Antibacterial and antibiotic-modifying activities of three food plants (Xanthosoma mafaffa Lam., Moringa oleifera (L.) Schott and Passiflora edulis Sims) against multidrug-resistant (MDR) Gram-negative bacteria

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.

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].

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].

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 MIC_{Antibiotic in combination}/MIC_{Antibiotic 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.

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.

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.

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).

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.