Fankam et al. BMC Complementary and Alternative Medicine (2015) 15:206 DOI 10.1186/s12906-015-0726-0 RESEARCH ARTICLE Open Access Antibacterial and antibiotic resistance modifying activity of the extracts from allanblackia gabonensis, combretum molle and gladiolus quartinianus against Gram-negative bacteria including multi-drug resistant phenotypes Aimé G. Fankam, Jules R. Kuiate and Victor Kuete* Abstract Background: Bacterial resistance to antibiotics is becoming a serious problem worldwide. The discovery of new and effective antimicrobials and/or resistance modulators is necessary to tackle the spread of resistance or to reverse the multi-drug resistance. We investigated the antibacterial and antibiotic-resistance modifying activities of the methanol extracts from Allanblackia gabonensis, Gladiolus quartinianus and Combretum molle against 29 Gram-negative bacteria including multi-drug resistant (MDR) phenotypes. Methods: The broth microdilution method was used to determine the minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of the samples meanwhile the standard phytochemical methods were used for the preliminary phytochemical screening of the plant extracts. Results: Phytochemical analysis showed the presence of alkaloids, flavonoids, phenols and tannins in all studied extracts. Other chemical classes of secondary metabolites were selectively presents. Extracts from A. gabonensis and C. molle displayed a broad spectrum of activity with MICs varying from 16 to 1024 μg/mL against about 72.41 % of the tested bacteria. The extract from the fruits of A. gabonensis had the best activity, with MIC values below 100 μg/mL on 37.9 % of tested bacteria. Percentages of antibiotic-modulating effects ranging from 67 to 100 % were observed against tested MDR bacteria when combining the leaves extract from C. molle (at MIC/2 and MIC/4) with chloramphenicol, kanamycin, streptomycin and tetracycline. Conclusion: The overall results of the present study provide information for the possible use of the studied plant, especially Allanblackia gabonensis and Combretum molle in the control of Gram-negative bacterial infections including MDR species as antibacterials as well as resistance modulators. Keywords: Efflux pumps, Extracts, Gram-negative bacteria, MDR bacteria, Resistance modulators Background mentioned an increase in the hospital dissemination of Infectious diseases caused by multi-drug resistant (MDR) bacterial strains specifically those expressing drug efflux Gram-negative bacteria are worldwide health concern, mechanism [3, 4]. Against Gram-negative bacteria, the causing increasingly morbidity and mortality particularly discovery of efflux pump inhibitors (EPIs) is an attractive in developing countries [1]. In Cameroon, previous studies strategy to combat MDR phenotypes [5]. EPI generally showed high levels of resistance to commonly used antibi- interact with specific efflux pump proteins to restore the otics in Gram-negative bacilli [2]. Several reports also susceptibility of MDR bacteria to antibiotics [6]. Medicinal plants constitute an important source of chemotherapeu- * Correspondence: [email protected] tic molecules, in regards to the chemical diversity found in Department of Biochemistry, Faculty of science, University of Dschang, several species [7, 8]. In recent years, some plants have P.O. Box 67, Dschang, Cameroon © 2015 Fankam et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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. Fankam et al. BMC Complementary and Alternative Medicine (2015) 15:206 Page 2 of 12 been successfully evaluated for their direct antibacterial Preliminary phytochemical investigation action, and for their antibiotic-modulation activity [9–12]. The plant extracts were screened for the presence of In the present work, we hypothesized that herbal medi- major secondary metabolite classes such as alkaloids, cines traditionally used for the treatment of infectious dis- anthocyanins, anthraquinones, flavonoids, phenols, eases could contain molecules acting as antibacterial and/ saponins, sterols and triterpenes according to common or antibiotic-resistance modulators. This study was there- phytochemical methods previously described [38]. The fore designed to investigate the in vitro antibacterial and tests were based on visual observation of the change in antibiotic-resistance modifying activities of the methanol color or formation of precipitate after the addition of extracts from Allanblackia gabonensis Pellegr. (Clusiaceae), specific reagents. Gladiolus quartinianus A. Rich (Iridaceae) and Combretum molle R. Br. ex G. Don (Combretaceae) against Gram- Antibacterial assays negative bacteria including multi-drug phenotypes. These MICs and MBCs of the plant extracts and chloram- plants are traditionally used to manage various ailments in- phenicol were determined by microdilution method cluding bacterial related infections. using rapid INT colorimetric assay [25, 39]. Briefly, the samples were first dissolved in 10 % Dimethyl-sulfoxide Methods (DMSO)/Mueller Hinton Broth (MHB). The solution Plant materials and extraction obtained was then added to MHB and serially diluted Medicinal plants used in this work were collected in dif- two fold (in a 96-well microplate). One hundred microli- ferent areas of Cameroon between January and April ters of inoculum (1.5× 106 CFU/mL) prepared in MHB 2012. The plants were identified at the National Herbar- were then added. The plates were covered with a sterile ium (Yaoundé, Cameroon), where voucher specimens plate sealer and then agitated with a shaker to mix the were deposited under the reference numbers (Table 1). contents of the wells and incubated at 37 °C for 18 h. The air-dried and powdered plant material was weighed The final concentration of DMSO was less than 2.5 %, (300 g) and soaked in 1 L of methanol (MeOH) for 48 h and did not affect the microbial growth. Wells contain- at room temperature. The filtrate obtained through ing MHB, 100 μL of inoculum, and DMSO at a final Whatman filter paper No. 1 was concentrated under concentration of 2.5 % served as the negative control. reduced pressure in vacuum to obtain the crude extracts. The MIC of each sample was detected after 18 h of in- All crude extracts were then kept at 4 °C until further uses. cubation at 37 °C following addition of 40 μLINT (0.2 mg/mL) and incubation at 37 °C for 30 min. Viable Chemicals for antibacterial assays bacteria reduced the yellow dye to a pink. The MIC was Eight commonly used antibiotics including tetracycline defined as the lowest sample concentration that pre- (TET), kanamycin (KAN), streptomycin (STR), ciprofloxa- vented this change and that resulted in the complete in- cin (CIP), norfloxacin (NOR), chloramphenicol (CHL), hibition of bacterial growth. The MBC of the sample ampicillin (AMP), erythromycin (ERY) (Sigma-Aldrich, St was determined by sub-culturing 50 μL of the suspen- Quentin Fallavier, France) were used. The p-Iodonitrotetra- sions from the wells which did not show any growth zolium chloride 0.2 % (INT) and phenylalanine arginine β- after incubation during MIC assays to 150 μl of fresh naphthylamide (PAβN) (Sigma-Aldrich) were used as bacter- broth, and re-incubated at 37 °C for 48 h before revela- ial growth indicator and efflux pumps inhibitor respectively. tion. The MBC was defined as the lowest concentration of sample which completely inhibited the growth of bac- Microorganisms and growth conditions teria [40]. Each assay was performed in three independ- Pathogenic bacteria used in the study were Gram-negative ent tests in triplicate. The samples were also tested in bacteria including MDR isolates (Laboratory collection) the presence of phenylalanine arginine β-naphthylamide and reference strains (American Type Culture Collection) (PAβN) at a final concentration of 20 μg/mL as previ- of Escherichia coli (ATCC8739, ATCC10536, AG100, ously described [41] on nine MDR bacteria. All assays AG100A, AG100ATet, AG102, MC4100 W3110), Entero- were performed three time in duplicate. bacter aerogenes (ATCC13048, CM64, EA27, EA3, EA289, EA298, EA294), Klebsiella pneumoniae (ATCC11296, Antibiotic-modulation assay KP55, KP63, K24, K2), Enterobacter cloacae (ECCI69, To evaluate the antibiotic resistance modifying activity BM47, BM67), Pseudomonas aeruginosa (PA01, PA124) of the extracts, the MIC of antibiotic was determined in and Providencia stuartii (ATCC29916, NEA16, PS2636, the presence or absence of the plant extracts. The 96- PS299645) were used. Their features were previously wells plate modulation method, as described by Stavri reported [37]. They were maintained at 4 °C and et al. [42] was used. Briefly, after serial dilutions of anti- sub-cultured on a fresh appropriate Mueller Hinton Agar biotics (256–0.5 μg/mL), the plant extracts at their sub- (MHA) for 24 h before any antibacterial test. inhibitory concentrations (MIC/2 and MIC/4; selected Fankam et al. BMC Complementary and Alternative Medicine Table 1 Plants used in the present study and evidence of their bioactivities Samples, family, and Traditional treatment Area of plant collection Known bioactive or potentially