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

  • Francesco K Touani1,
  • Armel J Seukep1,
  • Doriane E Djeussi1,
  • Aimé G Fankam1,
  • Jaurès A K Noumedem1 and
  • Victor Kuete1Email author
BMC Complementary and Alternative MedicineThe official journal of the International Society for Complementary Medicine Research (ISCMR)201414:258

DOI: 10.1186/1472-6882-14-258

Received: 18 April 2014

Accepted: 16 July 2014

Published: 21 July 2014

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 numbera

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, MMTS0 2 : 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 [2527]. 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.

Declarations

Acknowledgements

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

Authors’ Affiliations

(1)
Department of Biochemistry, Faculty of Science, University of Dschang

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  35. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/258/prepub

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© Touani et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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