Multidrug Resistant Bacteria Are Sensitive to Euphorbia Prostrata and Six Others Cameroonian Medicinal Plants Extracts Igor K
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Voukeng et al. BMC Res Notes (2017) 10:321 DOI 10.1186/s13104-017-2665-y BMC Research Notes RESEARCH ARTICLE Open Access Multidrug resistant bacteria are sensitive to Euphorbia prostrata and six others Cameroonian medicinal plants extracts Igor K. Voukeng1, Veronique P. Beng2 and Victor Kuete1* Abstract Background: Multidrug resistant (MDR) bacteria are responsible for therapeutic failure and there is an urgent need for novels compounds efcient on them. Methods: Eleven methanol extracts from seven Cameroonian medicinal plants were tested for their antibacterial activity using broth micro-dilution method against 36 MDR bacterial strains including Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Providencia stuartii, Pseudomonas aeruginosa and Staphylococ- cus aureus. Results: Euphorbia prostrata extract was found active against all the 36 tested bacteria including Gram-negative phe- notypes over-expressing efux pumps such as P. aeruginosa PA124, E. aerogenes CM64 and E. coli AG102. E. prostrata had minimal inhibitory concentrations values between 128 and 256 µg/mL on 55.55% of the studied microorganisms. Other plants extract displayed selective antibacterial activity. Conclusions: Results obtained in this study highlight the antibacterial potential of the tested plants and the possible use of E. prostrata to combat bacterial infections including MDR phenotypes. Keywords: Antimicrobial, Cameroon, Euphorbia prostrata, Medicinal plant, Multidrug resistance Background plants ofer a suitable alternative. According to WHO, Bacterial multidrug-resistance is the ability of bacteria to 80% of world population uses medicinal plants for their grow in the presence of antibiotics at concentrations that health needs; antibacterial potential against the multi- were previously inhibitory. Treating infections caused drug resistant phenotypes of many of them like Afro- by multidrug-resistant (MDR) bacteria is a challenge momum citratum, Afromomum melegueta, Imperata more and more difcult to solve within hospital units cylindricum, Cinnamomum zeylanicum, Dioscorea [1]. Although the resistance of bacteria to antibiotics is bulbifera, Dorstenia psilurus has already been demon- a natural adaptation phenomenon, the rapid emergence strated [3, 4]. In order to contribute to the discovery of of MDR phenotypes is mainly due to misuse of antibi- active substances from medicinal plants, this study was otics, which increases the selection pressure and favors designed to assess the antibacterial potential of difer- the appearance MDR microorganisms [2]. In hospitals, ent parts of Aloe buettneri A. Berger (Asphodelaceae), patients infected by these bacteria stay for long time, Alchornea foribunda Müll. Arg. (Euphorbiaceae), Cri- which impacts on the cost of treatment. Faced with this num purpurascens Herb. (Amaryllidaceae), Euphorbia crisis, it is important to develop new antibacterial mol- prostrata Ait. (Euphorbiaceae), Markhamia tomentosa ecules efective vis-à-vis of MDR bacteria and medicinal K. Schum. (Bignoniaceae), Viscum album L. (Loran- thaceae) and Rauwolfa macrophylla Ruiz & Pav. (Apoc- *Correspondence: [email protected] ynaceae) against MDR Gram-positive and Gram-negative 1 Department of Biochemistry, Faculty of Science, University of Dschang, phenotypes. Dschang, Cameroon Full list of author information is available at the end of the article © The Author(s) 2017. This 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. Voukeng et al. BMC Res Notes (2017) 10:321 Page 2 of 8 Methods incubated at 37 °C for 18 h. Bacterial growth was detected Plant material and sample preparation by adding 40 μL of INT (0.2 mg/mL) and the appear- Diferent part of the investigated plants including leaves, ance of pink color indicated bacterial growth; the lowest stem, stem bark, bark or whole plant (Table 1) were har- concentration of the extract where no color change was vested in diferent regions of Cameroon. Te plants were observed was recorded as MIC. then identifed at Cameroon National Herbarium where For the determination of MBCs, we used new 96-well the voucher specimens are available (Table 1). Te dry plates containing 150 μL of MHB in which we added an powders (200 g) of each part of plants were soaked in aliquot of 50 μL from the wells corresponding to MIC methanol for 48 h; the fltrates obtained after fltration as well as upper concentrations. Tose microplates were paper through Whatman No. 1 were concentrated under incubated at 37 °C for 48 h and revelation was done as reduced pressure and the obtained extracts were kept at mentioned above and the lowest concentration indicating 4 °C for further biological tests. the absence of bacterial growth was considered as MBC. Each of the experiments was carried out in triplicate. Phytochemical screening Te presence of compounds belonging to diferent classes Results of secondary metabolites was determined according to Te classes of secondary metabolites present in extracts described methods [5]. of diferent parts of plants were detected and results are summarized in Table 2. Alkaloids, triterpenes, sterols, Chemicals for antimicrobial assay favonoids, polyphenols and saponins were screened Te reference antibiotic (RA) used against bacteria were in all plant extracts. Other classes of compounds were chloramphenicol and ciprofoxacin (Sigma-Aldrich, selectively distributed. St. Quentin Fallavier, France) meanwhile the bacte- Te results of antibacterial tests are summarized in rial growth indicator was p-iodonitrotetrazolium chlo- Table 3. It appears that the plant extract from E. pros- ride ≥ 97% (INT, Sigma-Aldrich). trata was active against all tested bacterial strains (36/36) with MIC between 128 and 256 µg/mL on 55.55% (20/36) Microbial strains and culture media of the studied microorganisms. Te methanol extracts Microorganisms used in this study included 36 multi- of leaves and stems of V. album as well as other plant drug-resistant strains of Gram-negative belonging to extracts displayed selective activities on MDR Gram-neg- Escherichia coli, Enterobacter aerogenes, Enterobacter clo- ative as well as on methicillin-resistant S. aureus (MRSA) acae, Klebsiella pneumoniae, Providencia stuartii, Pseu- strains. Leaves extract of R. macrophylla were active domonas aeruginosa species and Gram-positive bacteria against 83.33% (30/36) of the tested bacteria while only belonging to and Staphylococcus aureus species (Table 3). 3/36 (8.33%) of the studied bacteria were sensitive to the Teir bacterial features were previous reported [6]. Muel- bark extract. Leaves extract of M. tomentosa was active ler–Hinton Agar (Sigma) was used to activate the micro- on 75% (27/36) of bacterial strains while the extracts of organisms whilst Mueller Hinton broth (MHB; Sigma) bark were active only on 30.55% (11/36). Te methanol was used for antibacterial assays. extract from the leaves of C. purpurascens showed activ- ity against all MRSA strains and 22/29 Gram-negative INT colorimetric assay for MIC and MBC determinations bacteria tested; except bacteria belonging to P. aerugi- Te minimal inhibitory concentrations (MIC) and mini- nosa species, all other bacterial species studied herein mal bactericidal concentrations (MBC) of diferent plant were susceptible to C. purpurascens leaves extract. E. coli, extracts were determined by a by the rapid INT colori- P. aeruginosa, E. aerogenes and P. stuartii strains were metric assay according to described methods [3]. Chlo- mostly resistant to the action of the extract of leaves of ramphenicol and ciprofoxacin were the reference drugs A. foribunda. Te extract of A. buettneri leaves had weak for Gram-negative and Gram-positive respectively. Each activity, its inhibitory efects being observed against 3/36 plant extract was dissolved in a 5% DMSO solution; an (8.33%) bacterial strains. aliquot of 100 μL was added to the wells of a microplate containing 100 μL of MHB; then serial dilution was per- Discussion formed. A bacterial suspension corresponding to 0.5 Te emergence of diseases due to MDR bacterial strains McFarland scale was prepared and diluted 100 times is a phenomenon of growing concern worldwide, being in the sterile MHB. Afterwards, 100 μL of the bacte- qualifed by WHO as a “slow-moving tsunami” [7]. rial suspension was added to all wells and the plate was Medicinal plants are a promising alternative for the Voukeng Voukeng Table 1 Information on studied species Plant species, (voucher Traditional use Part used Potential active constituents Previously screened activity number)/family traditionally (2017)10:321 ResNotes BMC et al. Aloe buettneri A. Berger Gastro-intestinal infections, chronic wounds, Leaves – Anti-ulcer, anti-infammatory, (59062/HNC)/Asphode- cutaneous infections, infammations, gastric ovarian steroidogenesis laceae ulcers, chronic skin ulcer, cough, dysmen- efect, sub-acute toxicity, orrhea, food poisoning, difcult delivery, Analgesic efect, Antipyretic dysentery, general stomachaches, lumbar activity [18–20] painregulation