In vitro antibacterial and antibiotic-potentiation activities of the methanol extracts from Beilschmiedia acuta, Clausena anisata, Newbouldia laevis and Polyscias fulva against multidrug-resistant Gram-negative bacteria

Background The present study was designed to investigate the antibacterial activities of the methanol extracts from different parts of Beilschmedia acuta Kosterm (Lauraceae), Clausena anisata (Willd) Hook (Rutaceae), Newbouldia laevis Seem (Bignoniaceae) and Polyscias fulva (Hiern) Harms (Araliaceae) as well as their synergistic effects with antibiotics against a panel of Gram-negative bacteria, including multi-drug resistant (MDR) phenotypes expressing active efflux pumps. Methods Broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) of the extracts, as well as those of antibiotics in association with the most active ones, B. acuta, N. laevis and P. fulva. Results MIC values obtained indicate that extracts from the bark of B. acuta were active on all the 26 tested Gram-negative bacteria, with MICs ranging from values below 8 to 256 μg/mL. Other samples displayed selective activities, their inhibitory effects being observed on 9 (34.62 %) of the 26 bacterial strains for N. laevis leaves extract, 6 (23.10 %) for both C. anisata leaves and roots extracts, 7 (26.9 %) and 4 (15.4 %) for leaves and roots extracts of P. fulva respectively. Extract from B. actua bark displayed the best antibacterial activity with MIC values below 100 μg/mL against 16 (61.5 %) of the 26 tested microorganisms. The lowest MIC values (below 8 μg/mL) were obtained with this extract against Escherichia coli W3110 and Klebsiella pneumoniae ATCC11296. The MIC values of this extract were lower than those of ciprofloxacin against E. coli W3110, Enterobacter aerogenes ATCC13048, CM64 and Providencia stuartii NAE16. At MIC/2, the best percentages of synergistic effects (100 %), were obtained with B. acuta bark extract and tetracycline (TET) as well as with P. fulva leaves extract and TET and kanamycin (KAN). Conclusion The overall results of the present study provide information for the possible use of the studied plants and mostly Beilschmedia acuta in the control of bacterial infections including MDR phenotypes. Electronic supplementary material The online version of this article (doi:10.1186/s12906-015-0944-5) contains supplementary material, which is available to authorized users.


Background
Fighting multi-drug resistant (MDR) Gram-negative (MDRGN) bacteria remains a challenging issue worldwide. Microbial infections involving MDRGN bacteria constitute a major public health problem in developing countries [1] where the high cost of antibiotics makes them unaffordable to the majority of the population. Clinically, the continuous emergence of MDRGN bacteria drastically reduced the efficacy of antibiotic arsenal and, consequently, increased the frequency of therapeutic failure [2]. Therefore, the discovery of new antimicrobial agents is still relevant nowadays. Also, the shortcomings of drugs available today and scarcity of novel antibiotics propel the discovery of new chemotherapeutic agents from medicinal plants [3]. Approximately 60 % of the world population still relies on medicinal plants for their primary healthcare [4]. Medicinal plants have been used as a source of remedies since ancient times in Africa. In addition, promising new concepts such as the efflux pump inhibitors [5,6], and synergy between antibiotics and phytochemicals are now being developed. The ability of several African medicinal plants to inhibit the growth of MDRGN bacteria, as well as their ability to potentiate the activity of commonly used antibiotics was previously reported. Some of these plants include Dorstenia psilurus, Dichrostachys glomerata and Beilschmiedia cinnamomea [7][8][9].

Plant material and extraction
All medicinal plants used in the present work were collected in different areas of Cameroon between January and April 2012. The plants were identified at the National Herbarium (Yaounde, Cameroon), where voucher specimens were deposited under the reference numbers (Table 1). Air-dried and powdered plant material was weighed (300 g) and soaked in 1 L of methanol (MeOH) for 48 h at room temperature. The filtrate obtained through Whatman filter paper No.1 was concentrated under reduced pressure in a vacuum to obtain the crude extract. All crude extracts were kept at 4°C until further use.

Microbial strains and culture media
The studied microorganisms included sensitive and resistant strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes, Escherichia coli obtained from the American Type Culture Collection (ATCC). Their bacterial features are summarized in Table 2. Nutrient agar was used to activate the tested Gram-negative bacteria [20].

INT colorimetric assay for MIC and MBC determinations
The MIC determination on the tested bacteria was conducted using rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay according to described methods [18] with some modifications [21,22]. The test samples and RA were first dissolved in DMSO/Mueller Hinton Broth (MHB). The final concentration of DMSO was lower than 2.5 % and does not affect the microbial growth [23,24]. The solution obtained was then added to Mueller Hinton Broth, and serially diluted two fold (in a 96-wells microplate). One hundred microlitre (100 μL) of inoculum 1.5 x 10 6 CFU/mL prepared in appropriate broth was then added [21,22]. The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a plate shaker and incubated at 37°C for 18 h. 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 and incubation at 37°C for 30 min. Viable bacteria reduced the yellow dye to pink. The MIC was defined as the sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth [18]. 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 Cancer and gastrointestinal infections [10].
Cytotoxicity towards leukemia, breast, glioblastoma, colon and liver cancer cell lines [10]. Cancers, spasms, infectious diseases, male infertility and diabetes [11,12], coagulant or anti-hemorrhagic properties; digestive threats, urogenital and pulmonary infections [13,14]; Dysentery, worms, malaria, sexually transmitted diseases, dental caries and diarrhea [15]. MBC was regarded as the lowest concentration of extracts, which did not produce a color change after addition of INT as mentioned above [21,22]. Samples were tested alone and the best four extracts (those from the leaves and bark of Beilschmedia acuta, and from the leaves of Newbouldia laevis and Polyscias fulva) were also selected and tested in association with antibiotics at the sub-inhibitory concentrations (MIC/2 and MIC/5) [7][8][9] against nine MDR bacteria. Fractional inhibitory concentration (FIC) was calculated as the ratio of MIC Antibiotic in combination /MIC Antibiotic alone and the results were discussed as follows: synergy (≤0.5), indifferent (0.5 to 4), or antagonism (>4) [25,26]. All assays were performed in triplicate.

Results
The antibacterial activities of methanol extracts from various parts of Beilschmedia acuta, Clausena anisata, Newbouldia laevis and Polyscias fulva are summarized in Table 3 (MIC values up to 1024 μg/mL are provided as supporting information; Additional file 1:         Table S2, supporting information). Five commonly used antibiotics (CIP, TET, KAN, AMP and CHL) were combined with extracts from B. acuta leaves and bark and those from the leaves of N. laevis and P. fulva at their MIC/2 and MIC/5, as obtained on each of nine tested bacterial strains (Tables 4 and 5). Synergistic effects were observed with all tested extracts and all studied antibiotics on at least one of the nine selected bacteria. The best percentages of synergistic effect (100 %) were obtained at MIC/2 with B. acuta bark extract in combination with TET (Table 5) as well as with P. fulva leaves extract in association with TET and KAN (Table 5).

Discussion
Phytochemicals are routinely classified as antimicrobials on the basis of susceptibility tests that produce MICs in the range of 100 to 1000 μg/mL [27]. Moreover, for crude extracts, the antimicrobial activity is considered to be significant if MIC values are below 100 μg/mL and moderate when 100< MIC <625 μg/mL [28,29]. Therefore, the activity recorded with B. acuta bark extract against the 26 tested bacterial strains can be considered as very important. If we consider the alternative criteria described by Fabry et al. [30], where extracts having MIC values less than 8000 μg/mL have noteworthy antimicrobial activity, the overall activity recorded with the leaves and fruit extracts of B. acuta, P. fulva and N. laevis leaves extracts can also be considered promising. A keen look of the results of MIC and MBC determinations (Table 3, Additional file 1: Tables S1 and S2) indicates that MBC/MIC ratios were mostly above four, suggesting that studied extracts, including the most active ones, generally displayed bacteriostatic effects (MBC/MIC > 4) [31][32][33]. Various classes of phytochemicals (Table 2) were previously detected in the extracts of the four tested plants [10] and this may explain their antibacterial activity.
The results obtained in this study, and mostly those obtained with the bark of B. acuta are very important when taking in consideration the fact that most of the bacterial strains used were MDR phenotypes expressing active efflux pumps [7-9, 34, 35]. In fact, the activity of antibiotics against the studied MDR bacteria was previously found to increase in the presence of phenylalanine arginine β-naphthylamide (PAßN), a potent inhibitor of RND efflux systems, particularly AcrAB-TolC (of Enterobaceriaceae) and MexAB-OprM (of Pseudomonas species) [7-9, 34, 35]. In the present study, we demonstrated that beneficial effects when combining four of the tested plant extracts [namely those from B. acuta (leaves and bark), N. leavis (leaves) and P. fulva (leaves)] with the first line antibiotics could be achieved. High percentages of synergistic effects (100 %) obtained with B. acuta bark extract and TET as well as P. fulva leaves extract in combination with TET and KAN, clearly suggest that such associations could improve the fight against MDR bacterial infections. This also suggests that some of the constituents of the corresponding plants can act as efflux pump inhibitors, as more than 70 % synergistic cases were observed with many combinations [26].
The antimicrobial potential of the genus Beilschmiedia has previously been documented. Chouna et al. [36] demonstrated that compounds such as beilschmiedic acid C isolated from B. anacardioides were significantly active against Bacillus subtilis, Micrococcus luteus and Streptococcus faecalis. Beilschmiedia cinnamomea was previously reported to have significant to moderate activities (64-1024 μg/mL) against the MDRGN tested in this work [7]. Beilschmedia obscura was also found to show a good and large spectrum of antibacterial activity against MDRGN [37]. Some compounds previously isolated from the genus Beilschmiedia and belonging to alkaloids, phenols, saponines, sterols and triterpenoids [36,38] were shown to possess antimicrobial activities [7]. The genus Beilschmiedia is also known traditionally to possess antimicrobial activities [7]. Beilschmedia acuta tested in this study is also used in Cameroon to treat gastrointestinal infections [10]. The obtained data highlight the importance of this plant in the control of microbial infections and mostly those involving MDR phenotypes. The antimicrobial activities of extracts and compounds from Newbouldia laevis towards sensitive bacteria and fungi were also reported [39,40], and the present study provides additional data on the potential of this plant to fight MDR bacteria. Also, the antimicrobial activity of essential oil from Clausena anisata was reported against Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus species, Salmonella typhimurium and Pseudomonas aeruginosa [41,42]. The present report provides more evidence of the antimicrobial potential of this plant.

Conclusion
The results of this study are very interesting, in regards to the medical importance of the studied microorganisms. These data provided evidence that crude extracts from the studied plants and mostly that from the bark of Beilschmedia acuta are potential sources of antimicrobial drugs to fight MDR bacterial infections. The purification of this plant will be carried out to isolate its active constituents. The cytotoxicity assays on normal cell lines constitute the limitation of the present work and will further be performed to ensure the safety of the tested extracts.