Background

The spread of multidrug resistant bacteria constitutes a major hurdle in chemotherapy (Kuete 2013). In Gram-negative 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 (Fankam et al. 2014), Capsicum frutescens (Touani et al. 2014), Fagara tessmannii (Tankeo et al. 2015). In our ongoing investigation of antibacterial plants, we designed the present work to investigate in vitro antibacterial activity of the methanol extracts of five medicinal plants, Canarium schweinfurthii Engl. (Burseraceae), Dischistocalyx grandifolius C. B. Clarke (Acanthaceae), Fagara macrophylla (Oliv.) Engl. (Rutaceae), Myrianthus arboreus P. Beauv. (Moraceae) and Tragia benthamii Bak. (Euphorbiaceae) (Table 1) against MDR Gram-negative bacteria.

Table 1 Information on the studied plants
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Methods

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.

Antimicrobial assays

Chemicals for antimicrobial assay

Chloramphenicol (CHL), (Sigma-Aldrich, St Quentin Fallavier, France) was used as a reference antibiotic (RA). p-Iodonitrotetrazolium chloride (INT) was used as microbial growth indicator (Eloff 1998; Mativandlela et al. 2006).

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, 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, 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, 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.

Table 2 MICs and MBCs (in μg/mL) of methanol extracts from the studied plants and chloramphenicol
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Several molecules belonging to classes of secondary metabolites previously reported in the tested plants (Table 1) have been reported to be active on pathogenic microorganisms (Awouafack et al. 2013; Cowan 1999; Ndhlala et al. 2013; Tsopmo et al. 2013). The presence of such metabolites in our extracts could explain their antibacterial activities. According to Kuete (2010), Kuete and Efferth (2010), the antibacterial activity of a plant extract is considered significant when the MICs are below 100 μg/mL, moderate when 100 ≤ MIC ≤ 625 μg/mL and weak if MIC >625 μg/mL. Consequently, the activity of Fagara macrophylla bark extract against Escherichia coli ATCC10536 and Enterobacter aerogenes EA27 and (MIC of 64 µg/mL) and Canarium schweinfurthii bark extract against K. pneumoniae KP63 (MIC of 32 µg/mL) can be considered important. The MIC values reported herein for the studies plants and mostly Fagara macrophylla, Canarium schweinfurthii and Myrianthus arboreus are moderate in general but can be considered important when regarding the medicinal importance of the tested MDR bacteria (Chevalier et al. 2000; Kuete et al. 2010, 2011a; Mallea et al. 1998, 2003; Pradel and Pages 2002; Tran et al. 2010). The antimicrobial properties compounds from Canarium schweinfurthii have been reported (Longanga Otshudi et al. 2000); also, the antibacterial activity of Myrianthus arboreus was also reported against Klebsiella pneumoniae, Proteus vulgaris, Staphylococcus aureus and Escherichia coli (Agwa et al. 2011). The present study provides additional data on the ability of this plant to fight MDR bacteria of these plants as well as information on the antibacterial potentcy of other extracts.

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.