Antibiofilm effects of N,O-acetals derived from 2-amino-1,4-naphthoquinone are associated with downregulation of important global virulence regulators in methicillin-resistant Staphylococcus aureus

Abstract

Despite the existing antibiotics, antimicrobial resistance is a major challenge. Consequently, the development of new drugs remains in great demand. Quinones is part of a broad group of molecules that present antibacterial activity besides other biological properties. The main purpose of this study was to evaluate the antibiofilm activities of synthetic N,O-acetals derived from 2-amino-1,4-naphthoquinone [7a: 2-(methoxymethyl)-amino-1,4-naphthoquinone; 7b: 2-(ethoxymethyl)-amino-1,4-naphthoquinone; and 7c: 2-(propynyloxymethyl)-amino-1,4-naphthoquinone] against methicillin-resistant Staphylococcus aureus (MRSA). The derivatives 7b and 7c, specially 7b, caused strong impact on biofilm accumulation. This inhibition was linked to decreased expression of the genes fnbA, spa, hla and psmα3. More importantly, this downregulation was paralleled by the modulation of global virulence regulators. The substitution of 2-ethoxymethyl (7b) in comparison with 2-propynyloxymethyl (7c) enhanced sarA-agr inhibition, decreased fnbA transcripts (positively regulated by sarA) and strongly impaired biofilm accumulation. Indeed, 7b triggered intensive autolysis and was able to eliminate vancomycin-persistent cells. Consequently, 7b is a promising molecule displaying not only antimicrobial effects, but also antibiofilm and antipersistence activities. Therefore, 7b is a good candidate for further studies involving the development of novel and more rational antimicrobials able to act in chronic and recalcitrant infections, associated with biofilm formation.

Figure 1

Hemocompatibility profile of 6a and its derivatives 7a, 7b, and 7c in concentrations of 25 µg/mL, 50 µg/mL, 100 µg/mL and 300 µg/mL. Cip, ciprofloxacin and Van, vancomycin were also used to control this test. C_neg, negative control (1% DMSO only); C_pos, positive control (1% Triton X-100).

Figure 2

Effect of 1/4MIC of 6a and N,O acetal naphthoquinone derivatives 7a, 7b, and 7c on biofilm development. (A) Biofilm formed on surfaces and stained with crystal violet. (B) Confocal laser scanning microscopy (CLSM). Effects of 1/4MIC of the derivatives 7b (32 µg/mL) and 7c (16 µg/mL) on biofilm formed by the MRSA strain BMB9393, compared with the untreated biofilm (C_neg). Van vancomycin (1/4 MIC; 0.5 µg/mL). NS not significant; **p value < 0.001; and ***p < 0.0001. The biofilms were treated with SYTO 9 and the image obtained in (I) two-dimensional axis (xy) and (II) three-dimensional plane (xyz). (III) Biofilm thickness. Red scale = 10 μm. For each set of experiments, a total of three independent experiments were performed in quadruplicate.

Figure 3

Effect of 7b and 7c derivatives in the expression of the biofilm-associated genes and global gene regulators. Gene expression was assessed by real-time qRT-PCR after treatment of the MRSA strain BMB9393 with 1/4MIC of derivatives 7b (32 µg/mL) or 7c (16 µg/mL). (A) Gene expression for biofilm-associated genes fnbA, hla, spa, psmα3, and mecA. (B) Gene expression for the biofilm regulators rnaIII, sarA, and sigB. Data were represented by the mean of three independent experiments with triplicates. The bar represents the standard deviation. C_neg, negative control (2% DMSO, only). NS not significant; *p values < 0.01; **p < 0.001; and ***p < 0.0001.

Figure 4

Effect of derivatives 7b and 7c on the expression of the major S. aureus autolysin (AtlA) using the MRSA strain BMB9393. (A) atlA expression was evaluated by real-time qRT-PCR after-treatment of the MRSA strain BMB9393 with 1/4MIC of derivatives 7b (32 µg/mL) and 7c (16 µg/mL). (B) eDNA was determined after-treatment of the MRSA strain BMB9393 with 1/4MIC of derivatives 7b and 7c. Data represent the mean of three independent experiments with triplicates. The bar represents the standard deviation. C_neg, negative control (2% DMSO only). NS not significant; **p values < 0.001 and ***p < 0.0001.

Figure 5

Effect of N,O-acetal derivatives against MRSA cells persistent to vancomycin. Persistent cells of the strain related to USA100 clone (CR15-071) were detected in plates containing 8 µg/mL vancomycin and high bacterial load. A dose dependent inhibition of persistent cells was observed for different combinations of vancomycin (8 µg/mL) with concentrations of 1/8MIC (16 µg/mL) to 1/2MIC (64 µg/mL) of each derivative. Note that 1MIC (128 µg/mL) of the derivative 7c was able to eliminate vancomycin-persistent cells completely. Data were represented by the mean of three independent experiments with triplicates. The bar represents the standard deviation. NS not significant; *p < 0.01; **p < 0.001; and ***p < 0.0001.

Figure 6

Antibiofilm and antipersistence effects of the N,O acetal derivative 7b in MRSA. (A) Upper panel: In concentrations as high as 4MIC (8 µg/mL), vancomycin does not impair biofilm formation. (A) Bottom panel. Contrarily to vancomycin, 1/4MIC derivative 7b (32 µg/mL) reduced biofilm development, importantly. The inhibition of biofilm formation was followed by reduced expression of the global virulence regulators agr and sarA, with concomitant inhibitions of biofilm-associated genes including fnbA and psmα3, among others. (B) Vancomycin-persistent cells formed at condition of high bacterial load (10⁹ CFU) could be eliminated by the association of derivative 7b to vancomycin in the culture media.