Pharmaceutical Microbiology Lecture 1 (PM 503) - Fall 2024 (PDF)

Pharmaceutical Microbiology (PM 503) Lecture 1 Sterilization Prof. Dr. Amal Emad Ali Dept. of Microbiology and Immunology Faculty of Pharmacy, FUE Dept of Microbiology and immunology Faculty of Pharmacy, Cairo University Overall Aim of Course...

Pharmaceutical Microbiology (PM 503) Lecture 1 Sterilization Prof. Dr. Amal Emad Ali Dept. of Microbiology and Immunology Faculty of Pharmacy, FUE Dept of Microbiology and immunology Faculty of Pharmacy, Cairo University Overall Aim of Course This course describes different methods for control of microbial growth in the field of pharmaceutical industry / hospitals. It includes different methods of sterilization, and validation of sterilization process. The course addresses classes and modes of action of chemotherapeutic agents and non-antibiotics antimicrobial agents including the new approaches to overcome bacterial resistance. The course includes microbiological evaluation of antibiotics and non- antibiotics. Laboratory procedures such sterility test and evaluation of antimicrobial effectiveness are also covered. Lecture 1 competency & LOs Competency LOs Integrate knowledge from 1. Describe different terms basic and applied for microbial control and pharmaceutical and clinical methods of sterilization sciences to standardize used in controlling materials, formulate and microbes giving examples manufacture products, and on their application. deliver population and patient-centered care. Lecture 1 competency & LOs 2. Discuss different methods Competency for evaluation of antibiotics Integrate knowledge from and non- antibiotics basic and applied antimicrobial agents. pharmaceutical and clinical sciences to standardize 3. Apply pharmaceutical materials, formulate and microbiology evaluation manufacture products, and assays for antimicrobial deliver population and patient-centered care. agents. Lecture 1 competency & LOs 4. Classify antibiotics & Competency non antibiotics Integrate knowledge from antimicrobials according to basic and applied their mechanism of action pharmaceutical and clinical sciences to standardize 5.Detect the most suitable materials, formulate and method of sterilization of manufacture products, and pharmaceutical product deliver population and patient-centered care. based on its properties. Lecture 1 competency & LOs 6. Determine the Competency characteristics of Actively share professional antibiotics, non-antibiotic decisions and proper antimicrobial agents, and actions to save patient’s life in emergency selective toxicity of situations including antibiotics. poisoning with various xenobiotics, and effectively work in forensic fields. Lecture 1 competency & LOs 7. Determine resistant Competency mechanisms to antimicrobial Provide counseling and agents. education services to patients 8. Point out hospital disinfection and communities about safe policy and rational use of medicines and medical devices. Essential Book Hugo & Russel Pharmaceutical Microbiology 8th Edition Assessment Schedule Assessment 1: First Midterm Exam Week 4. Assessment 2: Second Midterm Exam Week 9. Assessment 3: Practical Exam Week 10,11 Assessment 4: Final written Exam Week 15/16 Assessment 5: Oral Exam Week 15/16 Assessment 6: Class Work During the semester Weighting of Assessments First Midterm Examination 5% Second Midterm 15% Examination Participation & Attendance 5% Practical Examination 25% Class Work 10% Final-Term Examination 30% Oral Examination 10% Total 100%  ( Outline -Lecture 1) -Introduction to Sterilization - Different sterilization methods - Basis for selection of sterilization method - Kinetics of microbial death and survivor curve - Expressions of resistance to sterilization -Heat Sterilization - Factors affecting heat sterilization. - Mechanisms of heat sterilization -- Heat transfer -Heat sterilization process q Sterilization: is a process of freeing of an article of all living microorganism including spores, which are more resistant, either through killing or removing. The product may contain dead microorganisms (m.o.), except when filtration is used where the final product will not contain living or dead organisms. q Sterilization is absolute term, means the process is all or none i.e. system is sterile or none sterile, and an article should never be described as partially or relatively sterile. q However, sterility is uncertain and so it is more useful to describe the product subjected to a sterilization process as sterilized rather than sterile q Sterilization is an essential stage: ⚫ In the processing of any sterile product eg. product used for parenteral administration & eye preparations. ⚫ Microbiological materials. ⚫ Dressings and other contaminated items to minimize the health hazard associated with these articles. Sterilization is required in the following fields: 1) Microbiology: media - containers - suspending fluids and contaminated items. 2) Biotechnology: fermentation media and vessels. 3) Pharmacy: Injections - ophthalmics - drugs for contact with broken skin or internal organs. 4) Surgery: dressing, sutures, tools and equipment. 5)Medical devices: Syringes, gloves, canules, catheters, etc…  Sterilization Methods Physical sterilization Chemical sterilization Gaseous sterilization Heat sterilization Radiation sterilization Filtration sterilization Heating with bactericide Dry heat Moist heat  Sterilization Methods A) Physical (does not remain in the final preparation) 1) Dry heat 2) Moist heat 3) Radiation (gamma rays - accelerated electrons or b particles and to less extent C O L D S T E R I L I Z AT I O N Ultraviolet) 4) Filtration B) Chemical (remain in the final preparation except for gases) 1) Gases (ethylene oxide & formaldehyde-steam) 2) Heating with bactericide Official Sterilization methods u The European Pharmacopoeia recognizes five methods for the sterilization of pharmaceutical products: u (1) steam sterilization (heating in an autoclave) u (2) dry heat u (3) ionizing radiation u (4) gaseous sterilization u (5) filtration In addition, other approaches involving steam and formaldehyde and UV light have evolved for use in certain situations. Basis for successful sterilization process: ⚫ The success of the sterilization process depends on a suitable choice of treatment conditions: § Temperature § Duration of exposure ⚫ However, there is a potential risk of product damage due to sterilization (reduced therapeutic efficacy, stability or patient acceptability). ⚫ Thus, a suitable sterilization process should be selected to ensure maximum microbial kill/removal with minimum product deterioration. ⚫ There is a need to achieve a balance between the maximum acceptable risk of failing to achieve sterility and the maximum acceptable level of product damage. 1 üThus, The selection of most suitable sterilization method depends on: 1. The properties of the product to be sterilized. 2. The properties of the sterilizing agent. 3. The nature of the contaminant. Kinetics of microbial death & death (survivor) curve q In bacteria, death could be defined as the potential inability to multiply & not the lack of active metabolism. q The death of bacteria or their decrease in number when they suffer from chemical or physical antimicrobial activity is logarithmic (geometric) independent of the initial number of organisms. The rate of killing process is proportional to the initial number of viable cells (a) t = 1/K log a/b t = extinction time K = const. depending on the nature of the reaction a = initial number of viable cells (bioburden) b = number of remaining q Plotting log b against t, a straight line is observed with –ve slope representing the exponential or logarithmic phase of death i.e. the longer the contact time with the killing agent, the greater number of dead cells. The curve is usually sigmoid & only straight (continuous linearity) when the conc. of antimicrobial agent is high.  Survivor curve The sigmoid curve B shows 3 phases: 1. Initial lag phase (initial shoulder) for the diffusion of disinfectant or killing agent into the cells (also due to germination of spores if present). 2. Intermediate exponential or log phase. 3. Final irregular tailing due to presence of resistant cells (spores) or mutants. sigmoid Expression of resistance to sterilization: Thermal death point (TDP): lowest temp required to kill all M.O. in suspension within certain time (usually 10 min). Thermal death time (Extinction Time) (TDT): shortest time required to kill all M.O. in a suspension at certain temperature. - Factors such as pH, moisture, medium composition, and age of cells can influence the results and must be accounted for. Decimal reduction time (D-value): Time at fixed temp /or/ Radiation dose required to reduce the number of viable cells by 90% or one log cycle assuming linear curve and must be corrected in cases where there is variation from linearity. -It could be used as a measure for the degree of resistance of an organism at particular temperature. - D-value is usually determined with a reference microorganism known to be resistant to the sterilization method (biological indicator) To assess the influence of temperature change on thermal resistance Z value is used: Z-value: The increase in temp required to reduce the D-value of an organism by 90% or one log cycle. -It is a measure for the change in resistance upon changing the temperature. 1. Knowing that the D value for Bacillus stearothermophilus at 115 °C is 11 minutes and is 1.1 minutes at 125 °C, the Z value of the organism is: a.10 minutes b. 11 minutes c. 11°C d. 120° C e. None of the above 2. If the D value at 121 °C is 1.5 min & Z value is 10 °C , calculate the D value at 131 °C  Sterilization Methods Physical sterilization Chemical sterilization Gaseous sterilization Heat sterilization Radiation sterilization Filtration sterilization Heating with bactericide Dry heat Moist heat  STERILIZATION BY HEAT Each microorganism (M.O.) has an optimum temp for growth , below it growth decreases reversibly and above it decreases irreversibly due to degradation of vital molecules. The equation describing the effect of heat is the Higuchi and Busse equation: log t = 0.219 E + K T t = extinction time T = absolute temp K = constant depends on the No. and type of the most resistant M.O. E = heat of activation energy required to kill the most resistant M.O. (depends on the mode of action).  log t = 0.219 E + K T Plotting (Log t) against T a straight Log t line is observed i.e. the extinction time is inversely proportional to the temp Any small increase in temp greatly reduces the time required for sterilization. T  Mode of action of heat 1) Moist heat (water): - Death is through protein and nucleic acid denaturation due to coagulation, which is the loss of the tertiary structure of proteins. - small peptide chains & components with free SH groups are released. - It depends on the presence of water because of its high dipole moment and the mobility of the peptide chains. - This reaction requires low E and shorter t. 2) Dry heat (no water): death is due to slow protein denaturation through oxidation of certain groups in the protein molecule. This reaction requires high E and longer t. The polar groups in the peptide chains are less active, so that their mobility is much reduced, therefore it requires more energy to open the peptide molecules, hence the increase apparent resistance of the protein in the dry state (oxidation). Thus, at constant temp. bacteria are killed by moist heat in shorter time than dry heat. When applying heat in presence of a bactericide chemical agent, death is due to the chemical agent and heat only enhances the rate of reaction or decreases the resistance of the microorganism to the chemical agent.  Factors affecting heat sterilization 1) Time and temperature of exposure. 2) Number of microorganisms (bioburden). 3) Type of microorganisms (species, strain, spore formation). 4) Nature of the article to be sterilized.  Sensitivity of microorganisms to 33 sterilization Generally, microbial sensitivity to moist heat sterilization follows the following pattern: a. The most sensitive forms are vegetative bacteria, fungi, yeast and large viruses. Significant response to moist heat at 60-70 °C. b. Less sensitive forms are thermophilic bacteria, small viruses, resistant fungal spores. Significant response to moist heat at 70-90 °C. c. The most resistant forms are bacterial endospores which must be subjected to temperature in the range of 90-115 °C with full hydration before significant reduction in viability occurs. Why are endospores resistant to heat? 34 1. Contain the least amount of water (10% unbound water). 2. Spore wall contains calcium and dipicolinic acid which prevent the penetration of moisture but does not prevent the penetration of heat. 3. SH groups of proteins are protected through the formation of disulfide bond.  Heat Transfer: Direct Indirect e.g Incineration or flaming e.g through medium as air, steam or metals It is either through: 1. Radiation: using Infra- red source. 2. Conduction: usually through metals. 3. Convection: through air, water or oil, by currents of heat.  Heat transfer -Direct: e.g. incineration or flaming, only used for highly infective materials e.g. loop. -Indirect: through medium: e.g. air, water (steam) or metals. It is either through 1- Radiation: using infra red (I.R.) source or when the articles are placed near the heating sources. 2- Conduction: usually through metals. It is slow and needs intimate contact between the metal and both heat source and the article to be sterilized. 3- Convection: through air, water or oil by currents of heat.  Heat Sterilization Process Sterilization by heat proceeds through three successive periods: 1. Heating up period: time required for the temp to reach that required for sterilization. It requires high quality of activation energy. It depends on the type of the article to be sterilized. 2. Holding period: time during which the temperature of the object is kept at that required for sterilization. It requires small amount of activation energy. It also depends on the type of the article to be sterilized. 3. Cooling down period: time during which the temp decreases to that of ambient (room temp). Temperature Cooling stage Heating stage Holding stage Time Typical temperature profile of a heat sterilization process  Sterilization is an absolute term, needed in different fields Different methods of sterilization. Differentiate between different terms of expression of resistance to sterilization TDP, TDT, D value and Z value Kinetics of thermal sterilization ( non linear curve?) Mechanism of action of Dry heat and moist heat Heat transfer methods heat sterilization process curve.