PHARMACEUTICAL MICROBIOLOGY Subject Code: BP 303T* The word Microbiology was derived from Greek: mikros ("small") + bios ("life") * It is the branch of science that is concerned primarily with the biology of microorganisms and their effects on other living organisms. e.g., Bacteria, Protozoa, Algae, Fungus and Viruses. * Microbiology may be Pure or Applied: » Pure microbiology: Includes bacteriology, mycology, protozoology, phycology, parasitology virology, nematology, cellular microbiology, evolutionary microbiology, molecular microbiology, etc. > Applied microbiology: Includes medical microbiology, pharmaceutical microbiology, industrial obiology, food microbiology, soil microbiology, water microbiology, air microbiology and microbial biotechnology. * When microbiological concepts, processes and techniques are applied to pharmaceutical operations, the subject is then called ‘pharmaceutical microbiology’.* Objectives of pharmaceutical microbiology to ensure safety and efficacy of pharmaceutical products. * It embraces to validation of disinfectants, evaluation of the efficacy of disinfectants in suspension, on surfaces and through field trials. ¢ It offers protocols and techniques associated with the operation and assurance of clean-room, aseptic-room and controlled environments for preventing any possible microbial contamination, and introduces risk assessment and practical contamination control strategies. “Louis Pasteur is regarded as the father of microbiology whereas Robert Koch is the father of medical microbiology.iSiaaohy * 1673-1723, Antony Van Leeuwenhoek (Dutch) described live microorganisms that he observed in teeth scrapings and rain water. Location of specimen Lens * First person to actually see living microorganisms. — pyeuemnyeeens Seas er Cae oft te he SS SS (b) Microscope replicaHistory 1822-1895, Louis Pasteur A French organic chemist Considered as father of modern microbiology Fermentation process: beer/wine not produced without microbes Microbes causes spoilage Demonstrated guncotton experiment Performed swan neck flask experiment Diffrenet places/Diffrenet conc. of microbes Disproved spontaneous generation Developed Pasteurisation process(50- 60°C) Virulence power Developed Rabies/AnthraxaSINol as * 1843-1910, Robert Koch * German physician + Considered as father of medical microbiology * First to demonstrate the role of bi ria in causing di + First to isolate Bacillus anthracis * Discovered Mycobacterium tuberculosis, 1882 and Vibrio cholera, 1883, awarded a Nobel prize in 1905 * Developed relationship b/t microorganism and dis > Koch's postulates: I. The microorganism must be present in every case of disease I. The microorganisms must be isolated from the diseased animal and grown in pure culture II. The disease must be reproduced when a pure culture of the microorganisms is inoculated into a susceptible host IV. The same microorganism must be desolated from the experiment from the experimentally infected hostBranches of Microbiology Protozoology nN , onal Oe er a Microbiology eet Mycology | < Civica}Prokaryotic vs. Eukaryotic Cell Chromosomes : x y Nuclear , Membrane a No eee Internal membranes Prokaryate EukaryoteGlycocalyx Granules Cell Membrane endoplasmic —Ribosome reticulum Mesosomes — Pilus Cell Wall ai Nucleoid Flagellum General structure of Prokaryotic Cells Eukaryotic cellSI.No- a Re 10. 11. 12. Lat) CIN eh Nuclear membrane is absent Nucleolus is absent Single circular Chromosome Mitotic division is absent Deoxyribonucleoprotein is absent Cytoplasm streaming is absent Pinocytosis is absent Have Pili and fimbriae(for adhesion), flagella for propulsion Cell size ranges from 0.5-100um Contained only one copy of each gene(haploid) No histone in chromosome Asexual reproduction(binary fission) Differentiate between Prokaryotes and Eukaryoties erences Nuclear membrane is present Nucleolus is present More than one Linear chromosome Mitotic division is present Deoxyribonucleoprotein is present Cytoplasm streaming is present Pinocytosis is present Have cillia and flagella(for movement) Cell size ranges from 10-150m Some eukaryotics genome are organised into operons DNA bounded around Histone Sexual reproduction(mitosis & meiosis)154 Lysosomes are absent Golgi apparatuis is absent Endoplasmic reticulum absent Chloroplasts are absent Ribosomes is 70S, distributed in the cytoplasm In Cytoplasmic membranes sterols absent Cell wall contain peptidoglycan Locomotor organelles is simple fibril Pseudopodia absent Eg: Bacteria, rickettsiae, chlamydiae, actinomycetes, etc 14, Lysosomes are present Golgi apparatuis is present Endoplasmic reticulum present Chloroplasts are may be present 80S arrayed one membranes as in endoplasmic reticulum, 70S in mitochondria and chloroplasts Sterols present Peptidoglycan absent Locomotor organelles is multifibrilled Pseudopodia present Eg: Fungi, protozoa, algae, plants, animalsScopes and importance 1. Production of antibiotics: » Many antibiotics are isolated from natural microorganisms by the process of fermentation. >» Eg: Penicilli ; =2. Production of enzymes, vaccines and other pharmaceutical products: » Microbial cells produce intracellular and extracellular enzymes and these enzymes are important for the success of pharmaceutical fermentation process. Eg: amylase, protease, lipase, invertase, oxidase, catalase, cortison reductase, etc. > Bio-surfactants have lot of applications in agriculture, food industries, industrial cleaning, leather, paper and metal industries, textiles, cosmetics and the pharmaceutical industry. > Different types of bio-surfactants are synthezed by a number of microorganisms, eg: Acinetobacter speciesa, Bacillus species, Pseudomonas species, Rhodococcus species,etc.3. Diagnosis of diseases and treatment: > Different tests are used to detect infectious microorganisms, eg: ELISA, Widal test, TUBERCULLIN SKIN TEST, 4. Treatment of industrial waste material: > Most industrial processes produce waste water, salts, organic matter and spent media and these are toxic. >» Many microbial species are used for decomposition of such waste materials and organic components, eg: Actinomycetes, fungi, protozoa, etc.Plastic Microorganism Reference Polyethylene Brevibacillus borstelensis Hadad et al. 2005) Rhodococcus rubber Sivan etal, (2006); Gilan et al, (2004) Pseudomonas chlororaphis Zheng et a. (2005) Comamonas acidovorans TB-3S ‘Alkutsu et al (1998) Polyvinyl chloride Psexwdomonas putida AS Anthony et al. (2004) Ochrobacorum TD Mogitinitaki et al. (1987) ‘Pseudomonas fluorescens B~ 22 BIA copolyester, Thermomonospora fusca Kleeberg et al (1998) ‘Some biodeyradable/natural plastics and their degrading microorganisms Poly(3-hydroxybutyrate-co-3-meresplopropionste) Poly(3-hydroxybutyrate) Poly(3-hydroxybutyrate-co-3-meresplopropionste) Poly(3-hydroxybutyrate), poly(3-bydroxybutyrate- co-3-hydroxyvalerate) Poly(3-hydroxybutyrate-co-3-hydroxypropionate) Poly (3-hydrox butyrate), poly(3- hydroxypropionate), poly(4-hydroxybutyrate), polyethylene succinate, polyethylene adipate Poly(3-hydroxybutyrate) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Polycprolactone Polymer blends and its degrading microorganisms ‘Starch/polyester Schlegelella thermodepolymerans Pseudomonas lemoignet Pseudomonas indica K2 Streptomyces sp. SNC? ‘Ralstonia pikenit T1 Acidovorax sp. TPS Alcaligenes faecalis Pseudomonas stuceri Comamonas acidovorans Alealigenes faecalis Schlegelella thermadepotymerans Caenitacteriua 1 ion Clostridium bonatinum (Closeridium acetobutylicum Clostriivam botutinum Clostridium acetobutylicum Amycolatopsis sp. Bacillus brevis Ethanna et al. (2004) Jendrossek et al. (1998) Ethanna et al (2004) Mabrouk and Sabry (2001) Wang et al (2002) Kasuya et al. (1999) Kita et al. (1997) Romen et al 2004) ‘Abou-Zeid et al. (2001) ‘Abou-Zeid etal, (2001)5. Plant growth promotion: » Many microbial cells present in soil, play an important role in soil fertility, herbicidal resistance, insect resistance, change in protein/oil content and enhanced quality of plant products, eg: Rhizobium species, Rhodospirillum species, Azotobacter species, Agrobacterium rhizogenes, Pseudomonas species, Acetobacter species. >» Azotobacter chroococcum, living nitrogen fixing bacterium capable of synthezing and secreting plant growth promoting substances like thiamine, riboflavin, IAA, gibberellin,etc ss _— et <6. Sterile product preparation: » Ph. Microbiology plays a major role in preparation of sterile products. > Deals with sterile rooms, aseptic techniques, detection of microbes by sampling and sterility testing of different sterile preparations. 7. Sterilization: >» Any processes/methods that eliminates, removes, kills or deactivates all forms of life and other biological agents(bacteria, fungi, viruses, etc) called sterilization. >» Moist heat sterilization, dry heat sterilization, | membrane ___ filtration, gaseous sterilization and chemical sterilization are the methods used for killing microorganisms.8. Steroid bio-transformation: > Steroids are physiologically active compounds of complex structure, eg: cholesterol, ergosterol, testosterone, progesterone, etc. > Important steroids can be produced by microbiological transformations of naturally occuring steroids, eg: Steptomyces species, Rhizopus species, Aspergillus species, Penicillium species. 9, Identification of microorganisms: > One of the vital function of pharmaceutical microbiology is identification of microorganisms found in products and the manufacturing environment. The microorganisms are isolated and identified by morphological, biochemical, cultural, microscopic characteristics and genetic studies.CHa CHa c=o c=o HO. YY mgnicans of 3 I Progesterone la-Hydroxyprogesterone CHz OH tHe oH | c=0 b mu HO. a py aa oF ye Cortisol Prednisolone(OG E. coli Proteus mirabilis Enterococcus sp, (rose) (brown) (turquoise blue) © © Klebsiella pneumoniae Staphylococcus aureus 5. saprophyticus (metallic blue) (opaque cream) (opaque (pale) pink)10. Testing of pharmaceutical: » Raw material and finished products: * The Presence of microorganisms like E. coli, Salmonella species, Pseudomonas aeruginosa and Staphylococcus aureus, in the raw materials and finished products may deteriorate their efficacy and potency. Test the presence of microbes are described in the IP, USP, EP and BP. Limit on the total number of viable microbes in given product(TVC) and involved in exclusion of specific pathogens. * Water act as a vehicle-always tested for TMC.> Microbiological assays of antibiotics: * The antibiotic bioassay provides a potency for the over all biological activity of an antibiotic preparation. ¢ Agar diffusion(plate assay) and turbidometric assay. > Evaluation of disinfectant : * Effective cleaning and disinfection of manufacturing facilities are crucial to the achievement and maintenance of high quality standard required for medicine and medical devices. ¢« Phenol coefficient test, cup plate method, turbidometric method and kelsey Sykes test.> Antimicrobial Preservative efficacy testing: * Different types of pharmaceutical products and cosmetics are protected from attack by microorganisms by using preservatives. Eg: Methyl, ethyl, propyl and butyl Parabens, Sorbic acid. * 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 determination of anti-microbal efficacy of preservatives.> Endo-toxin testing: * Bacterial endotoxin or pyrogens have a number of physiological effects following intravenous injections such as fever, activation of cytokine system, endothelial cell damage and _ intravascular coagulation. * The Bacterial Endo-toxins Test(BET) is most important in microbiology laboratories involved with quality control of parenterals and medical devices. * LAL Test, gel-clot method, chromogenic end point, kinetic turbidometric assay and kinetic chromogenic assay are also used to detect endotoxins.> Test for support of the sterility assurance system: Sterility test mainly applicable in running an aseptic area and microbiology lab. It is mainly performed in laminar air flow cabinets for parenteral preparations and other sterile pharmaceuticals to detect presence of microorganism. Bioburden testing is used to estimate the number of microorganism in the product prior to sterilizationKnowledge will give you power, but character respect. Bruce Lee