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In vitro antibacterial and antibiotic-potentiation activities of the methanol extracts from Beilschmiedia acuta, Clausena anisata, Newbouldia laevis and Polyscias fulva against multidrug-resistant Gram-negative bacteria

BMC Complementary and Alternative MedicineThe official journal of the International Society for Complementary Medicine Research (ISCMR)201515:412

https://doi.org/10.1186/s12906-015-0944-5

Received: 1 March 2015

Accepted: 20 November 2015

Published: 22 November 2015

Abstract

Background

The present study was designed to investigate the antibacterial activities of the methanol extracts from different parts of Beilschmedia acuta Kosterm (Lauraceae), Clausena anisata (Willd) Hook (Rutaceae), Newbouldia laevis Seem (Bignoniaceae) and Polyscias fulva (Hiern) Harms (Araliaceae) as well as their synergistic effects with antibiotics against a panel of Gram-negative bacteria, including multi-drug resistant (MDR) phenotypes expressing active efflux pumps.

Methods

Broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) of the extracts, as well as those of antibiotics in association with the most active ones, B. acuta, N. laevis and P. fulva.

Results

MIC values obtained indicate that extracts from the bark of B. acuta were active on all the 26 tested Gram-negative bacteria, with MICs ranging from values below 8 to 256 μg/mL. Other samples displayed selective activities, their inhibitory effects being observed on 9 (34.62 %) of the 26 bacterial strains for N. laevis leaves extract, 6 (23.10 %) for both C. anisata leaves and roots extracts, 7 (26.9 %) and 4 (15.4 %) for leaves and roots extracts of P. fulva respectively. Extract from B. actua bark displayed the best antibacterial activity with MIC values below 100 μg/mL against 16 (61.5 %) of the 26 tested microorganisms. The lowest MIC values (below 8 μg/mL) were obtained with this extract against Escherichia coli W3110 and Klebsiella pneumoniae ATCC11296. The MIC values of this extract were lower than those of ciprofloxacin against E. coli W3110, Enterobacter aerogenes ATCC13048, CM64 and Providencia stuartii NAE16. At MIC/2, the best percentages of synergistic effects (100 %), were obtained with B. acuta bark extract and tetracycline (TET) as well as with P. fulva leaves extract and TET and kanamycin (KAN).

Conclusion

The overall results of the present study provide information for the possible use of the studied plants and mostly Beilschmedia acuta in the control of bacterial infections including MDR phenotypes.

Keywords

Antibacterial activities Beilschmedia acuta Gram-negative bacteriaMulti-drug resistanceLauraceae

Background

Fighting multi-drug resistant (MDR) Gram-negative (MDRGN) bacteria remains a challenging issue worldwide. Microbial infections involving MDRGN bacteria constitute a major public health problem in developing countries [1] where the high cost of antibiotics makes them unaffordable to the majority of the population. Clinically, the continuous emergence of MDRGN bacteria drastically reduced the efficacy of antibiotic arsenal and, consequently, increased the frequency of therapeutic failure [2]. Therefore, the discovery of new antimicrobial agents is still relevant nowadays. Also, the shortcomings of drugs available today and scarcity of novel antibiotics propel the discovery of new chemotherapeutic agents from medicinal plants [3]. Approximately 60 % of the world population still relies on medicinal plants for their primary healthcare [4]. Medicinal plants have been used as a source of remedies since ancient times in Africa. In addition, promising new concepts such as the efflux pump inhibitors [5, 6], and synergy between antibiotics and phytochemicals are now being developed. The ability of several African medicinal plants to inhibit the growth of MDRGN bacteria, as well as their ability to potentiate the activity of commonly used antibiotics was previously reported. Some of these plants include Dorstenia psilurus, Dichrostachys glomerata and Beilschmiedia cinnamomea [79].

In our continuous search of plant extracts with antibiotic-potentiating activity to combat MDR bacteria, the present work was designed to investigate the antibacterial activity of four Cameroonian medicinal plants used traditionally in the treatment of bacterial infections, namely Beilschmiedia acuta Kosterm (Lauraceae), Clausena anisata (Willd) Hook (Rutaceae), Newbouldia laevis Seem (Bignoniaceae) and Polyscias fulva (Hiern) Harms (Araliaceae), against MDRGN expressing active efflux via the Resistance-Nodulation Cell Division (RND)-type pumps. In the treatment of infectious diseases, Beilschmiedia acuta is traditionally used for gastrointestinal infections [10], Clausena anisata for fungal, bacterial and viral infections, Newbouldia laevis for bacterial and fungal infections [1114], dysentery, worms, malaria, dental caries and diarrhea [15] and Polyscias fulva for venereal infections [16, 17].

Methods

Plant material and extraction

All medicinal plants used in the present work were collected in different areas of Cameroon between January and April 2012. The plants were identified at the National Herbarium (Yaounde, Cameroon), where voucher specimens were deposited under the reference numbers (Table 1). Air-dried and powdered plant material was weighed (300 g) and soaked in 1 L of methanol (MeOH) for 48 h at room temperature. The filtrate obtained through Whatman filter paper No.1 was concentrated under reduced pressure in a vacuum to obtain the crude extract. All crude extracts were kept at 4 °C until further use.
Table 1

Information of plants used in this study

Plants samples (family) and Herbarium Voucher numbera

Part used and extraction yield (%)b

Area of plant collection (Geographic Coordinates)

Traditional treatment

Bioactive (or potentially active) compounds isolated from plants

Biological activities of crude extractc

Beilschmiedia acuta Kosterm (Lauraceae) 37335/HNC

Leaves (18.40 %), fruits (20.22 %) and barks (36.46 %)

Lebialem, South-West Region of Cameroon; (4°10′N 9°14′E/4.167°N 9.233°E)

Cancer and gastrointestinal infections [10].

Flavonoids, triterpenes, phenols, saponins, alkaloids [10].

Cytotoxicity towards leukemia, breast, glioblastoma, colon and liver cancer cell lines [10].

Clausena anisata (Willd) Hook (Rutaceae) 44242/ HNC

Leaves (16.31 %) and roots (13.%)

Lebialem, South-West region of Cameroon

Diabetes, anti-hypertensive, anti-nociceptive, malaria, fungal, bacterial and viral infections, inflammation, heart and mental disorders, constipation, convulsions, impotence and sterility [4346]

Essential oils (sabinene, β-pinene, pulegone, 1,8 -cineole, estragole, [42]; carbazole alkaloids, coumarins, limonoids [46, 47].

Antimicrobial: Essential oil active against Sa, Sp, Esp, St, Pa [41, 42]

Newbouldia laevis Seem. (Bignoniaceae) 29469/HNC

Leaves (18.75 %), and barks (19.35 %)

Melon, Littoral region of Cameroon (04°33'53"N 09°38'04"E)

Cancers, spasms, infectious diseases, male infertility and diabetes [11, 12], coagulant or anti-hemorrhagic properties; digestive threats, urogenital and pulmonary infections[13, 14]; Dysentery, worms, malaria, sexually transmitted diseases, dental caries and diarrhea [15].

Tannins, triterpenoids, mucilages and reducing compounds, flavonoids, steroids, alkaloids, cardiac glycosides [10, 14, 48].

Antimicrobial: active against Ca, Ck, Sa, Sf, Ec, Pa, Sp, Pv, Kp, St, Sd, Ng Mtb, Ms [14, 39, 49].

Polyscias fulva (Hiern) Harms. (Araliaceae) 60407/HNC

Leaves (15.62 %), roots (17.56 %) and barks (19.01 %)

Dschang, West region of Cameroon (6°30′N 10°30′E/6.500°N 10.500°E)

Malaria, fever, mental illness [50]; venereal infections and obesity [16, 17] and cancer [10]

Polysciasoside A, kalopanax-saponin B, alpha-hederin [51, 52]

Inhibition of microsomal lipid peroxidation [53]

aPlants were identified at the Cameroon National Herbarium (HNC); ICNA: Voucher with no identification code at the HNC; bThe percentage of the methanol extract; cMicroorganisms[Bs Bacillus subtilis, Ca Candida albicans, Ck Candida krusei, Mm Mucor miehei, Cv Chlorella vulgaris, Cs Chlorella sorokiniana, Ec Escherichia coli, Esp Enterococcus species, Mtb Mycobacterium tuberculosis, Ms Mycobacterium smegmatis, Ng Neisseria gonorrhoeae, Pa Pseudomonas aeruginosa, Sf Streptococcus faecalis, Pv Proteus vulgaris, Sa Staphylococcus aureus, Sp Streptococcus pneumoniae, St Salmonella typhimurium, Kp Klepsiella pneumoniae, Sd Shigella dysenteriae, Ss Scenedesmus subspicatus, Sv Streptomyces viridochromogeneu]

Antimicrobial assays

Chemicals for antimicrobial assays

Tetracycline (TET), ciprofloxacine (CIP), chloramphenicol (CHL), ampicillin (AMP) and kanamycin (KAN) (Sigma-Aldrich, St Quentin Fallavier, France) were used as reference antibiotics (RA). p-Iodonitrotetrazolium chloride (INT, Sigma-Aldrich) was used as a microbial growth indicator [18, 19].

Microbial strains and culture media

The studied microorganisms included sensitive and resistant strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes, Escherichia coli obtained from the American Type Culture Collection (ATCC). Their bacterial features are summarized in Table 2. Nutrient agar was used to activate the tested Gram-negative bacteria [20].
Table 2

Bacterial strains used and their features

Strains

Features and References

 

Escherichia coli

  

 ATCC10536

Reference strain

 

 AG100

Wild-type E. coli K-12

[54]

 AG100A

AG100 ΔacrAB::KANR

[34, 54, 55]

 AG100ATET

ΔacrAB mutant AG100, with over-expressing acrF gene; TETR

[54]

 AG102

ΔacrAB mutant AG100, owing acrF gene markedly over-expressed; TETR

[56, 57]

 MC4100

Wild type E. coli

[58]

 W3110

Wild type E. coli

[58, 59]

Enterobacter aerogenes

  

 ATCC13048

Reference strains

 

 CM64

CHLR resistant variant obtained from ATCC13048 over-expressing the AcrAB pump

[60]

 EA3

Clinical MDR isolate; CHLR, NORR, OFXR, SPXR, MOXR, CFTR, ATMR, FEPR

[61, 62]

 EA27

Clinical MDR isolate exhibiting energy-dependent norfloxacin and chloramphenicol efflux with KANR AMPR NALR STRR TETR

[61, 62]

 EA289

KAN sensitive derivative of EA27

[63]

 EA294

EA289 acrA::KANR

[63]

 EA298

EA 289 tolC::KANR

[63]

Enterobacter cloacae

  

 ECCI69

Clinical MDR isolates, CHLR

[7]

 BM67

Clinical MDR isolates, CHLR

[7]

Klebsiella pneumoniae

  

 ATCC12296

Reference strains

 

 KP55

Clinical MDR isolate, TETR, AMPR, ATMR, CEFR

[64]

 KP63

Clinical MDR isolate, TETR, CHLR, AMPR, ATMR

[64]

 K24

AcrAB-TolC, Laboratory collection of UNR-MD1, University of Marseille, France

[7]

 K2

AcrAB-TolC, Laboratory collection of UNR-MD1, University of Marseille, France

[7]

Providencia stuartii

 

[65]

 NEA16

Clinical MDR isolate, AcrAB-TolC

 ATCC29916

Clinical MDR isolate, AcrAB-TolC

 PS2636

Clinical MDR isolate, AcrAB-TolC

 PS299645

Clinical MDR isolate, AcrAB-TolC

Pseudemonas aeruginosa

  

 PA 01

Reference strains

 

 PA 124

MDR clinical isolate

[66]

aAMP, ATMR, CEFR, CFTR, CHLR, FEPR, KANR, MOXR, STRR, TETR. Resistance to ampicillin, aztreonam, cephalothin, cefadroxil, chloramphenicol, cefepime, kanamycin, moxalactam, streptomycin, and tetracycline; MDR Multidrug resistant

INT colorimetric assay for MIC and MBC determinations

The MIC determination on the tested bacteria was conducted using rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay according to described methods [18] with some modifications [21, 22]. The test samples and RA were first dissolved in DMSO/Mueller Hinton Broth (MHB). The final concentration of DMSO was lower than 2.5 % and does not affect the microbial growth [23, 24]. The solution obtained was then added to Mueller Hinton Broth, and serially diluted two fold (in a 96- wells microplate). One hundred microlitre (100 μL) of inoculum 1.5 x 106 CFU/mL prepared in appropriate broth was then added [21, 22]. 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. The MIC was defined as the sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth [18]. 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 extracts, which did not produce a color change after addition of INT as mentioned above [21, 22].

Samples were tested alone and the best four extracts (those from the leaves and bark of Beilschmedia acuta, and from the leaves of Newbouldia laevis and Polyscias fulva) were also selected and tested in association with antibiotics at the sub-inhibitory concentrations (MIC/2 and MIC/5) [79] against nine MDR bacteria. Fractional inhibitory concentration (FIC) was calculated as the ratio of MICAntibiotic in combination/MICAntibiotic alone and the results were discussed as follows: synergy (0.5), indifferent (0.5 to 4), or antagonism (>4) [25, 26]. All assays were performed in triplicate.

Results

The antibacterial activities of methanol extracts from various parts of Beilschmedia acuta, Clausena anisata, Newbouldia laevis and Polyscias fulva are summarized in Table 3 (MIC values up to 1024 μg/mL are provided as supporting information; Additional file 1: Table S1). It can be observed that extracts from the bark of B. acuta were active on all 26 tested Gram-negative bacteria, with MICs ranging from values below 8 to 256 μg/mL. Other samples displayed selective activities, their inhibitory effects being observed against nine (34.62 %) of the 26 bacterial strains for N. laevis leaves extract, six (23.10 %) for both C. anisata leaves and roots extracts, seven (26.9 %) and four (15.4 %) for leaves and roots extracts of P. fulva respectively. Extract from the bark of B. actua showed the best antibacterial activity with MIC values below 100 μg/mL against 16/26 (61.5 %) of the tested microorganisms. The lowest MIC values below 8 μg/mL were obtained with this extract against Escherichia coli W3110 and Klebsiella pneumoniae ATCC11296. MIC values of this extract were lower than those of ciprofloxacin against E. coli W3110, Enterobacter aerogenes ATCC13048 and CM64 and Providencia stuartii NAE16 (Table 3). The bactericidal activities of studied samples were mostly noted with the extract from B. acuta, with MBC values observed against 23/26 (88.5 %) tested bacteria (see Additional file 1: Table S2, supporting information).
Table 3

MICs (μg/mL) of the crude extracts and ciprofloxacin on the panel of tested bacteria

Bacterial strains

Studied samples and MIC (μg/mL)

Beilschmedia acuta

Clausena anisata

Newbouldia laevis

Polyscias fulva

 

Reference drug

L

B

F

L

R

L

B

L

B

R

CIP

Escherichia coli

           

 ATCC 10536

>256

64

>256

256

256

128

>256

>256

>256

>256

1

 AG 100A

>256

128

>256

>256

>256

256

>256

>256

>256

>256

<0.5

 AG 100

>256

16

>256

>256

>256

>256

>256

>256

>256

>256

16

 AG 100ATet

>256

256

>256

>256

>256

>256

>256

>256

>256

>256

64

 AG 102

>256

64

>256

>256

>256

>256

>256

>256

>256

>256

4

 MC 4100

256

128

256

256

256

128

>256

256

>256

256

16

 W 3110

256

<8

256

256

256

128

>256

128

>256

128

32

Enterobacter aerogenes

           

 ATCC 13048

>256

16

>256

>256

>256

>256

>256

256

>256

>256

32

 CM 64

>256

16

>256

>256

>256

>256

>256

>256

>256

>256

64

 EA3

>256

64

>256

>256

>256

>256

>256

>256

>256

>256

16

 EA27

>256

64

>256

>256

>256

128

>256

256

>256

>256

4

 EA 294

>256

64

>256

>256

256

256

>256

>256

>256

>256

2

 EA 289

>256

256

256

>256

>256

>256

>256

>256

>256

>256

128

 EA 298

>256

256

256

>256

>256

>256

>256

>256

>256

>256

16

Klebsiella pneumoniae

           

 ATCC11296

128

<8

256

256

256

128

>256

128

256

128

<0.5

 K2

>256

256

>256

>256

>256

>256

>256

>256

>256

>256

16

 KP55

>256

32

>256

>256

>256

>256

>256

>256

>256

>256

4

 KP63

128

64

128

256

>256

256

>256

128

>256

128

4

Providencia stuartii

           

 ATCC29916

>256

128

>256

>256

>256

>256

>256

>256

>256

>256

32

 PS2636

256

64

256

256

128

128

>256

256

>256

>256

64

 PS299645

>256

256

>256

>256

>256

>256

>256

>256

>256

>256

32

 NAE16

>256

32

>256

>256

>256

>256

>256

>256

>256

>256

128

Enterobacter cloacae

           

 ECCI69

>256

256

>256

>256

>256

>256

>256

>256

>256

>256

256

 BM67

>256

256

>256

>256

>256

>256

>256

>256

>256

>256

32

Pseudomonas aeruginosa

           

 PA01

>256

64

256

>256

>256

>256

>256

>256

>256

>256

16

 PA124

>256

32

>256

>256

>256

>256

>256

>256

>256

>256

32

The tested extracts were obtianed from the leaves (L), bark (B), roots (R) or fruits (F); CIP: ciprofloxacin]; MIC and MBC data with values up to 1024 μg/mL are provided as supporting information (Additional file 1: Table S1)

Five commonly used antibiotics (CIP, TET, KAN, AMP and CHL) were combined with extracts from B. acuta leaves and bark and those from the leaves of N. laevis and P. fulva at their MIC/2 and MIC/5, as obtained on each of nine tested bacterial strains (Tables 4 and 5). Synergistic effects were observed with all tested extracts and all studied antibiotics on at least one of the nine selected bacteria. The best percentages of synergistic effect (100 %) were obtained at MIC/2 with B. acuta bark extract in combination with TET (Table 5) as well as with P. fulva leaves extract in association with TET and KAN (Table 5).
Table 4

MIC of antibiotics after the association of the extract of Beilschmedia acuta at MIC/2 and MIC/5 against selected MDR bacteria

Antibioticsa

Extract and concentration

Bacterial strainsb, MIC (μg/mL) of antibiotics in the absence and presence of the extract and FIC in parenthesis

PBSS (%)

  

AG102

AG100ATET

EA27

CM64

KP55

NAE16

BM67

PA01

PA124

 

CIP

 

0

4

64

4

64

4

128

32

16

32

 
 

L

MIC/2

4 (1)I

64 (1)I

1 (0.25)S

64 (1)I

8 (2)A

128 (1)I

16 (0.50)S

16 (1)I

16(0.50)S

3/9 (27.27 %)

  

MIC/5

4 (1)I

64 (1)I

2 (0.50)S

64 (1)I

8 (2)A

128 (1)I

32 (1)I

16 (1)I

32(1)I

1/9 (11.11 %)

 

B

MIC/2

4 (1)I

32 (0.50)S

1 (0.25)S

64 (1)I

4 (1)I

128 (1)I

16 (0.50)S

16 (1)I

32(1)I

3/9 (27.27 %)

  

MIC/5

4 (1)I

64 (1)I

2 (0.50)S

64 (1)I

4 (1)I

128 (1)I

32 (1)I

16 (1)I

32(1)I

3/9 (27.27 %)

TET

 

0

8

64

64

32

2

64

32

64

16

 
 

L

MIC/2

2 (0.25)S

32 (0.50)S

32 (0.50)S

8 (0.25)S

2 (1)I

32 (0.50)S

32 (1)I

16 (0.25)S

8(0.50)S

7/9 (77.78 %)

  

MIC/5

4 (0.50)S

64 (1)I

64 (1)I

16 (0.50)S

2 (1)I

64 (1)I

32 (1)I

64 (1)I

8(0.50)S

3/9 (27.27 %)

 

B

MIC/2

4 (0.50)S

32 (0.50)S

16 (0.25)S

16 (0.50)S

1 (0.50)S

16 (0.25)S

16 (0.50)S

32 (0.50)S

8(0.50)S

9/9 (100 %)

  

MIC/5

4 (0.50)S

64 (1)I

64 (1)I

16 (0.50)S

1 (0.50)S

32 (0.50)S

32 (1)I

64 (1)I

16(1)I

4/9 (36.36 %)

KAN

 

0

-

16

128

4

16

16

64

4

128

 
 

L

MIC/2

8(˂0.03)S

4 (0.25)S

64 (0.50)S

2 (0.50)S

16 (1)I

16 (1)I

32 (0.50)S

4 (1)I

64(0.50)S

6/9 (66.67 %)

  

MIC/5

64 (˂0.25)S

16 (1)I

128 (1)

4 (1)I

16 (1)I

16 (1)I

64 (1)I

2 (0.50)S

64(0.50)S

3/9 (27.27 %)

 

B

MIC/2

8 (˂0.03)S

˂1 (˂0.06)S

32 (0.25)S

˂1 (˂0.25)S

<1 (<0.06)S

8 (0.50)S

16 (0.25)S

4 (1)I

32(0.25)S

8/9 (88.89 %)

  

MIC/5

128 (˂0.50)S

8 (0.50)S

128 (1)I

2 (0.50)S

4 (0.25)S

16 (1)I

32 (0.50)S

<1 (<0.25)S

64(0.50)S

6/9 (66.67 %)

AMP

 

0

-

-

-

-

-

-

-

-

-

 
 

L

MIC/2

128 (˂0.50)S

128 (˂1)S

-(≥1)

-(≥1)

-(≥1)

- (≥1)

-(≥1)

-(≥1)

-(≥1)

2/9 (22.22 %)

  

MIC/5

256 (˂1)S

-(≥1)

-(≥1)

-(≥1)

-(≥1)

- (≥1)

-(≥1)

128 (<0.50)S

-(≥1)

2/9 (22.22 %)

 

B

MIC/2

128 (˂0.50)S

-(≥1)

256 (˂1)S

-(≥1)

256 (<1)S

256 (<1)S

-(≥1)

-(≥1)

256(<1)S

5/9 (55.55 %)

  

MIC/5

256 (˂1)S

-(≥1)

-(≥1)

-(≥1)

-(≥1)

- (≥1)

-(≥1)

256 (˂1)S

-(>1)

2/9 (22.22 %)

CHL

 

0

32

64

64

-

8

64

128

16

128

 
 

L

MIC/2

16 (0.50)S

32 (0.50)S

64 (1)I

128 (˂0.50)S

16 (2)A

8 (0.13)S

64 (0.50)S

2 (0.13)

64(0.50)S

6/9 (66.67 %)

  

MIC/5

32 (1)I

64 (1)I

64 (1)I

-(˃1)

16 (2)A

16 (0.25)S

64 (0.50)S

8 (0.50)S

128(1)I

3/9 (27.27 %)

 

B

MIC/2

32 (1)I

32 (0.50)S

32 (0.50)S

256 (˂1)S

2 (0.25)S

16 (0.25)S

64 (0.50)S

4 (0.25)S

64(0.50)S

8/9 (88.89 %)

  

MIC/5

32 (1)I

32 (0.50)S

64 (1)I

-(˃1)

2 (0.25)S

32 (0.50)S

128 (1)I

8 (0.50)S

64(0.50)S

5/9 (55.55 %)

aAntibotics [TET tetracycline, CIP ciprofloxacin, KAN kanamycin, CHL chloramphenicol, AMP ampicillin]. bBacterial strains: Escherichia coli [AG102, AG100Atet], Pseudomonas aeruginosa [PA01, PA124], Enterobacter aerogenes [CM64, EA27], Enterobacter cloacae [BM67], Klebsiella pneumoniae [KP55], Providencia stuartii [NAE16]. cPBSS: percentage of bacteria strain on which synergism has been observed

(): fold increase in MIC values of the antibiotics after association with plants extract; S synergy, I indifference, na not applicable, B bart extract, L leaves extract, FIC fractional inhibitory concentration, (−): >256 μg/mL; 0: no extract (only antibiotic tested)

Table 5

MIC of antibiotics after the association of the extract of Newbouldia laevis and Polysicas fulva at MIC/2 and MIC/5 against selected MDR bacteria

Antibioticsa

Extract and concentration

Bacterial strainsb, MIC (μg/mL) of antibiotics in the absence and presence of the extract and FIC in parenthesis

PBSS (%)

  

AG102

AG100ATET

EA27

CM64

KP55

NAE16

BM67

PA01

PA124

 
 

Newbouldia laevis

          

CIP

0

4

64

4

64

4

128

32

16

32

 
 

MIC/2

4 (1)I

64 (1)I

2 (0.50)S

32 (0.50)S

4 (1)I

128 (1)I

16 (0.50)S

16 (1)I

32(1)I

3/9 (27.27 %)

 

MIC/5

4 (1)I

64 (1)I

2 (0.50)S

64 (1)I

4 (1)I

128 (1)I

32 (1)I

16 (1)I

32(1)I

1/9 (11.11 %)

TET

0

8

64

64

32

2

64

32

64

16

 
 

MIC/2

4 (0.50)S

16 (0.25)S

32 (0.50)S

4 (0.13)S

2 (1)I

32 (0.50)S

16 (0.50)S

32 (0.50)S

8(0.50)S

8/9 (88.89 %)

 

MIC/5

8 (1)I

32 (0.50)S

64 (1)I

8 (0.25)S

0.50 (0.25)S

64 (1)I

32 (1)I

64 (1)I

8(0.50)S

4/9 (36.36 %)

KAN

0

-

16

128

4

16

16

64

4

128

 
 

MIC/2

4 (˂0.03)S

8 (0.50)S

64 (0.50)S

2 (0.50)S

16 (1)I

8 (0.50)S

32 (0.50)S

2 (0.50)S

32(0.25)S

8/9 (88.89 %)

 

MIC/5

64 (˂0.50)S

16 (1)I

128 (1)I

4 (1)I

1 (0.06)S

8 (0.50)S

32 (0.50)S

4 (1)I

64(0.50)S

5/9 (55.55 %)

AMP

0

-

-

-

-

-

-

-

-

-

 
 

MIC/2

-(≥1)

-(≥1)

256 (˂1)S

-(≥1)

-(≥1)

256 (˂1)S

-(≥1)

-(≥1)

-(>1)

2/9 (22.22 %)

 

MIC/5

-(≥1)

-(≥1)

-(≥1)

-(≥1)

256 (˂1)S

- (≥1)

-(≥1)

-(≥1)

-(>1)

1/9 (11.11 %)

CHL

0

32

64

64

-

8

64

128

16

128

 
 

MIC/2

16 (0.50)S

32 (0.50)S

64 (1)I

64 (˂0.25)S

4 (0.50)S

16 (0.25)S

32 (0.25)S

4 (0.25)S

128(1)I

7/9 (77.78 %)

 

MIC/5

32 (1)I

64 (1)I

64 (1)I

128 (˂0.50)S

1 (0.13)S

32 (0.50)S

64 (0.50)S

16 (1)I

128(1)I

4/9 (36.36 %)

 

Polyscias fulva

          

CIP

0

4

64

4

64

4

128

32

16

32

 
 

MIC/2

4 (1)I

64 (1)I

2 (0.50)S

64 (1)I

4 (1)I

128 (1)I

2 (0.06)S

8 (0.50)S

32(1)I

3/9 (27.27 %)

 

MIC/5

4 (1)I

64 (1)I

2 (0.50)S

64 (1)I

4 (1)I

128 (1)I

4 (0.13)S

16 (1)I

32(1)I

2/9 (22.22 %)

TET

0

8

64

64

32

2

64

32

64

16

 
 

MIC/2

2 (0.25)S

32 (0.50)S

32 (0.50)S

4 (0.13)S

<0.50 (<0.25)S

32 (0.50)S

8 (0.25)S

2 (0.03)S

8(0.50)S

9/9 (100 %)

 

MIC/5

4 (0.50)S

64 (1)I

32 (0.50)S

16 (0.50)S

2 (1)I

32 (0.50)S

32 (1)I

32 (0.50)S

8(0.50)S

6/9 (66.67 %)

KAN

0

-

16

128

4

16

16

64

4

128

 
 

MIC/2

8(˂0.06)S

8 (0.50)S

64 (0.50)S

2 (0.50)S

2 (0.13)S

8 (0.50)S

32 (0.50)S

<1 (<0.25)S

64(0.50)S

9/9 (100 %)

 

MIC/5

64 (˂0.50)S

16 (1)I

128 (1)I

4 (1)I

16 (1)I

8 (0.50)S

32 (0.50)S

4 (1)I

64(0.50)S

4/9 (36.36 %)

AMP

0

-

-

-

-

-

-

-

-

-

 
 

MIC/2

128 (˂0.50)S

-(≥1)

256 (˂1)S

-(≥1)

256 (˂1)S

256 (˂1)S

-(≥1)

256 (˂1)S

-(>1)

5/9 (55.55 %)

 

MIC/5

256 (˂1)S

-(≥1)

-(≥1)

-(≥1)

-(≥1)

-(˃1)

-(≥1)

-(≥1)

-(>1)

1/9 (11.11 %)

CHL

0

32

64

64

-

8

64

128

16

128

 
 

MIC/2

16 (0.50)S

64 (1)I

64 (1)I

128 (˂0.50)S

4 (0.50)S

8 (0.13)S

64 (0.50)S

2 (0.13)S

64(0.50)S

7/9 (77.78 %)

 

MIC/5

32 (1)I

64 (1)I

64 (1)I

256 (˂1)S

4 (0.50)S

16 (0.25)S

64 (0.50)S

16 (1)I

64(0.50)S

5/9 (55.55 %)

aAntibotics [TET tetracycline, CIP ciprofloxacin, KAN kanamycin, CHL chloramphenicol, AMP ampicillin]. bBacterial strains: Escherichia coli [AG102, AG100Atet], Pseudomonas aeruginosa [PA01, PA124], Enterobacter aerogenes [CM64, EA27], Enterobacter cloacae [BM67], Klebsiella pneumoniae [KP55], Providencia stuartii [NAE16]. cPBSS percentage of bacteria strain on which synergism has been observed, NA not applicable

(): fold increase in MIC values of the antibiotics after association with plants extract, S synergy, I indifference, na not applicable, FIC fractional inhibitory concentration, (−): >256 μg/mL, 0: no extract (only antibiotic tested)

Discussion

Phytochemicals are routinely classified as antimicrobials on the basis of susceptibility tests that produce MICs in the range of 100 to 1000 μg/mL [27]. Moreover, for crude extracts, the antimicrobial activity is considered to be significant if MIC values are below 100 μg/mL and moderate when 100< MIC <625 μg/mL [28, 29]. Therefore, the activity recorded with B. acuta bark extract against the 26 tested bacterial strains can be considered as very important. If we consider the alternative criteria described by Fabry et al. [30], where extracts having MIC values less than 8000 μg/mL have noteworthy antimicrobial activity, the overall activity recorded with the leaves and fruit extracts of B. acuta, P. fulva and N. laevis leaves extracts can also be considered promising. A keen look of the results of MIC and MBC determinations (Table 3, Additional file 1: Tables S1 and S2) indicates that MBC/MIC ratios were mostly above four, suggesting that studied extracts, including the most active ones, generally displayed bacteriostatic effects (MBC/MIC > 4) [3133]. Various classes of phytochemicals (Table 2) were previously detected in the extracts of the four tested plants [10] and this may explain their antibacterial activity.

The results obtained in this study, and mostly those obtained with the bark of B. acuta are very important when taking in consideration the fact that most of the bacterial strains used were MDR phenotypes expressing active efflux pumps [79, 34, 35]. In fact, the activity of antibiotics against the studied MDR bacteria was previously found to increase in the presence of phenylalanine arginine β-naphthylamide (PAßN), a potent inhibitor of RND efflux systems, particularly AcrAB–TolC (of Enterobaceriaceae) and MexAB–OprM (of Pseudomonas species) [79, 34, 35]. In the present study, we demonstrated that beneficial effects when combining four of the tested plant extracts [namely those from B. acuta (leaves and bark), N. leavis (leaves) and P. fulva (leaves)] with the first line antibiotics could be achieved. High percentages of synergistic effects (100 %) obtained with B. acuta bark extract and TET as well as P. fulva leaves extract in combination with TET and KAN, clearly suggest that such associations could improve the fight against MDR bacterial infections. This also suggests that some of the constituents of the corresponding plants can act as efflux pump inhibitors, as more than 70 % synergistic cases were observed with many combinations [26].

The antimicrobial potential of the genus Beilschmiedia has previously been documented. Chouna et al. [36] demonstrated that compounds such as beilschmiedic acid C isolated from B. anacardioides were significantly active against Bacillus subtilis, Micrococcus luteus and Streptococcus faecalis. Beilschmiedia cinnamomea was previously reported to have significant to moderate activities (64–1024 μg/mL) against the MDRGN tested in this work [7]. Beilschmedia obscura was also found to show a good and large spectrum of antibacterial activity against MDRGN [37]. Some compounds previously isolated from the genus Beilschmiedia and belonging to alkaloids, phenols, saponines, sterols and triterpenoids [36, 38] were shown to possess antimicrobial activities [7]. The genus Beilschmiedia is also known traditionally to possess antimicrobial activities [7]. Beilschmedia acuta tested in this study is also used in Cameroon to treat gastrointestinal infections [10]. The obtained data highlight the importance of this plant in the control of microbial infections and mostly those involving MDR phenotypes. The antimicrobial activities of extracts and compounds from Newbouldia laevis towards sensitive bacteria and fungi were also reported [39, 40], and the present study provides additional data on the potential of this plant to fight MDR bacteria. Also, the antimicrobial activity of essential oil from Clausena anisata was reported against Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus species, Salmonella typhimurium and Pseudomonas aeruginosa [41, 42]. The present report provides more evidence of the antimicrobial potential of this plant.

Conclusion

The results of this study are very interesting, in regards to the medical importance of the studied microorganisms. These data provided evidence that crude extracts from the studied plants and mostly that from the bark of Beilschmedia acuta are potential sources of antimicrobial drugs to fight MDR bacterial infections. The purification of this plant will be carried out to isolate its active constituents. The cytotoxicity assays on normal cell lines constitute the limitation of the present work and will further be performed to ensure the safety of the tested extracts.

Declarations

Acknowledgements

Authors are thankful to the Cameroon National Herbarium (Yaounde) for the plant identification. Authors are also thankful to UMR-MD1 (Mediterranean University, Marseille, France) for providing some clinical bacteria.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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.

Authors’ Affiliations

(1)
Department of Biochemistry, Faculty of Science, University of Dschang
(2)
Laboratory of Natural Products Chemistry, Department of Chemistry, Faculty of Science, University of Dschang

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