Antibacterial activities of selected Cameroonian spices and their synergistic effects with antibiotics against multidrug-resistant phenotypes

Background The emergence of multi-drug resistant (MDR) phenotypes is a major public health problem today in the treatment of bacterial infections. The present study was designed to evaluate the antibacterial activities of the methanol extracts of eleven Cameroonian spices on a panel of twenty nine Gram negative bacteria including MDR strains. Methods The phytochemical analysis of the extracts was carried out by standard tests meanwhile the liquid micro-broth dilution was used for all antimicrobial assays. Results Phytochemical analysis showed the presence of alkaloids, phenols and tannins in all plants extracts. The results of the antibacterial assays indicated that all tested extracts exert antibacterial activities, with the minimum inhibitory concentration (MIC) values varying from 32 to 1024 μg/ml. The extracts from Dichrostachys glomerata, Beilschmiedia cinnamomea, Aframomum citratum, Piper capense, Echinops giganteus, Fagara xanthoxyloïdes and Olax subscorpioïdea were the most active. In the presence of efflux pump inhibitor, PAßN, the activity of the extract from D. glomerata significantly increased on 69.2% of the tested MDR bacteria. At MIC/5, synergistic effects were noted with the extract of D. glomerata on 75% of the tested bacteria for chloramphenicol (CHL), tetracycline (TET) and norfloxacin (NOR). With B. cinnamomea synergy were observed on 62.5% of the studied MDR bacteria with CHL, cefepime (FEP), NOR and ciprofloxacin (CIP) and 75% with erythromycin (ERY). Conclusion The overall results provide information for the possible use of the studied extracts of the spices in the control of bacterial infections involving MDR phenotypes.


Background
The emergence of MDR phenotypes is a major public health problem today in the treatment of bacterial infections. The multi-drug resistance of Gram negative bacteria is a major cause of morbidity and mortality in health care services [1]. The activation of bacterial efflux pumps also plays an important role in the appearance of resistance to antibiotics [2]. The real challenge for scientists worldwide today, is to continuously find new drugs to combat resistant microorganisms, or compounds which are able to inhibit the resistance mechanisms of pathogens, therefore restoring the activity of antibiotics. Medicinal plants are rich in compounds which may be potential natural drugs and serve as alternative, less expensive and safe antimicrobials for the treatment of common ailments. Plant drugs are widely used in Africa for the treatment of many ailments and constitute the first health recourse for about 80% of the population [3]. A number of pharmaceutical products in current use worldwide are derived from plants [4]. In Cameroon, many medicinal plants including spices are used as herbal medicines. The present work was therefore designed to investigate the antibacterial potential against MDR bacteria of some of the commonly used medicinal spices in Cameroon such as Fagara xantoxyloides Watern., Dichrostachys glomerata (Forsk) Chuov, Olax subscorpioïdea Oliv., Solanum melongeua L. Var inerme D.C Hiern, Piper capense Lin.f, Xylopia aethiopica Dunal A. Rich., Aframomum citratum (Pereira). Schum, Scorodophloeus zenkeri Harms., Beilschmiedia cinnamomea (Stapf) Robyns & Wilczek, Echinops giganteus A. Rich and Mondia whitei (Hook F). Skell. This study was also extended to the evaluation of the potencies of the above plant extracts to increase the activity of some antibiotics on MDR bacteria. The role of bacterial efflux pumps in resistance to the extracts was also studied.

Plant materials and extraction
The eleven edible spices used in this work were purchased from Dschang local market, West Region of Cameroon in January 2010. The collected spices materials were: the fruits of Fagara xanthoxyloides, Dichrostachys glomerata, Olax subscorpioïdea, Solanum melongeua, Piper capense and Xylopia aethiopica, the bark of Aframomum citratum, Scorodophloeus zenkeri, Beilschmiedia cinnamomea and the roots of Echinops giganteus and Mondia whitei. The plants were identified by Mr. Fulbert Tadjouteu of the National herbarium (Yaoundé, Cameroon) where voucher specimens were deposited under the reference numbers ( Table 1).
The air dried and powdered sample (1 kg) from each spice was extracted with methanol (MeOH) for 48 h at room temperature. The extract was then concentrated under reduced pressure to give residues which constituted the crude extracts. They were then kept at 4°C until further use.

Preliminary phytochemical investigations
The major classes of secondary metabolites; alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, tannins, steroids and triterpenes were screened according to the common phytochemical methods described by Harborne [5] with some modifications. Briefly, for alkaloids (5 mg plant extract in 10 ml methanol); a portion of 2 ml extract + 1% HCl + steam, 1 ml filtrate + 6 drops of Mayor's reagents/Wagner's reagent/ Dragendroff reagent; creamish precipitate/brownish-red precipitate/orange precipitate indicated the presence of respective alkaloids. For tannins (5 mg plant extract in 10 ml distilled water); a portion of 2 ml + 2 ml FeCl 3 ; blue-black precipitate indicated the presence of tannins. For saponins (frothing test: 0.5 ml filtrate + 5 ml distilled water); frothing persistence indicated presence of saponins. For steroids and triterpenoids (Liebermann-Burchard reaction: 5 mg plant extract in 10 ml chloroform, filtered); a 2 ml filtrate + 2 ml acetic anhydride + conc. H 2 SO 4 . Blue-green ring or pink-purple indicated the presence of steroids or triterpenoids. For flavonoids (5 mg plant extract in 10 ml methanol); a portion of 2 ml + conc. HCl + magnesium; ribbon pink-tomato red color indicated the presence of flavonoids. For anthocyanins (5 mg plant extract in 10 ml methanol); a portion 2 ml + 1% HCl +heating; orange color indicated the presence of anthocyanins. For anthraquinones (5 mg plant extract in 10 ml methanol); a portion of 2 ml + 2 ml ether-chloroform 1:1 v/v + 4 ml NaOH 10% (w/v); red color indicated the presence of anthraquinones. For phenols (5 mg plant material in 10 ml methanol); a portion of 2 ml + 2 ml FeCl 3 ; violet-blue or greenish color indicated the presence of phenols.

Bacterial strains and culture media
The studied microorganisms included reference (from the American Type Culture Collection) and clinical (Laboratory collection) strains of Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes and Enterobacter cloacae (Table 2). They were maintained on agar slant at 4°C and sub-cultured on a fresh appropriate agar plates 24 h 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 using rapid INT colorimetric assay [6,7]. 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-wells 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 h. The final concentration of DMSO was lower than 2.5% and did not affect the microbial growth. 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). The total volume in each well was 200 μl. Chloramphenicol was used as reference antibiotic. The MICs of samples were detected after 18 h incubation at 37°C, following addition (40 μl) of 0.2 mg/ml INT and incubation at 37°C for 30 minutes. 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 [8].
Samples were tested alone and then, in the presence of PAßN at 30 μg/ml final concentration. Two of the best extracts [those from D. glomerata and B. cinnamomea] 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 Additional file 1, Table A1). 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 (<0.5), indifferent (0.5 to 4), or antagonistic (>4) [9] (The FIC values available in Additional file 1, Tables A2 and A3).

Phytochemical composition of the spice extracts
The results of the phytochemical studies (Table 3) showed that all the tested extracts contain alkaloids, phenols and tannins. Anthocyanins, anthraquinones, flavonoids, saponins, sterols and triterpenes were selectively present.

Antibacterial activity of the spice extracts
The results of the antibacterial activity of the extract alone on a panel of Gram negative bacteria are summarized in Table 4. It appears that the extract from Piper capense Lin.f (Piperaceae)       Role of efflux pumps in susceptibility of Gram negative bacteria to the tested spice extracts The various strains and MDR isolates were tested for their susceptibilities to the spice extracts, and reference antibiotic, CHL in the presence of PAßN, a well-known efflux pump inhibitor. The results presented in Table 4 showed that the activity of the extract from D. glomerata significantly increased in the presence of PAßN on 18/26 (69.2%) of the tested bacteria. The MIC values below 100 μg/ml were noted with this extract against E. coli MC4100 and W3110 (< 8 μg/ml), K. pneumoniae KP63 and K24 (< 8 μg/ml and 32 μg/ml respectively) and P. stuartii NAE16 (32 μg/ml). Apart from the extract of D. glomerata, PAßN did not induce an increased activity of other tested extract.

Effects of the association of some spice extracts with antibiotics
To evaluate the possible synergistic effects of the extracts with antibiotics, four of the most active samples (F. xanthoxyloïdes, D. glomerata, B. cinnamomea and O. subscorpioïdea) were selected. A preliminary study using P. aeruginosa PA124, one of the MDR bacteria used in this work, was carried out with ten antibiotics (CLX, AMP, ERY, KAN, CHL, TET, FEP, STR, CIP and NOR) to select the appropriate sub-inhibitory concentrations to be used. The results (see Additional file 1, Table A1) allow the selection of MIC/2 and MIC/5 as the sub-inhibitory concentrations of the extracts from D. glomerata and B. cinnamomea, which were then tested on eight MDR bacteria, E. coli AG100, AG100 TET , K. pneumoniae KP55, E. aerogenes EA3, EA27, EA289, CM64 in addition to P. aeruginosa PA124. The results are summarized in Tables 5 and 6. Synergistic effects were observed with the association between D. glomerata ( Table 5, Additional file 1, Table A2) and B. cinnamomea (  (Table 6) also induced significant increase of the activity of several antibiotics, the synergistic effects being noted on 25% of the tested bacteria in the case of CLX and AMP, 50% in the case of KAN, 62.5% in the case of FEP and STR, 75% in the case of CHL, TET and CIP, 87.5% in the case of ERY and 100% for NOR. With this extract, synergistic effects were also observed at MIC/5 on 25% of the studied MDR bacteria in the case of CLX and AMP, 37.5% in the case of STR and KAN, 50% in the case of TET, 62.5% in the case of CHL, FEP, NOR and CIP and 75% in the case of ERY.

Phytochemical composition of the spice extracts
Phytochemical screening revealed the presence of several classes of secondary metabolites. Though the detection of such metabolites does not automatically predict the antimicrobial activity of a plant extract, it has clearly been demonstrated that several compounds belonging to the investigated classes of metabolites showed antibacterial activities [4,[10][11][12].
Antibacterial activity of the spice extract Phytochemicals are routinely classified as antimicrobials on the basis of susceptibility tests that produce MIC in the range of 100 to 1000 μg/ml [13]. Activity is considered to be significant if MIC values are below 100 μg/ml for crude extract and moderate when 100 < MIC < 625 μg/ml [11]. Therefore, the activity recorded with B. cinnamomea and E. giganteus respectively on E. aerogenes EA294 and K. pneumoniae K24, and X. aethiopica on E. coli ATCC10536 and K. pneumoniae KP63 can be considered significant. Alternative criteria have been described by Fabry et al. [14], which consider extracts having MIC values below 8000 μg/ml to have noteworthy antimicrobial activity. Under these less stringent criteria, and considering the fact that the spices tested are used as food ingredients with limited toxicity, the overall activity recorded with several extracts, most notably those of D. glomerata, B. cinnamomea, A. citratum, P. capense, E. giganteus, F. Xanthoxyloïdes and O. subscorpioïdea, could be considered important. Besides, some of the tested samples were more active than CHL used as reference antibiotic on some of the MDR bacteria such as E. cloacae ECCI69 and BM47, E. aerogenes EA27 and EA289, highlighting the importance of the results reported herein. It can be noted that all the investigated phytochemical classes were detected in the extracts of D. glomerata, S. melongena and M. withei. Contrary to D. glomerata extract that exhibited a good spectrum of activity, the inhibition potential of S. melongena and M. withei was lower and seems not in correlation with their chemical composition. This clearly confirms the fact that the presence of secondary metabolites does not automatically predict the antimicrobial activity of a plant  To the best of our knowledge, the antibacterial activity of B. cinnamomea and P. capense is being reported for the first time. Moreover, the present work reports for the first time the activity of the tested spices on MDR bacteria. Nevertheless, the antimicrobial potential of some of the plants or related genus were demonstrated on sensitive strains. Banso and Adeyemo [15] reported the presence of antibacterial tannins in the genus Dichrostachys. Chouna et al. [16] also demonstrated that Beilschmiedia anacardioides was significantly active against Bacillus subtilis, Micrococcus luteus and Streptococcus faecalis. Plants of the genus Echinops such as E. ellenbeckii and E. longisetus were found active on Staphylococcus aureus [17] meanwhile the antibacterial activity of the essential oils and alkaloids from F. xanthoxyloïdes was also documented [18,19]. The aqueous and ethanol extracts from O. subscorpioïdea were found active on both bacteria and fungi [20]. The results obtained in the present work therefore provide additional information on the studied plants and are in consistence with some of the previous reports.

Role of efflux pumps in susceptibility of Gram negative bacteria to the tested spice extracts
Tripartite efflux systems, mainly those clinically described as AcrAB-TolC in Enterobacteriaceae or MexAB-OprM in P. aeruginosa, are associated with a major human health problem as they play a central role in multidrug resistance of pathogenic Gram negative bacteria [21][22][23]. PAßN has been reported as a potent inhibitor of the RND efflux systems and is especially active on AcrAB-TolC and MexAB-OprM [22,24,25]. To determine the role of efflux pumps in this work, the concentration of PAßN used (30 μg/ml) had no intrinsic effect on the bacteria as previously determined [26]. In contrast, with these conditions significant increase of the antibacterial activity of D. glomerata extract was noted, showing that one or more active compounds from this plant could be substrate(s) of efflux pumps acting in resistant strains of E. coli, K. pneumoniae and P. stuartii. These data suggest that possible association of the extract of D. glomerata and efflux pump inhibitor can be envisaged to improve the fight against MDR phenotypes.
Effects of the association of extracts from D. glomerata and B. cinnamomea with antibiotics The association of natural products such as plant extracts and antibiotics constitutes an alternative in the fight against MDR bacteria. Significant synergistic effects were noted with both D. glomerata and B. cinnamomea extracts when they were associated with several antibiotics. Such effects might be due either to the action of the active compounds or possible inhibition of the efflux pumps by other compounds of the extracts. The lowest synergistic effects were observed with b-lactamines (CLX and AMP), obviously due to the fact their target are localized in the bacterial cell coat. However, the synergistic effects observed indicate that active compounds of the extract could also present different mode(s) of action from those of the studied antibiotics.

Conclusion
The overall results of the present work provide baseline information for the possible use of the studied spice extracts in the treatment of bacterial infections involving MDR phenotypes. In addition to these antibacterial activities, the data reported herein indicated that possible combinations of the extract of D. glomerata with an efflux pump inhibitor, and also the association of extract of this plant as well as that from B. cinnamomea with several antibiotics could be used in the control of bacterial infections involving MDR phenotypes.

Additional material
Additional file 1: Table S1. Activities of antibiotics in combination with the sub-inhibitory concentrations of some plants extracts on Pseudomonas aeruginosa PA124. Table S