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

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.

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].

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).

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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: 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).

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.