Principles of Antimicrobial Action and Resistance.pdf

Principles of Antimicrobial Action and Resistance Chapter 10 Bailey & Scott’s Diagnostic Microbiology Patricia M. Tille Fourteenth Edition Copyright © 2017 Elsevier Inc. All Rights Reserved Objectives • Define the following terminologies (susceptible, intermediate, resistant, breakpoint, MIC, M...

Principles of Antimicrobial Action and Resistance Chapter 10 Bailey & Scott’s Diagnostic Microbiology Patricia M. Tille Fourteenth Edition Copyright © 2017 Elsevier Inc. All Rights Reserved Objectives • Define the following terminologies (susceptible, intermediate, resistant, breakpoint, MIC, MBC, antimicrobial agents, and AST) • Compare the outcome categories of antimicrobial drugs combination (additive, synergistic, antagonistic, and indifferent) • Outline the factors needed for an antimicrobial agent to exert an effect and compare the different modes of action of antimicrobial agents (bacteriostatic vs bactericidal) • Compare the structural differences in the cell wall of Gram-negative and Gram positive bacterial species • Explain the different mechanisms of action of various antimicrobial agents • Categorize commonly used antibacterial agents accordingly to antimicrobial class, mode of action, spectrum of activity, resistance mechanisms • Differentiate between biological, clinical, environmentally-mediated, and microorganism-mediated (intrinsic/acquired) resistance • Explain the different mechanisms of antibiotic resistance and outline the role of plasmids in antibiotic resistance • Determine the factors that contribute to the emergence and dissemination of antimicrobial resistance among bacteria Terminologies Antimicrobial agent: A natural, semisynthetic, or synthetic substance that kills or suppresses the growth of microorganisms  Natural: Antibiotic (Produced by bacteria or fungi to inhibit growth of other organisms)  Synthetic: Man-made  Semisynthetic: Chemically modified Susceptible: -The organism “responds” to drug treatment at the recommended dosage - Antimicrobial agent is an appropriate choice of treatment Intermediate: -The organism is "moderately susceptible” to treatment - The antimicrobial agent is still effective but the response rates are lower than for susceptible isolates Resistant: -The isolate does not respond to a given drug, irrespective of the dosage and the location of the infection - Antimicrobial agent is not an appropriate choice for treatment Minimum bactericidal concentration (MBC): The minimum concentration of antimicrobial agent needed to yield 99.9% reduction in viable colony-forming units of a bacterial suspension Minimum inhibitory concentration (MIC): The minimum concentration of antimicrobial agent needed to prevent visible growth of a microorganism suspension Breakpoint: A chosen concentration (mg/L) of an antibiotic which defines whether a species of bacteria is susceptible or resistant to the antibiotic. If the MIC is less than or equal to the susceptibility breakpoint, the bacteria is considered susceptible to the antibiotic Antimicrobial susceptibility testing (AST) methods: Procedures used to detect antimicrobial susceptibility/resistance to therapeutic agents Drug Interactions Synergy: The activity of combining two antimicrobial drugs is greater than the activity of each single drug alone (1+2=4) Additive: The activity of combining two antimicrobial drugs gives a total effect that is the sum of the individual effects (1+2=3) Indifferent: The activity of combining two antimicrobial drugs is no better or worse than the single most active drug alone (2+1=2) Antagonism: The activity of combining two antimicrobial drugs gives a lower activity than that of the single most active drug alone (one drug interferes with the activity of the other) (1+2=1) Spectrum of Activity Narrow Spectrum: Antimicrobial agents are only active against a specific group of bacteria Broad spectrum: Antimicrobial agents are active against wide range of bacterial groups (both gram-negative and gram positive bacteria) Factors that Control Antimicrobial Activity For an antimicrobial agent to inhibit or kill an infecting microorganism: • The agent must be in an active form (take into account the route of administration; ex: orally, intramuscularly, intravenously) • The agent must be at sufficient levels or concentrations at the site of infection (in anatomic approximation with the infecting microorganism) • Several pharmacokinetic properties of the agent such as rate of absorption, distribution, metabolism, and excretion of the agent’s metabolites should be also considered Effect of Antimicrobial Agents  Bactericidal agents : kill bacteria (cide: kill)  Bacteriostatic agents: Inhibit bacterial growth (static: no change) Steps for antimicrobial acitvity The processes or structures most frequently targeted are: • Bacterial cell wall (peptidoglycan) synthesis • Cell membrane • DNA replication • DNA transcription • Metabolic pathways/Folate synthesis • mRNA translation/protein synthesis Bacterial Cell Wall • Not found in mammalian cells • Cell wall characteristics affect antibiotic spectrum of activity • Maintain Shape (provide rigidity) • Protect bacteria from osmotic pressure changes Structural differences in the cell wall of Gram-negative and Gram positive bacterial species • Gram-positive bacteria thick layer of peptidoglycan • Gram-negative bacteria thin layer of peptidoglycan • Compared to gram-positive bacteria, gram-negative bacteria are generally more resistant against antibiotics because of their impenetrable cell wall I. Cell Wall Synthesis 1. Cytoplasmic stage ( UDP-NAM-pp) (UDP-NAM-pp) 2. Membrane stage (UDP-NAM-pp) (Very Toxic) 3. Extracellular stage I. Inhibitors of Cell wall Synthesis • Selective Toxicity: A mammalian cell does not have a cell wall • Bactericidal Activity A. β-Lactam • Have a four-member, nitrogen-containing, β-lactam ring at the core of their structure -Antibiotics differ in ring structure and attached chemical groups • Comprises the largest group of antibacterial agents • Types of β-lactam agents include penicillins, cephalosporins, carbapenems, and monobactams • Inhibit peptidoglycan crosslinking by binding to penicillin binding proteins (PBPs) (transpeptidase) -cell wall synthesis is terminated and bacterial cell dies B. Fosfomycin • Synthetic, inactivates enzymes involved in the first step of peptidoglycan synthesis C. Glycopeptides • Bind to the end of the peptidoglycan -Interfere with transpeptidation and incorporation of the precursors into the growing cell wall • Examples: Vancomycin and Teicoplanin • Large in size -Cannot penetrate the outer membrane of most gram-negative bacteria → Ineffective against gram-negative bacteria • Vancomycin has a potential for toxicity D. Lipoglycopeptides -Examples: Oritavancin, Dalbavancin and Telavancin -Structurally similar to vancomycin -Semisynthetic molecules (glycopeptides with a hydrophobic groups) -Bind to the bacterial cell membrane → increase the inhibition of cell wall synthesis -Inhibit transglycosylation by complexing with the D-alanyl-D-alanine residues -Increase cell permeability and cause depolarization of the cell membrane potential E. Other Cell wall active antibiotics Example: Bacitracin II. Inhibitors of Cell Membrane Function Lipopeptides • Daptomycin binds to and disrupts the cell membrane (cytoplasmic membrane), increasing permeability in gram-positive bacteria -Large in size → unable to penetrate the outer membrane of gram-negatives and act against them -Rare occasions of eosinophilic allergic pneumonitis • Polymyxins (polymyxin B and polymyxin E/colistin) -Interact with the phospholipids in the cell membranes → increasing permeability → leakage of macromolecules and ions -Most effective against gram-negative bacteria -Risk of neurotoxicity and nephrotoxicity (since human host cells also have cell membranes) III. Inhibitors of DNA Synthesis • The primary antimicrobial agents that target DNA metabolism are fluoroquinolones (or quinolones) and metronidazole  Fluoroquinolones: -(Example: ciprofloxacin, moxifloxacin, levofloxacin….) -Broad spectrum of activity (gram-negative and gram-positive bacteria) -Some bind to and interfere with the DNA gyrase enzyme (in gram-negative bacteria) -Others inhibit topoisomerase IV (in gram-positive bacteria) -Bactericidal: Because they interfere with DNA replication and therefore cell division -Risk of Tendinitis and rupture of the Achilles tendon  Metronidazole: -Mechanism of activity is related to the presence of a nitro group in the chemical structure *Reduced by a nitroreductase to generate cytotoxic compounds and free radicals that disrupts DNA -Activation requires reduction under conditions of low redox potential (anaerobic environments) *Most potent against anaerobic and microaerophilic organisms (notably gram negatives) -Effective in the treatment of protozoans (Trichomonas, Giardia spp., Entamoeba histolytica) -Low toxicity/side effects generally include mild gastrointestinal symptoms and headaches IV. Inhibitors of RNA Synthesis  Rifamycins: -Semisynthetic antibiotics that bind to the enzyme DNA-dependent RNA polymerase and inhibit RNA synthesis -Include the drug rifampin (also known as rifampicin) *Rifampin does not effectively penetrate the outer membrane of most gram-negative bacteria → more active against grampositive bacteria *Spontaneous mutation, resulting in the production of rifampin-insensitive RNA polymerases, occurs at a relatively high frequency -Rifampin is typically used in combination with other antimicrobial agents *Rifampin side effects include gastrointestinal symptoms and hypersensitivity reactions V. Inhibitors of Folate Synthesis • Folic acid pathway provides precursors for DNA synthesis • Two key enzymes in the pathway (Dihydropteroate synthase and Dihydrofolate reductase)  Sulfonamides: -Target and bind to dihydropteroate synthase (competitive inhibitor of PABA) to disrupt the folic acid pathway -Active against a wide variety of bacteria, including the gram-positive and gram-negative (except P. aeruginosa) species -Moderately toxic causing vomiting, nausea, and hypersensitivity reactions -Antagonistic for several other medications (including warfarin, phenytoin, and oral hypoglycemic agents)  Trimethoprim: Inhibits dihydrofolate reductase -Active against several gram-positive and gram-negative species -Frequently, trimethoprim is combined with a sulfonamide (usually sulfamethoxazole) *Attack two targets of the folic acid metabolic pathway -Enhances activity against various bacteria -Helps against the emergence of bacterial resistance to a single agent -Mild toxicity/Side effects include gastrointestinal symptoms and allergic skin rashes Recall of protein synthesis process https://www.youtube.com/results?search_query=protein+synthesis VI. Inhibitors of Protein Synthesis • Antibiotic classes include aminoglycosides, macrolide-lincosamide-streptogramins (MLS group), ketolides, chloramphenicol, tetracyclines, glycylglycines, and oxazolidinones  Aminoglycosides: -Irreversibly bind to protein receptors on the microorganism’s 30S ribosomal subunit *Interrupts :-Formation of the ribosomal-mRNA complex & initial formation of the protein synthesis complex -Accurate reading of the messenger RNA (mRNA) code -Aminoglycosides include: gentamicin, tobramycin, amikacin, streptomycin, and kanamycin -Active against a wide variety of aerobic gramnegative and certain gram-positive bacteria (ex: S. aureus) *Typically no effect on anaerobic bacteria (unable to uptake these agents intracellularly) -Aminoglycosides uptake is accompanied by using them in combination of cell-wall active antibiotics -Major toxicities: nephrotoxicity and auditory or vestibular toxicity https://courses.lumenlearning.com/microbiology/chapter/mechanisms-of-antibacterial-drugs/  Macrolide-Lincosamide-Streptogramin (MLS) Group: -Most commonly used antibiotics in the MLS group are the macrolides (e.g. erythromycin, azithromycin, clarithromycin) and clindamycin (which is a lincosamide) Macrolides: -Bind to the 23srRNA on the bacterial 50S ribosomal subunit → disruption of the growing peptide chain by blocking the translocation reaction -Generally bacteriostatic -Generally not effective against gram-negative bacteria (due to outer membrane uptake difficulties) -Effective against gram-positive bacteria, mycoplasmas, treponemes, and rickettsiae - Toxicity is generally low Lincosamides: -Clindamycin and lincomycin -Bind to the 50s ribosomal subunit → prevent elongation by interfering with peptidyl transfer during protein synthesis -May exhibit bacteriocidal or bacteriostatic activity *Depends on bacterial species, size of the inoculum, and drug concentration -Generally effective against gram-positive cocci *Often used against anaerobic Gram-positive https://courses.lumenlearning.com/microbiology/chapter/mechanisms-ofantibacterial-drugs/ Streptogramins: -Naturally occurring and enter bacterial cells by passive diffusion -Bind irreversibly to the 50s subunit of the bacterial ribosome → conformational change in the ribosome structure →interferes with peptide bond formation during protein synthesis → disrupting elongation of the growing peptide - Quinupristin-dalfopristin is a dual streptogramin that targets two sites on the 50S ribosomal subunit -Effective against gram-positive and some gram-negative bacteria - Low toxicity/Localized phlebitis upon intravenous infusion  Ketolides: - Consists of chemical derivatives of erythromycin A and other macrolides - Bind to the 23s rRNA of the 50S ribosomal subunit → inhibiting protein synthesis - Telithromycin is the only available ketolide *Active against most macrolide resistant gram-positive organisms - Effective against respiratory pathogens and intracellular bacteria *Particularly against gram-positive and some gram-negative bacteria (e.g. Francisella tularensis), as well as Mycoplasma, Mycobacteria, Chlamydia, and Rickettsia spp. -Low toxicity/Side effects: gastrointestinal symptoms, including diarrhea, nausea, and vomiting  Oxazolidinones:  Chloramphenicol: -Currently represented by linezolid & tedizolid (synthetic agents) -Inhibits the addition of amino acids to the growing peptide chain/inhibits transpeptidation by reversibly binding to the 50S -Specifically interacts with the 23S rRNA in the 50S ribosomal ribosomal subunit subunit → interfering with the binding of tRNA for formylatedmethionine → inhibits initiation of translation of any mRNA→ -Highly active against a wide variety of gram-negative and grampreventing protein synthesis positive bacteria -Effective against most gram-positive bacteria and mycobacteria -Toxic/Bone marrow toxicity is the major side effect -Generally low toxicity/Side effects: gastrointestinal symptoms including diarrhea and nausea  Tetracyclines: -Broad spectrum bacteriostatic antibiotics -Binds reversibly to the 30S ribosomal subunit → interfering with tRNA-amino acid complexes to ribosomes → preventing peptide chain elongation -Effective against gram-negative bacteria, gram-positive bacteria, several intracellular bacterial pathogens (e.g. Chlamydia and Rickettsia spp.) and some protozoa -May successfully treat infections caused by Neisseria gonorrhoeae, mycoplasma, and spirochetes -Toxicity includes upper gastrointestinal effects (esophageal ulcerations, nausea, vomiting, and epigastric distress) and cutaneous phototoxicity causing photoallergic immune reactions  Glycylglycines: -Semi-synthetic tetracycline derivatives -Tigecycline is the first agent of this class approved for clinical use *Inhibits protein synthesis by reversibly binding to the 30S ribosomal subunit (Similar to the tetracyclines) *Refractory to the most common tetracycline resistance mechanisms -Most common side effects: nausea, vomiting, and diarrhea Nitrofurantoin: An antibiotic of diverse mechanism of action with several targets involved in protein and enzyme synthesis -Consists of a nitro group on a heterocyclic ring -Converted by bacterial nitroreductases to reactive intermediates that bind to bacterial ribosomal proteins & rRNA, disrupting synthesis of RNA, DNA, & proteins -Used to treat uncomplicated urinary tract infections caused by gram-positive and gram-negative bacteria -Toxicity primarily consists of gastrointestinal symptoms (diarrhea, nausea, and vomiting) *Chronic pulmonary conditions may develop (irreversible pulmonary fibrosis) ANTIBIOTIC RESISTANCE BIOLOGIC VERSUS CLINICAL RESISTANCE  Biologic resistance: Observably reduced susceptibility of an organism to a particular antimicrobial agent  Clinical resistance: Loss of antimicrobial susceptibility to the extent that the drug is no longer effective for clinical use ENVIRONMENTALLY-MEDIATED ANTIMICROBIAL RESISTANCE  Resistance directly resulting from physical or chemical characteristics of the environment *Either directly alter the antimicrobial agent or alter the microorganism’s normal physiologic response to the drug  Examples of environmental factors that mediate resistance include: pH, anaerobic atmosphere, cation concentrations, and thymidine content MICROORGANISM-MEDIATED RESISTANCE  Resistance that results from genetically encoded traits of the microorganism  Divided into two subcategories: intrinsic or inherent resistance and acquired resistance -Intrinsic Resistance: *Resulting from the normal genetic, structural or physiologic state of a microorganism *Natural and consistently inherited -Acquired resistance: *Resulting from altered cellular physiology and structure caused by changes in a microorganism’s genetic makeup *Acquired by: successful genetic mutation, acquisition of genes from other organisms via gene transfer mechanisms, combination of both Factors contributing to the emergence and dissemination of antimicrobial resistance among bacteria  Resistance genes are shared between bacteria via: -Transformation -Transduction -Conjugation -Plasmid-mediated resistance: transfer of antibiotic resistance genes via a plasmid -Transposable elements (TEs or transposons) -Integron  Expression of resistance could be Constitutive, Inducible, Homogenous, Heterogeneous Mechanisms of Drug Resistance Modified from https://www.sciencedirect.com/science/article/pii/S0960894X17308284  Decrease uptake or accumulation: - Membrane impermeability - Efflux  Enzymatic inactivation or modification  Target site Modification  Biofilms  Pathway bypass Resistance to β-lactam Antibiotics  Bacterial resistance to beta-lactams may be mediated by: 1. Enzymatic destruction of the antibiotics- Most common *β-lactamases open the β-lactam ring → altered structure prevents effective binding to PBPs → cell wall synthesis continues 2. Altered antibiotic targets → low affinity *Genetic mutation in PBP coding sequence/genetic recombination *Ex: Methicillin is a β-lactam antibiotic of the penicillin class -mecA gene is found in Methicillin-resistant S. aureus (MRSA) *β-lactamase is produced by both Gram-positive (mostly Staphylococci) and Gram-negative bacteria (Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp.) *β-lactamases also vary in their spectrum of substrates -Not all β-lactams are susceptible to hydrolysis by every β-lactamase -Extended spectrum β-lactamases (ESBLs): β-lactamases that inactivates both penicillins and cephalosporins *Penicillinase is the first identified β-lactamase *To circumvent resistance: -β-lactam/β-lactamase inhibitor combinations - Molecular alterations in the β-lactam structure -PBP2a is encoded by the gene mecA →MRSA produces an altered PBP called PBP2a →decreased binding between βlactam agents and PBP2a → cell wall synthesis proceeds 3. Decreased intracellular uptake (in gram-negative bacteria) or increased cellular efflux of the drug 4. Changes in number or characteristics of the outer membrane porins through which β-lactams pass (in gramnegative bacteria) Resistance to Glycopeptides Vancomycin resistance:  Acquired high-level resistance to vancomycin: *common among enterococci, rarely among staphylococci, and not at all among streptococci - vancomycin-resistant S. aureus from patients previously infected/colonized with enterococci  One mechanism of resistance: Production of altered cell wall precursors unable to bind vancomycin with sufficient avidity to allow inhibition of peptidoglycans synthesizing enzymes  Another mechanism of resistance with a lower level of resistance (only in staphylococci): Overproduction of the peptidoglycan layer →binds excessive amounts of glycopeptides→ diminished ability of the drug to exert its antibacterial effect Resistance to Aminoglycosides  Aminoglycoside resistance is accomplished by enzymatic modification, altered target, decreased uptake pathways, or porin alterations (in gram negative bacteria) *Gram-positive and gram-negative bacteria produce several aminoglycoside-modifying enzymes • Phosphorylation of hydroxyl groups • Adenylation of hydroxyl groups • Acetylation of amine groups Resistance to Quinolones  Most frequently mediated by a decrease in uptake (Components of the gram-negative cellular envelope can limit quinolone access to the cell’s interior), decrease in accumulation/efflux (staphylococci), or by production of an altered target -Primary resistant pathway involves mutational changes in the targeted subunits of DNA gyrase