1. Antibiotic Mechanisms of Action and Resistance MLAB 2434 – Microbiology Keri Brophy-Martinez

2. Overview Antimicrobial Therapy Broad term for use of chemical compounds to treat diseases caused by microorganisms Antimicrobial agents used to treat infections are directed by antimicrobial susceptibility testing (AST) Targets specific body sites or specific characteristics of microbes

3. Agents Antimycobacterials Treat mycobacterial diseases Antifungals Treat fungal disease Antiprotozoals Tread protozoal disease Antivirals Treat viral disease Antibiotics Treat bacterial disease

4. Antibiotics Antibiotics are naturally occurring substances produced by a fungus or bacteria Used to treat bacterial infections Alternate Forms Synthetic Totally manufactured or artificial Semi-synthetic compounds Naturally occurring substances that have been chemically altered

5. Definitions Bacteriocidal Kills the bacteria Bacteriostatic Inhibit microbial growth

6. Definitions (Cont’d) Spectrum of activity Range of susceptible organisms Narrow-spectrum Kill either Gram positive or Gram negative organisms Organism specifc Broad-spectrum Kill both Gram positive and Gram negative organisms Extensive Empirical therapy Initiation of therapy prior to organism ID

7. Definitions (Cont’d) Additive Effects Combining two antimicrobials causes twice the effect of the two drugs by themselves Indifference No effect of combining antimicrobial therapies Synergy Combined effect is greater than the two individual effects added together Antagonism One drug counteracts the other

8. Antimicrobial Agents: Factors to Consider What is the targeted bacteria? Where is it located? Can the antimicrobial reach that site in sufficient concentration? Can the antimicrobial be retained in the body long enough to be effective? What are the side effects? How is it excreted? What is the cost?

9. Antimicrobial Categories Mechanisms of action Effects on Cell Wall Synthesis Interruption of Cell Membrane Structure and Function Inhibition of Protein Synthesis Inhibition of Folate Synthesis Interference with Nucleic Acid Metabolism

10. Effects on Cell Wall Synthesis Cell wall protects the bacteria cytoplasmic membrance Cell wall primarily composed of a peptidoglycan layer Inactivating or interfering with enzymes that synthesize the cell wall can destroy the bacteria

11. β-Lactam Antibacterial Agents Effect cell wall synthesis Sizable portion of antibacterial agents used today Includes penicillins, monobactams, and carbapenems, and cephalosporins

12. β-Lactam Antibacterial Agents: Overview Bind specific enzymes known as penicillin- binding proteins (PBPs) PBPs mediate peptidoglycan cross- linking If PBPs are bound by the beta-lactam, the cross-linking of the cell wall is incomplete, results in cell death

13. β-Lactam Antibacterial Agents Penicillins Simple penicillins are effective against many streps, Neisseria, Pasteurella, and a number of anaerobes Monobactams Limited to aerobic Gram negative bacilli Carbapenems Broadest antimicrobial spectrum Effective against gram positive and negative organisms, and anaerobes Resistant to beta-lactamase Cephalosporins Classified by their spectrum of activity and are spoken of in terms of “generations”

14. Generations of Cephalosporins First-generation Have good GP and GN activity Second-generation Have better GN, and anerobes activity Third-generation Better with Enterobacteriaceae and Pseudomonas spp. Fourth –generation Effective against GNR that are resistant to 3 rd generation cephalosporins Fifth-generation Spectrum of activity includes the 3 rd and 4 th generation

15. β-Lactam/β-Lactamase Inhibitors Combination of a β-lactam and a β- lactamase inhibitor act in synergy Bind to beta-lactamase produced by certain microbes β-Lactamase Inhibitors Offer no antibacterial activity by themselves Examples include: clavulanic acid, sulbactam, tazobactam

16. Effects on Cell Wall Synthesis Glycopeptides Bind certain amino acids and inhibit enymes in the developing peptidoglycan layer Vancomycin Most clinically important Effective against MRSA, other GP organisms, and organisms resistant to penicillin

17. Interruption of Cell Membrane Structure and Function Damages the cytoplasmic membrane of the organism Bacitracin Prevents the addition of peptidogylcan to the cell wall Disrupts the cell membrane Primarily effective against GP organism Because of toxicity, these are limited to topical medications (ex. Neosporin, etc.)

18. Interruption of Cell Membrane Structure and Function Polymyxins Bind to outer surface of cell membrane, affecting phospholoid Leads to leakage of intracellular contents and cell death Effective against gram negative bacteria

19. Inhibition of Protein Synthesis These antimicrobials bind to ribosomal subunits This binding is either irreversible, resulting in cell death(bactericidal), or reversible, resulting in bacteriostatic effects Antibiotics Aminoglycosides, tetracyclines, macrolides, clindamycin chloramphennicol, and oxazolidinone

20. Antibiotics of Protein Synthesis Inhibition Aminoglycosides Bactericidal Used primarily against GN bacteria

21. Antibiotics of Protein Synthesis Inhibition Tetracyclines Bacteriostatic Broad spectrum Effective against GP and GN organisms Tetracycline is NOT used in young children or in pregnancy, as it affects tooth and bone development

22. Antibiotics of Protein Synthesis Inhibition Macrolides Bacteriostatic Broad spectrum Effective against GP and some GN organisms, spirochetes, Mycoplasma, Legionella, and Chlamydia Agents include: erythromycin, azithromycin, clarithromycin

23. Antibiotics of Protein Synthesis Inhibition Clindamycin Bacteriostatic Excellent activity against aerobic GP organisms Extremely potent against anaerobes “D” test Detects resistance to clindamycin based on past treatment with erythromycin

24. Antibiotics of Protein Synthesis Inhibition Chloramphenicol Bacteriostatic Has broad activity but is extremely toxic Oxazolidinone Linezolid Effective against MRSA, VRE, and mycobacteria

25. Inhibition of Folate Synthesis Folic acid pathway provides essential precursor molecules for DNA synthesis Antibiotics can block steps in this pathway resulting in cell death Agents: sulfonamides, trimethoprim Used in combination Active against broad spectrum, including GP and GN organisms, except for P. aeruginosa

26. Interference with Nucleic Acid Metabolism Interfere with either DNA or RNA metabolism Inhibit enzymes required in the replication process Agents: quinolones/fluoroquinolones, rifamycins

27. Antibiotics of Nucleic Acid Metabolism Interference RNA Synthesis Interference Rifampin Mainly used for M. tuberculosis and M. avium complex Has a broad spectrum of activity

28. Antibiotics of Nucleic Acid Metabolism Interference DNA Synthesis Interference Quinolones/Fluoroquinolones Bactericidal Used to treat GN organisms Agents- ciprofloxacin, levofloxacin Metronidzole Activates under anaerobic conditions Effective against anaerobes and protozoa, bacterial vaginosis Nitrofurantoin Used against GN and GN organisms Concentrates well in urine

29. Mechanisms of Antimicrobial Resistance Modify target If target is altered, reduction or prevention of antimicrobial binding can occur End result- antimicrobial is ineffective How does the microbe modify the target? Chromosomal mutations Transposons Plasmids

30. Mechanisms of Antimicrobial Resistance Inactivation of Antimicrobial Agent Genes of the microbe encode enzymes that convert active antimicrobial agents to an inactive form Encoding of enzymes via chromosomal or plasmid-mediated genes Example: beta-lactamase producing organisms

31. Mechanisms of Antimicrobial Resistance Blockage of antimicrobial entry into the cell Mechanisms Decreased permeability Decreased uptake Increased ability to pump antimicrobial out of cell

32. References Kiser, K. M., Payne, W. C., & Taff, T. A. (2011). Clinical Laboratory Microbiology: A Practical Approach. Upper Saddle River, NJ: Pearson Education. Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders. http://www.parn.org.pk/index_files/D.test.html