Antibacterial and Antibiotic-Modifying Activity of Methanol Extracts from Six Cameroonian Food Plants against Multidrug-Resistant Enteric Bacteria.

The present work was designed to investigate the antibacterial activities of methanol extracts from six Cameroonian edible plants and their synergistic effects with some commonly used antibiotics against multidrug-resistant (MDR) Gram-negative bacteria expressing active efflux pumps. The extracts were subjected to qualitative phytochemical screening and the microdilution broth method was used for antibacterial assays. The results of phytochemical tests indicate that all tested crude extracts contained polyphenols, flavonoids, triterpenes, and steroids. Extracts displayed selective antibacterial activities with the minimal inhibitory concentration (MIC) values ranging from 32 to 1024 μg/mL. The lowest MIC value (32 μg/mL) was recorded with Coula edulis extract against E. coli AG102 and K. pneumoniae K2 and with Mangifera indica bark extract against P. aeruginosa PA01 and Citrus sinensis extract against E. coli W3110 which also displayed the best MBC (256 μg/mL) value against E. coli ATCC8739. In combination with antibiotics, extracts from M. indica leaves showed synergistic effects with 75% (6/8) of the tested antibiotics against more than 80% of the tested bacteria. The findings of the present work indicate that the tested plants may be used alone or in combination in the treatment of bacterial infections including the multidrug-resistant bacteria.

1. Introduction

The discovery of antibiotics has inevitably fostered the development and emergence of resistances in bacteria, regardless of the mechanism of action of the antibiotic involved. For decades, and in order to reduce the development and epidemic spread of resistance, physicians and scientists have called for appropriate use of antibiotics [1]. Prudent use of antibiotics in humans demands that physicians establish that a bacterial infection is responsible for the patient's symptoms before an antibiotic prescription is made. By contrast, in agriculture, antibiotics are used in the absence of acute infection [2]. Bacteria faced with this fierce struggle against them have developed several mechanisms of resistance against antimicrobial agents whose main ones include enzymatic inactivation [3], modification of the drug target(s), and reduction of intracellular drug concentration by changes in membrane permeability or by the overexpression of efflux pumps [4]. With respect to efflux pumps, they provide a self-defense mechanism by which antibiotics are actively removed from the cell. For antibacterials, this results in sublethal drug concentrations at the active site that in turn may predispose the organism to the development of high-level target-based resistance [5]. In this way, efflux pumps inevitably become targets for the research and/or development of new, less toxic, and effective molecules capable, alone or in combination with the usual antibiotics, of effectively fighting infections involving multidrug-resistant pathogens. Medicinal plants in general and food plants in particular have been used for centuries to cure ailments of man. Today, there is a real revival of interest in these almost-exploited sources [6] of molecules whose pharmacological efficacy is no longer to be demonstrated [7]. The present work was designed to investigate the in vitro ability of methanol extracts from six Cameroonian edible plants (Psidium guajava Linn., Persea americana Mill., Camellia sinensis Linn., Mangifera indica Linn., Coula edulis Baill., and Citrus sinensis Linn.), to potentiate the activity of some commonly used antibiotics vis-à-vis Gram-negative multidrug-resistant bacteria.

2. Material and Methods

2.1. Plant Material and Extraction

The plant materials used in this work were collected in the period of March to April 2015 in two regions of Cameroon and included leaves and bark of pan class="Species">M. indica; fruits, leaves, and bark of span>n class="Species">P. guajava; leaves and bark of P. americana collected at Koung-Khi division (West Region); leaves of C. sinensis and the fruits of C. sinensis collected at Menoua division (West Region); and nuts of C. edulis collected at Mungo division (Littoral Region). 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 cleaned and air-dried and the powder (300 g) was extracted with methanol (MeOH, 1 L) for 48 h at room temperature. The extract was then concentrated under reduced pressure to give residues which constituted the crude extract. All extracts were then kept at 4°C until further use.

Table 1

Plants used in the present study and evidence of their bioactivities.

Species (family);voucher number∗	Traditional uses	Parts used traditionally	Bioactive or potentially bioactive components	Bioactivity of crude extract
Psidium guajava Linn. (Myrtaceae);2884/SRF/Cam	To treat wounds, lesions, ulcers, diarrhea, cholera, hypertension, and obesity and the control of diabetes mellitus [8].Anti-inflammatory effect, cough, diabetes, kidney problems, diarrhea, tonic, laxative, antihelminthic [9], conjunctivitis, oral care, and antispasmodic [10].	Leaves, fruits, flowers, bark, and roots	Tannins, flavonoids (myricetin, quercetin, luteolin and kaempferol), essential oils (caryophyllene, nerolidol, β-bisabolene, aromadendrene, p-selinene, α-pinene and 1,8-cineol), triterpenoids (oleanic acid, ursolic acid, catecholic acid, guayavolic acid, maslinic acid, ellagic acid) and β-sitosterol [11]; essential oil: propylbenzene, allyl benzene, and so forth [12].	Antibacterial activity of essential oil: Sa, Sf, Lspp, Esa, Aspp, Ec, Pv, Ea, St, Pa, andKp [12]; antioxidant activity of leaves essential oil [13]. Ethanolic extract of leaves: Ec, Kp, Pa, Sa, Ab, MRSA, and VRE [14]; antiproliferative, antiseptic and antifungal activity [9]; ethanolic extract of leaves: antioxidant, antitumor, and antibacterial (against Sm, Smt, and Sor) [15].
Persea americana Mill.(Lauraceae);57756/HNC	Anticancer activity, the food industry has grown tremendous interest in processing this crop and enhancing its value [16]. P. americana leaves' extracts have been used as analgesic, anti-inflammatory, hypoglycaemic, anticonvulsant, antidiabetic, and vasorelaxant [17].	Leaves, fruit, flowers, bark, and roots	Crude leaves extracts: glycosides, alkaloids, tannins, saponins, flavonoids, terpenoids, and steroids [18].	Methanol, chloroform, ethyl acetate, and petroleum ether leaves extracts were tested against Ec, St, Sa, Ca, Pa,and Bs; antioxidant activity [18]; aqueous leaves' extract: anti-inflammatory and analgesic activity [17].
Citrus sinensis Linn.(Rutaceae); 25859/HNC	Treat ailments like constipation, cramps, colic, diarrhea, bronchitis, tuberculosis, cough, cold, obesity, menstrual disorder, angina, hypertension, anxiety, depression, and stress [19]; used to soothe sore throats, indigestion, relieve intestinal gas and bloating and resolve phlegm and as an additive for flavoring to our foods [20].	Leaves, flowers, seeds, peel, fruits, and barks	Polyphenols (caffeic acid, p-coumaric acid, ferulic acid, and sinapinic acid), flavonoids (hesperidin, narirutin, naringin, eriocitrin) [21], essential oil composition: D-limonene, β-myrcene, α-pinene, β-pinene, γ-terpinene, α-terpinolene, α-caryophyllene, copaene, β-phellandrene [22] and many other constituents.	Ethanolic extracts of peels and juice (Q): Sta, Sau, Sm, Ss,Kp, and Ec [20]; ethanolic extracts of peels: Sta, Pv, Kp, Ec, Bs, and Pa [23]; crude extract of juice (Q): Ec, Sta, and MRSA [24];  crude extracts from different parts of the plants reduce high blood pressure, respiratory problems, rheumatism [25, 26], and anticancer, antimicrobial, and antioxidant activity [22].
Coula edulis Baill.(Olacaceae);African walnut22931 SRF/Cam	The stomachic bark decoction is used to treat dysentery in Liberia and the bark powder decoction is used in equatorial Africa to stimulate appetite and counteract anemia [27].	Leaves, stem bark, fruits, and roots	Ethanolic extract of leaves, stem bark and roots: alkaloids, flavonoids, tannins, saponins, terpenes, and cardiac glycosides [27].	Ethanolic extracts of bark and leaves: Pa, Sa, Ec, St, and Ca [27]. Methanolic extract of the bark: antiplasmodial activity [28].
Mangifera indica Linn.(Anacardiaceae);18646/HNC	Anti-syphilitic, vulnerary, antiemetic, anti-inflammatory, cough, hiccup, hyperdipsia, burning sensation, hemorrhages, haemoptysis, hemorrhoids, wounds, ulcers, diarrhoea, dysentery, pharyngopathy, scorpion string, wounds, ulcers, anorexia, and dyspepsia [29].	Leaves, flowers, barks, roots, stones, and fruits	Carotenoids (provitamin A compound, beta-carotene, lutein, and alpha-carotene), polyphenols [30] such as quercetin, kaempferol, gallic acid, caffeic acid, catechins, tannins, and mangiferin [31].	Mangiferin: anticancer (breast, renal, colon and leukemia cancer cell lines [32, 33]; antidiabetic [34]; antimalarial [35]; methanol and aqueous extracts: Sa, Sp, Pa, Ca, and Ef [36]; Se, Lm, Ec [37]; ethanol and benzene extracts: Pv, Pf, Sfx, Kp, and St [38]; antiamoebic [39].
Camellia sinensis Linn.(Theaceae);43103/HNC	Aiding digestion, blood purification, ensuring regularity, lowering body temperature, strengthening teeth and bones, boosting immune system, enhancing heart function, suppressing aging, deterring food poisoning, fighting virus, and lowering blood sugar levels [40].	Leaves, stem, bark, and roots	Polyphenols (catechins and their derivatives), methylxanthines (caffeine, theophylline and theobromine), carbohydrates, proteins, free amino acids, vitamins (vitamin C and carotenoids), volatile compounds, lipids, chlorophylls, saponins and inorganic elements (fluorine, manganese and aluminum) [40, 41].	Methanolic extract of leaves: antioxidant activity. These antioxidant properties are beneficial for several chronic diseases related to oxidative stress, including cancer and cardiovascular and neurodegenerative diseases [42].

HNC: Herbier National du Cameroun; SRF/Cam: Société des Réserves Forestières du Cameroun; Sm: Streptococcus mutans; Smt: Streptococcus mitis; Sor: Streptococcus oralis; Sta: Staphylococcus aureus; Sau: Staphylococcus auricularis; Ss: Streptococcus salivarius; Sp: Streptococcus pneumoniae; Kp: Klebsiella pneumoniae; Sa: Streptococcus aureus; Ec: Escherichia coli; Se: Salmonella enteritidis; Bs: Bacillus subtilis; St: Salmonella typhi; Ef: Enterococcus faecalis; Sf: Staphylococcus faecalis; Pv: Proteus vulgaris; MRSA: methicillin-resistant Staphylococcus aureus; Pf: Pseudomonas fluorescens; Sfx: Shigella flexneri; Pa: Pseudomonas aeruginosa; Ca: Candida albicans; Ea: Enterobacter aerogenes; Asp: Acinetobacter spp.; VRE:vancomycin-resistant Enterococcus; Lspp: Lactobacillus spp.; Ab: Acinetobacter baumannii; Esa: Enterococcus aerogenes; Lm: Listeria monocytogenes.

2.2. Preliminary Phytochemical Investigations

The major phytochemical classes such as pan class="Chemical">alkaloids (Dspan>n class="Chemical">ragendorff's and Mayer's tests), triterpenes (Liebermann-Burchard's test), flavonoids (aluminum chloride test), anthraquinones (Borntrager's test), polyphenols (ferric chloride test), sterols (Salkowski's test), coumarins (lactone test), saponins (foam test), and tannins (gelatin test) (Table 2) were investigated according to the commonly described phytochemical methods [43, 44].

Table 2

Extraction yields, aspects, and phytochemical composition of the plant extracts.

Extracts	Psidium guajava			Persea americana		Citrus sinensis	Coula edulis	Mangifera indica		Camellia sinensis
Parts used	Leaves	Bark	Fruits	Leaves	Bark	Peel	Fruits	Leaves	Bark	Leaves
Yield∗ (%)	5.47	4.17	6.12	5.15	4.47	6.56	7.25	5.36	4.26	5.8
Alkaloids	+	+	−	+	+	+	+	+	+	+
Polyphenols	+	+	+	+	+	+	+	+	+	+
Flavonoids	+	+	+	+	+	+	+	+	+	+
Anthraquinones	−	−	−	+	+	−	+	+	+	−
Coumarins	+	+	+	+	+	+	+	+	+	−
Tannins	+	+	−	+	+	+	+	+	+	+
Triterpenes	+	+	+	+	+	+	+	+	+	+
Sterols	+	+	+	+	+	+	+	+	+	+
Saponins	+	+	−	+	+	+	+	+	+	+

−: absent; +: present; yield calculated as the ratio of the mass of the obtained methanol extract/mass of the plant powder.

2.3. Bacteria Strains and Culture Media

The studied bacteria strains included reference (from American Type Culture Collection) and clinical (Labospan>n class="Species">ratory collection) strains of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Providencia stuartii, and Pseudomonas aeruginosa. Their bacterial features were previously reported [45, 46]. They were maintained on agar slant at 4°C and subcultured on a fresh appropriate agar plates 24 h prior to any antimicrobial test. Mueller-Hinton Agar (MHA) was used for the activation of bacteria, while Mueller-Hinton Broth (MHB) was used for the MIC determinations.

2.4. Chemicals for Antimicrobial Assays

The reference antibiotics used in the present work included pan class="Chemical">tetracycline (span>n class="Chemical">TET), cefepime (CEP), ciprofloxacin (CIP), streptomycin (STP), chloramphenicol (CHL), ampicillin (AMP), erythromycin (ERY), and kanamycin (KAN), all provided by Sigma-Aldrich, St Quentin Fallavier, France. p-Iodonitrotetrazolium (INT) chloride and phenylalanine-arginine-ß-naphthylamide (PAßN) were used as microbial growth indicator and efflux pumps inhibitor (EPI), respectively [47].

2.5. Bacterial Susceptibility Determination

pan class="Chemical">Rapid INT colorimetric assay [47] with some modifications [48] was used to determine the minimal inhibitory concentspan>n class="Species">rations (MIC) of the plant extracts and those of reference antibiotics (RA) against the studied bacteria. The test samples and RA were first of all dissolved in DMSO/Mueller-Hinton Broth (MHB). The final concentration of DMSO was lower than 2.5% and did not affect the microbial growth [49]. The solution obtained was then added to Mueller-Hinton Broth and serially diluted twofold (in a 96-well microplate). One hundred microliters (100 μL) of inoculum 1.5 × 106 CFU/mL prepared in appropriate broth was then added [48, 50]. 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 pink. MIC was defined as the sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth [47]. 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 [50].

The role of efflux pumps in the suspan class="Chemical">ceptibility of Gspan>n class="Chemical">ram-negative bacteria to M. indica, C. edulis, C. sinensis, leaves from P. guajava, P. americana, and C. sinensis, and crude extracts was evaluated by testing them in the presence of an efflux pump inhibitor (EPI), the PAβN (at 30 μg/mL) against fifteen selected MDR phenotypes (E. coli AG100A, AG102, and AG100ATet, E. aerogenes EA289, EA27, EA298, and CM64, K. pneumoniae KP55 and KP63, E. cloacae BM47, BM67, and ECCI69, P. aeruginosa PA124, and P. stuartii NAE16 and PS2636).

To evaluate the potentiating effect of tested extpan class="Chemical">racts, a preliminary combination at their subinhibitory concentspan>n class="Species">rations (MIC/2, MIC/4, MIC/8, and MIC/16) with antibiotics was assessed against P. aeruginosa PA124 strain (see Table S1 in Supplementary Material available online at https://doi.org/10.1155/2017/1583510). The appropriate subinhibitory concentrations were then selected on the basis of their ability to improve the activity of the maximum antibiotic [51, 52]. These subinhibitory concentrations for selected extracts were further tested in combination with antibiotics against more MDR bacteria. The fractional inhibitory concentration (FIC) of each combination was then calculated as the ratio of MIC of antibiotic in combination versus MIC of antibiotic alone (MICAntibiotic  in  combination/MICAntibiotic  alone) and the interpretation made was as follows: synergistic (≤0.5), indifferent (1 to 4), or antagonistic (>4) [53, 54]. All assays were performed in triplicate.

3. Results

3.1. Phytochemical Composition of Plant's Extracts

The results of the phytochemical screening (Table 2) showed that all the tested plant's extpan class="Chemical">racts contain span>n class="Chemical">polyphenols, flavonoids, triterpenes, and sterols. The other classes of secondary metabolites were selectively distributed. Also, the extract from the leaves and bark of P. americana as well as those of M. indica and the extract from C. edulis nuts contain all the classes of screened secondary metabolites.

3.2. Antibacterial Activity of the Extracts

The antibacterial activities of plants' extpan class="Chemical">racts alone and in some cases, in the presence of the span>n class="Chemical">PAβN on a panel of 29 Gram-negative bacteria, was investigated (Table 3). It appears that P. guajava leaves extract inhibited the growth of all (100%) the tested bacteria, and the recorded MICs ranged from 256 to 1024 μg/ml. Other samples showed very selective activity; their inhibitory activities were recorded on 26/29 (89.65%; P. guajava bark and P. americana leaves and bark), 25/29 (82.75%; C. sinensis), 23/29 (79.31%; C. edulis), 22/29 (75.86%; C. sinensis), 21/29 (72.41%; M. indica leaves and bark), and 11/29 (37.93%; P. guajava fruits). The lowest MIC value of 32 μg/mL was recorded with the crude extract of C. edulis against E. coli AG102 and K. pneumoniae K2, as well as with the extract from bark of M. indica against P. aeruginosa PA01 and the extract of C. sinensis against E. coli W3110. Extract from C. sinensis displayed the best MBC (256 μg/mL) value against E. coli ATCC8739.

3.3. Role of Efflux Pumps in the Susceptibility of Gram-Negative Bacteria to the Tested Plant Extracts

Fifteen of the studied MDR bacteria were also tested for their suspan class="Chemical">ceptibility to plant extspan>n class="Chemical">racts in the presence of the PAβN at 30 μg/ml. The results showed that when combined with extracts, PAβN improves the activity (decrease of MIC values) of C. sinensis on 11/15 (73.33%) tested MDR strains. EPI also improved the activity of other extracts against the selected bacteria (Table 3).

3.4. Effects of the Association of Extracts with Antibiotics

A preliminary study performed against pan class="Species">P. aeruginosa span>n class="Chemical">PA124 allowed us to select six out of ten extracts at the appropriate subinhibitory concentrations of MIC/2 and MIC/4 for further studies. All the six extracts were combined separately to eight antibiotics (CIP, STP, CHL, ERY, KAN, TET, CEF, and AMP) to evaluate their possible synergetic effects. The results summarized in Tables 4–9 showed that the synergic effects were noted with all the tested extracts with most of the tested antibiotics except β-lactams (CEF and AMP). At MIC/2, the extract from M. indica leaves showed synergistic effects with 7 of the 8 antibiotics (CIP, STP, CHL, ERY, KAN, TET, and AMP) (87.5%) against at least 70% MDR bacteria for each antibiotic. At that same MIC, C. sinensis extract improved the activity of all (8/8) tested antibiotics against at least 50% of the tested MDR bacteria. Synergies with FIC ranging from 0.5 to <0.007 were noted with the associations of each of the seven extracts with antibiotics. These low FIC values obtained in the majority of cases (FIC < 0.007 for associations of M. indica + β-lactams vis-à-vis almost all the bacterial strains and also with many other extracts with streptomycin against many bacterial strains) indicate that extracts have potentiated the activity of antibiotics vis-à-vis the studied bacteria.

Table 4

MIC of different antibiotics after the association of the extract of Citrus sinensis at MIC/2, MIC/4 against ten MDR bacteria strains.

Antibioticsa	Bacterial strains and MIC (μg/mL) of antibiotics in the absence and presence of the extractb
	Extractconcentration	PA124	AG102	AG100Atet	EA289	CM64	KP55	KP63	NEA16	PS2636	BM47	PBSSc (%)
CHL	0	128	8	128	256	256	32	32	16	64	128
	MIC/2	32 (0.25)S	<2 (<0.25)S	32 (0.25)S	256 (1)I	64 (0.25)S	32 (1)I	4 (0.125)S	16 (1)I	16 (0.25)S	128 (1)I	60
	MIC/4	128 (1)I	<2 (<0.25)S	128 (1)I	256 (1)I	128 (0.5)S	32 (1)I	4 (0.125)S	16 (1)I	64 (1)I	128 (1)I	30
CIP	0	64	2	>64	>64	>64	2	<0.5	4	8	<0.5
	MIC/2	16 (0.25)S	1 (0.5)S	>64 (≥1)I	64 (<1)S	16 (<0.25)S	1 (0.5)S	<0.5 (≤1)I	4 (1)I	<1 (<0.125)S	<0.5 (≤1)I	60
	MIC/4	32 (0.5)S	1 (0.5)S	>64 (≥1)I	>64 (≥1)I	16 (<0.25)S	1 (0.5)S	<0.5 (≤1)I	4 (1)I	2 (0.25)S	<0.5 (≤1)I	50
TET	0	16	16	32	64	4	32	2	4	2	32
	MIC/2	4 (0.25)S	<1 (<0.062)S	64 (2)I	64 (1)I	1 (0.25)S	32 (1)I	2 (1)I	4 (1)I	<1 (<0.5)S	32 (1)I	40
	MIC/4	8 (0.5)S	<1 (<0.062)S	64 (2)I	64 (1)I	1 (0.25)S	32 (1)I	2 (1)I	4 (1)I	<1 (<0.5)S	32 (1)I	40
ERY	0	128	8	128	256	8	16	4	16	16	16
	MIC/2	128 (1)I	<2 (<0.25)S	128 (1)I	256 (1)I	8 (1)I	16 (1)I	<2 (<0.5)S	16 (1)I	8 (0.5)S	16 (1)I	30
	MIC/4	128 (1)I	<2 (<0.25)S	128 (1)I	256 (1)I	8 (1)I	16 (1)I	<2 (<0.5)S	16 (1)I	16 (1)I	16 (1)I	20
KAN	0	128	4	8	32	32	32	<2	16	16	8
	MIC/2	128 (1)I	<2 (<0.5)S	8 (1)I	16 (0.5)S	16 (0.5)S	32 (1)I	<2 (≤1)I	8 (0.5)S	16 (1)I	8 (1)I	50
	MIC/4	128 (1)I	<2 (<0.5)S	8 (1)I	16 (0.5)S	16 (0.5)S	32 (1)I	<2 (≤1)I	8 (0.5)S	16 (1)I	8 (1)I	50
STP	0	256	>256	>256	128	8	64	8	16	>256	64
	MIC/2	32 (0.125)S	32 (≥0.125)S	<2 (≤0.007)S	128 (1)I	<2 (0.25)S	16 (0.25)S	<2 (<0.25)S	16 (1)I	<2 (≤0.007)S	32 (0.5)S	80
	MIC/4	128 (0.5)S	128 (≥0.5)S	<2 (≤0.007)S	128 (1)I	8 (1)I	16 (0.25)S	<2 (<0.25)S	16 (1)I	4 (≥0.062)S	64 (1)I	60
AMP	0	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
	MIC/2	>256 (≥1)I	<2 (≤0.007)S	>256 (≥1)I	>256 (≥1)I	<2 (≤0.007)S	>256 (≥1)I	<2 (≤0.007)S	<2 (≤0.007)S	<2 (≤0.007)S	>256 (≥1)I	50
	MIC/4	>256 (≥1)I	<2 (≤0.007)S	>256 (≥1)I	>256 (≥1)I	<2 (≤0.007)S	>256 (≥1)I	256 (<1)S	<2 (≤0.007)S	<2 (≤0.007)S	>256 (≥1)I	50
CEF	0	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
	MIC/2	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	<2 (≤0.007)S	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	10
	MIC/4	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	256 (<1)S	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	10

aAntibotics [TET: tetracycline, CIP: ciprofloxacin, STP: streptomycin, KAN: kanamycin, CHL: chloramphenicol, ERY: erythromycin, AMP: ampicillin, and CEF: cefepime]. bBacterial strains: Escherichia coli [AG102, AG100Atet], Pseudomonas aeruginosa [PA124], Enterobacter aerogenes [CM64, EA27, EA289], Klebsiella pneumonia [KP55, KP63], Providencia stuartii [NAE16], and Enterobacter cloacae [BM47]. cPBSS: percentage of bacteria strain on which synergism has been observed. Values in parenthesis are fractional inhibitory concentration (FIC) of the antibiotics after association with plants extract; S: synergy; I: indifference. The values in bold represent the cases of synergy between extract and antibiotic.

Table 5

MIC of different antibiotics after the association of the extract of Coula edulis at MIC/2, MIC/4 against ten MDR bacteria strains.

Antibioticsa	Bacterial strains and MIC (μg/mL) of antibiotics in the absence and presence of the extractb
	Extractconcentration	PA124	AG102	AG100Atet	EA289	CM64	KP55	KP63	NEA16	PS2636	BM47	PBSSc (%)
CHL	0	128	8	128	256	256	32	32	16	64	128
	MIC/2	64 (0.5)S	<2 (<0.25)S	128 (1)I	256 (1)I	64 (0.25)S	32 (1)I	32 (1)I	16 (1)I	8 (0.125)S	128 (1)I	40
	MIC/4	64 (0.5)S	4 (0.5)S	128 (1)I	256 (1)I	128 (0.5)S	32 (1)I	32 (1)I	16 (1)I	8 (0.125)S	128 (1)I	40
CIP	0	64	2	>64	>64	>64	2	<0.5	4	8	<0.5
	MIC/2	16 (0.25)S	<0.5 (<0.25)S	>64 (≥1)I	32 (<0.5)S	64 (<1)S	2 (1)I	<0.5 (≤1)I	<0.5 (<0.25)S	<0.5 (<0.125)S	<0.5 (≤1)I	60
	MIC/4	16 (0.25)S	<0.5 (<0.25)S	>64 (≥1)I	32 (<0.5)S	64 (<1)S	2 (1)I	<0.5 (≤1)I	<0.5 (<0.25)S	<0.5 (<0.125)S	<0.5 (≤1)I	60
TET	0	16	16	32	64	4	32	2	4	2	32
	MIC/2	16 (1)I	<1 (<0.062)S	64 (2)I	64 (1)I	<0.5 (<0.25)S	32 (1)I	2 (1)I	4 (1)I	<1 (<0.5)S	32 (1)I	30
	MIC/4	16 (1)I	<1 (<0.062)S	64 (2)I	64 (1)I	<0.5 (<0.25)S	32 (1)I	2 (1)I	4 (1)I	<1 (<0.5)S	32 (1)I	20
ERY	0	128	8	128	256	8	16	4	16	16	16
	MIC/2	128 (1)I	<2 (<0.25)S	128 (1)I	256 (1)I	16 (2)I	16 (1)I	4 (1)I	8 (0.5)S	8 (0.5)S	16 (1)I	30
	MIC/4	128 (1)I	<2 (<0.25)S	128 (1)I	256 (1)I	16 (2)I	16 (1)I	4 (1)I	8 (0.5)S	16 (1)I	16 (1)I	20
KAN	0	128	4	8	32	32	32	<2	16	16	8
	MIC/2	128 (1)I	<2 (<0.5)S	8 (1)I	32 (1)I	32 (1)I	32 (1)I	<2 (≤1)I	16 (1)I	16 (1)I	8 (1)I	0
	MIC/4	128 (1)I	<2 (<0.5)S	8 (1)I	32 (1)I	32 (1)I	32 (1)I	<2 (≤1)I	16 (1)I	16 (1)I	8 (1)I	20
STP	0	256	>256	>256	128	8	64	8	16	>256	64
	MIC/2	64 (0.25)S	8 (<0.031)S	<2 (≤0.007)S	128 (1)I	128 (16)A	64 (1)I	8 (1)I	16 (1)I	8 (<0.031)S	64 (1)I	40
	MIC/4	64 (0.25)S	>256 (≥1)I	64 (<0.25)S	128 (1)I	128 (16)A	64 (1)I	8 (1)I	16 (1)I	128 (<0.5)S	64 (1)I	30
AMP	0	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
	MIC/2	256 (<1)S	<2 (≤0.007)S	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	<2 (≤0.007)S	<2 (≤0.007)S	>256 (≥1)I	40
	MIC/4	>256 (≥1)I	<2 (≤0.007)S	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	<2 (≤0.007)S	<2 (≤0.007)S	>256 (≥1)I	30
CEF	0	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
	MIC/2	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	00
	MIC/4	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	00

aAntibotics [TET: tetracycline, CIP: ciprofloxacin, STP: streptomycin, KAN: kanamycin, CHL: chloramphenicol, ERY: erythromycin, AMP: ampicillin, and CEF: cefepime]. bBacterial strains: Escherichia coli [AG102, AG100Atet], Pseudomonas aeruginosa [PA124], Enterobacter aerogenes [CM64, EA27, EA289], Klebsiella pneumonia [KP55, KP63], Providencia stuartii [NAE16], and Enterobacter cloacae [BM47]. cPBSS: percentage of bacteria strain on which synergism has been observed. Values in parenthesis are fractional inhibitory concentration (FIC) of the antibiotics after association with plants extract; S: synergy; I: indifference; A: antagonism. The values in bold represent the cases of synergy between extract and antibiotic.

Table 6

MIC of different antibiotics after the association of the extract of leaves from Mangifera indica at MIC/2, MIC/4 against ten MDR bacteria strains.

Antibioticsa	Bacterial strains and MIC (μg/mL) of antibiotics in the absence and presence of the extractb
	Extractconcentration	PA124	AG102	AG100Atet	EA289	CM64	KP55	KP63	NEA16	PS2636	BM47	PBSSc (%)
CHL	0	128	8	128	256	256	32	32	16	64	128
	MIC/2	32 (0.25)S	<2 (<0.25)S	4 (0.031)S	64 (0.25)S	128 (0.5)S	4 (0.125)S	8 (0.25)S	4 (0.25)S	16 (0.25)S	32 (0.25)S	100
	MIC/4	32 (0.25)S	4 (0.5)S	4 (0.031)S	128 (0.5)S	128 (0.5)S	32 (1)I	32 (1)I	8 (0.5)S	64 (1)I	128 (1)I	60
CIP	0	64	2	>64	>64	>64	2	<0.5	4	8	<0.5
	MIC/2	32 (0.5)S	<0.5 (<0.25)S	8 (<0.125)S	2 (<0.031)S	64 (<1)S	2 (1)I	<0.5 (≥1)I	<0.5 (<0.125)S	<0.5 (<0.062)S	<0.5 (≥1)I	70
	MIC/4	32 (0.5)S	<0.5 (<0.25)S	>64 (≥1)I	2 (<0.031)S	64 (<1)S	2 (1)I	<0.5 (≥1)I	2 (0.5)S	4 (0.5)S	<0.5 (≥1)I	60
TET	0	16	16	32	64	4	32	2	4	2	32
	MIC/2	32 (2)I	4 (0.25)S	8 (0.25)S	<2 (<0.031)S	2 (0.5)S	8 (0.25)S	1 (0.5)S	<0.5 (<0.125)S	<0.5 (<0.25)S	8 (0.25)S	90
	MIC/4	32 (2)I	8 (0.5)S	16 (0.5)S	16 (0.25)S	2 (0.5)S	8 (0.25)S	1 (0.5)S	<0.5 (<0.125)S	<0.5 (<0.25)S	16 (0.5)S	90
ERY	0	128	8	128	256	8	16	4	16	16	16
	MIC/2	128 (1)I	<2 (<0.25)S	32 (0.25)S	32 (0.125)S	4 (0.5)S	8 (0.5)S	<2 (<0.5)S	4 (0.25)S	<2 (<0.125)S	<2 (<0.125)S	90
	MIC/4	128 (1)I	<2 (<0.25)S	32 (0.25)S	128 (0.5)S	4 (0.5)S	16 (1)I	<2 (<0.5)S	8 (0.5)S	4 (0.25)S	4 (0.25)S	80
KAN	0	128	4	8	32	32	32	<2	16	16	8
	MIC/2	128 (1)I	<2 (<0.5)S	4 (0.5)S	<2 (<0.062)S	8 (0.25)S	4 (0.125)S	<2 (≥1)I	<2 (<0.125)S	4 (0.25)S	8 (1)I	70
	MIC/4	128 (1)I	<2 (<0.5)S	8 (1)I	8 (0.25)S	16 (0.5)S	4 (0.125)S	<2 (≥1)I	8 (0.5)S	16 (1)I	8 (1)I	50
STP	0	256	>256	>256	128	8	64	8	16	>256	64
	MIC/2	128 (0.5)S	16 (<0.062)S	16 (<0.062)S	32 (0.25)S	4 (0.5)S	32 (0.5)S	4 (0.5)S	4 (0.25)S	64 (<0.5)S	4 (0.062)S	100
	MIC/4	128 (0.5)S	32 (<0.125)S	16 (<0.062)S	64 (0.5)S	8 (1)I	32 (0.5)S	8 (1)I	16 (1)I	>256 (na)	8 (0.125)S	60
AMP	0	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
	MIC/2	256 (<1)S	128 (<0.5)S	128 (<0.5)S	128 (<0.5)S	128 (<0.5)S	256 (<1)S	8 (<0.031)S	256 (<1)S	128 (<0.5)S	>256 (≥1)I	90
	MIC/4	>256 (≥1)I	256 (<1)S	256 (<1)S	256 (<1)S	256 (<1)S	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	40
CEF	0	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
	MIC/2	256 (<1)S	256 (<1)S	>256 (≥1)I	256 (<1)S	128 (<0.5)S	>256 (≥1)I	>256 (≥1)I	256 (<1)S	>256 (≥1)I	>256 (≥1)I	50
	MIC/4	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	256 (<1)S	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	10

aAntibotics [TET: tetracycline, CIP: ciprofloxacin, STP: streptomycin, KAN: kanamycin, CHL: chloramphenicol, ERY: erythromycin, AMP: ampicillin, and CEF: cefepime]. bBacterial strains: Escherichia coli [AG102, AG100Atet], Pseudomonas aeruginosa [PA124], Enterobacter aerogenes [CM64, EA27, EA289], Klebsiella pneumonia [KP55, KP63], Providencia stuartii [NAE16], and Enterobacter cloacae [BM47]. cPBSS: percentage of bacteria strain on which synergism has been observed. Values in parenthesis are fractional inhibitory concentration (FIC) of the antibiotics after association with plants extract; S: synergy; I: indifference. The values in bold represent the cases of synergy between extract and antibiotic.

Table 7

MIC of different antibiotics after the association of the extract of leaves from Psidium guajava at MIC/2, MIC/4 against ten MDR bacteria strains.

Antibioticsa	Bacterial strains and MIC (μg/mL) of antibiotics in the absence and presence of the extractb
	Concentration of the extract	PA124	AG102	AG100Atet	EA289	CM64	KP55	KP63	NEA16	PS2636	BM47	PBSSc (%)
CHL	0	128	8	128	256	256	32	32	16	64	128
	MIC/2	32 (0.25)S	4 (0.5)S	64 (0.5)S	256 (1)I	256 (1)I	32 (1)I	16 (0.5)S	8 (0.5)S	32 (0.25)S	32 (0.25)S	70
	MIC/4	32 (0.25)S	8 (1)I	128 (1)I	256 (1)I	256 (1)I	32 (1)I	32 (1)I	16 (1)I	64 (1)I	128 (1)I	10
CIP	0	64	4	>64	>64	>64	2	<0.5	4	8	<0.5
	MIC/2	32 (0.5)S	4 (1)I	>64 (≥1)I	16 (<0.25)S	64 (<1)S	2 (1)I	<0.5 (≥1)I	2 (0.5)S	1 (0.125)S	<0.5 (≥1)I	50
	MIC/4	32 (0.5)S	4 (1)I	>64 (≥1)I	>64 (≥1)I	64 (<1)S	2 (1)I	<0.5 (≥1)I	4 (1)I	4 (0.5)S	<0.5 (≥1)I	30
TET	0	16	16	32	64	4	32	2	4	2	32
	MIC/2	32 (2)I	4 (0.25)S	32 (1)I	32 (0.5)S	4 (1)I	16 (0.5)S	<0.5 (<0.25)S	<0.5 (<0.125)S	<0.5 (<0.25)S	16 (0.5)S	70
	MIC/4	32 (2)I	16 (1)I	32 (1)I	32 (0.5)S	4 (1)I	32 (1)I	1 (0.5)S	4 (1)I	<0.5 (<0.25)S	32 (1)I	30
ERY	0	128	8	128	256	8	16	4	16	16	16
	MIC/2	128 (1)I	4 (0.5)S	128 (1)I	128 (0.5)S	<2 (<0.25)S	16 (1)I	2 (0.5)S	16 (1)I	16 (1)I	16 (0.5)S	50
	MIC/4	128 (1)I	4 (0.5)S	128 (1)I	128 (0.5)S	<2 (<0.25)S	16 (1)I	4 (1)I	16 (1)I	16 (1)I	16 (1)I	10
KAN	0	128	4	8	32	32	32	<2	16	16	8
	MIC/2	128 (1)I	<2 (<0.5)S	8 (1)I	4 (0.5)S	32 (1)I	8 (0.25)S	<2 (≥1)I	8 (0.5)S	4 (0.25)S	8 (1)I	50
	MIC/4	128 (1)I	<2 (<0.5)S	8 (1)I	32 (1)I	32 (1)I	32 (1)I	<2 (≥1)I	8 (0.5)S	16 (1)I	8 (1)I	20
STP	0	256	>256	>256	128	8	64	8	16	>256	64
	MIC/2	128 (0.5)S	16 (<0.062)S	128 (<0.5)S	128 (1)I	4 (0.5)S	64 (1)I	4 (0.5)S	16 (1)I	128 (<0.5)S	16 (0.25)S	70
	MIC/4	128 (0.5)S	128 (<0.5)S	256 (<1)S	128 (1)I	8 (1)I	64 (1)I	8 (1)I	16 (1)I	>256 (≥1)I	16 (0.25)S	40
AMP	0	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
	MIC/2	256 (<1)S	>256 (≥1)I	>256 (≥1)I	256 (<1)S	128 (<0.5)S	>256 (≥1)I	128 (<0.5)S	>256 (≥1)I	256 (<1)S	>256 (≥1)I	50
	MIC/4	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	10
CEF	0	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
	MIC/2	256 (<1)S	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	10
	MIC/4	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	00

aAntibotics [TET: tetracycline, CIP: ciprofloxacin, STP: streptomycin, KAN: kanamycin, CHL: chloramphenicol, ERY: erythromycin, AMP: ampicillin, and CEF: cefepime]. bBacterial strains: Escherichia coli [AG102, AG100Atet], Pseudomonas aeruginosa [PA124], Enterobacter aerogenes [CM64, EA27, EA289], Klebsiella pneumonia [KP55, KP63], Providencia stuartii [NAE16], and Enterobacter cloacae [BM47]. cPBSS: percentage of bacteria strain on which synergism has been observed. Values in parenthesis are fractional inhibitory concentration (FIC) of the antibiotics after association with plants extract; S: synergy; I: indifference. The values in bold represent the cases of synergy between extract and antibiotic.

Table 8

MIC of different antibiotics after the association of the extract of Camellia sinensis at MIC/2, MIC/4 against ten MDR bacteria strains.

Antibioticsa	Bacterial strains and MIC (μg/mL) of antibiotics in the absence and presence of the extractb
	Extract concentration	PA124	AG102	AG100Atet	EA289	CM64	KP55	KP63	NEA16	PS2636	BM47	PBSSc (%)
CHL	0	128	8	128	256	256	32	32	16	64	128
	MIC/2	8 (0.0625)S	<2 (<0.25)S	64 (0.5)S	128 (0.5)S	64 (0.25)S	8 (0.25)S	16 (0.5)S	<2 (<0.125)S	4 (0.25)S	16 (0.25)S	100
	MIC/4	8 (0.0625)S	<2 (<0.25)S	64 (0.5)S	256 (1)I	128 (0.5)S	16 (0.5)S	16 (0.5)S	8 (0.5)S	64 (1)I	128 (1)I	70
CIP	0	64	4	>64	>64	>64	2	<0.5	4	8	<0.5
	MIC/2	32 (0.5)S	<0.5 (<0.125)S	16 (<0.25)S	>64 (≥1)I	16 (<0.25)S	<0.5 (<0.25)S	<0.5 (≥1)I	<0.5 (<0.125)S	<0.5 (<0.062)S	<0.5 (≥1)I	70
	MIC/4	32 (0.5)S	<0.5 (<0.125)S	>64 (≥1)I	>64 (≥1)I	16 (<0.25)S	<0.5 (<0.25)S	<0.5 (≥1)I	4 (1)I	2 (0.25)S	<0.5 (≥1)I	50
TET	0	16	16	32	64	4	32	2	4	2	32
	MIC/2	16 (1)I	<1 (<0.062)S	8 (0.25)S	16 (0.25)S	2 (0.5)S	32 (1)I	1 (0.5)S	<0.5 (<0.125)S	<0.5 (<0.25)S	16 (0.5)S	80
	MIC/4	16 (1)I	<1 (<0.062)S	16 (0.5)S	32 (0.5)S	4 (1)I	32 (1)I	2 (1)I	<0.5 (<0.125)S	<0.5 (<0.25)S	32 (1)I	50
ERY	0	128	8	128	256	8	16	4	16	16	16
	MIC/2	128 (1)I	<2 (<0.25)S	32 (0.25)S	64 (0.25)S	<2 (<0.125)S	16 (1)I	4 (1)I	4 (0.25)S	4 (0.25)S	<2 (<0.125)S	70
	MIC/4	128 (1)I	4 (0.5)S	64 (0.5)S	64 (0.25)S	<2 (<0.125)S	16 (1)I	4 (1)I	8 (0.5)S	4 (0.25)S	4 (0.25)S	70
KAN	0	128	4	8	32	32	32	<2	16	16	8
	MIC/2	64 (0.5)S	<2 (<0.5)S	4 (0.5)S	16 (0.5)S	8 (0.25)S	32 (1)I	<2 (≥1)I	<0.5 (<0.031)S	16 (1)I	8 (1)I	60
	MIC/4	64 (0.5)S	<2 (<0.5)S	8 (1)I	16 (0.5)S	16 (0.5)S	32 (1)I	<2 (≥1)I	8 (0.5)S	16 (1)I	8 (1)I	50
STP	0	256	>256	>256	128	8	64	8	16	>256	64
	MIC/2	64 (0.25)S	32 (<0.125)S	128 (<0.5)S	64 (0.5)S	4 (0.5)S	64 (1)I	<2 (<0.25)S	<2 (<0.125)S	64 (<0.25)S	4 (0.0625)S	90
	MIC/4	64 (0.25)S	64 (<0.25)S	128 (<0.5)S	64 (0.5)S	8 (1)I	64 (1)I	8 (1)I	8 (0.5)S	64 (<0.25)S	16 (0.25)S	70
AMP	0	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
	MIC/2	>256 (≥1)I	128 (<0.5)S	128 (<0.5)S	64 (<0.25)S	128 (<0.5)S	>256 (≥1)I	64 (<0.25)S	128 (<0.5)S	>256 (≥1)I	128 (<0.5)S	70
	MIC/4	>256 (≥1)I	128 (<0.5)S	>256 (≥1)I	256 (<1)S	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	20
CEF	0	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
	MIC/2	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	128 (<0.5)S	>256 (≥1)I	>256 (≥1)I	128 (<0.5)S	256 (<0.5)S	128 (<0.5)S	128 (<0.5)S	50
	MIC/4	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	00

aAntibotics [TET: tetracycline, CIP: ciprofloxacin, STP: streptomycin, KAN: kanamycin, CHL: chloramphenicol, ERY: erythromycin, AMP: ampicillin, and CEF: cefepime]. bBacterial strains: Escherichia coli [AG102, AG100Atet], Pseudomonas aeruginosa [PA124], Enterobacter aerogenes [CM64, EA27, EA289], Klebsiella pneumonia [KP55, KP63], Providencia stuartii [NAE16], and Enterobacter cloacae [BM47]. cPBSS: percentage of bacteria strain on which synergism has been observed. Values in parenthesis are fractional inhibitory concentration (FIC) of the antibiotics after association with plants extract; S: synergy; I: indifference; A: antagonism. The values in bold represent the cases of synergy between extract and antibiotic.

Table 9

M1IC of different antibiotics after the association of the extract of bark from Persea americana at MIC/2, MIC/4 against ten MDR bacteria strains.

Antibioticsa	Bacterial strains and MIC (μg/mL) of antibiotics in the absence and presence of the extractb
	Extractconcentration	PA124	AG102	AG100Atet	EA289	CM64	KP55	KP63	NEA16	PS2636	BM47	PBSSc (%)
CHL	0	128	8	128	256	256	32	32	16	64	128
	MIC/2	64 (0.5)S	4 (0.5)S	128 (1)I	256 (1)I	256 (1)I	32 (1)I	16 (0.5)S	16 (1)I	64 (1)I	64 (0.5)S	40
	MIC/4	64 (0.5)S	8 (1)I	128 (1)I	256 (1)I	256 (1)I	32 (1)I	16 (0.5)S	16 (1)I	64 (1)I	128 (1)I	20
CIP	0	64	8	>64	>64	>64	2	<0.5	4	8	<0.5
	MIC/2	32 (0.5)S	4 (0.5)S	>64 (≥1)I	>64 (≥1)I	32 (<0.5)S	<0.5 (<0.25)S	<0.5 (≥1)I	4 (1)I	8 (1)I	<0.5 (≥1)I	40
	MIC/4	32 (0.5)S	8 (1)I	>64 (≥1)I	>64 (≥1)I	>64 (≥1)I	<0.5 (<0.25)S	<0.5 (≥1)I	4 (1)I	8 (1)I	<0.5 (≥1)I	20
TET	0	16	16	32	64	4	32	2	4	2	32
	MIC/2	8 (0.5)S	<1 (<0.062)S	64 (2)I	32 (0.5)S	<0.5 (<0.125)S	16 (0.5)S	1 (0.5)S	<0.5 (<0.125)S	2 (1)I	32 (1)I	70
	MIC/4	8 (0.5)S	<1 (<0.062)S	64 (2)I	32 (0.5)S	<0.5 (<0.125)S	32 (1)I	2 (1)I	4 (1)I	2 (1)I	32 (1)I	40
ERY	0	128	8	128	256	8	16	4	16	16	16
	MIC/2	128 (1)I	4 (0.5)S	16 (0.5)S	128 (0.5)S	8 (1)I	4 (0.5)S	4 (1)I	16 (1)I	16 (1)I	8 (0.5)S	50
	MIC/4	128 (1)I	8 (1)I	128 (1)I	128 (0.5)S	8 (1)I	16 (1)I	4 (1)I	16 (1)I	16 (1)I	8 (0.5)S	20
KAN	0	128	4	8	32	32	32	<2	16	16	8
	MIC/2	128 (1)I	4 (1)I	4 (0.5)S	32 (1)I	16 (0.5)S	8 (0.25)S	<2 (≥1)I	16 (1)I	4 (0.25)S	8 (1)I	40
	MIC/4	128 (1)I	4 (1)I	8 (1)I	32 (1)I	16 (0.5)S	16 (0.5)S	<2 (≥1)I	16 (1)I	16 (1)I	8 (1)I	20
STP	0	256	>256	>256	128	8	64	8	16	>256	64
	MIC/2	64 (0.25)S	256 (<1)S	>256 (≥1)I	128 (1)I	4 (0.5)S	64 (1)I	4 (0.5)S	16 (1)I	256 (<1)S	32 (0.5)S	60
	MIC/4	64 (0.25)S	>256 (≥1)I	>256 (≥1)I	128 (1)I	8 (1)I	64 (1)I	8 (1)I	16 (1)I	>256 (≥1)I	32 (0.5)S	20
AMP	0	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
	MIC/2	256 (<1)S	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	128 (<0.5)S	>256 (≥1)I	256 (<1)S	>256 (≥1)I	30
	MIC/4	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	00
CEF	0	>256	>256	>256	>256	>256	>256	>256	>256	>256	>256
	MIC/2	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	128 (<0.5)S	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	10
	MIC/4	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	>256 (≥1)I	00

aAntibotics [TET: tetracycline, CIP: ciprofloxacin, STP: streptomycin, KAN: kanamycin, CHL: chloramphenicol, ERY: erythromycin, AMP: ampicillin, and CEF: cefepime]. bBacterial strains: Escherichia coli [AG102, AG100Atet], Pseudomonas aeruginosa [PA124], Enterobacter aerogenes [CM64, EA27, EA289], Klebsiella pneumonia [KP55, KP63], Providencia stuartii [NAE16], and Enterobacter cloacae [BM47]. cPBSS: percentage of bacteria strain on which synergism has been observed. Values in parenthesis are fractional inhibitory concentration (FIC) of the antibiotics after association with plants extract; S: synergy; I: indifference. The values in bold represent the cases of synergy between extract and antibiotic.

4. Discussion

Chemical Composition of the Tested Plant Extpan class="Chemical">racts. Phytochemical screening revealed the presence of sevespan>n class="Chemical">ral classes of secondary metabolites such as alkaloids, polyphenols, flavonoids, anthraquinones, coumarins, saponins, tannins, triterpenes, and steroids. Several in vitro experiments proved that molecules belonging to these classes were found active on pathogenic microorganisms [55].

Antibacterial Activity of the Tested Extpan class="Chemical">racts. After sevespan>n class="Chemical">ral decades of antibiotic use, pathogenic bacteria of human and animal origin have reached alarming levels of resistance. The reduced efficacy and increasing toxicity of synthetic drugs further aggravate this problem. Scientists are thus directed to seek more active natural or organic molecules [14]. It is imperative in developing countries that effective and less expensive antibacterials should be developed to accommodate all patients, regardless of financial status, in order to eliminate some human factors that can cause MDR. Medicinal plants have known to possess antimicrobial properties against MDR phenotypes [48–50, 56–59] and differences observed between antibacterial activities of the extracts in the present study could be due to the differences in the composition as well as the mechanism of action of their bioactive compounds [55, 60]. Different solvents extracts of leaves from P. guajava have been separately documented to be inhibitory against both Gram-positive and Gram-negative bacteria [14, 15, 61]. In the present study, methanol extract from P. guajava leaf demonstrated significant inhibitory activities against both sensitive and MDR bacteria, confirming the previous reports on its antimicrobial activity. According to Kuete and Efferth [62], the antibacterial activity of a plant extract is considered significant when MIC values are below 100 μg/mL, moderate when 100 ≤ MICs ≤ 625 μg/mL, and weak when MICs > 625 μg/mL. Consequently, the methanol P. guajava leaf extract tested herein had moderate activity. Besides, the activity (MIC of 32 and 64 μg/mL) observed with C. edulis nuts and M. indica bark as well as C. sinensis leaves extracts against E. coli AG102, P. aeruginosa PA01, and K. pneumoniae K2, respectively, can be considered important. The antibacterial activities of mango (M. indica) extracts against some microorganisms (Gram-positive and Gram-negative bacteria and Candida albicans yeast) have already been demonstrated [38, 63] and it is thought that the antibacterial activity of mango extract is due to the presence of gallotannin and mangiferin [64]. Different extracts of C. sinensis having antimicrobial activity were reported against Gram-positive and Gram-negative bacteria and fungi [65, 66] and it is thought that their antibacterial activities are due to the presence of catechin. In fact, catechin particularly affects the membrane fluidity in both hydrophilic and hydrophobic regions of lipid bilayers of the microorganism [67] or inhibits the action of DNA polymerases [68]. The present study provides additional data on the ability of this plant to fight MDR bacteria.

The leaves of pan class="Species">P. americana are widely used for ethnomedicinal purposes worldwide. In order to confirm its multiple medicinal properties, a study conducted by Nathaniel et al. [18] showed that the span>n class="Chemical">methanolic extract from leaves of P. americana had good antimicrobial activity against enteric microorganisms (Gram-negative and Gram-positive bacteria and also yeasts). This is in accordance with the present study as we also found that methanol extracts of both leaves and bark of the plant exhibited similar activities against several MDR bacteria, with leaves' extract having good inhibitor effects (64 μg/mL) against the E. coli AG102 strain. Considering the fact that some of the tested plants are used as food with limited toxicity, the overall activity recorded with several extracts, most notably those of C. sinensis, C. sinensis, and C. edulis or the fruits from P. guajava, could be considered important. However, a threshold for significant antibacterial effects has been proposed for MIC values ranging within 100–512 μg/mL when samples tested are edible parts of plants [69].

Role of Efflux Pumps in the Suspan class="Chemical">ceptibility of Gspan>n class="Chemical">ram-Negative Bacteria to the Tested Extracts. The studied bacterial strains used in this work mostly resist by efflux mechanism, which consists in expelling all toxic compounds (including antibiotics) out of the cytoplasm, thus preventing them from reaching their intracellular targets [70]. These efflux pumps can however be blocked competitively or not, in order to restore not only the intracellular concentration but also the activity of antibacterials acting on intracellular targets [71]. In the presence of PAβN (EPI), the antibacterial activity of C. sinensis extract was improved on 11/15 (73.37%) tested MDR strains. However, the antibacterial activities of other extracts were also improved on few selected bacteria. These results suggest that these extracts (C. sinensis especially) have an intracellular targets.

Effects of Association of Extracts with Antibiotics. The reduced efficiency of known antibiotics propels span>n class="Species">many researchers to search for novel compounds, as well as molecules capable of improving or at least restoring the efficiency of available antibiotics against resistant bacteria. The combination of both compounds can then help to prevent the emergence of resistant mutants and enable restoring the activity of antibiotics vis-à-vis resistant strains. Synergies with FIC ranging from 0.5 to <0.007 were noted with the associations of each of the extracts and antibiotics on at least one tested MDR bacteria. The extracts from leaves of M. indica and C. sinensis and also that from peels of C. sinensis had important synergistic effects with most of the tested antibiotics against the majority of bacteria. These results suggest that extracts inhibited the efflux pumps, increasing the activity of these antibiotics [72, 73]. Few cases of indifference (1 ≤ FIC < 4) were found in the majority of associations; meanwhile antagonistic effect was obtained when C. edulis extract was combined with streptomycin against E. aerogenes EA-CM64 strain. All plants tested in the present study (Table 1) are used traditionally to treat several affections or diseases with symptoms related to microbial infections [8–10, 16, 17, 19, 20, 27, 29, 40], and data obtained therefore consolidate their traditional use. The presence of previously reported secondary metabolites in the tested plants [11, 12, 18, 21, 22, 27, 30, 31, 40, 41] (Table 1) is also in accordance with the present studies and could in part justify their antibacterial effects. Natural compounds belonging to terpenoids (for example, camosic acid and isopimarane derivatives), flavonoids (for example, 5′-methoxyhydnocarpin) as well as alkaloids (for example, pheophorbide A or reserpine) and others have been identified as efflux pumps inhibitors [74, 75]. Extract from M. indica and C. sinensis having the best synergistic effects contain all these classes of secondary metabolites, probably explaining their antibiotic potentiation effects. These results could be explained by the interaction between active compounds of extracts and the active ingredients of antibiotics.

5. Limitations

Our study has limitations. It mainly reports the activity of crude plant extpan class="Chemical">racts and the identification of the active constituents of the plant would be necessary for better understanding of the reported effects. Though this study deals with food plants, the span>n class="Disease">cytotoxicity studies in normal cell lines will be performed to ascertain their safety.

6. Conclusion

The results of this study can provide some explanations on the tpan class="Chemical">raditional use of certain parts of the plants tested herein to combat span>n class="Disease">infections, particularly those caused by enteric bacteria. Evidence of the antibacterial activity of the studied plants, especially edible ones, as well as the ability of some of them to improve the activity of commonly used antibiotics, is an indication that there is a possibility of discovering alternative antibacterials in these plants.

Table S 1. Preliminary test of MIC of different antibiotics after the association of the extpan class="Chemical">racts at MIC/2, MIC/4, MIC/8 and MIC/16 against span>n class="Species">P. aeruginosa PA124 strain.

(a)

Bacterial strains	Tested samples and MIC and MBC and MIC in the presence of PAβN in parenthesis (μg/mL)
	P. guajava (leaves)			P. guajava (fruits)			P. guajava (bark)			P. americana (leaves)			P. americana (bark)
	MIC	MBC	R	MIC	MBC	R	MIC	MBC	R	MIC	MBC	R	MIC	MBC	R
Escherichia coli
ATCC8739	512	—	—	1024	—	—	1024	—	—	256	—	—	256	—	—
ATCC10536	512	—	—	1024	—	—	512	—	—	128	—	—	256	—	—
AG100	1024	—	—	1024	—	—	512	—	—	256	—	—	256	—	—
AG100A	256 (256)	—	—	512	—	—	1024	—	—	256 (256)	—	—	512 (512)	—	—
AG102	512 (512)	—	—	256	1024	4	1024	—	—	64 (16)	512	8	128 (64)	1024	8
AG100ATet	512 (128)	—	—	—	—	—	1024	—	—	512 (128)	—	—	512 (128)	—	—
MC4100	512	—	—	—	—	—	1024	—	—	128	—	—	512	—	—
W3110	1024	—	—	—	—	—	1024	—	—	256	—	—	256	—	—
Enterobacter aerogenes
ATCC13048	256	—	—	1024	—	—	512	—	—	512	—	—	512	—	—
EA-CM64	512 (512)	—	—	—	—	—	1024	—	—	512 (512)	—	—	512 (512)	—	—
EA3	512	—	—	—	—	—	512	—	—	256	—	—	128	—	—
EA27	128 (128)	—	—	—	—	—	—	—	—	128 (256)	—	—	256 (256)	—	—
EA289	256 (128)	—	—	—	—	—	512	—	—	256 (256)	—	—	512 (512)	—	—
EA294	1024	—	—	—	—	—	—	—	—	256	—	—	128	1024	8
EA298	1024 (1024)	—	—	—	—	—	—	—	—	128 (128)	1024	8	256 (256)	1024	4
Klebsiella pneumoniae
ATCC11296	1024	—	—	—	—	—	—	—	—	512	—	—	1024	—	—
K2	512	—	—	1024	—	—	1024	—	—	256	—	—	256	—	—
K24	512	—	—	—	—	—	1024	—	—	512	—	—	512	—	—
KP55	1024 (1024)	—	—	1024	—	—	512	—	—	— (—)	—	—	— (—)	—	—
KP63	256 (16)	—	—	—	—	—	512	—	—	512 (<8)	—	—	128 (<8)	1024	8
Providencia stuartii
ATCC29916	512	—	—	—	—	—	1024	—	—	—	—	—	—	—	—
ATCC299645	512	—	—	1024	—	—	1024	—	—	512	—	—	1024	—	—
PS2636	512 (512)	—	—	512	1024	2	512	—	—	256 (256)	1024	4	512 (512)	—	—
NEA16	1024 (1024)	—	—	—	—	—	512	1024	2	1024 (512)	—	—	256 (16)	1024	4
Enterobacter cloacae
BM47	256 (1024)	—	—	—	—	—	512	—	—	512 (512)	—	—	128 (128)	—	—
BM67	256 (256)	—	—	1024	—	—	512	—	—	512 (512)	—	—	256 (256)	—	—
ECCI69	512 (512)	—	—	—	—	—	512	—	—	1024 (1024)	—	—	— (—)	—	—
Pseudomonas aeruginosa
PA01	512	—	—	—	—	—	1024	—	—	—	—	—	128	—	—
PA124	1024 (1024)	—	—	—	—	—	1024	—	—	— (—)	—	—	1024 (512)	—	—

R: MIC/MBC; —: MIC > 1024 or not detected; ( ): values in parenthesis are MIC of substance in the presence of PAβN at 30 μg/mL; values in bold are the best MIC values for the plant extracts.

(b)

Bacterial strains	Tested samples and MIC and MBC and MIC in the presence of PAβN in parenthesis (μg/mL)
	Citrus sinensis			M. indica (leaves)			M. indica (bark)			Camellia sinensis			Coula edulis
	MIC	MBC	R	MIC	MBC	R	MIC	MBC	R	MIC	MBC	R	MIC	MBC	R
Escherichia coli
ATCC8739	64	256	4	—	—	—	256	—	—	512	—	—	64	256	4
ATCC10536	256	—	—	512	—	—	256	—	—	512	—	—	64	512	8
AG100	128	—	—	1024	—	—	—	—	—	512	—	—	64	—	—
AG100A	512 (256)	—	—	1024	—	—	— (—)	—	—	512 (256)	—	—	1024	—	—
AG102	128 (<8)	1024	4	1024 (1024)	—	—	1024 (1024)	—	—	256 (256)	—	—	32	256	8
AG100ATet	512 (8)	—	—	1024 (1024)	—	—	— (—)	—	—	256 (256)	—	—	1024	—	—
MC4100	64	—	—	1024	—	—	1024	—	—	1024	—	—	64	—	—
W3110	32	512	16	1024	—	—	1024	—	—	1024	—	—	64	—	—
Enterobacter aerogenes
ATCC13048	512	1024	2	512	—	—	512	—	—	512	—	—	512	—	—
EA-CM64	— (16)	—	—	— (1024)	—	—	— (—)	—	—	— (—)	—	—	64	—	—
EA3	256	—	—	1024	—	—	512	—	—	1024	—	—	128	—	—
EA27	512 (256)	—	—	512 (256)	—	—	512 (256)	—	—	1024 (1024)	—	—	256	—	—
EA289	512 (<8)	—	—	— (—)	—	—	512 (—)	—	—	512 (64)	—	—	512	—	—
EA294	128	512	4	—	—	—	—	—	—	1024	—	—	64	1024	16
EA298	128 (128)	512	4	— (—)	—	—	— (—)	—	—	512 (512)	—	—	256	—	—
Klebsiella pneumoniae
ATCC11296	512	—	—	—	—	—	—	—	—	512	—	—	—	—	—
K2	128	—	—	1024	—	—	1024	—	—	64	—	—	32	—	—
K24	—	—	—	1024	—	—	1024	—	—	512	—	—	1024	—	—
KP55	— (—)	—	—	1024 (1024)	—	—	1024 (1024)	—	—	— (—)	—	—	1024	—	—
KP63	256 (<8)	—	—	1024 (1024)	—	—	512 (256)	—	—	256 (64)	—	—	512	—	—
Providencia stuartii
ATCC29916	—	—	—	512	—	—	1024	—	—	512	—	—	—	—	—
ATCC299645	—	—	—	1024	—	—	—	—	—	512	—	—	128	1024	8
PS2636	1024 (<8)	—	—	512 (512)	—	—	512 (512)	—	—	— (—)	—	—	512	512	1
NEA16	128 (16)	1024	8	512 (512)	—	—	512 (512)	—	—	1024 (1024)	1024	1	128	1024	8
Enterobacter cloacae
BM47	256 (16)	—	—	512	—	—	512 (512)	—	—	1024 (1024)	—	—	1024	—	—
BM67	512 (512)	—	—	1024	—	—	512 (512)	—	—	256 (256)	1024	4	—	—	—
ECCI69	512 (512)	—	—	— (—)	—	—				512 (256)	—	—	—	—	—
Pseudomonas aeruginosa
PA01	128	512	4	1024	—	—	32	—	—	512	—	—	—	—	—
PA124	— (1024)	—	—	1024 (512)	—	—	512 (512)	—	—	1024 (1024)	—	—	—	—	—

R: CMB/CMI; —: MIC > 1024 or not detected; ( ): values in parenthesis are MIC of substance in the presence of PAβN at 30 μg/mL; values in bold are the best MIC values for the plant extracts.

(c)

Bacterial strains	Chloramphenicol
	MIC	MBC	R
Escherichia coli
ATTC8739	4	16	4
ATCC10536	4	128	32
AG100	8	64	8
AG100A	64	—	—
AG102	64	256	4
AG100ATet	128	—	—
MC4100	8	64	8
W3110	8	256	32
Enterobacter aerogenes
ATCC13048	8	64	8
EA-CM64	128	—	—
EA3	32	256	8
EA27	128	—	—
EA289	256	—	—
EA294	8	128	16
EA298	32	64	2
Klebsiella pneumoniae
ATCC11296	64	—	—
K2	32	128	4
K24	128	256	2
KP55	64	128	2
KP63	64	256	4
Providencia stuartii
ATCC29916	128	—	—
ATCC299645	64	256	4
PS2636	128	—	—
NEA16	32	256	8
Enterobacter cloacae
BM47	256	—	—
BM67	128	—	—
ECCI69	—	256	—
Pseudomonas aeruginosa
PA01	32	—	—
PA124	256	—	—

R: CMB/CMI; —: MIC > 1024 or not detected; values in parenthesis are MIC of substance in the presence of PAβN at 30 μg/mL; values in bold are the best MIC values for the plant extracts.