Bacterial strains and growth conditions

The MRSA (NCTC 12493) used in this study was supplied by the National Collection of Type Cultures (Public Health England, Colindale, London, UK). Mueller–Hinton (MH) agar or Mueller–Hinton (MH) broth was employed for preculturing and preparing the bacterial suspension for MRSA.

Preparation of medicinal plant extracts

The 68 dried commercial medicinal plants with specific parts of plant used for this study are listed in Table 1. The processing of plant materials involved grinding the dried medicinal plants into fine powder using a planetary ball mill (PM100, Retsch, UK). The fine plant material was suspended in distilled water at 1:1000 (w/v) and subjected to ultrasonic treatment (VWR Ltd, UK) at 45 kHz for 15 min, followed by immersion in a boiling water bath for an additional 30 min. The resulting extract was centrifuged at 12,000 rpm for 10 min at room temperature (RT) and the supernatant was filtered through a 0.45 μm syringe filter (Merck Millipore Ltd, Ireland) before testing.

Table 1 Medicinal plants (n = 68) screened for antibacterial activity against MRSA (NCTC 12493) and their MIC (mg/mL)Anti-MRSA activity screening

Following the guidelines set by the British Society for Antimicrobial Chemotherapy (BSAC), the microdilution method was employed to determine both MBC and MIC [8]. Extracts of plants and antibiotic vancomycin (CAS 1404-93-9; Sigma-Aldrich Ltd, Poole, UK) were serially diluted in MH broth at concentrations ranging from 0.9 to 1000 mg/L and 0.25 to 128 mg/L, respectively, and added to microtiter plates (M2311-100EA, Greiner, UK). The extracts exhibited a subtle green or yellow tint, which did not obstruct or interfere with the observation of bacterial growth manifested as turbidity within the well.

MRSA suspensions, initially adjusted to McFarland standards (0.5), were diluted at a ratio of 1:100 in MH broth. A sterile (negative) control containing MH broth only, a growth control with a bacterial suspension, and a positive control with vancomycin were included in each 96-well plate. The plates were incubated for 18–20 h at 37 °C. The MIC value was established as the lowest concentration of the test sample that completely inhibited bacterial growth, confirmed by the absence of visible growth under the specified experimental conditions. The determination of MBC involved sub-culturing 10 μL of each dilution from and above MIC wells and spotting onto Mueller–Hinton (MH) agar (Oxoid, CM0337; Hampshire, UK) plate and incubated for a further 24 h at 37 °C. The MBC was defined as the lowest micro-dilution of antimicrobial compound that prevents organism growth on the agar plate, with a 99.9% killing (3 log reduction) in CFU/mL compared to untreated organism (growth control) [9].

The kinetics of bacterial growth were studied to evaluate the efficacy of medicinal plant extracts in inhibiting bacteria. This assessment was carried out at three concentrations (MIC, 2MIC, and 4MIC) at 37 °C, using a microplate reader (SynergyTM HT, BioTek, Winooski, Vermont, USA) for accurate measurements. The OD at 600 nm of each well was automatically measured and recorded every 30 min over 24 h. Data were acquired using Gen5 1.10 software, exported to Microsoft Excel for processing, and expressed as the mean value of three replicates. Analysis was conducted using Microsoft Excel and GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA). Normalization for comparison involved aligning the same starting point of all datasets, with different values shown on the y-axis for each sample.

The time-kill assay was carried out to determine the rate and extent of microbial killing over time by the plant-derived substances. A bacterial suspension (1 × 106 CFU/mL) was treated with a medicinal plant extract (500 μL) at concentrations of 1, 2, 3 and 4 × MIC (7.8 μg/mL, 15.6 μg/mL, 23.4 μg/mL, 31.2 μg/mL) of R. palmatum, Arctium lappa L. and P. suffructicosa, respectively. The mixture was incubated at 37 °C with gentle agitation in a Labwit shaker (ZWY-100H, Australia). Samples (10 μL) were taken at 0, 1, 3, 6, 20 and 24 h, serially diluted, and plated on MH agar. After incubation for 24 h at 37 °C, a bactericidal effect was defined as a 3-log reduction in viable cell count. The time-kill assays were performed in triplicate, and GraphPad Prism 7.0 was used for graphical representation.

Evaluation of the synergistic effect

A broth checkerboard microdilution assay was employed to investigate combined treatments of vancomycin and plant extracts [10]. The assay featured a two-dimensional checkerboard with two-fold dilutions of the antibiotic vancomycin (0.25–128 mg/L) horizontally and plant extract (0.9–1000 mg/L) vertically. The bacterial suspension (1 × 106 CFU/mL) was added and incubated for 18–20 h at 37 °C in 96-well plates. Controls included bacterial suspension, antibiotics and plant dilution controls. The MIC was determined to be the lowest concentration of plant extracts and antibiotic without visible bacterial growth. Effective combinations were identified, and the fractional inhibitory concentration (FIC) was calculated for the first clear well containing both antimicrobial agents:

FIC of A (plant extract) = MICA+B in combination/ MICA alone.

FIC of B (antibiotic) = MICB+A in combination/ MICB alone.

The FIC index (FICI), the sum of both calculated FIC values, was interpreted as follows: synergistic (≤ 0.5), additive (> 0.5 and ≤ 1); indifferent (> 1 and ≤ 4); antagonistic (> 4) [10].

Cytotoxicity evaluation of potential plant candidates

The cytotoxicity of plant extracts against hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma (Caco-2) cell lines was assessed using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) colorimetric assay [11]. Cell lines were subcultured at 37 °C in a CO2 incubator with 5% CO2 gas and 95% humidity, using minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, 1 mM sodium pyruvate, and 2 mM L-glutamine. All reagents used were purchased from Life Technologies (Paisley, Scotland, UK). Cell suspension (100 μL) was seeded into BD Falcon 96-well microtiter plates (BD Biosciences, US) at a density of 1 × 104 cells/well and 5 × 103 cells/well for HepG2 and Caco-2 cells, respectively. After a 24 h cell attachment period, plant extracts (100 μL) at dilutions ranging from 0.0002 to 10 mg/mL were applied and incubated for 48 h. Following cell washing with phosphate buffered saline (PBS), 50 μL of 2 mg/mL MTT solution was added. After a 4 h incubation, the supernatant was discarded, and 200 μL of preheated dimethyl sulfoxide (DMSO) was introduced to dissolve formazan crystals. The plate was incubated at 37 °C with agitation for a further 10 min. Absorbance was measured at 570 nm with a reference filter at 630 nm using a TECAN microtiter plate reader (Safire II, BASIC). Viability was determined by calculating the percentage of sample absorbance relative to the untreated control. According to ISO 10993-5, the standard for test in vitro cytotoxicity, cell viability percentages less than 40% indicate significant cytotoxicity, values between 40 and 60% suggest moderate cytotoxic effects. Viability within the range of 60% to 80% is considered weak, and percentages above 80% are classified as non-cytotoxic [11].

FM analysis

Cultures of MRSA in the exponential phase were used to prepare a cell suspension at a concentration of 1 × 106 CFU/mL. Subsequently, 500 μL of each medicinal plant extracts of R. palmatum, A. lappa and P. suffructicosa at their MIC (7.8 μg/mL) diluted in MH broth was introduced into the prepared bacterial cell suspension (500 μL). The mixture was then incubated at 37 °C for 20 h before applying fluorescent dyes. As a growth control, a 500 μL cell suspension in 500 μL of MH broth was used. Following incubation, cells were rinsed, pelleted, and treated with 20 μl solution of 4% (v/v) formaldehyde for 10 min at room temperature in the absence of light. For nucleus staining, 20 μl Hoechst 33342 (Life Technologies, Scotland, UK) at 2 μM was added and incubated with the cells for 10 min at room temperature (RT) followed by three washes with PBS. Subsequently, the cells were counterstained with the red membrane dye FM 4–64 64 (Thermo Fisher Scientific, UK) at a concentration of 5 μg/mL for 5 min at RT, followed by additional 2 min on ice. The staining process was protected from light. FM images were captured immediately using the Olympus BX63 fluorescent microscope equipped with an Olympus DP74 camera with an excitation wavelength of 515 nm and an emission wavelength of 640 nm for the membrane, Excitation/Emission: 361/497 for the nuclear images, and co-staining using CellSens Dimension imaging acquisition software (Olympus, Center Valley, USA).

TEM analysis

Following a 24 h exposure to the medicinal extracts of R. palmatum, A. lappa and P. suffructicosa, bacterial cells were fixed for 2 h at RT in 2.5% (v/v) glutaraldehyde and 1.5% (v/v) paraformaldehyde buffered in PHEM (pH 7). The PHEM buffer comprised of 60 mM PIPES (piperazine-N, N'-bis), 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 10 mM EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and 2 mM MgCl2. Afterward, the cells underwent three times washes with PHEM buffer and resuspended in a 2% agarose solution. The agarose embedding technique, typically employed for tissue sample preparation, was repurposed with slight modifications for the current study to preserve bacterial cell integrity and ensure optimal image quality. The embedded cell pellets were processed using an automated tissue processor Leica EM-TP. Samples were first rinsed in PHEM buffer, then fixed, and stained with 1% (v/v) osmium tetroxide for 45 min, followed by dehydrated with a sequential ethanol series (30%, 50%, 75%, 95%, 3 × 100%), and finally three times washes in 100% acetone before embedding in Spurr resin (Electron Microscopy Sciences, Hatfield, USA). Semi-thin Sects (1 μm) were cut with a glass knife, followed by further cutting into ultrathin Sects. (90 nm) using a diamond knife via Ultracut-UCT ultramicrotome (Leica Microsystems, Vienna, Austria). Ultrathin sections were placed on formvar-coated, 300-mesh copper grids, and post-stained in the 2% (w/v) uranyl acetate and 1% (v/v) osmium tetroxide. The micrographs were captured using TEM (Jeol JEM-1400, USA), operated at 80 kV and magnification between × 1000 and × 50,000.