Antibacterial activities of Fagara macrophylla, Canarium schweinfurthii, Myrianthus arboreus, Dischistocalyx grandifolius and Tragia benthamii against multi-drug resistant Gram-negative bacteria

Bacterial infections caused by multidrug resistant phenotypes constitute a worldwide health concern. The present study was designed to evaluate the in vitro antibacterial activities of the methanol extracts of five medicinal plants: Fagara macrophylla, Canarium schweinfurthii, Myrianthus arboreus, Dischistocalyx grandifolius and Tragia benthamii against a panel of 28 multidrug resistant Gram-negative bacterial strains. The liquid broth microdilution was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the extracts. The best activity was recorded with Canarium schweinfurthii bark extract, MIC values ranging from 32 to 1024 µg/mL being recorded against 85.7 % tested bacteria. Broad spectra of antibacterial activities were also obtained with both bark and leaf extracts from Myrianthus arboreus (78.6 %) as well as the bark extract from Fagara macrophylla (75.0 %). The lowest MIC value of 32 µg/mL was obtained with Canarium schweinfurthii bark extract against Klebsiella pneumoniae KP63 strain. The results of this work provide baseline information for the use of the studied plants, and mostly Fagara macrophylla, Canarium schweinfurthii and Myrianthus arboreus in the treatment of bacterial infections including multidrug resistant phenotypes.


Background
The spread of multidrug resistant bacteria constitutes a major hurdle in chemotherapy (Kuete 2013). In Gramnegative bacteria, efflux pumps belonging to the resistance-nodulation-cell division (RND) family of tripartite efflux pumps are largely involved in multidrug resistance (Van Bambeke et al. 2006). The propagation of bacterial MDR phenotypes is a great challenge for scientist for the discovery of novel antibacterial agents. The role of medicinal plants as sources of anti-infective compounds has been largely documented (Cowan 1999;Kuete 2013;Ndhlala et al. 2013;Ngameni et al. 2013). It was reported that up to 80 % of the world population rely on plants or derived products for their treatment (WHO 1993). Several African medicinal plants previously displayed good antibacterial activities against Gram-negative MDR phenotypes. Some of them include Dichrostachys glomerata, Beilschmiedia cinnamomea and Olax subscorpioïdea (Fankam et al. 2011), Lactuca sativa, Sechium edule, Cucurbita pepo and Solanum nigrum (Noumedem et al. 2013b), Piper nigrum and Vernonia amygdalina (Noumedem et al. 2013a), Beilschmiedia obscura and Peperomia fernandopoiana , Capsicum frutescens (Touani et al. 2014), Fagara tessmannii (Tankeo et al. 2015

Plant material and extraction
The plants used in this work were collected in different localities of the West Region of Cameroon in January to April 2012. The plants were identified at the National herbarium (Yaounde, Cameroon) where voucher specimens were deposited under the reference numbers (Table 1). Each plant sample was air dried at 24 ± 2 °C, powdered (using a grinder) and a portion of each sample (200 g) was extracted with methanol (MeOH; 1 L) for 48 h at room temperature. The extract was then concentrated under reduced pressure to give residues which constituted the crude extract. All extracts were then kept at 4 °C until further use.

Microbial strains and culture media
Test organisms included sensitive and resistant strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes, Escherichia coli and Providencia stuartii obtained from the American Type Culture Collection (ATCC) (Lacmata et al. 2012;Seukep et al. 2013). Nutrient agar was used for the activation of the Gram-negative bacteria while the Mueller-Hinton Broth was used for antibacterial assays (Kuete et al. 2011b).

INT colorimetric assay for MIC and MBC determinations
MIC determinations were conducted using the rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay according to described methods (Eloff 1998) with some modifications (Kuete et al. 2008b(Kuete et al. , 2009). The test samples and RA were first of all dissolved in DMSO/Mueller-Hinton Broth (MHB) broth. The final concentration of DMSO was lower than 2.5 % and did not affect the microbial growth (Kuete et al. 2007(Kuete et al. , 2008a. The assay was repeated thrice. Wells containing adequate broth, 100 µL of inoculum and DMSO to a final concentration of 2.5 % served as negative control. The MIC of samples was detected after 18 h incubation at 37 °C, following addition (40 µL) of 0.2 mg/mL of INT. MIC was defined as the sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth (Eloff 1998). The MBC was determined by adding 50 µL aliquots of the preparations, which did not show any growth after incubation during MIC assays, to 150 µL of adequate broth. These preparations were incubated at 37 °C for 48 h. The MBC was regarded as the lowest concentration of extract, which did not produce a color change after addition of INT as mentioned above (Kuete et al. 2008b(Kuete et al. , 2009).

Results and discussion
The results the antibacterial assays as determined by broth microdilution are summarized in Table 2. Its appears that the tested extracts displayed selective antibacterial activities. The best activity was recorded with Canarium schweinfurthii bark extract, the obtained MIC values being ranged from 32 to 1024 µg/mL against 24 of the 28 (85.7 %) test bacteria. Broad spectra of antibacterial activities were also obtained with both bark and leaves extracts from Myrianthus arboreus [22/28 (78.6 %)] as well as the bark extract from Fagara macrophylla [21/28 (75.0 %)]. MIC values below or equal to 1024 µg/mL were noted with Fagara macrophylla leaves and whole-plant extracts from Dischistocalyx grandifolius and Tragia benthamii on respectively against 13/28(46.4 %), 12/28 (42.9 %) and 11/28 (39.3 %) tested bacteria. The lowest MIC value of 32 µg/mL was obtained with Canarium schweinfurthii bark extract against Klebsiella pneumoniae KP63 strain. MIC values lower than that obtained for the reference antibiotic chloramphenicol were recorded for Fagara macrophylla bark extract against Enterobacter aerogenes EA27 (64 µg/mL) and Canarium schweinfurthii bark extract (32 µg/mL) against K. pneumoniae KP63. The results presented in Table 2 also show that all extracts displayed poor bactericidal effect.

Conclusion
The results of this work suggest that the studied plant extracts, particularly those from Fagara macrophylla, Canarium schweinfurthii and Myrianthus arboreus, can be used to control some infections and especially those involving MDR bacterial species. Full purification of this plants in the future will be achieved to identified their antibacterial constituents.