This study was carried out to investigate possible bacteriostatic and bactericidal activities of Cnidoscolus aconitifolius leaf extract on multi-drug resistant micro-organisms. The antimicrobial property of C. aconitifolius leaf extract was carried out on the following multi-drug resistant microorganisms;Escherichia coli, Klebsiella species, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica Gallinarum and Candida albicans. The results from this study show that none of the multi-drug resistant micro-organisms was sensitive to the leaf extract of C. aconitifolius.Key words: Cnidoscolus aconitifolius, bioactivity, multi-drug resistant micro-organisms

Azeez, IO

Lawal, TO

Ogunleye, AO

Oyagbemi, AA

English

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African Journal of Biotechnology Vol. 10(3), pp. 413-415, 17 January, 2011 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB10.353 ISSN 1684–5315 © 2011 Academic Journals      Short Communication  The effect of Cnidoscolus aconitifolius on multi-drug resistant micro-organisms  Oyagbemi, A. A.1*, Ogunleye, A. O.2, Lawal, T. O.3 and Azeez, I. O.1  1Department of Veterinary Physiology, Biochemistry and Pharmacology, Faculty of Veterinary Medicine, University of Ibadan, Nigeria. 2Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Ibadan, Nigeria. 3Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Ibadan, Nigeria.  Accepted 20 August, 2010  This study was carried out to investigate possible bacteriostatic and bactericidal activities of Cnidoscolus aconitifolius leaf extract on multi-drug resistant micro-organisms. The antimicrobial property of C. aconitifolius leaf extract was carried out on the following multi-drug resistant micro-organisms; Escherichia coli, Klebsiella species, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica Gallinarum and Candida albicans. The results from this study show that none of the multi-drug resistant micro-organisms was sensitive to the leaf extract of C. aconitifolius.   Key words: Cnidoscolus aconitifolius, bioactivity, multi-drug resistant micro-organisms.   INTRODUCTION  Cnidoscolus aconitifolius is a perennial shrub belonging to the family Euphorbiaceae. It is commonly found in the tropic and sub tropical regions worldwide, including Africa, North and South America, India, etc. It is com-monly eaten as vegetable in soup (Ganiyu, 2005) in Southwestern Nigeria where it is called ‘Iyana Ipaja’. The edible parts of the plant, which taste like spinach when cooked, serve as important nutritional source of protein, vitamin (A and C), minerals (calcium, iron, and phosphorus), niacin, riboflavin and thiamine for popula-tions that cannot afford expensive foods rich in these nutrients (Yang, 1979). C. aconitifolius shoots and leaves have been taken as laxative, diuretic and circulatory stimulant, to improve digestion, stimulate lactation, and harden the fingernails (Rowe, 1994).  The high fibre content and antibacterial activities of this plant have been reported by various authors including Sarmiento-Franco et al. (2002) and Awoyinka et al. (2007). The anti-diabetic properties of leaf extract of C. aconitifolius in inbred type 2 diabetic mice  has  also been     *Corresponding author. E-mail: ademolaoyagbemi@yahoo.com, aa.oyagbemi@mail.ui.edu.ng or ademola.oyagbemi778@gmail .com. Tel: +23433639776. Fax: 02-8103043.       reported (Oladeinde et al., 2007). Recently, the ameli-orative effects of C. aconitifolius on anaemia and osmotic fragility induced by protein energy malnutrition have been reported (Oyagbemi et al., 2008). The current study was carried out to determine the antimicrobial activities of the aqueous leaf extract of C. aconitifolius on some multi-drug resistant clinical bacteria isolates and a laboratory stock fungus.                            MATERIALS AND METHODS  Collection of plant materials  Fresh matured leaves of C. aconitifolius were collected at the University Teaching Hospital, College of Medicine, Ibadan, Nigeria and were identified and authenticated at Department of Botany and Microbiology, University of Ibadan. The leaves were air-dried, reduced to powder and were kept separately in airtight containers until the time of use.   Extraction of plant material  Air-dried powder (1 kg) of fresh matured C. aconitifolius leaves was extracted by percolation at room temperature with 70% ethanol (EtOH) and later concentrated under reduced pressure (bath temperature 50°C) and finally evaporated to dryness. The dried mass yield obtained was 62.5 g. 414         Afr. J. Biotechnol.    Table 1. Antimicrobial activities of leaf extracts of C. aconitifolius to the micro organisms tested.  Organism Source Resistant to Antimicrobial activities of the extract Escherichia coli UCH091 Ci, cot, amx, tet, ca, chl, cf, gen, nal, aug and cxc No activity Escherichia coli VTH0919 Cf, cef, nor, tet, amp, gen and ofl No activity Escherichia coli VTH0911 Cef, tet, amp and nal No activity Klebsiella species UCH092 Amp, amx, cot, nit, nal, ofl, aug  and tet No activity Staphylococcus aureus UCH093 Chl, amx, ery and gen No activity Pseudomonas aeruginosa UCH 094 Aug, nal, cf, ca, gen and sf No activity Salmonella enterica VTH096 Cf, gen, ery, pen, cxc, amx and tet No activity Candida albicans PMUI lab. stock  No activity  Ci = Ceftriaxone; cot = cotrimoxazole; amx = amoxylin; tet = tetracycline; ca = ceftazidime; chl = chloramphenicol; cf = ciprofloxacin; gen = gentamycin; nal = nalidixic acid; aug = augumentin; Cxc = cloxacillin; cef = cefuroxime; amp = ampicillin; nit =nitrofurantoin; ofl = ofloxacin; ery = erythromycin; pen = penincillin G; sf =sulfisoxazole. UCH = University College Hospital, Ibadan; VTH = Veterinary Teaching Hospital, University of Ibadan; PHUI = Pharmaceutical Microbiology, University of Ibadan.     Microorganism used for the study   The microorganisms used for the study include three strains of multidrug resistant Escherichia coli, one Klebsiella species, one Staphylococcus aureus, one Pseudomonas aeruginosa, one Salmonella enterica Gallinarum and one laboratory stock of Candida albicans.   Antibiotic sensitivity test  The in vitro antibiotic sensitivity tests were carried out using conventional antimicrobial agents, namely: Sparfloxacin (5 mcg), chloramphenicol (30 mcg), gentamycin (10 mcg), amoxicillin (30 mcg ), cloxacillin (10 mcg), co-Trimoxazole (25 mcg), cefuroxime (30 mcg), nalidixic acid (30 mcg), penincilin (5 units), ampicillin (25 mcg), tetracycline (30 mcg), ofloxacin (5 mcg), ciprofloxacin (5 mcg), norfloxacin (10 mcg), nitrofurantoin (100 mcg), ceftazidime (30 mcg), ceftriaxone (30 mcg) and sulfisoxazole (300 mcg)  as described by Walton (1972) and modified by Adetosoye (1984). The isolates were multidrug resistant clinical isolates from University College Hospital, Ibadan and from Veterinary Teaching Hospital, Faculty of Veterinary Medicine, University of Ibadan having the resistant patterns shown in Table 1. Each of the isolates was inoculated into 5 ml sterile nutrient broth and incubated at 37°C for 8 h. A 1:2000 dilution of the culture was made with sterile nutrient broth, while the mixture was shaken vigorously. Subsequently, a diagnostic sensitivity test agar plate was inoculated by flooding with the 1:2000 diluted mixture of the culture. The excess fluid was drained off and the plate was allowed to stand on the bench for 1 h after which antibiotic sensitivity discs of the respective antibiotic discs (Fidson Healthcare LTD.6 Ilupeju Bypass, Ilupeju, Lagos, Nigeria) were aseptically applied. The test plates were allowed to stand on the bench for 1 h to allow the antimicrobial agents to diffuse into the agar. The plates were then incubated at 37°C for 18 h after which the results were recorded. The zone of inhibitions around each antibiotic disc in each test was compared with the corresponding zone of inhibition around the antibiotic disc for the control organism.   Determination of antimicrobial activities of leaf extracts of C. aconitifolius  The antimicrobial activity of the extracts was determined using  agar diffusion technique as earlier described by Adeniyi et al. (1996). Mueller Hinton agar plates were seeded each with 0.1 ml of a 1:100 dilution of tryptose soy broth (TSB) overnight culture of each clinical bacterial isolates. Sabouraud agar was carpeted with the fungi isolates. The seeded plates were allowed to dry in the incubator at 37°C for 20 min. A standard cork borer of 6 mm diameter was used to cut uniform well on the surface of the agar, 0.2 ml of the extract re-suspended in distilled water at a concentration of 20 mg/ml was added to the bored hole on the agar. The Mueller Hinton agar seeded with the bacteria, were incubated at 37°C for 24 h, while the Sabouraud agar was carpeted with the fungi isolates were incubated at 250C for three(3) days. Subsequently, the diameter of the zone of inhibition was measured. The distilled water used as solvent was included in each of the plates as solvent control, while conventional antibiotic discs were used as chemo-therapeutic control.   RESULTS AND DISCUSSION  Results from Table 1 show the resistant patterns of E. coli, Klebsiella species, Staph. aureus, P. aeruginosa and Salm. enterica Gallinarum to the conventional antimi-crobial agents. Also, the leaf extracts of the C. aconitifolius did not show any activity to the multidrug resistant micro organisms tested as shown in Table 1. In an earlier work, Awosika et al. (2007) reported the antimicrobial activities of the various extracts of C. aconitifolius. They observed that the control drug chlo-ramphenicol showed the largest and the most significant (P < 0.05) zone of inhibition (11.5 ± 0.1 mm) for Staph. aureus and (17 ± 0.1 mm) for Salm. enterica, compared to all other extracts on the two bacteria investigated in the study. The ethanolic extract was able to inhibit Salm. enterica within 1.5 ± 0.5 mm, while the dry and fresh leaf water extract showed no sensitivity on the organism. It was further reported that, the fresh leaf water and ethanolic extracts produced zones of inhibitions for Staph. aureus, while the dried leaf water extracts did not. The ethanolic extract was found to produce an inhibition zone of 3.00 mm ± 0.1 mm for the  Staph.  aureus  isolate      tested. Based on the work, it was concluded that the ability of C. aconitifolius to show sensitivity to two different strains of bacteria (gram positive and negative) points to its possible use as a broad spectrum antimicrobial agent with better efficacy from the ethanolic extract of the plant. The problem of drug resistance is a current global public health problem. For example, an increase in antimicrobial resistance to conventional antimicrobial agents in non-typhoidal Salmonella serotypes has been demonstrated since the early 1990’s and has grown to become a global problem (Alcaine et al., 2007). Recently, Ogunleye et al. (2008), reported occurrence of multidrug resistant E. coli of poultry origin in two Southwestern states of Nigeria. Observation from the work revealed that thirty nine E. coli isolates were recovered from the two hundred and fifty samples of intestine, kidneys, lungs, hearts, ovary, spleen and colorectum from diseased chickens that were examined. Each of the thirty nine isolates was resistant to 5 - 12 antimicrobial agents.  It has been noted that occurrence of antimicrobial resistance among bacteria pathogens usually makes the right choice of empirical antimicrobial treatment by clini-cians more difficult (Hubert et al., 1998; Walker, 2000). The continued search for new antimicrobial agents from natural products like plants is thus very relevant. Based on the earlier report of antimicrobial activities of the ethanolic fresh leaf extract of C. aconitifolius by Awosika et al. (2007), this study was carried out on the multidrug resistant isolates. The results from the current work how-ever, showed that the aqueous suspension of the ethanolic leaf extracts of C. aconitifolius did not produce antimicrobial activity for any of the seven multidrug resistant clinical bacteria isolate and one laboratory stock fungus used for this study. This finding is contrary to the report of Awosika et al. (2007). The observation in the current work may be attributed to the multidrug resistance nature of the clinical bacteria isolates obtained from both human and animals used for this study. From Table 1, it is observed that some of the isolates were resistant to wide range of conventional antimicrobial including the cephalosporin and fluoroquinolones group of drugs. It is imperative to take a decisive step in terms of policy and its enforcement in Nigeria to curtail indiscriminate abuse and misuse of conventional antibiotics that usually results     Oyagbemi et al.         415    in development of antimicrobial resistance. This becomes important because of the cost and long processes involved in the development of new, acceptable and safe antimicrobial agent.   REFERENCES  Adeniyi BA, Odelola HA, Oso BA (1996). Antimicrobial potentials of Diospyros mespiliformis (Ebenaceae). Afr. J. Med. Sci. 25: 221-224. Adetosoye AI (1984). Studies of Escherichial coli with special reference to drug resistance and enterotoxigenicity. A Ph.D thesis in the Department of Veterinary Microbiology and Parasitology, University of Ibadan. Alcaine SD, Warnick LD, WiedmannmM (2007). Antimicrobial resistance in nontyphoidal Salmonella. J. Food Prot. 70: 780-790. Awoyinka AO, Balogun IO, ogunnowo AA (2007). Phytochemical screening and in vitro bioactivity of Cnidoscolus aconitifolius (Euphorbiaceae). J. Med. Plant Res. 1(3): 063- 065. Ganiyu O (2005). Effect of Some Post-Harvest Treatment on the. Nutritional Properties of Cnidoscolus aconitifolus leaf. Pak. J. Nutr. 4(4): 226-230. Hubert SK, Nguyen PD, Walker RD (1998). Evaluation of a computerized antimicrobial susceptibility system with bacteria isolated from animals. J. Vet. Diagn. Invest. 10: 164-168. Ogunleye AO, Oyekunle MA, Sonibare AO (2008). Multidrug resistant Escherichia coli isolates of poultry origin in Abeokuta, South Western Nigeria. Vet. Arhiv. 78(6): 501-509. Oladeinde FO, Kinyua AM, Laditan AA (2007). Effect of Cnidoscolus aconitifolius leaf extract on the blood glucose and insulin levels of inbred type 2 diabetic mice. Cellular and molecular biology (Noisy-le-Grand, France, 53(3): 34-41. Oyagbemi AA, Odetola AA, Azeez OI (2008). Ameliorative effects of Cnidoscolus aconitifolius on anaemia and osmotic fragility induced by protein energy malnutrition. Afr. J. Biotechnol. 7(11): 1721-1726. Rowe L (1994).  Plant guards: Secret of good health. Valley Morning Star. 4: 12.   Sarmiento-Franco L, Pearson RA, Belmar-casso R (2002). Performance of broilers fed on diet containing different amount of chaya (Cnidoscolus aconitifoilus) leaf meal. Trop. Anim. Health Prod. 34(3): 257-269. Walker RD (2000). Antimicrobial susceptibility testing and interpretation of results. In: Prescott JF, Baggot JD, Walker RD (Eds). Antimicrobial therapy in veterinary medicine. Iowa State University Press. pp. 12-26. Walton JR (1972). Bacteriological, biochemical and virulence studies on Salmonella dublin from abortion and enteric disease in cattle and sheep. Vet. Rec. 90: 236-240. Yang YH (1979). Tropical home gardens as a nutritional intervention. In: Inglett GE, Charalambous G (eds). Tropical food chemistry. New York: Academic Press, pp. 417-436.       

The effect of Cnidoscolus aconitifolius on multi-drug resistant micro-organisms

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The effect of Cnidoscolus aconitifolius on multi-drug resistant micro-organisms

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