Chapter concepts: What are antibiotics, the different types, and how do they work? Antibiotics
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1 Chapter concepts: Antibiotics What are antibiotics, the different types, and how do they work? How do we decided on the most appropriate antibiotic treatment? What are some of the ways that bacteria are becoming resistant to antibiotics? In the US alone, 50,000 die each year due to antibiotic resistant strains of bacteria. How is this happening?? Two big reasons: Overuse of antimicrobials on everyday items and in agriculture Early investigators proposed the use of chemotherapy for the treatment of infections Bacteria naturally make their own antibiotics to fight off other bacteria. Natureal antibiotics are secondary metabolites Staphylococcus aureus Secondary metabolites are compounds not directly related to growth Natural antimicrobial = antibiotics made by organisms in nature. Paul Ehrlich Wrote about magic bullets that would destroy bacteria Discovered a arsenic compound that killed trypanosomes Alexander Flemming Observed penicillin released from Penicillium mold inhibits bacterial growth First discovered but not the first used Gerhard Domagk Discovered sulfanilamide, which was the first practical antimicrobial agent Zone where bacterial growth is inhibited Penicillium chrysogenum (fungus) Examples: Penicillium (natural) Amoxicillin (semisynthetic) Semisynthetic antimicrobials = chemically altered to make more stable, longer lasting, easy to administer Synthetic = completely synthesized 1
2 The key to an effective antimicrobial agent is selective toxicity Clinical Considerations in Prescribing Antimicrobial Drugs Selective toxicity means that the drug specifically targets a Ideal Antimicrobial Agent certain cell (pathogen) and not other cells Readily available There are more antibacterial drugs because bacteria have unique structures and metabolisms There are fewer antivirals because they have to target the host cell replication machinery and antifungals because they are eukaryotic Inexpensive Chemically stable Easily administered Nontoxic and nonallergenic Selectively toxic against wide range of pathogens No agent has all of these qualities! cdifffoundation.org Clinical Considerations in Prescribing Antimicrobial Drugs: Spectrum of action Summary of how antibiotics work (mechanisms of action) Narrow-spectrum effective against few organisms Broad-spectrum effective against many organisms May allow for secondary or superinfections to develop Killing of normal flora reduces microbial antagonism 6 general categories of mechanisms of action Inhibition of pathogen's attachment to, or recognition of, host Arildone Pleconaril Mechanisms of Action: Inhibition of cell wall Inhibition of cell wall Penicillins Cephalosporins Vancomycin Bacitracin Isoniazid Ethambutol Echinocandins (antifungal) Most in this group are considered narrow spectrum Inhibition of protein Aminoglycosides Tetracyclines Chloramphenicol Macrolides Antisense nucleic acids Human cell membrane Disruption of cytoplasmic membrane Polymyxins Polyenes (antifungal) Inhibition of DNA or RNA Actinomycin Nucleotide analogs Quinolones Rifampin Inhibition of general metabolic pathway Sulfonamides Trimethoprim Dapsone Prevent bacteria from increasing amount of peptidoglycan Can only act on actively growing bacteria Beta-lactams are most prominent in this group NAG NAM Crossbridge between NAM and NAM NAG-NAM chain New cross-links inhibited by beta-lactam A bacterial cell wall is made of peptidoglycan, which is made of NAG-NAM chains that are cross-linked by peptide bridges between the NAM subunits. Remember that the cell wall is made of peptidoglycan (NAG and NAM units) 2
3 Mechanisms of Action: Inhibition of cell wall Other examples of antibiotics that alter the cell wall in other ways: Vancomycin Disrupt NAM bridges in Gram positive bacteria Bacitracin Discovered in B. subtillis Blocks transport of NAG and NAM from cytoplasm to cell wall Isoniazid Disrupts formation of mycolic acid in Myobacterium tuberculosis Mechanisms of Action: Inhibition of protein Some antibiotics target prokaryotic ribosomes Prokaryotic ribosomes are 70S and eukaryotic are 80S Note: these drugs can damage the ribosomes of mitochondria A ribosome is made up of two parts: a large half and a small half Large subunit: Prokaryotes = 50S Eukaryotes = 60S Small subunit: Prokaryotes = 30S Eukaryotes = 40S Mechanisms of Action: Inhibition of protein Mechanisms of Antimicrobial Action: Inhibition of protein Most antibiotics that target protein are broad spectrum Ribosome can t continue to make polypeptide chain Tetracycline blocks docking site of trna. 30S 50S Aminoglycosides change the shape of the 30S subunit so ribosome cannot read codons on mrna Amino acids don t link Chloramphenicol acts on 50S subunit and blocks peptide bond formation Mechanisms of Antimicrobial Action: Inhibition of protein Mechanisms of Action: Inhibition of protein Macrolides bind to 50S subunit, blocking proper mrna movement through ribosome Ex: erythromycin 30S 50S Oxazolidinones block initiation of translation Used as last resort in treating infections of Gram positive bacteria (MSRAs) fmet 50S 30S 3
4 Mechanisms of Action: Disruption of the cytoplasmic membrane Drugs form a channel through the membrane, damaging its integrity EX: Polyenes attach to ergosterol, a lipid in fungi membranes Amphotericin B is a type of polyene that is considered a broad spectrum antifungal used intravenously for Aspergillus and Candida infections Mechanisms of Action: Inhibition of metabolic pathways Broad spectrum Unique metabolic reactions in microorganisms are good targets for antimicrobial drugs Ex: Sulfanilamide blocks the production of nucleotides in bacteria Structural analog (mimics) of which is required for the formation of nucleotides Phospholipid Amphotericin B Pore Ergosterol Amphotericin B (polyene) Bacterial don t have sterols in membrane so they are naturally resistant to these compounds Sulfanilamide Sulfonaminde is an analog for, and when incorporated into the biochemical pathway, essentially shuts down the ability of the cell to make DNA and RNA Other substrates Enzyme Dihydrofolic acid Dihydrofolic acid Enzyme Tetrahydrofolic acid (THF) Active site Enzymes Other substrates Purine and pyrimidine nucleotides Enzymes DNA and RNA Dihydrofolic acid not produced Mechanisms of Action: Inhibition of nucleic acid Broad spectrum Drugs that block DNA replication or RNA transcription can affect both prokaryotes and eukaryotes EX: Quinolones specific against prokaryotic DNA (acts on DNA helicase) Cipro is an example of this type of antibiotic which is used to treat gonorrhea Enzyme Enzyme Role of in folic acid in bacteria and protozoa Inhibition of folic acid by sulfonamide DNA gyrase/helicase unwind the DNA strand. If this process is blocked, replication cannot take place Mechanisms of Action: Inhibition of attachment (used for viruses) Antiviral agents can target the ability of virus to attach to host cell Ex: Pleconaril prevents rhinovirus (common cold) and piconavirus from binding to cell membrane, thus gaining access to cell Quinolones bind to gyrase, preventing it from working, so replication cannot take place Pleconaril prevents viruses from binding to the cell membrane and entering cell 4
5 Its important that the most appropriate drug is given to treat an infection Treatment: How to prescribe the correct antibiotic? Antibiotics are NOT effective against viruses!!! Viruses have no cell wall, no metabolism and they use the host cell they infect to duplicate their genomes Its important that the most appropriate drug is given to treat an infection How do we know which is the most effective? There are a variety of tests to ascertain the efficacy of antimicrobials Some antibiotics are also NOT Diffusion susceptibility test (Kirby Bauer test) effective against some species Looks at growth inhibition of bacteria EX: Gonorrhea (caused by Neisseria Bacterial lawn gonorrhoeae) was previously treated with beta-lactam antibiotics, now ~30% of infections are resistant (clinicians must test the strain to assess its Zone of inhibition Characteristic, Gram negative stain of diplococcus form of N. gonorrhoeae Disc containing drug sensitivity to antibiotics) Clinical Considerations in Prescribing Antimicrobial Drugs: Effectiveness Minimum inhibitory concentration test (MIC): smallest amount of drug that will inhibit growth and reproduction Zone of inhibition compared to standard table because drugs diffuse at different rates Increasing concentration of drug Figure An Etest, which combines aspects of Kirby-Bauer and MIC tests. Gradient of antimicrobial agent on strip placed on bacterial lawn. Tests minimum inhibitory concentration 5
6 Clinical Considerations in Prescribing Antimicrobial Drugs: Effectiveness Minimum bactericidal concentration (MBC) test: minimum amount of drug required to kill the microbe Tests minimum amount of drug needed to kill microbe Concentration of antibacterial drug (µg/ml) 8 µg/ml 16 µg/ml 25 µg/ml Bacterial colonies No colonies No colonies Drug-free media Though generally considered safe, antibiotics do have side effects and can be toxic Toxicity may cause of adverse reactions that are poorly understood (ex: Quinolones can cause birth defects, so Cipro cannot be used on pregnant women for Gonorrhea) Allergies - Allergic reactions are rare but may be life threatening 0.1% of Americans have an anaphylactic reaction to penicillin (300 deaths a year) Caused by antiprotozoan drug metronidazole Caused by tetracycline which chelates Calcium Antibiotics also have an affect on the normal microbiota in the body Antibiotics kill normal, beneficial microbes found in body Antibiotics may cause secondary infections, which are infections that you get after antibiotic treatment EX: getting a yeast infection after taking antibiotics for something else Antibiotic resistance is a significant health concern. How is this happening??? Overuse of antibiotics and antimicrobal products is killing off the susceptible bacteria, and making space for the naturally resistant bacteria to thrive Superinfections, which are infections on top of Drug-sensitive cells Drug-resistant mutant treated infection can occur and may be due to viruses or fungi Exposure to drug Remaining Population grows over time Many pharmacists are encouraging people to take probiotics during the course of their antibiotic treatment to re-populate normal bacteria in the body. Population of microbial cells Sensitive cells inhibited by exposure to drug Most cells now resistant Antibiotic resistance is a significant health concern. How is this happening??? Microbes that are naturally resistant can transfer their genetic information to other microbes Horizontal gene transfer: R plasmids (Resistant plasmids) can hop to bacteria via conjugation, transduction, or transformation Jumping genes called transposons can do this too! Spontaneous mutation can occur allowing microbes to gain resistance New evidence is also showing that some antibiotics can cause mutation What are these genes and mutations doing to allow bacteria to become resistant? Seven known mechanisms of resistance: Production of 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 own metabolic chemistry Pump antimicrobial drug out of the cell before it can act Bacteria in biofilms can resist antimicrobials 6
7 Example from MRSA: a gene found on a R plasmid codes for a enzyme that breaks down the beta lactam ring of penicillin and methicillin, preventing it from damaging the bacteria cell Lactam ring Some pathogens have developed resistance to multiple drugs Referred to in popular media as superbugs Often occurs when resistance (R) plasmids are exchanged Frequently develop in hospitals and nursing homes where lots of people are undergoing treatment with different antibiotics Penicillin β-lactamase (penicillinase) breaks this bond Inactive penicillin MRSA (methicillin-resistant) strains of S. aureus have developed this resistance to penicillin derived drugs. The enzyme frequently responsible is coded for on a R plasmid Example of a superbug C. difficle Normally found in your gut at low numbers, but can overgrow when you re treated with antibiotics because other species eliminated (this causes a superinfection) What can be done to retard microbial resistance to antimicrobial drugs? Maintain sufficiently high concentrations of drugs in patient s body Use antimicrobial agents in combination (synergism) Limit use of antimicrobials Development of new drugs that have different side chains to the original C. diff infection causes severe diarrhea, and can often be mistaken for something else (giving the patient time to spread it to others. molecule C. diff makes endospores = easy to spread and hard to kill. 7
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