Antibacterial activities of Beilschmiedia obscura and six other Cameroonian medicinal plants against multi-drug resistant Gram-negative phenotypes

Background The rapid spread of bacteria expressing multi-drug resistance propels the search for new antibacterial agents. The present study was designed to evaluate the antibacterial activities of the methanol extracts from Beilschmiedia obscura and six other Cameroonian plants against a panel of twenty nine Gram-negative bacteria including Multi-drug resistant (MDR) phenotypes. Methods The phytochemical investigations of the extracts were carried out according to the standard methods and the liquid micro-dilution assay was used for all antibacterial assays. Results Phytochemical analysis showed the presence of alkaloids in all studied extracts. Other chemical classes of secondary metabolites such as anthocyanines, anthraquinones flavonoids, saponins, tannins, sterols and triterpenes were selectively detected in the extracts. The extract from the fruits of Beilschmiedia obscura, Pachypodanthium staudtii leaves and Peperomia fernandopoiana (whole plant) displayed the best spectrum of activity with MIC values ranging from 16 to 1024 μg/mL against at least 65% and above of the tested bacteria. The extract from Beilschmiedia obscura was the most active with MIC values below 100 μg/mL against ten of the tested bacteria. This extract also showed MBC values below 1024 μg/mL against 55.17% of the studied microorganisms. Phenylalanine arginine β-naphthylamide (PAβN) significantly modulated the activities of extracts from the leaves and fruits of Pachypodanthium staudtii and Beilschmiedia obscura respectively, by increasing their inhibitory activity against Klebsiella pneumoniae KP55 strain at least four fold. Conclusion The overall results of the present investigation provide information for the possible use of the methanol extracts of the studied plant species, especially B. obscura to fight infectious diseases caused by Gram-negative bacteria including MDR phenotypes.


Background
Bacterial chemo-resistance is a worrisome health concern worldwide today [1 -3]. The development of multi-drug resistant (MDR) bacteria has severely compromised the efficacy of antimicrobial weapons and has dramatically increased the frequency of therapeutic failure [4]. Several reports highlighted the increased hospital dissemination of the bacterial strains expressing drug efflux mechanism [5,6]. Among the known efflux mechanisms of resistances in Gram-negative bacterial strains, Resistance-Nodulationcell Division (RND) pump is one of the most occurring systems [7]. A number of chemicals such as phenylalanine arginine β-naphthylamide (PAβN), 1-(1-naphthylmethyl)piperazine, some quinolone derivatives [8] as well as some natural products like reserpine [9] have been found to inhibit bacteria efflux pumps. The problem of bacterial resistance to commonly used antibiotics then shifted attention towards the discovery of new natural antibacterial compounds. Plants commonly used as herbal medicine may be a source of antibacterial, antifungal and antiviral activities [10][11][12]. In Cameroon, there is a rich tradition of using medicinal plants for the treatment of various infectious diseases, inflammations, injuries, and other diseases [13,14]. The aim of the investigation was to determine the antibacterial effects of twelve methanol crude extracts belonging to seven Cameroonian medicinal plants namely  Table 1). The role of efflux pumps was also investigated using pump-deleted bacteria and the efflux pump inhibitor (EPI) PAβN.

Plant materials and extraction
All medicinal plants used in the 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). The airdried 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 No1was concentrated under reduced pressure in a vacuum to obtain the crude extracts. All crude extracts were kept at 4°C until further uses.

Preliminary phytochemical investigations
The plant extracts were screened for the presence of major secondary metabolite classes such as alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, sterols and triterpenes according to common phytochemical methods previously described by Harbone [38].
Bacterial strains and culture media MDR isolates (Laboratory collection) and reference strains (from the American Type Culture Collection: Escherichia coli ATCC8739 and ATCC10536; Enterobacter aerogenes ATCC13048; Klebsiella pneumoniae ATCC11296; and Providencia stuartii ATCC29916) of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, and Providencia stuartii were used. Their features were previously reported [39,40]. They were maintained at 4°C and sub-cultured on a fresh appropriate Mueller Hinton Agar (MHA) for 24 h before any antibacterial test.

Microdilution assay for MIC and MBC determinations
The microdilution inhibitory concentration (MIC) of the seven plant extracts and chloramphenicol were determined using a rapid microdilution assay [41,42]. Briefly, the samples were first dissolved in 10% dimethyl sulfoxide (DMSO)/Mueller Hinton Broth (MHB). The solution obtained was then added to MHB and serially diluted two fold (in a 96-well microplate). One hundred microliters of inoculum (1.5 × 10 6 CFU/mL) prepared in MHB were then added. The plates were covered with a sterile plate sealer and then agitated with a shaker to mix the contents of the wells and incubated at 37°C for 18 h. The final concentration of DMSO was less 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 the negative control. The MIC of each sample was detected after 18 h of incubation at 37°C following addition of 40 μl INT (0.2 mg/mL) and incubation at 37°C for 30 min. The MIC was defined as the lowest sample concentration that prevented color change of the medium and that resulted in the complete inhibition of bacterial growth [43]. Viable bacteria reduced the yellow dye to a pink. The minimum bactericidal concentration (MBC) of the sample was determined by sub-culturing 50 μl of the suspensions from the wells which did not show any growth after incubation during MIC assays to 150 μl of fresh broth, and re-incubated at 37°C for 48 hours before re-evaluation. The MBC was defined as the lowest concentration of sample which completely inhibited the growth of bacteria [44,45]. Each assay was performed in three independent tests in triplicate.
The tested samples, the samples were also tested in the presence of PAβN at a final concentration of 20 μg/mL as previously described [46] against nine of the most resistant bacteria strains. The MIC was determined as described above.

Phytochemical composition of the plant extracts
The results of the qualitative phytochemical analysis indicated that alkaloids were present in all plant extracts. Each of the studied plant extract contained at least two classes of secondary metabolites (Table 2).

Antibacterial activity of the plant extracts
The data depicted in Table 3 show that all extracts were active on at least two bacterial strains with MIC values Fibromyomes [15], stomac ache and malaria [16,17].
The evidence on the puberty onset induction and ovarian folliculogenesis effect of the aqueous extract in immature female rat have been demonstrated [34].  (Table 4).

Phytochemical composition of the plant extracts
The selective distribution of the secondary metabolites in the plant extracts may be due to the difference in the plant genus and family or the plant parts used. In fact, the presence of a particular metabolite can be influenced by the metabolisms which take place in the different plant parts. Compounds belonging to alkaloids as well as phenolics and terpenoids are well documented for their antibacterial activities [11]. Their presence in the studied extracts could therefore explain the observed activities.

Antibacterial activity of the plant extracts
Phytochemicals are routinely classified as antimicrobials on the basis of susceptibility tests that produce MIC in the range of 100 to 1000 μg/mL [47]. Moreover, for crude extract, antimicrobial activity is considered to be significant if MIC values are below 100 μg/mL and moderate when 100 < MIC < 625 μg/mL [11]. Therefore, the activity recorded with B. obscura against ten of tested bacteria strains namely E. coli (ATCC10536, AG8739, AG100, W3110), K. pneumoniae ATCC11296, KP63), E. aerogenes (ATCC13048, EA27 and EA294) and P. stuarti ATCC29916 can be considered important. If we considered the alternative criteria described by Fabry et al. [48], where extracts having MIC values below 8000 μg/mL have noteworthy antimicrobial activity, the overall activity recorded with most of the         [39]. Some compounds previously isolated from the genus Beilschmiedia and belonging to alkaloids, phenols, saponines, sterols and triterpenoids [50][51][52] were reported to possess antimicrobial activities [53]. The genus Beilschmiedia is also known traditionally to possess antimicrobial activities [53]. The fruits of B. obscura used herein are also used as soup ingredient in Cameroun [54]. This highlights its importance in the control of microbial infections and mostly those involving MDR phenotypes. Compounds belonging to alkaloids, flavonoids, sterols and triterpenoids classes previously isolated from P. staudtii [21][22][23][24][25] may be responsible for their observed activities. Bioactive alkaloids like araliopsin were previously isolated from V. soyauxii [17].
To assess the implication of efflux pumps in the susceptibility of Gram-negative bacteria to the tested plant extracts, PAβN a potent inhibitor of RND efflux systems and particularly active on AcrAB-TolC (of Enterobaceriaceae) and MexAB-OprM (of Pseudomonas species) [8,55] has been used at a concentration of 20 μg/mL. This concentration had no intrinsic effect on the bacteria as previously determined [46,56]. A significant increase of the antibacterial activities of the extract from P. staudtii and B. obscura was observed against resistant bacteria K. pneumoniae Kp55 strain, showing that one or more active compounds present in these plant extracts could be substrate(s) of efflux pumps of this bacteria. However, little or no increase of activities observed with the remaining extracts in the presence of EPI may be an indication that either secondary metabolites of these extracts are not active against the studied bacteria or that RND efflux pumps are not the main resistance mechanisms involved. The tested bacteria are good models in investigating MDR as they expressed active efflux pumps as observed when choramphenicol was tested in the presence of PAβN.

Conclusions
The investigation provided informative data for the use of the crude medicinal plant extracts tested, especially those from Beilschmiedia obscura, Peperomia fernandopoiana and Pachypodanthium staudtii in the fight against MDR bacteria. The isolation of active constituents from these plants will further be performed in order to identify their active antibacterial ingredients.