Antibiotic-potentiation activities of four Cameroonian dietary plants against multidrug-resistant Gram-negative bacteria expressing efflux pumps

Background The continuous spread of multidrug-resistant (MDR) bacteria, partially due to efflux pumps drastically reduced the efficacy of the antibiotic armory, increasing the frequency of therapeutic failure. The search for new compounds to potentiate the efficacy of commonly used antibiotics is therefore important. The present study was designed to evaluate the ability of the methanol extracts of four Cameroonian dietary plants (Capsicum frutescens L. var. facilulatum, Brassica oleacera L. var. italica, Brassica oleacera L. var. butyris and Basilicum polystachyon (L.) Moench.) to improve the activity of commonly used antibiotics against MDR Gram-negative bacteria expressing active efflux pumps. Methods The qualitative phytochemical screening of the plant extracts was performed using standard methods whilst the antibacterial activity was performed by broth micro-dilution method. Results All the studied plant extracts revealed the presence of alkaloids, phenols, flavonoids, triterpenes and sterols. The minimal inhibitory concentrations (MIC) of the studied extracts ranged from 256-1024 μg/mL. Capsicum frutescens var. facilulatum extract displayed the largest spectrum of activity (73%) against the tested bacterial strains whilst the lower MIC value (256 μg/mL) was recorded with Basilicum polystachyon against E. aerogenes ATCC 13048 and P. stuartii ATCC 29916. In the presence of PAβN, the spectrum of activity of Brassica oleacera var. italica extract against bacteria strains increased (75%). The extracts from Brassica oleacera var. butyris, Brassica oleacera var. italica, Capsicum frutescens var. facilulatum and Basilicum polystachyon showed synergistic effects (FIC ≤ 0.5) against the studied bacteria, with an average of 75.3% of the tested antibiotics. Conclusion These results provide promising information for the potential use of the tested plants alone or in combination with some commonly used antibiotics in the fight against MDR Gram-negative bacteria.


Background
The spread of multidrug-resistant bacteria, partially due to the inappropriate use of common antibiotics, drastically reduced the efficacy of the antibiotic armory, increasing the frequency of therapeutic failure. The over-expression of efflux pumps is the main resistance mechanism observed in many bacteria [1]. In Gram-negative bacteria, many of these efflux pumps belong to the resistance-nodulation-cell division (RND), family of tripartite efflux pumps [2]. In the fight against microbial infections including those due to MDR bacteria, investigations are being carried out to discover new effective, none or less-toxic and available antibacterial drugs. Many scientist are also investigating synergistic compounds to potentiate the activity of the commonly used antibiotics [3]. The present work was designed to evaluate the in vitro ability of some edible plants namely Capsicum frutescens L. var. facilulatum (Solanaceae) or 'chili pepper' , Brassica oleacera L. var. italica commonly known as 'Broccoli' and Brassica oleacera L. var. butyris (Brassicaceae) or 'Cauliflower'; and Basilicum polystachyon (L.) Moench. (Lamiaceae) or 'Musk Basil' to potentiate the effect of common antibiotics against Gram-negative MDR phenotypes.

Plant material and extraction
The plants used in this study were collected in Douala (Littoral Region of Cameroon) in January 2013. The plants were further identified at the National Herbarium (Yaoundé, Cameroon) where voucher specimens were deposited under a reference number (Table 1). Air dried and powdered sample (0.1 g) of each plant was extracted by maceration with methanol (0.3 L) for 48 h at room temperature (25°C). After filtration using Whatman No. 1 filter paper, the filtrate of each plant was concentrated under reduced pressure in a rotary evaporator, and dried at room temperature to give the crude extract. The extraction yield was calculated ( Table 2). These extracts were then stored at 4°C until further use.

Preliminary phytochemical screenings
The secondary metabolite classes such as alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, tannins, sterols and triterpenes were screened according to the standard phytochemical methods described by Harbone [16].

Bacteria strains and culture media
The studied microorganisms included both reference (from the American Type Culture Collection, ATCC) and clinical (Laboratory collection) strains of Escherichia coli, Enterobacter aerogenes, Providencia stuartii, Pseudomonas aeruginosa and Klebsiella pneumoniae (Table 3). They were maintained at 4°C and sub-cultured on a fresh appropriate Mueller Hinton Agar (MHA) for 24 h before any antibacterial test. The Mueller Hinton Broth (MHB) was used for all antibacterial assays.
Strong activities of acidic extracts against Gram (+), but less activities against Gram;. Strong antifungal activities of ethanolic and methanolic extracts against An [15].  (PAβN) (Sigma-Aldrich) were used as bacterial growth indicator and efflux pumps inhibitor respectively. Dimethylsulfoxide 10% (DMSO) was used as solvent for all extracts.

Bacterial susceptibility determinations
The minimal inhibitory concentrations (MIC) of the plant extracts against the studied bacteria were determined by rapid INT colorimetric assay [19,20]. Briefly, the test samples were first dissolved in DMSO/MHB. The solution obtained was then added to MHB in a 96-well microplate followed by a two fold serial dilution. One hundred microliters (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 ranges were 8-1024 μg/mL for plant extracts and 2-512 μg/mL for reference antibiotic chloramphenicol (CHL). Wells containing MHB (100 μL), 100 μL of inoculum and DMSO at a final concentration of 2.5% served as negative growth inhibition control. MIC was detected after 18 h of incubation at 37°C, following addition (40 μL) of 0.2 mg/mL INT and incubation at 37°C for 30 min. 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 bacterial growth [21]. The minimal bactericidal concentrations (MBC) of the samples was determined by taking 50 μL of the suspensions from the wells which did not show any growth after incubation during MIC assays to a new 96-well microplate containing 150 μL of fresh broth per well. The plate was further re-incubated at 37°C for 48 hours the addition of INT. The MBC was defined as the lowest concentration of samples which completely inhibited the growth of bacteria. Samples were tested alone and in the presence of PAβN at 30 μg/mL final concentration [22].
To evaluate the potentiating effect of tested extracts, a preliminary combination at their sub-inhibitory concentrations (MIC/2, MIC/5, MIC/10 and MIC/20) with antibiotics was assessed against P. aeruginosa PA124 strain. The appropriate sub-inhibitory concentrations were then selected on the basis of their ability to improve the activity of the maximum antibiotic. These sub-inhibitory concentrations for selected extracts were further tested in combination with antibiotics against more MDR bacteria. The Fractional inhibitory concentration (FIC) of each combination was then calculated as the ratio of MIC of Antibiotic in combination versus MIC of Antibiotic alone [23,24].

Phytochemical composition of the tested plant's extracts
The results of the qualitative phytochemical analysis showed that each of the studied extract contained alkaloids, phenols, flavonoids, triterpenes and sterols. None of them contained anthocyanins and anthraquinones. Other phytochemical classes have been selectively detected as shown in Table 2.

Antibacterial activity of the plant's extracts
Bacterial strains and MDR isolates were tested for their susceptibility to plant extracts and chloramphenicol. The results summarized in Table 4 (22) studied MDR bacteria were also tested for their susceptibility to the plant extracts in the presence of PAβN ( Table 5). The largest spectrum of activity was recorded with B. oleacera var. butyris extract against 75% (6/8) tested MDR bacteria. This efflux pumps inhibitor (EPI) also improved the activity of C. frutescens extract against E. coli (AG100), K. pneumoniae (KP53) and E. aerogenes (EA27) as well as that of B. polystachyon against P. stuartii (NAE16).

Antibacterial activity of extract-antibiotic combination
A preliminary assay against P. aeruginosa PA124 strain allowed selecting MIC/2 and MIC/5 as appropriate subinhibitory concentrations to be used on other bacteria  ( Table 6). Synergistic effects were observed with all the tested extracts. Brassica oleacera var. italica and B. oleacera var. butyris extracts potentiate (0.125 < FIC < 0.5 and 0.031 < FIC < 0.5 respectively) the effects of the majority of antibiotics on most of the tested MDR bacteria (Table 7). Extracts from C. frutescens and B. polystachyon showed synergistic effects with six of the nine studied antibiotics, with 0.125 < FIC < 0.5 and 0.25 < FIC < 0.5 respectively.

Discussion
The Pharmacological potencies of plants' secondary metabolites are well demonstrated. The qualitative phytochemical screening of the plant extracts showed the presence of several classes of secondary metabolites, such as alkaloids, flavonoids, phenols, triterpenes, sterols, saponins, tannins and coumarins. Several antibacterial activities associated to the presence of compounds belonging to these various classes were shown [25][26][27]. It should however be mentioned that the detection of an alleged bioactive class of secondary metabolite in a plant is not a guarantee for any biological property, as this will depend on the nature of the compounds as well as their concentrations and the possible interactions with other constituents [12]. The differences observed between the antibacterial activities of the extracts as observed in the present work could be due to the differences in their phytochemical composition [9]. According to the criteria of classification of the antibacterial activity of the phytochemicals [28], the extracts used in this study were moderately and/or weak active (256 ≤ MIC < 1024 μg/mL). Their direct        Efflux pumps are responsible for the reduction of intracellular concentration of antibacterial compounds [29]. To tackle problems related to this phenomenon, an intensive search of efflux pumps inhibitors (EPI) is welcome [30]. The EPI blocks the efflux pumps and leads to the increase of the intracellular concentration of active principle contents of the extracts [29,31]. The activity of B. oleacera var. butyris extract against the tested bacteria in the presence of PAβN, increased in 75% of the cases. This suggests that some compounds present in this extract could be substrates of efflux pumps [31,32].
The extracts of B. oleacera var. butyris, B. oleracea var. Italica, Basilicum polystachyon and C. frutescens showed significant synergistic effects (0.031 < FIC < 0.5) with the majority of the tested antibiotics against the studied MDR strains. This suggests that the extracts might contain bioactive compounds that, combined with antibiotics, acted at different sites by various mechanisms [33,34]. These data indicate that a combination of these extracts with antibiotics could be envisaged to fight MDR bacteria.

Conclusion
These results provide promising baseline information for the potential use of Capsicum frutescens, Brassica oleacera var. italica, Basilicum polystachyon and Brassica oleacera var. butyris, independently or in combination with some commonly used antibiotics in the fight against MDR Gram-negative bacteria.