Antimicrobial agents
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1 Bacteriology Antimicrobial agents Learning Outcomes: At the end of this lecture, the students should be able to: Identify mechanisms of action of antimicrobial Drugs Know and understand key concepts about the antibiotics Master the concept of antibiotic combinations Define the basic terms frequently used in describing aspects of sterilization Antimicrobial agents Antimicrobial agents are any chemical substances that inhibit the growth or causing the death of microorganism. Antibiotic: Substance produced by a microorganism in small amounts and then inhibits microorganism Antimicrobial drugs interfere with the growth of microbes within a host. Chemotherapeutic agents is an antimicrobial agents synthesized artificially but not produced by microorganism Antimicrobials used to inhibit or destroy living organisms which are uninvited guests Drugs must harm invader without harming host Notion of selective toxicity (It must inhibit bacterial processes significantly more than it inhibits human cell processes). Chemotherapy: The use of drugs to treat a disease 1
2 Bactericidal drugs kill bacteria, whereas bacteriostatic drugs inhibit the growth of the bacteria but do not kill them. The silent features of the behavior of bacteriostatic drugs are that: 1. The bacteria can grow again when the drugs is withdrawn 2. Host defense mechanisms, such as phagocytosis, are required to kill the bacteria. Mechanisms of Action of Antimicrobial Drugs Main targets of antibacterial drugs: Cell wall Ribosomes Cell membrane Nucleic acids Inhibition of cell wall synthesis Penicillins (and cephalosporins) acting by inhibiting transpeptidase, the enzymes that catalyze the final cross-linking step in the synthesis of peptidoglycan. Additional factors are involved in the action of penicillin: 1. Penicillin-binding proteins (PBPs): Penicillin binds to a variety of receptors in the bacterial cell membrane and wall, called PBPs. Some PBPs are transpeptidase; the others function in the synthesis of peptidoglycan. 2. Autolytic enzymes: called murein hydrolases (murein is a synonym for peptidoglycan) are activated in penicillin-treated cells and degrade the peptidoglycan. If these autolytic enzymes are not activated (e.g., in certain strains of Staphylococcus aureus), the bacteria are not killed and the strain is said to be tolerant. 2
3 Penicillins kill bacteria when they are growing (i.e., they are synthesizing new peptidoglycan). Penicillins are therefore more active during the log phase of bacterial growth. Penicillins and cephalosporins are β-lactam drugs β-lactamases cleave the β-lactam ring and inactivate drug Vancomycin is a glycopeptide (i.e., it is not β-lactam drug), inhibits transpeptidase. Inhibition of protein synthesis ex: aminoglycosides (streptomycin, kanamycin, neomycin, gentamycin). Erythromycin, tetracycline and chloram pheniol streptomycin combine with the 30s component of the ribosome causing misreading of the genetic code so that the wrong amino acids are inserted into the growing polypeptide chain causing synthesis of non-functional protein & the cell eventually dies. Inhibition of Nucleic Acid Synthesis Rifampin inhibits RNA synthesis by blocking the RNA polymerase Sulfonamides and trimethoprim inhibit the synthesis of folic acid Inhibition of Cell Membrane Function E.g., colistion, triazoles, amphotericinb, imidazoles, nystatin, polymyxins. Polymyxin acts as a cationic detergent binding specifically to the cytoplasmic membrane of G-ve bacteria. As a result the semipermeable properties of the membrane are lost & the essential low-molecular weight intermediates & coenzymes pass from the cell to the environment causing cell death. 3
4 Resistance to antimicrobial drugs There are four major mechanisms that mediate bacterial resistance to drugs: 1. Bacteria produce enzymes that inactivate the drugs (e.g., β-lactamases can inactivate penicillins and cephalosporins by cleaving the β-lactam ring of the drug). 2. Bacteria synthesize modified targets against which the drug has a reduced effect (e.g., a mutant protein in the 30S ribosomal subunit can result in resistance to streptomycin). 3. Bacteria reduce permeability to the drug such that an effective intracellular concentration of the drug is not achieved (e.g., changes in porins can reduce the amount of penicillin entering the bacterium). 4. Bacteria actively export drugs using a multidrug resistance pump (MDR pump, or efflux pump). The MDR pump import protons and, in an exchange-type reaction, export a variety of foreign molecules including certain antibiotics, such as tetracyclines. How do Bacteria Become Resistant? Most drugs resistance is due to: Genetic basis of resistance Chromosomal-mediated resistance Chromosomal mutations typically either 1. Change the target of the drug so that the drug does not bind 2. Change the membrane so that the drug does not penetrate well into cell. Chromosomal mutations occur at a low frequency and often affect only one drug or one family of the drugs. 4
5 Plasmid-mediated resistance 1. It occurs in many different species, especially gram negative rods 2. Plasmids frequently mediate resistance to multiple drugs 3. Plasmids have a high rate of transfer from one cell to another by transduction, transformation, conjugation, and transposition. Transduction : plasmid DNA is enclosed in a bacterial virus (bacteriophage) & transferred by the virus to another bacteria of the same spp. E.g., the plasmid carrying the gene for β-lactamase production (in staph.) can be transferred from a p-resistant to susceptible staph. if carried by suitable bacteriophage. Transformation : naked DNA passes from one bacterial cell of a spp. To another cell thus altering its genotype, this occurs in laboratory manipulation or spontaneously. Bacterial conjugation: a unilateral transferring of genetic material between bacteria of the same or different genera occur during a mating process. This is mediated by a fertility (F) factor that result in the extension of sex pili from the donor (F+) cell to the recipient. Plasmid or other DNA will transfer through these tubules from donor to recipient cell. Such a resistance transfer factor (RTF) is the commonest method of spread of multidrug resistance in G-ve bacteria. Transposition: a transfer of gens that are transferred either within or between larger pieces of DNA such as the bacterial chromosome and plasmids. 5
6 Non genetic basis of resistance 1. Bacteria can be walled off within an abscess cavity that the drug cannot penetrate effectively. 2. Bacteria can be in a resting state (i.e., not growing); they are therefore insensitive to cell wall inhibitors such as penicillins and cephalosporins. 3. Under certain circumstances, organisms that would ordinarly be killed by penicillin can lose their cell walls, survive as protoplast, and be intensive to cell wall-active drugs. Later, if such organisms resynthesize their cell walls, they are fully susceptible to these drugs. 4. Presence of foreign bodies makes successful antibiotic treatment more difficult. This applies to foreign bodies such as a surgical implants and catheters as well as materials that enter the body at the time of penetrating injures, such as splinters and shrapnel. 5. Several artifacts can make it appear that the organisms are resistant (e.g., administratration of the wrong drugs or the wrong dose or failure of the drugs to reach the appropriate site in the body). Selection of antibiotics It depends on : 1. Etiological diagnosis (the causative microorganism). 2. Susceptibility tests of organism to the antibiotics in the lab. Antimicrobial activity in vitro Antimicrobial activity is measured in vitro in order to determine: 1. The potency of an antibacterial agent in 2. The concentration in body fluid or tissues 6
7 3. The sensitivity of a given M.O to known conc. of the drug by I. Dilution test: graded amounts of antimicrobial substance are incorporated in to liquid or solid bacteriologic media. Then the media is inoculated with test bacteria & incubated. The end point is taken as that amount of antimicrobial sub. required to inhibit the growth of or to kill the test bacteria (Figure 1). II. Diffusion method: a filter paper disk containing measured quantities of drug is placed on a solid media that have been heavily seeded with the test organisms, after incubation the diameter of the clear zone of inhibition surrounding the deposit of drug is taken as a measure of inhibitory power of the drug against the test organism (Figure 2) Figure 1: Antimicrobial activity (Dilution test) 7
8 Figure 2: Antimicrobial activity (Diffusion method) 8
9 Antimicrobial activity in vivo In vivo much more complex than in vitro. It involves: Drug, Host, parasite, and bacteria Host factors: 1. State of metabolic activity. 2. Distribution of agent. 3. Site of the infection 4. Absorption of drug. 5. Distribution of drug in the body Host pathogen relationship may be altered by antimicrobial drugs as: 1. Alteration of tissue response and then become chronic. 2. Alteration of immune response Antibiotic Combinations In most cases, the single best antimicrobial agent should be select for use because this minimizes side effects. However, there are several instances in which two or more drugs are commonly given: 1- To treat serious infections before the identity of the organism is known. 2- To achieve a synergistic inhibitory effect against certain organisms. 3- To prevent the emergence of resistance organisms. Synergism: Interaction of 2 or more agents so that their effects are combined and became higher than the sum of their individual effects (Figure 3). 1. Two drugs may block a microbial metabolic pathway, sulfonamide inhibit the use of extracellular ρ-aminobenzoic acid for the synthesis 9
10 of folic acid in some bacteria Trimethoprim inhibit the next metabolic step. the reduction of dihydro to tetrahydrofolic acid. 2. Pencillin + gentamicin or streptomycin. A cell wall inhibitor may enhance the entry of aminoglycoside gentamicin or streptomycin in to bacteria that act on protein synthesis. 3. Polymyxin + trimethoprim one drug affects cytoplasmic membrane & facilitates the entry of 2nd drug. 4. One drug may prevent the inactivation of a second (inhibitors of a βlactamase as calvulanic acid can protect amoxicillin for inactivation for inactivation by β- lactamases). Figure 3: Antibiotic combination (Synergism) Antagonism: Interaction of two or more agents that in combination have an overall effect which is less than the sum of their individual effects (Figure 4). (e.g., bacteriostatic drug as chloramphenicol or tetracycline + bactericidal as penicillin or aminoglycoside). 10
11 Figure 4: Antibiotic combination (Antagonism) Chemoprophylaxis Administration of antimicrobial drugs to prevent infection as in : 1. Persons exposed to a specific pathogen. 2. P. of increased susceptibility: a) Heart disease. b) RT disease. c) Recurrent UTI. 3. In surgery. Sterilization Sterilization The killing or removal of all viable organisms within a growth medium. Decontamination The treatment of an object to make it safe to handle. Disinfection Is reducing the number of bacteria to a level low enough that disease is unlikely to occur. 11
12 Disinfactant: This is a term applied to chemicals used to kill potentially infectious organisms. They are used in the treatment of inanimate objects, surface, water...etc. Their potential toxicity to man may not be important (since it s not such to man). Antiseptics (mild disinfectants) The term refers to relatively non-toxic & non-irritant antimicrobial agent that may be applied topically to the body surface either to kill or inhibit the growth of pathogenic microorganism. Sterilization may be accomplished by physical methods: 1. Filtration (in the case of liquid) 2. Heat 3. Radiation Chemical methods: 1. Alcohols: are toxic to cells in high conc. (ethyl & isoprophlatic) 70% they denaturize proteins. 2. Phenol: they denaturize proteins. 3. Heavy metal ions: mercury, copper, silver and salt. 4. Oxidizing agents: H2O2, chlorine, formaldehyde & ethylene, F-detergents and surface active. 12
13 Summary Antibiotic must be selective toxicity Cell wall, ribosomes, cell membrane, and nucleic acids are the targets of antibacterial drugs. Bactericidal drugs kill bacteria Bacteriostatic drugs inhibit the growth of the bacteria Mechanisms of antibiotic resistance (1) enzymatic degradation of the drugs (2) modification of the drug target (3) reduce permeability of the drugs. (4)active export Drug resistance is the result of a genetic change in the organism, or of a nongenetic basis Use of antibiotic combinations result in a synergistic effect and antagonism effect. Sterilization is the killing of all forms of microbial life. Further readings 1. Jawetz, Melnick and Adelberg s Medical Microbiology (Brooks, Butel,Morse). 25 th ed. Copyright Levinson W. Review of Medical Microbiology and Immunology.12 th ed. Copyright 2012, McGraw-Hill. 13
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