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ORIGINAL ARTICLE Antibacterial and Antibiotic-Potentiation Activities of Some Cameroonian Food Plants against Multi-Drug Resistant Gram-Negative Bacteria Simplice B. Tankeo, Stephen T. Lacmata, Jaures A. K. Noumedem, Jean P. Dzoyem, Jules R. Kuiate, and Victor Kuete Objective:: To evaluate the in vitro antibacterial properties and the ability to potentiate some common ABSTRACT Objective antibiotics effects of the methanol extracts of 11 Cameroonian food plants on 29 Gram-negative bacteria expressing Methods:: The antimicrobial activity of the extracts was performed using the broth multidrug resistant (MDR) phenotypes. Methods microdilution method. The phytochemical screening of these extracts was also performed using standard methods. Results:: Ocimum basilicum , Gnetum africanum and Eucalyptus robusta extracts possessed an antibacterial activity Results against all the 29 studied bacteria. The extracts from G . africanum and E . robusta were the most active with the lowest minimal inhibitory concentration of 64 μg/mL on Escherichia coli AG100A for both extracts and also against Klebsiella pneumoniae K24 for G. africanum. When tested in the presence of phenylalanine-arginine β-Naphtylamide (PAβN), an efflux pump inhibitor, the extract of Thymus vulgaris and E . Robusta showed the best activities on most tested strains. E. Robusta extract showed good synergistic effects, improving the activity of commonly used antibiotics Conclusion:: The overall results obtained provide the baseline information for the use of the in about 85% of cases. Conclusion tested plants in the treatment of bacterial infections. KEYWORDS food plants, Cameroon, multidrug resistant Gram-negative bacteria, efflux pumps

Infectious diseases are the main cause of mortality in the world with about 17 millions of victims each year. (1) The emergence of these infectious diseases in the developing world is mostly due to the poverty of the population or the inappropriate use of antibiotics.(2) The treatment of such diseases with antibiotics is continuously facing difficulties related to their high cost, the appearance of side effects and the resistance of pathogens. (3) One of the major mechanisms of bacteria resistance is the active efflux of substances which is a major problem limiting the drug's activity.(4-6) In Cameroon, many plants are used as traditional medicines to treat infectious diseases. (7) In the present work, we investigated the antibacterial activity of some of the commonly used food plants in Cameroon against some multidrug resistant (MDR) Gramnegative bacteria. These plants include Zingiber officinale Roscoe, Thymus vulgaris L., Cymbopogon citratus Stapf., Petroselinum crispum Ngn., Apium graveolens L., Ocimum basilicum Linn., Allium porrum Welus., Gnetum africanum L., Eucalyptus robusta L., Ocimum gratissimum L. and Capsicum annuum L. The study was also extended to the evaluation of the potencies of some of the above plant extracts to increase the activity of some

antibiotics against MDR strains. The role of bacterial efflux pumps in resistance to the extracts was also studied.

METHODS Plant Material and Extraction The 11 edible food plants used in this work were purchased from Dschang local market, West Region of Cameroon in January 2010. The collected plants material were the leaves of Thymus vulgaris , Cymbopogon citratus , Petroselinum crispum , Apium graveolens , Ocimum basilicum and Allium porrum , Gnetum africanum , Eucalyptus robusta and Ocimum gratissimum , the fresh and dry rhizomes of Zingiber officinale and the fruits of Capsicum annuum . The plants were identified by Mr. Victor Nana of the National Herbarium (Yaoundé, Cameroon) where voucher specimens were deposited under the reference number (Table 1).

© The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag Berlin Heidelberg 2014 Department of Biochemistry, Faculty of Science, University of Dschang, Cameroon Correspondence to: Dr. Victor Kuete, Tel: 237-77355927; Fax: 237-22226018, E-mail: [email protected] DOI: 10.1007/s11655-014-1866-7

Lamiaceae

Thymus vulgaris L.

–

Fruits

Leaves

Leaves, bark

Leaves

Seeds, leaves

Seeds, leaves

Seeds, leaves

Seeds, leaves

Seeds, leaves

Seeds, leaves

Rhizomes

Part of plant used

Antimicrobial activities of essential oil [thymol (46, 2%), γ-terpenene (14, 1%), p-cymene (9, 9%), linalool (4, 0%), myrcene (3, 5%), α-pinene (3, 0%), α-thujene (2, 8%)](29) against St, Ec, Sa, Cn, Asp,(29) Ao;(28) antioxidant(30)

Antimicrobial activities of ethanol extract against Sa, Sp, Spn, Hi;(25) Insecticidal (1,8-Cineole, Geranial, Camphene from rhizomes by coupled GC-MS analysis) against Sitophilus zeamais;(26) cytotoxic effect on leukemia cells CEM/ADR5000 and CCRFCEM, and on pancreatic MiaPaCa-2 cell lines(27)

Bioactive (or potentially active) compounds and screened activityb for crude plant extract

Antimicrobial activities of essential oils (β-caryophyllene, γ-terpinène, α-terpinéol, p-cymène, (Z)-α-bisabolène, thymol, eugénol, limonène, α-terpinolène)(49-51) of the leaves against Bc, Bs, Sa Cgl, Ec, Sf(49) and of ethanol extract (leaves) against Cn, An, Af, Fo, Rs, Bt, Pc.(50) Antibacterial and antiviral (Bs, Ec, Sc), fruits content [Capsinoides (dihydrocapsiate, nordihydrocapsiate, capsiate, capsacine) by HPLC method];(52,53) chemopreventive [QR inductive activity of methanol extracts (lutein, β-carotene) of the leaves](54)

Antioxidant, bacterial and viral infections(52)

Antibacterial and antifungal activities of essential oils extract (α-pinene, β-pinene, limonene, phellandrene, terpineol) of the leaves against Sa, Ec, Ca(26)

Antibacterial activities of ethanolic extracts of the leaves against Sa, Ec, St;(47) antiseptic and fungicical (seeds)(46)

Antimicrobial activities of methanolic extract against Sa, Bs, Bc, Ec, ethanolic extract against Cn;(45) cytotoxic, antioxidant (terponoids, flavonoids); Insecticidal, fungicide (S-methyl-L-cysteine sulfoxide, S-propyl-L-cysteine sulfoxide)(44,45)

Antimicrobial activities of methanolic and ethanolic extracts (leaves, flowers and stems) against Ac, Ec, Bm, Sa, Se, Ca;(43) anti-HIV and antioxidant (Chicoric acid from leaves), anti-inflammatory, antiproliferative of leaves extracts and essential oil(41,42)

Antibacterial activities of seeds methanol extract against St;(39) vasodilatory action (apigenin);(40) hepatoprotective activity of methanolic extracts of the seeds(38)

Pulmonary antiseptic, antifungal, anti-tussive, antispasmodic, antibacterial(49,50)

Anti-inflammatory, anti-bacterial, anti-parasitic and fungal infections, dysentery, fever, malaria(26-48)

Antidotes to some forms of poison and snake bite, antiseptic, anti-pains, diuretic, fungicide for dressing fresh and septic wounds, treats sore throats(46)

Antibacterial, antioxidant, cytotoxyc, insecticidal, fungicide(44,45)

Diarrhoea, hypoglycemic, anti-inflammatory, antibacterial, antioxidant, immunostimulation, antiviral(41,42)

Anti-inflammatory, anti-hypertensive(38)

Diuretic, emmenagogue, antimicrobial, ntioxidant(35) Antimicrobial (methanol and water extracts of leaves and stems) against Lp, Lm,(36) Ltm, Lti, Ec, Eca, Bs;(35-37) antioxidant (phenolics from methanol extraction)(35)

Central nervous system depressor, antioxidant, Antibacterial and antifungal activities of essential oil (geraniol, neral, geranial, citronellal, antimicrobial, antispasmodic, analgesic, antiseptic, myrcene, limonene), 3-tert-butyl-4-methoxy-phenol, Tert-Butylhydroquinone, 2, 6 -disedative, fever, diuretic(31,32) tert-butyl-4-Methylphenol(33,34) against Stm, Ao(29)

Carminative, antiseptic, antimicrobial, antioxidant, diuretic(28)

Analgesic, antioxidant, sedative, antipyretic, insecticidal, anticancer, microbial and respiratory tract infections, rheumatic diseases, vomiting, nausea, convulsion(25,26)

Traditional treatment

Plants Used in This Study with the Evidence of Their Activities

Notes: HNC or SRFK: Cameroon national herbarium code; –: Not reported; GC-MS: Coupled gas chromatography-mass spectrometry; HPLC: high-performance liquid chromatography; QR: Quinone reductase ; Af: Aspergillus flavus ; Ac: Acinetobacter calcoaceticus ; An: Aspergillus nige r; Ao: Aspergillus ochraceus ; Asp: Aspergillus species mutans; Bc: Bacillus cereus ; Bm: Bacillus macerans; Bs: Bacillus subtilis; Bt: Botryodiplodia theobromae ; Ca: Candida albicans ; Cgl: Corynebacterium glutamicum ; Cn: Candida néoformans; Eca: Escherichia carotovora ; Ec: Escherichia coli ; Fo: Fusarium oxsporium ; Hi: Haemophilus influenza ; Lm: Leuconostoc mesenteroides ; Lp: Lactobacillus plantarum ; Lti: Listeria innocua ; Ltm: Listeria monocytogenes ; Pc: Penicillium chrysogenum ; Rs: Rhizopus stolonifer; Sa: Staphylococcus aureus ; Sc: Saccharomyces cerevisiae ; Se: Staphylococcus epidermis ; Sf: Streptococcus faecalis ; Sp: Streptococcus pyogenes ; Spn: Streptococcus pneumoniae ; Stm: Streptococcus mutans ; St: Salmonella typhi ; Sz: Sitophilus zeamais ; b [Screened activity: significant (S: CMI4).(12,13) (The FIC values available in supplemental material SM2–4).

RESULTS Chemical Composition of the Plant Extracts The results of qualitative analysis showed that each of the studied plant extract contains various classes of secondary metabolite such as alkaloids, anthocyanins, anthraquinons, flavonoids, phenols, saponins, steroids, tannins and triterpenes (SM5).

Antibacterial Activity of the Plant Extracts Results summarized in SM6 showed the antibacterial activities of plants extracts tested alone and in combination with PAβN on a panel of Gramnegative bacteria. These results revealed that all the studied extracts were active on at least one bacterial strain. When tested alone, the extracts from O. basilicum , G . africanum and E. robusta showed the best activity, their inhibitory effect being recorded on all the 29 tested bacteria. Other samples showed selective activity in the absence of PAβN, their inhibitory effects being observed on 86.2% (25/29)

Chin J Integr Med 2014 Jul;20(7):546-554 Table 2. Strain

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Bacterial Strains Used and Their Features

Feature

Reference

Escherichia coli ATCC8739 and ATCC10536

Reference strains

AG100

Wild-type E. coli K-12

AG100A

AG100 △acrAB:KANR

(55, 56)

AG100ATET

△acrAB mutant AG100, with over-expressing acrF gene; TETR

(55)

AG102

△acrAB mutant AG100, owing acrF gene markedly overexpressed; TETR

(57)

MC4100

Wild type E. coli

W3110

Wild type E. coli

(55)

(58, 59)

Enterobacter aerogenes ATCC13048

Reference strains

EA-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)

ECCI69

Clinical isolates

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

BM47

Clinical isolates

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

BM67

Clinical isolates

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

Enterobacter cloacae

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

K2

AcrAB-TolC

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

Providencia stuartii NEA16

Clinical MDR isolate, AcrAB-TolC

ATCC29914

Clinical MDR isolate, AcrAB-TolC

PS2636

Clinical MDR isolate, AcrAB-TolC

PS299645

Clinical MDR isolate, AcrAB-TolC

(65)

Pseudemonas aeruginosa PA 01

Reference strains

PA 124

MDR clinical isolate

(66)

Notes : AMP, ATMR, CEFR, CFTR, CHLR, FEPR, KANR, MOXR, STRR, TETR meaning resistance to ampicillin, aztreonam, cephalothin, cefadroxil, chloramphenicol, cefepime, kanamycin, moxalactam, streptomycin, and tetracycline

of the tested bacteria for C. citratus , 82.7% (24/29) for T. vulgaris and O. gratissimum , 68.9% (20/29) for P. crispum , 65.5% (19/29) for dried Z. officinale and

A. graveolens , 51.2% (15/29) for C. annuum , 34.5 (10/29) for fresh Z. officinale and 20.7% (06/29) for A. porrum .

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Role of Efflux Pumps in Susceptibility of Gramnegative Bacteria to the Tested Plant Extracts Some of the reference bacterial strains as well as MDR isolated (SM6) were also tested for their susceptibilities to the plant extracts, and CIP used as reference antibiotic, in the presence of the efflux pumps inhibitor, PAβN. Preliminary tests in the present study showed that PAβN do not have any antibacterial activity at 20 μg/mL. When combined with extracts, the PAβN moderately increased the activity T. vulgaris and E. robusta on most of the tested bacteria. This EPI do not potentiate the activity of the other studied extracts.

Effects of the Association of Some Spice Extracts with Antibiotics Five of the studied plant extracts (T. vulgaris , C. citratus , O. basilicum , G . africanum and E. robusta ) were each combined with 10 antibiotics including CLX, AMP, ERY, KAN, CHL, TET, FEP, STR, CIP and NOR to evaluate the possible synergistic effects of the associations. A preliminary study (SM1) was carried out with these antibiotics using P. aeruginosa PA124, one of the MDR bacteria used in this work, to select the appropriate sub-inhibitory concentrations to be used. The results (SM1) allowed the selection of MIC/2 and MIC/5 as the sub-inhibitory concentrations. Three of these five extracts (O. basilicum , G . africanum and E. robusta ) which showed good activities against P. aeruginosa PA124 were then used in association with antibiotics against E. coli AG100, AG100ATET and AG102, E. aerogenes EA27, EA3, EA289 and CM64 and K. pneumoniae KP55 strains. E. robusta significantly increase of the antibiotics activity of >85% of the antibiotics on tested bacteria. The synergistic effects or significant decrease of MIC values were also observed when the extract of G . africanum was combined with antibiotics except with CLX, KAN and CEF. In some of the cases, indifference was observed between O. basilicum , G . africanum extracts and antibiotics meanwhile no antagonistic effect was observed between extracts and antibiotics (except the association of O. basilicum and TET on E. aerogenes EA3; supplemental material SM2–9).

DISCUSSION Chemical Composition of the Plant Extracts The phytochemical studies revealed the

presence of secondary metabolites such as alkaloids, anthocyanins, anthraquinons, flavonoids, phenols, saponins, sterols, tannins and triterpenes; several molecules belonging to these classes of secondary metabolites have being reported for their antimicrobial activities. (7-17) This can, in somehow, explain the activity of the studied spice extracts, but the isolation of the active compounds from each sample is still to be done.

Antibacterial Activity of the Plant Extract The antimicrobial activity of medicinal plants is linked to the presence of one or more classes of bioactive compounds. (17) Furthermore, pharmacologically active compounds of the plant extracts possess one or more functions and act on specific sites in the cell. As these compounds are of different chemical nature, they can differently affect the cellular metabolism of the microorganisms. (18) Compounds belonging flavonoids, quinones and tannins were found to act on bacterial cell wall. Alkaloids, coumarines and some phenolics have their targets within the cell.(19) This can explain the differences of antibacterial activities between plants extracts studied in this work. Ocimum basilicum , G . africanum and E. robusta extracts were active on all tested bacteria, suggesting the presence of either active compounds with a broad antibacterial spectra or the presence of several active constituents acting selectively. The weak activity of the most plant extracts as well as of the reference antibiotic (CIP) as observed herein is obviously due to the fact that most of studied bacteria were MDR phenotypes.

Role of Efflux Pumps in Susceptibility of GramNegative Bacteria to the Tested Plant Extracts The increase of the activity of T. vulgaris , G . africanum and E. robusta on most tested bacteria in the presence of PAβN indicates that their targets are located in the cytoplasm and suggest that the bioactive molecules of these plant extracts are substrate of efflux pumps. In fact, PAβN is the inhibitor of many efflux pumps including AcrAB-TolC from enterobacteriaceae and MexAB-OprM of P. aeruginosa .(4-20, 21)

Effects of the Association of Plant Extracts with Antibiotics Plant extracts from O. basilicum , G . africanum and E. robusta increased the activity of most the

Chin J Integr Med 2014 Jul;20(7):546-554

antibiotics against tested bacteria. This increase of the activity of some tested antibiotics with these plant extracts could result of the synergistic interactions (FIC 0.5) of thier active compounds which might inhibit bacteria by different mechanisms on different target sites of the bacteria.(22) Therfore, O. basilicum and G . africanum did not potentiate the activity of KAN and those of CLX and CEF, the two tested β-lactamines that act open on the bacteria cell wall synthesis, (23) meanwhile synergistic or indifference effects were observed when these two extracts were combined with the other antibiotics (SM7, 8). For E. robusta extract, synergistics effects were mostly observed when this extract was combined with antibiotics against almost all tested bacteria, except with CLX against E . coli AG100 and AG100A TET (SM9). It was shown that an EPI could enhance the activities of antibiotics that are effluxed in strains containing functioning pumps or could increase the level of accumulation and decrease the level of extrusion of efflux pump substrates.(20) Furthermore, it has being demonstrated that a substance can be qualified as an efflux pumps inhibitor if it exerts a synergistic effect with antibiotic on about 70% of bacteria. (12) In this work, E. robusta extract significantly potentiated the activity of TET, STR, CHL, CIP, NOR and ERY on 89%, 78%, 89%, 67%, 78% and 100% tested bacteria respectively. Meanwhile G . africanum extract significantly potentiated the activity of TET, STR and CHL on 78%, 69% and 78% of case respectively (SM1, 3, 4). This means that the intinsic resistance of tested bacteria to these antibiotics was decreased significantly, since bacteria strains used in this work were characterized to be MDR phenotypes overexpressing efflux pumps.(21) E. robusta was reported to have a high antibacterial activity. (24) The results obtained in the present work indicated that E. robusta and G . africanum can be used in association with certain antibiotics to overcome the problem of MDR bacteria.

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Supplementary Materials (see http://www.springer.com/medicine/journal/11655) SM1. Minimal Inhibitory Concentrations and Fractional Inhibitory Concentration (FIC) of AntibioticCrude Extract Association at Different MIC of Extracts on P. aeruginosa PA124 Strain. SM2. Fractional Inhibitory Concentrations (FIC) of Antibiotic-Ocimum Basilicum Extract Associations at MIC/2 and MIC/5 of the Extract. SM3. Fractional Inhibitory Concentration (FIC) of Antibiotic-Gnetum Africanum Extract Associations at MIC/2 and MIC/5 of the Extract. SM4. Fractional Inhibitory Concentrations of Antibiotic-Eucalyptus Robusta Extract Associations at MIC/2 and MIC/5 of the Extract. SM5. Extraction Yield, Physical Aspect and Chemical Composition of the Crude Extracts of Plants Used. SM6. Minimal Inhibitory Concentration of Crude Extracts in the Presence and Absence of PAβN. SM7. Minimal Inhibitory Concentrations of Antibiotic-Ocimum Basilicum Extract Association at MIC/2 and MIC/5 of the Extract. SM8. Minimal Inhibitory Concentrations of Antibiotic-Gnetum Africanum Extract Association at MIC/2 and MIC/5 of the Extract. SM9. Minimal Inhibitory Concentrations of Antibiotic-Eucalyptus Robusta Extract Association at MIC/2 and MIC/5 of the Extract.

Acknowledgements Authors are thankful to the Cameroon National Herbarium (Yaounde) for plants identification, and Mr Gerald N. Teke for technical support and language editing.

The overall results of the present work provide baseline information for the possible use of the studied plants and mostly E. robusta extract in the treatment of bacterial infections involving MDR phenotypes. In addition, the extracts of E. robusta and G . africanum could be used in association with common antibiotics to combat multidrug resistant pathogens.

Competing Interests The authors declare that they have no competing interests.

Authors' Contributions BST carried out the study; VK designed the experiments and wrote the manuscript; VK, SLT, JPD, JRK and JMP supervised the work; VK and JMP provided the bacterial strains;

Chin J Integr Med 2014 Jul;20(7):546-554

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All authors read and approved the final manuscript.

REFERENCES 1. WHO (World Health Organisation). 50 Facts: global health situation and trends. Partnership Forum in Africa, Moscou 2006;26-27.

Phytochemistry 1998;48:647-650. 15. Nascimento GGF, Locatelli J, Freitas PC, Silva GL. Antibacterial activity of plant extracts and phytochemicals on antibioticresistant bacteria. Braz J Microbiol 2000;31:247-256. 16. A l i y u A B , M u s a A M , A b d u l l a h i M S , O y e w a l e A O .

2. Carle S. Antibiotic resistance: an important public health

Phytochemical and antibacterial properties of Ludwigia

concern! Sponsorship of antimicrobial (2010). Pharmactuel

suffruticosa (Willd.) Oliv. ex. O. Ktze (Onagraceae ). Int J

2009;42:6-21.

Pure Appl Sci 2008;2:1-5.

3. Voukeng IK, Kuete V, Dzoyem JP, Fankam AG, Noumedem

17. Reuben KD, Abdulrahman FI, Akan JC, Usman H, Sodipo

JA, Kuiate JR, et al. Antibacterial and antibiotic-potentiation

OA, Egwu GO. Phytochemical screening and in vitro

activities of the methanol extract of some cameroonian

antimicrobial investigation of the methanolic extract of

spices against Gram-negative multi-drug resistant

Croton Zambesicus Muell ARG. stem bark. Eur J Sci Res

phenotypes. BMC Res Notes 2012;5:299. 4. Cattoir V. Efflux pumps and antibiotic resistance in bacteria. Pathol Biol 2004;52:607-616. 5. Poole K. Efflux-mediated multiresistance in Gram negative bacteria. Clin Microbiol Infec 2004;10:12-26. 6. Mesaros M, Bambeke FV, Glupczynski Y, Vanhoof R, Tulkens PM. Antibiotics efflux: a ubiquitous mechanism leading to resistance. State-o-the-art and microbiological

2008;23:134-140. 18. Ghannnoum MA, Rice LB. Antifungal agents: mode of action, mechanism of resistance and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev 1999;12:501-517. 19. Cowan MM. Plants products as antimicrobial agents. Clin Microbiol Rev 1999;12:564-582. 20. Lomovskaya O, Warren M, Lee A, Galazzo J, Fronko R,

and clinical implications. Louvain Med 2005;8:308-320.

Lee M, et al. Identification and characterization of inhibitors

7. Kuete V. Potential of Cameroonian plants and derived

of multidrug resistance efflux pumps in Pseudomonas

products against microbial infections: a review. Planta Med

aeruginosa : novel agents for combination therapy.

2010;76:1479-1491.

Antimicrob Ag Chemother 2001;45:105-116.

8. Harbone JB, ed. Phytochemical methods: a guide to

21. Kuete V, Ngameni B, Tangmouo JG, Bolla JM, Alibert-

modern techniques of plant analysis. London: Chapman

Franco S, Ngadjui BT, et al. Efflux pumps are involved in

and Hall Ltd.; 1973:49-188.

the defence of Gram-negative bacteria against the natural

9. Eloff JN. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med 1998;64:711-713.

products isobavachalcone and diospyrone. Antimicrob Ag Chemother 2010;54:1749-1752. 22. Adwan G, Mhanna M. Synergistic effects of plant extracts

10. Mativandlela SPN, Lall N, Meyer JJM. Antibacterial,

and antibiotics on Staphylococcus aureus strains

antifungal and antitubercular activity of Pelargonium

isolated from clinical specimens. Middle East J Sci Res

reniforme (CURT) and Pelargonium sidoides (DC) (Geraniaceae ) root extracts. S Afr J Bot 2006;72:232-237. 11. Kuete V, Ngameni B, Fotso SCC, Kengap TR, Tchaleu NB,

2008;3:134-139. 23. Tenover FC. Mechanism of antibacterial resistance in bacteria. Am J Med 2006;119:S3-S10.

Meyer JJM, et al. Antimicrobial activity of the crude extracts

24. Sartorelli P, Marquioreto AD, Amaral-Baroli A, Lima MEL,

and compounds from Ficus chlamydocarpa and Ficus

Moreno PRH. Chemical composition and antimicrobial

cordata (Moraceae ). J Ethnopharmacol 2008;120:17-24.

activity of the essential oils from two species of Eucalyptus .

12. Braga LC, Leite AAM, Xavier KGS, Takahashi JA,

Phytother Res 2007;21:231-233.

Bemquerer MP, Charton-Souza E, et al. Synergic interaction

25. Ngoupayo J, Tabopda TK, Shaiq AM. Antimicrobial and

between pomegranate extract and antibiotics against

immuno-modulatory properties of prenylated xanthones

Staphylococcus aureus . Can J Microbiol 2005;51:541-547.

from twigs of Garcinia staudtii . Bioorg Med Chemother

13. Shahverdi AR, Monsef-Esfahani HR, Tavasoli F, Zaheri A, Mirjani R. Trans-cinamaldehyde from Cinnamomum

2009;17:5688-5695. 26. Ukeh DA, Birkett MA, Pickett JA, Bowmana AS, Luntz

zeylanicum bark essential oil reduces the clindamycin

AJM. Repellent activity of alligator pepper, Aframomum

resistance of Clostridium difficile in vitro . J Food Sci

melegueta , and ginger, Zingiber officinale , against

2007;72:55-58. 14. Conrad J, Vogler B, Klaiber I, Roos G, Walter U, Kraus W. Two triterpene esters from Terminalia macroptera bark.

the maize weevil , Sitophilus zeamais . Phytochemistry 2009;70:751-758. 27. Kuete V, Krusche B, Youns M, Voukeng I, Fankam AG,

Chin J Integr Med 2014 Jul;20(7):546-554

• 553 •

Tankeo SB, et al. Cytotoxicity of some Cameroonian spices

42. Charvat TT, Lee DJ, Robinson WE, Chamberlin AR.

and selected medicinal plant extracts. J Ethnopharmacol

Design, synthesis, and biological evaluation of chicoric acid

2011;134:803-812.

analogs as inhibitors of HIVa integrase. Bioorg Med Chem

28. Nguefack J, Lekagne DJB, Dakole CD, Leth V, Vismer HF, Torp J, et al. Food preservative potential of essential

2006;14:4552-4567. 43. Adigüzel A, Güllüce M, SengüL M, Ögúütcü H, Sahün

oils and fractions from Cymbopogon citratus , Ocimum

F, Karaman I. Antimicrobial effects of Ocimum basilicum

gratissimum and Thymus vulgaris against mycotoxigenic

(Labiatae) extract. Turk J Biol 2005;29:155-160.

fungi. Int J Food Microbiol 2009;131:151-156. 29. Özcan M, Chalchat JC. Aroma profile of Thymus vulgaris L. growing wild in Turkey. Bull J Plant Physiol 2004;30:68-73.

44. Auger J Dugravot S, Naudin A, Abo-Ghalia A, Pierre D, Thibout E. Using Allium allelochemicals as insecticides. IOBC Bul 2002;25:1-13.

30. B a r a n a u s k i e n e R , V e n s k u t o n i s P R , V i s k e l i s P ,

45. Akroum S, Satta D, Lalaoui K. Antimicrobial, antioxidant,

Dambrauskiene E. Influence of nitrogen fertilizers on the

cytotoxic activities and phytochemical screening of some

yield and composition of thyme (Thymus vulgaris ). J Agric Food Chemother 2003;51:7751-7758. 31. Johnston JE, Sepe HA, Miano CL, Brannan RG, Aiderton AL. Money inhibits lipid oxidation in ready- to eat ground beef patties. Meat Sci 2005;70:627-631. 32. Negrelle RRB, Gomes EC. Cymbopogon citratus (DC.) Stapf: chemical composition and biological activities. Rev Bras Plants Med 2007;9:80-92. 33. Byrd SJ. Using antioxidants to increase shelf life of food products. Cereal Foods World 2001;46:48-53. 34. Barbosa LCA, Pereira UA, Martinazzo AP, Maltha

Algerian plants. Eur J Sci Res 2009;31:289-295. 46. Ekop AS. Determination of chemical composition of Gnetum

africanum (AFANG) seeds. Pak J Nut 2007;6:40-43. 47. Obiukwu CE, Nwanekwu KE. Evaluation of the antimicrobial potentials of 35 medicinal plants from Nigeria. Int Sci Res J 2010;2:48-51. 48. N a g p a l N , S h a h G , A r o r a M N , S h r i R , A r y a Y . Phytochemical and pharmacological aspects of eucalyptus

genus . Int J Pharm Sci Res 2010;1:28-36. 49. Ngassoum MB, Essia-Ngang JJ, Tatsadjieu LN, Jirovetz L, Buchbauer G, Adjoudjia O. Antimicrobial study of essential

CRÁ, Teixeira RR, Melo EC. Evaluation of the chemical

oils of Ocimum gratissimum leaves and Zanthoxylum

composition of Brazilian commercial Cymbopogon citratus

xanthoxyloides fruits from Cameroon. Fitoterapia

(D.C.) stapf samples. Molecules 2008;13:1864-1874.

2003;74:284-287.

35. Wong PYY, Kitts DD. Studies on the dual antioxidant and

50. Okigbo RN, Ogbonnaya UO. Antifungal effects of two

antibacterial properties of parsley (Petroselinum crispum )

tropical plant leaf extracts (Ocimum gratissimum and

and cilantro (Coriandrum sativum ) extracts. Food Chem

Aframomum melegueta ) on postharvest yam (Dioscorea

2006;97:505-515. 36. Kim OM, Kim MK, Lee SO, Lee KR, Kim SD. Antimicrobial

spp.) rot. Afr J Biotechnol 2006;5:727-731. 51. Lemos JA, Passos XS, Fernandes OFL, Paula JR, Ferri

effect of ethanol extracts from spices against Lactobacillus

PH, Hasimoto LK, et al. Antifungal activity from Ocimum

plantarum and Leuconostoc mesenteroides isolated from

gratissimum L. towards Cryptococcus neoformans . Mem

kimchi. J Kor Soc Food Sci Nutr 1998;27:455-460.

Inst Oswaldo Cruz 2005;100:55-58.

37. Manderfield MM, Schafer HW, Davidson PM, Zottola EA.

52. Lampe JW. Spicing up a vegetarian diet: chemopreventive

Isolation and identification of antimicrobial furocoumarins

effects of phytochemicals. Am J Clin Nut 2003;78:579S-583S.

from parsley. J Food Protect1997;60:72-77.

53. Singh S, Jarret R, Russo V, Majetich G, Shimkus J,

38. Singh A, Handa SS. Hepatoprotective activity of Apium

Bushway R, et al. Determination of capsinoids by

graveolens and Hygrophila auriculata against paracetamol

HPLC-DAD in Capsicum species. J Agric Food Chem

and thioacetamide intoxication in rats. J Ethnopharmacol 1995;49:119-126. 39. Rani P, Khullar N. Antimicrobial evaluation of some medicinal

2009;57:3452-3457. 54. Ku KM, Kang YH. Quinone reductase inductive activity of Capsicum annuum leaves and isolation of the active

plants for their antienteric potential against multi-drug

compounds. J Kor Soc Appl Biol Chem 2010;53:709-715.

resistant Salmonella typhi . Phytother Res 2004;18:670-673.

55. Viveiros M, Jesus A, Brito M, Leandro C, Martins M,

40. Ko FN, Huang TF, Teng CM. Vasodilatory action

Ordway D, et al. Inducement and reversal of tetracycline

mechanisms of apigenin isolated from Apium graveolens in

resistance in Escherichia coli K-12 and expression of

rat thoracic aorta. Biochim Biophys Acta 1991;1115:69-74.

proton gradient-dependent multidrug efflux pump genes.

41. Uma DP. Radioprotective, anticarcinogenic and antioxidant properties of the Indian holy basil, Ocimum sanctum (Tulasi). Ind J Exp Biol 2001;39:185-190.

Antimicrob Ag Chemother 2005;49:3578-3582. 56. Okusu H, Ma D, Nikaido H. AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of

• 554 •

Escherichia coli multiple-antibiotic- resistance (Mar) mutants. J Bacteriol 1996;178:306-308. 57. Elkins CA, Mullis LB. Substrate competition studies using whole-cell with the major tripartite multidrug efflux pumps of Escherichia coli . Antimicrob Ag Chemother 2007;51:923-929. 58. Baglioni P, Bini L, Liberatori S, Pallini V, Marri L. Proteome analysis of Escherichia coli W3110 expressing an heterologous sigma factor. Proteomics 2003;3:1060-1065. 59. Sar C, Mwenya B, Santoso B, Takaura K, Morikawa R,

Chin J Integr Med 2014 Jul;20(7):546-554 62. Malléa M, Mahamoud A, Chevalier J, Alibert-Franco S, Brouant P, Barbe J, et al. Alkylaminoquinolines inhibit the bacterial antibiotic efflux pump in multidrug-resistant clinical isolates. Biochem J 2003;376:801-805. 63. Pradel E, Pagès J-M. The AcrAB-TolC efflux pump contributes to multidrug resistance in the nosocomial pathogen Enterobacter aerogenes . Antimicrob Ag Chemother 2002;46:2640-2643. 64. Chevalier J, Pages J-M, Eyraud A, Mallea M. Membrane permeability modifications are involved in antibiotic

Isogai N, et al. Effect of Escherichia coli wild type or its

resistance in Klebsiella pneumoniae . Biochem Biophys Res

derivative with high nitrite reductase activity on in vitro

Commun 2000;274:496-499.

ruminal methanogenesis and nitrate/nitrite reduction. J Anim Sci 2005;83:644-652. 60. G h i s a l b e r t i D , M a s i M , P a g è s J M , C h e v a l i e r J . Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes . Biochem Biophys Res Commun 2005;328:1113-1118. 61. Malléa M, Chevalier J, Bornet C, Eyraud A, Pagès JM, Davin-Régli A. Porin alteration and active efflux: two in vivo drug resistance strategies used by Enterobacter aerogenes . Microbiology 1998;144:3003-3009.

65. Tran QT, Mahendran KR, Hajjar E, Ceccarelli M, DavinRegli A, Winterhalter M, et al. Implication of porins in betalactam resistance of Providencia stuartii . J Biol Chem 2010;285:32273-32281. 66. Lorenzi V, Muselli A, Bernardini AF, Berti L, Pagès JM, Amaral L, et al. Geraniol restores antibiotic activities against multidrug resistant isolate from Gram-negative species. Antimicrob Ag Chemother 2009;53:2209-2211. (Received November 25, 2012) Edited by GUO Yan