Analysis of the Antimicrobial and Anti-Biofilm Activity of Natural Compounds and Their Analogues against Staphylococcus aureus Isolates

Abstract

(1) Background: Staphylococcus aureus (S. aureus) is one of the most frequent causes of biofilm-associated infections. With the emergence of antibiotic-resistant, especially methicillin-resistant S. aureus (MRSA), there is an urgent need to discover novel inhibitory compounds against this clinically important pathogen. In this study, we evaluated the antimicrobial and anti-biofilm activity of 11 compounds, including phenyl propenes and phenolic aldehydes, eugenol, ferulic acid, sinapic acid, salicylaldehyde, vanillin, cinnamoyl acid, and aldehydes, against drug-resistant S. aureus isolates. (2) Methods: Thirty-two clinical S. aureus isolates were obtained from Alkhidmat Diagnostic Center and Blood Bank, Karachi, Pakistan, and screened for biofilm-forming potential, and susceptibility/resistance against ciprofloxacin, chloramphenicol, ampicillin, amikacin, cephalothin, clindamycin, streptomycin, and gentamicin using the Kirby-Bauer disk diffusion method. Subsequently, 5 representative clinical isolates were selected and used to test the antimicrobial and anti-biofilm potential of 11 compounds using both qualitative and quantitative assays, followed by qPCR analysis to examine the differences in the expression levels of biofilm-forming genes (ica-A, fnb-B, clf-A and cna) in treated (with natural compounds and their derivatives) and untreated isolates. (3) Results: All isolates were found to be multi-drug resistant and dominant biofilm formers. The individual Minimum Inhibitory Concentration (MIC) of natural compounds and their analogues ranged from 0.75−160 mg/mL. Furthermore, the compounds, Salicylaldehyde (SALI), Vanillin (VAN), α-methyl-trans-cinnamaldehyde (A-MT), and trans-4-nitrocinnamic acid (T4N) exhibited significant (15−92%) biofilm inhibition/reduction percentage capacity at the concentration of 1−10 mg/mL. Gene expression analysis showed that salicylaldehyde, α-methyl-trans-cinnamaldehyde, and α-bromo-trans-cinnamaldehyde resulted in a significant (p < 0.05) downregulation of the expression of ica-A, clf-A, and fnb-A genes compared to the untreated resistant isolate. (4) Conclusions: The natural compounds and their analogues used in this study exhibited significant antimicrobial and anti-biofilm activity against S. aureus. Biofilms persist as the main concern in clinical settings. These compounds may serve as potential candidate drug molecules against biofilm forming S. aureus.

Keywords: Staphylococcus aureus; anti-biofilm; antimicrobial; gene expression; natural compounds.

Figure 1

Qualitative and quantitative analysis of biofilm formation by S. aureus isolates: (A) biofilm staining intensity, for each isolate, measured using ImageJ software. The plot shows mean staining intensity (arbitrary unit) at the Y-axis, while the X-axis shows the isolates tested (S1–S32). (B) spectrophotometric analysis (OD630 nm) for the biofilm formation for each isolate. The plot shows mean OD values with standard deviation as an error bar on Y-axis, while isolates on X-axis.

Figure 2

Expression of genes associated with biofilm formation in treated and untreated samples. The expression (∆Ct) of (A) ica-A, (B) clf-A, (C) fnb-A, and (D) cna genes in sensitive (S11) and resistant (S25) isolates with and without all 11 test compounds treatment. The asterisk * indicates p-value < 0.05 and # indicates p-value <0.001.