Antibacterial and antibiotic-modulation activity of six Cameroonian medicinal plants against Gram-negative multi-drug resistant phenotypes

Background Bacterial Infections involving multi-drug resistant (MDR) phenotypes constitute a worldwide health concern. The present work was designed to assess the antibacterial properties of the methanol extracts of six medicinal plants (Anthocleista schweinfurthii, Nauclea latifolia, Boehmeria platyphylla, Caucalis melanantha, Erigeron floribundus and Zehneria scobra) and the effects of their associations with antibiotics on MDR Gram-negative bacteria over-expressing active efflux pumps. Methods The antibacterial activities and the ability to potentiate antibiotic effects of the methanol extracts the tested plants were evaluated in vitro against twenty eight Gram-negative bacteria expressing MDR phenotypes, using broth microdilution method. The phytochemical screening of these extracts was also performed using standard methods. Results All tested extracts displayed moderate to low antibacterial activity on at least 14.3 % of the 28 tested bacteria, with MIC values ranged from 128 to 1024 μg/mL. The best antibacterial spectrum was observed with Naulcea latifolia bark extract. Extracts from A. schweinfurthii fruits, N. latifolia stem bark, Z. scobra and N. latifolia leaves showed synergistic effects with many antibiotics against MDR bacteria. Conclusion The overall results of the present study provide information for the possible use of the studied plants, especially Nauclea latifolia in the control of Gram-negative bacterial infections including MDR species as antibacterials as well as resistance modulators. Electronic supplementary material The online version of this article (doi:10.1186/s12906-016-1105-1) contains supplementary material, which is available to authorized users.


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 Hernia, female sterility, stomach-ache in women, ovarian problems, venereal diseases, bronchitis, fever, purgative, malaria, hard abscesses anthelminthic, otitis, ophthalmia, pain, malaria, cancers, venereal diseases, bacterial diseases [21] Stem bark, roots, Sap of young leaves, leaves Bagangté, West region of Cameroon Polyphenols, alkaloids, terpenes and steroids [21], schweinfurthiin 1, bauerenone 2, bauerenol 3, 1-hydroxy-3,7,8 trimethoxy-xanthone 4 and 1, 8-dihydroxy-3, 7 dimethoxy-xanthone 5 [35] Antibacterial activity against Staphylococcus aureus and Escherichia coli [21] (Loganiaceae); 32389/HNC

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.

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× 10 6 CFU/ml) prepared in MHB were then added.

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)   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.

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:

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) (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).

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 antimicrobialresistant organisms of concern in healthcare facilities [18][19][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][23][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 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][27][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][30][31][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 betalactamine (AMP) acting in the cell wall (Tables 3, 4, 5 and 6).

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.

Additional file
Additional file 1: Table S1. Bacterial strains and features. Table S2. Minimal inhibitory concentration (MIC) and minimal bactericidal (MBC) of the plant extracts and CHL on the studied bacteria. Table S3. Effects of different concentrations of plant extracts on the MIC (μg/ml) of antibiotics against P. aeruginosa PA124. Table S4. Effects of different concentrations of plant extracts on the MIC (μg/ml) of antibiotics against E. aerogenes CM64 (DOC 320 kb)