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BMC Complementary and Alternative Medicine logoLink to BMC Complementary and Alternative Medicine
. 2014 Jul 21;14:258. doi: 10.1186/1472-6882-14-258

Antibiotic-potentiation activities of four Cameroonian dietary plants against multidrug-resistant Gram-negative bacteria expressing efflux pumps

Francesco K Touani 1, Armel J Seukep 1, Doriane E Djeussi 1, Aimé G Fankam 1, Jaurès A K Noumedem 1, Victor Kuete 1,
PMCID: PMC4223522  PMID: 25047005

Abstract

Background

The continuous spread of multidrug-resistant (MDR) bacteria, partially due to efflux pumps drastically reduced the efficacy of the antibiotic armory, increasing the frequency of therapeutic failure. The search for new compounds to potentiate the efficacy of commonly used antibiotics is therefore important. The present study was designed to evaluate the ability of the methanol extracts of four Cameroonian dietary plants (Capsicum frutescens L. var. facilulatum, Brassica oleacera L. var. italica, Brassica oleacera L. var. butyris and Basilicum polystachyon (L.) Moench.) to improve the activity of commonly used antibiotics against MDR Gram-negative bacteria expressing active efflux pumps.

Methods

The qualitative phytochemical screening of the plant extracts was performed using standard methods whilst the antibacterial activity was performed by broth micro-dilution method.

Results

All the studied plant extracts revealed the presence of alkaloids, phenols, flavonoids, triterpenes and sterols. The minimal inhibitory concentrations (MIC) of the studied extracts ranged from 256-1024 μg/mL. Capsicum frutescens var. facilulatum extract displayed the largest spectrum of activity (73%) against the tested bacterial strains whilst the lower MIC value (256 μg/mL) was recorded with Basilicum polystachyon against E. aerogenes ATCC 13048 and P. stuartii ATCC 29916. In the presence of PAβN, the spectrum of activity of Brassica oleacera var. italica extract against bacteria strains increased (75%). The extracts from Brassica oleacera var. butyris, Brassica oleacera var. italica, Capsicum frutescens var. facilulatum and Basilicum polystachyon showed synergistic effects (FIC ≤ 0.5) against the studied bacteria, with an average of 75.3% of the tested antibiotics.

Conclusion

These results provide promising information for the potential use of the tested plants alone or in combination with some commonly used antibiotics in the fight against MDR Gram-negative bacteria.

Keywords: Cameroonian dietary plants, Potentiation, Gram–negative bacteria, Multidrug resistant, Efflux pumps

Background

The spread of multidrug-resistant bacteria, partially due to the inappropriate use of common antibiotics, drastically reduced the efficacy of the antibiotic armory, increasing the frequency of therapeutic failure. The over-expression of efflux pumps is the main resistance mechanism observed in many bacteria [1]. In Gram-negative bacteria, many of these efflux pumps belong to the resistance-nodulation-cell division (RND), family of tripartite efflux pumps [2]. In the fight against microbial infections including those due to MDR bacteria, investigations are being carried out to discover new effective, none or less-toxic and available antibacterial drugs. Many scientist are also investigating synergistic compounds to potentiate the activity of the commonly used antibiotics [3]. The present work was designed to evaluate the in vitro ability of some edible plants namely Capsicum frutescens L. var. facilulatum (Solanaceae) or ‘chili pepper’, Brassica oleacera L. var. italica commonly known as ‘Broccoli’ and Brassica oleacera L. var. butyris (Brassicaceae) or ‘Cauliflower’; and Basilicum polystachyon (L.) Moench. (Lamiaceae) or ‘Musk Basil’ to potentiate the effect of common antibiotics against Gram-negative MDR phenotypes.

Methods

Plant material and extraction

The plants used in this study were collected in Douala (Littoral Region of Cameroon) in January 2013. The plants were further identified at the National Herbarium (Yaoundé, Cameroon) where voucher specimens were deposited under a reference number (Table 1). Air dried and powdered sample (0.1 g) of each plant was extracted by maceration with methanol (0.3 L) for 48 h at room temperature (25°C). After filtration using Whatman No. 1 filter paper, the filtrate of each plant was concentrated under reduced pressure in a rotary evaporator, and dried at room temperature to give the crude extract. The extraction yield was calculated (Table 2). These extracts were then stored at 4°C until further use.

Table 1.

Information on plants used in this study

Plants samples and herbarium voucher number a Parts used Popular names Traditional used Known antimicrobial activities of plants
Capsicum frutescens L. var. facilulatum (Solanaceae) 43079/HNC
Fruits
Green pepper
Antimitogenic [4], allergy, cancer and viral infection [5]
Antibacterial activities of aqueous and methanolic extracts against Sa, St, Vc [6,7], antifungal activities of lectin against Af, [8]; antifungal activities of saponin CAY-1 against Ca, Aspergillus Spp and dermatophytes Tm, Tr et Mc [9]
Brassica oleacera L. var. italica (Brassicaceae) 25686/SFR Cam
Leaves
Brocoli
Oxydative stress, cytotoxic [10]
Antibacterial activities of ethanolic extractsagainst Sa, Bc, Pa [11]. Antifungal activities against Sc, Te, Hm, Pm [12].
Brassica oleacera L. var. butyris (Brassicaceae) 25686/SFR Cam
Leaves
Flower cabbage
Cytotoxic effect, antiproliférative, Oxydative stress [13].
Antibacterial activities of sulfur compounds MMTSO, AITC, MMTSO2 against Pp, Lm, Lp, Lb Lm Sa, Ea, Ec, Bs, St and antifungal against strains Sc, Te, Hm, Pm [12].
Basilicum polystachyon (L.) Moench. (Lamiaceae) 38650/HNC Leaves Cotimandjo (Cameroon) Infectious diseases, gastroenteritis [14]. Strong activities of acidic extracts against Gram (+), but less activities against Gram;. Strong antifungal activities of ethanolic and methanolic extracts against An [15].

Af, Fm Ca, Tm, Tr, Tt, Mc, Sa, Bc, Ec, Pa, Sc, Te Hm, Pm, Pp, Lm, Lp, Lb, Lm, Bs, Ea, St, Te, Hm, An, Kp, Ec, Sm, Vc who are respectively : Aspergillus flavus, Fusarium moniliforme, Candidat albicans, trichophyton mentagrophytes, T. rubum, T.tonsuraus Microsporum canis, Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeroginosa, Saccharomyces cerevisiae, Torulopsis etchellsii, Hansenula mrakii, Pichia membranefaciens, Pediococcus pentosaceus, Leuconostoc mesenteroides, Lactobacillus plantarum , Lactobacillus brevis, Listeria monocytogenes, Bacillus subtilis, Enterobacter aerogenes, Salmonella. Typhimurium, Torulopsis etchellsii, Hansenula mrakii, Aspergillus niger, Klebsiella pneumoniae CI, Enterobacter cloacae CI, CIv Vibrio cholerae MMTSO: Méthylmethanethiosulfinate, AITC: Allyisothyocyanate, MMTS02: Méthylmethanethiosulfonat. SRFC: Company of Forest Reserve of Cameroon; HNC: Cameroon National Herbarium.

Table 2.

Extraction yields and phytochemical composition of the studied plants

Extract Capsicum frutescens Brassica oleacera var. butyris Brassica oleacera var. italica Basilicum polystachyon
Yield* (%)
7.22%
12.18%
7.31%
8.61%
Physical aspect
Oily brown and viscous
Oily brown and viscous
Oily brown and viscous
Compact
Alkaloids
+
+
+
+
Anthocyanins
-
-
-
-
Anthraquinones
-
-
-
-
Flavonoids
+
+
+
+
Phenols
+
+
+
+
Coumarins
-
-
-
+
Tannins
-
+
+
-
Triterpenes
+
+
+
+
Sterols
+
+
+
+
Saponins - + - +

(+): Present; (-): Absent; *yield calculated as the ratio of the mass of the obtained methanol extract/mass of the plant powder.

Preliminary phytochemical screenings

The secondary metabolite classes such as alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, tannins, sterols and triterpenes were screened according to the standard phytochemical methods described by Harbone [16].

Bacteria strains and culture media

The studied microorganisms included both reference (from the American Type Culture Collection, ATCC) and clinical (Laboratory collection) strains of Escherichia coli, Enterobacter aerogenes, Providencia stuartii, Pseudomonas aeruginosa and Klebsiella pneumoniae (Table 3). They were maintained at 4°C and sub-cultured on a fresh appropriate Mueller Hinton Agar (MHA) for 24 h before any antibacterial test. The Mueller Hinton Broth (MHB) was used for all antibacterial assays.

Table 3.

Bacterial strains and features

Bacteria and strains Features References
Escherichia coli
ATCC 8739
References strains
 
ATCC 10536
References strains
 
AG100 Atet
AG 100 sur-expressing AcrAB pumps, contaning TETR gène acrF
[14]
AG100
Wild-typeE. ColiK-12
[15]
AG102
AG100 Sur-exprissingAcrAB pumps.
[17]
MC4100
Wild typeE. coli
 
Enterobacter aerogenes
ATCC 13048
References strains
 
EA27
Clinical MDR isolate exhibiting energy-dependent norfloxacin and chloramphenicol efflux with KANR AMPR NALR STRR TETR
[18]
EA-3
Clinical MDR isolate CHLR, NORR, OFXR, SPXR, MOXR, CFTR, ATMR, FEPR
[18]
EA 289
KAN sensitive derivative d’EA27
[18]
EA 294
EA289 sur-expressing AcrA pumps Exhibiting KANR
[18]
EA 298
EA289 TolC KANR
[18]
CM64
CHLRresistant variant obtained from ATCC13048 over-expressing the AcrAB pump
[18]
Klebsiella pneumoniae
ATCC 11296
References strains
 
K-2
Clinical MDR isolate exhibiting energy-dependent norfloxacin and chloramphenicol efflux with KANR AMPR NALR STRR TETR
Laboratory collection of UNR-MD1, University of Marseille, France
K-24
AcrAB-Tolc
KP 55
Clinical isolate MDR, TETR, AMPR, ATMR, CEFR
[17]
KP 63
Clinical isolate du MDR, TETR, CHLRAMPR, ATMR
[17]
Pseudomonas aeruginosa
PA01
References strains
 
PA124
MDR Clinical isolate
[15]
Providencia stuartii ATCC 29916
References strains
 
NAE16 MDR clinical isolate AcrAB-TolC [15]

aAMP, ATMR, CEFR, CFTR, CHLR, FEPR, KANR, MOXR, STRR, TETR. Resistance to ampicillin, aztreonam, cephalothin, cefadroxil, chloramphenicol, cefepime, kanamycin, moxalactam, streptomycin, and tetracycline; OMPF and OMPC: Outer Membran Protein F and C respectively. AcrAB-Tol: Efflux pump of type AcrAB associated to one porine of type TolC.

Chemicals for antibacterial assays

Nine commonly used antibiotics including tetracycline (TET), cefepime (CEP), streptomycin (STR), ciprofloxacin (CIP), norfloxacin (NOR), chloramphenicol (CHL), ampicillin (AMP), erythromycin (ERY), kanamycin (KAN) (Sigma-Aldrich, St Quentin Fallavier, France) were used for potentiation assay. p-Iodonitrotetrazolium chloride 0.2% (INT) and phenylalanine arginine β-naphthylamide (PAβN) (Sigma-Aldrich) were used as bacterial growth indicator and efflux pumps inhibitor respectively. Dimethylsulfoxide 10% (DMSO) was used as solvent for all extracts.

Bacterial susceptibility determinations

The minimal inhibitory concentrations (MIC) of the plant extracts against the studied bacteria were determined by rapid INT colorimetric assay [19,20]. Briefly, the test samples were first dissolved in DMSO/MHB. The solution obtained was then added to MHB in a 96-well microplate followed by a two fold serial dilution. One hundred microliters (100 μL) of inoculum (1.5 × 106 CFU/mL) prepared in MHB was then added. The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a shaker and incubated at 37°C for 18 h. The final concentration ranges were 8-1024 μg/mL for plant extracts and 2-512 μg/mL for reference antibiotic chloramphenicol (CHL). Wells containing MHB (100 μL), 100 μL of inoculum and DMSO at a final concentration of 2.5% served as negative growth inhibition control. MIC was detected after 18 h of incubation at 37°C, following addition (40 μL) of 0.2 mg/mL INT and incubation at 37°C for 30 min. Viable bacteria reduced the yellow dye to pink. MIC was defined as the lowest sample concentration that prevented this change and exhibited complete inhibition of bacterial growth [21]. The minimal bactericidal concentrations (MBC) of the samples was determined by taking 50 μL of the suspensions from the wells which did not show any growth after incubation during MIC assays to a new 96-well microplate containing 150 μL of fresh broth per well. The plate was further re-incubated at 37°C for 48 hours the addition of INT. The MBC was defined as the lowest concentration of samples which completely inhibited the growth of bacteria. Samples were tested alone and in the presence of PAβN at 30 μg/mL final concentration [22].

To evaluate the potentiating effect of tested extracts, a preliminary combination at their sub-inhibitory concentrations (MIC/2, MIC/5, MIC/10 and MIC/20) with antibiotics was assessed against P. aeruginosa PA124 strain. The appropriate sub-inhibitory concentrations were then selected on the basis of their ability to improve the activity of the maximum antibiotic. These sub-inhibitory 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 [23,24].

Results

Phytochemical composition of the tested plant’s extracts

The results of the qualitative phytochemical analysis showed that each of the studied extract contained alkaloids, phenols, flavonoids, triterpenes and sterols. None of them contained anthocyanins and anthraquinones. Other phytochemical classes have been selectively detected as shown in Table 2.

Antibacterial activity of the plant’s extracts

Bacterial strains and MDR isolates were tested for their susceptibility to plant extracts and chloramphenicol. The results summarized in Table 4 the selectivity of the extracts towards the tested bacteria, with MIC values ranging from 256 to 1024 μg/mL on the majority of the 22 tested microorganisms. Capsicum frutescens extract displayed the largest spectrum of activity, 73% (16/22) against the tested bacteria; followed by Brassica oleacera var. italica, 50% (11/22); Basilicum polystachyon 41% (9/22) and Brassica oleacera var. butyris 27% (6/22) extracts. The lowest MIC value (256 μg/mL) was recorded with Basilicum polystachyon extract against P. stuartii (ATCC 29916) and E. aerogenes (ATCC 13048). No significant MBC value was recorded.

Table 4.

MIC and MBC of the tested plants extracts and CHL on the studied bacterial species

Strains bacterial Capsicum frutescens
Brassica oleacera var. varbutyris
Brassica oleacera var. italica
Basilicum polystachyon
Chloramphenicol
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
Escherichia coli
ATCC 8739
-
-
1024
-
-
-
-
-
8
512
ATCC 10536
512
-
-
-
1024
-
-
-
2
128
AG100 Atet
512
-
1024
-
1024
-
512
1024
64
64
AG100
-
-
-
-
-
-
-
-
16
128
AG102
1024
-
-
-
1024
-
1024
-
8
-
MC4100
1024
-
512
-
512
-
1024
-
128
128
Enterobacter aerogenes
ATCC 13048
1024
-
1024
-
1024
-
256
-
8
32
EA27
-
-
-
-
-
-
-
-
256
NT
EA-3
1024
-
-
-
1024
-
1024
-
-
-
EA294
-
-
-
-
-
-
-
-
256
512
EA298
512
-
-
-
-
-
-
-
4
16
EA 289
1024
-
-
-
-
-
-
-
128
-
CM64
1024
-
-
-
1024
-
-
-
128
-
Klebsiella pneumoniae
ATCC 11296
1024
-
1024
-
1024
-
-
-
8
512
K-2
512
-
-
-
1024
-
512
-
64
NT
K-24
1024
-
-
-
1024
-
1024
-
16
256
KP 55
1024
-
-
-
-
-
512
-
32
-
KP 63
512
-
-
-
-
-
-
-
128
NT
Pseudomonas aeruginosa
PA01
-
-
-
-
-
-
-
-
64
NT
PA124
-
-
-
-
-
-
-
-
512
NT
Providencia stuartii ATCC 29916
1024
-
1024
-
1024
-
256
-
4
32
NAE16 1024 - - - - - - - 256 NT

NT: Not determined; -: superior to 1024 μL for extracts and superior to 512 μg/mL for antibiotics; CHL: Chloramphenicol; Values in Bold are the lowest MIC values for the plant extracts.

Eight (8) of the twenty two (22) studied MDR bacteria were also tested for their susceptibility to the plant extracts in the presence of PAβN (Table 5). The largest spectrum of activity was recorded with B. oleacera var. butyris extract against 75% (6/8) tested MDR bacteria. This efflux pumps inhibitor (EPI) also improved the activity of C. frutescens extract against E. coli (AG100), K. pneumoniae (KP53) and E. aerogenes (EA27) as well as that of B. polystachyon against P. stuartii (NAE16).

Table 5.

Antibacterial activities of extracts alone and in the presence of PAβN

Bacterial strains Capsicum frutescens Brassica oleacera var. butyris Brassica oleacera var . italica Basilicum polystachyon CHL PAβN
AG100
1024 (256)
- (1024)
- (1024)
- (-)
16 (4)
>128
AG100 Atet
512 (512)
1024 (512)
1024 (1024)
- (-)
64(32)
>128
CM64
1024 (1024)
- (-)
1024 (512)
1024 (1024)
128 (64)
>128
EA27
- (512)
- (128)
- (512)
- (-)
256 (64)
>128
KP55
- (-)
- (1024)
- (1024)
- (-)
64(8)
>128
KP63
512 (256)
- (1024)
- (-)
- (-)
128(16)
>128
PA124
- (-)
- (-)
- (-)
- (-)
512(128)
>128
NAE16 - (-) - (1024) - (-) - (1024) 256(64) >128

( ): MIC value of extract in presence of PAβN; -: >1024 μg/mL for extracts and >512 μg/mL for antibiotic; CHL: Chloramphenicol.

Antibacterial activity of extract-antibiotic combination

A preliminary assay against P. aeruginosa PA124 strain allowed selecting MIC/2 and MIC/5 as appropriate sub-inhibitory concentrations to be used on other bacteria (Table 6). Synergistic effects were observed with all the tested extracts. Brassica oleacera var. italica and B. oleacera var. butyris extracts potentiate (0.125 < FIC < 0.5 and 0.031 < FIC < 0.5 respectively) the effects of the majority of antibiotics on most of the tested MDR bacteria (Table 7). Extracts from C. frutescens and B. polystachyon showed synergistic effects with six of the nine studied antibiotics, with 0.125 < FIC < 0.5 and 0.25 < FIC < 0.5 respectively.

Table 6.

MICs of antibiotics in combination with plant extracts against P. aeruginosa PA124

Plants’ extracts CEF AMP CIP ERY KAN TET STR CHL NOR
   ATB ALONE
- (-)
- (-)
64
512
128
64
64
512
256
Capsicum frutescens
MIC/2
- (-)
- (-)
32 (0,5) S
256 (0,5) S
128 (1)1
32 (0,5) S
256 (4)1
256 (0,5) S
128 (0,5) S
MIC/5
- (-)
- (-)
32 (0,5) S
256(0,5) S
128 (1)1
64 (1)1
256 (4)1
256 (0,5) S
128 (0,5) S
MIC/10
- (-)
- (-)
32 (0,5) S
256 (0,5) S
128 (1)1
64 (1)1
256 (4)1
256 (0,5) S
128 (0,5) S
MIC/20
- (-)
- (-)
64 (1) 1
256 (0,5) S
256 (2)1
64 (1)1
256(4)1
256 (0,5) S
128 (0,5) S
Brassica oleacera var. butyris
MIC/2
- (-)
- (-)
32 (0,5) S
256 (0,5) S
16 (0,125) S
16 (0,25) S
32 (0,5) S
256 (0,5) S
128 (0,5) S
MIC/5
- (-)
- (-)
32(0,5) S
256 (0,5) S
16 (0,125) S
16 (0,25) S
32 (0,5) S
256 (0,5) S
128 (0,5) S
MIC/10
- (-)
- (-)
32(0,5) S
256 (0,5) S
16 (0,125) S
32 (0,25) S
32 (0,5) S
256 (0,5) S
128 (0,5) S
MIC/20
- (-)
- (-)
32 (0,5) S
256 (0,5) S
32 (0,25) S
32 (0,25) S
64 (1)1
256 (0,5) S
128 (0,5) S
Brassica oleacera var. Italica
MIC/2
- (-)
- (-)
32 (0,5) S
256 (0,5) S
128 (1)1
32 (0,25) S
32 (0,5) S
256 (0,5) S
128 (0,5) S
MIC/5
- (-)
- (-)
64(1)1
256 (0,5) S
128 (1)1
32 (0,25) S
32 (0,5) S
256 (0,5) S
128 (0,5) S
MIC/10
- (-)
- (-)
64 (1)1
256 (0,5) S
128 (1)1
32 (0,25) S
64 (1)1
512 (1)1
256 (1)1
MIC/20
- (-)
- (-)
64(1)1
256 (0,5) S
128 (1)1
64 (1)1
64 (1)1
512 (1)1
256 (1)1
Basilicum polystachyon MIC/2
- (-)
- (-)
32 (0,5) S
128 (0,25) S
256(2)1
64 (1)1
64(1)1
256 (0,5) S
256 (1)1
MIC/5
- (-)
- (-)
32 (0,5) S
256 (0,5) S
256 (2)1
64 (1)1
64( 1)1
256 (0,5) S
256 (1)1
MIC/10
- (-)
- (-)
64 (1)1
256 (0,5) S
256 (2)1
64 (1)1
64 (1)1
256 (0,5) S
256 (1)1
MIC/20 - (-) - (-) 64 (1)1 256 (0,5) S 256 (2)1 64 (1)1 64 (1)1 256 (0,5) S 256 (1)1

s: Synergy; 1: Indifference; A: Antagonism; ( ): fractional inhibitory concentration or FIC; -: MIC > 512 μg/mL; ATB: Antibiotic; CIP: Ciprofloxacin, NOR: Norfloxacin, CHL: Chloramphenicol, STR: Streptomycin, TET: Tetracycline, KAN: Kanamycin, ERY: Erythromycin, AMP: Ampicillin and CEF: Cefepime; The values in bold represent the cases of synergy between extract and antibiotic.

Table 7.

MIC of antibiotics in combination with plant at their MIC/2 and MIC/5 against selected MDR bacteria strains

Antibiotics Plant extracts and MIC
Bacterial strains  
Capsicum frutescens
Brassica oleacera var. butyris
Brassica oleacera var. Italica
Basilicum polystachyon
MIC MIC/2 MIC/5 MIC/2 MIC/5 MIC/2 MIC/5 MIC/2 MIC/5
CEF
AG100
-
-
-
-
-
-
-
-
-
EA27
256
-
-
-
-
128 (0.5) s
256 (1)1
256 (1)1
256 (1)1
CM64
-
-
-
-
-
-
-
-
-
KP55
-
-
-
-
-
-
-
-
-
KP63
-
-
-
-
-
-
-
-
-
NAE16
-
-
-
-
-
-
-
-
-
PA124
 
 
 
 
 
 
 
 
 
AMP
AG100
-
-
-
-
-
-
-
-
-
EA27
-
-
-
-
-
-
-
-
-
CM64
-
-
-
-
-
-
-
-
-
KP55
-
-
-
-
-
-
-
-
-
KP63
-
-
-
-
-
-
-
-
-
NAE16
-
-
-
-
-
-
-
-
-
CIP
AG100
32
32 (1)I
64 (2)1
8 (0.25) S
8 (0.25) S
64 (2)1
64 (2)I
64 (2)1
128 (4)1
EA27
16
32 (2)1
32 (2)1
4 (0.25) S
4 (0.25) S
8 (0.5) S
8 (0.5) S
128 (8)A
128 (8)A
CM64
16
16 (1)I
16 (1)I
16 (1)I
16 (1)I
16 (1)I
16 (1)I
64 (4)1
128 (8)A
KP55
16
4 (0.25) S
8 (0.5) S
2 (0.125) S
4 (0.25) S
4 (0.25) S
16 (1)I
8 (0.5) S
8 (0.5) S
KP63
8*
4 (0.5) S
4 (0.5) S
1* (0.125) S
1* (0.125) S
1 (0.125) S
4 (0.25)S
16 (1)I
16 (1)I
NAE16
8*
2 (0.25) S
2 (0.25) S
2* (0.25) S
2* (0.25) S
2 (0.25) S
2 (0.25)S
8* (1)I
8 (1)I
PA124
64
32 (0.5) S
32 (0.5) S
32 (0.5) S
32 (0.5) S
32 (0.5) S
64 (1)1
32 (0.5) S
32 (0.5) S
ERY
AG100
32
16 (0.5) S
16 (0.5) S
8 (0.25) S
8 (0.25) S
16 (0.5) S
16 (0.5) S
64 (2)1
64 (2)1
EA27
32
32 (1)I
32 (1)I
64 (2)1
64 (2)1
64 (2)1
64 (2)1
64 (2)1
64 (2)1
CM64
32
64 (2)1
16 (0.5) S
32 (1)I
32 (1)I
32 (1)I
32 (1)I
64 (2)1
64 (2)1
KP55
128
128 (1) I
128 (1) I
64 (0.5) S
64 (0.5) S
64 (0.5) S
128 (1)I
256 (2)1
256 (2)1
KP63
128
32 (0.25) S
64 (0.5) S
32 (0.25) S
64 (0.5) S
64 (0.5) S
64 (0.5) S
256 (2)1
256 (2)1
NAE16
128
16 (0.125) S
16 (0.125) S
32 (0.25) S
64 (0.5) S
128 (1)I
128 (1)I
256 (2)1
256 (2)1
PA124
512
256 (0.5) S
256 (0.5) S
256 0.5) S
256 (0.5) S
256 (0.5)S
256 (0.5) S
128 (0.25) S
256 0.5) S
KAN
AG100
32
32 (1)I
64 (2)1
16 (0.5) S
16 (0.5) S
32 (1)I
32 (1)I
32 (1)I
32 (1)I
EA27
32
8 (0.25) S
8 (0.25) S
8 (0.25) S
8 (0.25) S
16 (0.5)S
16 (0.25) S
64 (2)1
64 (2)1
CM64
64
64 (1)I
64 (1)I
16 (0.25) S
32 (0.5) S
32 (0.5)S
32 (0.5) S
32 (0.5) S
32 (0.5) S
KP55
64
16 (0.25) S
16 (0.25) S
16 (0.25) S
16 (0.25) S
16 (0.25)S
16 (0.25) S
16 (0.25) S
16 (0.25) S
 
KP63
64
64 (1)I
64 (1)I
16 (0.25) S
16 (0.25) S
16 (0.25) S
32 (0.5) S
32 (0.5) S
32 (0.5) S
 
NAE16
64
64 (1)I
64 (1)I
32 (0.5) S
32 (0.5) S
64 (1)I
64 (1)I
64 (1)I
64 (1)I
 
PA124
128
128 (1)I
128 (1)I
16 (0.125) S
16 (0.125) S
128 (1)I
128 (1)I
256 (2)1
256 (2)1
TET
AG100
32
8 (0.25) S
8 (0.25) S
16 (0.5) S
16 (0.5) S
4 (0.25) S
4 (0.125) S
16 (0.5) S
32 (1)I
 
EA27
128
64 (0.5) S
64 (0.5) S
16 (0.125) S
16 (0.125) S
4 (0.031) S
32 (0.25) S
64 (0.5) S
64 (0.5) S
 
CM64
64
128 (2)1
128 (2)1
4 (0.062) S
8 (0.125) S
64 (1)I
64 (1)I
128 (2)1
256 (4)1
 
KP55
16
2 (0.125) S
4 (0.25) S
1 (0.062) S
1 (0.062) S
2 (0.125) S
2 (0.125) S
16 (1)I
16 (1)I
 
KP63
32
8 (0.25) S
8 (0.25) S
8 (0.25) S
16 (0.5) S
16 (0.5) S
8 (0.25) S
16 (0.5) S
16 (0.5) S
 
NAE16
128
64 (0.5) S
64 (0.5) S
128 (1)I
128 (1)I
64 (0.5) S
64 (0.5) S
-
-
 
PA124
64
32 (0.5) S
64 (1)I
16 (0.25) S
16 (0.25) S
32 (0.5) S
32 (0.5) S
64 (1)I
64 (1)I
STR
AG100
64
256 (4)1
256 (4)1
128 (1)I
128 (1)I
64 (0.5) S
128 (1)I
128 (1)I
128 (1)I
EA27
8
32 (4)1
32 (4)1
4 (0.5)S
8 (1)I
2 (0.25) S
2 (0.5) S
8 (1)I
8 (1)I
CM64
64
256 (4)1
256 (4)1
8 (0.125) S
16 (0.5) S
16 (0.5) S
16 (0.5) S
64 (1)I
64 (1)I
KP55
16
32 (4)1
32 (4)1
16 (1)I
16 (1)I
16 (1)I
16 (1)I
16 (1)I
16 (1)I
KP63
64
256 (4)1
256 (4)1
128 (1)I
128 (1)I
128 (1)I
128 (1)I
128 (1)I
128 (1)I
NAE16
64
256 (4)1
256 (4)1
64 (0.5) S
64 (0.5) S
64 (0.5) S
64 (0.5) S
128 (1)I
128 (1)I
PA124
64
256 (4)1
256 (4)1
32 (0.5) S
32 (0.5) S
32 (0.5) S
32 (0.5) S
64 (1)I
64 (1)I
CHL
AG100
16
4 (0.25) S
4 (0.25) S
4 (0.25) S
4 (0.25) S
16 (1)I
16 (1)I
16 (1)I
16 (1)I
EA27
256
-
-
64 (0.25) S
128 (0.5) S
32 (0.125) S
64 (0.25) S
256 (1)I
256 (1)I
CM64
128
32 (0.25) S
32 (0.25) S
16 (0.125) S
16 (0.125) S
64 (0.5) S
64 (0.5) S
256 (2)1
256 (2)1
KP55
64
32 (0.5) S
32 (0.5) S
16 (0.25) S
32 (0.5) S
32 (0.5) S
32 (0.5) S
64(1)1
64 (1)1
 
KP63
128
128 (1)I
128 (1)I
64 (0.5) S
64 (0.5) S
32 (0.125) S
32 (0.5) S
64 (0.5) S
64 (0.5) S
 
NAE16
256
16 (0.062) S
32 (0.125) S
8 (0.031) S
16 (0.062) S
32 (0.125) S
32 (0.125) S
128 (0.5) S
128 (0.5) S
 
PA124
512
256 (0.5) S
256 (0.5) S
256 (0.5) S
256 (0.5) S
256 (0.5) S
256 (0.5) S
256 (0.5) S
256 (0.5) S
NOR
AG100
16
16 (1)I
16 (1)I
4 (0.25) S
8 (0.5) S
8 (0.5) S
8 (0.5) S
16 (1)I
16 (1)I
EA27
16
128 (4)1
128 (4)1
8 (0.5) S
8 (0.5) S
2 (0.125) S
4 (0.25) S
8 (0.5) S
16 (1)I
CM64
128
256 (2)1
256 (2)1
8 (0.0625) S
16 (0.125) S
128 (1)I
128 (1)I
256 (2)1
256 (2)1
KP55
128
64 (0.5) S
64 (0.5) S
64 (0.5) S
64 (0.5) S
8 (0.0625) S
16 (0.125) S
64 (0.5) S
128 (1)I
KP63
8
32 (4)1
32 (4)1
4 (0.25) S
4 (0.25) S
8 (1)I
8 (1)I
8 (1)I
8 (1)I
 
NAE16
32
8 (0.25) S
16 (0.5) S
8 (0.25) S
8 (0.25) S
4 (0.125) S
4 (0.125) S
8 (0.25) S
8 (0.25) S
  PA124 256 128 (0.5) S 128 (0.5) S 128 (0.5) S 128 (0.5) S 128 (0.5) S 128 (0.5) S 256 (1)1 256 (1)1

s: Synergy; I: Indifference; A: Antagonism; ( ): FIC values; -: MIC > 512 μg/mL or not determined FIC; ATB: Antibiotic; CIP: Ciprofloxacin, NOR: Norfloxacin, CHL: Chloramphenicol, STR: Streptomycin, TET: Tetracycline, KAN: Kanamycin, ERY: Erythromycin, AMP: Ampicillin and CEF Cefepime; The values in bold represent the cases of synergy between extract and antibiotic.

Discussion

The Pharmacological potencies of plants’ secondary metabolites are well demonstrated. The qualitative phytochemical screening of the plant extracts showed the presence of several classes of secondary metabolites, such as alkaloids, flavonoids, phenols, triterpenes, sterols, saponins, tannins and coumarins. Several antibacterial activities associated to the presence of compounds belonging to these various classes were shown [25-27]. It should however be mentioned that the detection of an alleged bioactive class of secondary metabolite in a plant is not a guarantee for any biological property, as this will depend on the nature of the compounds as well as their concentrations and the possible interactions with other constituents [12]. The differences observed between the antibacterial activities of the extracts as observed in the present work could be due to the differences in their phytochemical composition [9]. According to the criteria of classification of the antibacterial activity of the phytochemicals [28], the extracts used in this study were moderately and/or weak active (256 ≤ MIC < 1024 μg/mL). Their direct use in the control of MDR bacterial infections could therefore be of limited importance. None-the-less, the obtained results can be considered as interesting when considering the fact that the extracts are obtained directly from edible plant materials.

Efflux pumps are responsible for the reduction of intracellular concentration of antibacterial compounds [29]. To tackle problems related to this phenomenon, an intensive search of efflux pumps inhibitors (EPI) is welcome [30]. The EPI blocks the efflux pumps and leads to the increase of the intracellular concentration of active principle contents of the extracts [29,31]. The activity of B. oleacera var. butyris extract against the tested bacteria in the presence of PAβN, increased in 75% of the cases. This suggests that some compounds present in this extract could be substrates of efflux pumps [31,32].

The extracts of B. oleacera var. butyris, B. oleracea var. Italica, Basilicum polystachyon and C. frutescens showed significant synergistic effects (0.031 < FIC < 0.5) with the majority of the tested antibiotics against the studied MDR strains. This suggests that the extracts might contain bioactive compounds that, combined with antibiotics, acted at different sites by various mechanisms [33,34]. These data indicate that a combination of these extracts with antibiotics could be envisaged to fight MDR bacteria.

Conclusion

These results provide promising baseline information for the potential use of Capsicum frutescens, Brassica oleacera var. italica, Basilicum polystachyon and Brassica oleacera var. butyris, independently or in combination with some commonly used antibiotics in the fight against MDR Gram-negative bacteria.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

FTK carried out the study; VK designed the experiments. FTK, AJS, AGF and VK wrote the manuscript; VK, JAKN and DED supervised the work; VK provided the bacterial strains; all authors read and approved the final manuscript.

Pre-publication history

The pre-publication history for this paper can be accessed here:

http://www.biomedcentral.com/1472-6882/14/258/prepub

Contributor Information

Francesco K Touani, Email: navires2000@yahoo.fr.

Armel J Seukep, Email: seukepp@yahoo.fr.

Doriane E Djeussi, Email: princessedoriane@yahoo.fr.

Aimé G Fankam, Email: agfankam@yahoo.fr.

Jaurès A K Noumedem, Email: jauresnoume@yahoo.fr.

Victor Kuete, Email: kuetevictor@yahoo.fr.

Acknowledgements

Authors are thankful to the Cameroon National Herbarium (Yaounde) for plants identification.

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