The microbiological quality of 24 samples of oral pharmaceuticals comprising antacids, cough and paracetamolsyrups purchased randomly from different drug stores operating in Abakaliki metropolis were assessed. They wereanalyzed by pour plate method. Their microbial load was determined using the viable cell count method. The resulting contaminating microorganisms were isolated and characterized by standard methods. The results revealed fungal and bacterial contaminations in 16 and 19 samples respectively. Contaminant bacteria include Bacillus spp.,Staphylococcus spp., E.coli, Proteus spp, Klebsiella spp. and Pseudomonas spp. with Staphylococcus spp. being the most predominant bacterial contaminant, while fungi contaminants were basically Mucor and Aspergillus species. The pH values of the analyzed drugs ranged from 5 to 9. The variations in the stated pH of sampled products were however, not justified in this work; thus queries the stated drug pH and why certain isolated organisms could grow on such pH outside their normal habitats’ pH. This study has shown therefore, that some oral pharmaceuticals sold in drug stores maybe heavily contaminated by varying microbial agents.Keywords: Oral, Pharmaceuticals, Bacteria, Fungi, Contamination

Azi, S

Emejuru, MC

Nwosu, NB

Ojiegbe, GC

English

AJOL - African Journals Online

International Journal of Community Research http://www.anrescentpub.comISSN: 2315 – 6562Emejuru et al., IJCR 2013; 2(3): 39-45 39MICROBIOLOGICAL LOAD OF SELECTED ORAL LIQUID PHARMACEUTICALS1EMEJURU M.C., 1OJIEGBE G.C., 1AZI S., 2NWOSU N.B.Department of 1Medical Laboratory, Ebonyi State University, Ebonyi State, Nigeria. 2Medical Microbiology, St Charles Hospital, Borromeo-Onictsha, Anambara State, Nigeria.Corresponding author: macchyke01@yahoo.comABSTRACTThe microbiological quality of 24 samples of oral pharmaceuticals comprising antacids, cough and paracetamol syrups purchased randomly from different drug stores operating in Abakaliki metropolis were assessed. They were analyzed by pour plate method. Their microbial load was determined using the viable cell count method. The resulting contaminating microorganisms were isolated and characterized by standard methods. The results revealed fungal and bacterial contaminations in 16 and 19 samples respectively. Contaminant bacteria include Bacillus spp., Staphylococcus spp., E.coli, Proteus spp, Klebsiella spp. and Pseudomonas spp. with Staphylococcus spp. being the most predominant bacterial contaminant, while fungi contaminants were basically Mucor and Aspergillus species. The pH values of the analyzed drugs ranged from 5 to 9. The variations in the stated pH of sampled products werehowever, not justified in this work; thus queries the stated drug pH and why certain isolated organisms could grow on such pH outside their normal habitats’ pH. This study has shown therefore, that some oral pharmaceuticals sold in drug stores maybe heavily contaminated by varying microbial agents.Keywords: Oral, Pharmaceuticals, Bacteria, Fungi, Contamination.Received: 1st April, 2013 Accepted: 18th July, 2013 Published: 31st July, 2013INTRODUCTION At the beginning of the 21st century, microbial contamination of non-sterile products became one of the major reasons for product recalls and production shutdown (Jimenez, 2004). So far, microbial contamination of non-sterile pharmaceutical liquid products is rapidly becoming a matter of worldwide concern (Sykes, 1971), considering the fact that oral liquid pharmaceuticals are expected by their nature, to achieve maximum degree of sterility (Baird and Petrie, 1981). Unfortunately, Adeshina et al. (2009) submitted that oral liquid drug formulations such as aqueous solutions, suspensions, emulsions, and syrups used for children are at a greater risk of microbial contamination during consumption due to its sweetening-agent content, reconstitution methods, improper storage, and handling defects, which may ultimately contribute to secondary bacterial and fungal infections in these patients. This indicates that microbial contamination of routine drugs is becoming a public health challenge.The United States Pharmacopoeia (USP) (1980) had suggested that liquid pharmaceuticals should not exceed 103 colony forming units per milliliter (cfu/ml) of bacteria, while yeast and mold should not exceed 102cfu/ml. Similarly, the National Agency for Food and Drug Administration and Control (NAFDAC) Hand Book (2000) had outlined the standard microbiological specifications for the certification of syrup oral suspensions. Thus, typical viable and fungal counts for bacterial and yeast cells must not exceed 1.0x103cfu/ml and 1.0x102cfu/ml for bacterial and fungal growth respectively.Furthermore, Mohammad et al. (2012) had admitted in their work that the presence of certain micro-organisms in non-sterile pharmaceutical products adversely affects the therapeutic activity of the product and even becomes detrimental to the health of the patient.With the exception of preparations which are terminally sterilized, the micro-flora of final products may represent the contamination from the raw materials used in their formulation, the equipment used, the atmosphere, the operator of the process or from the final containers into which it was packed.Indeed, as antacids, cough syrups and paracetamol syrups are the three widely used oral liquid formulations (amongst others) in Nigeria (Tukur et al., 2012), their microbiological safety is an important public health concern considering the fact that they are heavily commercialized and consumed by many people.International Journal of Community Research http://www.anrescentpub.comISSN: 2315 – 6562Emejuru et al., IJCR 2013; 2(3): 39-45 40This study therefore, is designed to evaluate the microbial load in some selected oral liquid pharmaceuticals in comparison with the permissible levels of contaminants officially allowed for such commercially available products.MATERIALS AND METHODSStudy area: This study was carried out in Abakaliki Metropolis, the Capital City of Ebonyi State, situated in the Southeastern geopolitical zone of Nigeria.Geographically, Abakaliki is located between 601511811N and 80051 5511E. The state owned University (Ebonyi State University), the Federal Teaching Hospital, and other governmental/non-governmental institutions are amongst the institutions located in Abakiliki.According to Nigeria Population Commission (NPC)census (2006), Abakaliki has a population of about 149,683 with the larger population being civil servants. The state is notable for her agricultural prowess and renowned for the production of salt; hence the state acronym “salt of the nation”.Sampling technique: Twenty-four (24) properly sealed and packed oral liquid drugs of antacids, cough syrups, and paracetamol syrups (2 samples each of 4 different brands) produced by NAFDAC approved manufacturing pharmaceutical companies, were randomly purchased from various operating drug stores in Abakaliki metropolis. They were checked for their batch numbers, production date, expiry dates and certified intact.The samples were then labeled for the purpose of this work and stored in a cool and dry laboratory shelf at room temperature (24-25oC) as recommended by the manufacture.LABORATORY ANALYSIS Preliminary assessment (Macroscopy): The NAFDAC number, colour, and taste of the samples were assessed. Drug pH and preservatives/constituents were also noted (Table 1).Enumeration of microbial contaminants (Microscopy): Inoculation by pour plate method was carried out after 1 in 100 serial dilutions of the various samples were carried out. 1ml of each of the diluted sample was then aseptically aspirated into each media (Nutrient Agar, MacConkey Agar, and Sabouraud Dextrose Agar) respectively. The media was poured aseptically into the sterile Petri dishes for each media at 40-45oC and then swirled following which they were allowed to solidify for incubation atthe appropriate temperature (Nutrient and MacConkey Agar plates at 370C for 24-48 hours;while Sabouraud Dextrose Agar plates at 25±20C for 3-5days) for the enumeration of total viable and fungal counts respectively. Typical colonies of microbial growth on plates were counted at the end of incubation and the total number of counts were multiplied by 10-2 (dilution factor) to get the total viable count for all samples as described by Fawole and Oso (1988) and Ochei and Kolhatkar (2008).The results obtained were after wards, compared with the standard microbiological specifications for certification of syrups and with the microbiological permissible levels as judged by USP (1980) and NAFDAC Handbook (2000).Characterization of isolates: The bacterial isolates were identified by Gram staining techniques and other biochemical tests (catalase, coagulase, oxidase, indole, methyl red and motility test by hanging drop method) as described by Fawole and Oso (1988)Cheesbrough (2000), Baker et al. (1998) while fungal isolation was based on growth rate and colonial morphology as described by Ochei and Kolhatkar(2008).Sterilizations of glassware’s and materials used for this research work were achieved by the use of direct heat and hot air oven as described by Isu and Onyeagba (2002).RESULTSResults from the microbiological analysis showed that out of the 24 samples of different brands of three oral pharmaceuticals (antacid, cough syrup and paracetamol syrup), 19 (79.17%) yielded growths while 5 (20.83%) yielded no growths (see tables 2 and figure 1).Also, a number of bacteria organisms were isolated from the various samples as shown in tables 3 below.For fungal isolates, 16 (66.67%) out of the 24 sample analyzed yielded growths while the rest 8 (33.33%) yielded no growth as shown in tables 4 below. Most recognizable fungal isolates were of Mucor and Aspergillus species. The growth pattern (in percentages) was graphically illustrated in figure 2below. International Journal of Community Research http://www.anrescentpub.comISSN: 2315 – 6562Emejuru et al., IJCR 2013; 2(3): 39-45 41Table 1: Preliminary Assessment of the samplesSample/brand NAFDAC NopH Colour Taste Constituents/Preservatives usedAntacidsEmtricil 04-1455 9.0 White Sour AMS, MT, MC, SB, SGestid 04-0480 8.0 Pink Sour Dried AH Gel, MH, Activated PMS.Mist mag 04-3165 9.0 White Sour MT, Light MC.Stopacid 04-4841 8.0 White Sour MH, Dried AH gel,Cough syrupsD-koff 04-2751 5.0 Amber Sweet DPH, BH, AC, SC, MethanolEmzolyn 04-0266 5.0 Amber Sweet DPH, AC, SC, MethanolBenylin 04-0887 5.0 Pink Sweet DPH, SC, PH EurCodrux A4-7704 5.0 Pink Sweet DPH, AC Methanol SC.Paracetamol syrupsAlbemol A4-7709 5.0 Pink Sweet Paracetamal BPAcepol A4-0704 5.0 Pink Sweet Paracetamal BPEmcap 04-7339 5.0 White Sweet Paracetamal BPMB 5 04-0247 5.0 Pink Sweet Paracetamal BPKEY: AMS - Aluminum Magnesium Silicate; MT - Magnesium Trisilicate,; MC - Methicone.; MH - Magnesium Hydroxide; Dried AH Gel - Dried Aluminum Hydroxide Gel; Activated PMS - Activated Polymethylsiloxane  ; DPH- Diphenhydramine Hydrogen chloride; BH- Bromohexine Hydrogen chloride; AC - Ammonium Chloride; SC- Sodium CitrateTable 2: Bacterial Growth Analysis on Samples and their PercentagesSample/brand No. sampledNo. of growthNo. of no growth% growthAntacidsEmtricil 2 2 - 100Gestid 2 - 2 0Mist mag 2 2 - 100Stopacid 2 1 1 50Cough syrupsD-koff 2 2 - 100Emzolyn 2 2 - 100Benylin 2 1 1 50Codrux 2 2 - 100Paracetamol syrupsAlbemol 2 1 1 50Acepol 2 2 - 100Emcap 2 2 - 100MB5 2 2 - 100International Journal of Community Research http://www.anrescentpub.comISSN: 2315 – 6562Emejuru et al., IJCR 2013; 2(3): 39-45 42Table 3: Bacteria’s isolated from each of the samplesSample/brand No. of brands with growth Organisms isolatedAntacidsEmtricil 2 Staph. spp.Mist Mag 2 E. coli, Klebsiella spp., Proteus spp. Stopacid 1 Klebsiella spp., Proteus spp., Bacillus spp., E.coli.Cough syrupsD- koff 2 Staph sppEmzolyn 2 Pseudomonas spp.,E.coli, Proteus spp. Benylin 1 Staph. spp.Codrux 2 E.coli. Paracetamol syrupsAlbemol 1 Staph. Spp.Acepol 2 Staph. spp., Pseudomonas spp. Emcap 2 Proteus spp. MB 5 2 Staph spp.Table 4: Fungal Growth Analysis on each of the Samples and their PercentagesSample/ brand No. sampledNo. that showed growthNo. that showed no growth% GrowthAntacidsEmtricil 2 2 - 100Gestid 2 1 1 50Mist Mag 2 2 - 100Stopacid 2 1 1 50Cough syrupsD-koff 2 2 - 100Emzolyn 2 - 2 100Benylin 2 1 1 50Codrux 2 1 1 50Paracetamol syrupsAlbemol 2 2 - 100Acepol 2 1 1 50Emcap 2 2 - 100MB 5 2 1 1 50DISCUSSIONThis study aligns with the argument by Sheikh et al., (1988) that sterility is not a requirement in the official compendia for oral pharmaceutical dosage forms. It also implies that contamination may occur during manufacturing, packaging, and/or handling by the consumer (Baird, 2004; Ibezim et al., 2002). Of course, this raises concern since some drug forms,even though stored in favorable environment, can serve as substrates for micro-organisms (Sapra et al., 2012). More so, variations in the stated pH of sampled products were not highly justified, thereby querying the authenticity of such stated (but questionable) pH; contrary to established normal habitat’s pH. Indeed, the ability of certain organisms(as seen in this study) to grow at such stated pH, makes it more worrisome. This implies that certain drug constituents (in addition to their normal roles), are microbial growth enhancers. Interestingly, Baird and Petrie (1981) states that liquid antacids often contain ingredients of high basic pH, which readily support the growth of a variety of micro-organisms,when appropriate precautions are not taken.International Journal of Community Research http://www.anrescentpub.comISSN: 2315 – 6562Emejuru et al., IJCR 2013; 2(3): 39-45 43Fig I: Pie- chart representation of percentage bacterial growth and no growthNB: Summary: No. of growths = 19(79.17%) No. of no growths = 5(20.85%)Fig 2: Pie- chart representation of percentage fungal growth and no growthNB. Summary: No of growths = 16 (66.67%) No of no growths = 8(33.33%)According to NAFDAC Handbook (2000), bacterial count should not exceed 1000 cfu/ml, but unfortunately, out of the 19 samples with growth, 8 (42.11%) samples exceeded this rule in Nutrient Agar, while 11 (57.89%) samples exceeded it in MacConkey Agar. Given the heavy contamination observed in this study, predominantly Staphylococcus aureus, it is a worrisome trend as such organism secretes toxins that contribute to serious gastrointestinal distress (Denyer, 1990). In addition, the presence of Escherichia coli is a good indicator of faecal contamination probably International Journal of Community Research http://www.anrescentpub.comISSN: 2315 – 6562Emejuru et al., IJCR 2013; 2(3): 39-45 44from the water used. This might be responsible for reported cases of infant diarrhea in some parts of Nigeria (Antai and Anozie, 1987).Similarly, NAFDAC Handbook (2000) stipulates that fungi count must not exceed 1.0x102cfc/ml, but the results on fungi count far exceeded this value in all the 16 samples with fungi growth. Given the hightoxin producing nature of some Aspergillus spp,especially Aspergillus flavus, serious problems could result in man due to its high aflatoxin production- a known carcinogenic agent (Fortnum, 1986; Payne and Brown, 1998; Abbas et al., 2004).Over all, the findings of this study imply that water supply, raw materials and lack of personal hygiene have remained the major sources of contamination in pharmaceuticals. Also implicated, are the high levels of sweetening and preservative agents (Mahboob et al., 2004). In fact, sweeteners of high sugar categoryin drugs, have been known to enhance microbial growth (Mahboob et al., 2004); hence the need for caution.It is our recommendation therefore, that health care providers should monitor this trend, while NAFDAC is encouraged to tighten regulations in this regard.ACKNOWLEDGEMENTThe authors are grateful to the Ebonyi State University, Department of Medical Laboratory Science Diagnostic and Research Laboratory for the research space and privileges granted to them in the course of the period of this research. No grants were received.REFERENCESAbbas, H.K., Zablotowicz, R.M., Weaver, M.A., Horn, B.W., Xie, W. and Shier, WT. (2004). Comparison of cultural and analytical methods for determination of aatoxin production by MississippiDelta Aspergillusisolates. Can J Microbiol.; 50: 193–199.Adeshina, G.O., Ajayi, S.O. and Onaolapo J.A., (2009). Microbiological quality of some commercially available paediatrics anti-malarial and cough preparations in ilorin, Nigeria. Nigerian J.Pharm. Sci.; 8(1):109-117.Antai, S.P. and Anozie, S.O. (1987). The incidence of infant diarrhea due to Enteropathogenic E. coli in Nigerian. J. Appl. Bacteriol.; 62:227-229.Baird, R.M. (2004). Microbial Spoilage, Infection Risk and Contamination Control. In: Stephen P. Denyer, Norman A. Hodges, Sean P. Gorman (eds). BlackWell Publishing Company, Massachusetts, USA. Pp. 263-284.Baird, R.M. and Petrie, P.S. (1981). A Study of Microbiological Contamination of Oral Medications. Pharm. J.; 226 (6105): 10-1.Baker, F.J., Silverton, R.E. and Pallister, C.J. (1998). Baker and Silverton's introduction to medical laboratory technology in conjunction with A. Hornby, R.W. Luxton, R.L. Griffin. 7th ed. Oxford; Woburn, MA : Butterworth-Heinemann, Pp. 260-261.Cheesbrough, M. (2005). District Laboratory Practice in Tropical Countries. Part 1. 2nd ed. Cambridge Low-Price Edition. Cambridge University Press, Cambridge, UK. Denyer, S.P. (1990). Guide to Microbiological Control. In: Pharmacentical  Chiccheste Eilis Horwood Taylor O., Silva B.O., Obi E.P. and Adedokun O. Substandard Commercial Trisilicate Mistures. 2: 46-49.Fawole, M.O. and Oso, B.A. (1988). Laboratory Manuals of Microbiology. Spectrum Books Limited. Pp. 16-35.Fortnum, B.A. (1986). Effect of environment on a-atoxin develop-ment in preharvest maize. In: Zuber MS, Lillehoj EB, eds. Aatoxin in Maize: A Proceedings of the Workshop. El Batan, Mexico: CIMMYT, Pp. 145–151.Ibezin, E.C., Simone, C.O., Ofoefule, S.I. and Chah,K.F. (2002). Evaluation of the Microbiological Quality of Some Commercially Available Syrups and Suspensions in Nigeria. J. Photomed. Therapy;7(1,2): 18-25.Isu, N. and Onyeagba, R. (2002). Basic Practical’s In:  Microbiology. 2nd ed. Fasmen Communication, Nigeria. Pp.126-128. Jimenez, L., (2004). Microbial Limits. In: Microbial Contamination Control in Pharmaceutical Industry (jimenez L., Ed). Marcel Dekker Inc., New York, USA. Pp. 15-44.Mahboob, H., Ara S. and Raham, M.Z., (2004). Quantization Examination of Aerobic Bacteria and Fungi in Locally available Antacid Suspension and International Journal of Community Research http://www.anrescentpub.comISSN: 2315 – 6562Emejuru et al., IJCR 2013; 2(3): 39-45 45Possible Contamination by Specific Bacteria. Pakistan J. Biol. Sci.; 7 (11):2014-2017.Mohammad, M., Farukh, F.A. and Zankaria, M. (2012). Qualitative and Quantitative Microbiological Studies of Antacid and Paracetamol Suspension from Different Drugstores of Dhaka. Dhaka University J.Biol. Sci.; 21(1):105-107.National Agency for Food and Drug Administration and Control (NAFDAC) (2000). Syrups, Suspensions, Linctures and Mixtures. J. Drug Processing; 8, 12-19.National Population Commission’s (NPC) (2006). Population Figures for Nigeria States. Population and Housing Census- Real or Imagined. Posted On : May 26th, 2013, Updated On : May 26th, 2013 Ochei, J. and Kolhatkar, A. (2008). Medical Laboratory science, Theory and Practice. Tata McGraw-Hill Publishing Company Limited, New Delhi  India. Pp. Payne, G.A. and Brown, M.P. (1998). Genetics and physiology of aatoxin  biosynthesis. Annu RevPhytopathol.; 36: 329–362.Sapra. A., Kakkar S. and Narasimhan, B., (2012). Sources of Impurities: A Review. Inter. Res. J.Pharmacy; 3(1).Sehaikh, D., Jamshed, T.A. and Sheikh, R. (1988). Microbial Contamination of Pharmaceutical Preparations. Pakistan J. Pharm. Sci.; 1(1): 61-66.Sykes, G. (1971). Pseudomonas aeruginosacontamination of liquid antacids: A Survey. J. Pharm. Sci.; 60: 604-605.Tukur, M.A.,  Muazu, J. and Mohammed, G.T. (2012). Microbial analysis of brands of multivitamin syrupsmarketed in Maiduguri, Northeast Nigeria. Adv. Appl. Sci. Res.; 3(5):3124-3128.United States Pharmacopoeia (USP) (2004). US Pharmacopeial Convention, Inc. United States Pharmacopeia 27. Chapter <797> “Pharmaceutical Compounding–Sterile Preparations.” Rockville, MD: USPharmacopeial Convention, Inc.; 2004: 2350-2370.AUTHOR’S CONTRIBUTIONAll the authors (Emejuru MC, Ojiegbe GC, Azi S.and Nwosu NB) contributed to the study from the design, data collection and analysis to the manuscript preparation and presentation of final draft.

Microbiological Load of Selected Oral Liquid Pharmaceuticals

https://www.ajol.info/index.php/ijcr/article/download/106243/96202

Microbiological Load of Selected Oral Liquid Pharmaceuticals

Abstract

Similar works

Full text

Available Versions

AJOL - African Journals Online