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Article

Acacia senegal Extract Rejuvenates the Activity of Phenicols on Selected Enterobacteriaceae Multi Drug Resistant Strains

by
René Dofini Magnini
1,2,3,
Adama Hilou
3,
Helana Millogo-Koné
2,
Jean-Marie Pagès
1 and
Anne Davin-Regli
1,*
1
Faculté de Pharmacie, UMR_MD1, U-1261, Aix-Marseille University, INSERM, SSA, IRBA, MCT, 13385 Marseille, France
2
Institut de Recherche en Sciences de la Santé (IRSS/CNRST), Département de Médecine et Pharmacopée Traditionnelle/ Pharmacie (MEPHATRA-PH), 03 BP 7047 Ouaga 03, Burkina Faso
3
Laboratoire de Biochimie et de Chimie Appliquée (LABIOCA), Université Ouaga I Pr Joseph Ki-Zerbo, 03 BP 848 Ouagadougou 03, Burkina Faso
*
Author to whom correspondence should be addressed.
Antibiotics 2020, 9(6), 323; https://doi.org/10.3390/antibiotics9060323
Submission received: 24 May 2020 / Revised: 8 June 2020 / Accepted: 9 June 2020 / Published: 13 June 2020
(This article belongs to the Special Issue Natural Antimicrobial Products from Plants and Fungi: Current Knowledge and Future Prospects)
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Abstract

:
This study reported the phytochemical composition of two hydroethanolic extracts of Acacia senegal and Acacia seyal trees from Burkina Faso and their activities, alone or in combination with selected antibiotics, against multidrug resistant bacteria. High performance thin layer chromatography (HPTLC) method was used for phytochemical screening. Total phenolic and total flavonoid ant tannins in leaves extracts contents were assessed by spectrophotometric method. The minimal inhibitory concentrations (MICs) of plant extracts and antibiotics were determined using the microdilution method and p-iodonitrotetrazolium chloride. Combinations of extracts and antibiotics were studied using checkerboard assays. Screening revealed the presence of phenolic compounds, flavonoids, and tannins in the hydroethanolic extract (HE) of the leaves. The HE of A. seyal showed the highest total phenolic (571.30 ± 6.97 mg GAE/g), total flavonoids (140.41 ± 4.01 mg RTE/g), and tannins (24.72 ± 0.14%, condensed; 35.77 ± 0.19%, hydrolysable tannins). However, the HE of A. senegal showed the lowest total phenolic (69.84 ± 3.54 mg GAE/g), total flavonoids (27.32 ± 0.57 mg RTE/g), and tannins (14.60 ± 0.01%, condensed; 3.09 ± 0.02%, hydrolysable). The MICs for HE and antibiotics were in the range of 2–512 and 0.008–1024 mg/L, respectively. All tested HE presented an MIC greater than 512 mg/L except HE of A. senegal. The lowest MIC value (128 mg/L) was obtained with HE of A. senegal against Klebsiella aerogenes EA298 and Escherichia coli AG100A. Interesting restoring effects on chloramphenicol and florphenicol activity were detected with alcoholic extracts of A. senegal against resistant E. coli and K. aerogenes strains that overproduce AcrAB or FloR pumps. The adjuvant effect of HE of A. senegal suggests that the crude extract of leaves could be a potential source of molecules for improving the susceptibility of bacteria to phenicols antibiotics.

1. Introduction

Traditional medicine occupies an important place in the care of African populations; it represents an easy and inexpensive therapeutic means in regions where access to health infrastructures (hospitals, dispensaries, pharmacies) are underdeveloped and/or expensive for the patient family. Consequently, about 80% of the population in developing countries uses traditional medicinal plants for primary care management [1,2]. Today, documentation of the medicinal uses of plants is imperative, following the rapid disappearance of some of these plants species due to human activities [3]. The overuse and the misuse of antibiotics has led to the emergence of multiresistant bacteria, inducing worrying public health problem. Consequently, the implementation of appropriate strategies to bypass therapeutic impasses are urgently needed, and medicinal plants are attractive ways for the research and development of original alternate therapeutic molecules [4]. Medicinal plants extracts usually contain mixtures of different chemical compounds that can act individually, additively, or synergistically to combat multi-drug resistant (MDR) infections [5]. Between 1981 and 2019, in the area of cancer and antibacterial therapies, more than a thousand new drug molecules were characterized, from which 60% were issued from natural products [6]. Moreover, many available antimicrobial drugs used in therapeutics are derived from bacterial or fungal molecules [7]. Medicinal plants from the African continent have been previously studied for biological properties, and they exhibit interesting antibacterial activity [8]. Preclinical data on traditional uses show that A. senegal treats respiratory tract infections, diarrhea, stomach aches, hemorrhoids, ulcers, trypanosomiasis, sexually transmitted diseases, wounds, malaria, abscesses, and boils [9,10,11,12]. Another traditional healer treats dysentery, gastrointestinal pain, leprosy, nervous sensory and digestive disorders, toothache, rheumatism, stomach ulcers, jaundice, intestinal parasites, and syphilis by roots, leaves, barks, and gum of A. seyal [10,13,14]. In recent studies, extracts of A. senegal and A. seyal have shown good activity against many agents responsible for infectious diseases [12,15,16,17] and have interesting chemical components such as phenolic compounds, flavonoids, tannins, or terpenes recognized as having a strong antibacterial potential [8,18]. There is no study regarding possible adjuvant propriety of their extracts and their capability to permeabilize the bacterial membrane. In this study, we aim to evaluate the anti-bacterial potential of two Burkinabè medicinal plants, namely Acacia Senegal and Acacia seyal, against Gram-negative Enterobacteriaceae strains. We investigated the activity spectrum of these extracts for restoring the efficacy of phenicols antibiotic families in MDR bacteria.

2. Materials and Methods

2.1. Plants Material and Extraction

The collection of plant samples was done in early June and August in the area of Saaba in Gonsé, mapped about twenty kilometers from Ouagadougou (Burkina Faso). The plants were certified by Pr A. Ouedraogo of Botany Section, University Joseph KI-ZERBO of Ouagadougou, and voucher specimens were deposited under references numbers 6896/17257 (A. senegal) and 6897/17258 (A. seyal). The different parts of each plant were dried under ventilation deprived of sunlight for 21 days in a drying room. The samples were then ground to fine powder using a blade mill (Gladiator Est., 1931 Type BN 1 Mach 40461 1083). The vegetable powders were placed in freezer bags and stored at 4 °C in the freezer for further use.

2.1.1. Hydroethanolic Extracts (HE)

Extracts were prepared by taking 100 g of A. senegal and A. seyal leaf and root bark powder and soaking it in ether petroleum (500 mL) for 24 h in the first step. The residue was filtered by Whatman filter N°1, and the marcs were dried and soaked again in ethanol (1000 mL) at 70% (V/V) overnight. Rotary vacuum evaporator at 50 °C removed the extraction solvent. This hydroethanolic extracts were freeze dried and store at 4 °C until use [19,20].

2.1.2. Aqueous Extract

One hundred grams of powder of A. senegal and A. seyal root bark were boiled in 1000 mL of distilled water for 30 min. After cooling, the extracts were first filtered on a nylon cloth and then centrifuged at 2000 rpm for 5 min. Supernatants were collected and then lyophilized and weighed. The extracts were stored at 4 °C until use [21,22].

2.1.3. Soxhlet Extraction

From each plant, 50 g of dried powdered leaves were put into the extraction filter that was covered with cotton and then transferred into a Soxhlet apparatus with a series of four solvents of increasing polarity. Extraction solvents (500 mL) were added to each flask, which was connected to the extractor. Each extraction was performed in triplicate during 8 h. The temperature of extraction corresponded with the boiling point of the different solvent in use. Each solvent was concentrated at 40 °C using rotary evaporator, and their excess was eliminated by drying at 40 °C for 1 h. The crude extracts were weighed before being stored at 4 °C for further analysis [23,24].

2.2. Phytochemical Composition

2.2.1. High-Performance Thin Layer Chromatography (HPTLC) Screening

Phytochemical screening of samples extracts was performed on 20 cm × 10 cm silica gel 60 F HPTLC (glass) plate (Merck, Darmstadt, Germany). Two µL of each extract were applied as 5 mm bands with a semi automatic plate spotter (CAMAG, Linomat V, Switzerland) set to dispense along a line 10 mm from the bottom edge of the plate. The distance between tracks was 10 mm. Distances from left and right edge of the plate were 20 mm. The plates were placed in a 20 × 20 cm vee bottomed TLC tank (saturation time 30 min) containing ethyl acetate:formic acid:acetic acid:water (100:11:11:26) and ethyl acetate:water:methanol:n hexane (11.9:1.6:1.4:3.5), respectively, for flavonoids and tannins. The developed plates were then dried with an air dryer (cold air) for 5 min. Concerning flavonoids, the plate was heated at 105 °C for 2 min and sprayed with the Neu reagent. Evaluation was performed under UV 366 nm. As for the tannins, the plate was sprayed with a 2% FeCl3 reagent. Evaluation was performed under white light [25].

2.2.2. Determination of Total Phenolic Content

Different plant extracts (25 µL, 100 µg/mL in methanol) were mixed with Folin Ciocalteu reagent (105 µL, 0.2 N) and 5 min later with sodium bicarbonate (100 μL, 75 g/L). After 1 h incubation, the absorbance of each mixture was measured (spectrophotometer UV, Shimadzu) at 760 nm against a blank. A standard calibration curve was plotted using Gallic acid (Y = 0.0664X − 0.0009; R2 = 0.9991). Polyphenol content was expressed as mg of Gallic acid equivalent per g of extract (mg GAE/g).

2.2.3. Determination of Total Flavonoids Content

The extract was prepared at a concentration of 1 mg/mL in methanol. Then, 1 mL of this extract was mixed with 3 mL of double-distilled water followed by 0.3 mL of NaNO2 at 5% (m/v); 5 min later, 0.3 mL of AlCl3 10% (m/v) was added. The whole was incubated at room temperature for 6 min. Subsequently, 1 mL of NaOH 1 N was added. The absorbance of the mixture was measured at 510 nm using a UV spectrophotometer (Shimadzu). Calculation was based on a calibration curve obtained with increasing concentration of rutin solution following the same procedure. The flavonoid content of the sample, expressed as milligrams of rutin equivalent per g of plant material (mg RT/g), was obtained by relating the absorbance read on the calibration curve [26].

2.2.4. Determination of Tannin Content

Hydrolyzable Tannins

One mL of the extract and 3.5 mL of the reagent (FeCl3 10−2 M in HCl 10−3 M) were mixed. The absorbance of the mixture was measured at 660 nm after 15 s [27]. The hydrolysable tannins content T (%) was determined using the following formula: T (%) = A × PM × V × FD/ε mole × P
A = absorbance, Ɛ mole = 2169 (for gallic acid), PM = weight of gallic acid (170.12 g/mol), V = volume of extract, P = sample weight and FD = dilution factor.

Condensed Tannins

The reagent was vanillin 1% (1 g of vanillin dissolved in 100 mL of 70% sulfuric acid); 2 mL of this reagent was added to 1 mL of extract. The absorbance of the mixture was measured at 500 nm after incubation of 15 min in a water bath at 20 °C [28]. The condensed tannins content T (%) was determined using the following formula: T (%) = (5.2 × 10-2 × A × V)/P
5.2 × 10−2 = equivalent constant of cyanidine, A = absorbance, V = extract volume and P = sample weight.

2.3. Bacterial Strains

Phenotypic and/or genotypic characteristics for identification and changes in membrane permeability of strains used in this study are presented in Table 1. Bacteria were routinely grown at 37 °C on Luria-Bertani agar (LB).

2.4. Determination of Minimal Inhibitory Concentration (MIC) of the Plant Extracts

Direct antibacterial activity was determined by the microdilution method as described [36]. In practice, 20 mg of each plant extract were dissolved in 100 μL of DMSO and then completed to 5 mL with broth to obtain a stock concentration of 4 mg/mL (2% DMSO). Then, this solution was diluted to obtain an entry concentration of 512 mg/L with a concentration of 0.2% in DMSO. This solution was transferred to the wells of the first column of the 96-well plates (200 μL/well) for serial dilution with Muller-Hinton II (MHII). One hundred (100 μL) of the bacterial suspension (5 × 105 CFU/mL) was added to each well to obtain a final volume of 200 μL, with a final DMSO concentration of 0.1%. The plates were incubated for 18 hours at 37 °C without agitation with closed lid. The minimal inhibitory concentrations (MICs) of samples were observed after the addition 40 µL of 0.2 mg/mL of iodonitrotetrazolium chloride. MIC values were recorded as the lowest concentration of the sample that totally inhibited bacterial growth. The reference antibiotics used in the present work included chloramphenicol, florfenicol, thiamphenicol, ciprofloxacin, cefepime, and ceftazidime (Sigma-Aldrich). Objectives were to detect molecules that were able to modulate the bacterial permeability of the bacterium in terms of penetration and efflux.

2.5. Combination with Antibiotics

The activity of the various combinations, antibiotics, and extracts of A. senegal and A. seyal were determined by the checkerboard test [37]. Serial dilutions of two antimicrobial agents were mixed so that each row (and column) contained a fixed concentration of the first agent (extract) and an increasing concentration of the second (antibiotics). The concentrations of the extracts were distributed vertically from 1 to 512 mg/L, and the antibiotic was dispatched from 1 to 1024 mg/L horizontally according to the sensibility of each bacteria. Thus, 190 µL of fresh bacterial suspension prepared in MH2 broth (5 × 105 CFU/mL) were added to each well. The first column was used to determine the MIC of the extract alone. The microplates were closed and incubated for 18 h at 37 °C. The MICs of samples were observed after the addition 40 µL of 0.2 mg/mL p-iodonitrotetrazoluim chloride (INT). MIC values were recorded as the lowest concentration of the sample that totally inhibited bacterial growth.

2.6. Outer Membrane Permeation Assay

An overnight culture of K. aerogenes 289 was diluted 100-fold into 10 mL MH2 broth. This strain was used since it normally expresses β-lactamase that is used to monitor outer membrane integrity [38]. After reaching an optical density (OD) of 600 nm of 0.5, cells were recovered by centrifugation (4000× g for 20 min) and washed twice in 20 mM potassium phosphate buffer (PPB pH 7) supplemented with 1 mM MgCl2 to reach an OD of 0.375. Then, 50 μL of each concentration was added to 100 μL of the cell suspension, yielding final concentrations ranging from 256 to 8 μg/mL. Then, 50 μL of nitrocefin was added to obtain a final concentration of 50 μg/mL. Absorbance at 490 nm was monitored by spectrophotometry using an Infinite M200 microplate reader (Tecan) over 60 min at 37 °C. Tazobactam (10 mg/mL) and clavulanic acid (1 mg/mL) were used as inhibitors and PPB as negative controls. Experiments were performed in triplicate. The effect on membrane permeability for extract was determined using the slope in the linear range and compared to the slope obtained with polymyxin B (used at 200 μM according to [37]).

2.7. Statistical Analysis

Quantification results for secondary compounds are expressed as mean ± SEM (n = 3). The analysis was performed using Graph Pad Prism 5 (Graph Pad Software, San Diego, CA, USA) followed by Dunett’s multiple comparison tests. Statistical procedures were performed using a p value less than 0.05.

3. Results

3.1. Phytochemical Screening by HPTLC

Phytochemical screening of (HE) of A. senegal and A. seyal resulted in the chromatoplates (Figure 1). The HPLC chromatographic profile showed blue, green, orange, yellow, and fluorescence spots under ultraviolet 366 nm, characterizing the presence of flavonoids in the HE of two extracts (Figure 1a). In addition, after spraying with the 2% FeCl2 reagent, the chromatoplate presented a much more intense brown spot in the HE of A. seyal compared to the HE of A. senegal in the visible section (Figure 1b). The brown spots were characteristic of the tannins.

3.2. Phytochemical Contents

Determination of phenolic, flavonoids, and tannins contents of HE of A. senegal and A. seyal leaves was performed by spectrophotometric method. Results of phenolic compounds, flavonoids, and tannins contents are presented in Figure 2. The total polyphenol and flavonoid contents of the hydroethanolic extract of A. seyal was 571.30 ± 6.97 mg GAE/g and 140.41 ± 4.01 mg RTE/g, respectively, and showed a very significant difference (p < 0.001) from the extract of A. senegal with respective values of 69.84 ± 3.54 mg GAE/g and 27.32 ± 0.57 mg RTE/g. In addition, the HE from the leaves of A. seyal showed higher percentages of condensed and hydrolyzable tannins (24.72 ± 0.14 and 35.77 ± 0.19, respectively) compared to the HE of A. senegal with percentages of 14.60 ± 0.01 and 3.09 ± 0.02, respectively. A statistical difference of p < 0.001 for condensed tannins and p < 0.01 for hydrolyzable tannins was obtained.

3.3. Antibacterial Effect of the Various Extract

Table 2 presents the MICs for the antibiotics tested in this study. The susceptibility data concerning the extracts showed that almost all of the extracts tested had a weak antibacterial activity (Table 3). The best activity was recorded with hydroethanolic (HE) extract from the leaves of A. senegal, with MIC values ranging from 128 to 512 mg/L. The lowest MIC value (128 mg/L) was recorded with HE from A. senegal leaves against K. aerogenes EA298 and E. coli AG100A, followed by E. coli AG100 and K. aerogenes ATCC 11296 with an MIC of 256 mg/L. The antibacterial activity of a plant extract is considered significant when the MIC values are less than 100 mg/L, moderate when the MIC comprises between 100 < MIC < 625 mg/L, and low when the MIC > 625 mg/L [39]. Interestingly, the activity (MIC of 128 mg/L and 256 mg/L) was observed with HE from A. senegal against Ea298, Ea ATCC 11296, AG100, and AG100A.

3.4. Combination with Different Antibiotics

A preliminary study performed against different strains cited in Table 1 allowed us to select two out of fourteen extracts at the appropriate subinhibitory concentrations for further studies. All the extracts were combined separately to six antibiotics (CIP, CHL, FLOR, THIAM, CEF, and CAZ) to evaluate their possible synergetic effects. Table 4 indicated that HE from the leaves of A. senegal and A. seyal were able to improve the antibacterial activity of the phenicols group against K. aerogenes, K. pneumoniae MDR strains, and E. coli strains. With 128 mg/L of HE leaf extract combined with the chloramphenicol, a significant decrease in the MIC with Ea289 (MIC CHL = 1024 mg/L), Ea CM64 (MIC CHL = 512 mg/L), AG100 (MIC CHL = 8 mg/L), and AG102 (MIC CHL = 64 mg/L) was observed and reached 128, 32, 2, and 4 mg/L, respectively. Significant effects on MIC were also observed when HE from the leaves of A. senegal was combined with florfenicol and thiamphenicol (Table 4). For A. seyal, HE at a concentration of 512 mg/L increased the susceptibility to chloramphenicol of AG100 (MIC = 8), AG102 (MIC = 64 µ/mL), Ea 289 (MIC = 1024 mg/L), and Ea CM64 (MIC = 512 mg/L) to 2, 16, 256, and 128 mg/L, respectively. Interestingly, susceptibility to other antibiotics (CAZ, CIP, and CEF) assayed in this study was not modified at all.

3.5. Effect on Membrane Permeability

The permeability test of the outer membrane was carried out with the most effective HE from the leaves of A. senegal. The nitrocefin hydrolysis was measured in the absence or the presence of an increased amount of extract to detect a direct effect on the membrane permeability. Polymyxin B that permeabilizes the bacterial outer membrane (OM) of Gram-negative bacteria was used as internal control and used as standard to compare the activity of various plants extracts [37,40]. The HE had a weak effect on the OM permeability at low concentrations (8–32 mg/L) compared to polymyxin B. A more significant effect was observed at 64 mg/L (12% of polymyxin effect) and increased at 128 mg/L (Table 5). At 256 mg/L, the OM permeability was seriously altered by HE A. senegal and may explain some antibacterial effects observed for high concentrations.

3.6. Combination Tests with Phenicols

The data in Table 4 identified the effects of plants extract on the susceptibility towards selected antibiotics. The HE from the leaves of A. senegal and A. seyal had interesting adjuvant effects by combining with chloramphenicol and florfenicol, which belong to the phenicol family. The MICs of the combinations indicated that HE from A. senegal has a better adjuvant effect on some strains (AG100, AG102, Ea289, and Ea CM64) than methanolic extract (Table 6). The presence of A. senegal HE (16 mg/L) in combination with chloramphenicol increased the susceptibility, e.g., MICs decreased from 64 to 4 mg/L for the AG102 and the Ea CM64 strains. These values are quite similar to those obtained with florfenicol. With methanolic extract, it was necessary to use 64 mg/L to observe the same effects. The direct MICs values obtained with methanolic extract from the leaves of A. seyal were greater than 512 mg/L (Table 3). The combination of the extract (256 mg/L) with chloramphenicol and florfenicol showed a decrease of the MIC from 512 to 128 mg/L for chloramphenicol and from 256 to 64 mg/L for florfenicol with strain CM64. The results were similar with the AG102 and the AG100 strains (data not shown). The associations of methanolic extracts of A. senegal and A. seyal (at 128 and 256 mg/mL, respectively) with other selected antibiotics (flerofloxacin, erythromycin, norfloxacin, and ciprofloxacin) showed no effect (Table S1).

3.7. FloR Pump and A. senegal HE

In order to define the effect of HE on phenicols transport, we measured the variation of susceptibility conferred by the FloR pump in the AG100A strain [41]. It was interesting to note that HE was able to potentialize the activity of chloramphenicol and florfenicol on the strain overproducing FloR (Table 7) In contrast, no significant modification was detected on the strain carrying only the empty plasmid. Moreover, the MICs to ceftazidime and norfloxacin were not modified at a concentration increasing phenicols susceptibility (Table 7). This suggests that HE was able to mask/protect the phenicols against the expel activity due to FloR pump.

4. Discussion

Phytochemical screening indicated the presence of several classes of secondary metabolites such as polyphenols, flavonoids, and tannins in the HE of A. senegal and A. seyal leaves. In vitro experiments showed that molecules belonging to these classes can be active on pathogenic microorganisms [8]. The results showed that the HE of A. senegal leaves increased the susceptibility of strains producing efflux pumps (Ea289, EaCM64, AG102, and AG100) for two antibiotics (chloramphenicol and florfenicol) with gains between 2 and 64 fold compared to assays performed without extracts. The adjuvant capability of natural extracts or compounds against resistant bacteria were already reported [41,42]. Previous studies have shown that HE from the root wood of A. senegal had a moderate direct activity against selected bacteria [43]. The same authors reported that dichloromethane extract of A. senegal root heartwood exhibited antibacterial activity against E. coli and S. aureus. The hexane fraction of the trunk bark of A. senegal is active against respiratory pathogenic bacteria such as K. pneumonia and Streptococcus pneumoniae. This observation would be due to the presence of tannins, steroids, cardiac glycosides, flavonoids, saponins, and alkaloids in A. senegal [44]. Plant extracts action can induce the alteration of the bacterial membrane facilitating the penetration of antibiotics into the Gram-negative bacteria that increases the intracellular concentration of antibiotics closed to the target [42,45,46]. As described, Epicatechin 3-gallate and caffeic acid disrupt the outer membrane of Gram-negative bacteria and increase the permeability of Pseudomonas aeruginosa [47]. A recent study showed that A. nilotica extracts disrupt the cell wall and the cytoplasmic membrane of Salmonella and E. coli, causing the leakage of cytoplasmic components [48]. Another hypothesis is that some plant-derived compounds may alter the efflux of usual antibiotics [43,49]. HE of A. senegal can permeabilize the OM when used at high amounts, but—interestingly—at low concentrations, it is able to potentialize mainly the phenicol antibacterial activity, suggesting an original synergy with this antibiotic group. Many extracts or compounds from plants have been recognized as efflux pump inhibitors when used as adjuvants in combination with specific antibiotics [43,50]. Flavonoids, polyphenols, terpenoids, and alkaloids potentially interact with antibiotics to improve their action on the target [51]. For example, conessin, a steroidal alkaloid, has been described as a MexAB-OprM efflux pump inhibitor in P. aeruginosa, restoring the antibiotic activity [52]. A chalcone (polyphenol) extracted from Dalea versicolor (Fabaceae) potentiates the action of berberine, erythromycin, and tetracycline on strains of S. aureus overexpressing NorA [53]. A Kaempferol derivative from Persea lingue showed inhibitory activity on the NorA efflux pump of S. aureus and restored ciprofloxacin susceptibility [54]. Carnosol and carnosic acid (diterpenoids) from Rosmarinus officinalis have also shown a modulating effect on erythromycin and tetracycline resistance in mutant strains of S. aureus [55]. In the present study, A. senegal extracts impair the activity of FloR pump without changing the susceptibility of other antibiotics, and this may suggest an affinity for this kind of transporter. This suggests that the extract contains compounds that may disturb the membrane stability and/or a compound targeting a selective efflux activity, thus increasing the antibiotic activity [56]. Importantly, the extract used at the same concentrations in combination showed no effect on other antibiotic groups. This indicates that one or more molecule present in the HE extract may have an affinity for the recognition/binding site of phenicol in the efflux process. This activity is also observed when FloR is overexpressed in an AcrAB minus strain; the extract is able to reverse the resistance level afforded by this selective pump against phenicols without altering other susceptibility. Recent phytochemical work on the leaves of A. senegal indicated the presence of several chemical groups such as sterols and steroids, phenols, alkaloids, flavonoids, leucoanthocyanins, anthocyanins, volatile oils, amino acid proteins, and carbohydrates [16]. Our preliminary study revealed the presence of flavonoids, phenolic, and tannins in the leaves of Acacia senegal and corroborates with previous plant studies. The presence of such phytochemical compounds in the extract of A. senegal can explain the antibacterial activities reported here. Phenicols are broad-spectrum drugs exhibiting a relative toxicity that seriously compromises its use in human medicine. For instance, in meningitis caused by Haemophilus influenzae or Neisseria meningitidis (in cases of penicillin allergy, for example), it diffuses easily into blood and tissues [57,58]. Combination with HE from the leaves of A. senegal could be an alternative to both restoring bacterial susceptibility to phenicols and to reducing the dose required for treatment, thus decreasing a hazard for adverse side effects.

5. Conclusions

This study explored the efficacy of the HE of A. senegal alone and in combination with antibiotics. Synergistic effects indicated that antibacterial combinations were more effective on characterized resistant strains. Many teams are looking for molecules that can inhibit the efflux pump. The tested combination of natural products with chloramphenicol and florfenicol pave the way for the development of efficient agents active against MDR bacteria. The discovery of natural compounds that impair the membrane-associated mechanisms of resistance in Gram-negative bacteria will increase the chances to combat resistant pathogens and stimulate the activity of usual antibiotics at low concentrations.

Ethics Approval and Consent to Participate

This collaborative work was performed in accordance with the Convention on Biological Diversity and the Nagoya protocol and compliance with laws in force in Burkina Faso. Provider (IRSS/CNRST) and users (U1261, UMR_MD1, France) have mutually agreed for equitable sharing benefits in case derivatives arising from this work will be patented.

Supplementary Materials

The following are available online at https://www.mdpi.com/2079-6382/9/6/323/s1, Table S1: Antibiotic resistance modulatory activity of methanolic extract of Acacia senegal leaves (μg /mL).

Author Contributions

R.D.M.: microbiologic and chemical experiments, writing and revising the manuscript; A.D.-R.: bacteriological methodology, writing, validation, and review; A.H.: review, supervision; H.M.-K.: conceptualization, chemical methodology, validation; J.-M.P.: investigation, coordination, and supported resources. All authors have read and agreed to the published version of the manuscript.

Funding

We thank Campus France and the French Embassy for their funding support.

Acknowledgments

We thank JM Brunel, J Vergalli and M Koala, who spent the countless hours helping us conduct this study.

Conflicts of Interest

The authors declare that they have no conflicts interests.

Abbreviations

DMSO: dimethyl sulfoxyde; HE: hydroethanolic extract; LB: Luria Bertani: MDR: multi drug resistant; MH: Mueller Hinton; MIC: minimal inhibitory concentration; OM: outer membrane. FLOR: florfenicol; CHL: chloramphenicol; CAZ: ceftazidime; CEF: cefepime; THIAM: thiamphenicol; CIP: ciprofloxacin; ATBs: antibiotics.

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Antibiotics 09 00323 g001 550
Figure 1. (a) Presence of flavonoids sprayed by Neu’s reagent at UV 365 nm (orange, yellow and green spots); (b) tannins sprayed with FeCl3 2% (blue-black, brown spots); ASY: hydroethanolic leaves of Acacia seyal; ASG: hydroethanolic leaves of Acacia senegal.
Figure 1. (a) Presence of flavonoids sprayed by Neu’s reagent at UV 365 nm (orange, yellow and green spots); (b) tannins sprayed with FeCl3 2% (blue-black, brown spots); ASY: hydroethanolic leaves of Acacia seyal; ASG: hydroethanolic leaves of Acacia senegal.
Antibiotics 09 00323 g001
Antibiotics 09 00323 g002 550
Figure 2. Phytochemical content of A. senegal and A. seyal. TP: Total phenols (exprimed in mg GAE/g; TF: Total flavonoids (exprimed in mg RTE/g); HT: Hydrolysable tannins; CT: Condensed tannins (exprimed in %). ** indicates statistically significant difference in values (p < 0.01); *** indicates statistically significant difference in values (p < 0.001).
Figure 2. Phytochemical content of A. senegal and A. seyal. TP: Total phenols (exprimed in mg GAE/g; TF: Total flavonoids (exprimed in mg RTE/g); HT: Hydrolysable tannins; CT: Condensed tannins (exprimed in %). ** indicates statistically significant difference in values (p < 0.01); *** indicates statistically significant difference in values (p < 0.001).
Antibiotics 09 00323 g002
Table
Table 1. Bacterial strains used in this study.
Table 1. Bacterial strains used in this study.
StrainsDescriptionReference
E. coli
AG100Parental E. coli K-12 Porin +; basal efflux[29]
AG100AAG100 acrAB::Kanr non (AcrAB-); Porin +[29]
AG102AG100 overexpressing the AcrAB pump, Porin +[29]
AG1004 plasmid
AG100A FloR		wid plasmid without floR
Expressing floR		[30]
[30]
K. aerogenes
Ea CM64CHL variant obtained from ATCC 13048 overexpressing the AcrAB pump; Porin+[31]
Ea 289KAN-sensitive derivative of EA27, Porin-[32]
Ea 298EA289 tolC::Kanr; Porin-[32]
EaATCC 15038Porin +; Normal efflux[33]
K. pneumoniae
ATCC 12296Reference strain; Porin +; Normal efflux[34]
KP 45Porin +; Normal efflux[35]
KP55Clinical MDR isolate; Tetr Ampr Atmr Cefr; Porin-; Normal efflux[35]
Table
Table 2. Minimal inhibitory concentrations (MICs, mg/L) of the different antibiotics against the tested bacterial species MIC.
Table 2. Minimal inhibitory concentrations (MICs, mg/L) of the different antibiotics against the tested bacterial species MIC.
AntibioticsE. coli K. aerogenesK. pneumoniae
AG100AG100AAG102Ea 289Ea298Ea ATCC 15038CM64KP45KP55KPATCC 11296
Chloramphenicol816410243245128168
Florfenicol161128256485128816
Thiamphenicol256161024>1024>102464>1024128256256
Ciprofloxacin0.030.0080.25324<0.1250.50.0640.06
Cefepime<0.125<0.1250.2528<0.1250.25<0.12516<0.125
Ceftazidime0.50.252>64>640.0320.25>640.5
Table
Table 3. Minimal inhibitory concentrations (MICs, mg/L) of the different extracts against the tested bacterial species.
Table 3. Minimal inhibitory concentrations (MICs, mg/L) of the different extracts against the tested bacterial species.
Plant ExtractE. coliK. aerogenesK. Pneumoniae
AG100AG100AAG102Ea 289Ea298Ea ATCC 15038CM64KP45KP55KP ATCC 11296
Soxhlet extraction
Acacia senegal(leaves)
Hexane extract>512>512>512>512>512>512>512>512>512>512
Dichloromethane extract>512>512>512>512>512>512>512>512>512>512
Ethyl acetate extract>512>512>512>512>512>512>512>512>512>512
Methanolic extract>512>512>512>512>512>512>512>512>512>512
Acacia seyal(leaves)
Hexane extract>512>512>512>512>512>512>512>512>512>512
Dichloromethane extract>512>512>512>512>512>512>512>512>512>512
Ethyl acetate extract>512>512>512>512>512>512>512>512>512>512
Methanolic extract >512>512>512>512>512>512>512>512>512>512
Cold-Maceration
Acacia senegal
Hydroethanolic extract (leaves) 256128>512>512128256>512>512512>512
Hydroethanolic (Root Bark)>512>512>512>512>512>512>512>512>512>512
Acacia seyal
hydroethanolic (Leaves)>512>512>512>512>512>512>512>512>512>512
Hydroethanolic (Root Bark)>512>512>512>512>512>512>512>512>512>512
Aqueous
Acacia senegal
Root bark>512>512>512>512>512>512>512>512>512>512
Acacia seyal
Root bark>512>512>512>512>512>512>512>512>512>512
ASGMOH: Methanolic extract of Acacia senegal leaves; ASYMOH: Methanolic extract of Acacia seyal leaves, ASGHE: Hydroethanolic extract of Acacia senegal leaves.
Table
Table 4. MIC of antibiotics associated to extracts at various concentrations.
Table 4. MIC of antibiotics associated to extracts at various concentrations.
E. coliK. pneumoniaeK. aerogenes
PlantsExtractsATBsExtracts (mg/L)AG100AG100AAG102KP45KP55KP ATCC 11296Ea289Ea298Ea ATCC 15038Ea CM64
A. senegalDecoction root barkCHL0816481681024324512
512816481681024644512
1281616481681024324512
161616481681024324512
CIP00.030.0080.250.0640.06644<0.1250.5
5120.030.0080.250.0340.06648<0.1251
1280.030.0080.250.0640.06644<0.1251
160.030.008ndnd4Nd644<0.1250.5
CAZ00.50.2520.25>640.25>64>640.032
51210.520.5>640.25>64>640.0.32
1280.50.2520.5>640.25>64>640.032
161<0.125ndnd>64nd>64>64>22
Hydroethanolic root barkCHL0816481681024324512
512 816481681024324512
128816481681024324512
168164816161024324512
CIP00.030.0080.250.0640.06644<0.1250.5
5120.030.0080.250.12540.06644<0.1250.5
1280.030.0080.250.0640.125644<0.1250.5
160.030.0080.250.0640.06644<0.1250.5
CEF00.06<0.030.5<0.12516<0.12528<0.1250.25
5120.06<0.030.50.58<0.12548<0.1250.25
1280.06<0.030.50.068<0.125216<0.1250.25
160.06<0.030.50.068<0.125216<0.1250.25
hydroethanolic leaves extractCHL0816481681024324512
5121<0.1252212644<216
12821424212816<232
16826481641024324512
CIP00.030.0080.250.0640.06644<0.1250.5
5120.03<0.0080.1250.0610.06641<0.125<0.125
1280.03<0.0080.250.0620.06642<0.125<0.125
160.030.0080.250.0640.03642<0.1250.5
CAZ00.50.2520.25>640.25>64>640.032
5120.25<0.1250.50.5>642>64>640.031
1280.50.2510.5>641>64>640.032
160.250.2510.25>640.5 >64>640.032
FLOR0161128881625648512
5120.5<0.125422280.25232
12820.58422161232
1616112888825628256
THIAM0256161024128256256>1024>102464>1024
5128<0.1251281286464>1024464512
128128425612864128>102410241281024
16128161024128128256>1024>1024>1024>1024
A. seyalDecoction root barkCHL0816481681024324512
512813281621024324256
128816481681024324512
16816416>641610243241024
CIP00.060.0080.250.0640.06644<0.1250.5
5120.060.0080.250.0640.06648<0.1250.5
1280.060.0080.50.0640.06644<0.1250.5
160.060.0080.250.580.06644<0.1250.5
CAZ00.50.2520.25>640.25>64>640.032
5120.5 0.522>640.125>64>640.032
1280.50.521>640.5>64>640.032
1610.2521>640.5>64>640.064
Hydroethanolic root barkCHL0816481681024324512
1288<0.1256481681024324512
1616<0.1256481681024648512
180.25648168102432512512
CIP00.030.0080.250.0640.06644<0.1250.5
1280.030.0080.250.0640.06644<0.1250.5
160.030.0080.250.0640.06642<0.1250.5
10.030.0080.50.0640.06642<0.1250.5
CEF0<0.125<0.1250.25280.125216<0.1250.25
128<0.125<0.1250.50.12580.12548<0.1250.25
16<0.125<0.1250.250.12580.12548<0.1250.25
10.06<0.1250.50.25160.125416<0.1250.25
Hydroethanolic leaves extractCHL0816481681024324512
5122116444256642128
1284132416810241284512
168164416810241284512
CIP00.030.0080.250.0640.06644<0.1250.5
5120.030.0160.250.0680.06>644<0.1250.5
1280.030.0080.1250.0640.125648<0.1250.5
160.030.0080.1250.0640.125644<0.1250.5
CAZ00.50.2520.25>640.25>64>640.032
5120.50.250.250.25>640.5>64>640.031
1280.50.2510.25>640.5>64>640.032
160.50.250.50.25>640.25>64>640.061
FLOR: florfenicol; CHL: chloramphenicol; CAZ: ceftazidime; CEF: cefepime; THIAM: thiamphenicol; CIP: ciprofloxacin; ATBS: antibiotics.
Table
Table 5. Effect of A. senegal hydroethanolic extract on bacterial outer membrane of K. aerogenes 289 expressed as percentage of polymyxin B activity.
Table 5. Effect of A. senegal hydroethanolic extract on bacterial outer membrane of K. aerogenes 289 expressed as percentage of polymyxin B activity.
Permeating Effect
Concentration extract (mg/L)2561286432168
% of polymyxin B50 (±18 *)31 (±13 *)12 (±4 *)7.3 (±2 *)2 (±1 *)2 (±1 *)
* The interval was calculated from three independent tests performed in triplicates.
Table
Table 6. Adjuvant activity of leaves extract of A. senegal, MIC in mg/L (Gain). Bacteria strains, MIC (mg/L) of antibiotics in the presence and absence of the extract.
Table 6. Adjuvant activity of leaves extract of A. senegal, MIC in mg/L (Gain). Bacteria strains, MIC (mg/L) of antibiotics in the presence and absence of the extract.
Hydroethanolic Extract of Acacia Senegal (mg/L)Methanolic Extract Acacia Senegal (mg/L)
ATBStrainMIC ATBMIC ASG8163264MIC ASG8163264
CHLE. coli
AG10082568 (-)4 (2)2 (4)1 (8)>25616 (-)8 (-)4 (2)2 (4)
AG100A11281 (-)2 (-)1 (-)0.5(2)>2561 (-)1 (-)1 (-)1 (-)
AG10264>25664 (-)16 (4)4 (16)4 (16) >25664 (-)64 (-)32 (2)8 (8)
K. aerogenes
Ea2891024>2561024 (-)512 (2)64 (16)64 (16)>2561024 (-)1024 (-)512 (2)128 (8)
Ea2983212832 (-)32 (-)16 (2)4 (8)>25632 (-)32 (-)32 (-)16 (2)
Ea ATCC 15038 42564 (-)2 (2)1 (4)1 (4)>2564 (-)4 (-)2 (2)2 (2)
Ea CM64512>256512 (-)128 (4)16 (32)16 (32)>256512 (-)256 (2)256 (2)32 (16)
FLORE. coli
AG100162568 (2)4 (4)2 (8)2 (8)>25632 (-)16 (-)8 (2)4 (4)
AG100A11281 (-)1 (-)1 (-)0.25 (4)>2561 (-)1 (-)1 (-)1 (-)
AG10264>256128 (-)32 (2)8 (8)4 (16)>25664 (-)64 (-)32 (2)16 (4)
K. aerogenes
Ea289128>25632 (4)64 (2)16 (8)8 (16)>256128 (-)64 (2)64 (2)32 (4)
Ea29841284 (-)2 (2)2 (2)1 (4)>2564 (-)4 (-)4 (-)2 (2)
Ea ATCC 1503842564 (-)2 (2)2 (2)2 (2)>2564(-)4 (-)4 (-)2 (2)
Ea CM64256>256256 (-)128 (2)16 (16)16 (16)>256256 (-)256 (-)64 (4)32 (8)
MIC: Minimal inhibitory concentration, ATB: Antibiotic, ASG: A. senegal, CHL: chloramphenicol, FLOR: florfenicol.
Table
Table 7. FloR expression and phenicols susceptibility in absence or in presence of HE extract.
Table 7. FloR expression and phenicols susceptibility in absence or in presence of HE extract.
ATBsStrainMIC (mg/L)Conc. HE Acacia senegal (mg/L)
E. coliATBsASG102030405064
CHLAG100 A2128222222
Plasmid 1128111111
FloR8128844221
FLORAG100 A1128111111
Plasmid 1128111111
FloR4 - 2128441110.5
THIAMAG100 A161281688844
Plasmid 161281688844
FloR512128256256128646432
CAZAG100 A0.251280.250.250.250.250.250.25
Plasmid 0.251280.250.250.250.250.250.25
FloR0.251280.250.250.250.250.250.125
NORAG100 A0.031280.030.030.030.030.030.03
Plasmid 0.0161280.0160.0160.0160.0160.0160.016
FloR0.0161280.0160.0160.0160.0160.0160.016
MIC: Minimal inhibitory concentration, ATB: antibiotic, ASG: A. senegal, CHL: chloramphenicol, FLOR: florfenicol, THIAM: thiamphenicol, CAZ: ceftazidime, NOR: norfloxacin.

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MDPI and ACS Style

Magnini, R.D.; Hilou, A.; Millogo-Koné, H.; Pagès, J.-M.; Davin-Regli, A. Acacia senegal Extract Rejuvenates the Activity of Phenicols on Selected Enterobacteriaceae Multi Drug Resistant Strains. Antibiotics 2020, 9, 323. https://doi.org/10.3390/antibiotics9060323

AMA Style

Magnini RD, Hilou A, Millogo-Koné H, Pagès J-M, Davin-Regli A. Acacia senegal Extract Rejuvenates the Activity of Phenicols on Selected Enterobacteriaceae Multi Drug Resistant Strains. Antibiotics. 2020; 9(6):323. https://doi.org/10.3390/antibiotics9060323

Chicago/Turabian Style

Magnini, René Dofini, Adama Hilou, Helana Millogo-Koné, Jean-Marie Pagès, and Anne Davin-Regli. 2020. "Acacia senegal Extract Rejuvenates the Activity of Phenicols on Selected Enterobacteriaceae Multi Drug Resistant Strains" Antibiotics 9, no. 6: 323. https://doi.org/10.3390/antibiotics9060323

APA Style

Magnini, R. D., Hilou, A., Millogo-Koné, H., Pagès, J. -M., & Davin-Regli, A. (2020). Acacia senegal Extract Rejuvenates the Activity of Phenicols on Selected Enterobacteriaceae Multi Drug Resistant Strains. Antibiotics, 9(6), 323. https://doi.org/10.3390/antibiotics9060323

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