Pharmaceutical microbiology may be defined as that part of microbiology that has special applications in the pharmaceutical field. This is one of the branches of applied microbiology. Applications of microbiology in the pharmaceutical area are described as follows:
Scope and Importance of Microbiology
(i) Production of antibiotics:
Today 2/3rd of antibiotics are produced from microorganisms. Pharmaceutical or industrial microbiology concerns itself with the isolation of antibiotic-producing microorganisms from natural environments such as soil or water. Many antibiotics are isolated from natural microorganisms by the process of fermentation. e.g. Penicillin from Penicillium species, Streptomycin from Streptomyces griseus, Tetracycline from Streptomyces aureofaciens, Chloramphenicol from Streptomyces venezuelae etc.
(ii) Production of enzymes, vaccines and other pharmaceutical products:
The detection and assay of pharmaceutical and chemical products of microbial activity is a part of pharmaceutical microbiology. Many microbial cells convert raw materials or substrates into valuable organic compounds such as butanol, ethanol, acetone etc. Microbial cells also produce intracellular and extracellular enzymes and these enzymes are important for the success of pharmaceutical fermentation processes. They are associated with the microorganism’s ability to attack, degrade and utilize components of the medium and to accumulate fermentation products e.g. amylases, proteases, lipases, invertases, oxidase, catalase, cortisone reductase, phospholipases, cellulases, chitinases etc. Biosurfactants have a lot of applications in agriculture, food industries, industrial cleaning, leather, paper and metal industries, textiles, cosmetics and the pharmaceutical industry. Different types of biosurfactants are synthesized by several microorganisms e.g. Acinetobacter species, Bacillus species, Pseudomonas species, Rhodococcus species etc.
(iii) Diagnosis of diseases and treatment:
Different tests are used to detect infectious microorganisms e.g. ELISA, and Widal test. Antimicrobial susceptibility testing is mainly used for the selection of antibiotics for the treatment of microbial infections.
(iv) Treatment of industrial waste material:
Most industrial processes produce waste water, salts, organic matter and spent media and these may be toxic. Many microbial species are used for the decomposition of such waste materials and organic components e.g. actinomycetes, fungi, protozoa etc. Anaerobic bacteria in anaerobic digestion tanks are active in the decomposition of organic matter by yielding simple organic molecules such as acids, alcohols, glycerol and amines. Cyanide is produced on an industrial scale for use in metal extraction, electroplating and the steel industry which is degraded by Streptomyces Lavandula.
(v) Plant growth promotion:
Many microbial cells present in the soil, play an important role in soil fertility, herbicidal resistance, insect resistance, change in protein/oil content and enhanced quality of plant products e.g. Rhizobium species, Rhodospirillum species, Azotobacter species, Agrobacterium rhizogenes, Pseudomonas species, Acetobacter species etc. Many nitrogen-fixing soil microorganisms are useful for providing nitrogen for the growth of crops and enzymes released from microorganisms are helpful for plant growth, germination of seeds, flavouring and fruiting of medicinal plants. Soil microbial populations are the key element in recycling plant nutrients and thus they play a major role in the maintenance of soil fertility and soil health. The rhizospheric bacteria may release various plant growth-promoting substances as secondary metabolites, which may promote plant growth directly or indirectly. Such beneficial rhizospheric bacteria are often referred to as plant growth-promoting rhizobacteria (PGPR). Pseudomonas is one of the extensively studied PGPR, which promotes the growth of plants by protecting plant health from soil-borne phytopathogens and their toxins. Azotobacter chroococcum is a well-known free living nitrogen fixing bacterium capable of synthesizing and secreting plant growth-promoting substances like thiamine, riboflavin, IAA, gibberellin etc. and is frequently used as a nitrogenous biofertilizer for several crops.
(vi) Sterile product preparation:
Pharmaceutical microbiology plays a major role in the preparation of sterile products. It deals with the preparation of sterile rooms, aseptic techniques, detection of microbes by sampling and sterility testing of different sterile preparations. Sterility testing is carried out to support batch releases of sterile pharmaceuticals and medical devices. Settle plates, contact plates and air sampling techniques are used for the detection of microorganisms in sterile rooms.
Different pharmaceutical products are sterilized by killing microorganisms. This process of killing microbes is called sterilization. Moist heat sterilization, dry heat sterilization, membrane filtration, gaseous sterilization and chemical sterilization are the methods used for killing microorganisms. Methods of sterilization are decided to depend on the properties of products and the general type of microbes present in the products.
All the sterilization processes may be validated by using biological indicators. A biological indicator (BI) is a preparation of microorganisms usually bacterial spores that are contained in filter papers, porcelain cylinders or test tubes which serves as a challenge to the effectiveness of a given sterilization process or cycle e.g. Moist heat sterilization (autoclave) – Bacillus stearothermophilus, Dry heat sterilization (hot air oven) – Bacillus subtilis, Radiation sterilization – Bacillus pumilis, Membrane filtration – Pseudomonas diminuta etc.
(viii) Steroid biotransformation:
Steroids are physiologically active compounds of complex structures e.g. cholesterol, ergosterol, testosterone, progesterone etc. Important steroids can be produced by microbiological transformations of naturally occurring steroids. Microbial conversions of steroids are brought about by enzymes secreted by selected strains of microorganisms e.g. Streptomyces species, Rhizopus species, Aspergillus species, and Penicillium species.
(ix) Identification of microorganisms:
Two vital functions of pharmaceutical microbiology and biotechnology are the enumeration and identification of microorganisms found in products and the manufacturing environment. There are many microbial cells present in the environment that are useful for the production of antibiotics, enzymes and other pharmaceutical substances. These microorganisms are isolated and identified by morphological, biochemical, cultural, and microscopic characteristics and genetic studies.
(x) Testing of pharmaceuticals:
(a) Raw material and finished products: Tests for the detection of Escherichia coli, Salmonella species, Pseudomonas aeruginosa and Staphylococcus aureus in pharmaceutical raw materials and finished products are described in the Indian Pharmacopoeia (IP), United States Pharmacopoeia (USP), European Pharmacopoeia (EP) and British Pharmacopoeia (BP). These organisms are given special attention because they represent particular infection hazards to the patient or their presence is a criterion for the quality of raw materials. This test is mainly applied to raw materials of natural or biological origin such as starch, gum, gelatin, talc etc. The microbiological quality of non-sterile pharmaceutical or cosmetic materials can be controlled by the adoption of one of the two types of standards or both standards. The first is a limit on the total number of viable micro-organisms in a given product (TVC, total viable count) and the second is the exclusion of specific pathogens. Water is always tested for the total microbial count and the presence of Escherichia coli.
(b) Microbiological assays of antibiotics: The antibiotic bioassay provides potency for the overall biological activity of an antibiotic preparation. Activity or potency is quoted in terms of units of a recognised international standard, specifically defined by pharmacopoeias. Agar diffusion (plate assays) and turbidometric assays permit an estimate of antibiotic potency through direct comparison of a test antibiotic with an approved, well-calibrated reference substance. Test microorganisms are used for the test that is non-pathogenic, sensitive to the action of antibiotics and capable of rapid growth.
(c) Evaluation of disinfectants: Effective cleaning and disinfection of manufacturing facilities are crucial to the achievement and maintenance of high-quality standards required for medicine and medical devices. The selection of cleaning agents (detergents) and disinfectants will be influenced by the range and composition of the materials, number and types of microorganisms, maximum residual levels acceptable on completion etc. The phenol coefficient test, cup plate method, turbidometric method and Kelsey Sykes test are mainly used for the evaluation of different disinfectants.
(d) Antimicrobial preservative efficacy testing: Different types of pharmaceutical products and cosmetics are protected from attack by microorganisms by using preservatives. This is to protect the user from dangerous infections and to prevent spoilage and deterioration of a product. Preservatives are intended to protect a product from spoilage due to microorganisms introduced by the user and those which unavoidably arise during the manufacturing process. There is an extensive range of chemical substances available for potential use in preservative systems. The preservatives should be non-toxic, easily available and have broad-spectrum activity. Cup-plate method, turbidometric method and total viable counting techniques are used for the determination of the antimicrobial efficacy of preservatives.
(e) Endotoxin testing: Bacterial endotoxin or pyrogens have several physiological effects following intravenous injections such as fever, activation of the cytokine system, endothelial cell damage and intravascular coagulation. The bacterial endotoxins test (BET) is most important in microbiology laboratories involved with the quality control of parenterals and medical devices. LAL test, gel-clot method, chromogenic end-point, kinetic turbidometric assay and kinetic chromogenic assay are used for the detection of endotoxins.
(f) Tests for support of the sterility assurance system: Sterility testing includes many of the basic principles applicable in running an aseptic area and microbiology laboratory that deal with the testing in support of the sterility assurance system. Sterility testing is mainly performed for parenteral preparations and other sterile pharmaceuticals to detect the presence of microorganisms. The interpretation of results from sterility testing can be confused by the risk of microbiological contamination during the test and the masking of a genuine non-sterile unit. Sterility tests are mainly performed in laminar air flow cabinets following all aseptic precautions. Bioburden testing is used to estimate the number of microorganisms in the product before sterilization. Pre-sterilization counts carried out in support of specific sterilization processes can involve selective sample treatments. The methods usually used for environmental monitoring in aseptic areas are settled plates, active air sampling, contact plates, and swabs for surfaces and garments. These methods provide an approximate guide to the level of the microorganisms present and contribute to the knowledge of the true microbiological state of a clean room, aseptic area and laminar air flow cabinet or isolator.
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