Antibacterial activities of selected edible plants extracts against multidrug-resistant Gram-negative bacteria

The herbal sample consisted of seven different Cameroonian dietary plants namely the leaves of Adansonia digitata and Anonidium mannii, the rhizomes of Aframomum alboviolaceum, Aframomum polyanthum, the whole plants of Hibiscus sabdarifa and Ocimum gratissimum, and the fruits of Tamarindus indica. The plants were purchased from markets in the West region of Cameroon in January 2011. They were further identified at the National Herbarium (Yaoundé, Cameroon) where the voucher specimens were deposited under reference numbers (Table 1). Each plant was dried at room temperature and the powdered material was then weighed (300 g), soaked in 1 L of methanol (MeOH) for 48 h and filtered using Whattman N^o1 filter paper. The filtrate obtained was concentrated under reduced pressure (at 68°C) in a rotary evaporator to obtain the crude extract. The crude extracts were kept at 4°C until further uses.

The plant materials were screened for the presence of different classes of secondary metabolites including alkaloids, flavonoids, phenols, saponins, tannins, anthocyanins, anthraquinones, sterols, and triterpenes using previously described methods [34].

The minimal inhibitory concentrations (MICs) of the seven plant extracts were determined using a rapid p-Iodonitrotetrazolium chloride (INT; Sigma-Aldrich, St Quentin Fallavier, France) colorimetric assay [35, 36]. Briefly, the test samples were first dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich)-Mueller Hinton Broth (MHB; Sigma-Aldrich). The solution obtained was then added to MHB and serially diluted two fold (in a 96-well microtilter plate). One hundred microliters of inoculums (1.5× 10⁶ CFU/ml) prepared in MHB were then added. The plates were covered with a sterile plate sealer and then agitated with a shaker to mix the contents of the wells and incubated at 37°C for 18 h. The final concentration of DMSO was less than 2.5%, and thus did not affect the microbial growth. Wells containing MHB, 100 μl of inoculum, and DMSO at a final concentration of 2.5% served as the negative control (this internal control with DMSO 2.5% was systematically added). Chloramphenicol (Sigma-Aldrich) was used as reference antibiotic. The MICs of each extract were detected after 18 h of incubation at 37°C following addition of 40 μl INT (0.2 mg/ml) and incubation at 37°C for 30 min. Viable bacteria reduced this yellow dye to pink. The MIC of each sample was defined as its lowest concentration that prevented this change and then resulted in the complete inhibition of microbial growth. The Minimum Bactericidal Concentration (MBC) was determined by sub-culturing samples from the wells with concentrations above the MIC on new plates of Mueller Hinton broth (MHB). The MBC was considered as the lowest concentration of the extract associated with no bacterial culture.

Each assay was performed three independent times in triplicate. In case where they were different, the MIC or MBC were taken as the most frequently occurring values. Chloramphenicol was tested alone and in the presence of Phenylalanine arginine-ß-naphtylamide (PAßN) at a final concentration of 30 μg/ml, as described previously [37].

Freshly prepared extracts were subjected to a preliminary phytochemical screening for various constituents. The results (Table 2) revealed the presence of phenols, polyphenols and triterpenes. Anthraquinones were not detected in any of the extracts while anthocyanins were found only in the extracts of the genus Aframomum (A. alboviolaceum and A. polyanthum).

The antibacterial activity of the plant extracts are depicted in Table 3. The results indicated that the plants extracts showed antibacterial activities at variable degrees against MDR bacteria, with MICs values varying from 32 to 1024 μg/ml. Extracts of A. digitata displayed the most important spectrum of activity, its inhibitory effects being observed against 81.48% of the bacterial strains, followed by the extracts of H. sabdarifa (66.66%), A. polyanthum ( 62.96%), A. alboviolaceum ( 55.55%) and O. gratissimum (55.55%). The extract of A. polyanthum showed the highest activity against E. aerogenes EA294 with a MIC value of 32 μg/ml. The extracts of T. indica and A. mannii did not show antibacterial activity against the majority of the bacteria tested, their inhibitory effect being noted against 6/27 (22.22%) and 7/27(25.92%) bacterial strains tested respectively. The microorganisms of the species P. aeruginosa (PA01 and PA124), known for their multi-resistance to drugs, were resistant to all the plant extracts tested in this work (with MIC > 1024 μg/ml).

Some of the studied extracts showed bactericidal effects on few numbers of bacteria. These effects were observed with the crude extracts of A. digitata, against E. coli MC4100 and K. pneumoniae KP55 with the ratios minimal bactericidal versus minimal inhibitory concentrations (MBC/MIC) equal to 1 and 2 respectively. For A. polyanthum’s extract, the ratio MBC/MIC was equal to 2 on K. pneumoniae KP55. O. gratissimum also showed ratios MBC/MIC equal to 1 on E. coli AG 102. The crude extract of H. sabdarifa was also bactericidal against E. coli MC4100 and W3110 and against E. cloacae BM67 with the ratio equal to 1; 1 and 4 respectively. Chloramphenicol used as reference antibiotic showed variable inhibitory activity on different strains of bacteria with MIC values ranging from 2 to 512 μg/ml. These activities of chloramphenicol was bacteristatic on the majority of bacteria (MBC/MIC > 4) and in some cases, its MICs were equal to those obtained with some plant extracts (A. digitata on K. pneumoniae KP55, H. sabdarifa on E. cloacae BM 67 and O. gratissinum on E. cloacae ECCI69).