Background

Infectious diseases remain the major cause of mortality amongst children and young adults worldwide, with higher prevalence in developing countries [1]. Despite the abundance of antibiotics used in chemotherapy, there is a drastic increase of resistant bacteria. Resistance to antibiotics occurs typically as a result of drug inactivation or modification, target alteration, or reduced accumulation associated with decreased permeability and⁄or increased efflux [2]. The scarcity of new antimicrobials active against MDR bacteria propels the search of chemotherapeutic agents. While 25–50 % of current pharmaceuticals are derived from natural products, it was reported that none are used as antimicrobials [3]. Investigation of substances which can potentiate the activity of commonly used antibiotics are also being intensified [4–7]. Previous studies documented the good antimicrobial potential of natural products from higher plants [8, 9]. Several food plants were also documented as potential candidate to fight MDR Gram-negative bacteria. Some of them include Dichrostachys glomerata, Beilschmiedia cinnamomea, Aframomum citratum, Piper capense, Echinops giganteus, Fagara xanthoxyloïdes and Olax subscorpioïdea [4], Lactuca sativa, Sechium edule, Cucurbita pepo and Solanum nigrum [10], Piper nigrum and Vernonia amygdalina [11], Beilschmiedia obscura, Pachypodanthium staudtii and Peperomia fernandopoiana [12], Capsicum frutescens [13]. In our continous search of functional food plants, we designed the present work to investigate in vitro antibacterial activity of the methanol extracts of three Cameroonian food plants, Moringa oleifera Lam. (Moringaceae), Xanthosoma mafaffa (L.) Schott (Araceae), Passiflora edulis Sims (Passifloraceae) against MDR bacteria. The study was extended to the ability of the studied extracts to potentiate the activity of some commonly used antibiotics against some of the tested MDR bacteria.

Methods

Plant material and extraction

The three food plants used in this work were purchased from the Bafoussam markets (West Region of Cameroon) in January 2014. The collected plant material were the leaves of Xanthosoma mafaffa, Moringa oleifera and the fruits of Passiflora edulis. The plants were identified at the National herbarium (Yaounde, Cameroon) where voucher specimens were deposited under the reference numbers (Table 1). Each plant sample was air dried and then powdered. The obtained powder (200 g) was extracted with methanol (MeOH; 1 L) for 48 h at room temperature with momentary shaking. Methanol was then removed under reduced pressure to give residues which constituted the crude extract. All extracts were then kept at 4 °C until further use.

Table 1 Information on plants used in the present study
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Preliminary phytochemical screening

The major phytochemical classes such as alkaloids (Dragendorff’s and Mayer’s tests), triterpenes (Libermann Burchard’s test), flavonoids (Aluminum chloride test), anthraquinones (Borntrager’s test), polyphenols (Ferric chloride test), sterols (Salkowski’s test), coumarins (Lacton test), saponins (Foam test) and tannins (Gelatin test) (Table 2) were investigated according to the commonly described phytochemical methods [14–17].

Table 2 Extraction yields and phytochemical composition of the plant extracts
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Antimicrobial assays

Chemicals for antimicrobial assay

Tetracycline (TET), cefepime (CEP), ciprofloxacin (CIP), norfloxacin (NOR), chloramphenicol (CHL), ampicillin (AMP), erythromycin (ERY), kanamycin (KAN) (Sigma-Aldrich, St Quentin Fallavier, France) were used as reference antibiotics (RA). p-Iodonitrotetrazolium chloride (INT; Sigma-Aldrich) and Phenylalanine-Arginine-ß-Naphthylamide (PAßN; Sigma-Aldrich) were used as microbial growth indicator and efflux pumps inhibitor (EPI) respectively [18, 19].

Microbial strains and culture media

The studied microorganisms included sensitive and resistant strains of Escherichia coli (ATTC8739, AG100, AG100A, AG102, AG100ATet, W3110), Enterobacter aerogenes (ATCC13048, EA289, EA27, EA298, CM64), Klebsiella pneumoniae (ATCC11296, KP55, KP63, K24), Pseudomonas aeruginosa (PA01, PA124), Providencia stuartii (ATCC29914, NEA16) obtained clinically or from the American Type Culture Collection (ATCC). Their resistance profiles have been previously reported [7, 13, 20]. Nutrient agar were used for the activation of the tested Gram-negative bacteria [21].

INT colorimetric assay for MIC and MBC determinations

The MIC determinations on the tested bacteria were conducted using rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay according to described methods [18] with some modifications [22, 23]. The test samples and RA were first of all dissolved in DMSO/Mueller Hinton Broth (MHB) or DMSO/7H9 broth. The final concentration of DMSO was lower than 2.5 % and does not affect the microbial growth [24, 25]. The solution obtained was then added to Mueller Hinton Broth, and serially diluted two fold (in a 96- wells microplate). One hundred microlitre (100 μL) of inoculum 1.5 x 106 CFU/mL prepared in appropriate broth was then added [22, 23]. The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a plate shaker and incubated at 37 °C for 18 h. The assay was repeated thrice. Wells containing adequate broth, 100 μL of inoculum and DMSO to a final concentration of 2.5 % served as negative control. The MIC of samples was detected after 18 h incubation at 37 °C, following addition (40 μL) of 0.2 mg/mL of INT and incubation at 37 °C for 30 min. Viable bacteria reduced the yellow dye to a pink. MIC was defined as the lowest sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth [18]. The MBC was determined by adding 50 μL aliquots of the preparations, which did not show any growth after incubation during MIC assays, to 150 μL of adequate broth. These preparations were incubated at 37 °C for 48 h. The MBC was regarded as the lowest concentration of extract, which did not produce a color change after addition of INT as mentioned above [22, 23].

To evaluate the role of efflux pumps in the susceptibility of Gram-negative bacteria to Xanthosoma mafaffa, Moringa oleifera and Passiflora edulis, crude extracts were tested in the presence of PAßN (at 30 μg/mL) against ten selected MDR phenotypes (E. coli AG100, AG102 and AG100ATet, E. aerogenes EA289, EA27 and CM64, K. pneumoniae KP55 and KP63, P. aeruginosa PA124 and P. stuartii NAE16).

Extracts from Xanthosoma mafaffa, Moringa oleifera and Passiflora edulis were also tested in association with antibiotics at their sub-inhibitory concentrations as obtained in each bacterium (MIC/2 and MIC/4) [4, 5, 11] against ten MDR phenotypes. Fractional inhibitory concentration (FIC) was calculated as the ratio of MICAntibiotic in combination/MICAntibiotic alone and the results were discussed as follows: synergy (≤0.5), indifferent (>0.5 to 4), or antagonism (>4) [26, 27]. All assays were performed in triplicate.

Results

Phytochemical composition

The results of the phytochemical screening (Table 2) showed that all the tested plant extracts contain polyphenols, triterpenes, sterols and saponins. The other classes of secondary metabolites were selectively distributed. Also, the extract from M. oleifera contains all the classes of screened secondary metabolites.

Antibacterial activity

Results of the antibacterial activities of the tested extracts alone and in some cases in the presence of the PAβN on a panel of 19 Gram-negative bacteria are summarized in Table 3. It appears that the extracts from P. edulis inhibited the growth of 17/19 (89.5 %) bacteria with a concentration ranged from 128 to 1024 μg/mL. The two other samples showed selective activities, their inhibitory activity being recorded on 13/19 (68.4 %) and 11/19 (57.9 %) tested bacteria for M. oleifera and X. mafaffa extracts respectively. The lowest MIC value (128 μg/mL) was obtained with P. edulis and M. oleifera extracts on Escherichia coli AG100.

Table 3 Minimal Inhibitory Concentration (MIC) in μg/mL of methanol extracts from the studied plants and chloramphenicol
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Role of efflux pumps in the susceptibility of Gram-negative bacteria

Ten of the studied MDR bacteria were also tested for their susceptibility to the plant extracts in the presence of the PAβN at 30 μg/mL. The results showed that when combined with the extracts, PAβN improves the activity (decrease of MIC values) of X. mafaffa on 4/10 (40 %) of tested MDR strains. The EPI also improved the activity of P. edulis against E. coli AG100 (Table 3).

Effects of the association of the extracts with antibiotics

A preleminary study was performed against P. aeruginosa PA124. This allowed selection of the appropriate sub-inhibitory concentrations of MIC/2 and MIC/4 for further studies. All the three extracts were combined separately with eight antibiotics (CIP, NOR, CHL, ERY, KAN, TET, CEF and AMP) to evaluate their possible synergetic effects. The results summarized in Tables 4, 5 and 6 showed synergic effects of the three tested extract with most of tested antibiotics except β-lactams (CEF and AMP). At MIC/2 of the extract from X. mafaffa, synergistic effects were observed with 6/8 (75 %) antibiotics (CIP, NOR, CHL, ERY, KAN, TET) against the tested MDR bacteria. Synergistic effects (FIC ranging from 0.5 to 0.03) were noted with the associations of each of the X. mafaffa, P. edulis and M. oleifera extracts and antibiotics. Low FIC values of 0.03 were obtained with the associations of M. oleifera extracts + ERY and M. oleifera extract + CHL against Enterobacter aerogenes EA27.

Table 4 MIC (FIC) of different antibiotics in association with the extract of Xanthosoma mafaffa at MIC/2, MIC/4 against ten MDR bacteria strains
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Table 5 MIC (FIC) of different antibiotics in association of the extract of Passiflora edulis at MIC/2, MIC/4 against ten MDR bacteria strains
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Table 6 MIC (FIC) of different antibiotics after the association of the extract of Moringa oleifera at MIC/2, MIC/4 against ten MDR bacteria strains
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Discussion

Phytochemical screening revealed the presence of several classes of secondary metabolites such as alkaloids, polyphenols, flavonoids, anthraquinones, coumarins, saponins, tannins, triterpenes and steroids. Several molecules belonging to these classes were found to be active on pathogenic microorganisms [3, 28–30]. The presence of such metabolites in the studied plant extracts can provide a preliminary explanation on their antibacterial activities. Differences were observed in the antibacterial activities of the extracts. These could be due to the differences in their chemical composition as well as in the mechanism of action of their bioactive constituents [3]. As shown in Table 2, all the extracts are rich in secondary metabolites especially the extract from M. oleifera (which contains all the tested classes); However, activity does not depend only on the presence of secondary metabolites in the plant extracts, but mostly on their concentration and the possible interactions with other constituents.

To the best of our knowledge, the antibacterial activity of X. mafaffa is being reported here for the first time. The inhibitory activity of M. oleifera was previously reported against some bacteria such as Escherichia coli, Pseudomaonas aeruginosa, Stapylococcus aureus and Salmonella typhii [31]. The present study confirmed the antimicrobial potential of this plant and provide additional information on it ability to inhibit the growth of MDR bacteria.

The results of this work are very important taking in account the medicinal importance of the tested MDR bacteria [32–36] and also the fact that samples used are edible plants. In the presence of PAβN (EPI), the antibacterial activity of some of the extracts increased, suggesting that some active constituents may have intracellular target. In the presence of the EPI, the activity of M. oleifera remain unchanged, indicating that the bioactive compounds of this extract are not the substrates of bacterial efflux pumps, as the tested MDR bacteria over-express efflux pumps [32–36]. However, it should be observed that in certain cases (Table 3), MIC values of Xanthosoma mafaffa and Passiflora edulis extracts increased in the presence of PAßN. A possible explanation is that some active constituents of these extracts may act in the cell coat, inhibiting the synthesis of peptidoglycan. In such case, in the absence of PAßN, such compounds are extruded from the cytoplasm of bacteria by efflux pumps, then re-cross the cell membrane to reach their target in the coat, explaining while the MIC value is lower; in presence of PAßN, the efflux pumps are blocked and such compounds could not be expelled from the cytoplasm, reducing their concentration in the cell coat and consequently their activity, explaining their higher MIC values.

In the recent years, scientists intensified the search of substances with ability to restore the activity of available antibiotics to MDR bacteria. In this work, synergistic effects were noted with the associations of X. mafaffa, P. edulis and M. oleifera extracts and some antibiotics, providing additional information of their possible use to combat MDR phenotypes.

Conclusion

The results of the present investigation show that X. mafaffa, P. edulis and M. oleifera may be useful in the control of many infectious diseases, particularly those caused by the multidrug resistant Gram-negative bacteria. These extracts may be used alone or in combination with certain antibiotics such as tetracycline, ciprofloxacin, norfloxacin, chloramphenicol, erythromycin, kanamycin but not beta-lactamines. The isolation of the active compounds from the three plants constitutes the limitation of the present study and will be further performed. Also, further investigations of the plant extracts are warranted in vivo to validate their use for the control of infectious diseases.