Disruption of microbial cell morphology by Buxus macowanii

Antimicrobial activity of the plant extract was evaluated using Staphylococcus aureus (ATCC 25923), Clostridium perfringens (ATCC 13126), Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Staphylococcus epidermidis (ATCC 12228), Bacillus cereus (ATCC 13061) and the fungal species Candida albicans (ATCC 90028) and Candida tropicalis (ATCC 756). All the bacterial and fungal species were supplied by the National Health Laboratory Services, Bloemfontein, South Africa. All the microbial species were maintained in Mueller Hinton agar plates at temperatures of 4 °C. Prior to antimicrobial testing, the microorganisms were inoculated in Mueller Hinton broth and placed in a shaking incubator (100 rpm) for 24 h at 37 °C. Thereafter, a microbial suspension was prepared by diluting one millilitre of the culture in 100 ml of Mueller Hinton broth (1:100).

Antibacterial and antifungal activity of the extract were evaluated using the broth microdilution method developed by Eloff [19]. The dried plant extract was dissolved in 5% dimethyl sulfoxide (DMSO). One hundred microliters of the bacterial suspension was pipetted into the 96 microwell plate already containing 100 μl of diluted plant extract to make a final volume of 200 μl in each well. The concentration of the plant extract ranged from 0.16 mg/ml to 2.5 mg/ml. The first control wells were filled with the culture medium only and the second control only contained the bacterial suspension. The third control was the solvent of extraction, methanol, at 5% [20, 21] and the plant extract alone was the fourth control. Chloramphenicol (0.125 mg/ml) [22] was used as a positive control in bacteria and Amphotericin B (0.03–1 μg/ml) [23] was used in fungi. The micro-well plates for bacteria were incubated for 24and 48 h for yeast. Thereafter, 40 μl of 4 mg/ml Iodonitrotetrazolium salt solution was added in each well. Microbial growth was indicated by a change of colour ranging from pink to violet after 10–30 min incubation. All samples were tested in triplicate. The MIC was defined as the lowest concentration at which no microbial growth was recorded.

The cell wall of Bacillus cereus (ATCC 13061) was examined using Scanning and Transmission Electron Microscopy after treatment with the methanol extract of Buxus macowanii. B. cereus was treated with different concentrations (0.2–2.5 mg/ml) of the methanol extract of B. macowanii. The untreated samples (control) and the treated samples were incubated at 37 °C for 24 h. After incubation, the cells were washed twice with 0.1 M phosphate buffer solution (PBS, pH 7.0) and were fixed using 3% glutardialdehyde and 1% osmiumtetroxide and kept for 2 h at − 4 °C. Thereafter, the cells were subjected to dehydration in ethanol at successive concentrations of 50, 70, 95, 2X100% followed by critical point drying using CO₂ to remove ethanol. The samples were finally mounted on a specimen stub and coated with gold under vacuum followed by microscopic examination using SEM [4, 24]. For TEM, dehydrated bacterial cells were embedded by replacing acetone with epoxy to make slim sections suitable for microscopy examination. Samples were also further embedded using epoxy for 8 h at 70 °C in special moulds. The samples were cut into sections using an ultramicrotome and stained with 6% Uranyl and lead citrate followed by TEM examination [25, 26].

The Sulforhodamine B (SRB) assay was carried out to evaluate the toxicity of the methanol extract of B. macowanii against WI-38 Normal human fetal lung fibroblast cells [27]. The WI-38 cells l from European Collection of Cell Culture (ECACC) were maintained at 37 °C, 5% CO₂, 95% air and 100% relative humidity as a monolayer cell culture in EMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 50 μg/ml gentamicin. For cytotoxicity screening, the cells were inoculated in 96-well microtiter plates at a plating density of 10,000 cells/well and incubated for 24 h. After 24 h the cells were treated with the plant extracts at five different concentrations ranging from 6.25–100 μg/ml (5 x two-fold serial dilutions) and further incubated for 48 h. The untreated cells served as a control, the blank contained the medium without cells and etoposide was used as a standard at concentrations ranging from 100 μg/ml – 0.05 μg/ml (8 × 3-fold serial dilutions). After 48 h incubation, viable cells were fixed to the bottom of each well with cold 50% trichloroacetic acid, washed, dried and dyed with SRB. The protein-bound dye was removed with 10 mM Tris base and the optical density determination done at 540 nm using a multi-well spectrophotometer. The IC₅₀ considered as 50% cell growth inhibition was determined by non-linear regression using GraphPad Prism 6.0. Cytotoxic activity was divided into 4 categories: Low Hazard (IC₅₀ > 100 μg/ml), weak Hazard (30 μg/ml < IC₅₀ < 100 μg/ml), moderate Hazard (5 μg/ml < IC₅₀ < 30 μg/ml) and High Hazard (IC₅₀ < 5 μg/ml). The Selectivity Index (SI) was expressed by dividing the IC₅₀ with the MIC (SI = IC₅MIC) [28].

The dried extract was dissolved in ethyl acetate, centrifuged and analysed by GCMS using an Agilent 6890 N linked to a Mass Detector 5975B. Total ion chromatograms (TIC) and their associated spectra were acquired in full scan mode using the Chemstation software linked to the GCMS. Peaks and their associated spectra were then searched using the Wiley 375 Mass Spectral Database. Due to the complexity of the chromatograms, there is a certain degree of spectral overlap. To overcome this, Automated Mass Spectral Deconvolution and Identification Software (AMDIS) was used to separate multiple overlapping spectra lying within single chromatographic peaks.

The methanol extract from the twigs and leaves of B. macowanii showed antimicrobial activity against all the tested bacterial and fungal species. B. macowanii inhibited the growth of Staphylococcus aureus, Staphylococcus epidermidis, Candida albicans, Candida tropicalis, Clostridium perfrengens and Pseudomonans aeruginosa at the MIC of 2.5 mg/ml. B. macowanii also showed antimicrobial activity against Enterococcus faecalis, Escherichia coli and Bacillus cereus at an MIC of 1.2 mg/ml.

The effects exerted by the methanol extract of B. macowanii on the cell shape and surface, the cell wall, the cytoplasmic membrane as well as on cell division and cell viability of Bacillus cereus are presented in Table 1. B. cereus cells treated with B. macowanii showed concentration-dependent morphological changes that increased in severity as the concentration of the increased (Fig. 1). Under normal conditions the cells of B. cereus under the Scanning Electron Microscopy (SEM) were uniformly rod-shaped and had smooth cell surfaces (Table 1 and Fig. 1a).

The Transmission Electron Microscopy (TEM) micrographs also showed that the cytoplasmic membrane and cell wall were intact under normal conditions (Fig. 3a). The SEM images (Fig. 2c and d) depicted structural changes such as damage on the cell wall (DCW), completely worn out cell walls and cytoplasmic membrane. The extract also caused incomplete cell division (ICD), swelling of the cell (CWS), cytoplasmic membrane damage and loss of intracellular contents (Table 1). Cells treated with the positive control (Chloraphenicol) at 0.025 mg/ml also showed bacterial structural changes similar to those seen in cells treated with B. macowanii such as roughness of the cell (RC), swollen cells (SC) and incomplete cell division (ICD) (Fig. 2b). B. cereus cells treated with B. macowanii at 2.5 mg/ml showed major structural changes on the cell wall that led to cell death in SEM (Figs. 1f and 2). The effects of B. macowanii on the morphology of the bacterial cells examined using TEM also confirmed the damaging effects of the plant extract on the bacterial cell structure (Table 1). The treated cells showed cell wall distortion (Fig. 3b). loss of intracellular or cytoplasmic contents, incomplete cell division, rough cell surfaces and separation of the cytoplasmic membrane from the cell wall when treated with B. macowanii. Additionally, the cells also showed partial to complete loss of electron density of both their cell wall and cytoplasm (LCD).

The SRB assay showed that the methanol extract of B. macowanii with an IC₅₀ of 20.4 μg/ml was moderately hazardous (5 μg/ml < IC₅₀ < 30 μg/ml) against the WI-38 cell line while etoposide was highly hazardous with an IC₅₀ of 5.1. μg/ml. The Selectivity Index was 0.017.

GCMS analysis of the plant extract was conducted using the National Institution of Standard Technology (NIST) software and the Wiley Online Library. The identification of the compounds was based on the molecular weight, peak area and molecular formula, and only prominent peaks were selected for identification. A compound was only regarded as positively identified when the percentage of similarities was more than 90%. Neophytadiene from B. macowanii was detected at a retention time of 10.92 (Fig. 4).