Lecture Week 7: Controlling Antimicrobial Growth in the Body PDF

Controlling Microbial Growth in the Body Week 7 Learning Objectives week 7 Explain to me the different mechanisms of antibiotic resistance. Explain the different mechanisms of action for antibiotics. Discuss how to test the efficacy of antimicrobial drugs. ...

Controlling Microbial Growth in the Body Week 7 Learning Objectives week 7 Explain to me the different mechanisms of antibiotic resistance. Explain the different mechanisms of action for antibiotics. Discuss how to test the efficacy of antimicrobial drugs.  Outline History of Antimicrobial Agents and what are antimicrobial agents. Mechanisms of Antimicrobial Action Why should you prescribe or not prescribe certain antimicrobials Antimicrobial Drug Resistance Antimicrobial Drugs  Outline History of Antimicrobial Agents and what are antimicrobial agents. Mechanisms of Antimicrobial Action Why should you prescribe or not prescribe certain antimicrobials Antimicrobial Drug Resistance Antimicrobial Drugs Giant Petri Dish https://www.youtube.com/watch?v=plVk4NVIUh8 Why should we care? What are antimicrobial agents? A drug is anything that effects the physiology of an organism. --- caffeine, alcohol, nicotine Chemotherapeutics are drugs that act against a disease. -- Lipator (cholesterol), Nexium (acid reflux), Humira (arthritis) Antimicrobial agents are drugs that treat an infection. Either killing or slowing the growth of a microbe.  History of Antimicrobial Agents Paul Ehrlich– coined the term chemotherapy to describe compounds that would selectively kill pathogens. (magic bullets) Salversan--Arsenic compounds that killed microbes One of the first screens to look at many different compounds to find a desired effect One of the first large scale systematic chemical modification strategies to optimize a biological effect  History of Antimicrobial Agents Gerhard Domagk German chemist who discovered sulfanilamide (Prontosil) which was the first commercially available antibiotic. Won the Nobel Prize but was not allowed to accept it and was jailed by the Nazis for even acknowledging the honor. Later found that cleavage of sulfanilamide left the active compound which was easier to produce, more effective, and had fewer side effects.  History of Antimicrobial Agents Alexander Fleming: Discovered that Penicillin released from Penicillium (fungi) can kill bacteria Two gripes: 1) The notion that Fleming was lucky 2) No one talks about Florey, Chain, and Heatly  History of Antimicrobial Agents Selman Waksman found that antibacterial compounds are made by other bacteria. Coined the term as antibiotics which only refer to compounds that come from other organisms, not synthetic compound made in the lab. Found that Streptomyces bacteria made a compound that was effective against Tuberculosis. “streptomycin”  Antibiotic vs Synthetic vs Semisynthetic Antibiotics Compounds made by microorganisms that kill other microorganism Semisynthetic Chemically altered antibiotics that are more effective than naturally occurring ones Synthetics Antimicrobials that are completely synthesized in a lab  The majority of antimicrobials that have been made are antibiotic or semisynthetic. Antibiotics Compounds made by microorganisms that kill other microorganism. Semisynthetic Chemically altered antibiotics that are more effective than naturally occurring ones Synthetics Antimicrobials that are completely synthesized in a lab Take home messages An antimicrobial agent is a drug that effects the physiology of a microorganism. An antibiotic specifies a drug that is made by another microbe. Synthetics and semisynthetic antibiotics are drugs that have been developed or modified in the lab.  Outline: History of Antimicrobial Agents and what are antimicrobial agents. Mechanisms of Antimicrobial Action Why should you prescribe or not prescribe certain antimicrobials Antimicrobial Drug Resistance Antimicrobial Drugs  The key to any antimicrobial being effective is selective toxicity! Compounds that will kill the microbe but not the fungi bacteria host. ~ =  Antibacterial drugs constitute largest number and diversity of antimicrobial agents Fewer drugs to treat eukaryotic (fungal) infections Even fewer antiviral drugs Viruses use host machinery to replicate.  Different classes of antimicrobials have distinct mechanisms by which they kill microbes. Inhibition of cell wall biosynthesis are quite popular mechanisms of action. Penicillins Cephalosporins Vancomycin Bacitracin Isoniazid Ethambutol Echinocandins (antifungal)  The most common way to inhibit bacterial wall synthesis is to prevent cross-linkage of NAM subunits. Bacterial cell walls are composed of peptidoglycan Peptidoglycan is composed of NAG-NAM chains that are cross-linked by peptide bridges between the NAM subunits. N-acetylglucosamine NAG N-acetylmuramic acid NAM New NAG and NAM subunits are inserted into the wall by enzymes, allowing the cell to grow. Normally, other enzymes link new NAM subunits to old NAM subunits with peptide cross-links.  The most common way to inhibit bacterial wall synthesis is to prevent cross-linkage of NAM subunits. Beta-lactams drugs are most prominent in this group Functional groups of these drugs are beta- lactam rings Beta-lactams bind to enzymes that cross- link NAM subunits  The lack of cell wall integrity causes bacteria to lyse (break open). Penicillin interferes with the enzymes that cross link the NAM subunits, Growth instead the NAM subunits remain New NAM-NAM unattached to their neighbors. cross-links inhibited by penicillin Previously formed cross-links remain However, the cell continues to grow as unchanged it adds more NAG and NAM subunits. The cell wall is very unstable and eventually will fall apart.  Penicillin was the founding member of this class and since a number of semisynthetic derivatives have been made. More stable in acidic environments Penicillins Cephalosporin Monobactam More readily absorbed -lactam ring Less susceptible to deactivation Penicillin G (natural) More active against more types of Cephalothin (semisynthetic)Aztreonam bacteria Methicillin (semisynthetic) (semisynthetic Simplest beta-lactams – effective only against aerobic Gram- positive  Antimicrobials that disrupt bacterial cell walls in a manner distinct of that used by beta- lactams. Vancomycin and cycloserine Interfere with bridges that link NAM subunits in many Gram-positives Bacitracin Blocks secretion of NAG and NAM from cytoplasm Isoniazid and ethambutol Disrupt mycolic acid formation in mycobacterial species The drugs described so far prevent bacteria from increasing amount of peptidoglycan Have no effect on existing peptidoglycan layer Effective only for growing cells Dormant cells are relatively unaffected The more quickly an organism is reproducing the great an effect drugs such as these can have. Echinocandins disrupt fungal cell wall biogenesis. Caspofungin is one aspergillus of only a few such C. albicans drugs It inhibits enzymes that produce glucan Long course of treatment, must be taken intravenou Glucan is essential for fungal cell wall biosynthesis  Different classes of antimicrobials have distinct mechanisms by which they kill microbes. Disruption of cytoplasmic membrane (fungi have different membranes then mammals. Polymyxins Polyenes (antifungal)  Some drugs form channels through cytoplasmic membranes and damage their integrity One of the biggest differences between mammals and fungi is in our cytoplasmic membranes. Fungi have ergosterol while humans have cholesterol. Amphotericin B attaches to ergosterol in fungal membranes Can be used orally for thrush Humans somewhat susceptible because cholesterol similar to ergosterol Very dangerous to take but the only known cure for some fungal infections. Bacteria lack sterols; not susceptible  Some drugs form channels through cytoplasmic membranes and damage their integrity One of the biggest differences between mammals and fungi is in our cytoplasmic membranes. Fungi have ergosterol while humans have cholesterol. Amphotericin B attaches to ergosterol in fungal membranes Can be used orally for thrush Humans somewhat susceptible because cholesterol similar to ergosterol Very dangerous to take but the only known cure for some fungal infections. Bacteria lack sterols; not susceptible Group Questions 1) What is the difference between antibiotic, semisynthetic, and synthetic antimicrobials. 2) How do fungal and bacterial cell walls and cytoplasmic membranes differ. How do these differences lead to different drug susceptibilities?  Different classes of antimicrobials have distinct mechanisms of action by which they kill microbes. Target differences of protein synthesis between bacteria and mammals. Aminoglycosides Tetracyclines Chloramphenicol Macrolides  Inhibition of protein synthesis is a powerful mechanism of antimicrobial action. Prokaryotic ribosomes are 70S (30S and 50S) Eukaryotic ribosomes are 80S (40S and 60S) Drugs can selectively target bacterial translation Mitochondria of animals and humans contain something like a 70S ribosome so such drugs have the potential to inhibit these ribosomes as well.  Inhibition of protein synthesis is a powerful method of antimicrobial action. Prokaryotic ribosomes are 70S (30S and 50S) Eukaryotic ribosomes are 80S (40S and 60S) Drugs can selectively target translation Mitochondria of animals and humans contain something like a 70S ribosome so these drugs have the potential to inhibit these ribosomesPrevents insertion of amino acids int as well. the growing protein chain.  Inhibition of protein synthesis is a powerful mechanism of antimicrobial action. Prokaryotic ribosomes are 70S (30S and 50S) Eukaryotic ribosomes are 80S (40S and 60S) Drugs can selectively target translation Mitochondria of animals and humans contain something like a 70S ribosome so these drugs have the potential to inhibit these ribosomes Cheap and easy to produce, side effects no longer as well. make it the first choice drug that it once was.  Inhibition of protein synthesis is a powerful mechanism of antimicrobial action. Prokaryotic ribosomes are 70S (30S and 50S) Eukaryotic ribosomes are 80S (40S and 60S) Drugs can selectively target translation Mitochondria of animals and humans contain something like a 70S ribosome so these drugs have the potential to inhibit these ribosomes as well.  Inhibition of protein synthesis is a powerful mechanism of antimicrobial action. Prokaryotic ribosomes are 70S (30S and 50S) Eukaryotic ribosomes are 80S (40S and 60S) Drugs can selectively target translation Mitochondria of animals and humans contain something like a 70S ribosome so these drugs have the potential to inhibit these ribosomes as well.  Inhibition of protein synthesis is a powerful mechanism of antimicrobial action. Prokaryotic ribosomes are 70S (30S and 50S) Eukaryotic ribosomes are 80S (40S and 60S) Drugs can selectively target translation Mitochondria of animals and humans contain something like a 70S ribosome so these drugs have the potential to inhibit these ribosomes as well. These drugs are the last resort for Gram positive inf Block methyl tRNA from initiating translation  Different classes of antimicrobials have distinct mechanisms by which they kill bacteria. Inhibition of DNA or RNA synthesis Actinomycin Nucleotide analogs Quinolones Rifampin  Inhibition of Nucleic Acid Synthesis Several drugs block DNA replication or mRNA quinolone (flouroquinolones) transcription Drugs often affect both eukaryotic and prokaryotic cells Not normally used to treat infections (quinolone are an exception) Used in research and perhaps to slow cancer cell replication Act against prokaryotic (bacter DNA gyrase Figure 10.7 Nucleotides and some of their antimicrobial analogs Dideoxyinosine (ddl) Ribavirin Penciclovir Valaciclovir Tenofovir Adefovir Adenosine arabinoside Adenosine Guanosine Acyclovir (ACV) Ganciclovir NUCLEOSIDES Stavudine Azidothymidine Thymidine Cytidine Dideoxycytidine (ddC) Lamivudine (d4T) (AZT) Iododeoxyuridine Trifluridine  Nucleotide or nucleoside analogs: Interfere with function of nucleic acids Distort shapes of nucleic acid molecules and prevent further replication, transcription, or translation Most often used against viruses Effective against rapidly dividing cancer cells  AZT is a nucleotide analog used to treat viral infections. It inhibits reverse transcriptase RNA Probably most noted for being a member of the AZ Reverse T transcrip HIV/AIDs drug cocktail tase Reverse transcriptase is necessary for HIV and other viruses to make a DNA DNA copy of their RNA genome. Insertion into the genomic DNA  Rifampin binds with greater affinity to bacterial RNA polymerase than eukaryotic RNA polymerase. Rifampicin binds to RNA polymerase at a site adjacent to the RNA polymerase active center and blocks RNA synthesis by physically blocking the formation of the phosphodiester bond in the RNA backbone. Mutations shown in red cause Rifampin not to bind and drug resistance. "Rifampicin RNApol" by Fdardel - Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons - https://commons.wikimedia.org/wiki/File:Rifampicin  Different classes of antimicrobials have distinct mechanisms in which they kill microbes. Inhibition of “general” metabolic pathway Sulfonamides (inhibit folic acid biosynthesis which is a precursor of nucleic acids) Trimethoprim Dapsone Bacteria synthesize folic acid, humans get it from their diet. Trimethoprim-binds to the enzyme responsible for dihydrofolic acid conversion to TH Inhibition of metabolic pathways can be effective when metabolic processes of pathogen and host differ. Quinolones interfere with (Prontosil) the first the metabolism of malaria commercially available parasites. Heavy metals inactivate antibiotic was a para-aminobenzoic acid enzymes. sulfanilamide. Agents that disrupt tubulin polymerization and glucose uptake by many protozoa and parasitic worms Drugs block activation of viruses Metabolic antagonists  Different classes of antimicrobials have distinct mechanisms by which they kill microbes. Inhibition of pathogen’s attachment to, or recognition of host Human cell membrane Arilone and pleconaril bind to the receptor of polio virus and cold virus Arildone Pleconaril Take Home Messages: Antimicrobials take advantage of differences between humans and microbial cellular structure. Protein and DNA synthesis, metabolism, cellular attachment, and RNA polymerization are biological processes that are targets of antibiotics. Majority of antimicrobial drugs act against bacteria, many fewer act against fungi, and even fewer act against viruses.  Outline: History of Antimicrobial Agents and what are antimicrobial agents. Mechanisms of Antimicrobial Action Why should you prescribe or not prescribe certain antimicrobials Antimicrobial Drug Resistance Antimicrobial Drugs So what would make an ideal antimicrobial agent? Readily available Inexpensive So what would make an ideal antimicrobial agent? Readily available Inexpensive Chemically stable: what if you need and antibiotic in jungle with no electricity So what would make an ideal antimicrobial agent? Readily available Inexpensive Chemically stable Easily administered: injections and other medical procedures can be difficult to administer. So what would make an ideal antimicrobial agent? Readily available Inexpensive Chemically stable Easily administered Nontoxic and nonallergenic Is penicillin allergy common? Penicillin antibiotics are the most common cause of drug allergies. Some people who are allergic to penicillin are also allergic to other closely related antibiotics, including cephalosporins, such as cefprozil, cefuroxime, and cephalexin. Many people who believe that they have an allergy to penicillin do not. They currently may be less sensitive to penicillin than they were in the past. Or they may have had an adverse reaction, such as a side effect, rather than an allergic reaction. So what would make an ideal antimicrobial agent? Readily available Inexpensive Chemically stable Easily administered Nontoxic and nonallergenic Selectively toxic against wide range of pathogens  So what would make an ideal antimicrobial agent? Readily available Inexpensive Chemically stable Easily administered Nontoxic and nonallergenic Selectively toxic against wide range of pathogens No drug is perfect, so Drs weigh the benefit/cost of each drug to decide on a course of treatment.  Toddler with an ear infection Drug A Drug B Slightly more effective Less effective IV Oral $10,000 per dose $100 Higher likelihood of side Fewer side effects effects  The spectrum of action is the number of different pathogens a drug acts against. Narrow-spectrum effective against few organisms Broad-spectrum effective against many organism May allow for secondary or superinfections to develop Group Questions 1) Define spectrum of action for a drug. Describe a time when you might want a broad spectrum of action drug. Describe a time when you might want a narrow spectrum drug. 2) Describe a mechanism of action other than cell wall or membrane inhibition. Describe a situation in which this mechanism of action or this class of drug would be useful. (There are many answers to this question, don’t over think it.) Efficacy can be ascertained by a number of different methods. Bacterial lawn Zone of inhibition Diffusion susceptibility test: Also known as Kirby Bauer tests. Minimum inhibitory concentration test Minimum bactericidal concentration test Compound on a piece of paper Efficacy can be ascertained by a number of different methods (broth dilution test). Diffusion Figure 10.10 Minimum inhibitory concentration (MIC) test in test tubes susceptibility test Minimum inhibitory concentration test Minimum bactericidal concentration test Efficacy can be ascertained by a number of different methods. E test Diffusion susceptibility test Minimum inhibitory concentration test Minimum bactericidal concentration test  Concentration of antibacterial drug (µg/ml) Minimum bactericidal concentration test: Clear MIC tube Allows one to determine the concentration of a drug that it takes to kill a microbe (bacteriocidal/fungic 8 µg/ml antibiotic 16 µg/ml antibiotic 25 µg/ml antibiotic idal) activity. Bacterial colonies No colonies No colonies Drug-free media Figure 10.12 A minimum bactericidal concentration (MBC) test  There are many clinical Administration method considerations when Oral prescribing antimicrobial Relative concentration of drug in blood drugs Routes of Administration Topical application of drug for Intramuscular external infections. (IM) Oral route requires no needles and is self-administered. Intramuscular administration delivers drug via needle into muscle. Continuous Intravenous administration delivers intravenous (IV) drug directly to bloodstream. Knowing how antimicrobial agent will be distributed to infected tissues is important. © 2012 Pearson Education Inc. Time (hours)  There are many clinical considerations when prescribing antimicrobial drugs. Safety and Side Effects Toxicity Cause of many adverse reactions poorly understood Drugs may be toxic to kidneys, liver, or nerves Consideration needed when prescribing drugs to pregnant women Allergies Allergic reactions are rare but may be life threateningMetronidazole, harmless Tetracycline, by breakdown of hemoglobin Anaphylactic shock damage tooth enamel  Scientists are beginning to realize that disruption of normal microbiota by antibiotics has profound effects on health. May result in secondary infections Overgrowth of normal flora causes superinfections Of greatest concern for hospitalized patients Take Home Messages: The ideal antibiotic does not exist, but Drs weigh the benefits vs risks of prescribing different antimicrobial drugs. There are many different tests that can be used to test the efficacy of antimicrobial drugs. The route of delivery is a key consideration when developing an antimicrobial drug.  Outline: History of Antimicrobial Agents and what are antimicrobial agents. Mechanisms of Antimicrobial Action Why should you prescribe or not prescribe certain antimicrobials Antimicrobial Drug Resistance Antimicrobial Drugs  The Development of Resistance in Populations Since antibiotics are made by microbes the genes/mechanism for resistance must exist. Some pathogens are naturally resistant. In the original papers by Fleming on Penicillin mention resistant mutants Resistance by bacteria acquired in two ways New mutations of chromosomal genes Acquisition of R-plasmids via transformation, transduction, and conjugation  The Development of Resistance in Populations Since antibiotics are made by microbes the genes/mechanism for resistance must exist. Some pathogens are naturally resistant. In the original papers by Fleming on Penicillin mention resistant mutants Resistance by bacteria acquired in two ways New mutations of chromosomal genes Acquisition of R-plasmids via transformation, transduction, and conjugation There are at least seven known mechanisms of microbial resistance to antibiotics Produce enzyme that destroys or deactivates drug Slow or prevent entry of drug into the cell Alter target of drug so it binds less effectively Alter their metabolic chemistry Pump antimicrobial drug out of the cell before it can act Biofilms retard drug diffusion and slow metabolic rate Mycobacterium tuberculosis produces MfpA protein There are at least seven known mechanisms of microbial resistance to antibiotics. Produce enzyme that destroys or deactivates drug Penicillin Inactive Penicillin Many bacteria have acquired genes that code for enzymes that breakdown penicillin (Beta-lactamases). Over 200 of these genes have been identified.  There are at least seven known mechanisms of microbial resistance to antibiotics. Slow or prevent entry of drug into the cell ansmembrane proteins that transport drugs There are at least seven known mechanisms of microbial resistance to antibiotics. Pump antimicrobial drug out of the cell before it can act There are at least seven known mechanisms of microbial resistance to antibiotics. Alter drug target so it binds less effectively Don’t necessarily need to alter the target site, can alter residues close by and still lead to resistance. There are at least seven known mechanisms of microbial resistance to antibiotics. Alter bacterial metabolic chemistry active site Enzy dru me g There are at least seven known mechanisms of microbial resistance to antibiotics. Alter bacterial metabolic chemistry Enzy dru me g Leads to microbe death! There are at least seven known mechanisms of microbial resistance to antibiotics. Alter bacterial metabolic chemistry Up regulation of enzyme either through Enzy transcription or translation. dru me g Enzy Enzy Enzy me Enzy Enzy me me Enzy Enzy me me me Enzy Enzy me Enzy me me Enzy Enzy me me me There are at least seven known mechanisms of microbial resistance to antibiotics. Biofilms retard drug diffusion and slow metabolic rate 20 mM thick biofilm in hotspring Staphylococcus on a catheter "Thermophilic bacteria" by Amateria1121 - Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons - https://commons.wikimedia.org/wiki/File:Thermophilic_bacteria.jpg#/media/ File:Thermophilic_bacteria.jpg Group questions 1) Explain to you neighbor how antimicrobial resistance occurs. 2) You are near death due to a microbial infection what would be the ideal method by which to give you a drug? 3) Describe your favorite drug resistance mechanism and why it is your favorite? We still don’t know exactly how antibiotic resistance develops or why some strains are more prone to developing resistance. https://www.whitehouse.gov/sites/default/files/microsites/ostp/PCAST/ pcast_carb_report_sept2014.pdf  Multiple Resistance and Cross Resistance is when a pathogen acquires resistance to more than one drug. R plasmids can contain numerous antibiotic resistance genes. Develop in hospitals and nursing homes because antibiotics are constantly being administered Constant use of drugs eliminates sensitive cells Resistance (R-plasmid) - contain antibiotic and toxin resistant genes; previously known as R- factors Virulence plasmid –  A superbug is a microbe that is resistant to three or more classes of antibiotics. Cross resistance is a phenomena of one antibiotic resistance mechanism leading to resistance to other similar antibiotics within in one class. For example resistance to streptomycin can confer resistance to other aminoglycoside drugs. All these drugs inhibit protein synthesis is similar ways. Kanamycin Streptomycin "Netilmicin structure" by Yikrazuul - Own work. Licensed under Public Domain via "Streptomycin2" by NEUROtiker ⇌ - Own work. Licensed under https://commons.wikimedia.org/wiki/File:Streptomycin2.svg#/media/File:Streptomycin2.s https://commons.wikimedia.org/wiki/File:Netilmicin_structure.svg#/media/File:Netilmicin_structure.svg vg  Methods to limit resistance are counterintuitive but necessary. Disk with semisynthetic Disk with semisynthetic Maintain high concentration of amoxicillin–clavulanic acid aztreonam drug in patient for sufficient time Kills all sensitive cells and inhibits others so immune system can destroy them Use antimicrobial agents in combination Synergism vs. antagonism (must be sure that the combination of drugs you are using work in synergy)  The best way to limit microbial antibiotic resistance is to use them only when absolutely necessary. When you feel sick in the winter or have flu like symptoms most of the time you have a virus in which there are very few drugs that will help. Antibacterials will not be helpful and in fact will likely be harmful. Develop new variations of existing drugs (next generation drugs) Search for new antibiotics, semisynthetics, and synthetics Take Home Messages: Antimicrobial drug resistance is as old as antimicrobial drugs. There are many different ways antimicrobial resistance can occur. Superbugs and multi-resistant bugs are resistant to multiple antimicrobial drugs. Taking your entire dose of antibiotic and taking antibiotics in combination are two of the best  Outline: History of Antimicrobial Agents and what are antimicrobial agents. Mechanisms of Antimicrobial Action Why should you prescribe or not prescribe certain antimicrobials Antimicrobial Drug Resistance Antimicrobial Drugs  So why don’t drug companies make more antibiotics?!!!!! It costs roughly $5,000,000,000 dollars to get a new drug to market. It does not matter if this drug is for high cholesterol or an antimicrobial. Antibiotics are supposed to be used sparingly so resistance does not occur. How can a company invest all of this money if they have no way of making it back.  So what is/could be done? More money for basic research and cutting back the overuse of antibiotics. Incentivize academia to find promising leads so drug companies don’t need to invest up front in discovery costs. Provide additional protections (orphan disease/orphan drug like status) to make it more profitable to make antibiotics. phan drug development The Orphan Drug Designation program provides orphan status to drugs and biologics which are defined as those intended for the safe and effective treatment, diagnosis or prevention of rare diseases/disorders that affect fewer than 200,000 people in the U.S., or that affect more than 200,000 persons but are not expected to recover the costs of developing and marketing a treatment drug. While antibiotic resistance as a whole is a large problem, development of drugs to treat each resistant isolate is a series of much smaller less profitable problems. There are at least seven known mechanisms of microbial resistance to antibiotics DNA MfpA protein Mycobacterium tuberculosis produces MfpA protein which binds to gyrase and blocks fluoroquinolone from binding. Protein is negatively charged and shaped very similarly to DNA. gyrase Even though it slows bacterial growth it is better than being killed by the antibiotic.