QUANTITATIVE ANALYSIS OF THE TOTAL VIABLE COUNT OF AEROBIC

BACTERIA AND FUNGI IN LIQUID PREPARATIONS AND IDENTIFICATION OF

SPECIFIC TYPES OF BACTERIA

Lungwani Tyson Muungo1, Ernest Simpemba

1, Aubrey N. Kalungia

1

1University of Zambia, School of Medicine, Department of Pharmacy, P.O. Box 50110

Corresponding Authors: Lungwani Tyson Muungo, University of Zambia, School of Medicine,

Department of Pharmacy, P.O. Box 50110, Tel. 00260 211 255852 / 00260 977775473, E-mail

tmuungo@yahoo.com or Lungwani.muungo@unza.zm

Abstract

Microbial contamination of non-sterile pharmaceutical liquid products is rapidly becoming a matter of worldwide concern (Sykes,G.,1971. Baird, R.M. and P.S.Petris,1981). Many failures in pharmaceutical processing have arisen because of the inability of those responsible for its design to be aware of the distribution and survival potential of micro organisms in the

environment and in the raw materials and equipment used in a pharmaceutical factory. In this study the microbiological status of liquid antacid suspension was studied. . Aerobic viable microbial count of 3 (25%) out of 12 samples studied were found to exceed the USP limit. Viable microbial count varied between 0 CFU per mL and 1000 CFU per mL .Out of the 12 samples 6 samples contained Fungi and 8 samples contained bacteria. None of the samples contained Pseudomonas. Out of 12 samples 2 (16.67%) samples of the same company having same manufacturing date contained E.coli and 1 sample (8.34%) contained Salmonella spp.5 samples (41.7%) contained Staphylococcus aureus. Proper sealing of containers is essential from the

microbiological point of view and sealing of the containers of all the companies were satisfactory. Manufacturing and expiry dates were printed in case of all the companies indicating the period in which the preparation was safe for consumption.

Key words: Antacid suspension, microbial contamination, colony forming unit

Introduction Microbial contamination of non-sterile pharmaceutical liquid products is rapidly becoming a matter of worldwide concern(Sykes,G.,1971. Baird, R.M. and P.S.Petris,1981). Many failures in pharmaceutical processing have arisen because of the inability of those responsible for its design to be aware of the distribution and survival potential of micro organisms in the environment and in the raw materials and equipment used in a pharmaceutical factory. The microbiological quality of pharmaceutical products is influenced by the environment in which they are manufactured and by the materials used in their formulation. With the exception of preparations that are terminally sterilized in their final container, the micro flora of the final product may represent the contaminants from the raw materials, from the equipment with which it was made, from the atmosphere, from the person operating the processor from the final container into which it was packed. Some of the contaminants may be pathogenic whilst others may grow even in the presence of preservatives and spoil the product. Any microorganisms that are destroyed by in-process heat treatment may still leave cell residues that may be toxic or pyrogenic, since the pyrogenic fraction, lipid A, which is present in the cell wall, is not destroyed under the same conditions as the organisms. An important group of pharmaceutical products, including those intended for parenteral administration or for instillation in the eye, are required to be free from living micro organisms, and with parenteral products, from those residues of the bacterial cell which may give rise to fever. Some pharmaceutical manufacturers consider liquid antacid to be a low cost item; as a result insufficient care is taken to ensure maximum control of microbiological aspects of manufacture. Since wrong or inadequate preservatives used in medicine cannot control the contamination (Gilbert, P. and N. Wright, 1987), it is imperative that correct preservatives should be used in appropriate amount. The formulation of an elegant, efficacious medicine which is both stable and acceptable to the patient may necessitate the use of a wide variety of ingredients in a complex physical state, aspects of which could create conditions conducive to the survival and even extensive replication, of contaminant microorganisms that might enter the product during its manufacture or with use by the patient or medical staff. Liquid antacids often contain ingredients which readily support the growth of a variety of micro organisms if appropriate precautions are not taken (Baird, R. M. and P.S. Petris 1981, Kallings, L.O., O.Ringertz, L.Silverstolpe and F. Ernerfeldt,1996). Antacid suspension containing organic components and being of neutral pH and processed under neutral appropriate preservation conditions is highly susceptible to microbial contamination. Pharmaceutical ingredients subject to microbial attack are as follows: Humectants; Glycerol and sorbitol in pharmaceuticals readily support microbial growth unless present in high concentrations. Fats and oils; These hydrophobic materials are attacked only when dispersed in aqueous formulations, although fungal growth is reported in condensed moisture films on the surface of oils, or if water droplets contaminate the

bulk phase during storage. Lipolytic rupture of triglycerides liberates glycerol and fatty acids, the later then

undergoing -oxidation of the alkyl chains with the production of odorous ketones. Sweetening, flavouring and colouring agents; many of the sugars and colouring agents used in pharmacy are ready substrates for microbial attack. Aqueous stock solutions of flavouring agents, such as peppermint water and chloroform water, and colouring agents such as amaranth readily support the growth of bacteria and yeasts. As the product is available as multi-dosed medicament there is further risk of contamination during the intake period. With their fastidious nature, contaminants acutely pathogenic to man are probably unable to replicate in most medicines but could remain viable and infective for an appreciable time. Although infrequently reported as pharmaceutical contaminants, they attract considerable attention when they are. For example, Salmonella infections have arisen from tablets and capsules of yeast, carmine pancreatic thyroid extract and powdered vegetable drugs. Of greater practical concern are a wider variety of common saprophytic contaminants, usually of limited pathogenicity to healthy individuals, which may replicate readily within some aqueous medicines. To certain groups of unhealthy patients, however, or to particularly damaged tissues some of these opportunistic microbes can represent a serious health hazard. For example, whilst the intact cornea is resistant to infection, when scratched, scarred, or surgically incised it offers little resistance to pseudomonads and some other Gram-negative bacteria. Many eyes have been lost following the use of improperly designed ophthalmic solutions containing actively growing Pseudomonas aeruginosa as contaminants. The skin of badly burned patients has become infected by pseudomonads contaminating antiseptic solutions, resulting in failure of skin-grafting operations, and even death. Gram- negative microbes in ointments and creams have infected eczematous skin, and Klebsiella contaminants in barrier creams have induced fatal respiratory disease in neonates. Oral mixtures and antacid suspensions have become colonized with Gram-negative bacteria, with probably limited consequence to many, but in patients immunocompromised as a result of Antineoplastic chemotherapy or as an aid to transplant surgery, life-threatening infections have resulted. Candida spp. Are a particular hazard to such patients and fatal septicaemias have been associated with these contaminants in oral and intravenous solutions. Extensive growth of bacteria in bladder washout solutions, including those containing quaternary ammonium antiseptics, has been responsible for many extremely painful infections. The major toxic metabolites found in medicines are pyrogens liberated from Gram-negative bacteria and blue-green algae (Cyanobacteria). These can induce acute febrile shock by the presence of miniscule quantities in infusion fluids entering the bloodstream or from contaminated haemodialysis fluids by diffusing through dialysis membranes. The acute bacterial toxins associated with food poisoning episodes have not been commonly reported in pharmaceutical products, although toxigenic fungi including aflatoxin-producing aspergilli and aflatoxins themselves have been detected. Many of the metabolic products of microbial deterioration are extremely unpleasant, if not toxic, and would deter use of a medicine if present even in small quantities. As a result inclusion of an antimicrobial preservative is a necessary part of the formulation process. The pharmaceutical manufacturers for the formulation of the medicines follow usually foreign Literatures. Hence, the antimicrobial agents used as preservatives may not be effective against the microbes of varied or subtropical habitat. In Zambia very little work has been carried out on microbial contamination and preservation of liquid antacid. Purpose of Study The purpose of this study was to grow awareness among the concerned persons so that microbial quality of liquid antacid preparation improves further. Literature review Salmonella

Figure 1. Salmonella enterica Salmonella is a Gram-negative facultative rod-shaped bacterium in the same proteobacterial family as Escherichia coli, the family Enterobacteriaceae, trivially known as "enteric" bacteria. Salmonellae live in the intestinal tracts of warm and cold blooded animals. Some species are ubiquitous. Other species are specifically

adapted to a particular host. In humans, Salmonella are the cause of two diseases called salmonellosis: enteric fever (typhoid), resulting from bacterial invasion of the bloodstream, and acute gastroenteritis, resulting from a foodborne infection/intoxication

Figure 2. Salmonella typhi, the agent of typhoid. Gram stain. (CDC) 2.1.1 Salmonella Nomenclature The genus Salmonella is a member of the family Enterobacteriaceae, It is composed of bacteria related to each other both phenotypically and genotypically. The bacteria of the genus Salmonella are also related to each other by DNA sequence. When serological analysis was adopted into the Kauffmann-White scheme in 1946, a Salmonella species was defined as "a group of related fermentation phage-type" with the result that each Salmonella serovar was considered as a species. Since the host-specificity suggested by some of these earlier names does not exist (e.g., S. typhi-murium, S. cholerae-suis are in fact ubiquitous), names derived from the geographical origin of the first isolated strain of the newly discovered serovars were next chosen, e.g., S. london, S. panama, S. stanleyville. Subsequently it was found that all Salmonella serovars form a single DNA hybridization group, i.e., a single species composed of seven subspecies, and the nomenclature had to be adapted. To avoid confusion with the familiar names of serovars, the species name Salmonella enterica was proposed with the following names for the subspecies:

enterica I

salamae II

arizonae IIIa

diarizonae IIIb

houtenae IV

bongori V

indica VI

Each subspecies contains various serovars defined by a characteristic antigenic formula. Since this formal Latin nomenclature may not be clearly understood by physicians and epidemiologists, who are the most familiar with the names given to the most common serovars, the common serovars names are kept for subspecies I strains, which represent more than 99.5% of the Salmonella strains isolated from humans and other warm-blooded animals. The vernacular terminology seems preferred in medical practice, e.g., Salmonella ser. Typhimurium (not italicized) or shorter Salmonella (or S.) Typhimurium. Antigenic Structure As with all Enterobacteriaceae, the genus Salmonella has three kinds of major antigens with diagnostic or identifying applications: somatic, surface, and flagellar. Somatic (O) or Cell Wall Antigens Somatic antigens are heat stable and alcohol resistant. Cross-absorption studies individualize a large number of antigenic factors, 67 of which are used for serological identification. O factors labeled with the same number are closely related, although not always antigenically identical. Surface (Envelope) Antigens Surface antigens, commonly observed in other genera of enteric bacteria (e.g., Escherichia coli and Klebsiella), may be found in some Salmonella serovars. Surface antigens in Salmonella may mask O antigens, and the bacteria will not be agglutinated with O antisera. One specific surface antigen is well known: the Vi antigen. The Vi antigen occurs in only three Salmonella serovars (out of about 2,200): Typhi, Paratyphi C, and Dublin. Strains of these three serovars may or may not have the Vi antigen.

Flagellar (H) Antigens Flagellar antigens are heat-labile proteins. Mixing salmonella cells with flagella-specific antisera gives a characteristic pattern of agglutination (bacteria are loosely attached to each other by their flagella and can be dissociated by shaking). Also, antiflagellar antibodies can immobilize bacteria with corresponding H antigens. A few Salmonella entericaserovars (e.g., Enteritidis, Typhi) produce flagella which always have the same antigenic specificity. Such an H antigen is then called monophasic. Most Salmonella serovars, however, can alternatively produce flagella with two different H antigenic specificities. The H antigen is then called diphasic. For example, Typhimurium cells can produce flagella with either antigen i or antigen 1,2. If a clone is derived from a bacterial cell with H antigen i, it will consist of bacteria with i flagellar antigen. However, at a frequency of 10

-3- 10

-

5, bacterial cells with 1,2 flagellar antigen pattern will appear in this clone.

Figure 2. Flagellar stain of a Salmonella Typhi. Like E. coli, Salmonella are motile by means of peritrichous flagella. A close relative that causes enteric infections is the bacterium Shigella. Shigella is not motile, and therefore it can be differentiated from Salmonella on the bais of a motility test or a flagellar stain. (CDC) Habitats The principal habitat of the salmonellae is the intestinal tract of humans and animals. Salmonella serovars can be found predominantly in one particular host, can be ubiquitous, or can have an unknown habitat. Typhi and Paratyphi A are strictly human serovars that may cause grave diseases often associated with invasion of the bloodstream. Salmonellosis in these cases is transmitted through fecal contamination of water or food. Gallinarum, Abortusovis, and Typhisuis are, respectively, avian, ovine, and porcine Salmonella serovars. Such host-adapted serovars cannot grow on minimal medium without growth factors (contrary to the ubiquitous Salmonella serovars). Ubiquitous (non-host-adapted) Salmonella serovars (e.g., Typhimurium) cause very diverse clinical symptoms, from asymptomatic infection to serious typhoid-like syndromes in infants or certain highly susceptible animals (mice). In human adults, ubiquitous Salmonella organisms are mostly responsible for Foodborne toxic infections. The pathogenic role of a number of Salmonella serovars is unknown. This is especially the case with serovars from subspecies II to VI. A number of these serovars have been isolated rarely (some only once) during a systematic search in cold-blooded animals. Salmonella in the Natural Environment Salmonellae are disseminated in the natural environment (water, soil, sometimes plants used as food) through human or animal excretion. Humans and animals (either wild or domesticated) can excrete Salmonella either when clinically diseased or after having had salmonellosis, if they remain carriers. Salmonella organisms do not seem to multiply significantly in the natural environment (out of digestive tracts), but they can survive several weeks in water and several years in soil if conditions of temperature, humidity, and pH are favorable. Isolation and Identification of Salmonella A number of plating media have been devised for the isolation of Salmonella. Some media are differential and nonselective, i.e., they contain lactose with a pH indicator, but do not contain any inhibitor for non salmonellae (e.g., bromocresol purple lactose agar). Other media are differential and slightly selective, i.e., in addition to

lactose and a pH indicator, they contain an inhibitor for nonenterics (e.g., MacConkey agar and eosin-methylene blue agar). The most commonly used media selective for Salmonella are SS agar, bismuth sulfite agar, Hektoen enteric (HE) medium, brilliant green agar and xylose-lisine-deoxycholate (XLD) agar. All these media contain both selective and differential ingredients and they are commercially available.

Figure 3. Salmonella sp. after 24 hours growth on XLD agar. Xylose Lysine (XL) agar is used when trying to culture and isolate Gram-negative enteric bacilli. When XL agar is supplemented with sodium thiosulfate, ferric ammonium citrate, and sodium deoxycholate, it is then termed XLD agar, and is then an even more selective medium than XL alone. The presence of any black colored area indicates the deposition of hydrogen sulfide, (H2S) under alkaline conditions. (CDC) Media used for Salmonella identification are those used for identification of all Enterobacteriaceae. Most Salmonella strains are motile with peritrichous flagella, however, nonmotile variants may occur occasionally. Most strains grow on nutrient agar as smooth colonies, 2-4 mm in diameter. Most strains are prototrophs, not requiring any growth factors. However, auxotrophic strains do occur, especially in host-adapted serovars such as Typhi and Paratyphi A.

Figure 4. Colonial growth Salmonella choleraesuis subsp. arizonae bacteria grown on a blood agar culture plate. Also known as Salmonella Arizonae, it is a zoonotic bacterium that can infect humans, birds, reptiles, and other animals. (CDC)

Figure 5. Colonial growth pattern displayed by Salmonella Typhimurium cultured on a Hektoen enteric (HE) agar. S. Typhimurium colonies grown on HE agar are blue-green in color indcating that the bacterium does not ferment lactose However it does produce hydrogen sulfide, (H2S), as indicated by black deposits in the centers of the colonies. (CDC) HE agar is the medium designed for the isolation and recovery of fecal bacteria belonging to the family, Enterbacteriaceae. S.Typhimurium causes 25% of the 1.4 million salmonellosis infections a year in the United States. Most persons infected with Salmonella sp. develop diarrhea, fever, and abdominal cramps 12 - 72 hours after infection. The illness usually lasts 4 - 7 days, and most people recover without treatment. However, in some cases, the diarrhea may be so severe that the patient needs to be hospitalized. Pathogenesis of Salmomella Infections in Humans Salmonella infections in humans vary with the serovar, the strain, the infectious dose, the nature of the contaminated food, and the host status. Certain serovars are highly pathogenic for humans; the virulence of more rare serovars is unknown. Strains of the same serovar are also known to differ in their pathogenicity. An oral dose of at least 10

5Salmonella Typhi cells are needed to cause typhoid in 50% of human volunteers, whereas at least

109 S. Typhimurium cells (oral dose) are needed to cause symptoms of a toxic infection. Infants,

immunosuppressed patients, and those affected with blood disease are more susceptible to Salmonella infection than healthy adults. In the pathogenesis of typhoid the bacteria enter the human digestive tract, penetrate the intestinal mucosa (causing no lesion), and are stopped in the mesenteric lymph nodes. There, bacterial multiplication occurs, and part of the bacterial population lyses. From the mesenteric lymph nodes, viable bacteria and LPS (endotoxin) may be released into the bloodstream resulting in septicemia Release of endotoxin is responsible for cardiovascular “collapsus and tuphos” (a stuporous state—origin of the name typhoid) due to action on the ventriculus neurovegetative centers. Salmonella excretion by human patients may continue long after clinical cure. Asymptomatic carriers are potentially dangerous when unnoticed. About 5% of patients clinically cured from typhoid remain carriers for months or even years. Antibiotics are usually ineffective on Salmonella carriage (even if salmonellae are susceptible to them) because the site of carriage may not allow penetration by the antibiotic. Salmonellae survive sewage treatments if suitable germicides are not used in sewage processing. In a typical cycle of typhoid, sewage from a community is directed to a sewage plant. Effluent from the sewage plant passes into a coastal river where edible shellfish (mussels, oysters) live. Shellfish concentrate bacteria as they filter several liters of water per hour. Ingestion by humans of these seafoods (uncooked or superficially cooked) may cause typhoid or other salmonellosis. Salmonellae do not colonize or multiply in contaminated shellfish. Typhoid is strictly a human disease.The incidence of human disease decreases when the level of development of a country increases (i.e., controlled water sewage systems, pasteurization of milk and dairy products). Where these hygienic conditions are missing, the probability of fecal contamination of water and food remains high and so is the incidence of typhoid. Foodborne Salmonella toxic infections are caused by ubiquitous Salmonella serovars (e.g., Typhimurium). About 12-24 hours following ingestion of contaminated food (containing a sufficient number of Salmonella), symptoms appear (diarrhea, vomiting, fever) and last 2-5 days. Spontaneous cure usually occurs. Salmonella may be associated with all kinds of food. Contamination of meat (cattle, pigs, goats, chicken, etc.) may originate from animal salmonellosis, but most often it results from contamination of muscles with the

intestinal contents during evisceration of animals, washing, and transportation of carcasses. Surface contamination of meat is usually of little consequence, as proper cooking will sterilize it (although handling of contaminated meat may result in contamination of hands, tables, kitchenware, towels, other foods, etc.). However, when contaminated meat is ground, multiplication of Salmonella may occur within the ground meat and if cooking is superficial, ingestion of this highly contaminated food may produce a Salmonella infection. Infection may follow ingestion of any food that supports multiplication of Salmonella such as eggs, cream, mayonnaise, creamed foods, etc.), as a large number of ingested salmonellae are needed to give symptoms. Prevention of Salmonella toxic infection relies on avoiding contamination (improvement of hygiene), preventing multiplication of Salmonella in food (constant storage of food at 4°C), and use of pasteurized and sterilized milk and milk products. Vegetables and fruits may carry Salmonella when contaminated with fertilizers of fecal origin, or when washed with polluted water. The incidence of foodborne Salmonella infection/toxication remains reletavely high in developed countries because of commercially prepared food or ingredients for food. Any contamination of commercially prepared food will result in a large-scale infection. In underdeveloped countries, foodborne Salmonella intoxications are less spectacular because of the smaller number of individuals simultaneously infected, but also because the bacteriological diagnosis of Salmonella toxic infection may not be available. However, the incidence of Salmonella carriage in underdeveloped countries is known to be high. Salmonella epidemics may occur among infants in pediatric wards. The frequency and gravity of these epidemics are affected by hygienic conditions, malnutrition, and the excessive use of antibiotics that select for multiresistant strains. Exotoxins Salmonella strains may produce a thermolabile enterotoxin that bears a limited relatedness to cholera toxin both structurally and antigenically. This enterotoxin causes water secretion in rat ileal loop and is recognized by antibodies against both cholera toxin and the thermolabile enterotoxin (LT) of enterotoxinogenic E. coli, but it does not bind in vitro to ganglioside GM1 (the receptor for E. coli LT and cholera ctx). Additionally, a cytotoxin that inhibits protein synthesis and is immunologically distinct from Shiga toxin has been demonstrated. Both of these toxins are presumed to play a role in the diarrheal symptoms of salmonellosis. Antibiotic Susceptibility During the last decade, antibiotic resistance and multiresistance of Salmonella spp. have increased a great deal. The cause appears to be the increased and indiscriminate use of antibiotics in the treatment of humans and animals and the addition of growth-promoting antibiotics to the food of breeding animals. Plasmid-borne antibiotic resistance is very frequent among Salmonella strains involved in pediatric epidemics (e.g., Typhimurium, Panama, Wien, Infantis). Resistance to ampicillin, streptomycin, kanamycin, chloramphenicol, tetracycline, and sulfonamides is commonly observed. Colistin resistance has not yet been observed. Until 1972, Typhi strains had remained susceptible to antibiotics, including chloramphenicol (the antibiotic most commonly used against typhoid) but in 1972, a widespread epidemic in Mexico was caused by a chloramphenicol-resistant strain of S. Typhi. Other chloramphenicol-resistant strains have since been isolated in India, Thailand, and Vietnam. Possible importation or appearance of chloramphenicol-resistance strains in the United States is a real threat. Salmonella strains should be systematically checked for antibiotic resistance to aid in the choice of an efficient drug when needed and to detect any change in antibiotic susceptibility of strains (either from animal or human source). Indiscriminate distribution and use of antibiotics should be discouraged. E. coli E. coli is a member of a group of micro organisms known as the enterobacteria, so called because they inhabit the intestines of humans and animals. It is a common infectant of the urinary tract and bladder in humans, and is a cause of pyelitis, pyelonephritis and cystitis. (Hugo, W.B. and A.D. Rusell, 1998) Escherichia coli is an emerging cause of food borne illness. Infection can also occur after drinking raw milk and after swimming in or drinking sewage-contaminated water. Most strains are harmless and live in the intestines of healthy humans and animals E. coli infection often causes severe bloody diarrhea and abdominal cramps; sometimes the infection causes nonbloody diarrhea or no symptoms. Usually little or no fever is present, and the illness resolves in 5 to 10 days. In some persons, particularly children under 5 years of age and the elderly, the infection can also cause a complication called hemolytic uremic syndrome, in which the red blood cells are destroyed and the kidneys fail. About 2%-7% of infections lead to this complication. In the United States, hemolytic uremic syndrome is the principal cause of acute kidney failure in children, and most cases of hemolytic uremic syndrome are caused by E. coli

Pseudomonas aeruginosa Pseudomonas aeruginosa is a Gram-negative bacterium that is noted for its environmental versatility, ability to cause disease in particular susceptible individuals, and its resistance to antibiotics. The most serious complication of cystic fibrosis is respiratory tract infection by the ubiquitous bacterium Pseudomonas aeruginosa. Cancer and burn patients also commonly suffer serious infections by this organism, as do certain other individuals with immune systems deficiencies. Unlike many environmental bacteria, P. aeruginosa has a remarkable capacity to cause disease in susceptible hosts. It has the ability to adapt to and thrive in many ecological niches, from water and soil to plant and animal tissues. The bacterium is capable of utilizing a wide range of organic compounds as food sources, thus giving it an exceptional ability to colonize ecological niches where nutrients are limited. P. aeruginosa can produce a number of toxic proteins which not only cause extensive tissue damage, but also interfere with the human immune Msystem's defense mechanisms. These proteins range from potent toxins that enter and kill host cells at or near the site of colonization to degradative enzymes that permanently disrupt the cell membranes and connective tissues in various organs. This bacterium is also noted for its resistance to many antibiotics and is biochemically very versatile, being able to use many disinfectants as food sources. (Hugo, W.B. and A.D. Rusell, 1998) Staphylococcus Staphyloccocus is a gram negative cocci. The spheres grow in aggregates which have been likened to a bunch of grapes. The organisms are none motile and none sporing;they can grow aerobically or anaerobically. Staphyloccocus aureus produces a golden yellow pigment. It is a cause of skin lesions such as boils, and can affect bone tissue in the case staphylococcal osteomyelitis. It produces a toxin which, if ingested with food in which the organism has been growing, can give rise to food poisoning. A common manifestation of its infection is the production of pus, i.e. the organism is pyogenic. Other common infections associated with staphylococcal infections are styes, impetigo and conjunctivitis(Hugo, W.B. and A.D. Rusell, 1998) Micro-Organisms and Microbial Toxins Microbial quality is an important issue. Materials of vegetal origin tend to show much higher levels of microbial contamination than synthetic materials. Thus, the requirements for microbial contamination in the European Pharmacopoeia allow higher levels of microbial contamination in herbal remedies than in synthetic pharmaceuticals. The European Pharmacopoeia also takes into account that the processing of the crude herb can reduce the number of microbes by allowing a higher contamination in herbal remedies to which boiling water is added before use (Anonymous 1996). The European Pharmacopoeia also specifies that Escherichia coli and Salmonella spp. should be absent (Anonymous 1996). The literature shows, however, that it is not always these two pathogenic bacteria which are causing clinical problems. The New England Journal of Medicine published a fatal case of listeriosis that could be traced to contamination of alfalfa tablets with the Gram-positive bacillus, Listeria monocytogenes (Faber J.M. 1996). Surprisingly, there has been a recent clinical report about viral contamination of an herbal remedy. Japanese researchers observed a man with acute hepatitis E (which is transmitted via the faecal/oral route) and they showed by nucleotide sequencing that he had become infected with the Chinese strain of the virus. The man had traveled for a few days to China four months prior to the onset of his symptoms, which is much longer than the average incubation period of six weeks. Detailed interviewing revealed that he had not come into contact with any persons with jaundice, but he had bought a liquid herbal medicine in China and had taken this medicine occasionally during the six weeks before his jaundice became manifest. Since no alternative causes could be found, the herbal medicine was deemed to be the most likely transmitter of the hepatitis E (Ishikawa k, Matsui k, Madarame T, Sato S, Oikawa K, Uchida T, 1995). In addition to the risk of bacterial and viral contamination, herbal remedies may also be contaminated with microbial toxins, such as bacterial endotoxins and mycotoxins. For instance, high levels of bacterial endotoxins could be demonstrated in the 1970s after United States Customs Services seized Mexican ampoules containing amygdalin (Laetrile) (De Smit PAGM 1992). German researchers assayed the bacterial and endotoxin content of four small-volume phytotherapeutical injection fluids on the West German market after the use of such preparations had been associated with pyrogen-like side effects. None of the studied samples showed bacterial contamination, but each of the four brands tested had batches with substantial endotoxin levels. Their findings most certainly illustrate that parenteral phytotherapeutical preparations should be controlled for the absence of pyrogens, even when their volume does not exceed 15 ml ( De Smit PAGM 1992). Care should be taken, however, to avoid false results. It has been shown that the lectins in mistletoe preparations from Viscum album cause false positive limulus amebocyte lysate tests, resulting in falsely high contents of endotoxins ( Scheer R 1993). Not only are bacterial toxins important, but for botanicals, fungal toxins may also be at issue. There is evidence, for instance, that medicinal plants from India and Sri Lanka can be contaminated with toxigenic fungi (Aspergillus,

Fusarium). Since aflatoxin B has sometimes been recovered from such materials in potentially unsafe amounts, it would certainly be prudent to improve their storage conditions (De Smit PAGM 1992). A last and unusual microbial health hazard of herbal products is that certain plant constituents are susceptible to chemical transformation by contaminating micro-organisms. An example is that the moulding of dried sweet clover (Melilotus officinalis) can result in serious hemorrhagic activity. This medicinal herb contains coumarin, 3,4-dihydrocoumarin (= melilotine), o-coumaric acid, o-hydroxycoumaric acid, and the O-glycoside of o-coumaric acid (= melilotoside). Since withering leads to enzymatic glycoside hydrolysis and the resulting o-coumaric acid are spontaneously transformed to coumarin, the dried herb strongly smells of coumarin. Coumarin itself is devoid of anticoagulant effects in man, because an intact 4-hydroxycoumarin residue with a carbon constituent at the 3-position are essential characteristics for the anticoagulant potential of coumarin derivatives. Coumarin can be metabolized, however, by Penicillium moulds, such as P.nigricans and P.jensi, and the transformation product dicoumarol is a 4-hydroxycoumarin derivative with potent anticoagulant effects (De SMIT pagm 1992). Although poisoning by moulded sweet clover has been observed primarily in cattle, there is an unusual case report about abnormal clotting function and mild clinical bleeding in a young woman who had been drinking large amounts of an herbal tea prepared from sweet clover and other coumarincontaining ingredients (Hogan III R.P. 1983). Thus, this may not be just a theoretical problem. Aim

To analyse liquid preparations in respect to microbial contamination. Objectives

To find out the total viable aerobic count(sum of the bacterial count and the fungal count) in various antacid liquid preparations.

To compare the total viable aerobic count with the standard values given in the USP.

To find out the specific type of bacteria found in the liquid antacids(ie.Staphyloccocus aureus, Pseudomonas aeruginosa, Eschericia coli,Salmonella spp)

To check the labeling on the containers. Methodology / Material Antacid suspension: Liquid antacid preparation of various companies having different manufacturing date were purchased from various drug stores of Lusaka. Samples were checked for their batch number, production and expiry date. Culture media: Before culturing in selective media, the microorganisms were grown in Casein soybean digest broth or lactose monohydrate broth depending on the type of bacteria to be detected. Enterobacteriaceae enrichment broth-mossel (EE broth) was used to enhance the growth of bacteria belonging to Enterobacteriaceae such as Escherichia coli, Salmonella spp. and Pseudomonasa aeruginosa. Eosin methylene blue was used to detect the presence of gram negative bacteria. Triple Iron Sugar, Lysine Iron Agar, Urease, Citrare and Sulphide and Motility was used for the identification of Escherichia coli. Biochemical tests were performed for primary identification of salmonella. Sallmonela antisera was used as confirmatory test. Oxidase reagent was used for primary identification of Pseudomonas aeruginosa.* Pseudomonas aeruginosa is oxidase positive(purplish colour).Then biochemical tests were performed. Catalase was used to rule out Streptococcus. All Staphylococcus are catalase positive to rule out Streptococcus. Coagulase test was done since Staphylococcus aureus is the only one which is coagulase positive. MSN (Manitol Salt Agar ) Staphylococcus aureus is the only one which can ferment manitol(appearance of colonies is golden yellow in the culture plate). Triple sugar iron agar and Motility Indole Urea (MIU) agar media were used to identify all the above mentioned microorganisms except Staphylococcus aureus. Organisms: Standard organisms such as Salmonella typhi, Escherichia coli Staphylococcus aureus and Pseudomonas aeruginosa were used as controls for testing against the expected bacteria. Quantitative enumeration of mesophilic bacteria and fungi Preparation of the sample suspension: Prior to bacteriological study, the inhibitory effects of antimicrobial agents used as preservatives were removed by dilution of the antacids with buffered sodium chloride peptone solution(Rawlings,E.A.,1996) .

Ten milliliter of liquid antacid was added to 90 mL of sterile buffered sodium chloride peptone solution pH 7.0 in100 mL conical flasks. Detection of total microbial count: The present total microbial count were performed by pour plate method. Using petridishes of 9 cm diameter 1 mL of the diluted sample (prepared as described above) was added to each Petri dish previously sterilized in hot-air oven. For each sample plating was done in duplicate. The average value obtained was multiplied by the dilution factor to get the total microbial count. Approximately 15 to 20 mL of the liquified Soyabean Casein Digest (SCD) agar and Czapek-Dox medium with antibiotic was added to each petridishes at 46°C. Samples with SCD agar and CDM were incubated at 37 and at 25°C, respectively. Both media were incubated for 5 days. Two petri dishes for each level of dilution were prepared for each medium. Samples were diluted 100 or 1000 or more times depending on number of Colony Forming Units (CFU) observed in the plate. Arithmetic average of the counts were taken and the number of colony forming units per milliliter calculated. Detection of specified bacteria: Ten milliliter of liquid antacid preparation was added to 90 mL of lactose monohydrate broth, homogenized and incubated at37ºC to revive the bacteria for about 18-24 h. After incubation the conical flask was shaken well and 1 mL of the product transferred to 100 mL of Enterobacteria enrichment broth-mossel (EE broth) and incubated at37°C for 48 h. After incubation, it was subcultured on Eosin methylene blue, plate and was incubated at 37°C for 24 h. Negative growth indicated the product was not contaminated by gram negative bacteria. Test for the detection of specific bacteria: Tests for the detection of Escherichia coli, Salmonella spp., was done according to the microbiological standard procedures (Anonymous,1998). Interpretation of results: The bacterial number was considered to be equal to the average number of Colony Forming Units (CFU) found on Soyabean Casein Digest agar (SCD). The fungal counts will be considered to be equal to the average number of colony forming units on Czapek-Dox medium. The total viable aerobic count was the sum of the bacterial count and the fungal count. The growth of identical microorganisms in different media was counted once. Sample Size 12 samples manufactured by various pharmaceutical companies were collected from various outlets in Lusaka. In this study microbiological quality of 12 samples of various pharmaceutical industries was investigated. Proper sealing of containers is essential from the microbiological point of view and sealing of the containers of all the companies were satisfactory. Manufacturing and expire date were printed in case of all the companies. The microbial quality of pharmaceutical products is influenced by the environment in which they are manufactured and by the materials used in their formulations. Marked spoiled pharmaceuticals can often yield surprisingly low numbers of spoilage organism. To get the real picture of the antacid quality, samples were collected from various parts of the city. Aerobic viable microbial count of 3 (25%) out of 12 samples studied were found to exceed the USP limit. Viable microbial counts varied between less than 10 CFU per mL and 1000 CFU per mL (Table 1). To avoid any false reading both positive (medium inoculated with standard microorganisms) and negative control (medium only) were used. Since media composition and incubation temperature greatly influence the resultant microbial counts, media used to detect the total microbial count were nutritious. It is not possible to detect 100% microorganisms present in the sample because considerable variation in estimated microbial count may be obtained from the same sample using different media or even different batches of the same medium. Most oral liquid medicines are prepared as mixtures, suspensions, syrups or emulsions. These often contain ingredients which readily support the growth of a variety of microorganisms if appropriate precautions are not taken.( Baird, R.M. and P.S. Petris, 1981. Kallings, L.O., O. Ringertz, L. Silverstolpe and F. Ernerfeldt, 1996).Particularly, liquid antacids containing neutral pH which is processed under partial asceptic conditions, is highly susceptible to microbial contamination. In this study it was observed that the number of fungal colonies were much lower those of bacterial colonies. This may be due to less number of fungal contamination in the samples studied. Another reason could be the preservative commonly used in antacid such as methyl paraben and propyl paraben act against wide range of fungi but less effective against bacteria. Moreover, parabens are active at pH between 7 and 9.( Hugo, W.B and A.D. Rusell, 1983 ),inappropriate use of preservatives and formulations of the drug may be responsible the high microbial count. Absence of indicator microorganisms( Pseudomonas aeruginosa, Eschericia coli, Staphylococcus aureus and Salmonella spp). is an absolute requirement However, 5 samples contained coagulase positive Staphylococcus aureus and none of the samples was found to contain Pseudomonas aeruginosa (Table 2). 2 of the 12 samples were found to contain Escherichia coli and 1 of the 12 samples contained Salmonella. Presence of Escherichia coli and Salmonella spp. in some of the samples studied indicates that the water used by those pharmaceutical industries may be contaminated by coliform

bacteria. The organisms of these types are water borne and frequently contaminate liquid pharmaceutical product (Sykes, G., 1971.). Moderate to heavy contamination by microbes in the liquid preparations of the 25% of the samples studied indicate that the excipients used might be contaminated or the antacid preparation room were not satisfactorily cleaned. Antacid suspension acts as weak bases and as it contains organic components, it is highly susceptible to microbial contamination. As a result, it is necessary to add anti-microbial preservatives in the formulation process. Inadequate preservation may lead to the microbes getting exposed to sub-lethal concentration of preservatives and develop resistant variants (Gilbert, P. and N. Wright, 1987. ) However, high concentration may prove to be toxic for consumer’s health. Therefore, it is necessary to determine the appropriate preservation conditions for efficacy and usefulness of the product during its storage period. Lower microbial count in the antacid samples of some companies than those of many other companies does not necessarily indicate that the samples with less microbial count are better. It is important to note which preservatives they use. Use of toxic preservatives to reduce the microbial load is not acceptable. Rather to reduce microbial load in the antacid suspension, the ingredients must be examined to determine whether there is any pathogenic microbe and the microbial count within acceptable limit. Preservative should not be expected to sterilize formulations that are heavily contaminated as a result of low quality raw materials and poor manufacturing procedures (Hugo, W.B and A.D. Rusell, 1983) Maintenance of sterility in liquid antacid preparation is a common problem in many settings. In this study also it appears to be a problem. Although fewer numbers of samples have been analyzed without recourse of statistical design, the results indicate the antacid manufacturing conditions and or the preservative(s) used were not effective. Study can be extended in the laboratory setting to determine appropriate preservative(s), its level and conditions that may be required under the conditions of production. Constraints

Far reaching sample places were not included in the study

Some outlets were not reached out.

The apparatus capacity was a limiting factor for limited size of the samples considered.

The cost of some samples was a limiting factor to consider all the available study material Conclusion All the samples were labeled with the batch number, manufacturing date and expiry date except for samples 3, 4,11and12. Some of the samples analysed were found to contain bacteria alone or fungi alone while others contained both bacteria and fungi. The levels of contamination ranged from 1CFU per ml to 400CFU per ml for fungi. However, only 2 samples contained fungal counts above the USP Limit. Bacterial contamination ranged from 3CFU per ml to 500CFU per ml. However, only 3 samples had a contamination above the USP Limit. The total viable aerobic count of 25% of the samples exceeded the USP Limit. The actual point at which the suspensions got contaminated could not be established from the methodology and was not part of the study. However, the results suggested that a significant percent of the products on the market are contaminated by microorganisms and considerations should be made to ensure that the qualities of substances entering the country are checked by laboratory procedures. The actual organisms found in the samples were E.coli, Salmonella, and staphylococcus aureus. These organisms were only representative of the many other organisms, which could have been there but were not investigated. Recommendations

The study should be extended be extended to laboratory setting in order to determine appropriate preservatives.

The government should find ways in which medicines entering the country will be thoroughly investigated in order to reduce the health hazards posed by microbial contamination as well as other adulterants.

Site of the Project

The project was conducted in Lusaka at University of Zambia School of Natural Sciences Biological Sciences Department.

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