Antibacterial and Antibiotic-Potentiating Activities of Thirteen Cameroonian Edible Plants against Gram-Negative Resistant Phenotypes

This work was designed to investigate the antibacterial activities of methanol extracts from thirteen Cameroonian edible plants and their antibiotic-potentiating effects against Gram-negative multidrug-resistant (MDR) phenotypes. The broth microdilution method was used to evaluate the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the extracts, as well as their antibiotic-potentiating activities. The phytochemical screening of the extracts was carried out according to the standard methods. The results of phytochemical tests revealed the presence of sterols, polyphenols, and tannins in most of the tested extracts, with the other classes of secondary metabolites being selectively distributed. Tested extracts showed variable antibacterial activities with MIC values ranging from 64 to 1024 μg/mL. However, some extracts were significantly active against certain bacterial strains: seeds extract of Theobroma cacao (64 μg/mL) against Escherichia coli AG100Atet and Klebsiella pneumoniae K24, and the bark extract of Uapaca guineensis against E. coli ATCC 8739. The leaves extract of T. cacao displayed the best MBC values (256 μg/mL) against E. aerogenes EA27. Some tested extracts included extracts from the leaves of T. cacao and P. vulgaris, and the seeds of D. edulis and barks A. indica has selectively improved (2- to 64-fold) the antibacterial activities of some of the tested antibiotics, chloramphenicol (CHL), tetracycline (TET), kanamycin (KAN), streptomycin (STR), and erythromycin (ERY), against more than 70% of tested MDR bacteria. The findings of this work showed that tested plant extracts and particularly those from T. cacao and Phaseolus vulgaris can be used alone or in combination with conventional antibiotics in the treatment of infections involving multiresistant bacteria.


Introduction
The advent of antibiotics has been a tremendous therapeutic progress as it has significantly reduced mortality due to bacterial infections [1]. However, their misuse in humans and animals has contributed to the emergence of drugresistant bacteria [2]. These bacteria are often more difficult to be combatted, and the infections they caused are more difficult and expensive to be treated. This can therefore lead to serious disability and even death [3,4]. According to World Health Organization [4], antibiotic resistance has drastically increased to high levels all over the world. The multidrug-resistant (MDR) genes may be carried on the bacterial chromosome, plasmid, or transposons, and their expression allows bacteria to overcome the effects of many antibiotics [5]. Among the antibiotic resistance mechanisms, the most common are enzymatic inactivation of antibiotics, changes in cell permeability, and induction/activation of efflux pumps [6]. The Gram-negative bacteria are among the bacteria that drastically impair the efficacy of antibacterial agents and therefore limit their clinical use [3,7]. In fact, to guide research, discovery, and development of new antibiotics against MDR bacteria, WHO has developed a global priority list of antibiotic-resistant bacteria where Gram-negative multidrug-resistant (MDR) bacteria, particularly Pseudomonas aeruginosa and Enterobacteriaceae, constitute the most critical group [4]. These groups of bacteria are particularly characterized by the expression of efflux pumps belonging to the Resistance Nodulation-cell Division (RND) family, which constitutes one of their main resistance 2 The Scientific World Journal mechanisms [8,9]. Tackling antibiotic resistance mechanisms like efflux pumps expression is therefore a high priority for WHO and scientists over the world. Plant sources used since ancient time to fight microbial infections appear as an interesting alternative for the discovery of new antibacterial substances against MDR bacteria [10][11][12]. For instance, a number of plant-deriving compounds with possible efflux pump inhibitor (EPI) activities such as reserpine [13], berberine [13,14], and curcumin [15] have been discovered, but they are still unused clinically. In our previous studies, we have also shown that edible plants could be used alone and/or in combination with commonly used antibiotics to fight infections involving Gram-negative MDR bacteria [16][17][18][19]. In the continuous search for natural substances effective in MDR bacteria, this study was aimed at investigating the in vitro antibacterial and antibiotic-potentiating activities of methanol extracts of thirteen Cameroonian edible plants  Table S1).

Plant Material and Extraction.
Plants used in this study were collected in West, Southwest, and Centre regions of Cameroon from March to April 2016. All plants collected were identified at the National Herbarium (Yaoundé, Cameroun) where the voucher specimens were deposited. The names as well as the reference numbers of the studied plants are shown in Table S1 (Supplementary Materials). For the extraction, each plant material was cleaned and air-dried, and the powder (200 g) was soaked in methanol (MeOH, 1 L) for 48 h at room temperature. The extract obtained was collected by filtration using Whatman filter paper n ∘ .1 and concentrated under reduced pressure using a rotary evaporator to yield a residue which constituted the plant extract. All the extracts were then kept at 4 ∘ C until further use.  Table S2). These bacteria were maintained at 4 ∘ C and subcultured on a fresh Mueller Hinton Agar (MHA) for 24 h before any antibacterial assay. Mueller Hinton Broth (MHB) was used for antibacterial assays [23].

Antibacterial
Assays. MIC and MBC values of the different samples were determined by microdilution using INT colorimetric assay as previously described [22,24]. Briefly, the samples were dissolved in 10% dimethyl-sulfoxide (DMSO)/Mueller Hinton Broth (MHB) and serially diluted twofold (in a 96-well microplate). Then, 100 L of inoculum (2 × 10 6 CFU/mL) prepared in MHB was added in each well. Chloramphenicol was used as reference drug and the well containing the vehicle (DMSO 2.5%) as control. The plates were then covered with a sterile plate sealer and gently shaked to mix the contents of the wells. After 18 h of incubation at 37 ∘ C, the MIC value of each sample, defined as the lowest sample concentration that inhibited complete bacteria growth, was detected following addition of 40 L INT (0.2 mg/mL) and incubation at 37 ∘ C for 30 min. Viable bacteria reduced the yellow dye to pink. The MBC value was determined by adding 50 L aliquots of the preparations, which did not show any growth after incubation during MIC assays, to 150 L of MHB. Then, these preparations were incubated at 37 ∘ C for 48 h. The MBC was regarded as the lowest concentration of samples, which did not produce a color change after addition of INT as mentioned above [24]. Each assay was performed in three independent tests in triplicate. In case there was difference, the MIC or MBC values were taken as the most frequently occurring values.

Antibiotic Resistance Modifying
Assay. The resistance modifying activity of the extracts was evaluated by determining the MICs of antibiotics in the presence or absence of the plant extracts in the 96-wells modulation assay as previously described [13,25]. Briefly, after serial dilutions of antibiotics (256-0.5 g/mL), the plant extracts were added at their subinhibitory concentrations (MIC/2 and MIC/4) selected after preliminary study assessed against P. aeruginosa PA124 (Supplementary Materials, Table S3). The MIC of each treatment was determined as described above. Each assay was performed in three independent tests in duplicate. Modulation factors (MF), calculated as MIC of antibiotic alone/MIC of antibiotic + extract, were used to express the antibiotic-potentiating effects of the plant extracts [11,26,27].
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Qualitative Phytochemical Composition of the Tested
Extracts. The results of the phytochemical screening (Table 1) showed that only Theobroma cacao broad bean (TCBB) extract contained all the classes of screened secondary metabolites. These metabolites were selectively distributed in other tested plant extracts. In addition, results showed that polyphenols, tannins, triterpenes, and steroids were the most represented metabolites in the tested extracts.

Antibacterial Activity of the Tested Extracts.
Nineteen extracts from thirteen plants as well as chloramphenicol were tested for their antibacterial activities on a panel of 21 Gram-negative bacteria. The results of

Antibiotic Resistance-Modifying Activities of the Extracts.
The antibacterial activity of 6 commonly used antibiotics was evaluated in the presence of plant extracts at the concentrations equivalent to MIC/2 and MIC/4. The results obtained are summarized in Tables 3-9. From these tables, it was observed that some extracts selectively improved the antibacterial activities of tested antibiotics against the selected MDR bacteria (2-to 64-fold decrease of MIC). D. edulis seeds extract has significantly improved the antibacterial activities of CHL and KAN against 90% (8/10) and 80% (8/10) of the tested MDR bacteria, respectively ( Table 4). The bark extract of A. indica (Table 3) and leaves extract of P. vulgaris (Table 6) also improved the activities of CHL, STP, and TET to about 80% (8/10) and 70% (7/10) of the tested MDR bacteria, respectively. The modulating effects were also observed after the combination of T. cacao leaves extract with STP, CHL, CIP, TET, and STP against 80% to 70% of the tested MDR bacteria (Table 8) whilst other extracts were less active.

Discussion
Plants constitute an undeniable source of substances named secondary metabolites which are known for their direct or indirect antimicrobial activities; some examples include flavonoids, phenols, terpenoids and sterols, saponins, and tannins [28][29][30]. The results of the phytochemical screening carried out on the tested extracts indicated the presence of at least one of these metabolites in each of the tested extracts (Table 1). This may explain the antibacterial activities of the extracts tested.

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The Scientific World Journal According to Tamokou et al. [31], an edible plant extract is very active if it has a MIC<100 g/mL, significantly active if 100 ≤ MIC<512 g/mL, moderately active when 512<MIC ≤ 2048 g/mL, and weakly active for a MIC>2048 g/mL.
Thus, many of the tested extracts presented significant to moderate activities, 100≤ MIC<2048 g/mL. Therefore, two extracts were very active (MIC<100 g/mL), including the extract from seeds of Theobroma cacao, active against E. coli ATCC8739, AG100Atet, and K. pneumoniae K 24 and the extract from bark of Uapaca guineensis which was very active on E. coli ATCC 8739 (Table 2). Several other studies have already demonstrated the in vitro antibacterial activity of at least one of the parts of these two plants or those belonging to the same genus. Previous study has already demonstrated the antibacterial potential of extracts of bark and pulp of T. cacao on many bacterial strains including E. coli [32]. Singh et al. [33] have also demonstrated the antibacterial activity of T. cacao seed extract against K. pneumoniae. In addition, the bark of several species of the Uapaca genus has shown very good antibacterial activity against certain sensitive and resistant strains [34,35]; the results obtained in this work reinforce those previous works done with some of the tested extracts. The extracts of seeds of Theobroma cacao and that from bark of Uapaca guineensis could be used to fight infections involving multidrug-resistant bacteria.
In addition to their direct antibacterial activities, secondary metabolites have been found to act indirectly as modulators of the activity of antibacterial agents [10,28,36]. In this work, some antibiotics (CHL, TET, KAN, STR, and ERY) activities were improved (2 to 64 times) on more than 70% of the multidrug-resistant bacteria tested in the presence of T. cacao leaves, P. vulgaris leaves, D. edulis seeds, and A. indica barks extracts (Tables 3-9). The bacteria used in this work are multiresistant and overexpress efflux pumps as a resistance mechanism (Supplementary Materials, Table S2). This suggests that aforesaid extracts could contain substances which are able to inhibit the efflux pumps expressed in these bacteria [37], thus leading to an increase in the effectiveness of antibiotics [38]. Several studies have shown that polyphenols, especially flavonoids, could improve the activity of antibiotics against resistant bacterial strains [39,40]. Thus, the presence of these metabolites in the most active extracts may be the origin of the observed antibiotic-potentiating activity. Many cases of antagonism were also observed and this could be due to the negative interactions between the antibiotics and the compounds of the plant extract, leading, for example, to the inhibition of the active groups of the antibiotics. These results of this study indicate, for the first time, the potential of the tested plant extracts, mainly extracts from D. edulis, P. vulgaris, A. indica, and T. cacao, to reverse antibiotic resistance.

Conclusion
This work has provided informative data related to the antimicrobial activity of the tested plant extracts. It suggests that plant extracts and particularly those from Theobroma cacao and P. vulgaris can be used alone or in combination with conventional antibiotics in the treatment of bacterial infections involving multiresistant phenotypes.

Data Availability
The data used to support the findings of this study are included within the article.