Evaluation of antimicrobial activity of chondrillasterol isolated from Vernonia adoensis (Asteraceae)

Abstract

Background: Bacteria have developed resistance to most of the current antibiotics. There is evidence suggesting that plant-derived compounds have a potential for interacting with biological processes. One of the plants commonly used in African ethnomedicine is Vernonia adoensis from the Asteraceae family. The leaves of the plant have been reported to have antimicrobial activity. Hence, the aim of this study was to isolate the bioactive compounds from the leaf extract and evaluate their antibacterial activity on Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa. In addition, the effect of the isolated compound on biofilms of P. aeruginosa was determined.

Methods: Isolation of phytochemicals from the leaves of V. adoensis was done using column chromatography. Preparative TLC was used to further isolate mixed compounds in the fractions. Nuclear magnetic resonance spectroscopy and mass spectrometry was used to identify the isolated pure compounds. The broth microdilution assay was carried out to evaluate the antibacterial activity of the isolated compound on P. aeruginosa, S. aureus and K. pneumoniae. Crystal violet staining technique was used to evaluate the effect of the isolated compound on biofilms of P. aeruginosa.

Results: The compound isolated from V. adoensis was identified as chondrillasterol. Chondrillasterol exhibited 25, 38 and 65% inhibition of growth on S. aureus, K. pneumoniae and P. aeruginosa respectively. At 1.6 μg/mL chondrillasterol completely disrupted mature biofilm of P. aeruginosa while at 100 μg/mL the compound completely inhibited formation of biofilms of the bacteria.

Conclusion: Chondrillasterol isolated from V. adoensis has antibacterial properties against S. aureus, K. pneumoniae and P. aeruginosa. The compound also has biofilm inhibition and disruption activity against P. aeruginosa biofilms. Thus, the active phytochemical could be a useful template for the development of new antimicrobial agents with both antibacterial and antibiofilm activity.

Keywords: Antibacterial; Biofilms; Chondrillasterol; Vernonia adoensis.

Fig. 1

Plate layout for testing effect of chondrillasterol on mature biofilm. A two-fold serial dilution was carried out for chondrillasterol starting from 200 μg/mL. Increasing concentrations of chondrillasterol were added starting from column C1. Controls included: (i) the extract being incubated alone (rows 1 and 2), (ii) media alone (column 10 and 11) and (iii) viable bacterial cells incubated alone (column 12)

Fig. 2

TLC profile of compounds from column chromatography. Single spot represents a pure compound while more than one spot represents a mixture of compounds isolated from V. adoensis

Fig. 3

Structure of Chondrillasterol. Data to elucidate the structure was obtained by NMR and MS studies

Fig. 4

The effect of chondrillasterol on growth of bacteria pathogens. The pathogens were susceptible to chondrillasterol and (a) is % remaining viable cells for P. aeruginosa, and (b) is % of remaining viable cells for K. pneumoniae while (c) is % of remaining viable cells for S. aureus following exposure to chondrillasterol. Concentrations of 1.6 × 10⁶ cfu/mL of bacteria were used. Values are expressed as mean cell density at 590 nm wavelength ± the standard deviation (n = 4). TSB is tryptic soy broth

Fig. 5

The effect of chondrillasterol on nucleic acid leakage in P. aeruginosa. Cells and control (0.1% SDS) were exposed to chondrillasterol in triplicate and the nucleic acid leakage was quantified. The test for significance was carried out by comparing the fluorescence of the samples to untreated cells (cells only), ***P < 0.0001. SDS: sodium dodecyl sulphate

Fig. 6

Effect of chondrillasterol on P. aeruginosa biofilm formation. P. aeruginosa (1.6 × 10⁶ cfu/mL) was incubated with different concentrations of the pure compound for 72 h. at 37 °C and the resulting biofilm mass was stained using crystal violet staining. The dye was extracted using ethanol and quantified spectrophotometrically at 590 nm. The absorbance values of crystal violet are directly proportional to the amount of biofilm that had formed. The error bars indicate the standard deviation from mean (n = 4). Dunnet multiple comparison test was used to compare columns using GraphPad Prism (GraphPad Software Inc., San Diego, California, USA) version 5.03. The asterisks (*) indicate statistically significant differences with the positive control (0.0) which represented cells only without extract * < 0.05 *** < 0.0001. TSB is tryptic soy broth.

Fig. 7

Effect of chondrillasterol on preformed biofilms by P. aeruginosa. Cells were incubated for 72 h. at 37 °C. Planktonic cells were aspirated and different concentrations of chondrillasterol added to the preformed biofilm. The plate was further incubated for 24 h. after which it was washed to remove planktonic cells. Remaining biofilm mass was stained using crystal violet staining and quantified at 590 nm. The absorbance values of crystal violet are directly proportional to the amount of biofilm that remained intact after exposure to extract. The error bars indicate the standard deviation from mean (n = 4). TSB is tryptic soy broth