated control. (limit of detection < 50 CFU/mL).
Fig 8.
Disruption of A. baumannii biofilms.
Biofilms were treated with colistin (30 μg mL-1), Qn-prAP and QCybuAP (both at 30 μg mL-1), erythromycin (Ery, 30 μg mL-1), tobramycin (Tobra, 30 μg mL-1), erythromycin + Qn-prAP/QCybuAP (30 μg mL-1 + 30 μg mL-1). Confocal laser scanning microscopy (CLSM) stained with SYTO9 dye. z-stack images were processed using Zeiss LSM software and 3D representation was processed using ImageJ. Scale bars, 5 μm.
Fig 9.
Dispersed cells of A. baumannii biofilms.
O.D.600 of the planktonic growth of bacteria due to dispersed cells from biofilms in presence of colistin (30 μg mL-1), Qn-prAP and QCybuAP (both at 30 μg mL-1), erythromycin (Ery, 30 μg mL-1), tobramycin (Tobra, 30 μg mL-1), erythromycin + Qn-prAP/QCybuAP (30 μg mL-1 + 30 μg mL-1).
Fig 10.
Development of bacterial resistance.
(A) A. baumannii did not develop resistance to QCybuAP but a very high and rapid resistance was observed for known antibiotics with an increase in MIC up to 64 fold after 28 passages. Macromolecules delay the development of resistance to both the known antibiotics with only 8-fold increase in MIC even after 28 passages. (B) E. coli developed rapid resistance to colistin and tetracycline but not to Qn-prAP and Qn-prAP + tetracycline.
Fig 11.
In-vivo A. baumannii burn wound infection.
(A). Experimental plan. (B). Mice (n = 4) were treated with erythromycin (Ery, 20 mg kg-1), QCybuAP (50 mg kg -1), Qn-prAP (50 mg kg-1), Qn-prAP + Ery (50 mg kg-1 + 20 mg kg-1), QCybuAP + Ery (50 mg kg-1 + 20 mg kg-1) and colistin (5 mg kg-1). Combination treatment and colistin showed decrease below detection limits (< 50 CFU mL-1). P value was calculated using one way ANOVA (Dunnett's Multiple Comparison Test) between the control group and treatment groups and a value of P < 0.05 was considered significant.
Fig 12.
Scale bar = 100 μm (inset, 20 μm).
Fig 13.
In-vivo A. baumannii burn wound infection.
(A) Mice (n = 4) were treated with rifampicin (rif, 5 mg kg-1), QCybuAP (50 mg kg-1) and QCybuAP + rif (50 mg kg-1 + 5 mg kg-1). P value was calculated using one way ANOVA (Dunnett's Multiple Comparison Test) between the control group and treatment groups and a value of P < 0.05 was considered significant. (B) Histopathology analysis. Scale bar = 100 μm (inset 20 μm).
Fig 14.
In-vivo anti-infective activity against K. pneumoniae (carbapenem and tetracycline resistant clinical isolate, KPC) surgical wound infection.
a. Experimental model. b. Mice (n = 4) were treated with colistin (5 mg kg-1), tetracycline (Tet) (100 mg kg-1), QCybuAP (50 mg kg-1) and QCybuAP + Tet (50 mg kg-1+ 100 mg kg-1). P value was calculated using one way ANOVA (Dunnett's Multiple Comparison Test) between the control group and treatment groups and a value of P < 0.05 was considered significant. c. Histopathology analyses of mice wounds untreated (A and E) and treated with colistin (B and F), Qn-prAP (C and G) and Qn-prAP + Tetracycline (D and H).