In Vitro antibacterial and antibiotic-potentiation activities of four edible plants against multidrug-resistant gram-negative species

Background The present study was designed to investigate the antibacterial activities of the methanol extracts of four Cameroonian edible plants, locally used to treat microbial infections, and their synergistic effects with antibiotics against a panel of twenty nine Gram-negative bacteria including Multi-drug resistant (MDR) phenotypes expressing active efflux pumps. Methods The broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of the extracts [alone and in the presence of the efflux pumps inhibitor (EPI) Phenylalanine-Arginine β-Naphtylamide (PAβN)], and those of antibiotics in association with the two of the most active ones, Piper nigrum and Telfairia occidentalis. The preliminary phytochemical screening of the extracts was conducted according to the standard phytochemical methods. Results Phytochemical analysis showed the presence of alkaloids and flavonoids in all studied extracts. Other chemical classes of secondary metabolites were selectively present in the extracts. The results of the MIC determination indicated that the crude extracts from P. nigrum and V. amygdalina were able to inhibit the growth of all the twenty nine studied bacteria within a concentration range of 32 to 1024 μg/mL. At a similar concentration range (32 to 1024 μg/mL) the extract from T. occidentalis inhibited the growth of 93.1% of the tested microorganisms. At MIC/2 and MIC/5, synergistic effects were noted between the extracts from P. nigrum and T. occidentalis and seven of the tested antibiotics on more than 70% of the tested bacteria. Conclusion The overall results of the present study provide information for the possible use of the studied edible plants extracts in the control of bacterial infections including MDR phenotypes.


Background
Despite the impressive scientific progress in vaccination and chemotherapy, infectious diseases remain a serious health issue. Following the massive and inappropriate use of antibiotics, bacteria have developed various mechanism of resistance; consequently, infectious diseases remain one of the leading causes of morbidity worldwide [1]. Microbial infections constitute a major public health problem in developing countries [2] where the high cost of antibiotics makes them unaffordable to the majority of the population. Therefore, the discovery of new antimicrobial agents is still relevant nowadays. Among the bacterial resistance mechanisms, efflux of antibiotics plays an important role; In fact it is widely recognized that the expression of efflux pumps encoded by housekeeping genes in bacteria is largely responsible for the phenomenon of intrinsic antibiotic resistance [3]. Also, the shortcomings of the drugs available today and the scarcity of novel antibiotics propel the discovery of new chemotherapeutic agents from medicinal plants [4]. The medicinal properties of many phytochemicals have been demonstrated [5]. In addition, promising new concepts such as the efflux pump inhibitors [6,7], and synergy between antibiotics and phytochemicals are now been explored.
The present work was therefore designed to investigate the antibacterial potential of four Cameroonian edible plants used traditionally in the treatment of bacterial infections, namely the fruits of Piper nigrum L (Piperaceae), the leaves of Telfairia occidentalis Hook.

Plant material and extraction
The four edible plants used in this work were purchased from Dschang local market, West Region of Cameroon in June 2010. The collected plants material were the fruits of Piper nigrum, the fruits of Syzygium aromaticum, the leaves of Telfairia occidentalis and the leaves of Vernonia amygdalina. These plants were identified by M. Victor Nana of the National Herbarium (Yaounde-Cameroon) where all the voucher specimens were available under the reference numbers (see Table 1). The air dried and powdered sample (1 kg) from each plant was extracted with methanol (MeOH) for 48 h at room temperature. The extracts were then filtered and concentrated under reduced pressure to give the crude extracts. All extracts were kept at 4°C until further investigations.

Preliminary phytochemical investigations
The major classes of secondary metabolites such as alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, tannins, sterols and triterpenes were screened according to the common phytochemical methods described by Harbone [44].

Bacterial strains and culture media
The studied microorganisms included the reference (from the American Type Culture Collection) and clinical (Laboratory collection) strains of Providencia stuartii, Pseudomonas aeruginosa, K. pneumoniae, Escherichia coli, Enterobacter aerogenes and Enterobacter cloacae (See supporting information Additional file 1: Table S1 for their features). They were maintained in a Nutrient Broth at 4°C and activated on a fresh appropriate Mueller Hinton Agar plates 24 h prior to antimicrobial test. The Mueller Hinton Broth (MHB) was also used for all the antibacterial assays.

Bacterial susceptibility determination
The MICs were determined using the rapid INT colorimetric assay [45,46]. 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 set at 2.5% (a concentration at which DMSO does 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). Chloramphenicol was used as reference antibiotic. The MICs of samples were detected after 18 h incubation at 37°C, following addition of 40 μL of a 0.2 mg/mL INT solution and incubation at 37°C for 30 minutes. Viable reduce the yellow dye to pink. MIC was defined as the lowest sample concentration that exhibited complete inhibition of microbial growth and then prevented this change [47].
Samples were tested alone and the best three extracts (those from the seeds of P. nigrum, T. occidentalis and V. amygdalina) were also tested in the presence of PAβN at 30 mg/L final concentration. After a preliminary assay on one of the MDR bacteria, P. aeruginosa PA124 (See supporting information Additional file 1: Table S2), the two best extracts were those from P. nigrum and T. occidentalis. They were then selected and tested at MIC/2 and MIC/5 in association with antibiotics. 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) [48,49]. All assays were performed in triplicate.

Effect of the association of extracts with antibiotics
A preliminary study (See supporting information; Additional file 1: Table S2) (Table 5).

Antibacterial activities and chemical composition of the tested extracts
Many secondary metabolites belonging to alkaloids, anthocyanins, anthraquinons, flavonoids, phenols, saponins, sterols, tannins and triterpenes were detected in the tested plant extracts. Several compounds from the investigated classes of phytochemicals were reported for their antibacterial activities [50,51], and their presence in the tested extracts could explain their antibacterial effects. The differences in bacterial susceptibility to the extracts may be either due to the differences in cell wall composition and/or genetic content of their plasmids [52] or to the differences in the composition and the mechanism of action of the bioactive compounds [53]. As shown in Table 3, the three most active plants (P. nigrum, T. occidentalis and V. amygdalina) possess more classes of phytochemicals than the extract from S. aromaticum. Each of the three most active plant extracts contains at least four classes of secondary metabolites namely alkaloids, phenols, flavonoids and tannins. However, it should be noted that the activity does not depend on the number of classes of detected bioactive compounds, but mostly on their concentration. The inhibitory activity of P. nigrum was previously reported against some bacteria such as Staphylococcus aureus, Bacillus cereus, Streptococcus faecalis, Pseudomonas aeruginosa, Salmonella typhi and Escherichia coli [54], and the data reported in this study confirms the anti-infective potential of this plant. It has also been demonstrated that the acetone-ethanol extract of the leaves from V. amygdalina was weakly active against K. pneumoniae, E. coli, S. aureus, B. cereus, S. dysentriae and S. typhimurium [35] with MIC values ranged from 7.5 mg/mL to 25 mg/mL [42]. These activities are in accordance with the results obtained in the present work, but we observed higher antibacterial activity of this plant on all 29 bacteria including MDR phenotypes (with MIC values ranging between 256 and 1024 μg/mL).

Role of efflux pumps in the susceptibility of Gram-negative bacteria to the tested extracts and effects of the association of some extracts with antibiotics
All the bacterial strains tested with a combination of plant extract and PAβN were proven to possess multidrug resistance efflux pumps [55][56][57][58][59]. Tripartite efflux systems, mainly those clinically described such as AcrAB-TolC in Enterobacteriaceae or MexAB-OprM in P. aeruginosa play a central role in multidrug resistance of pathogenic Gramnegative bacteria [55,56]. PAßN, a potent inhibitor of the RND efflux systems is especially active on AcrAB-TolC and MexAB-OprM [57,58] and does not present any intrinsic effect on the bacteria at the concentration of 30 μg/mL used in this work [59]. In the presence of PAßN at this concentration, significant increase of the activity of the extract from P. nigrum was noted against 13/14 of the tested MDR bacteria. This shows that at least one active compound from this plant, acting inside the bacteria cell could be the substrate of efflux pumps. From this observation, it can be suggested that the association of the extract of P. nigrum and efflux pump inhibitors could be helpful in the fight against infections due to MDR bacteria [5]. Moreover, we demonstrated in this study that the beneficial effect of the combination of two of the tested plant extracts namely those from P. nigrum and T. occidentalis, with the first line antibiotics could be achieved. Their synergistic effects with antibiotics were noted on more than 70% of the tested MDR bacteria (with seven antibiotics), also suggesting that some of their constituents can act as efflux pump inhibitor [49]. This hypothesis is emphasized by the fact that these extracts were more synergistic with antibiotics acting inside the bacteria cells. Besides, it has already been proved that the extract from P. nigrum can also act by  improving the penetration of antibiotics in cells via membrane alteration [54]. However, further phytochemical investigations will be done to isolate the active constituents of P. nigrum, T. occidentalis and V. amygdalina. Besides, toxicological studies will be carried out to evaluate their safety.

Conclusion
The overall results of the present study provide baseline information for the possible use of the tested plants and mostly P. nigrum, T. occidentalis and V. amygdalina in the control of infections due to MDR Gram-negative bacteria. In addition, the extracts from P. nigrum and T. occidentalis could be used in association with antibiotics to combat multidrug resistant pathogens.