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Ethnobotanical Leaflets 14: 876-88. 2010. Susceptibility
of Bacterial Isolates of Wound Infections to Chromolaena
odorata T.O. Agbabiaka,
T. Samuel and I. O. Sule Department of
Microbiology, E-mail: toyinagbabiaka@yahoo.com Issued: August 1,
2010 Abstract Hot ethanolic extract, cold ethanolic extract and hot aqueous extract of Chromolaena odorata
were assayed for in vitro antibacterial activity using agar diffusion method.
The extracts produced measurable zones of inhibition against Staphylococcus aureus,
Proteus vulgaris and Escherichia coli. However, some organisms such as Streptococcus pyogenes,
Pseudomonas aeruginosa and Klebsiella species were found to be resistant to the extracts. Potency of
the extracts depends on the solvent used for the extraction and method of
extraction. Hot ethanolic extraction was the most
effective which has its highest activity at low pH.
The minimum inhibitory concentration of the extracts ranged from 15 mg/ml to
50mg/ml. Hot ethanolic was found to be bacteriocidal at 25 mg/ml on E. coli. Key
words: Chromolaena odorata,
ethanolic extracts, bacteriocidal,
bacteriostatic. Introduction A wound is an abrasion in the skin and the exposure of subcutaneous tissue following the loss of skin integrity which provide moist, warm and nutritious environment that is conducive for microbial colonization and proliferation (Duguid and Colle, 1975). Wounds disrupt the protective barrier of the skin and provide a portal of entry through which microorganisms can enter the circulatory system and deep body tissue. Wound and other open lesions are liable to infection with a
multiplicity of organism from the body surface or environment. Infection
occurs when one or more of the contaminants evade the cleaning effect of the
host’s defenses, replicate in large number, attack and harm to the host and
may best be described as colonization (Simchen et al., 1991). Wound infection may be
endogenous or exogenous. Endogenous infection or auto-infection is caused by
organism that has been living a commensal existence
in the patient’s body. On abdominal surgical wound for instance, may become
infected from the large bowel after an operation involving incision of the
colon. While exogenous infections are spread from person to person, this may
occur after accident or intentional trauma of the skin or other tissue which
is also called surgical or post-operative sepsis. Exogenous infection
involves an effected person providing source from which other patient may
acquire the organism. There are various ways by which wound may become infected by different types of microorganisms. It may be directly from the patient, a member of the operating room staff or from other person in the hospital ward. Wound may be infected outside hospital ward via exposure to dust carrying infecting organism either in vegetative form or in form of spores. In fresh wound, bacteria have little time to multiply and there is practically no evidence of inflammatory tissue response. Hence, with few exceptions, bacteria are regarded as contaminants. But after a few hours, however, if sign of inflammation or other tissue response appear, then the bacteria must no longer be considered as contaminant but as infecting bacteria (Topley and Wilson, 1988). The commonest pyogenic bacteria often associated with infected wounds are Staphylococcus aureus, Streptococcus pyogenes, Pneumococcus sp and coliform bacilli, such as Escherichia coli, Proteus sp, Pseudomonas aeruginosa and other enteric bacilli. Aerobic organisms particularly Clostridium perfringes and other species of bacteriodes and aerobic cocci are present (Klein et al. 1995). Pseudomonas aeruginosa causes a wide variety of septic infections in man and other vertebrates (Hare and Wilits, 1962). Among species much less commonly encountered in wound infections are Pasteurella multicida in animal bites, Corynebacterium diphtheria and Bacillus anthracis in malignant pustules of skin. In chronic infection that are slow to heal and in pus showing no other microbes, the possibility of infection with Mycobacterium tuberculosis and other Mycobacteria is there (Colle et al., 1963). Despite the fact that antibiotics are inhibiting to pathogenic microorganisms, Russell and Hugo (1983) reported that a large number of antibiotics that have been discovered are now useless in the treatment of infections due to selective toxicity apart from the problem of microbial resistance. In view of this and other problems associated with the use of antibiotics, scientists have reverted to the initial form of chemotherapy which was by the use of natural plant and plant products. These plants are usually regarded as medicinal plants (Owonubi, 1988). Medicinal plant is defined as any plant which one or more of its organs
contain substance that can be used for therapeutic purpose or which can be
used as precursors for the synthesis of useful drugs (Sofowora,
1982). There are thousand species of medicinal plants used globally for the
cure of different infections. These plants are used as antimicrobial agents
and several works has been carried out by scientists to find out its
scientific basis (Omotayo, 1998). Some of these
plants include: Anacardium occidentale, Pilostigma recticulatum, Anogeissus leiocarpa, Enantia chlorantha, Senna occidentalis and Azadiracha indica. The use of medicinal plants predates the introduction of antibiotics and other modern drugs in the African continent. Herbal medicine has been shown to be effective and over 60% of the Nigerian population depend on traditional medicine for their health care needs (Ghani et al., 1989). Traditional medicine practitioner in Nigeria use a variety of herbal preparations to treat different kinds of ailments including typhoid fever, paratyphoid fever, dysentery, malaria, diarrhoea and wound infections. In recent past, attention has been directed to medicinal research to substantiate the claims of cure made by the traditional healers and thus provide a scientific basis for their efficiency (Olukoya et al., 1993). Plants synthesize a large variety of chemical substances which include alkaloids, tannin, saponin, steroid, phlobatannin, flavonoid, cardic glycoside and a host of other chemical compounds referred to as secondary metabolites. These compounds are of no importance to plant’s own life (Sofowora, 1993). Many of these metabolites have prominent effects on the animal systems and microbial cells (Oyagade et al., 1999). Some of these plant metabolites possess important therapeutic properties which can be and have been utilized in the treatment of human and other animal diseases worldwide. For example, the roots of Zanthoxyllus sp. which were used for cleaning teeth and for treatment of toothache, urinary problem and throat infection contain chemical with anaesthetic, anti-tumor, anti-sickling and antimicrobial properties ( Oyagade et al., 1999). Chromolaena odorata
formerly called Eupatorium odoratum and also
known as It is important and expedient to analyze the effectiveness of antibiotics in common use today. This is because a large part of the population will be affected by their short-coming. Many local medicinal plants are being researched into. This can help to reinstate them since they have been proven in some cases to be curative to a wide variety of infections. They are readily available, cheap and are believed to have fewer side effects since they come from natural sources (Owonubi, 1988). For as long as man’s existence depends on the control of the pathogenic microbial populations, he must continue to monitor the effectiveness of the antibiotics he uses especially in the light of the limitations of these drugs. Materials and Methods Collection of Clinical Isolates Pure cultures of clinical isolates of Escherichia coli, Klebisiella sp, Staphylococcus aureus,
Pseudomonas aeruginosa, Streptococcus pyogenes, Klebsiella sp and
Proteus vulgaris were collected from Department
of Microbiology and Parasitology, Collection of Chromolaena
odorata plant Fresh leaves of Chromolaena odorata were collected from Danialu,
near Agbabiaka area, Plant Extracts Preparation Different forms of extraction were carried out on the powder of the test plant by using water and ethanol. a. Cold Ethanolic
Extraction 30g of the powdered plant material was soaked in 150ml of 95% ethanol in
a conical flask. This was placed on a shaker for about 5 days at about b. Hot Ethanolic
Extraction 30g of the
powdered plant material was soaked in 150ml of 95% ethanol in a conical
flask. This was placed in water bath at c. Hot Aqueous Extraction The same quantity of the plant material and sterile distilled water was
used as in hot ethanolic extractions, but the
duration of extraction in the water bath at Assay of Plant Extracts Each of the above extracts was tested for growth or contaminants by
plating them on nutrient agar and incubated at Determination of pH of Extract of Chromolaena odorata The pH of the extract of Chromolaena odorata was determined using pH meter Wag WT 3020 dipped into conical flasks containing the extracts. The constant reading on the pH meter was taken as the pH of the antimicrobial substance of extract of Chromolaena odorata. Sensitivity Test of extract of Chromolaena odorata
using Ditch Plate Method Overnight broth culture of the test organisms were swabbed on sterile
Muller Hinton agar in petri dishes using sterile cotton
swabs. A sterile cork borer of size 6mm in diameter was used to make ditches
on the plates.0.3ml of the respective leaf extract were then put into each
appropriately labeled ditches using sterile syringes. The incubated petri dishes were left for 1 hour for the extract to
diffuse into the agar. The plates were incubated at Determination of Effect of varying
concentration of the Plant Extract on the Isolates Overnight broth culture of the test organisms were swabbed on the
sterile Mueller Hinton agar in the petri dishes
using sterile cotton swabs. A sterile cork borer of size 6mm in diameter was
used to make ditches on the plate. Stock concentration of the extract of Chromolaena odorata was
diluted from 200mg/ml to 150mg/ml to 100mg/ml and finally to 50mg/ml with
extraction solvent. Each of this concentration was introduced into ditches on
the inoculated petri dishes. The plates were left
for 1 hour for the extract to diffuse. The plates were subsequently incubated
at Determination of Minimum Inhibitory
Concentration (MIC) Each test organism that is susceptible to the extract of Chromolaena odorata was
inoculated unto one sterile nutrient broth and incubated overnight at Determination of Minimum Bacteriocidal Concentration (MBC) Samples from tubes used in the MIC assays which did not show signs of
turbidity after period of incubation were streaked out on solidified sterile
nutrient agar plates using sterile cotton swab and incubated at Results and Discussion Zone of inhibition given by the extract of C. odorata were measured and the results are shown in Table 1. No inhibition zone was formed by the plant extract on Pseudomonas aeruginosa, Streptococcus pyogenes and Klebsiella sp. Staphylococcus aureus, Proteus vulgaris and Escherichia coli were sensitive to extract of C. odorata with varying degree. The stock concentration is 200mg/ml. The effect of different concentration of the extract of Chromolaena odorata on the susceptible organisms is shown on Table 2. Table 2a, 2 b and 2c show patterns of different concentration of HEE, CEE and HAE. Results of minimum inhibitory concentration of the extracts are shown in Table 3a, 3b and 3c. The ethanolic extracts were observed to show more antibacterial activity than the hot aqueous extract. This may be due to the fact that accessibility of ethanol to the bioactive component of the leaf is greater than that of water. Brain and Turner (1975) indicated that the efficiency of the extraction procedure depends on the accessibility of the constituents to the solvent. The fact that ethanolic extraction of leaf of Chromolaena odorata is more active against test organisms than aqueous extraction may suggest that the active principle of the plant is more soluble in ethanol than in water. The herbalists claim that the plant is better extracted using local gin referred to as ‘Ogogoro’. All forms of the leaf extracts viz HEE, CEE and HAE showed antibacterial effect on P. vulgaris, S. aureus and E. coli. These bacteria fall in gram positive and gram negative groups which may suggest that the active principles in the extracts probably possess a broad spectrum antibacterial activity (Jafri and Jalis-Subhani 1999; Samy and Ignacinauthu 2000). Both hot and cold ethanolic extracts were found to be 5.7 and that of hot aqueous extract was 6.5. Pseudomonas aeruginosa, Klebsiella sp and Streptococcus pyogenes were found to be resistant to all forms of the extractions. Susceptible bacterial isolates (P. vulgaris, S. aureus and E. coli) were subjected to varying concentration of the different extractions as shown in Table 3. Table 3a showed the pattern of inhibition zone of susceptible isolates to different concentration of hot ethanolic extract. Result showed decrease in zone of inhibition with decrease in concentration of the extract except for Proteus vulgaris which showed no significant decrease in zone of inhibition with decrease in concentration. This suggests that the inhibition of growth of Proteus vulgaris by the extracts of Chromolaena odorata is not a function of its concentration. Tables 3b and table 3c also followed the same trend. The trend followed by P. vulgaris is contrary to the finding of Umeze and Abarikwu (1986) that stated that the potency of plant extract depends on the concentration and also method of extraction. The result obtained for S .aureus and E. coli however agreed with this fact. It can also be deduced from Table 1 and Table 2 that activity of the plant extract also depends on the pH. Hot and cold ethanolic extracts which were found to both have pH of 5.7 is more potent than hot aqueous extract which has pH of 6.5. This agrees with Alade and Irobi (1993) which confirmed that the antibacterial activity of Chromolaena odorata significantly reduces with increase in pH. Pseudomonas aeruginosa, Klebsiella sp, Streptococcus pyogenes were not subjected to varying concentration because they are not susceptible to the plant extracts. This may suggest the ability of P .aeruginosa to metabolise many organic substrates, and viability and possession of extra cellular component (capsular material around the cell wall) by Klebsiella sp. Table 3 showed various result of minimum inhibitory concentration of the extraction. Table 3a showed that the MIC for hot ethanolic extract for each organism varies. The smallest concentration that shows no turbidity was taken as the MIC. Both S. aureus and P. vulgaris have the same MIC of 25mg/ml while E. coli has MIC of 15mg/ml. Table 3b showed the result of cold ethanolic extraction’s MIC. It followed the same trend as Table 3a except that the MIC for P. vulgaris. 50mg/ml. hot aqueous extraction’s minimum inhibitory concentration is presented in Table 3c. P. vulgaris was found to have no MIC while S. aureus and E. coli have MIC of 50mg/ml. All the tubes with MIC were plated out on sterile nutrient agar to determine the minimum bacteriocidal concentration (MBC). All the tubes were found to grow on the plate except the tubes that contained hot ethanolic extract and E. coli. The MBC for E. coli was found to be 15mg/ml. In conclusion, all the clinical bacterial isolates challenged with the extract of Chromolaena odorata are common pathogens associated with wound infections. Researches conducted in the past have shown that the rate at which pathogenic bacteria are developing resistance to common conventional antibiotics is alarming (Montefijore et al., 1983; Olayemi and Oyabade, 1987). Broad spectrum activity of extract of C. odorata tested appears to be asset for the development of antibacterial drugs. References Alade, P.I. and Irobi, O.N. 1993. Antimicrobial activities of crude leaf extracts of Acalypha wilkesiana. Journal of Ethnopharmacology 39:171-174. Awe, S. and Omojasola,P.F. 2003.
Antibacterial Screening of three medicinal plants used for diarrhoea treatment in Bhat, B., Adeloye,
A.A. and Etejere, E.O. 1985. Some Medicinal Plants
in Brain, K.R. and Turner, I.D.1975. Basic Medical Microbiology, Little Brown and Co, Boston. Colle, J.G., Franser,
A.G., Mabinion, B.P. 1963. Practical Medical Microbiology, Little Brown and Co, Duguid, R. R. and Colle, W. F. 1975. Occurrence of Serious Bacterial wound infection since introduction of antibacterial agents. Journal of American Medicine 170:2197-2199. Ghani, A., Abdulrahman,
E. M., and Onaolapo, J. A. 1989. Clinical and
Microbiological Evaluation of some Nigerian Preparations. The Reporter, Hare, A.R. and Willitis, P.O. 1962. Causative Agents due to Community Wound infections. Journal of Canadian Society for Microbiology 28:288-300. Jafri, M.A. and Jalis-Subhani, M.1999. Hepatoproctective activity of leaves of Senna occidentalis against paracetamol and ethyl alcohol intoxication in rats. Journal of Ethnopharmacology 66(3):355-361. Klein, R.S., 15:182-184. Montefijore, M., Alausa, K.O., Cooke, A. R., Fankus, J. B., Ojo, S. and Soyanwo, M. A. Epidemiological Surveillance of Hospital acquired wound infection. Nigerian Medical Journal 9(3):289-293. Olayemi, A.B. and Oyabade, J.O. 1987. Incidence of antibiotics resistance among Escherichia coli isolates from clinical source and river water. Nigerian Medical Journal 17(4):207-209. Olukoya, D.K., Idika,
N. and Odugbemi, Omotayo, A.E. 1998. Antibacterial activity of some anti-malarial plants. Proceedings of NSM 1(1): 31-41. Owonubi, M. 1988. Use of local herbs for
curing disease. Clinical Pharmacy and
Herbal Medicine 4(2):23. Oyagade, J.O., Awotoye, O. O., Adewumi, J. T. and Thorpe, H. T. 1999. Antimicrobial activity of some Nigerian Medicinal Plants: Screening for antimicrobial activity. Bio. Res. Comm. 11(3):193-197. Russell, A.D. and Hugo, W.B. 1983. Pharmaceutical
Microbiology, Blackwell Scientific Publication, Samy, R. P. and Ignacinauthu,
S. 2000. Antibacterial Activity of Some Folklore Medicinal Plants Used by
Tribal in Western Ghats of Simchen, E., Michael, A.N., Epistein, M., Franser, A.G.
1991. Infection in the Surgical Department Teaching Hospital in Sofowora, A. 1993. Medicinal plants and Traditional Medicine in Africa, Spectrum
Books Ltd, Sofowora, E. A. 1982. Medicinal Plants and Traditional Medicine in Topley, K.Y. and Table 1:
Susceptibility pattern of bacterial isolates to leaf extract of Chromolaena odorata.
Key HEE—Hot ethanolic extraction CEE—Cold ethanolic extraction HAE—Hot aqueous extraction - No zone of inhibition Table 2a: Hot Ethanolic Extraction (HEE)
Table 2b: Cold Ethanolic Extraction (CEE)
Table 2c: Hot
Aqueous Extraction (HAE)
Table 3a: Hot ethanolic extraction (HEE)
Key NT-Not Turbid T-Turbid Table 3b: Cold Ethanolic Extraction (CEE)
Key NT-Not Turbid T-Turbid Table 3c: Hot
Aqueous Extraction (HAE)
Key NT-Not Turbid T-Turbid |
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