MPharm Programme: Microbial Growth and Evolution PDF

MPharm Programme Microbial growth and evolution Dr Callum Cooper [email protected] Learning Objectives • How microbes are cultured • Different stages of bacterial and viral growth • Introduce bacterial evolution and where it can be important Top 10 elements making up a bacterium El...

MPharm Programme Microbial growth and evolution Dr Callum Cooper [email protected] Learning Objectives • How microbes are cultured • Different stages of bacterial and viral growth • Introduce bacterial evolution and where it can be important Top 10 elements making up a bacterium Element Carbon % Dry Weight Source Organics or CO2 50 Oxygen 20 H2O, Organics, CO2, and O2 Nitrogen 14 NH3, NO3, organics, N2 Hydrogen 8 H2O, organics, H2 Phosphorus 3 Inorganic phosphates Sulfur 1 SO4, H2S, So, organic sulfur compounds Potassium 1 Potassium salts Magnesium 0.5 Magnesium salts Calcium 0.5 Calcium salts Iron 0.2 Iron salts Function Main constituent of cell Cell material and water; electron acceptor in aerobic respiration amino acids, nucleotides, and coenzymes Organic compounds and cell water Nucleic acids, nucleotides, phospholipids Proteins, several coenzymes Main inorganic cations and enzymatic cofactor Inorganic cations, enzymatic cofactor Inorganic cations, enzymatic cofactor, endospores Cytochrome component, enzymatic cofactor Sources of Carbon, Energy & Electrons Carbon Source • Autotrophs • Heterotrophs Energy Sources • Phototrophs • Chemotrophs Electron Sources • Lithotrophs • Organotrophs • CO2 sole or principle carbon source • Obtained from other organisms • Light • Compound oxidation • Reduced inorganic compounds • Organic molecules N.B. These terms may be joined together e.g.“chemoorganoautotroph” In-vitro microorganism culture • Two ways to culture microorganisms • Liquid media (broth) • Solid media (agar plates) • Originally grown by Koch (late C19th) on potato slices and gelatine • In liquid media bacteria grow as individual cells until available nutrients are exhausted • Produces a suspension of cells (unable to distinguish between multiple cell types without further testing) • On solid media bacteria and fungi form colonies with distinctive appearances • In theory each colony derives from a single cell (makes culture purification easier) • Formula of media can influence colony appearance (selective and differential media) • Solid media formulas generally the same as liquid media with the addition of a gelling agent (agar) Undefined Vs Defined media • Undefined media contains chemically undefined yeast/vegetable/meat extracts and digested proteins • Batch-batch variation and reproducibility • Useful for routine growth applications • Defined media (synthetic media) all components are chemically defined • Highly reproducible • Can be rich or minimal depending on requirements Undefined Media Defined Media TSA agar Sabouraud dextrose agar M9 minimal media Tryptone 15g Dextrose 40g M9 Salts (Na2HPO4, KH2PO4, NaCl, NH4Cl) Soytone 5g Peptone 10g MgSO4 NaCl 5g Agar 20g CaCl2 Agar 15g dH2O to 1L Carbon Source (e.g. glucose) dH2O to 1L pH 5.6 dH2O pH 7.5-8 Atmospheric requirements 1. Obligate aerobe e.g. Mycobacterium tuberculosis • Cannot survive without oxygen 2. Obligate anaerobe e.g. Clostridium difficilie • Cannot survive (killed) in the presence of oxygen 3. Facultative aerobe e.g. Staphylococcus aureus and E. coli • Can use grow in the presence of oxygen or produce energy by fermentation 4. Microaerophile e.g. Camplylobacter jejuni • Requires reduced oxygen content (increased CO2 content) in order to survive 5. Aerotolerant anaerobe e.g Streptococcus mutans • Can tolerate oxygen in the air but produces energy by fermentation Anaerobic microorganisms • Several methods available for Anaerobic (or aerotolerant) culture: • Anaerobic cabinet • Basically big isolator cabinets • 95% N2 5%H2 with a palladium catalyst • Can be under positive pressure • GasPak sachets • Produces CO2 and H2 from breakdown of citric acid, cobalt chloride and sodium borohydride. • Candle extinction • Uses up oxygen by burning of a candle in the jar • GasPak and Candle methods rarely produce a true anaerobic environment Bacterial cell division • Each cell is able to survive and reproduce independently • Most bacteria reproduce by DNA binary fission • Some reproduce by budding • Time taken to reproduce is called generation time • Varies wildly between species • Escherichia coli ~30 min • Mycobacterium leprae ~14 days • 4 distinct phases to bacterial growth https://www.youtube.com/watch?v=gEwzDydc iWc Bacterial growth Lag phase Log number viable cells • No immediate increase in cell number • Old cells depleted & need time for synthesis of new cell components / metabolites lag Time Bacterial growth Exponential (log) phase Log number viable cells • Growth & division at maximum possible rate given genetic potential & environmental conditions • Regular doubling time log Time Stationary phase Bacterial growth • In closed system nutrients become depleted & waste products build-up • Growth ceases (or is balanced by death) • Morphological and metabolic changes (e.g. secondary metabolism) Log number viable cells stationary Time Bacterial growth Death phase (senescence) Log number viable cells • Severe nutrient deprivation • Build-up of toxic waste products • Viable cell numbers decline at an exponential rate death Time Log number viable cells Why does this matter? Secondary metabolism stationary death log lag Time Microbial metabolism Primary metabolism • Includes major metabolic pathways • Energy production and release • Cell component synthesis • Enzyme production Secondary metabolism • Non-essential metabolic pathways • Includes production of natural products e.g. antibiotics • Production of secondary metabolites in disease states which can increase pathogenicity e.g. pyocyanin Laboratory scale culture- batch culture Flask cultures- closed systems • Used for optimisation of steps • Nutrient availability limited • Atmosphere limited due to diffusion at liquid surface • Limited product production • Not suitable for industry • Lab scale only Laboratory scale culture- Continuous culture Chemostats- Open systems • Allows for highly controlled growth • Nutrients supplied at constant rate • When at steady state  = D = F/V Scaling up production Bulk culturing Three growth modes: • Batch - full at start • Fed batch - fill until vessel full • Continuous - fill and overflow Important criteria: • Maintain adequate mixing • Maintain high oxygen levels - if aerobic • Control pH • Control temperature • Control foam • Initial starting concentration Viral replication • Viral replication relies on the subversion of host replication machinery • True in both prokaryotic and eukaryotic viruses • In bacteriophages, subversion leads to cell destruction • Lytic replication • In Eukaryotes, viruses often bud rather than destroy the cell • Influenza • HIV Viral replication • Some bacteriophages can also integrate their genome into the host • Lysogenic replication • Replicates alongside host • Can enter lytic replication in favourable conditions • Viruses which can lie dormant can also cause human disease; • Herpes simplex virus • Human papillomavirus • Important in the spread of bacterial genes (horizontal gene transfer) Bacterial evolution •Evolution: changes in population over time •Can arise due to a variety of causes • Acquisition of new genes • Mutation of existing genes • Results of bacterial evolution can be a good or bad thing; • Increased product yield • Become pathogenic • Increase in resistance to treatment •Rate of evolution varies by organism •More complex the organism, slower the rate of evolution Bacterial evolution: Mutation Mutation: Permanent change in a single cell, does not necessarily cause any noticeable change or get passed on; • UV irradiation • Chemical exposure • Poor genome copy • Different types of mutation; • • • A harmful, or deleterious, mutation decreases organism fitness. A beneficial, or advantageous mutation increases organism fitness. • Mutations that promote desirable traits (e.g increased product yield) • Can include things which are harmful to other organisms A neutral mutation has no harmful or beneficial effect. Such mutations occur at a steady rate Bacterial evolution: DNA acquisition Three main mechanisms by which bacteria can evolve; • Transformation: Direct uptake of DNA through cell membrane • Transduction: Introduction of genetic material via a viral vector • Conjugation: Transfer of genetic material between two directly connected bacteria WHY SHOULD I CARE?? Bacterial evolution: Antimicrobial resistance Bacterial evolution: Antimicrobial resistance Bacterial evolution: Antimicrobial resistance • Antibiotic usage can act as a selective pressure on bacteria • Removes competition for resistant cells by killing susceptible cells • Unlikely to be due to a single mutation; • Harvard Med School: https://www.youtube.com/watch?v=plVk4NVIUh8 Strain Improvement • Initial strains may produced products at low concentrations • Need to boost efficiency of production • More cost effective • Bacterial strains can improve naturally • Spontaneous mutation (usually random and infrequent) • Can also make things worse! • Exposure to mutagens can increase frequency of mutation • UV/Chemical exposure • Random mutagenesis • Mutants can be picked and assessed for increased production • Genetic modification of organisms • Targeted mutagenesis: Add/remove/alter genes to improve overall yield Improvement in yields: Penicillium chrysogenum 1943 NRRL 1951 [120] S NRRL 1951 B25 [250] X X-1612 [500] S = Spontaneous mutants X = X-ray mutagenesis UV = UV mutagenesis N = mustard gas UV Q176 [900] UV, N, S 53-399 [2658] Today • Used a combination of spontaneous and random mutagenesis to increase production by a factor of 20 • Production is in units of activity/mL Heterologous Gene Expression • Insulin originally derived and purified from animal sources • First human-identical insulin (humulin) produced in 1978 by Genetech then licenced to Eli Lilly • Cloned human insulin gene into E.coli Human insulin gene GMO E. coli Plasmid • Humulin and similar products been in use > 25 years Humulin Summary • • • • Requirements for bacterial growth Different stages of bacterial and viral replication How bacteria can evolve Examples of why bacterial evolution is important • Link between evolution and antibiotic resistance Extra Reading • Prescotts Microbiology: Part II; Section 6 & 7 • Brock Microbiology: Part I; Section 3 and 5 MPharm Programme Sources of Contamination and Sampling Dr Callum Cooper [email protected] Hugo & Russell’s Pharmaceutical Microbiology • • Useful reference for this section of Mpharm Also useful for future Micro. courses Learning Objectives • Microbial contamination of pharmaceutical products – – – – Origins / sources of contamination Reducing risks of contamination Testing for contamination Results of contamination How did this sample get contaminated? How can we reduce the risk of contamination? Types of contamination in pharmaceuticals Types of Contamination Chemical Biological Bacterial Fungal Viral Physical Sources of biological contamination Utilities Facilities Process Contaminated Product Materials Equipment Personnel Controlling Microbial Contamination • Control measures to reduce risk • Environmental controls • Clean or aseptic preparationareas • Clean room • Laminar flow cabinets • Isolators • Air/water controls • Personnel controls • PPE • Hand hygiene (antisepsis) • Disinfection/antisepsis • Cleaning / disinfection of working environment/personnel • Sterilisation • Destruction of potential contaminants prior to release • Preservation • Reduces risk of longer term contamination and spoilage Aseptic Technique • Under normal conditions, working areas will be constantly contaminated with microorganisms • Free floating or carried on dust particles • Personnel • Risk of contamination can be reduced using; • Bunsen burner • Used for heat sterilising metal and glass tools on lab bench • Biosafety Cabinets • Laminar flow cabinet Pharm. Micro. • Glovebox/isolator Aseptic Technique Bunsen Burner Laminar airflow cabinet Biosafety Cabinet Isolator Cabinet Environmental controls Aseptic production areas For the manufacture of sterile medicinal products normally 4 grades can be distinguished: • Grades C and D: Clean areas for carrying out less critical stages in the manufacture of sterile products. Less stringent • Grade B: In case of aseptic preparation and filling, the background environment for grade A zone. • Grade A: The local zone for high risk operations, e.g. filling zone, stopper bowls, open ampoules and vials, making aseptic connections. More stringent Environmental controls • Air drawn from outside aseptic area • How does this reduce contamination? • High efficiency particulate air (HEPA) filtration; • Defined in US as removal of at least 99.97% of 0.3 µm diameter airborne particles • EU has multiple classifications based on level of filtration Environmental controls • Two standards for water in pharmaceutical manufacturing • Purified • Water for injection • Purified water used for non-sterile applications • Media preparation • Basic preparations e.g cough syrup • Water for injection (WFI) is used in sterile applications • Stricter quality guidelines than purified water • Endotoxin levels Reducing contamination from personnel • Humans are disgusting meat bags covered in microbes • Facial skin has ~100 million microbes/cm3 • Common to find feacal microbes on peoples hands and worksurfaces • A single sneeze contains about 40, 000 droplets • Saliva has up to 1x108 microbes/ml • A sneeze can tracel about 6 meters! • Droplets can remain in the air for up to 2 hours Reducing contamination from personnel Hand washing • Reduces risk of transmission of contaminants between hands and products, surfaces • Reduces risk of transmission to sterile gloves • Use of alcohol hand gels to further reduce bacterial numbers Reducing contamination from personnel Protective equipment • Includes items for non-sterile manufacturing; • Gloves • Hairnets • Overshoes • Different levels of equipment for different levels of production • Sterile production often wear oversuit and face mask Microbial sampling Product Sampling / Clinical Samples • Filtration Broth / Agar • Direct inoculation Environmental Sampling • • • • Surface swabbing Contact plates Air sampling Liquid sampling Broth / Agar Counting microbes • Serial dilution and plate counts • • • Optical density (OD)/ turbidity • • • Counts everything Inaccurate at high and low OD Direct microscopy • • Only shows viable cells (colony forming units; CFU) 3 different methods • Pour plate • Spread plate • Drop count Total number of cells in a defined area Flow cytometry • Uses fluorescence Microbiological Calculations: Total microbial count • Uses a haemocytometer • Enables the counting of bacterial cells in a known area • Requires multiple fields of viewing • VERY time consuming • Shows intact bacterial cells • Nothing about viability • Good for difficult to culture and polymicrobial Microbiological Calculations: Total viable count • For bacteria ,yeasts and moulds • Can see individual • Count colony forming units (CFU) • For viruses you don’t see colonies • Absence of growth (plaques) • Count plaque forming units (PFU) • Shows presence of culturable organisms • Can get organisms that are difficult to grow • Believed to be that only 1-5% of human microbiota can be cultures Microbiological Calculations: Total viable count Calculate CFU/mL of the original inoculum from the plate; • Number of colonies: 12 • Initial dilution • Volume of sample added: 0.1 mL CFU/mL = Number of colonies X (dilution in tubes X no. aliquots in 1mL) CFU/mL = 12 X (1000 X 10) = 12 X 10000 CFU/mL = 120000 = 1.2x105 Regulating Contamination: QC Standards British Pharmacopoeia • Official standards for UK medicinal products and pharmaceutical substances (includes Ph. Eur.) • Specifies acceptable limits for microbial contamination of non-sterile products AdministrationRoute Max TotalAerobes (CFU / g or /ml) Specified Absences Oral (non-aqueous) 103 E. coli Oral (aqueous) 102 E. coli Rectal 103 - Mucosal / Cutaneous 102 S. aureus; P.aeruginosa • Sterile products have to contain no microbial contaminants and also have additional criteria What can microbial contamination do? • Spoilage • • Chemical & physicochemical deterioration • Rate of breakdown will depend on; • Molecular structure • Environmental conditions • Type and number of microbial contaminant • Inactivation of product e.g. penicillin breakdown by βlactamase • Breakdown of thickening/suspending agents e.g. starch breakdown by amylases • Synthetic packing materials (e.g nylon) more resistant than naturally derived (e.g. cellophane) Important to understand where and how product is to be used • Manipulate formulation to create resistance to spoilage e.g add preservatives What can microbial contamination do? • Health hazards to patients • Product contamination with pathogens →infections in susceptible patients • Response varies; No reaction Local infection GI infection Systemic/bloodstream infection • • • Most serious are from injected products or immunocompromised patients Contamination with toxic microbial metabolites Most serious effects from contaminated injectable products; • • General bacteraemic shock Death What happens when it all goes wrong? What happens when it all goes wrong? • Can lead to; • Product recall • Litigation https://www.fda.gov/Safety/Recalls/default.htm https://www.gov.uk/drug-device-alerts Summary • Looked at different sources of contamination and how they interconnect • Methods which can be used to reduce the risk of contamination • Introduction to how to test for contamination • Introduced regulations around contamination • What happens when it goes wrong Extra reading • Hugo and Russell's pharmaceutical microbiology; Part 3, Section 16, 21 MPharm Programme Purification & sterilization Dr Callum Cooper [email protected] Russell, Hugo & Ayliffe • • Useful reference for this section of Mpharm Also useful for future Micro. courses Learning Objectives • Introduction to Downstream processing • Purification • Sterilisation • Different processes of sterilisation • Sterility checking • Sterility testing Recap Recap Purification • Separation of products from production mixtures/removal of unwanted components/contaminants – Sedimentation and precipitation e.g. heat, pH, organics – Centrifugation – Adsorption e.g. ion exchange, immuno-affinity – Micro-filtration with specified molecular weight cut-off (MWCO) Why purify? Yield at different recovery % • Reduces risk of side effects while maintaining yield 1. 2. How many steps are used Loss of product at each step % Yield Yield depends on: 120.0 100.0 95 80.0 90 60.0 85 40.0 80 20.0 0.0 1 2 3 4 5 6 Step 7 8 9 10 Downstream processing: Sedimentation and precipitation Sedimentation: • Speed depends on cell size, density and mixing speed * * * * * * * * * ** * * * * * * * * * ** * * * * * * * * * ** * * * * * * * * * ** ******************** ******************** *************** * * * * * * * ** Precipitation: • Lowers solute (media) solubility and causes product to fall out of solution • Can be done through various routes • Chemical, temperature, pH etc • Used in production of recombinant DNA polymerases Downstream processing: Centrifugation • Application of centrifugal force to separate out products • Denser particles move to outside first • Requires components to have a different density from the medium • Sedimentation speed depends on cell size, density and rpm By Zuzanna K. Filutowska Own work, CC BY-SA 3.0, https://commons.wikimedia. org/w/index.php?curid=2976 4058 Downstream processing: Adsorption • Principle is based on that of chromatography • Passage of a liquid phase through a semi-solid phase • Ion exchange: Binds proteins based on protein charge • Can be used to capture or allow passage of proteins of interest Charged + - + - proteins + + + + + + + + ++ + + + + + + + + + + + + • Immuno-affinity: Uses antigenic regions to bind unwanted components • Usually targeting specific contaminants • Industrial removal of bacterial endotoxins (LPS) + -+ + -+ + + + + + + + + + + + - + + + + + + + -- Sterilization • Process that removes / kills everything – Normally refers to bacteria & fungi • Viruses must be removed from biologically- derived therapeutics – e.g. monoclonal antibodies, plasma components • Modern usage may include disabling/destruction/removal of infectious proteins e.g. Prions (TSE) When? Any medical product where use will breach normal bodily defences against infection: 1. 2. 3. Parenteral (IV) administration Contact with broken skin (e.g. wounddressings) Contact with mucosalsurfaces or internal organs Microbial Sensitivity to sterilisation • Different microbes have different levels of sensitivity to sterilisation • Generally independent of sterilising method used but will influence choice of method Prions More resistant Spores Gram negative bacteria Small non-enveloped viruses Fungi Large non-enveloped viruses Gram positive bacteria Lipid enveloped viruses Less resistant • Prions exhibit exceptional resistance to all known sterilizing agents – may even survive 18 minutes @ 134-138 °C Selection of Sterilization Method • All sterilization methods involve some risk of product damage • Especially harsh ones • Product damage can reduce therapeutic efficiency, stability, patient acceptability • Limit to level of microbial reduction • Important to minimise microbial contamination Product Damage Sterilization Failure • 5 methods recognised by European Pharmacopoeia(2002): Gas sterilization Steam (autoclave) Filtration Dry heat (oven) Ionising radiation Downstream processing & Sterilisation: Filtration • Removes rather than destroys microorganisms • Filter grades vary by size and ability to remove microbes • Filtration efficacy assessed by reduction in bacterial count • Pore size plays a role in retaining contaminants • Composition of the membrane etc will also play a role • 0.22µm pore size generally used • MWCO filters can remove on basis of atomic mass (Da) • Major uses; • Heat sensitive solutions • Biological products • Air and other gases • Water Filtration • Membrane filters: particles retained on filter surface (sieving) • Depth filters: particles trapped within the filter • Depth (prefilter) and membrane (sterilizing) filters can be combined Heat-Based Sterilization Methods Moist Heat Methods • Uses hydrolytic action • Steam at >120oC • > 1 atmosphere pressure Dry Heat Methods • Uses oxidative action • Temperature >150oC • Broad spectrum antimicrobial • Standard method for inactivating bio hazardous waste • Compatibility Issues Gas-Based Sterilization Methods • Chemically reactive gases – Ethylene oxide (CH2)2O – Formaldehyde H.CHO • Packaging materials must be permeable • Not as reliable as heat-based methods • Generally reserved for temperature sensitive items: – Reusable surgical instruments, medicaldiagnostic, electrical equipment, powders • Broad spectrum antimicrobials • Mechanism of action assumed to be alkylation of various protein functional groups • Ethylene oxide is flammable, toxic and carcinogenic Sterilization Using Radiation • Two types of radiation used; • Ionising: γ-rays, accelerated electrons, X rays • Non-Ionising: UV light (optimum  = 260 nm) • Ionising radiation • Facility must be heavily shielded • Can damage some materials (e.g. radiolysis of water) • UV light only used for air/surface/ shallow water sterilisation • Lower energy than ionising • Both primarily target microbial DNA Sterilisation • Should we automatically assume that sterilisation is successful? NO • Various factors which can influence outcome of these processes; • Poor circulation of steam • Poor equipment maintenance/cleaning What do we do? • Sterilisation can be checked in one of three ways; • Physical indicators • Chemical indicators • Biological indicators Physical / Chemical Sterilisation Indicators • Temperature/pressure record chart of each heat sterilization cycle • Thermometer probe (thermocouple) located at coolest part of loaded sterilizer or inserted into test packs • Chemical indicators based on ability to visibly alter chemical characteristics • e.g. autoclave tape • Also available for gaseous and radiation based sterilisation Biological Sterilization Indicators • Standardised bacterial spore preparations; • Non-pathogenic • Possess good thermal resistance • Geobacillus stearothermophilus • Placed in dummy packs located around sterilizer • After processing spores are grown in nutrient medium • Delay from incubation time can be reduced by using visible indicator • pH decrease causes purple →yellow change (steam) sterile nonsterile Sterility Assurance • On exposure to sterilization process, microbial populations loose viability exponentially, independent of initial numbers • REMEMBER: Sterile means no survivors 1 Surviving Fraction 10-1 10 • Achieving true sterility could 10 take forever • Microbial safety index: 10 chance of a single surviving 10 organism ExposureTime • The probability of a non-sterile unit is no more than 1 in 1 million (10-6) -2 -3 -4 -5 The nature of the contaminant will have a direct impact on the success of sterilisation Sterility Testing Assesses whether a sterilized product is free from microbial contamination by incubation of a sample in nutrient medium Membrane Filtration • Method of choice for pharmaceutical products • Microorganisms from liquid product collected on sterile filter • Filter transferred to appropriate media • Long incubation times e.g. 14 days • Visual inspection for turbidity What happens when it all goes wrong? • Can lead to; • Product Recall • Litigation https://www.fda.gov/Safety/Recalls/default.htm Summary • Looked at latter stages of manufacturing • Purification • Sterilisation • Different purification methods • Risks Vs Benefits of purification • Introduced concept of Sterilisation and Sterility • Different methods used • Suitability of different methods for different types of product Extra reading • Russell, Hugo & Ayliffe's principles and practice of disinfection, preservation and sterilization: Section 2 Chapter 15 • Hugo and Russell's pharmaceutical microbiology; Part 3, Section 17,19, 20, 21