Microbial, phytochemical, toxicity analyses and antibacterial activity against multidrug resistant bacteria of some traditional remedies sold in Buea Southwest Cameroon

This was a laboratory-based experimental study. A selection of traditional medicine remedies were purchased based on information provided by the sellers on the infectious diseases treated. The bacterial loads of the preparations were determined to ascertain their microbial safety. Clinical isolates and control strains of bacteria were characterised for antibiotic susceptibility to identify resistant strains. Methanol crude extracts of the powders were prepared, phytochemical constituents determined and together with the liquid preparations (not extracted) were screened for activity against the characterised bacteria. Toxicity of active extracts was investigated on a cell line and in vivo in mice. Experimental data were analysed by comparison with reference data.

We approached sellers of traditional medicine remedies in the local markets (Muea and Central markets) in Buea municipality, Southwest Cameroon, who consented to provide information on their remedies displayed for sale. Information on the name, source, disease(s) treated, component(s) of the remedy and their local name(s) were noted. Ten anti-infective remedies were purchased. Where taxonomic nomenclature of component(s) was provided, this was crosschecked in online plant databases and publications on ethnobotanical surveys in the area of origin of the remedy. The seller or respondent was requested to provide specimens (leaves, seeds, and flower of plants) for identification. Preliminary identification of the samples was done by botanists, Professor Chuyong George and Dr. Neba Godlove in the Department of Plant and Animal Physiology, University of Buea, Cameroon. The identities were confirmed by Mr. Ndive Elias using voucher specimens in the Biodiversity and Conservation Centre in Limbe and the National Herbarium of Cameroon in Yaounde and the voucher specimen numbers noted. Where the taxonomic names of the components could not be established such remedies were excluded from the study; but their identification will be pursued further to the source of the product subsequently.

Presence of bacterial contaminants in the remedies was determined as described [10]. Five dilutions of 100, 50, 25, 10 and 1% (v/v) of the liquid remedy were made. For the powders, 10 mg of each was put in 1 mL of 0.85% saline, vortexed and allowed to stand. The supernatant was collected and diluted same as liquid remedies. Then 100 μL of each dilution was uniformly spread on nutrient agar and incubated (DHP-9052, England) at 37 °C for 24 h. The number of colony forming units (CFUs) were counted and the microbial load calculated in CFUs/g or CFUs/mL. Gram staining was done for microscopic analysis as described [17] using a loop of cells and visualized under oil immersion.

Extracts of the remedies were prepared as described [18]. Briefly, weighed amounts (14 to 80 g) of the powders were macerated three times in methanol for 72 h with intermittent stirring, filtered and the filtrate concentrated by rotary evaporation (Buchi Rotavapor R200, Switzerland). The extracts were dried at room temperature and stored at 4 °C until used. Phytochemical analysis was performed for each extract to determine chemical classes of secondary metabolites present in them and their relative amounts based on qualitative observations produced by specific chemical tests for alkaloids, flavonoids, steroids, tannins and triterpenoids [19].

Ten resistant strains of nine pathogenic bacteria species (Citrobacter freundii, Citrobacter youngae, Enterobacter cloacae, Escherichia coli, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Salmonella typhi, Salmonella sp. and Methycillin-resistant Staphylococcus aureus (MRSA) were obtained from clinical specimens in Buea Regional Hospital annex, a regional referral health facility in the Southwest region of Cameroon. The cells were characterized by bacteriologists of the hospital by microscopy, susceptibility to reference antibiotics, appropriate culture methods and biochemical tests using the API 20E kit (Biomerieux SA, France). Pure stocks of cells were then stored in 10% glycerol in Mueller Hinton broth (Liofilchem, Italy) at − 20 °C during the study period. The following control strains were obtained from BEI Resources and American Type Culture Collection (USA): Escherichia coli (NR 32771), P. aeruginosa (ATCC 27853), Staphylococcus aureus (NR-46003), Staphylococcus epidermidis (NR-46376), Salmonella enterica (NR-515) and Salmonella enterica (NR-4311).

For the purpose of this study, the clinical isolates were re-tested for antibiotic susceptibility to determine the magnitude of their resistance, together with the control strains. For experimental use, working near the freezer the stored cells were retrieved, the frozen stock rapidly scraped using an autoclaved toothpick and deposited on the surface of nutrient agar; and the stock returned immediately into the freezer. A wire loop was flamed and then used to streak on nutrient agar. Susceptibility was done on the clinical isolates as described by the Clinical and Laboratory Standard Institute [20], using discs of 12 standard antibiotics (Liofilchem, Italy), from eight chemical classes selected for this study: Amikacin (30 μg), Ceftriaxone (30 μg), Cefuroxime (30 μg), Cefotaxime (30 μg), Ciprofloxacin (5 μg), Chloramphenicol (30 μg), Gentamicin (10 μg), Imipenem (10 μg), Nitrofurantoin (300 μg), Norfloxacin (10 μg), Tetracycline (30 μg), Trimethoprim (5 μg).

All experiments were performed under sterile conditions using sterilized glassware and materials. Freshly sub-cultured bacterial cells were used in all experiments. The disc diffusion test was performed as described [18] with some modifications. Stock solutions of the extracts (10 mg/100 μL dimethylsufoxide, DMSO) were prepared while the liquid remedies were tested undiluted. Bacterial suspension (0.5 McFarland) was spread on Mueller Hinton agar (Liofilchem, Italy) and allowed for 3–5 min to dry. Six millimetre sterile Whatman filter paper discs (number 9) were gently fixed at labelled positions on the agar surface. Then 10 μL of test solution (1 mg extract) was transferred onto each disc. Positive (appropriate standard antibiotic) and negative (10 μL DMSO) controls were included, the plates kept for 30 min at room temperature, incubated as above and zones of inhibition measured.

The minimum inhibitory concentrations (MIC) of extracts were determined using the microdilution method as described [18] with one modification: experimental changes in microtitre plate wells were recorded by spectrophotometry. A stock solution of each extract was prepared (40 mg dissolved in 200 μL DMSO and 800 μL Mueller Hinton broth added); then diluted to obtain test solutions from 40 to 1 mg/mL. The assay was set up in a 96 well microtitre plate in duplicate wells as follows: 50 μL test solution, 130 μL Mueller Hinton broth followed by 20 μL bacterial cell suspension (6 × 10⁵ CFU/mL final density). Positive (20 μg/mL Gentamicin) and negative (0.85% saline) controls were included. Final test concentrations were 10, 8, 6, 4, 2, 1, 0.5 and 0.25 mg/mL respectively containing 5% DMSO. The optical densities (ODs) of wells were read, the plate incubated as above and the OD read again at 595 nm (Emax-Molecular Devices Corporation, USA).

The MIC was calculated from % growth of bacteria relative to the negative control using the formula below:

where change in OD = OD at 24 h – OD at 0 h. MIC is taken as the lowest concentration at which % growth becomes approximately constant with no further decrease in growth. To determine minimum bactericidal concentration (MBC), a 1:10 dilution in fresh broth of MIC well contents were re-incubated and percentage growth calculated from ODs before and after incubation. The lowest concentration without bacterial growth was recorded as MBC using the formulas: change in OD = OD at 24 h ̶ OD at 0 h and

The cytotoxicity of the extracts was investigated as described [21] using monkey kidney epithelial cells, LLCMK2 Original (ATCC® CCL7™), (Virginia, USA). All reagents were from Sigma Aldrich (USA). The cells were cultured to confluence in complete RPMI-1640 medium, CCM (containing NaHCO₃, supplemented with 25 Mm HEPES, 0.3 g γ-irradiated L-glutamine powder, 10% heat inactivated new born calf serum, 200 units/mL penicillin and 200 μg/mL streptomycin and 0.25 μg/mL amphotercin B, pH 7.4) in a T-shaped flask in a 5% CO₂ incubator in humidified air at 37 °C (Heracell 150i, USA). The medium was decanted, washed off twice with incomplete (calf serum-free) RPMI-1640 medium (ICM), and cells dislodged with × 5 trypsin-EDTA. The cells were then centrifuged at 125 x g for 10 min (Eppendorf 5810R, Germany). The supernatant was discarded; the cells re-suspended in fresh ICM and quantified using a haemocytometer (Hausser Scientific, USA) and an inverted microscope (Nikon Eclipse TS100, China). The cells were diluted to 30,000/mL with CCM, 100 μL seeded in a 96-well flat bottom microtitre plate in duplicate and incubated as above for 3 days for cells to grow and become fully confluent.

Stock solutions of extracts (25 mg/mL DMSO) were prepared and diluted with CCM to 2 mg/mL in duplicate microtitre wells. The seeded cells were checked for confluence by microscopy. Then 100 μL of extract was transferred into respective wells giving final concentration of 1 mg/mL. Positive (30 μM auranofin) and negative (2% DMSO in CCM) controls were included. The plates were incubated as above for 5 days with daily microscopic examination for toxicity. Extracts that were cytotoxic were serially diluted in CCM and re-incubated same as above with freshly cultured cells at final extract concentrations of 15–1000 μg/mL. Thereafter the medium was discarded and wells decolourised by shaking with ICM (IKA Labortechnik KS125 basic shaker) at 600 rpm for 5 min three times. Then 100 μL of 5 mg/mL MTT in ICM was added, the plate incubated as above for 30 min and 100 μL DMSO added to dissolve formazan precipitate. Well contents were gently mixed by shaking and optical densities read same as above at 595 nm. Percentage inhibition was calculated using the formula below:

CC₅₀ of the cytotoxic extract was determined by plotting a graph, using Graph pad prism, of % inhibition against log of concentration of extracts.

This was done following guidelines of the Organization for Economic Cooperation and Development version 423 [22]. This was done for the bactericidal extract TP2 as described [23]. Five adult female BALB/c mice (nine weeks old) were selected in the animal house of the study laboratory and the experiment conducted in the adjacent test room. One animal was weighed, fasted overnight (no food; water only), and administered a dose of 2000 mg/kg body weight by oral gavage from a freshly prepared solution of TP2 in 7% Tween 80. The animal was fasted for a further 2 h, then provided food and water. The treated animal was observed for any obvious acute signs of toxicity hourly during the day while the other four were fasted overnight. Following the survival of the treated animal after 24 h, the other four fasted animals were weighed and treated as above and all animals observed for gross changes such as loss of appetite, piloerection, lacrimation, tremors, convulsions, salivation, diarrhoea, mortality and other signs of overt toxicity for 14 days. The animals were then sacrificed by cervical dislocation.

Zone diameters recorded in the susceptibility test and antibacterial screening by disc diffusion were interpreted based on CSLI reference data for the corresponding antibiotics [20]. The zone diameters of the extracts were compared with corresponding values for the Gentamycin positive control at a significance of P < 0.05 using the T-test (data with normal distribution determined with the Kolmogorov-Smirnov-Test) or Welch test (no normal distribution of data). In the microdilution assay, the % growth of bacteria calculated from optical densities were plotted against concentration of extract, then MIC and MBC interpolated at the lowest of the concentrations at which bacterial growth was near constant and no longer decreasing. Data from cytotoxicity assay was analysed using Graph pad prism version 6.0, the concentration of extract that produced 50% cytotoxic effect (CC₅₀) on Monkey kidney epithelial cells obtained.

Of the ten remedies purchased, two (200 Disease cure and German powder), which could not be identified were excluded from the study. Of the eight remedies investigated, six were powder and two in liquid form. Information on the remedies obtained from the sellers and the corresponding taxonomic information are shown on Table 1. The sources revealed that some remedies originate far from the local markets where sold, within Cameroon and abroad from neighbouring Nigeria and further from the republic of Niger, West Africa. Following solvent extraction of the powders high yields were obtained for most of the extracts ranging from 31 to 81%. The taxonomic information of the plant species contained in four remedies were established as described under the methods section, based on their corresponding names (Madachi, Bagaruwa, Gewaya tsamiya and Gesa) in the Hausa vernacular of the source areas and specimens provided.

All eight remedies were contaminated with both Gram- positive and negative bacilli and cocci. The bacterial load ranged from 1.8 × 10³ to 2.1 × 10⁵ CFUs/mg or CFUs/mL. None had a microbial count within the acceptable range of microbial contamination of 10⁵ CFU/g (10² CFU/mg) for aerobic bacteria as stipulated in the WHO guidelines on the quality of herbal materials [24]. TP3 (Madigest 2) and TP4 (Desert war 2) had the highest microbial counts of 1.8 × 10⁵ CFU/mg and 2.1 × 10⁵ CFU/mg respectively while TP7 (Fever medicine) had the lowest microbial count of 1.8 × 10³ CFU/mg.

Based on reference data (diameter of inhibition zones) for antibiotic susceptibility of the Clinical and Laboratory Standards Institute [20], both the control and clinical isolates were sensitive to the aminoglycosides (Amikacin, Gentamicin), fluoroquinolones (Ciprofloxacin, Norfloxacin), Nitrofurantoin and Chloramphenicol. Resistance was observed for the carbapenem (Imipenem), cephems (Cefotaxime, Ceftraixone, Cefuroxime), folate pathway inhibitor (Trimethoprim) and Tetracycline. Each bacterial strain was resistant to at least one antibiotic. Multidrug resistance was confirmed in nine of the ten clinical isolates (resistant to at least one antibiotic of three chemical classes) [2], presented on Table 2. Proteus vulgaris, Providencia rettgeri and Salmonella typhi were the most resistant to 5, 6 and 5 classes of antibiotics respectively. The resistance of the clinical isolate of S. aureus to methicillin was confirmed.

Extracts of four remedies (TP 1, 2, 4, 6a, 6b) were active against all the clinical isolates. Extracts of three remedies (TP 1, 2, 4, 6a, 6b) were active against at least four of the control strains. All extracts showed activity against S. enterica (NR-515). The liquid remedies (TL 5 and 7) tested undiluted were less active; TL7 showed relatively low activity against only two control strains with no activity against the clinical isolates (Table 3). The zones of inhibition for the extracts ranged from 8 to 27 mm against 17 to 24 mm for Gentamycin positive control. Two extracts (TP1 and 4) produced zones ≥20 mm against multidrug resistant clinical isolates (Citrobacter freundii and Escherichia coli). The highest zone against clinical isolates was 24 mm produced by TP1 against E. coli; and the highest zone against control strains was 27 mm produced by TP4 against Staphylococcus aureus (NR-46003). However, when zone diameters were compared using statistical tests, Gentamycin was more active than each of the four most active extracts (P < 0.0001–0.0014). Four of the eight remedies (TP 1, 2, 4, 6) recorded MIC values from 1 to 4 mg/mL. TP1 had the lowest MIC of 1 mg/mL against S. typhi clinical isolate shown on Table 4. Of these 4 extracts, only TP2 showed MBC at 8 mg/mL against MRSA. The MBC:MIC ratio gave a value of 2 which is less than 4 indicating bactericidal effect of TP2 against this strain (Table 4) [25]. Figure 1 shows inhibition of growth at higher concentrations of TP2 with higher growth at the lower concentrations giving the MIC and MBC values of 4 and 8 mg/mL respectively.

The crude extracts and undiluted liquid remedies were found to contain alkaloids, flavonoids, steroids, tannins and triterpenoids from the phytochemical tests. The four most active extracts (TP 1, 2, 4, 6a, 6b) had relatively high amounts of alkaloids and flavonoids (Table 1).

The cytotoxicity assay on monkey kidney cells, LLCMK2 Original (ATCC® CCL7™), showed that 2 extracts (TP 3 and 6a) were non-cytotoxic at a high dose of 1000 μg/mL since their percentage inhibition of formazan formation was less than 50%. Six extracts (TP 1, 2, 4, 6b, 8 and TL7) had CC₅₀ values greater than the cut-off point for lack of cytotoxicity (CC₅₀ > 30 μg/mL), [26]. One extract (TL5) had CC₅₀ value lower than 30 μg/mL indicating mild cytotoxicity (Table 4). Only piloerection was observed in the first two days in the oral toxicity test for TP2 with no mortality recorded. The body weight of all the animals increased; average body weights at the start and after day 14 were 26.6 and 29.4 g respectively with an average increase of 2.8 g.