Antimicrobial and Antibiofilm Effect of 4,4'-Dihydroxy-azobenzene against Clinically Resistant Staphylococci

Abstract

The spread of antibiotic resistance among human and animal pathogens is one of the more significant public health concerns. Moreover, the restrictions on the use of particular antibiotics can limit the options for the treatment of infections in veterinary clinical practice. In this context, searching for alternative antimicrobial substances is crucial nowadays. In this study, 4,4'-dihydroxy-azobenzene (DHAB) was tested for its potential in vitro as an antimicrobial agent against two relevant human and animal pathogens, namely Staphylococcus aureus and Staphylococcus pseudintermedius. The values of minimal inhibitory concentration (MIC) were 64 and 32 mg/L respectively, and they comparable to other azo compounds of probed antimicrobial activity. In addition, the minimal bactericidal concentrations (MCB) were 256 and 64 mg/L. The mechanism by which DHAB produces toxicity in staphylococci has been investigated. DHAB caused membrane damage as revealed by the increase in thiobarbituric acid reactive substances (TBARS) such as malondialdehyde. Furthermore, differential induction of the enzymes peroxidases and superoxide dismutase in S. aureus and S. pseudintermedius suggested their prevalent role in ROS-scavenging due to the oxidative burst induced by this compound in either species. In addition, this substance was able to inhibit the formation of biofilms by both bacteria as observed by colorimetric tests and scanning electron microscopy. In order to assess the relevance of DHAB against clinical strains of MRSA, 10 clinical isolates resistant to either methicillin or daptomycin were assayed; 80% of them gave values of CMI and CMB similar to those of the control S. aureus strain. Finally, cutaneous plasters containing a composite formed by an agar base supplemented with DHAB were designed. These plasters were able to inhibit in vitro the growth of S. aureus and S. pseudintermedius, particularly the later, and this suggests that this substance could be a promising candidate as an alternative to antibiotics in the treatment of animal skin infections, as it has been proven that the toxicity of this substance is very low particularly at a dermal level.

Keywords: Staphylococcus aureus; Staphylococcus pseudintermedius; azo compounds; biofilms; clinical samples; cutaneous plasters; oxidative stress.

Figure 1

Semi-quantitative analysis of the inhibitory effect of DHAB on the growth of Staphylococcus spp. Using the disks test. Data are mean ± standard deviation of three determinations.

Figure 2

Structure of azo compounds tested as antimicrobial substances.

Figure 3

Levels of thiobarboturic acid reactive substances (TBARS) in cells of S. pseudintermedius and S. aureus treated with DHAB for 24 h at two different doses: MIC and MCB. C: control cells grown in TSB. Data are means of three determinations in two independent cultures, and significant differences at p < 0.05 are indicated by different letters; (a) correspond to the level of MDA in the strains cultivated in the absence of DHAB; (b, c) indicate significant differences in the presence of DHAB.

Figure 4

Effect of DHAB on the formation of biofilms in vitro by S. pseudintermedius and S. aureus. Data are means ± standard deviations of three independent determinations.

Figure 5

Observations by SEM of biofilms formed by staphylococcal species after 24 h on glass slides and the effect of DHAB on biofilm formation. (A) Biofilm formed by S. pseudintermedius in the absence of DHAB; (B) closer view of colonies of S. pseudintermedius in the absence of DHAB. Yellow arrows point out extracellular material (particulated material and fibers) secreted by bacteria for attachment; (C) small colonies of S. pseudintermedius in the presence of DHAB at MIC; (D) individual cells of S. pseudintermedius in the presence of DHAB at MBC. Note the presence of small and deformed cells; (E) biofilm formed by S. aureus in the absence of DHAB; (F) colonies of S. aureus in the absence of DHAB. Yellow arrows show extracellular material (particulated material and fibers) secreted by bacteria for attachment; (G) small colonies of S. aureus in the presence of DHAB at MIC; and (H) individual cells of S. pseudintermedius in the presence of DHAB at MBC. Note the presence of small and deformed cells at a high DHAB concentration.

Figure 6

Analysis of the genetic diversity of ten clinically relevant isolates of Staphylococcus aureus by PFGE.

Figure 7

(A) Dermal sterile cotton plaster loaded with composite material made from 0.9% agar containing DHAB. (B) Aspect of the plaster from the bottom of the plate. (C) Growth inhibition halo observed for S. pseudintermedius after 24 h of incubation at 37 °C. (D) Growth inhibition observed for S. aureus after 24 h of incubation at 37 °C.