Antibacterial and antibiotic-potentiation activity of the constituents from aerial part of Donella welwitshii (Sapotaceae) against multidrug resistant phenotypes

Background The rise of multidrug-resistant (MDR) bacteria is a real public health problem worldwide and is responsible for the increase in hospital infections. Donella welwitschii is a liana or shrub belonging to the family Sapotaceae and traditionally used to cure coughs. Objective This study was conducted with the objective to validate the medicinal properties of this plant, the aerial part was studied for its phytochemical composition using column and PTLC chromatography and exploring its antibacterial and antibiotic-modifying activity as well as those of its phytochemicals. Methods The structures of the compounds were elucidated from their physical and spectroscopic data in conjunction with literature. The antibacterial activity of the isolated metabolites was performed toward a panel of MDR Gram negative and Gram-positive bacteria. The broth micro-dilution method was used to determine antibacterial activities, efflux pump effect using the efflux pump inhibitor (EPI) (phenylalanine-arginine-ß-naphthylamide (PAβN)), as well as the modulating activity of antibiotics. Monitoring the acidification of the bacterial growth medium was used to study the effects of the samples on the bacterial proton-ATPase pumps and cellular ATP production. Results Eleven compounds were isolated including pentacyclic triterpenes, C-glucosyl benzophenones. With a MIC value < 10 μg/mL, diospyric acid (7) significantly inhibited the growth of Escherichia coli AG102, Enterobacter aerogenes ATCC13048, Klebsiella pneumoniae KP55, Providencia stuartii NEA16 and Staphylococcus aureus MRSA3. 28-hydroxy-β-amyrin (8) significantly impaired the growth of Enterobacter aerogenes EA27, Klebsiella pneumoniae ATCC11296 and Staphylococcus aureus MRSA6; and oleanolic acid (9) strongly impaired the growth of Escherichia coli AG 102, Enterobacter aerogenes EA27 and Providencia stuartii PS2636. Diospyric acid (7) and 28-hydroxy-β-amyrin (8) induced perturbation of H+-ATPase pump and inhibition of the cellular ATP production. Moreover, at MIC/2 and MIC/4, compounds 7, 8, and 9 strongly improved the antibacterial activity of norfloxacin, ciprofloxacin and doxycycline with antibiotic-modulating factors ranging between 2 and 64. Conclusion The overall results of the current work demonstrate that diospyric acid (7), 28-hydroxy-β-amyrin (8) and oleanolic acid (9) are the major bioactive constituents of Donella welwitschia towards Gram-negative bacteria expressing MDR phenotypes. Supplementary Information The online version contains supplementary material available at 10.1186/s12906-022-03673-3.


Introduction
According to the World Health Organization (WHO), high levels of resistance to many pathogenic bacteria remains a serious health concern globally [1]. The rapid emergence of bacterial multidrug-resistant (MDR) phenotypes is a critical issue in the fight against infectious diseases [2]. Consequently, there is a need to search for new antimicrobial agents that can efficiently tackle bacterial resistance. The WHO has stated that most of the developing world still benefits from the use of traditional medicines derived from medicinal plants [3]. Moreover, many studies revealed the antibacterial inhibition activity of botanicals from African medicinal plants against MDR bacteria [4][5][6][7][8][9]. Several constituents including triterpenoids, phenolics, as well as alkaloids isolated from African medicinal plants were also documented for their antibacterial activities [10][11][12][13]. The present study focusses on the antibacterial activity of the constituents of Donella welwitschii (Engl.) Pierre ex Aubrev. & Pellegr.
(Sapotaceae). The antibacterial activity of many secondary metabolites from plants belonging to the Sapotaceae family such as Tridesmostemon omphalocarpoides [14], Omphalocarpum elatum [15], Chrysophyllum lacourtianum [16], Synsepalum msolo [17], and Manilkara zapota [18] have been reported. The infusion of the shrub of D. welwitschii is traditionally used in the Central African Region and is used as an antitussive to relieve coughs and a stiff neck [19]. Previous phytochemical studies on species of the genus Donella reported cyclopropane type triterpene di-acids [20]. Herein, we reported the isolation of 11 secondary metabolites from D. welwitschii, and their antibacterial potential against a panel of MDR bacteria.

Chemicals for antibacterial assays
A few reference antibiotics (RA), namely chloramphenicol (CHL), streptomycin (STR), erythromycin (ERY), norfloxacin (NOR), ciprofloxacin (CIP), ampicillin (AMP), and doxycycline (DOX) (Sigma-Aldrich, St. Quentin Fallavier, France) were predominantly used for the sample association tests, and only one was used as a positive control. Dimethyl sulfoxide (Sigma-Aldrich) was used to dissolve the samples before any tests. p-Iodonitrotetrazolium chloride (INT; Sigma-Aldrich) and phenylalaninearginine-ß naphthylamide (PAβN; Sigma-Aldrich) were used as microbial growth indicator and efflux pump inhibitor (EPI), respectively. Solvents used for extraction and purification of bioactive compounds were of analytical grade.

Plant materials and extraction procedure
The aerial part of D. welwitschii was collected from Manjo in Bertoua, the East region of Cameroun on May 2018. The appropriate authorisation has been obtained for the collection of the plant and its use has been carried out in accordance with the relevant guidelines. The identification of the plant was carried out by Dr. Tchiengue Barthelemy at the Cameroon National Herbarium (Yaoundé) where a voucher specimen was conserved under specimen No: 10708SFC / 56630HNC. The airdried and powdered twig and leaf (1160 g) of D. welwitschii were macerated in a mixture of CH 2 Cl 2 -MeOH (1:1, 5 L) for 48 h, three times. The filtrate was evaporated at 40 °C under vacuum to yield 42.7 g of dark brown extract. The structures of compounds (Fig. 1) were determined by means of modern spectroscopic techniques (NMR and MS) and comparison with available literature (SM1). All 1 H and 13 C NMR spectra and major chemical shifts of these compounds are shown in the supplementary file (SM1).

Bacteria strains, culture media and growth conditions
Thirteen pathogenic strains consisting of Gram-negative and Gram-positive bacteria were recruited to measure the bacteriostatic and bactericidal power of the samples used in the study. Gram-negative bacteria included multidrug-resistant (MDR) isolates (laboratory collection) and reference strains of Escherichia coli (ATCC 10536, and AG102), Enterobacter aerogenes (ATCC 13048 and EA27), Klebsiella pneumoniae (ATCC 11296 andKP55), Providencia stuartii (ATCC 29916 and NEA16) and Pseudomonas aeruginosa (PA01 and PA124). Note that the clinical strains came from the laboratory collection of the UMR-MD1, University of Marseille, France. The Grampositive bacterial strains were specifically Staphylococcus aureus including a reference strain obtained from the American Type Culture Collection (ATCC) (ATCC   [21,22]. Susceptibility patterns of the tested bacteria are given in the Supplementary file (Table S1, SM2).

Evaluation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The ability of the isolated compounds to stop growth and kill bacteria was achieved through the determination of minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). This was possible using microdilution methods as described by Eloff (1998) [23] with some modifications and improvements [22,24]. Briefly, series of dilutions of the samples were performed in a 96-well microplate after dissolving the samples in 10% dimethyl sulfoxide (DMSO)/Mueller Hinton broth (MHB). Then 100 μL of bacterial inoculum (2 × 10 6 colony forming units (CFU)/mL) was added to each well. Chloramphenicol was used as a reference substance. The final concentrations ranged 4-512 μg/mL. Plates were then covered with a sterile plate sealer and incubated at 37 °C for 18 h. Revelation was done by adding 40 μL of INT (0.2 mg/mL) followed by incubation at 37 °C for 30 min. The MIC of each sample was defined as the lowest concentration of the sample that completely inhibited the growth of the bacteria. The MICs obtained were classified as follows: significant activity MIC < 100 μg/mL (MIC < 10 μg/mL for pure compounds), moderate activity 100 μg/ mL < MIC ≤625 μg/ mL (10 < MIC ≤100 μg/ mL for pure compounds), weak activity MIC> 625 μg/mL (MIC> 100 μg/mL for pure compounds) [22,25]. After MIC readings, minimum bactericidal concentrations (MBC) were determined by introducing 150 μL of MHB into a new 96-well microplate, after addition of 50 μL of the microplate contents, where no microbial growth was observed, and which did not receive INT during the MIC reading. The MBC of each sample was determined after 48 h of incubating at 37 °C, by adding 40 μL of 0.2 mg/mL INT, as previously described [22]. MBC was the lowest concentration of the samples, which did not produce a colour change after adding INT. Each experiment was performed in triplicate and repeated three times.

Evaluation of the role of efflux pumps in the antibacterial activity of the samples
After testing the isolated compounds alone, the same compounds were put in the presence of PaβN (at 30 μg/ mL) against 13 bacterial strains including five MDR phenotypes (E. coli AG102, E. aerogenes EA27, K. pneumoniae KP55, P. stuartii PS2636 and S. aureus MRSA3) as previously described [25,26]. The activity improvement factor (AIF) or fold increase of the activity was determined as ratio of MIC (sample alone) /MIC (sample + PAβN) [22]. Each assay was performed in duplicate and repeated three times.

Assessment of the antibiotic-modifying activity of the samples
The combination assay was performed as previously described, with some modifications made during the procedure [4,7]. The test was briefly performed by introducing 100 μL of MHB into the wells of a 96-well microplate, followed by the addition of 100 μL of the test antibiotics in the first well of each column and progressive dilution. 50 μL of solution of test compounds at sub-inhibitory concentrations (MIC/2 and MIC/4) were then introduced into the wells, followed by the addition of 50 μL of bacterial inoculum. The microplates were sealed and incubated for 18 h at 37 °C. MICs were determined using the INT as previously described. The antibiotic modulation factor (AMF), calculated as MIC (antibiotic alone) /− MIC (antibiotic + compound) , was used to express the modulating effects of compounds on antibiotics [5,22,27]. Each test was performed in duplicate and repeated three times.

H + -ATPase-mediated proton pumping assay
The ability of compounds 7 and 8 to inhibit the H + − ATPase mediated proton pumps of E. coli AG102 was assessed by monitoring the acidification of the bacterial growth medium as previously described [28] with slight modifications [22]. Briefly, 100 mL of bacteria suspension was cultured in MHB for 18 h at 37 °C. The resulting culture was centrifuged at 3000 tr/min for 10 min at 4 °C. The pellet was first washed twice in distilled water, then in 50 mM KCl and suspended in 50 mL of 50 mM KCl. Then, the cell suspension was incubated overnight (18 h) at 4 °C for glucose starvation. In 4 mL of the cell medium, 0.5 mL of the tested samples (MIC/2, MIC, and 2 × MIC) were added, and pH adjusted to 6.8 with 1 M HCl or 0.1 M NaOH. Upon 10 min pre-incubation at 37 °C, medium acidification was initiated by adding 0.5 mL of 20% glucose, followed by pH measurement every 10 min for 1 h using a pH-meter. Tube containing MHB, inoculum, and DMSO was used as negative control. Each essay was performed in triplicates.

Measurement of cellular ATP concentration
The ability of compounds 7 and 8 to inhibit the cellular ATP production of E. coli AG102 was assessed as previously described [28] with slight modifications. Briefly, 100 mL of bacterial culture (1:100 v/v) was grown in

Statistical analysis
Data from each experimental group was expressed as mean ± SD. One-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test for multiple comparisons were used for statistical analysis of data using GraphPad Prism Version 8.1.0 (GraphPad Software, CA, USA). Differences were considered significant at a probability level of 5% (p < 0.05), ** p-values < 0.01, and *** p-values < 0.001.

MICs and MBCs of phytochemicals
The antibacterial activity of the isolated compounds 1-4 and 7-10 has been evaluated against E. coli (ATCC 10536, and AG102), E. aerogenes (ATCC 13048 and EA27), K. pneumoniae (KP55 and ATCC 11296), P.   6, 7, 8 and 9 in the presence of PaβN showed improved antibacterial activity (MIC reduction) against the tested bacteria by 2 to > 64 times. These isolated compounds were more effective against MDR bacteria and mainly against Gram-negative bacteria. Moreover, PAβN did not affect the activity of chloramphenicol against Gram-positive bacteria (S. aureus: ATCC 25923 and MRSA3) ( Table 2).

Antibiotic-modulating activity of compounds
The modulatory activity of active phytochemicals (7, 8 and 9) was investigated on six classical antibiotics including streptomycin (STR), erythromycin (ERY), norfloxacin (NOR), ciprofloxacin (CIP), ampicillin (AMP) and doxycycline (DOX). The overall results showed a considerable increase in the activity of the tested antibiotics. The best potentiating effects were recorded with NOR, CIP and DOX, in combination with phytochemicals 7, 8 and 9 at MIC/2 and MIC/4. It was found that the overall activity enhancement factors (AEFs) obtained with the antibiotics ranged from 2 to 64 (Table 3, Table 4 and Table 5), indicating a 2 to 64 fold increase in antibacterial activity of the antibiotics. These activities were predominantly  22:194 recorded on the MDR strain E. aerogenes EA-CM64. The reference strain E. coli ATCC 10536 was also sensitive to the same combination (Table 3, Table 4 and Table 5). Combination of the other antibiotics and the compounds associations has mainly presented indifference or antagonistic effects. Contrary to the control whose pH decreases from the beginning of the experiment until reaching pH 6.2 at the end (T = 60 min) (Fig. 2a). In compound 8, a constant progression of pH (pH 7.3) can be observed until the end of the experiment, at MIC and 2MIC concentrations. At CMI/2, a decrease in pH is observed until reaching 6.4 at t = 20 min, then remains constant until the end of experiment. Contrary to the control whose pH decreases from the beginning of the experiment until reaching pH 6.2 at the end (t = 60 min) (Fig. 2b). Figure 3a and b show the results of the effects of actives compounds 7 and 8 on the activity of ATP synthase. The differences observed in compounds 7 and 8 tested at MIC/2, MIC and 2MIC were significant compared to the control. For compound 7, an increase in pH can be observed from the second minute of the experiment (pH 7.9), at concentrations of MIC/2, MIC and 2MIC. Contrary to the control whose pH decreases from the beginning of the experiment until reaching pH 6.1 at the end (T = 160 min) (Fig. 3a). Virtually the same behaviour was observed with compound 8 (Fig. 3b).

Discussion
Chemical compounds produced by plants, generally help them to resist fungi, bacteria, and plant virus infections, and consumption by insects and other animals. However, some phytochemicals' class have been used as medicine to cure various ailments. In the current work, we explored the anti-MDR bacteria's chemicals of Donella welwitschii, as well as the mechanism of action through which they exhibited their antibacterial effects.  22:194 The chemical investigation of the methylene chloridemethanolic extract of D. welwitschii aerial part, led to isolation of eleven compounds belonging to various class of secondary metabolites known for their antimicrobial activities through specific mechanisms of actions i.e. terpenoids, phenolic compounds and alkaloids [29,30]. Vanillic acid 3 [31] is a phenolic acid belonging to phenolic compounds' group, taraxerol 4 [32], taraxeryl acetate 5 [33], ursolic acid 6 [34], diospyric acid 7 [35], 28-hydroxy-β-amyrin 8 [36] and Oleanolic acid 9 [37] are all triterpenes belonging to terpenoids group.
Against a panel of MDR Gram-negative and Grampositive bacteria, D. welwitschii's compounds 5 and 11 were found to be inactive, while other compounds (1-4, 6-10) were selectively significantly active, moderately active and/or weakly active. Diospyric acid (7) significantly inhibited (MIC < 10 μg/mL) the growth of E. coli AG102, E. aerogenes ATCC 13048, K. pneumoniae KP55, P. stuartii NEA16 and S. aureus MRSA3. To the best of our knowledge, this study is illuminating for the first time the antibacterial activity of diospyric acid against the Gram-negative and Gram-positive   [40] showed the effect of oleanolic acid against E. faecalis ATCC 28212. In the present work, we also investigated the effect of diospyric acid (7) and 28-hydroxyβ-amyrin (8) on the functioning of H + -ATPase pump and the cellular ATP production of E. coli AG102. Let's note that from the studies of Futai and Kanazawa [41] and those of Senior and Wise [42], it is well established that the H + -ATPase pump of Escherichia coli membranes catalyses ATP synthesis and the formation of ATP-driven proton electrochemical gradients, and resembles analogous enzymes from mitochondria, chloroplasts, and other bacteria. The D. welwitschii's compounds might induce bacteria growth inhibition through significant perturbation of H + -ATPase pump and inhibition of the cellular production of ATP. Clinically tripartite efflux systems, AcrAB-TolC for Enterobacteriaceae or MexAB-OprM for P. aeruginosa, are associated with multidrug resistance of  22:194 pathogenic Gram-negative bacteria [43][44][45]. PAßN has been reported as a potent inhibitor of the RND efflux systems and is especially active on AcrAB-TolC and MexAB-OprM [46][47][48][49]. PAßN has been used to demonstrate the role of efflux pumps in MDR of the tested microorganisms. In presence of the efflux pump inhibitor PAβN, the MIC values of phytochemicals 7-9 decreased, showing that these compounds could be substrates of efflux pumps acting in resistant strains used in our study. These data suggested that combination of these compounds with antibiotics could improve the antibacterial activity of conventional antibiotics against MDR phenotypes. Combining phytoconstituents 7-9 with antibiotics against MDR bacteria at MIC/2 and MIC/4 strongly improved the antibacterial activity of norfloxacin, ciprofloxacin and doxycycline with antibiotic-modulating factors ranging between 2 and 64. This indicated that compounds 7, 8 and 9 behave as natural efflux pumps inhibitor. Martins et al. [39] revealed the antibiotic potentiating capacity (tetracycline) of β-amyrin (8) and Oleanolic acid (9) against E. coli AG100 TET8 . Moreover, our results are in accordance with those of Abreu et al. [50] which highlighted synergism effect against drug-resistant Staphylococcus aureus when oleanolic acid was combined to tetracycline or erythromycin, as well as those of Basri et al. [51] showing the synergistic interaction arising from the combination of the oleanolic acid and norfloxacin against SA1199B and MRSA274829 [51] .

Conclusion
The overall results of the current work provide baseline information for the possible use of some compounds, diospyric acid (7), 28-hydroxy-β-amyrin (8) and Oleanolic acid (9), of Donella welwitschii extract to fight against bacterial infections involving MDR phenotypes. In addition, the data reported herein indicated that compounds diospyric acid (7), 28-hydroxy-β-amyrin (8) and Oleanolic acid (9) behave like efflux pump inhibitors. They are the major antibacterial constituents of D. welwitschii against MDR Gram-negative bacteria as well as Staphylococcus aureus.