Background

Infectious diseases still represent one of the major health concern worldwide [1]. According to the National Institute of Health, infectious diseases are the second cause of death and the leading cause of loss of productive life years worldwide. Bacterial infections are responsible of about 70 % of cases of death related to microorganisms [1]. The use of antibiotics and hygiene rules helped to fight infectious diseases in the past. However, they are becoming increasingly difficult to control as results of the spread of resistant phenotypes. The resistance to antibiotics has increased in recent decades, mainly due of their inappropriate use [2]. Bacteria have developed several mechanisms of resistance including active efflux which plays an important role in multi-drug resistance (MDR), mainly in Gram-negative bacteria [3]. There is a need for the discovery of new active antimicrobials to combat MDR microorganisms. Amongst the new areas explored to overcome infectious diseases caused by MDR bacteria, medicinal plants seem to offer an ideal alternative since they are readily available source of bioactive agents and are well accepted by about 80 % of the world population. Many African medicinal plants and their metabolites were previously found active against MDR Gram-negative bacteria [4, 5]. Also the synergistic activities of some African medicinal plants with antibiotics against MDR Gram-negative bacteria were reported [5, 6]. It was demonstrated that several naturally occurring efflux pump inhibitors can restore the activity of antibiotics against MDR bacteria [7, 8]. The present study was therefore designed to investigate the antibacterial potential against MDR Gram-negative phenotypes expressing active efflux pumps of six Cameroonian medicinal plants used traditionally in the treatment of bacterial infections, namely Anthocleista schweinfurthii Gilg.(Loganiaceae), Boehmeria platyphylla D. Don (Urticaceae), Caucalis melanantha (Hochst/Hien) (Urticaceae), Erigeron floribundus (H.BK) (Asteraceae), Nauclea latifolia Smith (Rubiaceae) and Zehneria scobra (cf) Sondev (Cucurbitaceae).

Methods

Plant materials and extraction

The plant materials used in this study were collected on April 2013 in West and South West regions of Cameroon and identified by a specialist of the National Herbarium (Table 1). The plants included two trees namely Anthocleista schweinfurthii and Nauclea latifolia, and four herbs namely Boehmeria platyphylla, Caucalis melanantha, Erigeron floribundus and Zehneria scobra. The whole plant was collected for herbs whilst leaves, fruits and stem bark were collected for trees. Each plant material was dried at room temperature and powdered using a grinder. One hundred grams of each powder was then macerated in 1 L of pure methanol (MeOH) for 48 h and filtered through Whatman filter paper no.1. The filtrate obtained was concentrated under reduced pressure in a rotary evaporator to obtain the crude extract. All crude extracts were then kept at 4 °C until further uses.

Table 1 General informations and report on evidence of biological activities and chemistry of the studied plants
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Phytochemical screening

The major phytochemical classes such as phenols tannins, flavonoids, saponins, alkaloids, anthraquinones, cardiac glycosides, steroids and triterpenes (Table 2) were investigated according to the common described phytochemical methods [9–13].

Table 2 Preliminary chemical composition of the studied plant extracts
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Chemicals for antibacterial assays

Seven 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. 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 microorganisms used in the present 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, 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). The clinical strains were the laboratory collection from UMR-MD1, University of Marseille, France. Their features are reported in Additional file 1: Table S1. They were maintained at 4 °C and sub-cultured on a fresh appropriate Mueller Hinton Agar (MHA) for 24 h before any antibacterial test.

Antibacterial assays

The MICs of the tested extracts were determined using a rapid INT colorimetric assay [14]. Briefly, test samples were first dissolved in dimethylsulfoxide/ Mueller Hinton Broth (DMSO/MHB). The solution obtained was then added to MHB and serially diluted two fold (in a 96-well microtilter plate). One hundred microliters of inoculums (1.5× 106 CFU/ml) prepared in MHB were then added. The plates were covered, 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 2.5 %, a concentration at which DMSO does not affect bacterial growth. Wells containing MHB, 100 μl of inoculum, and DMSO at a final concentration of 2.5 % served as the negative control. Chloramphenicol was used as reference antibiotic. The MICs of each extract were detected after 18 h of incubation at 37 °C after addition of 40 μl INT (0.2 mg/ml) and incubation at 37 °C for 30 min. Viable bacteria reduce INT with appearance of a pink dye. The MIC of each sample was defined as its lowest concentration that prevented this change and resulted in the complete inhibition of microbial growth. The Minimum Bactericidal Concentration (MBC) was determined by sub-culturing samples from the wells with concentrations above or equal to the MIC on new plates of Mueller Hinton broth (MHB). The MBC was considered as the lowest concentration of the extract which prevented appearance of pink color after addition of INT. Each assay was performed in triplicate at three different days.

Antibiotic-modulation assay

To evaluate the antibiotic resistance modifying activity of the extracts, the MIC of antibiotics were determined in the presence or absence of the plant extracts using the broth microdilution technique as described above. After a preliminary assay on two MDR bacteria, P. aeruginosa PA124 and E. aerogenes CM64 (Additional file 1: Tables S3 and S4), extracts from A. Schweinfurthii fruits, N. Latifolia leaves and stem bark, and from the whole plant of Z. scobra were selected and tested at their MIC/2 and MIC/5 in combination with seven antibiotics (CHL, AMP, KAN, NOR, ERY, TET and STR) on six MDR bacterial strains (P. aeruginosa PA124, E. aerogenes EA289 and CM64, E. coli AG100, P. stuartii NAE16 and K. pneumoniae K24).

The reverse of Fractional Inhibitory concentration (1/FIC) was calculated as follows:

$$ \mathsf{1}/\mathrm{F}\mathrm{I}\mathrm{C} = \kern0.5em \mathrm{M}\mathrm{I}{\mathrm{C}}_{\mathrm{Antibiotic}\ \mathrm{alone}}/\mathrm{M}\mathrm{I}{\mathrm{C}}_{\mathrm{Antibiotic}\ \mathrm{in}\ \mathrm{combination}\ \mathrm{with}} $$

The interpretation was made as follows: Synergistic (≥2), Indifferent (1 to 0.5), or Antagonistic (≤0.25) [5, 15]. All assays were performed in triplicate and repeated thrice.

Results

Phytochemical composition of the tested extracts

The main classes of secondary metabolites for each extract were screened and the results are summarized in Table 2. It appears that all the plant extracts of this study possess at least 3 classes of screened secondary metabolites. Only three classes of the screened phytochemicals were detected in the extracts from Z. scobra, E. floribundus and A. schweinfurthii leaves. Extracts from N. latifolia leaves and stem bark contained six phytochemical classes. All the extracts contained phenols and cardiac Glycosides.

Antibacterial activity

The results (Additional file 1: Table S2) showed that all extracts displayed antibacterial activity against at least 4/28 (14.3 %) tested bacterial strains, with MIC values ranged from 128 to 1024 μg/mL. Extracts from A. schweinfurthii leaves, fruits and bark exerted inhibitory effects respectively against 14/28 (50 %), 13/28 (46.4 %) and 8/28 (28.6 %) studied bacteria. The extracts from the fruits and leaves of N. latifolia were active respectively on 6/28 (21.4 %) and 7/28 (25 %) tested bacteria whilst the bark extract displayed the best spectrum of activity [active on 22/28 (78.6 %) tested bacteria]. Extracts of B. platyphylla and Erigeron floribundus also showed large spectra of antibacterial activity with MIC values recorded on 17/28 (60.7 %) and 21/28 (75 %) bacterial strains respectively. Causalis melanantha and Z. scobra extracts displayed low antibacterial spectra [MIC recorded respectively on 4/28 (14.3 %) and 7/28 (25 %) tested bacterial]. P. aeuginosa PA124 appeared to be the most resistant bacteria strain with the sensitivity observed only towards N. latifolia bark extract.

Antibiotic resistance modifying activities of the plant extracts

Preliminary results obtained in two most resistant strains, P. aeruginosa PA124 and E. aerogenes CM64 (results presented in Additional file 1: Tables S3 and S4) allowed selecting the following extracts: A. schweinfurthii fruits, N. latifolia leaves and bark and Z. scobra as well as the appropriate sub-inhibitory concentrations of MIC/2 and MIC/5 for further studies. From the results summarised in Tables 3, 5, 5 and 6, it appears that all the four extracts improved the activities of antibiotics, from 2 to more than 64 folds. The highest activities were observed with A. schweinfurthii fruits (Table 3) and Z. scobra (Table 6). A. schweinfurthii fruits potentiated the activities of TET on 66.7 % and 50 % of the bacteria strains at MIC/2 and MIC/5 respectively. It also increased the activity of KAN (MIC/2 and MIC/5) and STR (MIC/2) in 50 % of the tested bacterial strains while Z. scobra improved the activities of STR on 66.7 % and 50 % of the bacteria strains at MIC/2 and MIC/5 respectively. Z. scobra also improved the activity of CHL on 50 % of the tested bacterial strains at the two sub-inhibitory concentrations of MIC/2 and MIC/5 (Table 6). Synergistic effects (50 % of antibiotic activity potentiating at MIC/2 and MIC/5) were observed with N. Latifolia leaves extract (Table 5) on TET and STR. The highest rate of improvement of antibiotic activity by N. Latifolia stem bark extract was rather noticed on TET and KAN with a rate of 33.3 %. Among the four extracts, this later displayed the lowest antibiotic potentiating effect. Moreover, no synergistic effect was observed with NOR, while synergy between the studied extracts and antibiotics were observed with Ampicilin, with a rate of only 16.67 % (Table 5).

Table 3 Effect of sub-inhibitory concentrations of Anthocleista schweinfurthii fruits extract on the activities of first line antibiotics against Gram-negative MDR bacteria
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Discussion

Medicinal plants are potential source of antimicrobial agents used in the treatment of infectious diseases [16]. According to Rios and Recio [17], and Kuete et al. [17], the antibacterial activity of a plant extract is considered significant when the MICs are below 100 μg/mL. The activity is considered moderate when 100 ≤ MIC ≤ 625 μg/mL and weak when MIC are above 625 μg/mL [17]. Therefore, the antibacterial activities reported in the present study can mostly be regarded as moderate or low. This could be explained by the fact that the tested bacteria are mostly MDR phenotypes. In fact, P. aeruginosa and MDR Enterobacteriaceae ( K. pneumoniae, E. aerogenes, E.cloacae and P. stuartii and E. coli) tested in the present study have been classified as antimicrobial-resistant organisms of concern in healthcare facilities [18–20]. The previously reported activities of A. schweinfurthii include antibacterial inhibitory effects of n-hexane, dichloromethane, ethyl acetate and methanol extracts from leaves and stem bark against Staphylococcus aureus ATCC 33591 and E. coli ATCC 27195 [21]. The MIC values obtained in the present study were respectively 62.5 and 125 μg/ml against S. aureus and E. coli. Such values were higher than those previously documented, highlighting the MDR feature of the studied bacteria. MBC values were obtained in few cases (Additional file 1: Table S2). A keen look of data (Additional file 1: Table S2) indicates that, in most of the cases, the tested extract exerted bacteriostatic effects with a ratio MBC/MIC above 4. The overall antibacterial activity of the tested extracts could be due their phytochemical composition. However, the presence of a specific class of second metabolite could not guarantee the antibacterial activity of the plant, as this will depend on nature of the compounds, its concentration as well as the possible interactions with other constituents of the extract. It is also surprising that saponins, known to possess antibacterial activities were not detected in the tested extracts; However, this does means that the extract were completely devoid of this class of secondary metabolite; One of the most understandable explanation should that saponins could be present in very little amounts in the tested extract, and therefore could not be detected using the qualitative phytochemical methods. Some cardiac glycosides such as bufalin, oubain, digoxin are toxic meanwhile many of them have therapeutic uses and these primarily involve the treatment of cardiac failure [22–24]. Their utility results from an increased cardiac outpout by increasing the force of contraction. By increasing intracellular calcium, cardiac glycosides increase calcium-induced calcium release and thus contraction [23, 24]. The traditional use of the studied plants could suggest that their cardiac glycoside could be not toxic and have very low toxic effects.

To the best of our knowledge, the present work describes for the first time the antibacterial activity of B. platyphylla. This activity could be due to the presence of the detected phytochemicals. In fact, antibacterial compounds such as acetophenone [25] and cryptopleurine [26–28] were previously isolated from B. Platyphylla. The antibacterial activity of C. melanantha and E. floribundus is also reported here for the first time. However these plants were previously reported for their antifungal activities [29–32]. The antibacterial activities of extracts from Zehneria scobra and Nauclea latifolia [33, 34] were reported on some bacteria: The present study therefore provides additional information on the activity of these plants against MDR Gram-negative phenotypes.

The synergistic effects between antibiotics and the tested plants are also reported here for the first time. The observed synergistic effects could be due to possible interaction between plant constituents and the tested antibiotics. As the strains used in this study are known to actively expressed efflux pumps, one of the possible explanations for the observed synergistic effects could be the ability of the constituents of the extracts to act as efflux pumps inhibitor. This can explain why the effect of antibiotics with intracellular targets such as STR, CHL and KAN increased contrary to that of beta-lactamine (AMP) acting in the cell wall (Tables 3, 4, 5 and 6).

Table 4 Effect of sub-inhibitory concentrations of Nauclea latifolia stem bark extract on the activities of first line antibiotics against Gram-negative MDR bacteria
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Table 5 Effect of sub-inhibitory concentrations of Nauclea latifolia leaves extract on the activities of first line antibiotics against Gram-negative MDR bacteria
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Table 6 Effect of sub-inhibitory concentrations of Zehneria scobra extract on the activities of first line antibiotics against Gram-negative MDR bacteria
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Conclusion

The overall results of the present study provides baseline information for the possible use of the tested plants, especially A. schweinfurthii, N. Latifolia, B. platyphylla and E. floribundus in the control of infections due to Gram-negative bacteria. The present study indicates that the tested plant extracts alone could not be used efficiently to tackle MDR bacterial infections. However, it was demonstrated that extracts from A. schweinfurthii fruits and Z. scobra could be used in combination with some antibiotics to fight bacterial multi-drug resistance.

Availability of data and materials

The datasets supporting the conclusions of this article are presented in this main paper and supporting material. Plant materials used in this study have been identified at the Cameroon National Herbarium where voucher specimens are deposited.

Consent for publication

Not applicable in this section.

Ethic approval and consent to participate

Not applicable in this section.