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Int J Biol Med Res.2017;8(1):5843-5850 Int J Biol Med Res www.biomedscidirect.com Volume 6, Issue 2, April 2015 Contents lists available at BioMedSciDirect Publications International Journal of Biological & Medical Research Journal homepage: www.biomedscidirect.com BioMedSciDirect International Journal of Publications BIOLOGICAL AND MEDICAL RESEARCH Original article Antibacterial activity of Daniellia oliveri, Lophira alata, and Lannea welwitschii against plasmid-carrying enteric pathogens from Lagos, Nigeria KO Akinyemia*CO Fakoredea, BA Iwalokun bAO B Oyefolua aDepartment of Microbiology, Lagos State University, Ojo P.M.B 1087, Apapa, Lagos, Nigeria bBiochemistry Unit and Malaria research Laboratory , Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria A R T I C L E I N F O A B S T R A C T Keywords: Aim: Three medicinal plants Daniellia oliveri, Lophira alata and Lannea welwitschii were Plant, tested for in vitro antibacterial activity and their plasmid eviction potentials on enteric extract, MDR, pathogens. Method: Fresh plant materials collected from herbs seller were sun dried and pathogens, grounded to fine powder. Water and methanol extracts of these plants were obtained by plasmid, standard methods. Enteric bacterial agents from clinical samples were used. In-vitro curing, antimicrobial susceptibility testing of the bacterial isolates was carried out using commercial photochemical antibiotics and the crude plant extracts by standard procedures. Phytochemical screening of the plant extracts was carried by the standard procedure. Plasmid DNA extraction was performed using the alkaline lyses method. Result: The crude methanol extracts of all the plants were effective on MDR strains of Salmonella enterica serova Typhi, S. Typhimurium, Escherichia. coli, Shigella spp and Klebsiella spp used in this study MIC and MBC values of 0.625 to 10 mcg/ml and 5.0 to 20 mcg/ml respectively were recorded for L. welwitschii,, 10 to 20 mcg/ml and 20 to40 mcg/ml for D. oliveri, and 10 to 20 mcg/ml and 20 to40mcg/ml were recorded for L. alata respectively. The plasmid eviction potentials of the crude plants extracts indicated that only crude extract of L. Welwitschii exerted anti-plasmidic effect on Shigella spp, Klebsiella spp, S. Typhimurium, and S. Typhi , as over 60% of these bacterial strains loss their plasmid DNA. Conclusion: This study revealed that methanol extracts of the three medicinal plants were active against enteric bacterial pathogens. The extracts of L. alata and D. oliveri were not active against MDR pathogens thus; their continuous usage should be discouraged. The methanol crude extract of L. welwitschii exerted antiplasmidic effect on MDR enteric pathogens. This finding suggests possibility of a new approach in drug formulation using anti- plasmidic plant extracts for effective management of plasmid encoded drug resistant enteric bacteria-associated diseases c Copyright 2010 BioMedSciDirect Publications IJBMR - ISSN: 0976:6685. All rights reserved. 1. Introduction Enteric infection is an increasingly recognized array of antibiotics has played roles in the development and the spread of bacterial, parasitic and viral pathogens which can profoundly multidrug resistance among different bacteria species. Also, extra- disrupt intestinal function with or without causing overt chromosomal genetic elements (plasmids) harboured by some dehydrating diarrhoea. Some of the organisms implicated in bacterial agents had been responsible for increased resistant enteric infection include Salmonella spp., Shigella spp., Proteus development either horizontally or vertically [2]. The use of spp., Klebsiella spp., Staphylococcus aureus and Escherichia coli. medicinal plants and herbs in curing diseases has always been part Enteric pathogen has been reported as the major causative agent of human culture and has transcended all social, cultural, economic, of sporadic and endemic diarrhoea both in children and in adult religious and other barriers created by man. It has been established [1]. Drugs like aminoglycosides, cephalosporins, and that the plants which naturally synthesis and accumulate some fluoroquinolone have antibacterial effects against enteric secondary metabolites possess medicinal properties [3]. pathogens [2]. The evolutionary pressure from the use of In Nigeria many hundred of plants are used in traditional * Corresponding Author : Akinyemi K.O (PhD) medicine as treatment for bacterial infections, some of these plants Department of Microbiology, Lagos State University have been subjected to an in-vitro screening but the efficacy of such PMB 1087, Apapa, Lagos, Nigeria. herbal medicine has seldom been rigorously tested in controlled Phone: +2348029088676 E-mail: [email protected] clinical trials. Plants have been major source of medicine and plants secondary metabolite has been attributed for most plant therapeutic c Copyright 2011. CurrentSciDirect Publications. IJBMR - All rights reserved. KO Akinyemi et al./Int J Biol Med Res.8(1):5843-5850 5844 was shaken vigorously and left to stand overnight (24 hours) in a activities [4-,]. Daniellia oliveri is of medicinal important value refrigerator maintained at 2 to 8°c. The mixture was filtered using a being potent in treating gastrointestinal ailment [6], as clean muslin cloth. The filtrate was evaporated to dryness in a hot air antibioficient agent in pregnancy, pain killer, skin, mucosa and oven maintained at 40°c for 72 hours. The crude extracts were sedative [7] for the cure of rheumatism pain [8] and active as an scrapped and weighed to determine the yield. The crude extracts antimicrobial agent [9,10]. In traditional African medicine, yield were dispensed in a sterile container and labelled as water Lannea welwitschii is used for swellings, oedema, gout, extracts and designated as AW (D. oliveri), BW (L. welwitschii), and haemorrhoids and emesis [11] antidiarrheal [12] and CW (L. alata) respectively. antipurgative [13]. While in South West Nigeria Lophira alata is use for the treatment of typhoid fever [13] and for the treatment of Bacterial cultures fever, cough, jaundice and gastrointestinal disorder [14]. The bacteria used in this work were obtained from the Nigerian Therefore a need to search and develop newer, safer, effective and institute of Medical Research (NIMR), Yaba, Lagos, Nigeria and were cheaper antimicrobial agent from natural products such as all subjected to standard microbiological methods maintained on medicinal plants that will compliment synthetic antimicrobial Tryptone soya agar petri dishes at 2 to 8°C for 24 hrs before use. drugs against multiple drug resistant pathogens is paramount These bacteria include Salmonella enterica serovarTyphi, importance. This study was undertaken with a view to Salmonella Typhimurium, Escherichia coli ATTCC 25922, Shigella determining the antimicrobial activity of three medicinal plants spp, and Klebsiella spp, and were isolated from stool samples of and their plasmid curing ability on plasmid-mediated multiple patients diagnosed of gastro intestinal infections. drug resistant enteric bacterial pathogens. Antimicrobial susceptibility testing Materials and methods Agar disc diffusion assay: In-vitro susceptibility of the bacterial Plant materials isolates to the plant extract was tested for lead antimicrobial Fresh plant materials of Lannea welwitschii, Daniellia oliveri response by the standard disc diffusion technique using guideline and Lophira alata were collected from herbs seller in Itoku established by the NCCLS 2002 [16]. A standardized Inoculum of 1.0 market, Abeokuta south local government Area of Ogun state x 107 with 0.5 McFarland standards was used to evenly inoculate the Nigeria. The identification and authentication of these plants surface of Mueller-Hinton agar plate (Oxoid, UK). Sterile paper discs were carried out by Mr. OK Oluwa and voucher specimens' previously soaked in known concentration (5, 20, and 40 mg/ml) of numbers were deposited in the herbarium of the Botany extracts were carefully placed at different spot on agar plate department of the Lagos State University. containing the organisms used and the discs were gently pressed down to ensure contact. The plates were incubated aerobically at Extraction 37°C for 18 to 24 hours. The diameter of the zones of inhibition were Stem bark of Daniellia oliveri (A) Rolfe. Hutch & Dalz, Lannea measured with a ruler and interpreted according to the guidelines welwitschii (B) Hiern, and Lophira alata (C) Bank ex f. Gaertn.f provided by the National Committee for Clinical Laboratory were sun dried for one week and were grounded to fine powder Standards [16]. using pestle and mortar and were stored at room temperature in a Plasmid DNA extraction/Analysis clean dry air tight bottle for further use. Plasmid extraction was performed by a simplified alkaline lyses Methanol extracts (Cold) method described by Birnboim and Doly(1979) [17]. Overnight The methanol extractions of these plants were carried as culture (1.5 ml) was centrifuged at 5,000 rpm for 1 minute to pellet described by Akinyemi et al [14].In brieftwenty grams of the the cells. The supernatant was gently decanted. After washing, the powdered plant materials A, B, and C were weighed and soaked in c e l l p e l l e t w a s r e - s u s p e n d e d i n 3 0 0 μ l T r i s separate beakers containing 200 ml of 70% methanol each. The ( t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ) - E D T A beaker was wrapped with aluminium foil and sealed with tape. (ethylenediaminetetraacetic acid)-NaOH-SDS (sodium dodecyl The mixture was shaken vigorously and left to stand overnight (24 sulfate) (TENS) buffer solution and mixed by gentle inversion (five hours) in a refrigerator maintained at 2 to 8°c. The mixture was times) within 3–5 minutes of incubation on ice to obtain a straw-like, filtered using a clean muslin cloth. The filtrate was evaporated to sticky lysate. This was followed by neutralization by adding 200 μl of dryness in a hot air oven maintained at 40°c for 72 hours. The 5 M potassium acetate buffer (pH 5.2), followed by incubation on ice crude extracts were scrapped and weighed to determine the yield.