In Vitro Antibacterial, Anti-Adhesive and Anti-Biofilm Activities of Krameria lappacea (Dombey) Burdet & B.B. Simpson Root Extract against Methicillin-Resistant Staphylococcus aureus Strains

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) represents a serious threat to public health, due to its large variety of pathogenetic mechanisms. Accordingly, the present study aimed to investigate the anti-MRSA activities of Krameria lappacea, a medicinal plant native to South America. Through Ultra-High-Performance Liquid Chromatography coupled with High-Resolution Mass spectrometry, we analyzed the chemical composition of Krameria lappacea root extract (KLRE). The antibacterial activity of KLRE was determined by the broth microdilution method, also including the minimum biofilm inhibitory concentration and minimum biofilm eradication concentration. Besides, we evaluated the effect on adhesion and invasion of human lung carcinoma A549 cell line by MRSA strains. The obtained results revealed an interesting antimicrobial action of this extract, which efficiently inhibit the growth, biofilm formation, adhesion and invasion of MRSA strains. Furthermore, the chemical analysis revealed the presence in the extract of several flavonoid compounds and type-A and type-B proanthocyanidins, which are known for their anti-adhesive effects. Taken together, our findings showed an interesting antimicrobial activity of KLRE, giving an important contribution to the current knowledge on the biological activities of this plant.

Keywords: A549 cells; Krameria lappacea; adhesion and invasion; biofilm; methicillin-resistant Staphylococcus aureus; proanthocyanidins.

Figure 1

Schematic model of the virulence factors of S. aureus. (A). Cell structure of S. aureus. The cell-wall contains several proteins, including Protein A which binds the Fc fragment of immunoglobulin-G (IgG), blocking its phagocytic activity, and the adhesins, which mediate the adhesion and invasion of the bacterium to the host cell. Besides, S. aureus synthesizes different exoenzymes, which facilitate its dissemination and tissue invasion. This pathogen is also able to secrete a large variety of toxins, responsible for severe diseases. (B). Molecular mechanisms involved in the adhesion and invasion of S. aureus to host cell. The binding of surface adhesins with specific cellular receptors or with the component of extracellular matrix triggers a signalling cascade that culminates with bacterial internalization. (C). Biofilm formation stages. Favorable environment conditions promote the formation of biofilm, which consists of three different stages: attachment of free bacteria (planktonic cells) to the abiotic surface (e.g., medical devices); initial synthesis of biofilm and formation of bacterial colony inside the biofilm (sessile bacteria) and, finally, complete biofilm formation. The bacteria embedded in the biofilm can reduce their metabolic activity, becoming quiescent. Under specific environment signals, the sessile bacteria can switch to planktonic form, provoking reinfection.

Figure 2

Biofilm quantification assay of methicillin-resistant Staphylococcus aureus strains. (A). Representative figure of biofilm quantification by crystal violet assay in 96-well microplate. (B). Quantitative analysis of biofilm formation. Bacteria were categorized as non-adherent (O.D. ≤ 0.120: yellow histograms), weakly adherent (O.D. > 0.120: red histograms) and strongly adherent (O.D. > 0.240: blue histograms) [30]. In the x-axis are reported the tested strains for biofilm production: one standard (S. aureus ATCC 6538) and ten MRSA clinical isolates (progressively numbered); in the y axis, the OD₅₇₀ values of the tested bacterial strains. The bars represent the means ± SD of three independent experiments (SD = standard deviation).

Figure 3

Minimal biofilm inhibitory concentration (MBIC) of ciprofloxacin and K. lappacea root extract (KLRE) on S. aureus ATCC 6538 and MRSA 8. S. aureus ATCC 6538 and MRSA 8 were exposed to different concentrations of ciprofloxacin (A,C), ranging from 0.12 to 64.00 μg/mL or KLRE (B,D), ranging from 0.50 to 256.00 μg/mL, for 24 h. In the x-axis are reported the biofilm formation and metabolic activity of untreated (positive control) and ciprofloxacin/KLRE treated bacterial strains. The values are expressed as a percentage of positive control. The bars represent the means ± SD of three independent experiments (SD = standard deviation). Statistically significant differences, determined by two-way analysis of variance ANOVA, are indicated ^§ p ≤ 0.0001 versus positive control.

Figure 4

Minimal biofilm eradication concentration (MBEC) of ciprofloxacin and K. lappacea root extract (KLRE) on S. aureus ATCC 6538 and MRSA 8. S. aureus ATCC 6538 and MRSA 8 were exposed to different concentrations of ciprofloxacin (A,C), ranging from 0.12 to 64.00 μg/mL or KLRE (B,D), ranging from 0.50 to 256.00 μg/mL, for 24 h. In the x-axis are reported the biofilm eradication and metabolic activity of untreated (positive control) and ciprofloxacin/KLRE treated bacterial strains. The values are expressed as a percentage of positive control. The bars represent the means ± SD of three independent experiments (SD = standard deviation). Statistically significant differences, determined by two-way analysis of variance ANOVA, are indicated: * p ≤ 0.05, ^§ p ≤ 0.0001 versus positive control.

Figure 5

Growth curves of standard S. aureus ATCC 6538 and MRSA 8 exposed to different concentrations of Krameria lappacea root extract (KLRE) or ciprofloxacin, for 72 h. Growth trend of S. aureus ATCC 6538 (A) and MRSA 8 (C) untreated (positive control) and treated with increased concentrations of ciprofloxacin (Cip) ranging from 0.12 to 64.00 μg/mL. Growth trend of S. aureus ATCC 6538 (B) and MRSA 8 (D) untreated (positive control) and treated with increased concentrations of KLRE, ranging from 0.50 to 256.00 μg/mL. In the x-axis is reported the incubation time; in the y axis, the OD₅₇₀ values of the two tested bacterial strains.

Figure 6

Effect of K. lappacea root extract (KLRE) on the adhesion of S. aureus ATCC 6538 and MRSA 8 to human lung A549 cell line. (A1, A2). Representative photographs of plate bacterial count. (B). Quantitative analysis of bacterial adhesion to A549 cells. Light grey histograms: A549 cells infected with S. aureus ATCC 6538 or MRSA 8 strains (positive controls). Violet histograms: A549 cells infected with standard or clinical strains and simultaneously treated with 32.00 μg/mL of KLRE. Pink histograms: A549 cells infected with standard or clinical strains and simultaneously treated with 64.00 μg/mL of KLRE. The values are expressed as colony-forming unit mL⁻¹ (CFU/mL). The bars represent means ± SD of three independent experiments performed in triplicate (SD = standard deviation). Statistically significant differences, determined by one-way analysis of variance ANOVA, are indicated: * p ≤ 0.05, § p ≤ 0.0001 versus positive control.

Figure 7

Effect of K. lappacea root extract (KLRE) on the invasion of S. aureus ATCC 6538 and MRSA 8 to human lung A549 cell line. (A1, A2). Representative photographs of plate bacterial count. (B). Quantitative analysis of bacterial invasion to A549 cells. Light grey histogram: A549 cells infected with MRSA 8 strain (positive control). Violet histogram: A549 cells infected with MRSA 8 and simultaneously treated with 32.00 μg/mL of KLRE. Pink histogram: A549 cells infected with MRSA 8 and simultaneously treated with 64.00 μg/mL of KLRE. The values are expressed as colony-forming unit mL⁻¹ (CFU/mL). The bars represent means ± SD of three independent experiments performed in triplicate (SD = standard deviation). Statistically significant differences, determined by one-way analysis of variance (ANOVA), are indicated: * p ≤ 0.05, p ≤ 0.0001 versus positive control.

Figure 8

S. aureus ATCC 6538 (panels A-D) and MRSA 8 (panels E-G) adhesion to human lung A549 cells visualized using Gram staining (40× magnification A-G; 100X A’-G’). (A,A’): uninfected cells (negative control); (B,B’): A549 cells infected with S. aureus ATCC 6538 (positive control); (C,C’): A549 cells infected with S. aureus ATCC 6538 and simultaneously treated with 32.00 μg/mL of K. lappacea root extract (KLRE); (D,D’): A549 cells infected with S. aureus ATCC 6538 and simultaneously treated with 64.00 μg/mL of KLRE; (E,E’): A549 cells infected with MRSA 8 (positive control); (F,F’): A549 cells infected with MRSA 8 and simultaneously treated with 32.00 μg/mL of KLRE; (G,G’): A549 cells infected with MRSA 8 and simultaneously treated with 64.00 μg/mL of KLRE. Adherent colonies of S. aureus to A549 cell surface (black arrows).

Figure 9

Chemical structure of the detected flavonoid and proanthocyanidin compounds in KLRE.

Figure 10

Krameria lappacea roots.