Antibacterial activities of the methanol extracts of seven Cameroonian dietary plants against bacteria expressing MDR phenotypes

The morbidity and mortality caused by bacterial infections significantly increased with resistance to commonly used antibiotics. This is partially due to the activation of efflux pumps in Gram-negative bacteria. The present work designed to assess the in vitro antibacterial activities of seven Cameroonian dietary plants (Sesamum indicum, Sesamum radiatum, Cinnamomum zeylanicum, Corchous olitorius, Cyperus esculentus, Adansonia digitata, Aframomum kayserianum), against multidrug resistant (MDR) Gram-negative bacteria over expressing active efflux pumps. The standard phytochemical methods were used to detect the main classes of secondary metabolites in the extracts. The antibacterial activities of the studied extracts in the absence or presence of an efflux pump inhibitor (PAβN) were evaluated using liquid microbroth dilution method. The results obtained indicated that apart from the extract of C. esculentus, all other samples contained alkaloids, phenols and polyphenols meanwhile other classes of chemicals were selectively present. The studied extracts displayed antibacterial activities with minimal inhibitory concentrations (MICs) values ranged from 64 to 1024 μg/mL on the majority of the 27 tested microbial strains. The extract of S. indicum was active against 77.77% of the tested microorganisms whilst the lowest MIC value (64 μg/mL) was recorded with that of A. kayserianum against E. aerogenes EA294. The results of the present work provide baseline information on the possible used of the tested Cameroonian dietary plants in the treatment of bacterial infections including multi-drug resistant phenotypes.


Introduction
The continuous emergence of multidrug-resistant (MDR) bacteria drastically reduces the efficacy of our antibiotic armory, and consequently increases the frequency of therapeutic failure Falagas and Bliziotis (2007). The resistance of bacteria to chemically unrelated antimicrobial agents may be associated with the over-expression of efflux pumps (Poole 2004, Li andNikaido 2009). In Gram-negative bacteria, many of these efflux pumps belong to the resistance-nodulation-cell division (RND) family of tripartite efflux pumps. Among those efflux pumps, those belonging to the AcrAB-TolC family are detected in many clinical bacterial isolates and are reported to be a key factor in the expression of the MDR phenotypes (Mallea et al. 2003;Lomovskaya et al. 2004). This efflux pumps mechanism can be blocked by various inhibitors which restore the intracellular concentration as well as the activities of the antibiotics (Chollet et al. 2004;Pagès and Amaral 2009). The scarcity of original synthetic antibiotics has stimulated the search for new antibacterial agents from medicinal plants. This explains our endeavor to evaluate in vitro the antibacterial activities of Cameroonian dietary plants namely the beans of Sesamum indicum, the stem and leaves of Sesamum radiatum, Corchous olitorius and Cyperus esculentus, the bark of Cinnamomum zeylanicum, the fruits of Adansonia digitata and Aframomum kayserianum against Gram-negative bacteria expressing MDR phenotypes. The role of efflux pumps in the activity of our plants extracts was also investigated using a previously described efflux pump inhibitor.

Plant materials and extraction
The seven edible plants used in this work were purchased from Bafoussam local market, West Region of Cameroon in January 2012. The collected plant samples were the beans of Sesamum indicum, the stem and leaves of Sesamum radiatum, Corchous olitorius and Cyperus esculentus, the bark of Cinnamomum zeylanicum, the fruits of Adansonia digitata and Aframomum kayserianum. The plants were further identified at the National Herbarium (Yaoundé, Cameroon) where voucher specimens were deposited under a reference number (Table 1). The powdered air-dried sample from each plant was extracted with methanol for 48 h at room temperature. The extract was then concentrated under reduced pressure to give a residue that constituted the crude extract. They were then kept under 4°C until further use.

Bacteria strains and culture media
The studied microorganisms included references (from the American Type Culture Collection) and clinical (Laboratory collection) strains of Escherichia coli, Enterobacter aerogenes, Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterobacter cloacae (Table 2). They were maintained on agar slant at 4°C and subcultured on a fresh appropriate agar plates 24 h prior to any antimicrobial test. Mueller Hinton Agar (MHA) was used for the activation of bacteria and the Mueller Hinton Broth (MHB) was used for the MIC determinations.

Bacterial susceptibility determinations
The respective MICs of samples on the studied bacteria were determined by using rapid INT colorimetric assay Mativandlela et al. (2006). Briefly, the test samples were first dissolved in DMSO/MHB. The solution obtained was then added to MHB, and serially diluted two fold (in a 96-well microplate). One hundred microlitres (100 μL) of inoculum (1.5 × 10 6 CFU/mL) prepared in MHB was then added. The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a shaker and incubated at 37°C for 18 hrs. Wells containing MHB, 100 μL of inoculum and DMSO at a final concentration of 2.5% served as negative control (this internal control was systematically added). Chloramphenicol (CHL) was used as reference antibiotic. The MICs of samples were detected after 18 hrs of incubation at 37°C, following addition (40 μL) of 0.2 mg/mL INT and incubation at 37°C for 30 min Kuete et al. (2008). Viable bacteria reduced the yellow dye to pink. MIC was defined as the lowest sample concentration that prevented this change and exhibited complete inhibition of bacterial growth. For the determination of MBC, he microplates ones were filled by 150 μL of MHB without extract of plant; for wells not having received a INT (during the reading of the MIC), 50 μL of the contents of the wells corresponding to the concentrations higher or equal to the MIC was taken and introduced into these microplates. These were then incubated during 48 h à 37°C followed by revelation with the INT. All the concentrations to which we did not observe pink coloring were taken as bactericides and smallest of those, was noted as MBC. Samples were tested alone and then, in the presence of PAβN at 30 μg/mL final concentration.

Phytochemical composition of the plant extracts
The results of qualitative analysis showed that all the crude extracts tested, except that for C. esculentus, contained alkaloids, phenols and polyphenols; others phytochemicals classes being selectively detected (Table 3).

Antibacterial activity of the plant extracts
The various strains and MDR isolates were tested for their susceptibilities to plants extracts and reference antibiotic (chloramphenicol). Chloramphenicol was also tested the presence of PAßN, a well-known efflux pump inhibitor to confirm the role of efflux pumps in the resistance of the studied microorganisms. Assays were performed using the broth microdilution method. The results depicted in Table 4 indicate that the plant extracts exhibited activities depending of bacteria strains, with MICs values ranged from 64 to 1024 μg/mL on the majority of the 27 tested microbial strains. At the tested concentration range (8 to 1024 μg/mL), the extracts which displayed activities against the majority of bacteria strains were those from S. indicum (active against 77.77% of the tested bacteria), C. zeylanicum (70.37%), S. radiatum (66.66%), C. olitorius (62.96%), A. kayserianum (51.85%), C. esculentus (18,52%) and A. digitata (14,81%). The lowest MIC value (64 μg/mL) was recorded with the extract of A. kayserianum against E. aerogenes EA294. Other extracts exhibited weak activities against a limited number of strains studied. A keen look of the results of Table 4 also shows that the extract of S. indicum displayed the best spectrum of bactericidal  ), antimicrobial Osibote et al. 2010).
Methanol and ethanol extracts S : Ca  effect with a ratio MBC/MIC ≤ 4 on 6 bacterial strains, followed by that of S. radiatum (2/27) and those of C zeylanicum, C. olitorius, A. kayserianum (1/27). Only the extract of A. digitata did not present any bactericidal activity.

Role of efflux pumps in susceptibility of Gram-negative bacteria to the tested plants extracts
The various strains and MDR isolates were also tested for their susceptibility to CHL in the presence of PAβN. As shown in Table 4, PAβN improved the activity of CHL to all studied bacteria.

Discussion
The extract from A. digitata, others extracts exhibited antibacterial activities against at least one of the tested bacteria. The differences in antibacterial activities were noted between the various extracts and could be related to the differences in their phytochemical composition as shown in Table 2. Except in the extract of C. esculentus, the alkaloids, phenols and polyphenols were detected in all extracts. The antibacterial activities of many molecules belonging to these classes of compounds were shown (Cowan 1999;Kuete et al. 2009). Sharma and Singh (2011) also associated the antibacterial activities of the medicinal plants to the presence of flavonoids, tannins and alkaloids. It should however be mentioned that the detection of the bioactive phytochemical classes in a plant is not a guarantee for any biological property, as this will depend on the types of compounds, as well as their concentrations and possible interaction with other constituents. Alkaloids, phenols, sterols, polyphenols, triterpenes, anthraquinones and saponins were detected in the extract of S. indicum. The presence of the anthocyanins and the flavonoids in this plant was previously reported Awobajo et al. (2009), though such classes of chemicals were not detected in this work. This could be explained by fact that the presence of the secondary metabolites in a plant depend on the environmental factors such as climate, chemical nature of the ground in which plant grow, the period of harvest, conditions of drying and extraction method Bruneton (1999). To the best of our knowledge, no antibacterial activity of this plant was shown up to now, but presence of the various classes of secondary metabolites could explain its activity on majority of tested strains obtained in this work. In addition the antibacterial activities of the essential oil of S. radiatum (Shittu et al. 2006Konan et al. 2008). Voukeng and collaborators (2012) documented the antibacterial activities of methanol extract of the sheets of C. zeylanicum against MDR Gram-negative bacteria used in this work. The presence of the phenolic compounds detected in thebark of this plant could explain its activities. Significantantibacterial activities of the methanol extract of C. olitorius were shown against the Gram-positive bacteria Pal et al. (2006). Also, the roots of this plant showed a significant antibacterial activity Ramadevi and Ganapaty (2011). The antibacterial activities of this plant as shown in this work provide therefore additional data on its antimicrobial potentials. Plants of the genus Aframomum are known for their antibacterial activities, and this has been assigned to the presence of terpenoids such as aframodial Ayafor et al. (1994). This could also explain the activity observed with the extract of A. kayserianum in the present work. Several former studies showed the presence of the terpenoids, phenolic and alkaloids in the extract of A. digitata (Wickens 1979;Chadare et al. 2009). The aqueous, ethanol, and petroleum-ether extracts of this plant have shown an antibacterial activity against E. coli Yagoub (2008). However, we observed a weak antibacterial activity of this plant. The extract of C. esculentus contains less secondary classes of metabolites. Only sterols, triterpenes and saponins were detected. To the best of our knowledge, the antibacterial of this plant is being reported for the first time. Generally, the weak antibacterial activity observed with the majority of the extracts could be explained by the fact that bacterial strains used are MDR phenotypes expressing active efflux pumps Cattoir (2004), as shown with the increase of the activity of CHL in the presence of PAβN (Table 4). It was demonstrated that efflux pumps decrease the intracellular concentration of chemicals and consequently their activities (Bambeke and Pagès 2010;Kuete 2010). These efflux pumps can be blocked in a competitive way or not, by an efflux inhibitor, which restore therefore not only intracellular concentration, but also the activity of antibiotics (Pagès and Amaral 2009;Kuete 2010). Finally, the results obtained with the studied plants are encouraging not only because we are dealing with MDR bacteria, but also the fact investigated plant materials are food plants which are relatively non toxic.

Conclusion
The results of the present work provide baseline information on the possible used of the tested Cameroonian dietary plants in the treatment of bacterial infections including MDR phenotypes.