Background

Infectious diseases caused by multi-drug resistant (MDR) Gram-negative bacteria are worldwide health concern, causing increasingly morbidity and mortality particularly in developing countries [1]. In Cameroon, previous studies showed high levels of resistance to commonly used antibiotics in Gram-negative bacilli [2]. Several reports also mentioned an increase in the hospital dissemination of bacterial strains specifically those expressing drug efflux mechanism [3, 4]. Against Gram-negative bacteria, the discovery of efflux pump inhibitors (EPIs) is an attractive strategy to combat MDR phenotypes [5]. EPI generally interact with specific efflux pump proteins to restore the susceptibility of MDR bacteria to antibiotics [6]. Medicinal plants constitute an important source of chemotherapeutic molecules, in regards to the chemical diversity found in several species [7, 8]. In recent years, some plants have been successfully evaluated for their direct antibacterial action, and for their antibiotic-modulation activity [9–12]. In the present work, we hypothesized that herbal medicines traditionally used for the treatment of infectious diseases could contain molecules acting as antibacterial and/or antibiotic-resistance modulators. This study was therefore designed to investigate the in vitro antibacterial and antibiotic-resistance modifying activities of the methanol extracts from Allanblackia gabonensis Pellegr. (Clusiaceae), Gladiolus quartinianus A. Rich (Iridaceae) and Combretum molle R. Br. ex G. Don (Combretaceae) against Gram-negative bacteria including multi-drug phenotypes. These plants are traditionally used to manage various ailments including bacterial related infections.

Methods

Plant materials and extraction

Medicinal plants used in this work were collected in different areas of Cameroon between January and April 2012. The plants were identified at the National Herbarium (Yaoundé, Cameroon), where voucher specimens were deposited under the reference numbers (Table 1). The 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 vacuum to obtain the crude extracts. All crude extracts were then kept at 4 °C until further uses.

Table 1 Plants used in the present study and evidence of their bioactivities
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Chemicals for antibacterial assays

Eight commonly used antibiotics including tetracycline (TET), kanamycin (KAN), streptomycin (STR), ciprofloxacin (CIP), norfloxacin (NOR), chloramphenicol (CHL), ampicillin (AMP), erythromycin (ERY) (Sigma-Aldrich, St Quentin Fallavier, France) were used. The p-Iodonitrotetrazolium chloride 0.2 % (INT) and phenylalanine arginine β-naphthylamide (PAβN) (Sigma-Aldrich) were used as bacterial growth indicator and efflux pumps inhibitor respectively.

Microorganisms and growth conditions

Pathogenic bacteria used in the study were Gram-negative bacteria including MDR isolates (Laboratory collection) and reference strains (American Type Culture Collection) of Escherichia coli (ATCC8739, ATCC10536, AG100, AG100A, AG100ATet, AG102, MC4100 W3110), Enterobacter aerogenes (ATCC13048, CM64, EA27, EA3, EA289, EA298, EA294), Klebsiella pneumoniae (ATCC11296, KP55, KP63, K24, K2), Enterobacter cloacae (ECCI69, BM47, BM67), Pseudomonas aeruginosa (PA01, PA124) and Providencia stuartii (ATCC29916, NEA16, PS2636, PS299645) were used. Their features were previously reported [37]. They were maintained at 4 °C and sub-cultured on a fresh appropriate Mueller Hinton Agar (MHA) for 24 h before any antibacterial test.

Preliminary phytochemical investigation

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 [38]. The tests were based on visual observation of the change in color or formation of precipitate after the addition of specific reagents.

Antibacterial assays

MICs and MBCs of the plant extracts and chloramphenicol were determined by microdilution method using rapid INT colorimetric assay [25, 39]. 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× 106 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. Viable bacteria reduced the yellow dye to a pink. The MIC was defined as the lowest sample concentration that prevented this change and that resulted in the complete inhibition of bacterial growth. The 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 h before revelation. The MBC was defined as the lowest concentration of sample which completely inhibited the growth of bacteria [40]. Each assay was performed in three independent tests in triplicate. The samples were also tested in the presence of phenylalanine arginine β-naphthylamide (PAβN) at a final concentration of 20 μg/mL as previously described [41] on nine MDR bacteria. All assays were performed three time in duplicate.

Antibiotic-modulation assay

To evaluate the antibiotic resistance modifying activity of the extracts, the MIC of antibiotic was determined in the presence or absence of the plant extracts. The 96-wells plate modulation method, as described by Stavri et al. [42] was used. Briefly, after serial dilutions of antibiotics (256–0.5 μg/mL), the plant extracts at their sub-inhibitory concentrations (MIC/2 and MIC/4; selected after preliminary study; Table 2), were added and inoculation was done. The MIC was determined as described above. Modulation factors (MF), calculated as MIC Antibiotic alone/MIC Antibiotic alone + Extract; was used to express the modulating or synergy effects of the plant extracts.

Table 2 MIC of antibiotics in combination with extracts at sub-inhibitory concentrations against P. aeruginosa PA124
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Results

Phytochemical Screening of the plant extracts

The main classes of secondary metabolites for each extract were screened and the results are summarized in Table 3. Tannins, flavonoids, alkaloids and phenols were present in all tested extracts. Others classes of botanicals were selectively distributed in different plant extracts.

Table 3 Qualitative phytochemical composition of the plant extracts
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Antibacterial activity of the plant extracts

The results summarized in Table 4 show that all extracts were active on at least three of bacterial strains, with MIC values varying from 16 to 1024 μg/mL. Extracts from Combretum molle leaves (CML) and Allanblackia gabonensis displayed the most important spectrum of activity. Their inhibitory effects were observed on 72.41 % (27/29) for CML of the tested bacteria, 58.62 % (17/29) for leaves (AGL), 75.86 % (22/29) for flower (AGFl) and bark (AGB), 79.31 % (23/29) for bark and 86.20 % (25/29) for fruits (AGF) extracts from Allanblackia gabonensis. AGF was the best extract with MIC values below 100 μg/mL on 38 % (11/29) of the tested bacteria. CML mostly showed moderate activity with MIC values ranging from 128–512 μg/mL. The activity of CHL was comparable to that of plant extracts in certain cases. This was the case with AGF, AGFl and AGR against K. pneumoniae Kp55 (64 μg/mL); AGF against K. pneumoniae Kp53 (64 μg/mL), and AGFl against P. stuartii PS2636 (32 μg/mL). MICs values equal or above 1024 μg/mL were obtained with the extract from G. quartinianus (GQW). MBCs values were generally equal or below 1024 μg/mL (Table 4).

Table 4 Minimal inhibitory concentration (MIC) and minimal bactericidal (MBC) of the plant extracts and CHL on the studied bacterial species
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Antibacterial activity of the extracts in the presence of an Efflux Pumps Inhibitors

In the present work, extracts were combined with PAβN; However, no significant increase of the activities of the tested plant extracts was generally observed. Only AGL showed 4 times decrease of MICs against E. coli AG102 and E. cloacae BM67. In contrast, PAβN significantly improved the activity of the reference drug, CHL (more than 16 times) on MDR bacteria used (Table 5).

Table 5 MIC of the samples in the absence and presence of PAβN on the selected MDR bacterial species
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Antibiotic resistance modifying activities of the plant extracts

Tables 2, 6 and 7 highlights the potentiating effects of the extracts on the activity of eight commonly used antibiotics. The most important modulating effects were observed of association CML with aminoglycosides (kanamycin and streptomycin), the potentiation effects varying from 77.78 to 88.89 % and from 66.67 to 77.78 % at MIC/2 and MIC/4 respectively; and with tetracycline (100 % and 77.78 % at MIC/2 and MIC/4 respectively) (Table 6). The modulating effects also ranged between 50 to 67 %, with the extract from A. gabonensis fruits (AGF) when combined at (MIC/2) with the some antibiotics. At MIC/4, AGF showed synergy less than 50 % on the tested bacteria with all antibiotics (Table 7). The most significant increases of antibiotic activity in the presence of plant extracts were noted with the association of streptomycin and CML and AGF on E. coli AG100ATet, with more than 128 fold and 64-fold decreases of MIC respectively. No increase of activity was noted with ampicillin, a β-lactamine when it was combined with plant extracts.

Table 6 Resistance modulating effect of the methanol leaves extract from Combretum molle at its sub-inhibitory concentrations on selected
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Table 7 Resistance modulating effect of the Fruits methanol extract from Allanblackia gabonensis at its sub-inhibitory concentrations on selected MDR bacteria
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Discussion

Medicinal plants are potential source of antimicrobial agents used in the treatment of infectious diseases [43, 44]. According to Kuete et al. [45, 46], the antibacterial activity of a plant extract is considered significant when the MICs are below 100 μg/mL, moderate when 100 ≤ MIC ≤ 625 μg/mL and weak when MIC are above 625 μg/mL. Consequently, the antibacterial activities of the tested extracts particularly those from A. gabonensis (AGF, AGR, AGB and AGFl) and C. molle (CML) were generally moderated (Table 4). Significant activities were recorded with AGF, AGR, AGB, and AGFl respectively on 37.93 %, 24.14 %, 20.70 % and 17.24 %. This highlights the good antibacterial potential of the tested extracts. The overall activity recorded with most of the studied extracts could be considered significant, especially those from A. gabonensis and C. molle [47]. When analyzing carefully the MIC and MBC results for the extract, it can be noted that MBC ≤ 4 were obtained with these samples on most of the tested bacterial species, suggesting their killing effects [48].

PAβN is a potent inhibitor of RND systems like AcrAB-TolC in Enterobacteriaceae or MexAB-OprM in P. aeruginosa used in the present work [49, 50]; The activity observed when chloramphenicol was tested in the presence of PAβN increased significantly, confirming that the tested bacteria are good models of efflux pumps-expressing bacteria.

Reversal of multi-drug resistance appears today as another attempt to mitigate the spread of resistance in bacteria. In recent years, many plants extracts and secondary metabolites have been evaluated as modulators of the antibiotic activity in efflux pumps in MDR bacteria [9–11, 37, 51–54]. Herein, we demonstrated that a beneficial effect of the combination of the extracts from the leaves of C. molle (CML) and fruits of A. gabonensis (AGF) with CHL, KAN and STR could be achieved. Synergistic or modulating effects of the plant extracts particularly C. molle extract with antibiotics were noted on more than 70 % of the tested MDR bacteria, suggesting that some of their constituents can act as efflux pump inhibitors [51]. These constituents might act by blocking the efflux pumps located in the cell membrane in the tested bacteria, preventing the extrusion of antibiotics in the cytoplasm and therefore restoring their activity as observed in this study [55, 56]. This is the first time to report the potential of the studied extracts, particularly those from the leaves of C. molle (CML) and the fruits of A. gabonensis (AGF) to reverse antibiotic resistance in MDR bacteria.

Conclusion

This study provides informative data on the antimicrobial potential of the tested plant extracts and suggests that extracts from Allanblackia gabonensis could be a source of natural antibacterial products whilst Combretum molle leaves extract could contain both antibacterial substances and antibiotic-modulation agents. These data indicate that these plants can be used to fight bacterial infections and especially those involving MDR phenotypes.