CSLO3. Distinguish between mechanisms of physical and chemical agents to control microbial populations.
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1 PowerPoint Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University CSLO3. Distinguish between mechanisms of physical and chemical agents to control microbial populations. C H A P T E R 10 Controlling Microbial Growth in the Body: Antimicrobial Drugs
2
3 bacteria fungi HOW? WHAT? virus protozoa
4 Figure 10.1 Antibiotic effect of the mold Penicillium chrysogenum. Staphylococcus aureus (bacterium) Penicillium chrysogenum (fungus) Zone where bacterial growth is inhibited
5 FACT 10.1 Antibiotics CONCEPT 10.1 Antimicrobial agents produced naturally by (micro)organisms Common usage: made by microorganisms Common usage: antibiotic = antibacterial Natural Antibiotics Semisynthetics Antibiotics Chemically altered antibiotics that are more effective, longer lasting, or easier to administer than naturally occurring ones Synthetics Antibiotics Antimicrobials that are completely synthesized in a lab
6 Are Antibiotic Antiseptic? Typically used for treatment of disease and not for environmental control (like chemicals from Chapter 9) But also used for antimicrobial control outside the body What would they be called then? Nisin (kill bacteria in cheese) Natamycin (kill fungi in cheese)
7 Figure 10.2 Mechanisms of action of microbial drugs. Inhibition of pathogen's attachment to, or recognition of, host Arildone Pleconaril Human cell membrane Inhibition of cell wall synthesis Penicillins Cephalosporins Vancomycin Bacitracin Isoniazid Ethambutol Echinocandins (antifungal) CONCEPT 10.2 FACT 10.2 Inhibition of protein synthesis Aminoglycosides Tetracyclines Chloramphenicol Macrolides Antisense nucleic acids Inhibition of DNA or RNA synthesis Actinomycin Nucleotide analogs Quinolones Rifampin Disruption of cytoplasmic membrane Polymyxins Polyenes (antifungal) Inhibition of general metabolic pathway Sulfonamides Trimethoprim Dapsone
8 CONCEPT 10.2 FACT 10.2 Targets of Antimicrobial Agents on the microbe Mechanisms of Antimicrobial Action 1) Inhibition of Cell Wall Synthesis 2) Disruption of Cytoplasmic Membranes 3) Inhibition of Protein Synthesis 4) Inhibition of Metabolic Pathways/Nucleic Acid Synthesis 5) Prevention of Virus Attachment
9 1) Inhibition of Cell Wall Synthesis CONCEPT 10.3 Beta-lactams (Penicillin) are most prominent in this group Beta-lactams are chemicals with functional groups called beta-lactam rings Beta-lactams bind to enzymes that cross-link NAM subunits and inactivate them Bacteria have weakened cell walls and eventually lyse Prevent bacteria from increasing amount of peptidoglycan Have no effect on existing peptidoglycan layer Effective only for growing cells - Dormant cells are not affected
10 How cell walls are made and maintained 1) NAG-NAM polylinker 2) NAM-NAM crosslink NAG-NAM polylinker NAG NAM NAG NAM NAG-NAM chain Crossbridge between NAM and NAM Growth Divide New NAG and NAM subunits New NAG and NAM subunits are inserted into the wall by enzymes, allowing the cell to grow. Other enzymes link new NAM subunits to old NAM subunits with peptide cross-links. 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. CONCEPT 10.3
11 Beta-lactams (Penicillins) CONCEPT 10.3 Beta-lactams are chemicals with functional groups called beta-lactam rings
12 Figure 10.3c-e Bacterial cell wall synthesis and the inhibitory effects of beta-lactams on it. Beta-lactams (Penicillin) Functional groups are beta-lactam rings inhibit enymes which make new NAM-NAM crosslinks Penicillin G (natural) Penicillins Methicillin (semisynthetic) Cephalothin (natural) Cephalosporins New cross-links inhibited by beta-lactam Previously formed cross-link Growth Beta-lactam interferes with the linking enzymes, and NAM subunits remain unattached to their neighbors. However, the cell continues to grow as it adds more NAG and NAM subunits. The cell bursts from osmotic pressure because the integrity of peptidoglycan is not maintained. Osmotic pressure causes water to move in and burst the cell CONCEPT 10.3
13 CONCEPT 10.3 Semisynthetic derivatives of beta-lactams More stable in acidic environments (methicillin) More readily absorbed in intestine Less susceptible to deactivation by bacteria More active against more types of bacteria
14 Lysozyme NAG-NAM polylinker is Lysozyme an antibiotic?
15 Other Inhibitors of synthesis of bacterial walls Vancomycin (against MRSA) Interfere with particular NAM-NAM crosslinks (Ala-Ala bridges) present in many Gram-positive bacteria FACT 10.3 Bacitracin Blocks transport of NAG and NAM from cytoplasm NAG-NAM polylinker NAG NAM
16 Acid Fast bacteria cell wall Peptidoglycan layer (cell wall) Cytoplasmic membrane mycolic acid Gram-positive cell wall Lipoteichoic acid Teichoic acid Some gram positive bacteria have up to 60% mycolic acid (waxy lipids) which helps cells survive desiccation called Acid Fast bacteria Integral protein Acid Fast bacteria cell wall
17 Isoniazid and ethambutol Disrupt mycolic acid formation in mycobacterial species Because mycolic acid takes a long time to synthesize the generation time of mycobacterial species is long - 12 to 24 hrs FACT 10.4 mycolic acid Therefore treatment against the mycobacterium extends for months or years
18 Inhibitors of synthesis of fungal walls FACT 10.5 Fungal cells composed of various polysaccharides not found in mammalian cells (1,3 D-glucan) Echinocandins (caspofungin) inhibit the enzyme that synthesizes glucan
19 Targets of Antimicrobial Agents on the microbe Mechanisms of Antimicrobial Action 1) Inhibition of Cell Wall Synthesis 2) Disruption of Cytoplasmic Membranes 3) Inhibition of Protein Synthesis 4) Inhibition of Metabolic Pathways/Nucleic Acid Synthesis 5) Prevention of Virus Attachment
20 Disruption of Cytoplasmic Membranes in bacteria FACT 10.6 Polymyxin disrupts cytoplasmic membranes of Gramnegative bacteria Pseudomonas
21 Disruption of Cytoplasmic Membranes in Fungus FACT 10.7 Polyenes (Nystatins/Amphotericin B) attaches to ergosterol in fungal membranes form channel through cytoplasmic membrane and damage its integrity (Humans show side-effects because cholesterol similar to ergosterol)
22 Disruption of Cytoplasmic Membranes in Fungus FACT 10.8 Azoles (fluconazole), Allylamines (terbinafine) and Tolnaftate (Tinactin) inhibit ergosterol synthesis Ergosterol (like cholesterol) needed for integrity of cytoplasmic membrane
23 Disruption of Cytoplasmic Membranes in parasitic worms FACT 10.9 Some parasitic worm drugs act against cytoplasmic membranes (praziquantel and ivermectin) ivermectin The drug binds to channels (GluCls) in the membranes of invertebrate neurons and myocytes.
24 Targets of Antimicrobial Agents on the microbe Mechanisms of Antimicrobial Action 1) Inhibition of Cell Wall Synthesis 2) Disruption of Cytoplasmic Membranes 3) Inhibition of Protein Synthesis 4) Inhibition of Metabolic Pathways/Nucleic Acid Synthesis 5) Prevention of Virus Attachment
25 Mechanisms of Antimicrobial Action Inhibition of Protein Synthesis CONCEPT 10.4 Prokaryotic ribosomes are 70S (30S and 50S) Eukaryotic ribosomes are 80S (40S and 60S) Mitochondria of animals and humans contain 70S ribosomes Side-effect (liver and bone marrow affected) WHY?
26 Figure 10.4 The mechanisms by which antimicrobials target prokaryotic ribosomes to inhibit protein synthesis. FACT Incorrect amino acids 30S Ribosome Streptomycin/gentamicin mrna Some aminoglycosides cause change in 30S shape; mrna is misread. Lincosamides or macrolides bind to 50S subunit, blocking proper mrna movement through ribosome. Synthesis stops. 50S erthromycin mrna 50S Tetracycline and some aminoglycosides block docking site of trna. mrna 50S Antisense nucleic acid fomiversen mrna Amino acids Chloramphenicol binds enzymatic site 30S 50S mrna fmet 50S Mupirocin binds Isoleucine trna 30S Oxazolidinone mrna
27 Targets of Antimicrobial Agents on the microbe Mechanisms of Antimicrobial Action 1) Inhibition of Cell Wall Synthesis 2) Disruption of Cytoplasmic Membranes 3) Inhibition of Protein Synthesis 4) Inhibition of Metabolic Pathways/Nucleic Acid Synthesis 5) Prevention of Virus Attachment Antiviral agents
28 Mechanisms of Antimicrobial Action CONCEPT 10.5 FACT Inhibition of Metabolic Pathways Antimetabolic agents can be effective when pathogen and host metabolic processes differ 1) Microbial Non-Specific Inhibition of Nucleic Acid Synthesis Actinomycin D binds DNA block DNA replication or RNA transcription Not normally used to treat human infections WHY? RNA A potent anti-cancer drug
29 Atovaquone interferes with electron transport in protozoans which cause malaria and toxoplasmosis 1) Microbial Specific FACT 10.12
30 Antiviral agents CONCEPT 10.6 FACT Antiviral agents can target unique aspects of viral metabolism 1) Amantadine, rimantadine prevent viral uncoating 2) Protease inhibitors interfere with an enzyme that HIV needs in its replication cycle
31 3) Inhibition of Viral Nucleic Acid Synthesis 3a) Nucleotide or nucleoside analogs Interferance with function of nucleic acids AZT Used against viruses because 1) proof reading by viral DNA polymerase not as effective as human DNA polymerase 2) rapid synthesis of viral template so more incorporation
32 3) Inhibition of Viral Nucleic Acid Synthesis 3b) Reverse transcriptase inhibitors Act against an enzyme HIV uses in its replication cycle NO SIDE-EFFECT Why?
33 Mechanisms of Antimicrobial Action 5) Prevention of Virus Attachment Attachment antagonists (peptide/sugar analogs) block viral attachment or receptor proteins e.g. pleconaril binds surface of cold virus and prevents its binding to host cells
34 Maraviroc: A Coreceptor CCR5 Antagonist for Management of HIV Infection
35 Clinical Considerations in Prescribing Antimicrobial Drugs Ideal Antimicrobial Agent Spectrum of Action/Effectiveness Routes of Administration/Safety and Side Effects Resistance to Antimicrobial Drugs The Development of Resistance in Populations Mechanisms of Resistance Multiple Resistance and Cross Resistance Retarding Resistance
36 Clinical Considerations in Prescribing Antimicrobial Drugs CONCEPT 10.7 What is an Ideal Antimicrobial Agent? Readily available Inexpensive Chemically stable Easily administered Nontoxic and nonallergenic Selective toxicity
37 Clinical Considerations in Prescribing Antimicrobial Drugs Selective toxicity against microbes means killing the microbial cells but not the host's cells. CONCEPT 10.8 e.g. Inhibition of Metabolic Pathways specific to pathogens
38 Clinical Considerations in Prescribing Antimicrobial Drugs CONCEPT 10.9 Spectrum of Action Number of different pathogens a drug acts against 1) Narrow-spectrum effective against few organisms 2) Broad-spectrum effective against many organisms Killing of normal flora reduces microbial antagonism Superinfection is a possibility
39 Figure 10.8 Spectrum of action for selected antimicrobial agents. CONCEPT 10.9 Which are narrow/broad spectrum?
40 Clinical Considerations in Prescribing Antimicrobial Drugs CONCEPT Effectiveness of Antimicrobial Drugs is Determined by 3 tests 1) Diffusion susceptibility test (KIRBY BAUER) 2) Minimum inhibitory concentration test (MIC) 3) Minimum bactericidal concentration test (MBC) (for bacteriocidal/static check)
41 Figure 10.9 Zones of inhibition in a diffusion susceptibility (Kirby-Bauer) test. Bacterial lawn Zone of inhibition 1) Kirby-Bauer test Diffusion susceptibility test
42 Figure Minimum inhibitory concentration (MIC) test in test tubes. Turbid tubes Clear tubes 2) MIC smallest amount that inhibits growth Increasing concentration of drug
43 Figure A minimum bactericidal concentration (MBC) test. Concentration of antibacterial drug ( g/ml) Clear MIC tube Modified MIC test 8 g/ml 16 g/ml 25 g/ml 3) Minimum bactericidal concentration test (MBC) Bacterial colonies No colonies No colonies Drug-free media
44 Figure An Etest, which combines aspects of Kirby-Bauer and MIC tests. Etest = MIC+Kirby-Bauer type
45 Clinical Considerations in Prescribing Antimicrobial Drugs Routes of Administration CONCEPT Topical/local application of drug for external infections Oral route requires no needles and is self-administered Intramuscular administration delivers drug via needle into muscle Intravenous administration delivers drug directly to bloodstream
46 Figure The effect of route of administration on blood levels of a chemotherapeutic agent.
47 Clinical Considerations in Prescribing Antimicrobial Drugs Safety and Side Effects 1) Drug Toxicity (1/3) Drugs may be toxic to kidneys, liver, or nerves Consideration needed when prescribing drugs to pregnant women CONCEPT Therapeutic index is the ratio of the dose of a drug that can be tolerated to the drug's effective dose (higher TI = safer) Therapeutic Range/Window (safe range of drug conc.) FACT 10.14
48 Figure Some side effects resulting from toxicity of antimicrobial agents. Metronidazole antiprotozoan Black hairy tongue hemoglobin breakdown products Accumulate in tongue Tetracyline-Calcium complexes get incorporated in bones and teeth
49 2) Allergies (2/3) CONCEPT Allergic reactions are rare but may be life threatening Anaphylactic shock (0.1 % American penicillin allergy 300 deaths/year) Tightening of the airways and throat, causing trouble breathing Nausea or abdominal cramps Vomiting or diarrhea Dizziness or lightheadedness Weak, rapid pulse Drop in blood pressure Seizures Loss of consciousness
50 3) Disruption of normal microbiota Pathogen dies so primary infection is gone But normal microbiota also dies and an opportunistic pathogen may overgrow causing secondary infection Of leads to superinfections greatest concern for hospitalized patients Use Narrow Spectrum Antibiotics Broad Spectrum Antibiotics Normal Microbiota Opportunistic Pathogen Pathogen CONCEPT 10.14
51 Development of Resistant Microbes antibacterial hand CONCEPT Little evidence that products containing antiseptic and disinfecting chemicals add to human or animal health Use of antibiotics promotes development of resistant microbes FDA bans antibacterial soaps - Sep2016 Rule removes triclosan and triclocarban from overthe-counter antibacterial hand and body washes could pose health risks, such as bacterial resistance or hormonal effects.
52 Figure The development of a resistant strain of bacteria. Drug-sensitive cells Drug-resistant mutant CONCEPT Exposure to drug Population of microbial cells Sensitive cells inhibited by exposure to drug Remaining Population grows over time Most cells now resistant X X X X X
53 Genes prs in Genome antibiotic-resistance gene Genes prs in small genetic elements called plasmids antibiotic-resistance gene R plasmid
54 The Development of Resistance in Populations 1) Some pathogens are naturally resistant to antibiotics CONCEPT ) Those pathogens which are not naturally resistant can gain resistance Resistance by bacteria acquired in two ways 1) New mutations of chromosomal genes 2) Acquisition of R plasmids via transformation, transduction, and conjugation.
55 CONCEPT Different Mechanisms of Resistance to antimicrobial agents 1) Production of enzyme that destroys or deactivates drug 2) Slow or prevent entry of drug into the cell 3) Alter target of drug so it binds less effectively 4) Alter their own metabolic chemistry 5) Pump antimicrobial drug out of the cell before it can act 6) Bacteria in biofilms can resist antimicrobials
56 Figure 10.3c-e Bacterial cell wall synthesis and the inhibitory effects of beta-lactams on it. Beta-lactams Functional groups are beta-lactam rings Penicillin G (natural) Penicillins Methicillin (semisynthetic) Cephalothin (natural) Cephalosporins New cross-links inhibited by beta-lactam Previously formed cross-link Growth Beta-lactam interferes with the linking enzymes, and NAM subunits remain unattached to their neighbors. However, the cell continues to grow as it adds more NAG and NAM subunits. The cell bursts from osmotic pressure because the integrity of peptidoglycan is not maintained. Osmotic pressure causes water to move in and burst the cell
57 Figure How -lactamase (penicillinase) renders penicillin inactive. CONCEPT ) Production of enzyme that destroys or deactivates drug Lactam ring Penicillin -lactamase (penicillinase) breaks this bond Inactive penicillin 200 lactamases known many coded by R plasmids
58 Resistance to Antimicrobial Drugs Multiple Resistance CONCEPT Pathogen can acquire resistance to more than one drug Multi-drug-resistant (MDR) pathogens are resistant to at least three antimicrobial agents Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that causes infections in different parts of the body. sometimes called a "super bug.
59 Resistance to Antimicrobial Drugs CONCEPT Cross resistance resistance to one confers resistance to similar drugs e.g. streptomycin and other aminoglycoside-related drugs Resistance to 1 drug affecting ribosome activity because of change in ribosomal structure But now the ribosome also resistance to other similar drugs
60 4 ways for Retarding development of Resistance 1) Use antimicrobials only when necessary 2) Maintain high concentration of drug in patient for sufficient time (Complete the dose) - Inhibit the pathogen long enough so immune system can eventually eliminate it 3) Develop new drugs e.g. Vancomycin against MRSA 4) Use antimicrobial agents in combination Synergism of drugs penicillin and streptomycin CONCEPT 10.21
61 Figure An example of synergism between two antimicrobial agents. Disk with semisynthetic amoxicillin-clavulanic acid Disk with semisynthetic aztreonam CONCEPT Synergism of drugs
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