Lecture 5 Control of Microbial Growth and Microbial Metabolism PDF

General Microbiology MLAB 213 Control of microbial growth and Microbial Metabolism Charbel Al-Bayssari, PhD University of Balamand Faculty of Health Sciences 1 Medical Laboratory Sciences Department ...

General Microbiology MLAB 213 Control of microbial growth and Microbial Metabolism Charbel Al-Bayssari, PhD University of Balamand Faculty of Health Sciences 1 Medical Laboratory Sciences Department  The effectiveness of antimicrobial treatments depends on four factors Ø Number of Organisms The number of organisms in the starting population determines how long it takes to reduce the number of organisms to a given level at a logarithmic death rate. Ø Environmental Influences Organic matter may interfere with heat treatments and chemical control agents. (blood, vomitus, feces, biofilm) Disinfectant work better under warm conditions Heat is more effective at lower pH. Ø Time of Exposure Chemical agents require longer exposures to control more resistant organisms. Longer exposure to lower heat can produce the same effect as shorter time at higher heat Ø Microbial Characteristics Microbial species and life cycle phases (endospores) have different susceptibilities to physical and chemical controls. 2 3 Control of microbial growth Physical ü Heat (Moist versus Dry) ü Cold ü Osmotic Pressure ü Radiation ü Desiccation ü Filtration Chemicals 4  Heat Ø Temperature and time, determine the effectiveness of heat for sterilization. Ø The thermal death point (TDP) of any particular species of microorganism is the lowest temperature that will kill all the organisms in a standardized pure culture within a specified period Ø The thermal death time (TDT) is the length of time necessary to sterilize a pure culture at a specified temperature. 5  Moist Heat Sterilization Ø Moist heat kills microorganisms primarily by coagulating proteins (denaturation), which is caused by breakage of the hydrogen bonds that hold the proteins in their three- dimensional structure Ø Heat applied in the presence of moisture Ø It is is faster and more effective than dry heat Ø kills most viruses , bacteria, and fungi Ø It can be accomplished at a lower temperature; thus, it is less destructive to many materials that otherwise would be damaged at higher temperatures. 1- Boiling ü Time required: 10 min ü The endospores of the bacteria that cause anthrax, tetanus, gas gangrene, and botulism, as well as hepatitis viruses, are especially heat resistant and often resist boiling 6 2- Autoclave is like a large meta pressure cooker that uses steam under pressure to completely destroy all microbial life 7 8 3-Pasteurization q High temperature short time pasteurization ü Kill pathogens and reduce the total bacterial count ü mild heating (72 degree) for short time (15 sec) Ex: milk that keeps well under refrigeration q Ultra-high-temperature (UHT) treatments ü It can then be stored for several months without refrigeration. ü Sterilizing temperatures are reached almost instantaneously. ü After reaching a temperature of 140°C for 4 seconds, the fluid is rapidly cooled in a vacuum chamber 9 10  Dry Heat Sterilization Ø Items must be baked at 160°C to 165°C for 2 hours or at 170°C to 180°C for 1 hour. Ex 1: Flaming/Incineration Ex 2: Hot-air sterilization: Items to be sterilized by this procedure are placed in an oven (170°C for 2 hours) 11  Cold Ø Most microorganisms are not killed by cold temperatures and freezing, but their metabolic activities are slowed, greatly inhibiting their growth (bacteriostatic effect). Ø Rapid freezing, using liquid nitrogen, is a good way to preserve foods, biologic specimens, and bacterial cultures. Ø Slow freezing and thawing is harmful to the bacteria 12  Other physical inhibition mechanism Ø High Pressure If the pressure is high enough, it alters the molecular structures of proteins and carbohydrates, resulting in the rapid inactivation of vegetative bacterial cells. Endospores are relatively resistant to high pressure (versus high T, high pressure/ alternate pressure) Ø Desiccation is the process of extreme dryness: lyophilization, or freeze-drying Ø Osmotic pressure High concentration of salts and sugar to preserve food 13 Ø Radiation Ionizing radiation: X-rays and gamma and beta rays of certain wavelengths from radioactive materials may be lethal or cause mutations in microorganisms and tissue cells, because they damage DNA and proteins within those cells. ex: sterilization medical and dental supplies Nonionizing radiation: has a wavelength longer than that of ionizing radiation ex: ultraviolet (UV) light. UV light damages the DNA of exposed cells by causing bonds to form between adjacent pyrimidine bases, usually thymines, in DNA chains Microwaves: do not have much direct effect on microorganisms 14 Ø Filter sterilization § Heat-sensitive liquids are sterilized by filtration § The selection of filters for sterilization must account for the size range of contaminants to be excluded § Filters of various pore sizes are used to filter or separate cells, larger viruses, bacteria, and certain other microorganisms from the liquids or gases. Ø Depth filter Ø Membrane filter 15 1-Depth filters: a fibrous sheet or mat made from a random array of overlapping paper (asbestos or borosilicate fibers) Used as prefilters to remove large particles in a liquid suspension so that the final filter in sterilization is not clogged Used for filter sterilization of air (most forced air heating and cooling systems use a simple depth filter to trap particulate matter like dust, spores and allergens) High-efficiency particulate air filters (HEPA filters) are used in biological safety cabinets with air flow. These filters generally remove 0.3 µm particles with 99.97% efficiency 16 17 2- Membrane filters § Composed of polymers with high tensile strength such as cellulose acetate, cellulose nitrate and manufactured in such a way as to contain a large number of tiny whole (~ 80% of membrane surface area consist of pores) § The most common type of filter for liquid sterilization in microbiology laboratories 18 Membrane filters used in liquid sterilization 19 Agent Mechanisms of Action Comments Moist Heat, boiling Denatures proteins Kills vegetative bacterial cells and viruses Endospores survive Moist Heat, Denatures proteins 121°C at 15 p.s.i. for 30 min kills everything Autoclaving Moist Heat, Denatures proteins Kills pathogens in food products Pasteurization Dry Heat, Flaming Incineration of contaminants Used for inoculating loop Dry Heat, Hot air oven Oxidation & Denatures proteins 170°C for 2 hours; Used for glassware & instrument sterilization Filtration Separation of bacteria from liquid Used for heat sensitive liquids (HEPA: from air) Cold, Lyophilization Desiccation and low temperature Used for food & drug preservation; Does not (also desiccation) Decrease metabolism necessarily kill so used for Long-term storage of bacterial cultures Cold, Refrigeration Decreased chemical reaction rate Bacteriostatic Osmotic Pressure, Plasmolysis of contaminants Used in food preservation (less effective against Addition of salt or fungi) sugar Radiation, UV DNA damage (thymine dimers) Limited penetration Radiation, X-rays DNA damage Used for sterilizing medical supplies 20 21 22  Chemical antimicrobial control An antimicrobial agent is a natural or synthetic chemical that kills or inhibits the growth of microorganisms v Cidal agents agents kill microorganisms (bacteriocidal, fungicidal, viricidal) v Static agents: agents that do not kill but only inhibit growth (bacteriostatic, fungistatic and viristatic) v Sepsis: Bacterial contamination v Aseptic: Object free of pathogens 23  Chemical antimicrobial agents for external use The chemical antimicrobial agents intended to prevent growth of human pathogens in inanimate environments are 4 categories: a. Sterilants b. Disinfectants c. Sanitizers d. Antiseptics and germicides 24 A- Sterilants 1. Also called sterilizers, they kill all forms of microbial life even endospores 2. Used to sterilize heat and radiation-sensitive equipment (ex: catheters, lensed instruments, thermometers etc..) 3. Used in gaseous or liquid form Ø The gaseous form is called cold sterilization where gaseous chemicals like formaldehyde, ethylene oxide, hydrogen peroxide are used to treat enclosed devices Ø Liquid sterilants are used for equipment that can’t withstand gas treatment 25 Mode of function of common sterilant a- Ethylene oxide (C2H4O-gas): a colorless flammable gas or refrigerated liquid § Usually stored as pressurized or refrigerated liquid § Used for sterilization of food (mainly spices), medica l supplies (sutures, bandages, surgical implements) § Toxic by inhalation, hence sterilization occurs in closed chambers § kills microbes by alkylating the nucleic acids b- Formaldehyde : 37 % formalin § Toxic, irritant and carcinogenic § Acts as an alkylating agent In gaseous form (obtained by heating formalin) used as: § sterilant / area fumigant § at 3-8% aqueous solution is used as a disinfectant 26 c- Hydrogen peroxide (vapour) Ø Used in the vapour state Ø Less toxic to the environment than the previous sterilants Ø Oxidizing agent Ø Its sterilization mechanism is based on production of HO free radicals that are highly reactive and disrupt proteins 27 MERS OUTBREAK in KOREA 28 B- Disinfectants § Chemicals that kill microorganisms but not endospores § Used to disinfect medical instruments, food and dairy equipment, water supplies Ex: ethanol (60-85%), cationic detergents (quaternary ammonium compounds), chlorine compounds, iodine-containing iodophore compounds 29  Mode of function of common disinfectants a- Ethanol (60-85%) Ø Denatures proteins and dissolves lipids Ø Inactive against spores b- Cationic detergents Ø Interact with phospholipids Ø Not active against mycobacteria (because of the waxy nature of their cell walls) and non-enveloped viruses c. Chlorine compounds Ø oxidizing agents (ex: sodium hypochloride) c. Iodine-containing compounds Ø oxidizing agent (can substitute for H on OH, NH, CH and SH groups on molecules 30 C- Antiseptics and germicides § Agents that kill or inhibit microbial growth § Can be used topically (non-toxic) § Used for handwashing or treating surfaces of wounds Ex: ethanol (60-85%), iodine compounds (Betadine), hydrogen peroxide (3%) D-Sanitizers § Agents that reduce (but may not eliminate) microbial numbers to levels considered safe § Widely used in food industry to treat surfaces of cooking equipment Ex: cationic detergents (soap), chlorine compounds 31  Antimicrobial efficacy Several factors affect the efficacy of these chemical antimicrobial agents. Examples: ü Organic materials can neutralise many disinfectants ü Capsules around pathogens ü Formation of biofilms which contain several layers of cells è The penetration of the chemical agent to all viable cells will be limited 32 33 The characteristics of an ideal chemical antimicrobial agent include : It should have a wide or broad antimicrobial spectrum It should be fast-acting It must be nontoxic to human tissues and noncorrosive and nondestructive to materials on which it is used It should leave a residual antimicrobial film on the treated surface It must be soluble in water and easy to apply It should be inexpensive and easy to prepare, with simple, specific directions It must be stable both as a concentrate and as a working dilution, so that it can be shipped and stored for reasonable periods It should be odorless Ø Some disinfectants (e.g., surface- active soaps and detergents, alcohols, and phenolic compounds) target and destroy cell membranes. Others (e.g., halogens, hydrogen peroxide, salts of heavy metals, formaldehyde, and ethylene oxide) destroy enzymes and structural proteins. Others attack cell walls or nucleic acids. 34  High level disinfectant kill all microbes (including viruses) except large numbers of bacterial spores Intermediate-level disinfectants might kill mycobacteria, vegetative bacteria, most viruses and most fungi, but do not necessarily kill bacterial spores Low level disinfectants kill most vegetative bacteria, some fungi, some viruses within 10 min of exposure q A few disinfectant will kill bacterial spores with prolonged exposures times (3-12hours) 35 q Critical items surgical instruments Cardiac and Urinary catheters Implants Ultrasound probes Ø Items in this category could be purchased as sterile or be sterilized using steam, ethylene oxide gaz (mixed with Nitrogen or CO2), hydrogen peroxide gas q Semi critical items Semicritical contact mucous membranes or nonintact skin and require high-level disinfection. Semicritical items include respiratory therapy and anesthesia equipment , some endoscopes, laryngoscope blades, esophageal manometry probes, cytoscopes, anorectal catheters, and diaphragm fitting rings Ø Semicritical items minimally require high-level disinfection using glutaraldehyde, hydrogen peroxide, ortho-phtalaldehyde or peracetic acid with hydrogen peroxide. 36 q Noncritical items Noncritical items are those that come in contact with intact skin, but not mucous membranes. Such items are divided into two subcategories: noncritical patient care items (eg, bedpans, blood pressure cuffs, crutches, computers) non critical environmental surfaces (e.g bed rails, some food utensils, bedside tables, patient furniture, floors) Ø Low level disinfectant may be used: 70% to 90% ethyl or isopropyl alcohol sodium hydrochloride (household bleach diluted 1:500) phenolic germicidal detergent solution iodophor germicidal detergent quaternary ammonium germicidal detergent solution 37 Agent Mechanisms of Action Comments Surfactants Membrane Disruption; Soaps; detergents increased penetration Quats (cationic Denature proteins; Antiseptic - benzalconium chloride, detergent) Disrupts lipids Cepacol; Disinfectant Organic acids and High/low pH Mold and Fungi inhibitors; e.g., bases benzoate of soda Heavy Metals Denature protein Antiseptic & Disinfectant; Silver Nitrate Halogens Oxidizing agent Antiseptic - Iodine (Betadine) Disrupts cell membrane Disinfectant - Chlorine (Chlorox) Alcohols Denatures proteins; Antiseptic & Disinfectant Disrupts lipids Ethanol and isopropyl Phenolics Disrupts cell membrane Disinfectant Irritating odor Aldehydes Denature proteins Gluteraldehyde - disinfectant (Cidex); Formaldehyde - disinfectant Ethylene Oxide Denaturing proteins Used in a closed chamber to sterilize Oxidizing agents Denature proteins Hydrogen peroxide – antiseptic; Hydrogen peroxide – disinfectan; 38 Benzoyl peroxide – antiseptic Evaluation of disinfectant by disk diffusion method 39 40 41 42  Definition Sterilization Destruction or removal of all forms of Usually done by steam under pressure microbial life including endospores but with or a sterilizing gas such as ethylene the possible exception of prions. oxide. Commercial Sufficient heat treatment to kill endospores More-resistant endospores of Sterilization of Clostridium botulinum in canned food. thermophilic bacteria may survive but they will not germinate and grow under normal storage conditions Disinfection Destruction of vegetative pathogens. May make use of physical or chemical methods. Antisepsis Destruction of vegetative pathogens on Treatment is almost always by chemical living tissue. antimicrobials. Degerming Removal of microbes from a limited area, Mostly a mechanical removal by an such as the skin around an injection site. alcohol-soaked swab. Sanitization Treatment intended to lower microbial May be done with high-temperature counts on eating and drinking utensils to washing or by dipping into safe public health levels. a chemical disinfectant. 43 |Resistance of microorganisms to chemicals 44 45 Microbial metabolism 46 Ø Metabolism is the sum of the chemical reactions in an organism Ø Metabolic reaction: are enhanced and regulated by enzymes known as metabolic enzymes Ø Metabolic enzymes enhances and regulates metabolic reaction Ø Metabolic pathway: is a sequence of enzymatically catalyzed chemical reactions in a cell Ø Metabolite: any molecule that is a nutrient, an intermediary or end product of metabolism 47 48 49 ØCatabolism vIs the energy-releasing processes (Destructive metabolism). vBreakdown of complex organic molecules into simpler compounds ADP + Pi + energy (released) à ATP ØAnabolism vThe energy-using processes (Constructive metabolism) vThe building of complex organic molecules from simpler ones ATP à ADP + Pi + energy (to be used) 50  Types of Enzymes Endoenzymes: Enzymes produced within a cell that remain within the cell to catalyze reactions (digestive enzymes) Exoenzymes: Enzymes produced within the cell and. released from the cell (cellulase) 51  Metabolic Enzymes An enzyme does not become altered during a chemical reaction that it catalyzes At the end of the reaction è enzyme remains unchanged Enzyme activity is affected by multiple factors – Temperature: optimal temperature for most disease- producing bacteria in the human body is 35°C - 40°C – PH: Most enzymes have an optimum pH – Substrate concentration – Inhibitors: Competitive and non competitive inhibitors 52  4 Stages of Aerobic Respiration Glycolysis Formation of acetyl CoA Krebs cycle Electron transport chain (ETC) and chemiosmosis 54  Glycolysis § A ten-step biochemical path, involving ten separate biochemical reactions, each of which requires specific enzymes. § Six-carbon molecule of glucose is broken down into three- carbon molecules of pyruvic acid. § Can take place with or without oxygen. § Produces very little energy: only 2 ATP (and 2 NADH) § Takes place in the cytoplasm of both prokaryotic and eukaryotic cells. 55  Formation of acetyl CoA Occurs in the mitochondria after the 2 pyruvate enters the mitochondria. Each of the 2 pyruvate (3 carbon) will be catabolised into one acetyl coenzyme A ( 2 carbon).. This step allows the net yield of 2 NADH and 2 Glycolysis CO2 No ATP production during this phase 56  Krebs Cycle The pyruvic acid molecules produced during glycolysis are converted into acetyl-CoA molecules. Krebs cycle The Krebs Cycle is consists of eight separate reactions, each of which is controlled by a different enzymes. Only 2 ATP produced (6 NADH, 2 FADH2) Krebs cycle takes place in mitochondria (eukaryotes); inner surface of cell membrane (prokaryotes). 57  At the end of citric acid cycle 1 Glucose has been catabolized (degraded) into: 4 ATP ( substrate level phosphorylation), 10 NADH and 2 FADH2 58  Electron Transport Chain and Chemiosmosis (oxidative phosphorylation) Certain of the products produced during the Krebs cycle enter the electron transport chain. It consists of a series of oxidation-reduction reactions, whereby energy is released as electrons are transferred from one compound to another. Oxygen is at the end of the chain; referred to as then final or terminal electron acceptor. Electron Transport Chain: is a group of complexes in the inner membrane of the mitochondria that will transport H atoms (or electrons) from NADH/ FADH2 to reduce molecular oxygen O2 ( final acceptor of electrons) forming water. Chemiosmosis: protons (H+) leave the matrix to the intermembrane space then diffuse down their gradient, back to the matrix, through the ATP synthase forming ATP (ADP + Pi) 59 Through oxidative phosphorylation: Each NADH will allow the production of 3 ATPs ( 10 NADH will produce 30 ATPs) Each FADH2 will allow the production of 2 ATPs (2 FADH2 will produce 4 ATPs) In total, from oxidative phosphorylation, 34 ATPs are produce per each molecule of Glucose. (34 in prokaryotic cells and 32 in eukaryotic cells) So in Aerobic Respiration, both prokaryotes and eukaryotes produce the same amount of ATP which is thought to be 38 ATPs. Some textbooks state that the net yield is 36 ATPs in eukaryotes because 2 ATPs are used up to power cellular respiration itself in moving 2 NADH molecules into a mitochondrian (38 ATPs - 2 ATPs = 36 ATPs). 60 Sugar fermentation and acidic end product 61  Anaerobic respiration In anaerobic respiration, the final electron acceptor is an inorganic substance other than oxygen (02) Bacteria can use Ø Nitrate ion: the nitrate ion is reduced to a nitrite ion, nitrous oxide or nitrogen gas (N2). Ex: Pseudomonas and Bacillus Ø Sulfate S04 2- as the final electron acceptor to form hydrogen sulfide (H2S) Ø Carbonate (C03 2- ) to form methane (CH4) 62  Nitrate test Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase. It also tests the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen Reagents: Sulphalinic acid reagent, Alpha napthylamine reagent, zinc dust 63 Ø The nitrate reduction test is based on the detection of nitrite and its ability to form a red compound when it reacts with sulfanilic acid to form a complex (nitrite-sulfanilic acid) which then reacts with a α-naphthylamine to give a red precipitate Media with bacteria (prontosil), which is a water-soluble azo dye. Pink /red color develops No pink/red color develops Good reducing agent Nitrite is present, Pinch of zinc Bacteria are nitrate is added (+) Pink/red color No pink/red color develops All Nitrate completely Nitrate present in reduced to nitric oxide, media before nitrous oxide, or addition of Zn nitrogen Bacteria are nitrate Bacteria are nitrate (+) (-) 64  Anaerobic Fermentation of Glucose No oxygen is used Involves two steps: 1. Glycolysis è produces two ATP molecules 2. Conversion of Pyruvic acid into an end product – Ethyl alcohol in alcoholic fermentation – Lactic acid in lactic acid fermentation End energy product = 2 ATP molecules 65  Fermentation During fermentation: There is release of energy from sugars or other organic molecules, such as amino acids, organic acids, purines, and pyrimidines oxygen is not required (but sometimes can occur in its presence) Fermentation does not require the use of the Krebs cycle or an electron transport chain uses an organic molecule as the final electron acceptor produces only small amounts of ATP (only one or two ATP molecules for each molecule of starting material) Fermentation produces only 2 ATP 66 Industrial Use For Fermentation 67 68 Ø End product depends on specific organism involved 69 70 71 72 Summary 73  Cell Requirements All organisms require a source of energy, a source of carbon, and additional nutrients Essential nutrients – Materials that organisms are unable to synthesize – Required for sustaining life Essential nutrients must be continually supplied in order for an organism to survive Essential nutrients vary from species to species 74 75  Categorizing Microorganisms Microorganisms are categorized according to their energy and carbon sources Four major categories/groups I. Photoautotrophs II. Photoheterotrophs III. Chemoautotrophs IV. Chemoheterotrophs 76 77  Terms Relating to Organism’s Carbon Source § Autotrophs: use CO2 as their carbon source (plants, algae, and cyanobacteria) § Photoautotrophs: use light energy and CO2 § Chemoautotrophs: use chemicals as energy source and CO2 as carbon source § Heterotrophs: use organic compounds as their carbon source (Humans, animals, fungi, bacteria and protozoa) § Photoheterotrophs: use light and organic compounds § Chemoheterotrophs: use inorganic energy sources to synthesize organic compounds from carbon dioxide 78