Control of Microorganisms Unit 7 PDF

Control of Microorganisms Unit 7 1  Objectives Describe physical treatments used to control the growth of microorganisms. Describe the chemical treatments used to control the growth of microorganisms. Explain how chemotherapeutic agen...

Control of Microorganisms Unit 7 1  Objectives Describe physical treatments used to control the growth of microorganisms. Describe the chemical treatments used to control the growth of microorganisms. Explain how chemotherapeutic agents such as antibiotics control microbial growth. 2  Control of Microorganisms Outline: Definitions Conditions Influencing Microbial Activities Physical Methods Chemical Methods Chemotherapeutic Agents 3  Definitions Sterilization: A treatment that kills or removes all living cells, including viruses and spores, from a substance or object. Disinfection: A treatment that reduces the total number of microorganisms on an object or surface, but does not necessarily remove or kill all of the microorganisms. 4  Definitions Sanitation: Reduction of the microbial population to levels considered safe by public health standards Antiseptic: A mild disinfectant agent suitable for use on skin surfaces -cidal: A suffix meaning that “the agent kills.” For example, a bacteriocidal agent kills bacteria 5  Definitions static: A suffix that means “the agent inhibits growth.” For example, a fungistatic agent inhibits the growth of fungi, but doesn’t necessarily kill it. 6 Conditions Influencing Antimicrobial Activity – Population size – Types of organisms – Concentration of the antimicrobial agent – Duration of exposure – Temperature – pH 7  Antimicrobial Targets Cell membrane Enzymes & Proteins DNA & RNA 8  Established methods of microbial control 1. Physical agents used exclusively on objects outside the body 2. Chemical agents used on inanimate objects as, well as on the body surface 3. Chemotherapeutic agents most often used inside the living body 9  Physical Methods of Control Moist Heat Dry Heat Low Temperatures Filtration Irradiation Drying Osmotic Strength 10  Moist Heat Mechanism: protein/nucleic acid denaturation and membrane disruption Presence of spores- more difficult to kill Effectiveness dependent on: type of cells present environment (type of medium or substrate) 11  Moist Heat Measurements of killing: Thermal Death Point (TDP) – lowest temp. at which all microorganism in a liquid culture are killed in 10 minutes Thermal Death Time (TDT) – time required to kill a known population of microorganisms in a specific suspension at a particular temperature does not account for the logarithmic nature of the death curve (theoretically not possible to get down to zero) 12  Methods of Moist Heat Methods: Boiling at 100 degrees Celsius Autoclaving Pasteurisation Tyndallisation 13  Boiling Kills most vegetative bacteria and viruses within 10 minutes( not ideal for heat sensitive chemical etc). Generally done at 100 °C for 30 minutes. Bacterial endospores can survive boiling temperatures Some bacterial toxins are heat resistant e.g. Staphylococcal enterotoxin 14  Autoclaving (steam under pressure) Preferred method of sterilization Water boils at 100 C higher temp. may be obtained under pressure. Increasing the pressure:15 psi raises the Temp. 121C 121 C for 15-30 min. NB!! validated autoclave by testing with spores of Clostridium or Bacillus stearothermophilus 15  Schematic diagram of a laboratory autoclave in use to sterilize microbiological culture medium The sterilization process is a 100% kill, and guarantees that the medium will stay sterile unless exposed to contaminants. 16  Pasteurization Reduces microbial count in milk/beverages Eliminates the transmission of Coxiella burnetti, Mycobacterium tuberculosis, Brucella, Staphylococcus, Salmonella and E. coli. Initially food was heated at 66ºC for 30 minutes Flash pasteurization 71 ºC for 15 seconds. 140 – 150ºC for 1-3 sec (UHT) Does not sterilize 17  Tyndallisation Boiling a solution for 30 minutes boiled medium is cooled incubated for a period of hours boiled again and this cycle is repeated three times. – Cooling facilitates germination of endospores into heat-sensitive vegetative cells. 18  Dry Heat Incineration – Burner flames, electric loop incinerators Oven sterilization – glassware & heat-resistant metal equipment – generally 2 hr at 160°C is required to kill bacterial spores by dry heat-does not include penetration nor cooling time 19  Low Temperature Refrigeration – Temperature: 4°C – inhibits growth of mesophiles and thermophiles but not psychrophiles Freezer: – “ordinary” freezer around -10 to -20°C – “ultracold” laboratory freezer typically -80°C – Generally inhibits all growth; many survive freezing temperatures 20  Low Temperature Low temperature is not damaging to most microorganisms For most when brought up to suitable temperatures, will begin growing again. Bacterial cells are too small for ice crystals to form within them, they are not killed by the mechanical destruction of cellular structures. Instead they are killed by the high osmotic strength that develops as water in the environment freezes 21  Filtration mechanical device for removing microorganisms from a solution. The organisms are trapped in the pores of the filter, and the filtrate is decontaminated or possibly sterilized. – culture media – enzymes – vaccines – antibiotics 22  Filtration Three types of Filters: Depth filters: fibrous sheet or mat forming a randomly arranged lattice of paper, asbestos etc – Traps particles in network Membrane filters: most common type made of polymers with high tensile strength, functioning like a sieve eg. Nitrocellulose, nylon, polyvinylidene difluoride 23  Filtration HEPA filters: High efficiency particulate air filters used in laminar flow biological safety cabinets. Filtration does not remove viruses from solution -too small – In essence the solution is nonsterile 24  Irradiation high energy electromagnetic radiation used in the reduction of microbial load. Types of electromagnetic radiation: UV light X-rays Gamma rays Electrons beams 25  Irradiation Types of Radiation : 1. ionizing radiation 2. Non-ionizing radiation. Ionizing Radiation; has enough energy to remove electrons from a target molecule causing it to form ions. Eg Xrays, gamma rays and electron beam.  Powerful sterilizing agent; penetrates and damages both DNA and protein; effective against both vegetative cells and spores 26  Irradiation 2. Non-Ionizing Radiation – UV Light has, a wavelength between 100 and 400 nm, and the energy at about 265 nm is most destructive to bacteria. The spectrum of visible and invisible energies. Exposure to UV damages the DNA. UV radiations are used to reduce air contamination. 27  Drying removal of H2O involve removal of water from product by heat, evaporation, freeze-drying Frequently used to preserve perishable materials such as proteins, blood products and reference cultures of microorganisms, Often used to preserve foods (e.g. fruits, grains, etc.). 28  Osmotic Strength Utilises high concentrations of salt or sugar High osmotic strength of salt and sugar solution - damage cells by plasmolysis. Method not used routinely in laboratory 29  Chemical Methods of Control Antimicrobial agents employs the use of natural or synthetic chemicals that kills or inhibit microbial growth Employs technique of selective toxicity Denature protein and disrupt membranes Rarely achieve sterilization as in physical methods. The process of removal is called disinfection. If the object is non living, the chemical is known as disinfectant, if the object is living, as a tissue of human body, then the chemical is an antiseptic. 30  Chemical Agents Phenolics Alcohols Halogens Heavy metals Quaternary Ammonium Compounds Aldehydes Sterilizing Gases Evaluating Effectiveness of Chemical Agents 31 Table showing: Antiseptics, sterilants, disinfectants and sanitizers 32  Chemical Agents: Disinfectants Chemicals used to kill pathogenic microorganisms May or may not kill endospores Used on inanimate objects Include: chlorine compounds such as hypochlorites, copper sulfate quaternary ammonium compounds. Uses: Important in infection control Decontaminate surfaces eg tables, floors etc 33  Chemical Agents: Antiseptics inhibit or kill microorganisms and are safe to use on the skin and mucous membranes, but are normally not taken internally. Examples include: Mercurials silver nitrate iodine solution Alcohol Used to reduce but not eliminate microbial load to a safe number. 34  Chemical Agents: Sterilants Sterilizers or sporicides Destroy vegetative cells and endospores Ideally used when the use of heat or radiation is not practical 35  Types of agents High level Germicides – These are generally alkylating agents, which kill by adding alkyl groups to nucleic acids or proteins. Intermediate-Level Germicides – little activity against endospores. 36  Phenols Aromatic organic compounds with attached -OH Denature protein & disrupt membranes Phenol, orthocresol, orthophenylphenol, hexachlorophene Commonly used as disinfectants (e.g. “Lysol”); are tuberculocidal, effective in presence of organic matter, remain on surfaces long after application 37  Phenol is the standard disinfectant, which coagulates the proteins, particularly cell membrane enzymes. especially useful against Gram-positive bacteria. An alternative of phenol, cresol has become more popular in modern medicine as it is cheaper than phenol. Because of its toxicity, this compound is generally used as a solution between 2% to 5% in concentration 38  Halogens Act as oxidizing agents; oxidize proteins & other cellular components Most commonly used halogens chlorine and iodine Chlorine compounds – Used in disinfecting municiple water supplies (as sodium hypochlorite, calcium hypochlorite, or chlorine gas) – Sodium Hypochlorite (Chlorine Bleach) used at 10 - 20% dilution as bench top disinfectant 39  Iodine lethal to all vegetative forms of microorganisms, can inactivate viruses. Pure iodine is caustic to tissues, so it is diluted with other compounds: Iodine Compounds – Tincture of iodine (iodine solution in alcohol) – Potassium iodide in aqueous solution – Iodophors: Iodine complexed to an organic carrier; e.g. Wescodyne, Betadyne – Used as antiseptics for cleansing skin surfaces and wounds 40 3. Low level Germicides- ineffective against M. tuberculosis but are active against other vegetative cells, fungi and some viruses. Hydrogen Peroxide a weak acid, with strong oxidizing properties powerful bleaching agent, disinfectant, antiseptic, Mechanism of action: releases the peroxide ion which is a strong oxidizing agent, and the water released provides hydroxide ions which strip hydrogen from biological molecules (oxidizing them). 41  Alcohols effective antiseptic applied to skin. The most common is ethyl alcohol, though propyl, butyl and pentyl alcohols have a greater germicidal ability. Ethanol acts particularly on vegetative bacterial cells. It is strong dehydrating agent. Ethyl alcohol (70%) is mostly used. Mechanisms: kill microorganisms by denaturing proteins, dehydration (100% concentration), and as solvents which disrupt the phospholipid structure of the cell membrane. Also proteins are not soluble in high concentrations of alcohol, hence won’t coagulate hence do not use 100% alcohol as germicide. 42  Heavy Metals The activity on microorganisms is termed oligodynamic action. Metals as silver, mercury(as mercuric chloride) (HgCI2) and copper are used. In products like mercurochrome, mercury is combined with organic carrier compounds, that reduces its toxicity to skin. Copper is particularly active against algae. 43  Mechanism: combine with sulfur groups in the proteins of microorganisms, causing them to denature. Silver is also toxic, and is applied as silver nitrate (AgNO3) in a 1% solution, was commonly used to inhibit the growth of Neisseria gonorrhoeae in the eyes of newborn infants- antibiotics are used instead. Copper, in the form of copper sulfate used to limit the growth of algae in ponds and lakes. 44  Detergents and soaps Detergents and soaps (surfactants)are compounds that have hydrophillic and hydrophobic parts. Detergents are synthetic chemicals developed for their ability to be strong wetting agents and surface tension reducers. They destabilize the plasma membrane of microorganisms. To some extent it also destroys microorganisms due to alkalinity- about ph 8.0 Soap is used for mechanical washing of the skin surface. 45  Quartenary ammonium compounds (QUATS) A major class of surfactant germicides. Quats such as benzalkonium chloride (Zephiran ) – have broad-spectrum inhibitory activity against bacteria, fungi, and protozoa – are mildly antiseptic and disinfecting when used as cleaning agents for laboratory fomites and on the surface of skin – remain active after drying, but lose much of their activity when mixed with soaps. 46  Factors affecting effectiveness of disinfectants Biofilms, which are populations of microorganisms that grow on surfaces and encase themselves in excreted layers of polysaccharide. Biofilms can greatly retard or even prevent the diffusion of disinfectants to the microorganisms, eliminating the effectiveness of the compound. The composition of the item being disinfected can also alter the activity of a disinfectant. High concentrations of organic compounds decrease the potency of disinfectants and it is usually prudent to clean a surface before adding disinfectant. Endospores are characteristically more resistant to disinfectants than are vegetative cells. However, certain agents kill spores. 47  Antibiotics and chemotherapeutic agents Antibiotics are low-molecular weight substances that are produced as secondary metabolites by certain groups of microorganisms, especially Streptomyces, Bacillus, and a few molds (Penicillium and Cephalosporium) that are inhabitants of soils. Many are now synthetic or semi-synthetic compounds produced by the pharmaceutical industry. (that is, natural metabolites are isolated and then chemically modified) 48  A standard assay often used to gauge the effectiveness of an antimicrobial is the minimum inhibitory concentration (MIC) test. The MIC is the lowest concentration of a compound that still inhibits the growth of a microorganism. The MIC of a given compound for a certain bacterial species is determined using a series of test tubes containing medium in which the microbe will normally grow. 49  Each tube contains progressively lower concentrations of the test compound. Each tube is inoculated with the microbe and after incubation the tubes in which growth does not occur are noted. The lowest concentration of the antimicrobial compound that prevents growth defines the MIC. 50  In some situations it is important to determine the minimum concentration at which a compound is lethal to a microorganism and this is defined as the minimal lethal concentration (MLC). Antimicrobials that are cidal will normally kill a microorganism at two to four times their inhibitory concentration. A static agent will require a much higher concentration and may not ever be lethal. 51  Another commonly used assay to assess the potency of an agent is the agar diffusion method. A microbial culture is spread evenly on the top of an agar plate containing medium that will support its growth. Disks impregnated with antimicrobial compounds are then placed onto the agar and the plate incubated at an appropriate temperature for that microbe. During incubation the antimicrobial compound diffuses away from the disk and into the agar creating a concentration gradient that is highest near the disk and decreases as one moves away from the disk. 52  If a microbe is inhibited by the agent, it will be unable to grow near the disk, which we see as a zone of clearing in the lawn of growth. Farther away from the disk, where the concentration of the antimicrobial compound is much lower, growth will be evident. The size of the zone of clearing around the disk is an indication of the potency of the antimicrobial for the tested microbe. 53  Antibiotics The range of bacteria or other microorganisms that is affected by a certain antibiotic is expressed as its spectrum of action. Antibiotics effective against prokaryotes which kill or inhibit a wide range of Gram-positive and Gram- negative bacteria are said to be broad spectrum. If effective mainly against Gram-positive or Gram- negative bacteria, they are narrow spectrum. If effective against a single organism or disease, they are referred to as limited spectrum. 54 A clinically-useful antibiotic should have as many of these characteristics as possible: It should have a wide spectrum of activity with the ability to destroy or inhibit many different species of pathogenic organisms. It should be nontoxic to the host and without undesirable side effects. It should be nonallergenic to the host. 55  It should not eliminate the normal flora of the host. It should be able to reach the part of the human body where the infection is occurring. It should be inexpensive and easy to produce. It should be chemically-stable (have a long shelf-life). Microbial resistance is uncommon and unlikely to develop 56  Mode of Action of antibiotics Cell wall synthesis inhibitors: include two widely used classes of antibiotics, the penicillins and cephalosporins. Both contain a β- lactam ring. They act on various Gram positive and Gram negative rods and cocci, responsible for various diseases. They inhibit the formation of peptide cross linkages within the peptidoglycan backbone of the cell wall. 57  The beta lactam antibiotics are stereochemically related to D-alanyl-D-alanine which is a substrate for the last step in peptidoglycan synthesis, the final cross-linking between peptide side chains. Penicillins bind to and inhibit the carboxypeptidase and transpeptidase enzymes that are required for this step in peptidoglycan biosynthesis. Beta lactam antibiotics are normally bactericidal and require that cells be actively growing in order to exert their toxicity. 58  Semi Synthetic Penicillins In the late 1950s, the betalactum nucleus of the penicillin molecule was identified and synthesised. Various groups then could be attached to this nucleus, creating a number of new penicillins. At present thousands of penicillins are prepared by this semi-synthetic process. E.g. Ampicillin, Amoxicillin 59  Cephalolsporins Are beta lactam antibiotics with a similar mode of action to penicillins that are produced by species of Cephalosporium. They have a low toxicity and a somewhat broader spectrum than natural penicillins. They are often used as penicillin substitutes, against Gram-negative bacteria 60  Cell membrane inhibitors These antibiotics disorganize the structure or inhibit the function of bacterial membranes. The integrity of the cytoplasmic and outer membranes is vital to bacteria, and compounds that disorganize the membranes rapidly kill the cells. However, due to the similarities in phospholipids in eubacterial and eukaryotic membranes, this action is rarely specific enough to permit these compounds to be used systemically. 61  The only antibacterial antibiotic of clinical importance that acts by this mechanism is polymyxin, produced by Bacillus polymyxis Polymyxin is effective mainly against Gram- negative bacteria and is usually limited to topical usage. Polymyxin binds to membrane phospholipids and thereby interferes with membrane function. 62  Protein synthesis inhibitors Many therapeutically useful antibiotics owe their action to inhibition of some step in the complex process of protein synthesis. Their attack is always at one of the events occurring on the ribosome and never at the stage of amino acid activation or attachment to a particular tRNA. Most have an affinity or specificity for 70S (as opposed to 80S) ribosomes, and they achieve their selective toxicity in this manner. 63  The most important antibiotics with this mode of action are the tetracyclines, chloramphenicol, the macrolides (e.g. erythromycin) and the aminoglycosides (e.g. streptomycin). 64  Effects on Nucleic Acids Some antibiotics and chemotherapeutic agents affect the synthesis of DNA or RNA, or can bind to DNA or RNA so that their messages cannot be read. They block the growth of cells. The majority of these drugs are unselective, however, and affect animal cells and bacterial cells alike and therefore have no therapeutic application. Two nucleic acid synthesis inhibitors which have selective activity against prokaryotes and some medical utility are the quinolones (eg. Nalidixic acid and rifamycins (eg. Rifampicin ). 65  Competitive Inhibitors Many of the chemotherapeutic agents are competitive inhibitors of essential metabolites or growth factors that are needed in bacterial metabolism. Hence, these types of antimicrobial agents are sometimes referred to as anti-metabolites or growth factor analogs, since they are designed to specifically inhibit an essential metabolic pathway in the bacterial pathogen. At a chemical level, competitive inhibitors are structurally similar to a bacterial growth factor or metabolite, but they do not fulfil their metabolic 66 function in the cell.  Some are bacteriostatic and some are bactericidal. Their selective toxicity is based on the premise that the bacterial pathway does not occur in the host. The sulfonamides (e.g. Gantrisin) are examples of inhibitors of the bacterial enzymes required for the synthesis of tetrahydofolic acid (THF), the vitamin form of folic acid essential for 1-carbon transfer reactions. 67  Reference http://www.textbookofbacteriology.net/contr ol.html http://microbiology.suite101.com/article.cfm/ control_of_microorganisms http://www.microbiologyprocedure.com/micr obial-control/microbial-control.htm http://www.microbiologyprocedure.com/micr obial-control/microorganisms-controlling-by- heavy-metals.htm 68  References Ingraham, J. L. (2000). Introduction to microbiology (2nd ed.). Pacific Grove, CA: Brooks/Cole. Chapter 9. Kelly Cowan, M. M. & Talaro, K. P. (2006). Microbiology: a systems approach (1st ed.). New York, NY: McGrawhill. Chapter 11. Madigan, M. T., Martinko, J. M., Dunlap, P. V. & Clarke, D. P. (2009). Brock biology of microorganisms (11th ed.). San Francisco, CA: Pearson Education. Chapter 27. Tortora, G. J., Funke, B. R. & Case, C. L. (2010). Microbiology: an introduction (10th ed.). San Francisco, CA: Pearson Education. Chapter 7. 69