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Evidence-Based Complementary and Alternative Medicine
Volume 2012 (2012), Article ID 623723, 11 pages
doi:10.1155/2012/623723
Research Article

Antibacterial Activities of Selected Cameroonian Plants and Their Synergistic Effects with Antibiotics against Bacteria Expressing MDR Phenotypes

Stephen T. Lacmata,1 Victor Kuete,1 Jean P. Dzoyem,1 Simplice B. Tankeo,1 Gerald Ngo Teke,1 Jules R. Kuiate,1 and Jean-Marie Pages2

1Department of Biochemistry, Faculty of Science, University of Dschang, Dschang, Cameroon
2Transporteurs Membranaires, Chimioresistance et Drug Design, UMR-MD1, IFR 88, Université de la Méditerranée, Aix-Marseille II, Marseille, France

Received 20 October 2011; Accepted 13 December 2011

Academic Editor: Namrita Lall

Copyright © 2012 Stephen T. Lacmata et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The present work was designed to assess the antibacterial properties of the methanol extracts of some Cameroonian medicinal plants and the effect of their associations with currently used antibiotics on multidrug resistant (MDR) Gram-negative bacteria overexpressing active efflux pumps. The antibacterial activities of twelve methanol extracts of medicinal plants were evaluated using broth microdilution. The results of this test showed that three extracts Garcinia lucida with the minimal inhibitory concentrations (MIC) varying from 128 to 512  g/mL, Garcinia kola (MIC of 256 to 1024  g/mL), and Picralima nitida (MIC of 128 to 1024  g/mL) were active on all the twenty-nine studied bacteria including MDR phenotypes. The association of phenylalanine arginine -naphthylamide (PA N or efflux pumps inhibitor) to different extracts did not modify their activities. At the concentration of MIC/2 and MIC/5, the extracts of P. nitida and G. kola improved the antibacterial activities of some commonly used antibiotics suggesting their synergistic effects with the tested antibiotics. The results of this study suggest that the tested plant extracts and mostly those from P. nitida, G. lucida and G. kola could be used alone or in association with common antibiotics in the fight of bacterial infections involving MDR strains.

1. Introduction

Bacterial infections are responsible for 90% of infections found in health care services. The emergence of MDR bacterial strains appears as the major cause of treatment failure [1]. Among the known mechanisms of resistances, active efflux via resistance-nodulation-cell division (RND) pumps is one of the most occurring system in Gram-negative bacterial strains [2]. Efflux pumps are transport proteins involved in the extrusion of toxic substrates (including virtually all classes of clinically relevant antibiotics). The present work was therefore designed to investigate the antibacterial potential against MDR bacteria expressing active efflux though RND pumps. Medicinal plants of Cameroon used in this study include the fruits of Citrus medica L. (Rutaceae), the bulbs of Allium sativum L. (Liliaceae) and Allium cepa L. (Liliaceae), the seeds of Carica papaya Linn (Caricaceae), Cola acuminata (P. Beauv.) Schott and Endl. (Sterculiaceae), Buchholzia coriacea Engl. (Capparidaceae), Garcinia kola Heckel (Guttifeare), and Garcinia lucida Vesque (Guttifeare), the seeds and fruits of Picralima nitida; the potential of the extract from the above plant extracts to increase the activity of some antibiotics on MDR bacteria was also investigated as well as the role of bacterial efflux pumps in the resistance to the tested plant extracts.

2. Material and Methods

2.1. Plant Materials and Extraction

The nine edible plants used in this work were purchased from Dschang local market, west region of Cameroon in January 2010. The collected vegetal material were the fruits of Citrus medica, the bulbs of Allium sativum and Allium cepa, the seeds of Carica papaya, Cola acuminata, Buchholzia coriacea, Garcinia kola, and Garcinia lucida, the seeds and fruits of Picralima nitida. The plants were identified by Mr. Tadjouteu Fulbert (Botanist) of the National Herbarium (Yaoundé, Cameroon) where voucher specimens were deposited under a reference number (Table 1).

tab1
Table 1: Plants used in the present study and evidence of their activities.

The fresh or powdered air-dried sample (1 kg) from each plant was extracted with methanol (MeOH) for 48 h at room temperature. The extract was then concentrated under reduced pressure to give a residue that constituted the crude extract. They were then kept under 4°C until further use.

2.2. Preliminary Phytochemical Investigations

The presence of major secondary metabolite classes, namely, alkaloids, flavonoids, phenols, saponins, tannins, anthocyanins, anthraquinones, sterol, and triterpenes was determined using common phytochemical methods as described by Harborne [3].

2.3. Chemicals for Antimicrobial Assays

Ciprofloxacin (CIP), chloramphenicol (CHL), streptomycin (STR), tetracycline (TET), norfloxacin (NFX), cloxacillin (CLX), ampicillin (AMP), erythromycin (ERY), kanamycin (KAN), and cefepim (CEF) (Sigma-Aldrich, St Quentin Fallavier, France) were used as reference antibiotics. p-Iodonitrotetrazolium chloride (INT) and phenylalanine arginine β-naphthylamide (PAβN) were used as microbial growth indicator and efflux pumps inhibitor (EPI), respectively.

2.4. Bacterial Strains and Culture Media

The studied microorganisms include references (from the American Type Culture Collection) and clinical (Laboratory collection) strains of Escherichia coli, Enterobacter aerogenes, Providencia stuartii, Pseudomonas aeruginosa, Klebsiella pneumonia, and Enterobacter cloacae (Table 2). They were maintained on agar slant at 4°C and subcultured on a fresh appropriate agar plates 24 hrs prior to any antimicrobial test. Mueller Hinton Agar was used for the activation of bacteria. The Mueller Hinton Broth (MHB) was used for the MIC determinations.

tab2
Table 2: Bacterial strains and features.
2.5. Bacterial Susceptibility Determinations

The respective MICs of samples on the studied bacteria were determined by using rapid INT colorimetric assay [4]. Briefly, the test samples were first dissolved in DMSO/MHB. The solution obtained was then added to MHB, and serially diluted twofold (in a 96-well microplate). One hundred microlitres (100 μL) of inoculum (1.5 × 106 CFU/mL) prepared in MHB was then added. The plates were covered with a sterile plate sealer, then agitated to mix the contents of the wells using a shaker and incubated at 37°C for 18 hrs. The final concentration of DMSO was lower than 2.5% and does not affect the microbial growth. Wells containing MHB, 100 μL of inoculums, and DMSO at a final concentration of 2.5% served as a negative control. Ciprofloxacin was used as reference antibiotic. The MICs of samples were detected after 18 hrs of incubation at 37°C, following addition (40 μL) of 0.2 mg/mL INT and incubation at 37°C for 30 minutes [5]. Viable bacteria reduced the yellow dye to pink. MIC was defined as the lowest sample concentration that prevented this change and exhibited complete inhibition of microbial growth.

Samples were tested alone and then, in the presence of PAβN at 30 μg/mL final concentration. Two of the best extracts, those from seeds of Garcinia kola and Picralima nitida fruits were also tested in association with antibiotics at MIC/2 and MIC/5. These concentrations were selected following a preliminary assay on one of the tested MDR bacteria, P. aeruginosa PA124 (see Supplemental Material S1 available online at doi:10.1155/2012/623723.). All assays were performed in triplicate and repeated thrice. Fractional inhibitory concentration (FIC) was calculated as the ratio of MICAntibiotic  in  combination/MICAntibiotic  alone and the interpretation made as follows: synergistic (FIC ≤ 0.5), indifferent (0.5 < FIC < 4), or antagonistic (FIC ≥ 4) [6]. (The FIC values are available in Supplemental Material S2).

3. Results

3.1. Phytochemical Composition of the Plant Extracts

The results of qualitative analysis showed that each plant contains various phytochemicals compounds such as alkaloids, anthocyanins, anthraquinons, flavonoids, phenols, saponins, tannins, and triterpenes as shown in Table 3.

tab3
Table 3: Extraction yields, aspects, and phytochemical composition of the plant extracts.
3.2. Antibacterial Activity of the Plant Extracts

Extracts were tested for their antibacterial activities alone and in combination with PAβN on a panel of Gram-negative bacteria by the microdilution method. Results summarized in Table 4 showed that the most active extracts were those from Garcinia lucida (MIC ranged from 128 to 512 μg/mL), Garcinia kola (MIC from 128 to 1024 μg/mL), and the fruits of Picralima nitida (MIC from 256 to 1024 μg/mL). The antibacterial activities of these plant species were recorded against all the 29 studied microorganisms. Other extracts exhibited weak activities against a limited number of strains studied.

tab4
Table 4: Minimal inhibitory concentration (μg/mL) of methanol extracts from the studied plants and ciprofloxacin.
3.3. Role of Efflux Pumps in Susceptibility of Gram-Negative Bacteria to the Tested Plants Extracts

The various strains and MDR isolates were also tested for their susceptibility to the plants extracts, and reference antibiotic (ciprofloxacin) in the presence of PAβN, an EPI. Preliminary tests showed that PAβN did not have any antibacterial activity at 30 μg/mL. The association of the PAβN with the extracts reduced the MIC values of some of the extracts on some tested bacteria (Table 4). However, most of the studied extracts are not the substrates of the active efflux pumps.

3.4. Effects of the Association of Some Plants Extracts with Antibiotics

The strain P. aeruginosa PA124 was used to find the appropriate subinhibitory concentration of the antibiotic-crude extract to be tested on other bacteria strains. The association of the extracts of P. nitida and G. kola reduced the MIC of ten antibiotics (CLX, AMP, ERY, KAN, CHL, TET, FEP, STR, CIP, and NOR) at MIC/2 and/or MIC/5 explaining the use of such concentrations. The associations of the extracts of P. nitida fruits and G. kola with antibiotics did not show any case of antagonism (FIC ≥ 4) meanwhile indifference was observed in some cases of the associations of the extracts with FEP, CLX, and AMP (see Tables 5 and 6, Supplemental Material S2). Many cases of synergy were observed in most of the strains with the associations G. kola/ERY against CM64, P. nitida/NOR against KP63, and P. nitida/ERY against PA124.

tab5
Table 5: MIC of different antibiotics after the association of the extract of Picralima nitida fruits at MIC/2, MIC/5 against ten MDR bacteria strains.
tab6
Table 6: MIC of different antibiotics after the association of the extract of Garcinia kola seeds at MIC/2, MIC/5 against ten MDR bacteria strains.

4. Discussion

4.1. Antibacterial Activities and Chemicals Compositions of the Tested Extracts

The phytochemical studies revealed the presence of at least two classes of secondary metabolites in each of the plant extracts. Several alkaloids, flavonoids, phenols, saponins, anthocyanins, anthraquinones, sterols, tannins, and triterpenes have been found active on pathogenic microorganisms [44, 45]. Some of these compounds were found to be present in the plant species under this study, and they could contribute to the observed antimicrobial activities of some plant extracts. The results of the phytochemical test on G. kola are in accordance with those obtained by Onayade et al., [46, 47]. Many compounds have been isolated from G. kola, such as kolaflavone and 2-hydroxybiflavone [48–50] but their antimicrobials activities have not been evaluated. However, Adegboye et al. [51] reported the activity of G. kola on some streptomycin-sensitive Gram-positive bacteria strain. The present study therefore provides additional information on the antibacterial potential of this plant on MDR bacteria.

The previous phytochemical analyses on hexane extract from the seeds of G. lucida revealed several types of compounds [8, 23]. These include terpenoids, anthocyanins, flavonoids, and saponins derivatives. This report therefore agrees well with the phytochemical data being reported herein.

The results of the phytochemical analysis of the extract of fruits of P. nitida are similar to those obtained by Kouitcheu [52]. Several alkaloids previously isolated from this plant include akuammicine, akuammine, akuammidine, picraphylline, picraline, and pseudoakuammigine [32, 53]. Their antibacterial activities have not yet been demonstrated but many alkaloids are known to be active on Gram-negative bacteria [33]. Differences were noted in the chemical composition of the seeds and fruits of P. nitida, evidently explaining the differences in the antibacterial activity of the two parts of this plant. In fact, the presence of tannins in the fruits may contributes to its better activity compared to the seeds as they were reported to inactivate the microbial adhesins, enzymes, transports proteins and cellular envelop [54].

Extracts from C. papaya, C. medica, B. coriacea, A. cepa, and C. acuminata showed weak activities against a limited number of strains. Nonetheless, the extracts from B. coriacea were rather reported to have good antibacterial activities. Their weak activities as observed in the present paper could therefore be due to the multidrug resistance of the studied bacteria.

4.2. Effects of the Association of Some Plants Extracts with Antibiotics

Three of the most active plants extracts (G. kola, G. lucida, and P. nitida) were associated with antibiotics with the aim to evaluate the possible synergistic effects of their associations. A preliminary study using P. aeruginosa PA124, one of the ten MDR bacteria used in this paper, was carried out with ten antibiotics (CLX, AMP, ERY, KAN, CHL, TET, FEP, STR, CIP, and NOR) to select the appropriate sub-inhibitory concentrations of the extract to be used. The results (see Supplemental Material S1) allowed the selection of G. kola, G. lucida and their MIC/2 and MIC/5 as the sub-inhibitory concentrations. No antagonistic effect (FIC ≥ 4) was observed between extracts and antibiotics meanwhile indifference was observed in the case of CLX, FEP, AMP, which are β-lactams acting on the synthesis of the bacteria cell wall [55] (Tables 5 and 6, Supplemental Material S2). Many studies demonstrated that efflux is the mechanism of resistance of bacteria for almost all antibiotic classes [56]. It is well demonstrated that the efflux pumps reduce the intracellular concentration of antibiotics and consequently their activities [57]. The MDR bacteria strains used in this paper are known for their ability to overexpress active efflux [58]. At MIC/2, synergistic effects were noted with the association of NOR, CHL, TET (on 100% the studied bacteria), ERY (on 80%), CIP (on 70%), and P. nitida extract meanwhile G. kola extract also increased the activity of NOR, TET (on 100%), ERY, and CIP (on 70%). Plant can be considered as an efflux pumps inhibitor if a synergistic effect with antibiotics is induced on more than 70% bacteria expressing active efflux pumps [6]. Therefore, the extracts from P. nitida and G. kola probably contain compounds that can acts as EPI. The results of the present paper corroborate with those of Iwu et al. [7] reporting the existence of synergy effects between G. kola extract and gatifloxacin (G. kola/gatifloxacin in the proportions of 9/1, 8/2, 7/3, and 6/4) against Bacillus subtilis and the proportions of G. kola/gatifloxacin (at 9/1, 2/8, and 1/9) against Staphylococcus aureus.

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

Acknowledgments

The authors are thankful to the Cameroon National Herbarium (Yaounde) and University of Dschang Herbarium for plants identification.

References

  1. J. Koyama, “Anti-infective quinone derivatives of recent patents,” Recent Patents on Anti-infective Drug Discovery, vol. 1, no. 1, pp. 113–125, 2006. View at Publisher · View at Google Scholar · View at Scopus
  2. O. Lomovskaya and W. Watkins, “Inhibition of efflux pumps as a novel approach to combat drug resistance in bacteria,” Journal of Molecular Microbiology and Biotechnology, vol. 3, no. 2, pp. 225–236, 2001. View at Scopus
  3. J. B. Harborne, Phytochemical Methods, Chapman and Hall, New York, NY, USA, 1973.
  4. S. P. N. Mativandlela, N. Lall, and J. J. M. Meyer, “Antibacterial, antifungal and antitubercular activity of (the roots of) Pelargonium reniforme (CURT) and Pelargonium sidoides (DC) (Geraniaceae) root extracts,” South African Journal of Botany, vol. 72, no. 2, pp. 232–237, 2006. View at Publisher · View at Google Scholar · View at Scopus
  5. V. Kuete, B. Ngameni, C. C. F. Simo et al., “Antimicrobial activity of the crude extracts and compounds from Ficus chlamydocarpa and Ficus cordata (Moraceae),” Journal of Ethnopharmacology, vol. 120, no. 1, pp. 17–24, 2008. View at Publisher · View at Google Scholar · View at Scopus
  6. A. Athamna, M. Athamna, A. Nura et al., “Is in vitro antibiotic combination more effective than single-drug therapy against anthrax?” Antimicrobial Agents and Chemotherapy, vol. 49, no. 4, pp. 1323–1325, 2005. View at Publisher · View at Google Scholar · View at Scopus
  7. M. W. Iwu, A. R. Duncan, and C. O. Okunji, “News antimicrobials of plant origin,” in Perspectives on New Crops and New Uses, J. Janick, Ed., pp. 457–462, ASHS Press, Alexandria, Va, USA, 1999.
  8. N. M. Guedje and R. Fankap, “Utilisations traditionnelles de Garcinia lucida et Garcinia kola (Clusiaceae) au Cameroun, Phytogeography for the Understanding of African Plant Systematics and Biodiversity, Systematics and Geography of Plants,” National Botanic Garden of Belgium, vol. 71, no. 2, pp. 747–758, 2001.
  9. I. A. Abdou, A. A. Abou-Zeid, M. R. El-Sherbeeny, and Z. H. Abou-El-Gheat, “Antimicrobial activities of Allium sativum, Allium cepa, Raphanus sativus, Capsicum frutescens, Eruca sativa, Allium kurrat on bacteria,” Plant Foods for Human Nutrition, Chemistry and Material Science, vol. 22, pp. 29–35, 2010.
  10. E. Quisumbing, Medicinal Plants of the Philippines, Katha, Manila, Philippines, 1978.
  11. N. K. Lohiya, N. Pathak, P. K. Mishra, and B. Manivannan, “Contraceptive evaluation and toxicological study of aqueous extract of the seeds of Carica papaya in male rabbits,” Journal of Ethnopharmacology, vol. 70, no. 1, pp. 17–27, 2000. View at Publisher · View at Google Scholar · View at Scopus
  12. J. A. Osato, A. Librado, M. R. Gemma, et al., Antimicrobial and Antioxidant Activities of Unripe Papaya, Sun-O International, Gifu, Japan, 1993.
  13. T. I. Mbota, C. M. Duru, and H. A. Onwumelu, “Antibacterial activity of crude seed extracts of Buchholzia coriaceae on some pathogenic bacteria,” Journal of Developmental Biology and Tissue Engineering, vol. 1, no. 1, pp. 001–005, 2009.
  14. E. O. Ajaiyeoba, P. A. Onocha, S. O. Nwozo, and W. Sama, “Antimicrobial and cytotoxicity evaluation of Buchholzia coriacea stem bark,” Fitoterapia, vol. 74, no. 7-8, pp. 706–709, 2003. View at Publisher · View at Google Scholar · View at Scopus
  15. http://en.wikipedia.org/wiki/Citrus_medica. citron, 2010.
  16. V. Kuete, V. Penlap, F. X. Etoa, et al., “Activités antimicrobienne de l'extrait total et des fractions de jus de fruit de Citrus medica Linn. (Rutaceae),” Pharmacopee et Medecine Traditionelle Africaine, vol. 13, pp. 91–101, 2004.
  17. “Cola acuminata,” http://en.wikipedia.org/wiki/Cola_acuminata, 2010.
  18. O. J. Uko, A. Usman, and A. M. Ataja, “Some biological activities of Garcinia kola in growing rats,” Veterinarski Arhiv, vol. 71, no. 5, pp. 287–297, 2001. View at Scopus
  19. V. Kuete, B. Krusche, M. Youns, et al., “Cytotoxicity of some Cameroonian spices and selected medicinal plant extracts,” Journal of Ethnopharmacology, vol. 134, no. 3, pp. 803–812, 2011.
  20. M. M. Iwu, Handbook of African Medicinal Plants, CRC Press, London, UK, 1993.
  21. R. A. Hussain, A. G. Owegby, and P. G. Waterman, “Kolanone, a novel polyisoprenylated benzophenone with antimicrobial properties from the fruit of Garcinia kola,” Planta Medica, vol. 44, no. 2, pp. 78–81, 1982. View at Scopus
  22. J. F. Akoachere, R. N. Ndip, R. B. Chenwi, L. M. Ndip, T. E. Njock, and D. E. Anong, “Antibactirial effect of Zingiber officinale and Garcinia kola on respiratory track pathogens,” East African Medicinal Journal, vol. 79, no. 11, pp. 588–592, 2002.
  23. A. M. Nyemba, T. N. Mpondo, J. D. Connolly, and D. S. Rycroft, “Cycloartane derivatives from Garcinia lucida,” Phytochemistry, vol. 29, no. 3, pp. 994–997, 1990. View at Scopus
  24. J. Fotie, D. S. Bohle, M. Olivier, M. A. Gomez, and S. Nzimiro, “Trypanocidal and antileishmanial dihydrochelerythrine derivatives from Garcinia lucida,” Journal of Natural Products, vol. 70, no. 10, pp. 1650–1653, 2007. View at Publisher · View at Google Scholar · View at Scopus
  25. J. Gangoué-Piéboji, Caractérisation des β-Lactamases et leur inhibition par les extraits de plantes médicinales, Thèse présenté en vue d’obtention du diplôme de Doctorat ès Sciences en Biochimie, Université de Liège, Centre d’Ingénierie des Protéines, 2007.
  26. G. C. Kirby, N. B. Khumalo-Ngwenya, B. A. Grawehr, T. W. Fison, D. C. Warhurst, and J. D. Phillipson, “Antimalarial activity from 'Mhekara' (Uapaca nitida Mull-Arg.), a Tanzanian tree,” Journal of Ethnopharmacology, vol. 40, no. 1, pp. 47–51, 1993. View at Scopus
  27. K. Likhitwitayawuid, C. K. Angerhofer, G. A. Cordell, J. M. Pezzuto, and N. Ruangrungsri, “Traditional medicinal plants of Thailand. 20. Cytotoxic and antimalarial bisbenzylisoquinoline alkaloids from Stephania erecta,” Journal of Natural Products, vol. 56, 38 pages, 1993.
  28. M. E. Xu, S. Z. Xiao, Y. H. Sun, Y. Ou, C. Guan, and X. X. Zheng, “A preadipocyte differentiation assay as a method for screening potential anti-type II diabetes drugs from herbal extracts,” Planta Medica, vol. 72, no. 1, pp. 14–19, 2006. View at Publisher · View at Google Scholar · View at Scopus
  29. H. Shittu, A. Gray, B. Furman, and L. Young, “Glucose uptake stimulatory effect of akuammicine from Picralima nitida (Apocynaceae),” Phytochemistry Letters, vol. 3, no. 1, pp. 53–55, 2010. View at Publisher · View at Google Scholar · View at Scopus
  30. G. François, L. Aké Assi, J. Holenz, and G. Bringmann, “Constituents of Picralima nitida display pronounced inhibitory activities against asexual erythrocytic forms of Plasmodium falciparum in vitro,” Journal of Ethnopharmacology, vol. 54, no. 2-3, pp. 113–117, 1996. View at Publisher · View at Google Scholar · View at Scopus
  31. M. Viveiros, A. Jesus, M. Brito et al., “Inducement and reversal of tetracycline resistance in Escherichia coli K-12 and expression of proton gradient-dependent multidrug efflux pump genes,” Antimicrobial Agents and Chemotherapy, vol. 49, no. 8, pp. 3578–3582, 2005. View at Publisher · View at Google Scholar · View at Scopus
  32. C. K. Nkere and C. U. Iroegbu, “Antibacterial screening of the root, seed and stembark extracts of Picralima nitida,” African Journal of Biotechnology, vol. 4, no. 6, pp. 522–526, 2005. View at Scopus
  33. V. Kuete, “Potential of Cameroonian plants and derived products against microbial infections: a review,” Planta Medica, vol. 76, no. 14, pp. 1479–1491, 2010. View at Publisher · View at Google Scholar · View at Scopus
  34. H. Okusu, D. Ma, and H. Nikaido, “AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants,” Journal of Bacteriology, vol. 178, no. 1, pp. 306–308, 1996. View at Scopus
  35. C. A. Elkins and L. B. Mullis, “Substrate competition studies using whole-cell accumulation assays with the major tripartite multidrug efflux pumps of Escherichia coli,” Antimicrobial Agents and Chemotherapy, vol. 51, no. 3, pp. 923–929, 2007. View at Publisher · View at Google Scholar · View at Scopus
  36. P. Baglioni, L. Bini, S. Liberatori, V. Pallini, and L. Marri, “Proteome analysis of Escherichia coli W3110 expressing an heterologous sigma factor,” Proteomics, vol. 3, no. 6, pp. 1060–1065, 2003. View at Publisher · View at Google Scholar · View at Scopus
  37. D. Ghisalberti, M. Masi, J. M. Pagès, and J. Chevalier, “Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes,” Biochemical and Biophysical Research Communications, vol. 328, no. 4, pp. 1113–1118, 2005. View at Publisher · View at Google Scholar · View at Scopus
  38. M. Malléa, A. Mahamoud, J. Chevalier et al., “Alkylaminoquinolines inhibit the bacterial antibiotic efflux pump in multidrug-resistant clinical isolates,” Biochemical Journal, vol. 376, no. 3, pp. 801–805, 2003. View at Publisher · View at Google Scholar · View at Scopus
  39. M. Mallea, J. Chevalier, C. Bornet et al., “Porin alteration and active efflux: two in vivo drug resistance strategies used by Enterobacter aerogenes,” Microbiology, vol. 144, no. 11, pp. 3003–3009, 1998. View at Scopus
  40. E. Pradel and J. M. Pagès, “The AcrAB-TolC efflux pump contributes to multidrug resistance in the nosocomial pathogen Enterobacter aerogenes,” Antimicrobial Agents and Chemotherapy, vol. 46, no. 8, pp. 2640–2643, 2002. View at Publisher · View at Google Scholar · View at Scopus
  41. J. Chevalier, J. M. Pagès, A. Eyraud, and M. Malléa, “Membrane permeability modifications are involved in antibiotic resistance in Klebsiella pneumoniae,” Biochemical and Biophysical Research Communications, vol. 274, no. 2, pp. 496–499, 2000. View at Publisher · View at Google Scholar · View at Scopus
  42. Q. T. Tran, K. R. Mahendran, E. Hajjar et al., “Implication of porins in β-lactam resistance of Providencia stuartii,” Journal of Biological Chemistry, vol. 285, no. 42, pp. 32273–32281, 2010. View at Publisher · View at Google Scholar · View at Scopus
  43. V. Lorenzi, A. Muselli, A. F. Bernardini et al., “Geraniol restores antibiotic activities against multidrug-resistant isolates from gram-negative species,” Antimicrobial Agents and Chemotherapy, vol. 53, no. 5, pp. 2209–2211, 2009. View at Publisher · View at Google Scholar · View at Scopus
  44. A. L. Otshudi, A. Vercruysse, and A. Foriers, “Contribution to the ethnobotanical, phytochemical and pharmacological studies of traditionally used medicinal plants in the treatment of dysentery and diarrhoea in Lomela area, Democratic Republic of Congo (DRC),” Journal of Ethnopharmacology, vol. 71, no. 3, pp. 411–423, 2000. View at Publisher · View at Google Scholar · View at Scopus
  45. R. Havagiray, C. Ramesh, and K. Sadhna, “Study of antidiarrhoeal activity of Calotropis gigantean in experimental animals,” Journal of Pharmacology and Pharmaceutical Science, vol. 7, no. 1, pp. 70–75, 2004.
  46. O. A. Onayade, A. M. G. Looman, J. J. C. Scheffer, and Z. O. Gbile, “lactone and other volatile constituents of the oleoresin from seeds of Garcinia kola hechel,” Flavour Frangrance Journal, vol. 13, no. 6, pp. 409–412, 1998.
  47. R. U. B. Ebana, B. E. Madunagu, E. D. Ekpe, and I. N. Otung, “Microbiological exploitation of cardiac glycosides and alkaloids from Garcinia kola, Borreria ocymoides, Kola nitida and Citrus aurantifolia,” Journal of Applied Bacteriology, vol. 71, no. 5, pp. 398–401, 1994. View at Scopus
  48. C. O. Okunji and M. M. Iwu, “Molluscicidal activity of Garcinia kola biflavanones,” Fitoterapia, vol. 62, no. 1, pp. 74–76, 1991. View at Scopus
  49. K. Terashima, Y. Kondo, M. Aqil, M. Waziri, and M. Niwa, “A study of biflavanones from the stems of Garcinia kola (Guttiferae),” Heterocycles, vol. 50, no. 1, pp. 283–290, 1999. View at Scopus
  50. C. O. Okunji, A. W. Tantalia, R. P. Hicks, M. M. Iwu, and D. J. Skanchy, “Capilary determination of biflavonones from Garcinia kola in three traditional African medical formulations,” Planta Medica, vol. 68, pp. 440–444, 2002.
  51. M. F. Adegboye, D. A. Akinpelu, and A. I. Okoh, “The bioactive and phytochemical properties of Garcinia kola (Heckel) seed extract on some pathogens,” African Journal of Biotechnology, vol. 7, no. 21, pp. 3934–3938, 2008. View at Scopus
  52. M. L. B. Kouitcheu, Evaluation des propriétés anti-diarrhéiques et de la toxicité de Cylecodicus gabunensis (Mimosaceae) et Picralima nitida (Apocynaceae), plantes médicinales utilisées dans le traitement des maladies diarrhéiques, Thèse de Doctorat/PHD en Biochimie, Université de Yaoundé, Yaoundé, Cameroun, 2007.
  53. G. François, L. Aké Assi, J. Holenz, and G. Bringmann, “Constituents of Picralima nitida display pronounced inhibitory activities against asexual erythrocytic forms of Plasmodium falciparum in vitro,” Journal of Ethnopharmacology, vol. 54, no. 2-3, pp. 113–117, 1996. View at Publisher · View at Google Scholar · View at Scopus
  54. M. M. Cowan, “Plant products as antimicrobial agents,” Clinical Microbiology Reviews, vol. 12, no. 4, pp. 564–582, 1999. View at Scopus
  55. X. Z. Li and H. Nikaido, “Efflux-mediated drug resistance in bacteria,” Drugs, vol. 64, no. 2, pp. 159–204, 2004. View at Publisher · View at Google Scholar · View at Scopus
  56. F. Van Bambeke, J.-M. Pages, and V. J. Lee, “Inhibitor of bacterial efflux pumps as adjuvants in antibacterial therapy and diagnostic tools for detection of resistance by efflux,” Frontier in Anti-Infective Drug Discovery, vol. 1, pp. 170–175, 2010.
  57. V. Kuete, B. Ngameni, J. G. Tangmouo et al., “Efflux pumps are involved in the defense of gram-negative bacteria against the natural products isobavachalcone and diospyrone,” Antimicrobial Agents and Chemotherapy, vol. 54, no. 5, pp. 1749–1752, 2010. View at Publisher · View at Google Scholar · View at Scopus
  58. K. C. Ofokansi, A. N. Mbanefo, M. N. Ofokansi, and C. O. Esimone, “Antibacterial interaction of crude methanol extract of Garcinia kola seed with gatifloxacin,” Tropical Journal of Pharmacological Research, vol. 7, no. 4, pp. 1159–1165, 2008.