Antibacterial Activities of Selected Cameroonian Plants and Their Synergistic Effects with Antibiotics against Bacteria Expressing MDR Phenotypes

The present work was designed to assess the antibacterial properties of the methanol extracts of some Cameroonian medicinal plants and the effect of their associations with currently used antibiotics on multidrug resistant (MDR) Gram-negative bacteria overexpressing active efflux pumps. The antibacterial activities of twelve methanol extracts of medicinal plants were evaluated using broth microdilution. The results of this test showed that three extracts Garcinia lucida with the minimal inhibitory concentrations (MIC) varying from 128 to 512 μg/mL, Garcinia kola (MIC of 256 to 1024 μg/mL), and Picralima nitida (MIC of 128 to 1024 μg/mL) were active on all the twenty-nine studied bacteria including MDR phenotypes. The association of phenylalanine arginine β-naphthylamide (PAβN or efflux pumps inhibitor) to different extracts did not modify their activities. At the concentration of MIC/2 and MIC/5, the extracts of P. nitida and G. kola improved the antibacterial activities of some commonly used antibiotics suggesting their synergistic effects with the tested antibiotics. The results of this study suggest that the tested plant extracts and mostly those from P. nitida, G. lucida and G. kola could be used alone or in association with common antibiotics in the fight of bacterial infections involving MDR strains.


Introduction
Bacterial infections are responsible for 90% of infections found in health care services. The emergence of MDR bacterial strains appears as the major cause of treatment failure [1]. Among the known mechanisms of resistances, active efflux via resistance-nodulation-cell division (RND) pumps is one of the most occurring system in Gram-negative bacterial strains [2]. Efflux pumps are transport proteins involved in the extrusion of toxic substrates (including virtually all classes of clinically relevant antibiotics). The present work was therefore designed to investigate the antibacterial potential against MDR bacteria expressing active efflux though RND pumps. Medicinal plants of Cameroon used in this study include the fruits of Citrus medica L. (Rutaceae), the bulbs of Allium sativum L. (Liliaceae) and Allium cepa L. (Liliaceae), the seeds of Carica papaya Linn (Caricaceae), Cola acuminata (P. Beauv.) Schott and Endl. (Sterculiaceae), Buchholzia coriacea Engl. (Capparidaceae), Garcinia kola Heckel (Guttifeare), and Garcinia lucida Vesque (Guttifeare), the seeds and fruits of Picralima nitida; the potential of the extract from the above plant extracts to increase the activity of some antibiotics on MDR bacteria was also investigated as well as the role of bacterial efflux pumps in the resistance to the tested plant extracts.

Plant Materials and Extraction.
The nine edible plants used in this work were purchased from Dschang local market, west region of Cameroon in January 2010. The collected vegetal material were the fruits of Citrus medica, the bulbs of Allium sativum and Allium cepa, the seeds of Carica papaya, Cola acuminata, Buchholzia coriacea, Garcinia kola, and Garcinia lucida, the seeds and fruits of Picralima nitida. The plants were identified by Mr. Tadjouteu Fulbert (Botanist) of the National Herbarium (Yaoundé, Cameroon) where voucher specimens were deposited under a reference number ( Table 1).
The fresh or powdered air-dried sample (1 kg) from each plant was extracted with methanol (MeOH) 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.

Bacterial Strains and Culture
Media. The studied microorganisms include references (from the American Type Culture Collection) and clinical (Laboratory collection) strains of Escherichia coli, Enterobacter aerogenes, Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumonia, and Enterobacter cloacae (Table 2). They were maintained on agar slant at 4 • C and subcultured on a fresh appropriate agar plates 24 hrs prior to any antimicrobial test. Mueller Hinton Agar was used for the activation of bacteria. 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 [4]. Briefly, the test samples were first dissolved in DMSO/MHB. The solution obtained was then added to MHB, and serially diluted twofold (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. The final concentration of DMSO was lower than 2.5% and does not affect the microbial growth. Wells containing MHB, 100 μL of inoculums, and DMSO at a final concentration of 2.5% served as a negative control. Ciprofloxacin 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 minutes [5]. 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 microbial growth.
Samples were tested alone and then, in the presence of PAβN at 30 μg/mL final concentration. Two of the best extracts, those from seeds of Garcinia kola and Picralima nitida fruits were also tested in association with antibiotics at MIC/2 and MIC/5. These concentrations were selected following a preliminary assay on one of the tested MDR bacteria, P. aeruginosa PA124 (see Supplemental Material S1 available online at doi:10.1155/2012/623723.). All assays were performed in triplicate and repeated thrice. Fractional inhibitory concentration (FIC) was calculated as the ratio of MIC Antibiotic in combination /MIC Antibiotic alone and the interpretation made as follows: synergistic (FIC ≤ 0.5), indifferent (0.5 < FIC < 4), or antagonistic (FIC ≥ 4) [6]. (The FIC values are available in Supplemental Material S2).

Phytochemical Composition of the Plant Extracts.
The results of qualitative analysis showed that each plant contains various phytochemicals compounds such as alkaloids, anthocyanins, anthraquinons, flavonoids, phenols, saponins, tannins, and triterpenes as shown in Table 3.

Antibacterial Activity of the Plant Extracts.
Extracts were tested for their antibacterial activities alone and in combination with PAβN on a panel of Gram-negative bacteria by the microdilution method. Results summarized in Table 4 showed that the most active extracts were those from Garcinia lucida (MIC ranged from 128 to 512 μg/mL), Garcinia kola (MIC from 128 to 1024 μg/mL), and the fruits of Picralima nitida (MIC from 256 to 1024 μg/mL). The antibacterial activities of these plant species were recorded against all the 29 studied microorganisms. Other extracts exhibited weak activities against a limited number of strains studied.

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 the plants extracts, and reference antibiotic (ciprofloxacin) in the presence of PAβN, an EPI. Preliminary tests showed that PAβN did not have any antibacterial activity at 30 μg/mL. The association of the PAβN with the extracts reduced the MIC values of some of the extracts on some tested bacteria (Table 4). However, most of the studied extracts are not the substrates of the active efflux pumps.

Antibacterial Activities and Chemicals Compositions of the Tested Extracts.
The phytochemical studies revealed the presence of at least two classes of secondary metabolites in each of the plant extracts. Several alkaloids, flavonoids, phenols, saponins, anthocyanins, anthraquinones, sterols, tannins, and triterpenes have been found active on pathogenic microorganisms [44,45]. Some of these compounds were found to be present in the plant species under this study, and they could contribute to the observed antimicrobial activities of some plant extracts. The results of the phytochemical test on G. kola are in accordance with those obtained by Onayade et al., [46,47]. Many compounds have been isolated from G. kola, such as kolaflavone and 2-hydroxybiflavone [48][49][50] but their antimicrobials activities have not been evaluated. However, Adegboye et al. [51] reported the activity of G. kola on some streptomycin-sensitive Grampositive bacteria strain. The present study therefore provides additional information on the antibacterial potential of this plant on MDR bacteria. The previous phytochemical analyses on hexane extract from the seeds of G. lucida revealed several types of compounds [8,23]. These include terpenoids, anthocyanins, flavonoids, and saponins derivatives. This report therefore agrees well with the phytochemical data being reported herein.
The results of the phytochemical analysis of the extract of fruits of P. nitida are similar to those obtained by Kouitcheu [52]. Several alkaloids previously isolated from this plant include akuammicine, akuammine, akuammidine, picraphylline, picraline, and pseudoakuammigine [32,53]. Their antibacterial activities have not yet been demonstrated but many alkaloids are known to be active on Gram-negative bacteria [33]. Differences were noted in the chemical composition of the seeds and fruits of P. nitida, evidently explaining the differences in the antibacterial activity of the two parts of this plant. In fact, the presence of tannins in the fruits may contributes to its better activity compared to the seeds as they were reported to inactivate the microbial adhesins, enzymes, transports proteins and cellular envelop [54].
Extracts from C. papaya, C. medica, B. coriacea, A. cepa, and C. acuminata showed weak activities against a limited number of strains. Nonetheless, the extracts from B. coriacea were rather reported to have good antibacterial activities. Their weak activities as observed in the present paper could therefore be due to the multidrug resistance of the studied bacteria.

Effects of the Association of Some Plants Extracts with
Antibiotics. Three of the most active plants extracts (G. kola, G. lucida, and P. nitida) were associated with antibiotics with the aim to evaluate the possible synergistic effects of their associations. A preliminary study using P. aeruginosa PA124, one of the ten MDR bacteria used in this paper, was carried out with ten antibiotics (CLX, AMP, ERY, KAN, CHL, TET, FEP, STR, CIP, and NOR) to select the appropriate sub-inhibitory concentrations of the extract to be used. The results (see Supplemental Material S1) allowed the selection of G. kola, G. lucida and their MIC/2 and MIC/5 as the subinhibitory concentrations. No antagonistic effect (FIC ≥ 4) was observed between extracts and antibiotics meanwhile indifference was observed in the case of CLX, FEP, AMP, which are β-lactams acting on the synthesis of the bacteria cell wall [55] (Tables 5 and 6, Supplemental Material S2). Many studies demonstrated that efflux is the mechanism of resistance of bacteria for almost all antibiotic classes [56]. It is well demonstrated that the efflux pumps reduce the intracellular concentration of antibiotics and consequently their activities [57]. The MDR bacteria strains used in this paper are known for their ability to overexpress active efflux [58]. At MIC/2, synergistic effects were noted with the association of NOR, CHL, TET (on 100% the studied bacteria), ERY (on 80%), CIP (on 70%), and P. nitida extract meanwhile G. kola extract also increased the activity of NOR, TET (on 100%), ERY, and CIP (on 70%). Plant can be considered as an efflux pumps inhibitor if a synergistic effect with antibiotics is induced on more than 70% bacteria expressing active efflux pumps [6]. Therefore, the extracts from P. nitida and G. kola probably contain compounds that can acts as EPI. The results of the present paper corroborate with those of Iwu et al. [7] reporting the existence of synergy effects between G. kola extract and gatifloxacin (G. kola/gatifloxacin in the proportions of 9/1, 8/2, 7/3, and 6/4) against Bacillus subtilis and the proportions of G. kola/gatifloxacin (at 9/1, 2/8, and 1/9) against Staphylococcus aureus.
The overall results of the present work provide baseline information for the possible use of the studied plants and mostly G. Lucida, G. Kola, and P. Nitida extracts in the treatment of bacterial infections involving MDR phenotypes. In addition, the extracts of these plants could be used in association with common antibiotics to combat multidrug resistant pathogens.