Antimicrobial efficacy of selected disinfectants

Transcription

1 American Journal of Biology and Life Sciences 2014; 2(2): Published online April 10, 2014 ( Antimicrobial efficacy of selected disinfectants Chioma C. Okore 1, *, Ogechukwu N. Mbanefo 1, Bright C. Onyekwere 2, Simon C. Onyewenjo 1, Agaptus U. Ozurumba 1, Chinyere A.M. Abba-Father 1 1 Biology Department, Federal Polytechnic Nekede Owerri, Imo-State, Nigeria 2 Chemistry Department, Federal Polytechnic Nekede Owerri, Imo-State, Nigeria address chiemeziechioma@gmail.com (C. C. Okore), chiomakore@yahoomail.com (C. C. Okore) To cite this article Chioma C. Okore, Ogechukwu N. Mbanefo, Bright C. Onyekwere, Simon C. Onyewenjo, Agaptus U. Ozurumba, Chinyere A.M. Abba-Father. Antimicrobial Efficacy of Selected Disinfectants, American Journal of Biology and Life Sciences. Vol. 2, No. 2, 2014, pp Abstract The antimicrobial effectiveness of four selected disinfectants (Dettol, Izal, Z-Germicide and Jik) was determined using agar diffusion (paper disc) method. Phenol coefficient was used to compare the effectiveness of these disinfectants against phenol. The test organisms used include Escherichia coli, Staphylococcus aureus, Streptococcus spp and Mucor spp. The undiluted concentrations of the disinfectants showed varying zones of inhibition against the test organisms, on E.coli it ranged from 17mm (Izal) to 25mm (Jik), on Staph aureus, 9mm (Jik) to 28mm (Dettol), whereas on Streptococcus spp it ranged from 8mm (Jik) to 30mm (Dettol). The different dilutions 2 1 to 2 3 of the disinfectants on E. coli ranged from 0mm (Izal) to 17mm (Jik), 7mm (Z-Germicide) to 24mm (Dettol) on Staph aureus and it ranged from 7mm (Z-Germicide, Jik) to 25mm (Dettol) on Streptococcus spp. For the fungi the zones of inhibitions of the diluted disinfectants ranged from 43mm to 53mm (Z-Germicide), 65mm to 100mm (Dettol), from 35mm to 110mm (Jik). The undiluted concentrations of the disinfectants ranged from 70mm (Z-Germicide) to 140mm (Dettol). The result showed that Dettol was most effective against the test organisms than the other disinfectants. Izal recorded the least antimicrobial activity. The phenol coefficient values also showed that Dettol was better when compared with the other tested disinfectants. Disinfectant for external uses is necessary to avoid related infections or diseases caused by these test microorganisms. Keywords Antimicrobial, Disinfectants, Dettol, Izal, Jik, Phenol-Coefficient, Z-germicide 1. Introduction The term control of microorganisms refers to the reduction in number or activity of the total microbial load. The principal reasons for controlling micro organisms are as follow, to prevent transmission of diseases and infection, to prevent contamination by the growth of undesirable microorganisms, to prevent deterioration and spoilage of materials by microorganisms [1], [2]. The control directed at destroying harmful microorganisms is called disinfection. It usually refers to the destruction of vegetative (non-endospore forming) pathogens. The term most commonly applies to the use of a disinfectant to free an inert surface or substance. When this treatment is directed at living tissue, it is called antisepsis, and the chemical is then called antiseptic. Therefore, in practice the same chemical might be called a disinfectant for one use and antiseptic for another [3], [4]. According to [5] and [6], disinfectants are toxic not only for microbial pathogens but for host cells as well and because of this, they can only be used to inactivate microorganisms in the inanimate environment or to a limited extent, on skin surfaces but cannot be administered systemically. These disinfectants

2 54 Chioma C. Okore et al.: Antimicrobial Efficacy of Selected Disinfectants cause distraction either by coagulating the protein of the bacteria, by destroying its cell membrane or by removal of a sulphonhydric group from the organisms [7], [8]. Also according to [9] and [10], the mode of action of disinfectants is thought to be linked to destruction of proteins, lipids or nucleic acids in the cells or its cytoplasmic membrane, although microorganisms differ in their sensitivity to chemical germicide. The gram positive and gram negative microorganisms are implicated in different diseases and infections [1], [2], [3]. The content of many chemical agents can be expressed by more than one notation. In dilutions, a small volume of the liquid chemical (solute) is diluted in a large volume of solvent to achieve a certain ratio. For example, a common laboratory phenolic disinfectant such as Lysol is usually diluted 1:200 parts of water by volume. In general, solutions of low dilution or high percentage have more of the active chemical (are more concentrated) and tend to be more biocide, but expense and potential toxicity can necessitate using the minimum strength that is effective [11]. The compositions of Dettol include Chloroxylenol B.PC (4.8% w/v), Oleum/Pini aromaticum (8.38% w/w), Isorophyl alcohol (9.43% w/w), Sapo vegetalis (5.60%w/w), Saccharum ustum q.s., Aqua ad (100 volumes); Z-germicide include 7% Tar acid phenol, 2% Cresylic creosote; Jik includes 3.85% m/v Sodium hypochlorite; Methylated spirit include 5% V/V Wood naphtha, 95% Alcohol. The effectiveness of a given disinfectant can be evaluated using the Phenol co-efficient test which is the best known disinfectant screening test in which the potency of a disinfectant is compared with that of phenol. The series of dilutions of phenol and the disinfectant being tested are prepared. A standard amount of the test organism is added to each dilution, the dilutions are then placed in a 20 0 C or 37 0 C water bath. At interval of 5, samples are taken from each dilution and inoculated in a growth medium and then incubated for hours at 37 0 C. The tubes will be examined for growth. If there is no growth in the growth medium, the dilution at that particular time of sampling killed the bacteria. The highest dilution (i.e. the lowest concentration) that kills the bacteria after 10 of exposure, but not 5, is used to calculate the phenol coefficient. This is done by dividing the reciprocal of the appropriate dilution for the disinfectant being tested, by the reciprocal of the appropriate dilution of the phenol. A value greater than one means that the disinfectant is more effective than phenol. Phenol coefficient, though a useful initial screening procedure, can be misleading if taken as a direct indication of disinfectant potency during normal use. This is because the phenol coefficient is determined under carefully controlled conditions with pure bacteria strains whereas disinfectants are normally used on complex populations in the presence of organic matter and with significant variations in environmental factors like ph, temperature and presence of salts [3]. The aims of this study are to know the effectiveness of different disinfectants under different trade names on selected tests microorganisms: Escherichia coli, Staphylococcus aureus, Streptococcus spp, and Mucor spp; to know if they have any antimicrobial activity against the test microorganisms; to know the concentrations at which they were effective; the susceptibility of the test gram positives and gram negative to the test disinfectants, and to help know the most effective disinfections to use for household and hospital cleanings. 2. Materials and Methods 2.1. Materials Dettol, Izal, Z-germicide, Jik and Phenol were obtained from Ekeonuwa Market, Owerri, Imo State, Nigeria Source of Microorganisms Cultures of the test organisms Staphylococcus aureus, Escherichia coli, Streptococcus spp, Mucor spp, were collected from Umezurike hospital Owerri, Imo State, Nigeria Organism Suspension Suspension of each of the test organisms was made by collecting a loopful of colony from each plate and inoculating in a nutrient broth. The tubes of the subcultured organisms were incubated at 37 o C for 24 hours Inoculation of the Organisms Using different sterile swab sticks, 24 hour old culture of each of the test organisms was collected. The swab sticks containing the different bacterial cultures were swirled into different test tubes containing 10ml of sterile water. The content of each of the tubes was properly homogenized before the inoculation. Another set of sterile swab sticks were dipped into each of the bacterial solution and were used to inoculate the solidified Nutrient agar plates ensuring that the plates were completely covered for uniform growth Preparation of the Disinfectants The disinfectants were poured into different sterile test tubes and these became the stock solutions. A 2-fold serial dilution of each of the disinfectant was prepared as follow: 3 sterile test tubes were placed in a test tube rack; 1ml of distilled water was pipetted into each of the 3 test tubes using a sterile pipette; 1ml of disinfectant was pipetted from the stock into test tube 1, and this was labelled 2-1, the content was properly mixed; 1 ml of solution was collected from tube 1 (2-1 ) and transferred into tube 2 (2-2 ) and the content was properly mixed; 1ml was collected from tube 2(2-2 ) and transferred to tube 3 (2-3 ) and the content was properly mixed; 1ml was collected from tube 3 (2-3 ) and discarded. This procedure was repeated for all the disinfectants.

3 American Journal of Biology and Life Sciences 2014; 2(2): Paper Disc Diffusion method This involves a heavy inoculation of an agar plate with the test organism. Sterile colour coded filter paper discs were impregnated with the different antiseptics or disinfectants and equally spaced on the inoculated plate. Following incubation, the agar plate was examined for zones inhibition (areas of no growth) surrounding the discs. A zone of inhibition is indicative of microbial activity against the organism. Absence of zone of inhibition indicates that the antiseptic or disinfectant was ineffective against the test organism [3] Impregnation of the Discs The sterile filter paper discs were impregnated with 0.1ml each of the dilutions of the disinfectant using different sterile pipettes Inoculation of Impregnated Disc Using sterile forceps, the different discs impregnated with different dilution of the disinfectants were placed on each of the plates inoculated with the test organisms. The forceps was used to press down each of the disc gently against the agar surface so as to ensure good contact. The plates were incubated in an inverted position at 37 o C for 24 hours. The zones of inhibition were observed, and then measured accurately Method for Determining the Phenol Coefficient of the Disinfectants The phenol coefficient of the disinfectants was determined using standard microbiological method. Different dilutions of the phenol stock solution were made (that is 1:80, 1: 90 and 1:100) in sterile test tubes. 0.1ml each of the suspension of the test organisms was introduced into each of the dilutions and mixed properly. 0.1ml was inoculated into tubes of (2ml each) sterile nutrient broth after 5, 10 and 15, for each of the dilutions. The same procedure was repeated for each of the test disinfectants using dilutions 1:400, 1:450 and 1:500. The tubes were incubated for 24 hours at 37 0 C and then observed for growth (turbidity). Phenol coefficient for each of the test disinfectants was calculated using the formula: 3. Results The result obtained in this study of the zone diameter of inhibition of the disinfectants on the various test microorganisms is presented in Table 1. The result of the anti fungal activities of the disinfectants is presented in Table 2; Table 3 is the result of phenol coefficients of the various disinfectants used. microorganism Escherichia coli Staphylococcus aureus Streptococcus spp Table 1. Anti-bacterial sensitivity of the disinfectants. Diameter zone of inhibition Disinfectants (mm) Undiluted Dettol Izal Z-germicide Jik Dettol Izal Z-germicide Jik Dettol Izal Z-germicide Jik The undiluted and diluted concentrations of the disinfectants showed varying zones of inhibition on the test microorganisms. The undiluted showed higher zones of inhibition than the diluted disinfectants. The zones of inhibition of the undiluted ranged from 17mm (Izal) to 25mm (Jik) on Escherichia coli; 9mm (Jik) to 28mm (Dettol) on Staphylococcus aureus whereas on Streptococcus spp it ranged from 8mm (Jik) to 30mm (Dettol). The higher the dilution factor the lower the zones of inhibition on the test microorganisms. microorganis m Mucor spp Table 2. Anti-fungal activity of the disinfectants. Disinfectants Diameter zone of inhibition (mm) Undiluted Dettol Izal Z-germicide Jik Example: If the dilution of the disinfectant that killed the organism in 10 is 1:450, and that of phenol is 1:100, calculation of the phenol coefficient, Pc will be as follows: The zones of inhibition of the undiluted on the fungus is higher 7.0mm (Izal), 12.0mm (Jik), 14.0mm (Dettol) when compared to the different dilutions of the disinfectants. But the diluted and undiluted concentrations of the Z-germicide did not inhibit the growth of Mucor spp.

4 56 Chioma C. Okore et al.: Antimicrobial Efficacy of Selected Disinfectants Table 3. Phenol coefficients of the disinfectants against the test organisims. microorganism Escherichia coli Staphylococcus aureus Streptococcus spp Mucor spp Key: Pc = Phenol coefficient Disinfectant Pc comparism with phenol Dettol 500/80 = 6.25 Izal 400/100 = 4 Jik 450/100 = 4.5 Dettol 6.25 Izal 0 = No clearance Jik No clearance after 10 Dettol 500/80 = 6.25 Izal 400/100 = 4 Z Germicides 0 = No clearance after 10 Jik 0 = No clearance after 10 Dettol 500/100 = 5 Izal 0 = No clearance Jik 500/80 = 6.25 In this study, the phenol coefficient obtained with each of the disinfectants on Escherichia coli ranged from 4.0 (Izal and Z germicide) to 6.25 (Dettol), on Staphylococcus aureus it ranged from 0 (Izal and Jik) to 6.25 (Dettol), on Streptococcus spp it ranged from 0 (Jik and Z- germicide) to 6.25 (Dettol) also on the Mucor spp the phenol coefficient ranged from 0 (Izal) to 6.25 (Jik). From Table 3, the disinfectant that is most effective is that which the ratio of the phenol to disinfectant is > 1. Dettol is the most effective of the other disinfectants on E.coli (6.5), followed by Jik (4.5), then Izal and Z-germicide (4). On Staph aureus Dettol is the most effective (6.25), followed by Z- germicide (4), both Izal and Jik are less effective on Staph aureus than phenol being < 1. Dettol is the most effective on Streptococcus spp (6.25), followed by Izal (4), but Jik and Z-germicide are less effective on Strep spp as they are < 1. Jik is the most effective disinfectant for inhibiting Mucor spp (6.25), followed by Dettol (5), then Z-germicide (4). Izal is less effective than phenol < 1 in controlling Mucor spp. zones of inhibitions when compared with the undiluted concentrations. The disinfectants showed remarkable zones of inhibition against the Mucor spp except Z-germicide. The test microorganisms differ in their susceptibilities to the disinfectants. Dettol had broad spectrum activity as it inhibited the growth of gram positive (Staph aureus; Streptococcus spp) and gram negative (E.coli). The antimicrobial activity was more on the gram positives; Streptococcus spp (30mm) and Staph aureus (28mm). Izal showed narrow spectrum activity on gram negative bacteria E.coli 17mm. Z germicide and Jik showed broad spectrum activity on both gram positive and gram negative test microorganisms but their activity was more on the gram negative microorganism (E.coli) 19mm and 25mm respectively. The antifungal activity of the undiluted disinfectants ranged from the zones of inhibition of 7.0mm (Z germicide) to 14.0 mm (Dettol). The different dilutions of the disinfectants gave different zones of inhibition of the range 3.5mm to 11.0 mm (Jik). The antifungal effect of Jik was also reported by [12.] No zones of inhibition were recorded with Izal in all the concentrations used on the test microorganisms. The outcome of this study proves Dettol to be the strongest antimicrobial agent irrespective of the dilutions when compared with the other disinfectants used in this study. Also the report of [13] and [9] showed that Dettol is a strong antimicrobial agent. It also showed that the dilutions of the other disinfectants exhibited remarkable growths of the test microorganisms, (that is the form in which these disinfectants are used). The antimicrobial effects of these disinfectants against the test microorganisms showed the biocide effects of these disinfectants against these microorganisms that are associated with water. Escherichia coli is an indication of water contamination of faecal origin which is the cause of many diseases. Staphylococcus aureus is well known to cause wound infection, and Streptococcus spp is well implicated to cause sour throat [2]. Mucor spp is also well known to cause mycosis in an immunocompromised person [10]. Mucor is a moisture lover and inhibits such surface as towel, panties etc. The use of these disinfectants may be means to reduce cases of acquired diseases caused by the test microorganisms. 4. Discussion The results obtained in this study showed that the disinfectants screened for antimicrobial activity have considerable antibacterial and antifungal effects on the test microorganisms. The antimicrobial properties of Dettol, Izal, Z-germicide, Jik, have been described by [14], [15], [16], [17], [18], [19], [20]. The different dilutions of Jik recorded no antimicrobial activity on Staph aureus and Streptococcus spp. Dettol recorded the highest zones of inhibition against Streptococcus spp (30 mm) while Izal and different dilutions of Jik recorded no growth inhibition. The dilutions of different disinfectants showed remarkable 5. Conclusion The potency of disinfectants is very important to enhance the antimicrobial activity of these disinfectants towards controlling microbial population which includes prevention of diseases transmission and infection. It also prevents deterioration and spoiling of materials which could also lead to microbial infection. Determination of antimicrobial effectiveness of disinfectants is essential to achieve total disinfection of pathogens. The use of good disinfectants should be encouraged to reduce cases of skin and wound infections caused by most microorganisms.

5 American Journal of Biology and Life Sciences 2014; 2(2): References [1] J.M. Pelezar, E.C.S. Chan and R.N. Krieg, Microbiology, 5th ed, New Delhi: Tata McGraw-Hill publishing Company Ltd, pp , [2] J.M. Willey, L.M. Sherwood and C.J. Woolverton, Prescott, Harley, and Klen's Microbiology, 7th ed., New York: McGraw-Hill Higher Education, pp , [3] G.J. Tortora, B.R. Funke, C.J. Case, Microbiology: An Introduction, 8th ed., New York: Pearson Education and Dorling Kindersley Pvt, Ltd, pp , [4] K.J. Ryan and C.G. Ray, Bacteria, In: Sherris Medical Microbiology, 4th ed., Mc Gram Hill, ISBN , [5] G.F. Brooks, S.J. Butel, and A.S. Morse, Jawetz, Melnick and Adelberg's Medical Microbiology, 23rd ed., United States: McGraw-Hill Companies, pp , 2004 [6] World Health Organization (WHO), Manual for the laboratory identification and antimicrobial susceptibility testing of bacterial pathogens of public health importance in the developing world, [7] Omoruyi MI, Idemudia MI. Comparative analysis of the antiseptic properties of some disinfectants on bacteria and fungi of public health importance isolated from barbing clippers. J. of Asian Scientific Research 2011; 2(1): [8] Boyce JM, Pittet D. Guidelines for hand hygiene in healthcare settings, Recommendation of the healthcare infection control practices, Advisory committee and Hand Hygiene Task Force, HICPAC/SHSA/APIC/IDSA, Infec control Hosp Epidemiol, Suppl. 2002; 23, S. 3-S. 40, available at [9] Olowe OA, Olayemi AB, Eniola KIT, Adeyeba O.A. Antibacterial activity of some selected disinfectants regularly used in hospitals. African J. of clinical and experimental microbiology 2004; 5(1): ISSN X, AJCEM/ /2412. [10] M. Cheesbrough, District Laboratory Practice in Tropical Countries, 2nd ed., New York: Cambridge University Press, pp , [11] K.P. Talaro, Foundation in Microbiology, 5th ed, New York: McGraw-Hill companies, pp , [12] Awodele O, Emeka PM, Agbamuche HC, Akintowa A. The antimicrobial activities of some commonly used disinfectants on Bacillus subtilis, Pseudomonas aeruginosa and Candida albicans. African J. of Biotechnology 2007; 6(8): [13] Olasehinde GI, Akinyanju JA, Ajayi AA. Comparative antimicrobial activity of commercial disinfectants with naphtholics. Research J. of Microbiology 2008; 3(4): , ISSN [14] El-Mahmood AM, J.H. Doughari JH. Bacteriological examination of some diluted disinfectants routinely used in the specialist hospital Yola, Nigeria. African J. of pharmacy and Pharmacology 2009; 3(5): , ISSN [15] Thomas BT, Adeleke AJ, Raheem-Ademola RR, Kolawole R, Musa OS. Efficiency of some disinfectants on bacterial wound pathogens. Life Science J 2012; 9(2): , [16] Otokunefor TV, Usoh CU. Microbial contamination of inuse disinfectants in small private medical facilities in Owerri, in south east Nigeria, Scientia Africana 2009; 8(2):17-25, ISSN [17] Iruoha IR, Oji AE, Nwosu OK, Amadi ES. Antimicrobial activity of Savlon, Izal and Z-germicide against clinical isolates of Pseudomonas aeruginosa from hospital wards. European J. Dentistry and Medicine 2011; 3(1):32-35, ISSN /doi: /ejdm [18] Ghotaslou R, Bahrami N. Antimicrobial activity of chlorhexidine, peracetic acid/peroxide hydrogen and alcohol based compound on isolated bacteria in Madani Heart Hospital, Tabriz, Azerbaijan, Iran. Advanced Pharmaceutical Bulletin 2012; 2(1): [19] Rutala WA, Barbee SL, Agular NC, Sobsey MD, D.J. Weber DJ. Antimicrobial activity of home disinfectants and natural products against potential human pathogens. Infection control and hospital epidemiology 2000; 21(1):