Antimicrobial Agent Mechanisms of Action and Resistance

Antimicrobial Agent Mechanisms of Action
and Resistance
Copyright © 2019 by Elsevier Inc. All rights reserved.

Learning Objectives
 To identify the sources of antibiotics.
 To compare broad-spectrum antimicrobials with
narrow-spectrum antimicrobials.
 To distinguish bacteriostatic agents from
bactericidal agents.
 To explain the mechanism of the antibiotic
action.
 To differentiate intrinsic resistance from acquired
resistance.
 To describe B-lactamase and how they could
disseminate antimicrobial resistance.
2
Historical Context and Examples
 The discovery of potent, relatively nontoxic
antimicrobial therapeutic agents
 Considered as the foremost medical advance in the
20th century
 Examples of antimicrobial agents that all have
the capacity of killing or suppressing growth of
microorganisms
 Antibacterials, antifungals, antiseptics, antibiotics,
preservatives, sterilants, disinfectants

3
Antibiotics Description
 Traditionally reserved for compounds that are
naturally produced by living microorganisms (in
particular bacteria and fungi)
 Now more widely applied to any natural, semi-
synthetic, or synthetic molecule used to treat or
prevent disease
 Numerous classes have been discovered with
different modes of action.
 Bacteria can evolve and counteract the action of
antibiotics.

4
Antimicrobial Agents, Antibiotics,
Resistance
 Antimicrobial agents
 Chemical compounds to kill or suppress
microorganisms
 Antibiotics
 Natural, semi-synthetic, or synthetic molecules used to
treat or prevent disease
 Resistance
 Intrinsic resistance
• Naturally found in bacteria (chromosomal)
 Acquired resistance
• Acquired from exogenous DNA (plasmid or through
conjugation)

5
Antimicrobial Drug Resistance
 Natural consequence of drug exposure and results
from the use and inappropriate use of antimicrobial
agents
 Some microorganisms acquire multidrug
resistance (MDR) and as such they can
 withstand the toxic effects of antimicrobial agents.
 utilize acquired elaborate mechanisms to mobilize
and disseminate genes to aid in the resistance effort.

6
Antibiotic Targets and
Mechanisms of Action

7
Antibiotics
 Approximately 23 unique classes and 18
subclasses of clinically useful antibiotics
 Two basic activities
 Cell wall
• Interference or disruption of cell integrity by disrupting cell
wall or cell membrane
 Metabolic functions
• Interruption of basic metabolic functions such as protein
synthesis, nucleic acid metabolism, and inhibition of essential
metabolites

8
Antimicrobial Agent Targets and
Pathways

9
Pathways

10
Pathways

11
Pathways

12
Pathways

13
Pathways

14
Pathways

15
Pathways

16
Primary Sites of Antibacterial Action for
Major Classes of Antimicrobial Agents

17
Inhibition of Bacterial Cell Wall
Biosynthesis
 Cell wall protects bacteria.
 Specific integral enzymes are necessary to build and
shape the cell wall.
• Transpeptidases: cross-link the cell wall
 These enzymes are also called penicillin binding proteins
(PBPs).
 Specific to each bacteria; therefore drugs may have difference
in binding to each PBP and thus have different levels of
effectiveness
 Drugs are designed to inhibit and inactivate these
enzymes.

18
The Cell Envelope Structure of a Gram-
Positive (Left) and Gram-Negative (Right)
Bacterium

19
Peptidoglycan Biosynthesis Stages
 Synthesis of precursors in the cytoplasm
 Transport of lipid-bound precursors across the
cytoplasmic membrane
 D-Cycloserine and bacitracin inhibit steps 1 and 2.
 Insertion of glycan units into the cell wall
 Transpeptidation linking and maturation
 β-Lactams and glycopeptides inhibit steps 3 and 4.

20
Peptidoglycan Layer in the Cell Wall of
Escherichia coli

21
β-Lactam Antibiotics and Role of the β-
Lactam Ring
 β-lactam antibiotics
 Natural and semi-synthetic
 Penicillins, cephalosporins, carbapenems,
monobactams
 Based on chemical structure, the β-lactam ring
 Binds to and inhibits the transpeptidases (known as
penicillin-binding proteins, PBPs)

22
β-Lactam Description and Example
 β-lactams must pass through cell wall porins in
gram-negative cells to reach cell PBPs.
 The binding of the type of β-lactam to specific
PBPs influences effectiveness of certain drugs
on specific organisms.
 Example
 Monobactam (aztreonam)
• Binds primarily to PBP of gram-negative aerobes
• No activity against gram-positive because incorrect PBPs

23
Examples of β-Lactam Antibiotics

24
Chemical Structures of Major Classes
β-Lactam Antibiotics

25
Narrow Spectrum Versus Broad
Spectrum Antimicrobial Activity
 Narrow spectrum
 Work only on gram-positives (cannot penetrate the
outer cell membrane)
 Exception: Doripenem, carbapenem
• Used to treat gram-positive and gram-negative bacillus
(including Pseudomonas aeruginosa)
 Broad spectrum
 Work on a broad variety of bacteria (can penetrate the
outer membrane)

26
Glycopeptides
 Examples
 Vancomycin
 Teicoplanin
 Block transpeptidation step
 Narrow spectrum antibiotic
 Useful for staphylococci, streptococci, and
enterococci

27
Inhibition of Folate Synthesis
Folic Acid
 Necessary for the synthesis of bacterial DNA
 Mediated by two enzymes
 Dihydropteroate synthase
 Dihydrofolate reductase
 Most make their own; very few can acquire it
from the environment

28
SMZ and TMP
 Sulfamethoxazole (SMZ)
 Competitively inhibits dihydropteroate synthetase
 Needs constant levels of drug to inhibit enzyme
 Trimethoprim (TMP)
 Competitively inhibits dihydrofolate reductase
 Needs constant levels of drug to inhibit enzyme

29
Combination of SMZ and TMP
 Using these in combination as a synergistic
effect
 Synergy
• Combined effect is greater than the additive effect.
• Enhanced activity
 Spectrum of activity of folate pathway inhibitors
• Provides activity against the Enterobacteriaceae that cause
urinary tract infections

30
Sites of Action of Sulfonamides and
Trimethoprim: Affects on the Synthesis of
Amino/Nucleic Acids

31
Interference with DNA Replication
 Disruption of DNA synthesis
 Disruption of replication
• Nalidixic acid and fluorinated quinolones
 Target type II (DNA gyrase) and IV topoisomerase enzymes
 Uses in treatment of Enterobacteriaceae, pseudomonads,
staphylococci, enterococci, neisseria, and streptococci species
other than S. pneumoniae
 Traps enzymes as stable reaction intermediates
inhibiting DNA replication through bacteriostasis

32
Interference with DNA Transcription
 Rifampin
 Synthetic derivative of rifamycin B, targets DNA
transcription
 Interferes with production of mRNA
• Thus prevents protein synthesis via blocking RNA
polymerase
 Principle: therapeutic use is in combination with other
antibacterial classes to treat Mycobacterium
tuberculosis.

33
Interference of mRNA Translation
 Bind to the 50S or 30S ribosomal subunit
 Can be reversible or irreversible
 Aminoglycosides
 Irreversibly binds to 30S subunit
• Prevents docking of aminoacyl-tRNA
 Also contributes to misreading the genetic code
 Spectinomycin
 Binds 30S subunit
• Interferes with the stability of peptidyl tRNA by inhibiting
elongation factor

34
Tetracyclines and Macrolides
 Tetracyclines
 Tetracycline, doxycycline, minocycline
 Reversible binding to 30S subunit
• Inhibit rotation of bound tRNA into the A site
• Causes premature release of peptides
 Macrolides
 Erythromycin, clarithromycin, azithromycin
 Reversibly binds to the 50S subunit
• Prevent elongation by blocking exit site
• Prevent ribosome assembly
• Interference and antagonism can occur if used in
combination with each other.
35
Ketolides
 Semi-synthetic compound
 Telithromycin
 Binds to the 50S subunit
 Inhibits RNA-dependent protein synthesis
 Evade macrolide-resistance mechanisms
 Through improved ribosome binding affinity
 Evasion of macrolide efflux mechanisms

36
Oxazolidinones and Streptogramin
 Oxazolidinones
 Linezolid
• Activity against gram-positive bacteria by binding 50S subunit
and blocking initiation and translocation
 methicillin-resistant S. aureus (MRSA), streptococcal
pneumonia, M. Tuberculosis
 Streptogramin
 70:30 mixture of A and B molecules called
Dalfopristin–quinupristin
• Disrupt translation by blocking elongation and binding of
aminoacyl-tRNA
• Dalfopristin helps quinupristin bind with greater affinity.
37
Glycylcycline
 Tigecycline
 Higher affinity for 30S ribosomal subunit than
tetracycline
 Increased effectiveness against tetracycline-
resistant organisms
 Wide spectrum of activity against gram-positive,
gram-negative, atypical, and anaerobic pathogens

38
Mechanisms of Antimicrobial
Resistance

39
Definitions
 Intrinsic resistance
 Naturally found in bacteria (chromosomal)
 Transmitted to progeny vertically
 Predictable once the organism is identified
 Acquired resistance
 Acquired from exogenous DNA (plasmid, conjugation,
transposons, bacteriophage, etc.)

40
41
Origins of Antibiotic Resistance
 Some resistance is 2000 years old.
 Canadian glaciers
 Some resistance in deep sea areas is 10,000
years old.
 Near Papua New Guinea
 Evolution of resistance
 May have occurred as a way to prevent autotoxicity

42
Intrinsic Mechanisms
Resistance

43
Definition and Possible Causes
 Innate ability of a bacterial species to resist the
activity of a particular antimicrobial agent
 Possible causes
 Lack of affinity of the drug for the bacterial target
 Inability of the drug to enter the bacterial cell
 Removal of the drug by chromosomally encoded
efflux pumps
 Innate production of enzymes that inactivate the drug

44
Biofilms
 Sessile bacterial communities that are
irreversibly attached to a solid surface and are
embedded in an exopolysaccharide matrix
 Biofilms are highly resistant to antimicrobial
agents.
 Resistance is not attributed to typically acquired
genetic mechanisms but instead is determined
by chemical and physical characteristics of
biofilm formation.

45
Impermeability and Efflux
 Impermeability
 Antibiotics are unable to penetrate the bacteria cell
wall.
 Efflux
 Involves efflux pumps
• Proteins located in the bacterial cell membrane that transport
molecules out of the cell
 Increase secretion of drug
• Five major superfamilies based on amino acid sequence and
energy source used to export their substrates

46
Active Efflux in Gram-Negative Bacteria

47
Active Efflux in Gram-Negative Bacteria

48
Enzymatic Inactivation
 Produce enzymes that destroy the drug before
they are able to reach their targets
 Example
β-lactamases

49
Hydrolysis of β-Lactamase

50
Current β-Lactamase Classification
Schemes

51
Acquired Mechanisms
Resistance

52
Efflux Pumps and Target Site
Modification
 Efflux pumps
 Increase secretion of drug
 Target site modification
 Usually occurs by chromosomal mutation
 Mutations that reduce effectiveness of drug to act on
its target
 Enzymatic alteration of antibiotic target sites
• Reduce effectiveness of drug to act on its target

53
Chromosomal and Ribosomal
Mutations
 Chromosomal mutation
 Targets DNA gyrase and topoisomerase IV
 Inhibits DNA synthesis
 Generally localized to the amino terminal domains of
GyrA and ParC
 Ribosomal mutation
 Amino acid changes

54
Enzymatic Target Site Alteration and
Acquisition of New Targets
 Enzymatic target site alteration
 Enzymes alter antibiotic targets resulting in reduced
affinity and effectiveness.
 Acquisition of new targets
 Microorganisms acquire new cellular targets with
reduced affinity to antibiotic.
• Transfer of mobile genetic elements
 Target site substitution
• Acquisition of a new enzyme that is unaffected by drugs or
creates a new pathway

55
Enzymatic Inactivation of Antibiotics
 Enzymes produced by the microorganisms

inactivate antibiotics directly.
 Destroy antibiotic
 Modification rendering it ineffective

56
β-Lactamase Inhibitors
B-Lactamase Inhibitors

57
Description
 β-Lactamase inhibitors (BLIs)
 Prevent the degradation of β-lactam antibiotics
 Extends the range of bacteria the β-lactam
antibiotics are effective against
 Important in treating gram-negative bacterial
infections
 In the case of S. aureus (gram-positive pathogen),
the cause of antibiotic resistance is mostly due to
variant PBPs.
 BLIs have little antimicrobial activity of their own.
 Used in combination with specific antibiotics

58
β-Lactamase Inhibitors

59
Dissemination of Resistant
Determinants

60
LGT and Mechanisms of Transfer
 Lateral gene transfer (LGT)
 Two processes
• Physical movement
• Incorporation into genome
 Mechanisms of transfer
 Conjugation
 Transformation
 Transduction

61
 Conjugation

62
 Transformation

63
 Transduction

64
Plasmids
 Conjugative and self-transmissible or
 Nonconjugative, requiring mobilization by
conjugative plasmids

65
Integrons
 Genetic elements that capture mobile gene
cassettes by site-specific recombination
composed of
 A gene (intl) that encodes a specific recombinase
(IntI) and an adjacent primary recombination site (attI)
 Gene cassettes can be integrated into this site.

66
Insertion Sequences
 Short DNA sequences that act as simple
transposable elements
 Two major characteristics: relatively small and
only code for proteins implicated in the
transposition activity

67
Nanotechnology to Deliver
Therapeutic Agents

68
Nanoparticles and Components
 Nanoparticles
 Less than 100 nm in size
 Unique biological, physical, and chemical properties
 Nanomaterial can be loaded with
 drugs.
 biomolecule diagnostic tools.
 contrasting agents.

69
Technology Potential and Drug Delivery
Mechanisms
 Technology potential
 New ways to deliver drugs
 Improve circulation time
 Improve drug localization within the body
 Improve solubility and pharmacokinetic profile
 Drug delivery mechanisms
 Liposomes
 Nanoshells
 Cochleates

70

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 Enzymes produced by the microorganisms

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