Open Access

Antibacterial and antibiotic-potentiation activities of the methanol extract of some cameroonian spices against Gram-negative multi-drug resistant phenotypes

  • Igor K Voukeng1,
  • Victor Kuete1Email author,
  • Jean P Dzoyem1,
  • Aimé G Fankam1,
  • Jaures A K Noumedem1,
  • Jules R Kuiate1 and
  • Jean-Marie Pages2
BMC Research Notes20125:299

DOI: 10.1186/1756-0500-5-299

Received: 27 January 2012

Accepted: 4 June 2012

Published: 15 June 2012

Abstract

Background

The present work was designed to evaluate the antibacterial properties of the methanol extracts of eleven selected Cameroonian spices on multi-drug resistant bacteria (MDR), and their ability to potentiate the effect of some common antibiotics used in therapy.

Results

The extract of Cinnamomum zeylanicum against Escherichia coli ATCC 8739 and AG100 strains showed the best activities, with the lowest minimal inhibitory concentration (MIC) of 64 μg/ml. The extract of Dorstenia psilurus was the most active when tested in the presence of an efflux pump inhibitor, phenylalanine Arginine-β- Naphtylamide (PAβN), a synergistic effect being observed in 56.25 % of the tested bacteria when it was combined with Erythromycin (ERY).

Conclusion

The present work evidently provides information on the role of some Cameroonian spices in the fight against multi-resistant bacteria.

Keywords

Multi-Drug Resistant bacteria Spices Methanol extract Cameroon

Background

Infectious diseases are one of the leading causes of morbidity and mortality worldwide, especially in developing countries [1, 3]. Following the massive use of antibiotics in human therapy, bacteria have developed several resistance mechanisms including the efflux of antibiotics [3]. Several Cameroonian spices are known to possess medicinal values [4]. In our previous report, we demonstared that several medicinal spices inhibited the growth of MDR bacteria and were also able to improve the activity of commonly used antibiotics [5]. In our continuous search of antimicrobial drugs from medicinal plant, we designed the present work to investigate the antibacterial potential against Gram-negative MDR bacteria of some of the commonly used medicinal spices in Cameroon such as Aframomum citratum (Pereira) K. Schum. (Zingiberaceae), Aframomum melegueta (Roscoe) K. Schum. (Zingiberaceae), Scorodophloeus zenkeri Harms (Caesalpiniaceae), Tetrapleura tetraptera (Schum. & Thonn) Taub. (Mimosaceae), Fagara leprieurii (Guill and Perr) Engl. (Rutaceae), Monodora myristica Dunal (Annonaceae), Piper guineense (Schum and Thonn) (Piperaceae), Dorstenia psilurus Welwitch (Moraceae), Imperata cylindricum Beauv. var. koenigii Durand and Schinz (Gramineae), Pentadiplandra brazzeana Baill. (Capparaceae) and Cinnamomum zeylanicum (Linn) Cor. (Lauraceae).

Material and methods

Plant materials and extraction

The eleven edible spices used in this work were purchased from Dschang local market, West Region of Cameroon in January 2010. The collected spices material were the fruits of Aframomum citratum Aframomum melegueta, Scorodophloeus zenkeri, Tetrapleura tetraptera, the seeds of Fagara leprieurii Monodora myristica and Piper guineense, the roots of Dorstenia psilurus Imperata cylindricum and Pentadiplandra brazzeana and the leaves of Cinnamomum zeylanicum. The plants were identified by Mr. Victor Nana of the National herbarium (Yaoundé, Cameroon) where voucher specimens were deposited under a reference number (Table 1). The extracts were obtained by methanol (MeOH) maceration as previously described [5].
Table 1

Spices used in the present study and evidence of their activities

Spice samples (Family)

Herbarium Voucher numbera

Traditional Treatment

Part used

Bioactive (or potentially active) compoundsband screened activitycfor crude plant extract

Aframomum citratum (Pereira) K. Schum. (Zingiberaceae)

37 736/HNC

Malaria, aphrodisiac, cancer [4, 6]

Fruits. leaves. seeds

Antimicrobial: Ethylacetate extract of fruits on Ec. Pa. Sa[7]

    

Cytotoxicity of fruits crude methanol extract [weak activity on leukemia CCRF-CEM and CEM/ADR5000 cells, and pancreatic MiaPaCa-2 cell lines] [4]

Aframomum melegueta (Roscoe) K. Schum. (Zingiberaceae)

39 065/HNC

Malaria, dysentery, carminative, dysmenorrheal, fertility, rubella, leprosy, cancer [6, 8]

Fruits, leaves

Antimicrobial : Aqueous and ethanol extract of leaves on Fo. An[9] Methanol extract of fruits (Q) on Sa. Bs. Ec. Pa. Ca[8]

    

Cytotoxicity of fruits crude methanol extract [weak activity on leukemia CCRF-CEM and pancreatic MiaPaCa-2 cell lines and significant activity on CEM/ADR5000 cells with IC50 value of 7.08 μg/ml] [4]

Cinnamomum zeylanicum (Linn) Cor. (Lauraceae)

22 309/SRFC

Cancer [4]

Fruits, leaves. bark

Antimicrobial : Cd, Cm, Lt, Fp[10, 11]

    

Cytotoxicity of leaves crude methanol extract [weak activity on leukemia CCRF-CEM and CEM/ADR5000 cells, and pancreatic MiaPaCa-2 cell lines] [4]

Dorstenia psilurus Welwitch (Moraceae)

44 839/HNC

Snake bite, rheumatism, head and stomach ache, hypertension, cancer [4, 12, 13].

Leaves, roots

Cytotoxicity of roots crude methanol extract [Significant activity with IC50 values of 7.18; 7.79 and 9.17 μg/ml respectively on leukemia CCRF-CEM cells, CEM/5000 cells and pancreatic MiaPaCa-2 cell lines] [4]

Fagara leprieurii (Guill and Perr) Engl. (Rutaceae)

37 632/HNC

Gastritis, gingivitis. bilharzias, antidiarrhoeal, cancer, laxative, antimicrobial, ulcer, gonorrhea, kidney ache., sterility [4, 14, 15]

Bark, leaves. roots

Antimicrobial : Ethanol extract of the seeds on Ca. Cn. Mg. Tm. Tr. Bci. Af. Afl. Sb[6]Essential oil : Sa[15]

    

Cytotoxicity of seeds crude methanol extract [weak activity on leukemia CCRF-CEM and pancreatic MiaPaCa-2 cell lines and significant activity on CEM/ADR5000 cells with IC50 value of 8.13 μg/ml] [4]

Imperata cylindricum Beauv. var. koenigii Durand et Schinz (ramineae)

30 139/SRFC

Diuretic, anti-inflammatory, dysentery, urinary tract infections, cancer [4, 16, 17]

Leaves, roots

Cytotoxicity of roots crude methanol extract [Significant activity with IC50 values of 8.4; 7.18 and 12.11 μg/ml respectively on leukemia CCRF-CEM cells, CEM/5000 cells and pancreatic MiaPaCa-2 cell lines] [4]

Monodora myristica Dunal (Annonaceae)

2 949/SRFC

Insecticidal, diuretic, constipation, anti-hemorrhage, headache, wounds, worm infections,cancer [4, 15, 18, 19]

Fruits, leaves. seeds

Antimicrobial : Fm. Afl. Af[18]; Essential oil : Af. Bc. Bs. Cgl. Ec. Kp. Sa. Sf[15]. Cytotoxicity of fruits seeds methanol extract [weak activity on leukemia CCRF-CEM and CEM/ADR5000 cells, and pancreatic MiaPaCa-2 cell lines] [4]

Pentadiplandra brazzeana Baill. (Capparaceae)

42 918/HNC

Gastric ulcer, cancer [4, 20]

Fruits, leaves

Cytotoxicity of roots crude methanol extract [weak activity on leukemia CCRF-CEM and CEM/ADR5000 cells, and pancreatic MiaPaCa-2 cell lines] [4]

Piper guineense (Schum and Thonn) (Piperaceae)

6 018/SRFC

Cough, bronchitis, rheumatism, insecticidal, anemia, carminative, stomach ache, cancer [4, 8, 21]

Fruits, leaves. bark

Insecticidal : Cs[20]Antimicrobial (Q); Ec. Sa. Bs. Pa. Ca. An[8, 22]

Scorodophloeus zenkerii Harms (Caesalpiniaceae)

44 803/HNC

Cancer [4]

Leaves. roots

Antimicrobial : Essential oil of stem bark on Ec, Sa, Bs, Cu [23].

    

Cytotoxicity of fruits crude methanol extract [weak activity on leukemia CCRF-CEM and CEM/ADR5000 cells, and pancreatic MiaPaCa-2 cell lines] [4]

Tetrapleura tetraptera (Schum. & Thonn) Taub. (Mimosaceae)

12 117/SRFC

Pain, arthritis, epilepsy, convulsion, gastric ulcer, cancer [4, 20]

Bark, leaves. roots

Cytotoxicity of fruits crude methanol extract [weak activity on leukemia CCRF-CEM and CEM/ADR5000 cells, and pancreatic MiaPaCa-2 cell lines] [4]

a(HNC): Cameroon National Herbarium; (SRFC): Société des reserves forestières du Cameroun; b(/): Not reported.

c[Screened activity: significant (S: CMI < 100 μg/ml). moderate (M : 100 < CMI ≤ 625 μg/ml). Weak (W: CMI > 625 μg/ml) Q: Qualitative activity based on the determination of inhibition zone [15]; Af : Aspergillus fumigatus. Afl : Aspergillus flavus. An: Aspergillus niger. Bc : Bacillus cereus. Bci : Botrytis cinerea. Bs : Bacillis subtilis. Bt: Botryodiploidia theobromae. Ca : Candida albicans. Cd : Clostridium difficile. Cm : Colletotrichum musae. Cn : Cryptococcus neoformans. Cs: Callosobruchus subinnotatus. Cu : Candida utilis. Ec : Escherichia coli. Fm :Fusarium moniliforme. Fo :Fusarium oxysporum. Fp : Fusarium proliferatum. Lt : Lasiodiplodia theobromae. Mg : Microsporum gypseum. Pa: Pseudomonas aeruginosa. Sa : Staphylococcus aureus. Sb: Scopulariopsis brevicaulis. Tm: Trichophyton mentagrophytes. Tr: Trichophyton rubrum.

Preliminary phytochemical investigations

The major secondary metabolites classes were screened according to the common phytochemical methods described by Harborne [24].

Chemicals for antimicrobial assays

Tetracycline (TET), cefepime (FEP), streptomycin (STR), ciprofloxacin (CIP), norfloxacin (NOR), chloramphenicol (CHL), cloxacillin (CLX), ampicillin (AMP), erythromycin (ERY), kanamycin (KAN) (Sigma-Aldrich, St Quentin Fallavier, France) were used as reference antibiotic. p-Iodonitrotetrazolium chloride (INT) and phenylalanine arginine β-naphthylamide (PAßN) were used as microbial growth indicator and efflux pumps inhibitor (EPI) respectively.

Bacterial strains and culture media

The studied microorganisms included reference (from the American Type Culture Collection) and clinical (Laboratory collection) strains of Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes and Enterobacter cloacae The bacterial strains and their features were previously reported [5]. The preliminary treatment of these organisms as well as the culture media were conducted as previously described [5].

Bacterial susceptibility determinations

The respective MICs of samples on the studied bacteria were determined using rapid INT colorimetric assay [25, 26] with some modifications as previously reported [5]. The inoculum concentration used was 1.5 x106 CFU/ml and the samples were incubated at 37 °C for 18 h [5]. The final concentration of DMSO was lower than 2.5 % and this concentration also served as negative control [5]. Chloramphenicol was used as reference antibiotic. The MICs of samples were detected after 18 h incubation at 37 °C, following addition (40 μl) of 0.2 mg/ml INT and incubation at 37 °C for 30 minutes [5]. MIC was defined as the lowest sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth [27].

Samples were tested alone and then, in the presence of PAßN at 20 mg/L final concentration as previously reported [5]. Four of the best extracts, those from A. citratum, C. zeylanicum, D. psilurus and T. tetraptera were also tested in association [5] at the concentrations selected following a preliminary assay on P. aeruginosa PA124 (See Additional file 1: Table S1). All assays were performed in triplicate and repeated thrice. Fractional inhibitory concentration (FIC) [5] were calculated and the interpretations were made as follows: synergistic (< 0.5), indifferent (0.5 to 4), or antagonistic (> 4) [28] (The FIC values available in Additional file 1: Table S2 and S3).

Results

Phytochemical composition of the spice extracts

The results of qualitative analysis showed that each plant contains various phytochemicals compounds such as alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, steroids, tannins and triterpenes as shown in Table 2.
Table 2

Extraction yields, aspects and phytochemical composition of the plant extracts

Spice samples

Extraction yield (%)*

Physical aspect

Phytochemical composition

   

Alkaloids

Anthocyanins

Anthraquinons

Flavonoids

Phenols

Saponins

Sterols

Tannins

Triterpenes

Aframomum citratum

2.6

Oily, dark green

+

-

-

+

+

-

-

+

+

Aframomum melegueta

7.3

Brown powder

+

-

-

-

-

+

-

-

+

Cinnamomum zeylanicum

8.4

Oily, dark green

+

-

-

+

+

-

+

+

-

Dorstenia psilurus

10.3

Oily, brown

+

+

+

+

+

+

-

+

+

Fagara leuprieurii

26.2

Creamy, brown

+

-

+

+

+

-

-

+

+

Imperata cylindricum

8.2

Creamy, brown

+

+

+

+

+

-

-

-

+

Monodora myristica

23.5

Oily, brown

+

-

+

+

+

-

-

-

+

Pentadiplandra brazzeana

4.6

Creamy, brown

+

-

-

+

+

-

-

-

-

Piper guineense

17.5

Creamy, brown

+

-

+

+

+

-

-

-

-

Scorodophloeus zenkeri

9.2

Creamy, dark green

+

-

-

+

+

-

-

+

-

Tetrapleura tetraptera

29.4

brown

+

-

+

+

+

+

-

+

+

(+): Present; (−): Absent; *The yield was calculated as the ratio of the obtained methanol extract according to the initial mass of the spice powder.

Antibacterial activity of the spice extracts

The results summarized in Table 3 summarize the MIC of the extract tested alone or in combination with PAβN on the tested microorganisms. Its shows that all the studied extracts were active on at least one microbial strain. A. citratum showed the best activity, it inhibitory effect being recorded on 85% (24/28) of the tested bacteria. Other samples were less active, their inhibitory potencies being observed on 75% of tested bacteria (21/28) for I. cylindricum and C. zeylanicum, 67.9 % (19/28) for A. melegueta, D. psilurus, F. leprieuri and T. tetraptera; 64.3% (18/28) for M. myristica and S. zenkeri; 50 % (14/28) for P. guineense and 42.9 % (12/28) for P. brazzeana.
Table 3

Minimal inhibitory concentration (MIC) of the studied plants extracts and chloramphenicol on the studied bacterial species

Bacterial strains

Tested samples and MIC in μg/ml in the absence and presence of PAßN (in parenthesis)

 

Aframomum citratum

Aframomum melegueta

Imperata cylindricum

Cinnamomum zeylanicum

Dorstenia psilurus

Fagara leprieuri

Monodora myristica

Pentadiplandra brazzeana

Piper guineense

Scorodophloeus zenkeri

Tetrapleura tetraptera

CHL

E. coli

            

ATCC8739

512

512

512

64

-

512

1024

1024

1024

1024

1024

1

ATCC10536

1024

512

1024

512

128

256

1024

512

1024

512

1024 (1024)

32 (<2)

AG100

1024 (1024)

1024 (1024)

1024 (256)

- (64)

- (1024)

1024 (1024)

512

1024 (1024)

1024 (512)

1024 (1024)

1024 (1024)

4 (<2)

AG100A

512 (128)

1024 (1024)

1024 (128)

512 (128)

512 (128)

512 (512)

1024 (1024)

- (−)

1024 (1024)

1024 (1024)

1024 (1024)

<2 (<2)

AG100ATET

512 (512)

1024 (1024)

1024 (1024)

512 (512)

512 (128)

1024 (1024)

-

-

1024

512

1024

32 (<2)

AG102

1024

-

1024

1024

512

1024

-

-

-

-

-

16 (<2)

MC4100

512 (512)

512 (256)

1024 (1024)

1024 (1024)

512 (256)

512

- (−)

1024 (1024)

1024 (1024)

1024 (1024)

512 (512)

4 (<2)

W3110

512 (256)

512 (512)

512 (512)

512 (512)

512 (256)

256

512

1024 (1024)

1024 (128)

512

512 (512)

1 (<2)

E. aerogenes

            

ATCC13048

1024

-

1024

1024

1024

1024

1024

-

-

-

-

8 (<2)

CM64

1024 (1024)

1024 (1024)

512 (128)

1024 (512)

512 (256)

1024 (1024)

1024 (1024)

1024 (1024)

1024 (1024)

1024 (1024)

512 (512)

32

EA27

512 (512)

1024 (1024)

512 (512)

512 (512)

- (−)

1024 (1024)

1024 (1024)

1024 (1024)

-(−)

1024 (1024)

1024 (512)

64 (32)

EA289

-

1024

-

1024

-

-

1024

1024

-

-

1024

256

EA298

1024

512

-

-

1024

-

256

256

512

256

1024

256

EA3

-

-

-

-

-

1024

-

-

-

-

-

256

E. cloacae

            

BM47

512 (512)

1024 (1024)

1024 (1024)

1024 (512)

1024 (128)

1024 (1024)

1024 (1024)

1024

1024

1024

1024

- (8)

BM67

512 (512)

1024 (1024)

1024 (1024)

1024 (1024)

1024 (128)

- (−)

- (−)

- (−)

- (−)

- (−)

- (−)

- (32)

ECCI69

512 (512)

1024 (1024)

1024 (1024)

1024 (1024)

-(−)

-(−)

1024 (1024)

- (−)

1024 (1024)

1024 (1024)

1024 (512)

- (32)

K. pneumoniae

            

ATCC12296

1024

1024

1024

1024

1024

1024

512

-

-

-

1024

4

K2

1024

-

1024

1024

1024

-

1024

-

-

-

-

-

K24

1024

1024

1024

1024

1024

1024

512

-

-

1024

1024

32 (<2)

KP55

512

1024

256

512

512

1024

1024

-

-

-

1024

32(<2)

KP63

512 (512)

1024 (1024)

1024 (1024)

512 (512)

512 (128)

1024 (512)

1024 (1024)

512

1024 (1024)

1024 (512)

1024 (1024)

64(<2)

P. stuartuii

            

ATCC29916

1024 (1024)

- (−)

-(−)

-(−)

1024 (1024)

-(−)

-(−)

-(−)

1024 (1024)

1024 (1024)

1024 (1024)

8

NEA16

1024 (512)

- (−)

1024 (1024)

512 (512)

512 (256)

1024

1024

-

-

1024

-

64(<2)

PS2636

1024

-

-

-

-

-

-

-

-

-

-

-

PS299645

512

512

1024

1024

1024

1024

-

1024

1024

512

1024

128

P. aeruginosa

            

PA01

-

-

-

-

-

-

-

-

-

-

-

-

PA124

-

-

-

-

-

-

-

-

-

1024

-

32(<2)

(−): MIC not detected at up to 1024 μg/ml for the les extracts and 256 μg/ml for chloramphenicol. () : values in parenthesis are MIC of substance in the presence of PAßN at 20 μg/ml. The MIC of PAßN was 64 μg/ml on E. coli. AG100A. 512 μg/ml on ATCC11296. BM67. EA27. EA289; 1024 μg/ml on AG100ATET. ATCC13048. CM64; and > 1024 μg/ml on other bacteria. CHL: chloramphénicol; (in bold): significant MIC value.

Role of efflux pumps in susceptibility of gram negative bacteria to the tested spice extracts

Potentiating effect of EPI was not observed on tested bacteria when associated with M. myristica, P. brazzeana, T. tetraptera and S. zenkeri. PAβN weakly increased the activity of A. citratum, A. melegueta, F. leprieuri, I. cylindricum, C. zeylanicum and P. guineense. The activity of D. psilurus in the presence of EPI significantly increased on most of the tested bacteria (except against P. stuartii ATCC29916, E. cloacae ECCI69 and E. aerogenes EA27) (see Table 3).

Effects of the association of some spice extracts with antibiotics

A. citratum, C. zeylanicum, D. psilurus and T. tetraptera (Tables 4, 5, 6 and 7) were associated to antibiotics in view of evaluating the possible synergistic effect of these associations. A preliminary study using P. aeruginosa PA124 was carried out with ten antibiotics (CLX, AMP, ERY, KAN, CHL, TET, FEP, STR, CIP and NOR) to select the appropriate sub-inhibitory concentrations to be used. MIC/2.5 and MIC/5 were then selected as the sub-inhibitory concentrations (see Additional file 1: Table S1). All of these four extracts were then tested in association with antibiotics previously listed on strains of E. coli AG100ATET and AG102, E. aerogenes CM64, K. pneumonia KP63 and P. aeruginosa PA124. No antagonistic effect (FIC > 4) was observed between extracts and antibiotics meanwhile indifference was observe between T. tetraptera and antibiotics in most of the case (see Tables 5, 6, and 7, Additional file 1: S2, S3, S4 and S5). Significant increase of the activity was observed with the association of the extracts of A. citratum and D. psilurus on E. aerogenes CM64 and K. pneumoniae KP63, and with C. zeylanicum against K. pneumoniae KP63. A significant decrease (synergy effect) of MIC values was also observed when ERY was associated with various extracts, and when extracts of A. citratum and C. zeylanicum were each combined with aminoglycosides (KAN, STR), the best activity being noted against E. aerogenes CM64.
Table 4

Minimal inhibitory concentration (MIC) in μg/ml of antibiotics in the absence and presence sub-inhibitory concentrations of Aframomum citratum extract against some MDR bacteria

Bacterial strains

Antibiotics and MIC in absence and presence ofAframomum citratum extract

Ampicillin

Cefepime

Chloramphenicol

Ciprofloxacin

Cloxacillin

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

AG100Atet

-

-

-

-

-

-

256

32 (8) S

64 (4) S

256

128 (2) S

256 (1) I

-

-

-

AG102

-

-

-

128

128 (1) I

128 (1) I

16

8 (2) S

8 (2) S

<2

<2

<2

-

-

-

CM64

-

256 (1)I

-

-

64 (>4) S

-

nt

nt

nt

nt

nt

nt

-

-

-

KP63

-

32 (>8)S

-

256

32 (8) S

256 (1) I

-

64 (>4) S

256 (>1)S

64

64 (1) I

64 (1) I

-

256 (>1)S

-

PA124

128

16 (8) S

64 (2) S

128

128 (1) I

256 (0.5) I

32

16 (2) S

16 (2) S

16

4 (4) S

16 (1) I

-

-

-

Bacterial strains

Erythromycin

Kanamycin

Norfloxacin

Streptomycin

Tetracyclin

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

AG100Atet

64

16 (4) S

32 (2) S

-

32 (>8)S

256

128

16 (8) S

128 (1) I

<2

<2

<2

2

<2 (>1)S

2 (1) I

AG102

32

16 (2) S

16 (2) S

<2

<2

<2

<2

<2

<2

-

128 (>2)S

256 (>1)S

<2

<2

<2

CM64

-

128 (>2)S

256 (>1)S

4

<2

<2

4

<2 (>2)S

4 (1) I

32

4 (8) S

8 (4) S

nt

nt

nt

KP63

16

<1 (>16)S

4 (4) S

32

16 (2) S

32 (1) I

-

128 (>2)S

256 (>1)S

<4

<4

<4

<2

<2

<2

PA124

128

64 (2) S

64 (2) S

128

16 (8) S

64 (2) S

64

8 (8) S

32 (2) S

nt

nt

nt

8

2 (4) S

2 (4) S

MIC/2.5: concentration of plant extract added equal to 204.8 μg/mL for AG100ATET. KP63; and to 409.6 μg/mL for PA124. CM64. AG102.

MIC/5: concentration of plant extract added equal to 102. 4 μg/mL for AG100ATET. KP63; and to 204.8 μg/mL for PA124. CM64. AG102.

(): Folds decreasing of MIC. S: synergy. I: indifference. nt: not tested; (−): MIC > 256.

Table 5

Minimal inhibitory concentration (MIC) of antibiotics in absence and presence of Cinnamomum zeylanicum extract (μg/mL)

Bacterial strains

Antibiotics and MIC in absence and presence ofCinnamomum zeylanicum extract

Ampicillin

Cefepime

Chloramphenicol

Ciprofloxacin

Cloxacillin

Alone

MIC/2.5

MIC/5

Alone

MIC/2

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

AG100Atet

-

-

-

-

-

-

256

16 (16) S

32 (8) S

256

64 (4) S

128 (2) S

-

-

-

AG102

-

-

-

128

256 (0.5) I

256 (1) I

16

8 (2) S

16 (1) I

<2

<2

<2

-

256 (>1)S

-

CM64

-

256 (>1) S

-

-

256 (>1) S

-

nt

nt

nt

nt

nt

nt

-

-

-

KP63

-

32 (>8) S

-

256

32 (8) S

256 (1) I

-

32 (>8) S

256 (>1) S

64

128 (0.5) I

128(0.5)I

-

64 (>4)S

256 (>1)S

PA124

128

16 (8) S

64 (2) S

128

128 (1) I

128 (1) I

32

2 (16) S

8 (4) S

16

8 (2) S

16 (1) I

-

-

-

Bacterial strains

Erythromycin

Kanamycin

Norfloxacin

Streptomycin

Tetracycline

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

AG100Atet

64

16 (4) S

32 (2) S

-

16 (>16)S

128 (>2)S

128

128 (1) I

128 (1) I

<2

<2

<2

2

2 (1) I

2 (1) I

AG102

32

16 (2) S

16 (2) S

<2

<2

<2

<2

<2

<2

-

256 (>1) S

256 (>1)S

<2

<2

<2

CM64

-

128 (>2)S

256 (>1)S

4

<2 (>2) S

<2 (>2) S

4

<2 (>2) S

4 (1) I

32

4 (8) S

8 (4) S

nt

nt

nt

KP63

16

1 (16) S

4 (4) S

32

32 (1) I

32 (1) I

-

128 (>2)S

256 (>1)S

<4

<4

<4

<2

<2

<2

PA124

128

16 (8) S

32 (4) S

128

8 (16) S

32 (4) S

64

32 (2) S

64 (1) I

nt

nt

nt

8

2 (4) S

2 (4) S

MIC/2.5: concentration of plant extract added equal to 204.8 μg/mL for AG100ATET. KP63; and to 409.6 μg/ml for PA124. CM64. AG102.

MIC/5: concentration of plant extract added equal to 102. 4 μg/mL for AG100ATET. KP63; and to 204.8 μg/ml for PA124. CM64. AG102.

(): Folds decreasing of MIC. S: synergy. I: indifference. nt: not tested; (−): MIC > 256 μg/ml.

Table 6

Minimal inhibitory concentration (MIC) of antibiotics in absence and presence extracts Dorstenia psilurus (μg/ml)

Bacterial strains

Antibiotics and MIC in absence and presence ofDorstenia psilurus extract

Ampicillin

Cefepime

Chloramphenicol

Ciprofloxacin

Cloxacillin

Alone

MIC/2.5

MIC/5

Alone

MIC/2

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

AG100Atet

-

-

-

-

-

-

256

128 (2) S

256 (1) I

256

64 (4) S

128 (2) S

-

-

-

AG102

-

-

-

128

256 (0.5) I

256 (0.5) I

16

4 (4) S

4 (4) S

<2

<2

<2

-

-

-

CM64

-

256 (>1)S

-

-

64 (>4) S

64 (>4) S

nt

nt

nt

nt

nt

nt

-

256 (>1) S

-

KP63

-

32 (>8) S

-

256

64 (4) S

128 (2) S

-

64 (>4) S

256 (>1) S

64

64 (1) I

64 (1) I

-

64 (>4) S

256 (>1) S

PA124

128

64 (2) S

64 (2) S

128

128 (1) I

128 (1) I

32

16 (2) S

32 (1) I

16

16 (1) I

16 (1) I

-

-

-

Bacterial strains

Erythromycin

Kanamycin

Norfloxacin

Streptomycin

Tetracycline

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

AG100Atet

64

32 (2) S

32 (2) S

-

128 (>2)S

256 (>1) S

128

128 (1) I

256 (0.5) I

<2

<2

<2

2

2 (1) I

2 (1) I

AG102

32

64 (0.5) I

64 (0.5) I

<2

<2

<2

<2

<2

<2

-

256 (>1)S

256 (>1)S

<2

<2

<2

CM64

-

64 (>4) S

256 (>1)S

4

4 (1) I

8 (0.5) I

4

<2 (>2)S

<2 (>2)S

32

8 (4) S

32 (1) I

nt

nt

nt

KP63

16

1 (16) S

8 (2) S

32

32 (1) I

64 (0.5) I

-

-

-

<4

<4

<4

<2

<2

<2

PA124

128

64 (2) S

128 (1) I

128

4 (32) S

16 (8) S

64

32 (2) S

64 (1) I

nt

nt

nt

8

2 (4) S

8 (1) I

MIC/2.5: concentration of plant extract added equal to 204.8 μg/mL for AG100ATET. CM64. KP63. AG102 and to 409.6 μg/ml for PA124.

MIC/5: concentration of plant extract added equal to 102. 4 μg/mL for AG100ATET CM64. KP63. AG102; and to 204.8 μg/ml for PA124.

(): Folds decreasing of MIC. S: synergy. I: indifference. nt: not tested; (−): MIC > 256 μg/ml.

Table 7

Minimal inhibitory concentration (MIC) of antibiotics in absence and presence extracts Tetrapleura tetraptera (μg/ml)

Bacterial strains

Antibiotics and MIC in absence and presenceTetrapleura tetraptera extract

Ampicillin

Cefepime

Chloramphenicol

Ciprofloxacin

Cloxacillin

Alone

MIC/2.5

MIC/5

Alone

MIC/2

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

AG100Atet

-

-

-

-

-

-

256

256 (1) I

-

256

128 (2) S

128 (2)S

-

-

-

AG102

-

-

-

128

256 (0.5)I

256 (0.5) I

16

8 (2) S

8 (2) S

<2

<2

<2

-

-

-

CM64

-

-

-

-

-

-

nt

nt

nt

nt

nt

nt

-

-

-

KP63

-

-

-

256

256 (1) I

-

-

256 (>1) S

256 (>1) S

64

64 (1) I

64 (1) I

-

128 (>2) S

256 (>1) S

PA124

128

64 (2)S

128 (1)I

128

128 (1)I

128 (1) I

32

4 (8) S

8 (4) S

16

16 (1) I

16 (1) I

-

-

-

Bacterial strains

Erythromycin

Kanamycin

Norfloxacin

Streptomycin

Tetracycline

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

Alone

MIC/2.5

MIC/5

AG100Atet

64

64 (1) I

64 (1) I

-

256 (>1) S

256 (>1) S

128

128 (1) I

256 (0.5) I

<2

<2

<2

2

2 (1) I

2 (1) I

AG102

32

64 (0.5) I

64 (0.5) I

<2

<2

<2

<2

<2

<2

-

256 (>1) S

256 (>1) S

<2

<2

<2

CM64

-

256 (>1) S

-

4

4 (1) I

8 (0.5) I

4

4 (1) I

8 (0.5) I

32

16 (2) S

32 (1) I

nt

nt

nt

KP63

16

<1 (>16) S

8 (2) S

32

32 (1) I

64 (0.5) I

-

256 (>1) S

-

<4

<4

<4

<2

<2

<2

PA124

128

64 (2) S

64 (2) S

128

64 (2) S

64 (2) S

64

32 (2) S

64 (1) I

nt

nt

nt

8

2 (4) S

2 (4) S

MIC/2.5: concentration of plant extract added equal to 204.8 μg/mL for CM64 and to 409.6 μg/ml for AG100ATET. PA124. KP63. AG102.

MIC/5: concentration of plant extract added equal to 102. 4 μg/mL for CM64; and to 204.8 μg/ml for AG100ATET.PA124. KP63. AG102.

(): Folds decreasing of MIC. S: synergy. I: indifference. nt: not tested; (−): MIC > 256 μg/ml.

Discussion

Phytochemical composition of the spice extracts

The phytochemical studies revealed the presence of secondary metabolite such as alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins, sterols, tannins and triterpenes; several molecules belonging to these classes of secondary metabolites were found active on pathogenic microorganisms [29].

Antibacterial activity of the spice extract

Although this is the first time that plants used in this work are studied for their activities vis-à-vis multi-resistant bacteria, plants belonging to some of the genus studied herein, like the Aframomum genus are well documented for their antimicrobial activity [6]. Some antibacterial compounds, such as acridone and chelerythrine have previously been isolated from the fruits of F. leprieurii[14, 30]. The antimicrobial activity of P. brazzeana and S. zenkeri is mainly due to some sulfur compounds. In fact, sulfur compounds with antimicrobial properties have previously been isolated from the two plants [7, 31]. Several alkaloids of the genus Piper proved to be responsible for the activity of P. guineense[32]. The detection of this class of secondary metabolites in the extract studied herein can explain the observed activities. According to Krishnaiah et al. [16], the antimicrobial activity of I. cylindricum can be due to the presence of tannins in this plant. However, tannins were not detected in the extract of I. cylindricum as found in the present work (Table 2), suggesting that other classes of secondary metabolites might be responsible for the antibacterial activity of this plant.

Role of efflux pumps in susceptibility of gram negative bacteria to the tested spice extracts

The significant increase of the activity of the extract of D. psilurus in the presence of EPI, indicates that bioactive constituents of this plant extract are substrate of efflux pumps. Efflux through AcrAB-TolC pumps was reported as essential mode of resistance of several Gram-negative MDR bacteria to a number of flavonoids isolated from plants of the genus Dorstenia, such as isobavachalcone, kanzonol C, stipulin, etc. [4, 15, 3335]. This suggests that possible combination of the extract of D. psilurus with EPI can be envisaged to overcome MDR bacteria.

Effects of the association of extracts with antibiotics

The results obtained by combining the antibiotic with the extracts of A. citratum, C. zeylanicum, D. psilurus and T. tetraptera indicate that these extracts contain chemical compounds that can modulate the activity of antibiotics against bacteria expressing MDR phenotypes. The methanol extracts of A. citratum, C. zeylanicum and D. psilurus showed a synergistic effect with antibiotics inhibiting bacterial cell wall synthesis (AMP and CEF) on K. pneumoniae KP63. The intrinsic mode of action of the active extracts is to be investigated.

Conclusion

The present work evidently provides information in the role of some Cameroonian spices in the fight against multi-resistant bacteria. The study also highlights the potential of D. psilurus as a strong antibacterial agent when the extract is combined with efflux pump inhibitor and several antibiotics.

Declarations

Acknowledgements

Authors are thankful to the Cameroon National Herbarium (Yaounde) for plants identification, Mr Simplice R. Mouokeu for its technical support, and Mr. Paul K. Lunga for language editing.

Authors’ Affiliations

(1)
Department of Biochemistry, Faculty of science, University of Dschang
(2)
Transporteurs Membranaires, Chimiorésistance et Drug Design, UMR-MD1

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