1. Control of Microbial Growth Tim Ho University of Alberta, Canada * The materials are mostly based on Dr. Brian Lanoil’s Microb 265 tim3@ualberta.ca 1 Part II

2. Objectives 1.Know 3 methods of microbial control 2.Know the strategies on how drugs control the growth of microorganisms. 3.Understand how do bacteria become resistant to antibiotics. tim3@ualberta.ca 2 Physical agents Mechanical removal methods Chemical agents

3. tim3@ualberta.ca 3 1.With minimal side effects 2.Therapeutic dose: the amount of drug required for treatment or the desired effect. 3.Broad spectrum activity: against a wide variety of pathogens or do not know the specific bacteria that want to target. 4.Chemotherapeutic agents can be synthetic or semi-synthetic. Characteristics of antimicrobial drugs:

4. tim3@ualberta.ca 4 Dilution Susceptibility test Each test tube containing different concentrations of drug - MIC: minimum inhibitory concentration - MLC: minimum lethal concentration Low [drug]high [drug] + + + + + + + - - -

5. tim3@ualberta.ca 5 Image:http://www.biotopics.co.uk/microbes/penici.htmlhttp://www.biotopics.co.uk/microbes/penici.html Disk diffusion test Kirby-Bauer method Drug diffuses from disk into agar, establishing concentration gradient Measure the diameter of clear zone (no growth) around disks -> determine MIC and MLC Large clear zone = sensitive No or small clear zone = resistant

6. tim3@ualberta.ca 6 Disk diffusion test Image:http://www.biotopics.co.uk/microbes/penici.htmlhttp://www.biotopics.co.uk/microbes/penici.html

7. tim3@ualberta.ca 7 How different types of antibiotics affect cell functions

8. Folic acid synthesis inhibitors 8 tim3@ualberta.ca

9. 9 Folic acid synthesis inhibitors Sulfanilamide: - Competitive inhibitor of PABA - [PABA] ↑= rate of folic acid biosynthesis ↓ Image: Fdardel, 2011

10. DNA gyrase inhibitors 10 tim3@ualberta.ca

11. DNA gyrase inhibitors Quinolones: - inhibit bacterial DNA gyrase - effective against G- urinary tract infections and respiratory infections - (eg. Bacillus anthracis) - [PABA] ↑= rate of folic acid biosynthesis ↓ tim3@ualberta.ca 11 Image: Drug Reference - Encyclopedia Ciprofloxacin DNA gyrase: the enzyme that introduces negative supercoils into DNA

12. RNA synthesis inhibitors 12 tim3@ualberta.ca

13. RNA synthesis inhibitors 13 tim3@ualberta.ca Rifamycin/ Rifampin: - block transcription by binding RNA polymerase - Not selectively toxic → Prokaryotes and eukaryotes synthesize nucleic acids in pretty much the same way - [PABA] ↑= rate of folic acid biosynthesis ↓

14. Cell wall synthesis inhibitors 14 tim3@ualberta.ca - Ampicillin is protected from lactamases by co-treatment with clavulanic acid WHY: ß-lactamases have higher binding affinity for clavulanic acid than ampicillin Image: Dengler et al. BMC Microbiology 2011 11:16 doi:10.1186/1471-2180-11-16

15. Cell wall synthesis inhibitors 15 tim3@ualberta.ca Image: Insilico Genomics Lab Technologies. http://insilicogenomics.in/penicillin.asphttp://insilicogenomics.in/penicillin.asp It breaks ß-lactam rings: antibiotic resistance - G+ cells: ß-lactamases are located on outside surface Activity is blocked by binding to transpeptidases G- cells: ß-lactamases are in periplasmic space (transpeptidase) G + bacteria are more susceptible to ß-lactam antibiotics!!

16. blocks transpeptidization blocks dephosphorylation of bactoprenol phosphate blocks D-Ala peptidization Cell wall synthesis inhibitors tim3@ualberta.ca 16 Image: Dengler et al. BMC Microbiology 2011 11:16 doi:10.1186/1471-2180-11-16

17. Protein synthesis inhibitors 17 tim3@ualberta.ca

18. Protein synthesis inhibitors 18 tim3@ualberta.ca Aminoglycosides: -Binding to the small subunit ribosome - effective against G- cells Macrolides: -Binding to the large subunit ribosome Tetracyclines: - First broad-spectrum antibiotics -Blocking tRNA attachment to ribosome - effective against G- and G+ cells

19. Cytoplasmic membrane inhibitors 19 tim3@ualberta.ca

20. Cytoplasmic membrane inhibitors 20 tim3@ualberta.ca Daptomycin: - Cyclic lipopeptide - Makes pore on cytoplasmic membrane - Resistance from changes in cell membrane structure - Primarily targets G+ cells (G- cells have extra outer membrane: protection)

21. tim3@ualberta.ca 21 Anti-fungal Drugs Fungal infections are difficult to treat - host and pathogen have biological similarity → drug can harm host at the same time Target against chitin (fungal cell wall) mostly - animals (host) don’t have chitin Nystatin: first discovered antifungal antibiotic in 1949 by Hazen and Brown Superficial mycoses - Infections of outer layers of skin - Treatment (drugs): Miconazole, Nystatin, and Griseofulvin - Minimizes toxic systemic side effects (e.g. liver damage)

22. tim3@ualberta.ca 22 Antiviral Drugs Many drugs are still in development stage Mainly target against either RNA or DNA synthesis of viral pathogen - Structural analogs of purine or pyrimidine bases - difficulties: viruses use metabolic machinery of the host Protease inhibitors: against virus-specific enzymes Interferons: stimulate production of host anti-viral proteins