In vitro activity against multi-drug resistant bacteria and cytotoxicity of lichens collected from Mount Cameroon

2.1 Preparation and phytochemical analysis of extracts

All the lichens were collected in April 2016 from Mount Cameroon found in the Southwest of Cameroon and one of them also collected at Ekona, a village at the foot of the mountain. They were collected by Dr. Ayuk Elizabeth Orock, a Lichenologist in the Department of Botany, Faculty of Science, University of Buea. The lichens include Usnea articulata, Usnea florida, Leptigium gelatinisum, Physcia parietina, Ramalina sinensis and Xanthoparmelia plitti. Crude extracts were prepared as previously described (Mbah et al., 2012). The lichens were chopped, air-dried under laboratory conditions at room temperature for two weeks. Each dried material was ground to fine powder, weighed and macerated in methanol then filtered through Whatman filter paper No.1 into weighed bottles and kept to dry at room temperature. Maceration was done four times for three days each for plant materials available in small quantities to increase yield. The filtrate was evaporated (Rotavapor BÜCHI R-200), the extracts kept at room temperature to dry to constant mass, then weighed and stored at 4 ^°C until tested. Phytochemical tests were performed for presence of secondary metabolites in the extracts as described by Hossain et al., (2013). Briefly about 1 g of each extract was dissolved in the appropriate solvent and reacted with corresponding reagents to detect specific functional groups. The Dragendorff test showed an orange spot for alkaloids; the cyanidin test produced a dark red coloration for flavonoid; the Liebermann-Burchard test showed blue green indicating steroids and reddish coloration for terpenoids; persistent foam was seen in the frothing test showing saponins and the ferric chloride test produced a blue black coloration indicating presence of tannins.

2.2 Bacterial cells

Clinical strains of bacteria were obtained from the Regional Hospital Annex Buea, South West Region of Cameroon. They were identified by culture and biochemical tests using API 20E test kit (Biomérieux, France), and the cells stored in 10% sterile glycerol in Mueller Hinton Broth (Liofilchem, Italy) at −20 °C until use. Six control strains were included namely Escherichia coli KTE 181 (NR 32771), NR-515 Salmonella choleraesius and NR-4311 Salmonella enterica, NR-46003 Staphylococcus aureus strain 315, NRS-848 Staphylococcus epidermidis, were obtained from BEI Resources and P. aeruginosa Boston 41501 (ATCC 27853) from American Type Culture Collection (Manassas, USA).

2.3 Antibacterial susceptibility test

The Kirby Bauer disc diffusion method as recommended by the Clinical and Laboratory Standard Institute (CLSI, 2012) was employed as previously described (Akoachere et al., 2014) with some modifications. A uniform spread of bacterial cells on Mueller–Hinton agar was made using 100 μL of McFarland 0.5 (approximately 1.5 × 10⁸ CFUs/mL) bacterial suspension. Plates were allowed to dry for 3–5 minutes. Discs of twelve reference antibiotics (LiofilChem, Italy), were embedded on the surface of the bacteria spread. Plates were incubated at 37 °C in a heating incubator (DHP-9052, England) for 18 to 24 h and diameter of zones of inhibition measured.

2.4 Determination of antibacterial activity

This was done as previously described by Mbah et al., (2012) with some modifications. A 10 mg/100 μL solution of each lichen methanol extract was prepared in dimethyl sulfoxide (DMSO). Sterile paper discs (6 mm diameter) were gently fixed on agar plates prepared as in the Kirby-Bauer method above. Ten (10) microlitres of test solution containing 1 mg extract was transferred on to the corresponding disc. Gentamicin standard disc (10 μg) and 10 μL of DMSO were included as positive and negative controls respectively. The plate was incubated at 37 °C (DHP-9052, England) for 18–24 h and diameter of zone of inhibition measured.

The minimum inhibitory concentration (MIC) was determined as previously described (Mbah et al., 2012) with some modifications. Briefly, extract stock solutions were prepared by completely dissolving 40 mg of crude extract in 200 μL of DMSO and 800 μL of Mueller Hinton (MH) broth added and mixed. Each stock was diluted in MH broth to give 32, 24, 16, 8, 4, 2, and 1 mg/mL test solutions (final test range 0.25 to 10 mg/mL). The microtire plate wells comprised 50 μL of extract test solution or gentamicin (50 μg/mL) positive control,130 μL of MH broth containing bromothymol blue indicator followed by 20 μL of bacteria suspension (6.0 × 10⁶ CFUs/mL) to all required wells. Saline (50 μL of 0.85%) was added to the negative control. The optical densities (OD) of wells were read at 595 nm (Emax microplate reader, Molecular Devices, USA). The plates was incubated at 37 °C (DHP-9052, England) for 24 h, read visually for inhibition and the OD read again. All experiments were done in duplicate. Percentage inhibition of bacteria cell growth after 24 h of incubation was calculated using the formula: $$\%\text{Inhibition =}\frac{\mathrm{\Delta }\text{OD}\left(\text{NC}\right)-\mathrm{\Delta }\text{OD}(\text{test}\text{substance})}{\mathrm{\Delta }\text{OD}\left(\text{NC}\right)}$$ where ΔOD (test substance) = OD 24 h – OD baseline (0 h), ΔOD (NC) = OD 24 h – OD baseline in the negative control well (NC); ΔOD: change in optical density.

MIC was taken as the lowest concentration which showed >50% inhibition of bacterial growth. For minimum bactericidal concentration (MBC) assay required well contents were mixed and 20 μL of MIC well diluted in 180 μL of fresh medium. The OD was read before and after incubation as above. The percentage growth of bacteria cells was calculated using the following formula and MBC was recorded as the lowest concentration of MIC wells with <50% growth. $$\%\text{Growth}=\left[\mathrm{\Delta }\text{OD}/\text{OD}\left(\text{baseline}\right)\right]\times 100,$$ where (ΔOD) = OD 24 h – OD baseline (0 hour)

2.5 Cytotoxicity test

All reagents used were purchased from Sigma Aldrich (USA). RPMI-1640 complete culture medium (CCM) containing sodium bicarbonate was supplemented with 25 mM HEPES, 0.3g gamma-irradiated L-glutamine powder, 5%heat inactivated new born calf serum (NBCS), 200 units/mL penicillin and 200 μg/mL streptomycin and 0.25 μg/mL amphotericin B, pH 7.4. Monkey kidney epithelial cells (LLC-MK2; ATCC, USA) were cultured (37 °C, 5% CO₂ in humidified air; Heracell150i, USA) to confluence using standard protocol and trypsinized to dislodge, centrifuged in incomplete culture medium (ICM, without NBCS) as above and re-suspended in CCM and counted by microscopy. The cells (3000 cells/ 100 μL in CCM) were seeded into 96-wells flat bottom microtitre plate (Becton Dickinson,) and incubated same for 3 days to grow and become fully confluent.

Cytotoxicity assay was done as described (Nondo et al., 2015) with some modifications. Briefly, 25 mg/mL extract stock was prepared in DMSO and 2 mg/mL serially diluted to 1000–15.625 µg/mL test concentration range. Then 100 µL test solution was added to corresponding wells with cells and incubated for five days. Negative control, NC (cells in medium and 2% DMSO without extract) and positive control (30 μM auranofin) were also included. The shape and appearance of the cells were examined microscopically daily for five days. Viability of the cells was assessed using the formazan assay. The plate was washed with ICM to decolorize wells by shaking three times at 600 RPM for five minutes at 37 °C in an incubator. MTT [3-(4,5-dimethylthiazol-2-yl-diphenyl tetrazolium bromide] solution (100 μL) was added into the wells, the plate incubated for 2 h and 100 μL DMSO added to solubilise the formazan precipitate. The plate was shaken at 400 RPM for five minutes at 37 °C inside an incubator and optical density (OD) of wells read at 595 nm (Emax microplate reader). Percentage inhibition of formazan formation was calculated using the formula $$\%\text{Inhibition =}\frac{\text{OD}\left(\text{NC}\right)-\text{OD}(\text{test})}{\text{OD}\left(\text{NC}\right)}\times 100$$

2.6 Data analysis

Zone diameters from susceptibility test and antibacterial screening by disc diffusion were interpreted based on Clinical and Laboratory Standards Institute reference data for the corresponding antibiotics (CLSI, 2012). The T- test performed using the MedCalc software version v8.0.0.1 was used to compare the zone diameters of the extracts to that of the gentamicin positive control with significance taken at P < 0.05. To determine minimum inhibitory concentrations (MICs), the experiment was considered valid only when both bacterial growth in the negative control was >50% and the positive control showed an inhibition of >50% after 24 h incubation compared to the baseline at 0 h. The MIC was determined by plotting percentage inhibition against concentration using Microsoft Excel 2010 and the MIC value interpolated from the curve. Data from cytotoxicity assay were analyzed using Graph pad prism version 6.0 and the concentration of extract that produced 50% cytotoxic effect on Monkey kidney epithelial cells CC₅₀ recorded.

3.1 Yield and phytochemical composition of extracts

The weights of the dried extracts were recorded and yield calculated as shown on Table 1. U. articulata is rich in four classes of phytochemicals (alkaloids, flavonoids, terpenoids and steroids). U. florida has high amounts of saponins and steroids while Xanthoparmelia plitti similarly is rich in terpenoids and saponins.

3.2 Antibiotic susceptibility of test bacteria

The sixteen bacterial strains (six controls and ten clinical isolates) showed inhibition zones ranging from 7 to 37 mm for twelve antibiotics belonging to eight chemical classes (Table 2). Nine of the ten clinical isolates were multidrug resistant with P. rettgeri being the most resistant to at least one molecule of six antibiotics classes. All clinical isolates were susceptible to the aminoglycosides (gentamicin and amikacin) while resistant to the cephalosporins (cefuroxime and cefotaxime). Eight and seven of the isolates were susceptible to the quinolones, ciprofloxacin and norfloxacin, respectively while 7 of the isolates were susceptible to nitrofurantoin and chloramphenicol. Five and four isolates were susceptible to imipenem and ceftriaxone respectively. Three of the bacterial isolates were susceptible to tetracycline and trimethoprim. Four control strains, E. coli, S. enterica, S. aureus and S. epidermidis, were multidrug resistant but were also susceptible to the aminoglycosides and fluoroquinolones like the clinical isolates.

3.3 Antibacterial activity of crude extracts

All the six extracts were active against at least two bacterial strains with diameter of zones of inhibition ranging from 9 to 30 mm for clinical isolates and 7 to 29 for control strains (Table 3). Extracts of three lichens (U. articulata, U. fllorida and R. sinensis) were highly active (zones ≥ 20 mm,) against at least five of both control and clinical strains; the other three showed weak activity. Overall, U. articulata, and U. fllorida, were the most active. All bacterial isolates were susceptible (zones ≥ 20 mm) to gentamicin except for one which was moderately sensitive (zone = 18 mm) as shown on Table 3. Statistical analyses (T-test) showed that the activities of U. articulata, U. fllorida and R. sinensis in the diffusion test was similar to the gentamicin positive control (P = 0.1018 to 0.6699) whereas the gentamicin was more active than the other three extracts (P < 0.0001) shown on Table 3. In the MIC experiments, growth of negative control ranged from 124 to 200%; and inhibition in positive control containing gentamicin ranged from 79.3 to 91.6%. Based on the criteria stated under data analysis above, the MICs ranged from 4 to 10 mg/mL. The lowest MIC (4 mg/mL) was produced by two lichens, U. articulata and P. parietina. Overall the highest number of MICs was recorded for U. articulata and U. florida, against five organisms while no MIC was observed for L. gelatinisum in the concentration range tested (Table 4). U. articulata and U. florida, produced MICs against four clinical and one control strain each. MICs were recorded for at least two lichen extracts against each strain and the highest was four extracts with MIC values against E. coli (Table 4). The active extracts showed broad spectrum bacteriostatic activity against seven Gram-negative bacteria which was dose-dependent as illustrated for the lowest MICs in Fig. 1. No MIC was recorded against Gram-positive S. aureus (not shown on Table 4). No MBC was recorded for all the extracts.

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Fig. 1

Dose-dependent inhibition of bacterial growth by methanol crude extracts of lichens. Minimum inhibitory concentration is the lowest concentration which shows > 50% inhibition of growth. Ua: Usnea articulata against C. youngae; Uf: Usnea florida against C. freundi; Pp: Physcia parietina against S. typhi; Rs: Ramalina sinensis against P. rettgeri; Xp: Xanthoparmelia plitti against P. rettgeri.

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3.4 Cytotoxicity of extracts

Cytotoxicity profile was evaluated on monkey kidney epithelial cells in the range 15.625 to 1000 μg/mL. The CC₅₀ of the extracts were all >30 μg/mL, the cut point for lack of cytotoxicity (Ogbole et al., 2017) as shown on Table 4. This suggests a low risk of toxicity on mammalian cells. U. articulata and U. fllorida, the most active lichens, had relatively lower CC₅₀ values while L. gelatinisum and X. plitti had the highest CC₅₀ values hence were least cytotoxic.