Pharmacy 754, Spring 2007 INTRDUCTIN T ANTIMICRBIAL AGENTS Dr. Mark Zabriskie ak Creek Bldg, Rm 238 Ph: 737 5774 Mark.Zabriskie@oregonstate.edu ffice ours: by appointment Recommended reading: G&G 10 th ed., pp 1143-1170 Phar 754: Antimicrobial Agents verview Introduction Agents affecting bacterial cell wall or cell membrane Agents that inhibit bacterial protein biosynthesis Agents that interfere with bacterial nucleic acid synthesis and/or function Agents that block essential bacterial metabolic pathways Antitubercular drugs Antifungal agents Antimalarial Drugs bjectives Understand the basic principles and concepts of antimicrobial chemotherapy Know and be able to describe mechanisms of action of the drug groups Know and be able to describe the basis for selective toxicity of each drug group Know the primary mechanism of resistance to a drug or class of drugs Be able to assign all drugs discussed to a category on a "recognition" basis Know/recognize structural features that affect spectrum of activity 1
The Battle Against Infectious Disease - 1960s ne can think of the middle of the twentieth century as the end of one of the most important social revolutions in history the virtual elimination of the infectious disease as a significant factor in social life. Macfarlane Burnet, 1962 (Nobel prize in Medicine, 1960) It is time to close the book on infectious diseases, declare the war against pestilence won, and shift national resources to such chronic problems as cancer and heart disease." W.. Stewart, 1967 (United States Surgeon General) The Battle Against Infectious Diseases Continues Emerging Infectious Diseases (>40 since 1975) Legionnaires disease (Legionnella pneumophila) (1977) Ebola virus (1977) anta virus (1977) Escherichia coli 157:7 (1982) Lyme disease (Borrelia burgdorferi) (1982) elicobacter pylori (1983) IV (1983) Community-acquired Methicillin-resistant Staph. Aureus (CA-MRSA) (2000) Vancomycin-resistant MRSA (2002) SARS (2003) Extensively Drug-resistant Mycobacterium tuberculosis (XDR-TB) (2006) According to most recent US Centers for Disease Control statistics: World-wide, 3 out of 10 deaths are caused by infectious diseases 2 million patients in the United States get an infection in the hospital each year ~ 90,000 of those patients die as a result of their infection up from 13,300 patient deaths in 1992 > 70% of the bacteria that cause hospital-acquired infections are resistant to at least one of the antibiotics most commonly used to treat them Patients with antibiotic-resistant infections are have longer hospital stays and require treatment with second- or third-choice drugs that may be less effective, more toxic, and more expensive Antimicrobial resistance is driving up health care costs and increasing the severity of disease 2
Antimicrobial Agents Goal of antimicrobial chemotherapy Assist the body (host) in ridding itself of the infecting organism A healthy human body is well equipped to combat infection Antibody production Complement system series of enzyme complexes in normal serum that destroy antibody-tagged cells and bacteria Inflammatory response Phagocytosis Most infections do not require therapy and often go unnoticed Chemotherapy is required when the infecting organism is particularly virulent the host s defense systems are compromised Definitions and Concepts Antimicrobial Agents Introduction Some confusion in the usage of various terms describing microbe-inhibiting products. You will see/hear the terms antibiotic, antibacterial and 'antimicrobial' used interchangeably and sometimes as distinct terms. Antibiotic: Formal definition A substance produced by a microorganism with the capacity to inhibit the growth of, or kill, other microorganisms (bacteria, fungi and protozoa) Expanded definition includes purely synthetic compounds and NTE: Antibiotics do NT inhibit or destroy viruses Antibacterial: Natural or synthetic agents that specifically inhibit or kill bacteria Antimicrobial: Natural or synthetic agents that inhibit or kill bacteria, fungi and protozoa Ideal antibiotic: lethal to the microbe with no effect on the host Closest is Penicillin G in persons not allergic to it Lethal to Gram-(+) bacteria Acts by inhibiting cell wall biosynthesis a process that does not take place in mammalian cells 3
Definitions and Concepts Antifungal: Drugs that specifically inhibit or kill fungi Antiviral: Agents that specifically inhibit or destroy viruses Antiseptic: Substances (like isopropanol or ethanol) applied topically to living tissues that prevent or inhibit the growth of microorganisms Disinfectant: Substances (like chlorine bleach and ammonia compounds) applied to inanimate surfaces (counter tops and cutting boards) to destroy or inhibit microorganisms of all kinds, including viruses Selective Toxicity - Central Concept to Antibiotic Action - Inhibit the growth of, or kill, an infecting organism without damaging the host s cells. Selectivity is usually achieved by exploiting differences between the biochemistry of the infecting organism and the host Bactericidal vs Bacteriostatic Agents Antibiotics can be bactericidal, bacteriostatic, or both Bactericidal agents cause bacterial cell death Preferred if the patient is immunocompromised or in the case of particularly virulent and life-threatening infections Bacteriostatic agents inhibit bacterial growth and replication but do not kill the organism. They slow/stop bacterial growth allowing the host defenses to catch up and eradicate the infection. Some compounds can be -cidal or -static depending on the organism the site of infection (blood, lungs, urinary tract, bone, CNS, etc) The concentration that can be achieved at the site of infection 4
Targets of antibiotic action Factors in Choosing an Antibiotic ost Factors Issues affecting a patient s response to an antibiotic or adverse reaction to it Functional state of the host s defense mechanisms e.g., healthy person vs immunocompromised patient Local factors abscess cavities Age Susceptibility or tolerance to side effects Genetics Race Metabolic deficiencies Allergies β-lactams, sulfonamides Pharmacokinetic Factors demonstrated in vitro sensitivity does not guarantee efficacy must get the drug to the site of infection and in sufficient concentrations route of administration dosage, duration of therapy need to consider patient s ability to clear drug Age, pathophysiology 5
Bacterial Factors: Antibiotic Resistance Resistance is known for every clinically-used antibiotic Nothing can be done to prevent the emergence of resistance it is a natural consequence of antibiotic use and begins to develop as soon as the antibiotic is used Preventing the spread of antibiotic-resistant bacteria is the only way to combat the problem Types of resistance Intrinsic resistance - the inherent insensitivity of a microorganism to a compound For an antibiotic to function, the target must be present and accessible e.g., Gram-(+) vs Gram-(-) bacteria Protein targets of the penicillins are found in both types of bacteria but many penicillins are generally not effective to treat Gram-(-) infections Gram-(-) bacteria have an outer membrane with narrow porin channels e.g., isoniazid - inhibits mycolic acid biosynthesis Process is unique to mycobacteria ver 60% of antibiotics are derived from bacterial natural products In general, bacteria that produce antibiotics also have mechanisms for selfprotection (self-resistance or autoimmunity) from their own antibiotics ne form of intrinsic resistance Most of the antibiotic-producing bacteria are non-pathogenic. But, the genes encoding the resistance factors can be acquired by pathogens. Mutational resistance Exposure of bacteria to an antibiotic exerts selective (evolutionary) pressure to survive and out compete other bacteria Development of resistance Mutations likely because of large number (10 9-10 10 ) of bacteria in a fulminant infection and high mutation frequency Bacteria replicate rapidly (as short as 20-30 min) A bacterial genome contains 2-4 10 6 base pairs (~2500-4000 genes) Frequency of spontaneous mutation is ~1 cell per 10 7 In a population of 10 10 bacteria, this corresponds to 1000 mutants if randomly dispersed among 1000 different genes 25-40% of total Exposure to an antibiotic will kill most of the cells, BUT if a mutation occurs that confers resistance in some way, then the mutant has a competitive edge for survival and will eventually dominate the bacterial population That which does not kill me, makes me stronger" F. Nietzsche This mutational resistance is a stable genetic change that is inherited by subsequent generations 6
Transferable (Acquired) Resistance Resistance to an antibiotic can be conferred by a single gene (penicillin, erythromycin) or can require several genes (vancomycin) Genes directing some forms of antibiotic resistance can be transferred between individual bacteria Lateral or horizontal gene transfer (GT) There are three possible mechanisms of GT. These are transduction, transformation or conjugation. Transduction occurs when bacteria-specific viruses (bacteriophages) transfer DNA between two closely related bacteria. Transformation is a process where parts of DNA are taken up by the bacteria from the external environment. This DNA is normally present in the external environment due to the death of another bacterium. Conjugation occurs when there is direct cell-cell contact between two bacteria (which do not have to be closely related) and transfer of plasmids (small pieces of non-chromosomal DNA) takes place. This is thought to be the main mechanism of antibiotic resistant gene transfer. Types of acquired antibiotic resistance Enzymatic modification or destruction of the antibiotic Most often seen with natural product-derived antibiotics - e.g., β-lactams Rare with synthetic agents - e.g., fluoroquinolones, sulfonamides Product will not inhibit the target Antibiotic ydrolysis Destruction of β-lactams by β -lactamases RN N S C 2!-lactamase 2 RN N S C 2 R 1 N S!-lactamase R 1 N S N C 2 R 2 2 N C 2 R 2 ydrolysis of macrolides hydrolase Et R' R 2 Et R' R 7
Types of acquired antibiotic resistance Antibiotic structure modification - usually group transfer enzymes Aminoglycosides can be modified with acetyl, phosphate or nucleoside groups AMP P 3 Ac C 2 N 2 N 2 2 N Ac N 2 2 C 2 N P 3 or AMP Chloramphenicol can be -acetylated N CCl 2 Ac-CoA N CCl 2 2N 2N Ac Reduce uptake of the antibiotic into the cell Mutation in transport protein - common mutational resistance, can be acquired Blocks antibiotic from gaining access to the target e.g., chloramphenicol, aminoglycosides, fluoroquinolones Types of acquired antibiotic resistance Active efflux of the antibiotic out of the cell Efflux pumps are very common in antibiotic producers Toxic concentrations of antibiotic in the cell are not reached Clinically relevant for macrolides, some peptide antibiotics, fluoroquinolones, and tetracyclines Some efflux pumps have narrow substrate specificity (Tetracycline pumps) others recognize many antibiotics and confer multidrug resistance (MDR) phenotypes. Efflux pumps are common to all bacteria and are used to pump out specific metabolites and byproducts and toxins can confer intrinsic resistance Many forms of resistance are the result of efflux pumps encoded on plasmids or transposons 8
Types of acquired antibiotic resistance Increase production of drug target Increase levels of enzyme targeted by the antibiotic Concentrations of drug can t reach levels high enough to inhibit all copies of the target e.g., dihydrofolate reductase (DFR), the trimethoprim target Reduce affinity of target for the antibiotic Mutation of the target so that drug does not bind, or binds with low affinity Alterations in the sequence of the target protein or nucleic acid Alter the structure of other cellular components e.g., the region of Lipid II and peptidoglycan recognized by vancomycin Enzymatic modification of the target Covalent modification of the protein/nucleic acid target e.g., methylation of one adenosine base in 23S rrna blocks erythromycin binding Same as the self-resistance mechanism used by erythromycin producing bacteria Types of acquired antibiotic resistance verproduction of the natural substrate for a target enzyme igh concentration of the substrate will out compete the inhibitor for the target binding site e.g., high levels of p-aminobenzoate, one substrate for dihydropteroate synthase, are found in some bacteria resistant to the sulfonamides New pathway/enzyme to synthesize the product of the antibiotic target Metabolic detours/bypass pathways Can evolve, mutate or acquire the necessary metabolic machinery Reduce metabolic activation of the drug Flucytosine must be converted to fluorouracil after it is taken into the fungal cell Isoniazid must be converted to an active metabolite in M. tuberculosis 9
Common antibiotics, their targets and resistance mechanism(s) Ways to combat development of antibiotic resistance Reserve certain antibiotics for last resort use do not make available for routine ambulatory use Identify new targets aided by genomics, examples include Sortase involved in the attachment of proteins to the surface of Gram-(+) cells. Without the surface proteins, the bacteria cannot adhere to human tissues and are therefore noninfectious. Deformylase removes the formyl group from N-terminal methionine found on bacterial proteins. fmet is initiator amino acid for bacterial protein synthesis but the formyl group must be removed for the proteins to function. Methyl Erythritol Phosphate (MEP) pathway for isoprenoid biosynthesis Some pathogens use a pathway for sterol precursor biosynthesis that is different from the mammalian mevalonate route. Block efflux pumps Co-administer agents that prevent bacteria from exporting antibiotics that enter the cell Rotate antibiotic use Cycle through various structural/mechanistic classes for fixed periods of time Wise use of combination therapy to slow emergence of resistance 10
Combination Therapy Simultaneous use of multiple antibiotics usually not necessary increase risk of drug toxicity increase cost possible selection of bacteria resistant to drugs that might not have been necessary possible antagonism (lessening) of antibiotic action Combined therapy is called for in certain situations treatment of mixed infections e.g., Gram-(-) and Gram-(+) prevention of resistance different mechanisms of action usually require different mechanism of resistance combination therapy should decrease emergence of resistant strains e.g., if 1 in 10 7 cells is resistant to β-lactams and 1 in 10 7 cells is resistant to aminoglycosides, statistically, only 1 in 10 14 cells should be resistant to both Initial therapy of severe infections where causative organism is unknown most common clinical indication for combined therapy when cultures have been grown and identified may need to switch therapy fever in immunocompromised patients is treated empirically with a combination of antibiotics Combining antibiotics can reduce toxicity allows for smaller doses of each antibiotic reducing dose-dependent toxic side effects Combination Therapy: Three Possible Responses Additive response The combination is more effective than either drug alone the response is roughly the sum of the two drugs effects Synergistic Response ccurs when the combination provides a greater response than the sum of the two individual drugs effects usually defined as inhibition of growth with a combination of drugs at concentrations that are 25% of the minimum inhibitory concentration (MIC) of each drug alone. i.e., 1/4 of the usual concentration ften indicates one drug is affecting an organism in a way that it becomes more sensitive to the other drug. e.g., use of a penicillin and an aminoglycoside evidence that the action of the penicillin facilitates passage of the aminoglycoside through the cell wall Synergistic toxicity is also possible Antagonistic Response A combination of two drugs yields a response less than that seen for one of the drugs alone. more likely to occur when a bacteriostatic drug is combined with a bactericidal agent Possible explanation the cidal compounds only have a killing effect on organisms that are actively dividing or synthesizing protein Antagonism is seldom less than the static agent alone 11
Antibiotic Misuse Frequently misused and overused by the patient and the MD Prescribed for untreatable infections Used in noninfectious conditions due to misdiagnosis e.g., fever of undetermined origin Inappropriate prophylactic use 30-50% of antibiotics used to prevent infections ospital acquired infections nosocomial infections contributes to resistant organism selection applies evolutionary pressure selects for the survival of organisms resistant to the antibiotic Improper dosage fear of toxicity dose is too low to be clinically effective contributes to resistant organism selection Too much reliance on chemotherapy omission of surgical drainage Antibiotic Misuse Lack of microbiological information Improper use of combinations and broad spectrum ABX contributes to superinfections disruption of normal balance of body s microbial flora one organism may grow unchecked unsusceptible organisms can flourish because normal microflora not present to fight these organisms off. Massive release of antibiotics into the environment agriculture feed additives to promote growth many are closely related to clinically used ABX but had certain unfavorable properties animals fed sub-therapeutic levels of ABX grow faster in 1994, Denmark imported 24 kg of vancomycin for medical use and 24,000 kg of the closely related avoparcin as a livestock and poultry feed additive Vancomycin-resistant bacteria transferred to poultry farmers and meat processors similar results observed in Taiwan with fluoroquinolone use in swine The problem is NT the ABX in the food, the issue is the ABX-resistant bacteria that evolve unmetabolized antibiotics Patient misuse Failure to initiate therapy Failure to complete a course of therapy Self treatment with antibiotics obtained for a previous infection Use of another person s antibiotics 12