(Finals) MICROBIOLOGY / Principles of Antimicrobial Action and Resistance

Medical Intervention eradicate infecting pathogen

Antibiotics substances that actively inhibit or kill the
organism

Antibiotics maybe:
1. Obtained and purified from other microorganisms
2. Chemically synthesized

Antimicrobial agents
natural and synthesized substances
play a central role in the control and management
of infectious diseases
1. Antibacterial
2. Antifungal
3. Antiparasitic
4. Antiviral
ANTIMICROBIAL ACTION

Principles

Key Steps for antimicrobial agent to inhibit/kill

microorganism:
1. Must be in active form
Ensured through pharmacodynamic design of
drug
2. Must be able to achieve sufficient levels or
concentrations at site of infection
Depends on pharmacokinetic properties of agent
3. Remaining steps relate to direct interactions
between agent and bacterial cell.
Pharmacokinetic properties
1. Distribution
2. Excretion of agents metabolites
3. Rate of absorption
4. Metabolism

** Bactericidal agents are more effective against

organisms that are more difficult to control in combination
with hosts immune system.
** Primary goal is to optimize a drugs ability to achieve
steps for antimicrobial activity.
** Antimicrobial agents are frequently
according to their mode of action.

1.
2.

3.
4.

Anatomic Distribution of Some Common

Antibacterial Agents
Norfloxacin and Nitrofurantoin
Therapeutic levels generally not achievable at
Serum-Blood
Gentamicin,
Clindamycin,
Norfloxacin,
Nitrofurantoin
Therapeutic levels generally not achievable at
CSF
Vancomycin and Ciprofloxacin
Moderate
to
poor
therapeutic
levels
achievable at CSF
Urine
Therapeutic levels not achievable at Urine
Bacteriostatic agents inhibit bacterial growth but do
not kill the organism

categorized

Mode of Action of Antibacterial Agents

** Most targets of antibacterial agents are intracellular.

Basic Steps Required for Antimicrobial Activity
1. Active Drug
2. Anatomic approximation
3. Surface binding (Adsorption)
4. Intracellular uptake
5. Target Binding
6. A. Growth Inhibition
B. Lysis and Death

Bactericidal agents usually kill target organisms

Inhibitors of cell wall synthesis

o Beta-lactams
o Glycopeptides
Inhibitors of Cell Membrane Functions
o Lipopeptides
o Polymixins
Inhibitors of Protein Synthesis
o Binds to 30S

Aminoglycosides

Tetracyclines

Glycylcyclines
o Binds to 50S

Macrolide-LincosamideStreptogramin

Oxazolidinone

Chloramphenicol
Inhibitors of DNA & RNA Synthesis
o Fluoroquinolone
o Ciprofloxacin
o Metronidazole
o Rifampin
Inhibitors of Folic Acid Synthesis
o Sulfonamides
o Trimethoprim

A.

Inhibitors of Cell Wall Synthesis

1.

Beta-Lactams
Core - four-member, N containing, Beta-lactam
ring
Largest group of antibacterial agents
Lacks toxicity to humans
Beta-lactam ring
key to the mode of action of Betalactams
similar to acyl-D-alanyl-D-alanine
Beta-lactam binds enzyme, inhibiting
transpeptidation and cell wall synthesis
Penicillin-binding proteins (PBPs)

Acyl-D-Alanyl-D-Alanine normal substrate for synthesis

of linear glycopeptide in the bacterial cell wall.
-

Types:
1. Penicillin
a. Penicillin
b. Ampicillin
c. Meziocillin
d. Piperacillin
2.

Cephalosporins

(Finals) MICROBIOLOGY / Principles of Antimicrobial Action and Resistance

a. Cefazolin
b. Cefuroxime
c. Cefotetan
d. Cefotaxime
e. Ceftriaxone
f. Ceftazidime
g. Cefepime
3. Carbapenems
a. Meropenem
b. Imipenem
c. Doripenem
4. Monobactams
a. Aztreonam
Penicillin-binding Proteins (PBP)
Enzymes essential to produce and maintain their
peptidoglycan layer
B-lactam + PBP = cell wall synthesis is halted due to
osmotic instability
Spectrum of Activity type of bacteria against which a
particular antimicrobial agent does and does not have
activity.

Spectrum of Activity of B-lactams: gram + and gram

bacteria

4 major categories of B-lactamases

1. Class A & D
o Serine peptidases
2. Class C
o Cephalosphorinases
3. Class B
o Requires zinc
o Metallo-B-lactamases

B-lactamase should be located on:

1. Plasmids
2. Transposons
3. within integrons
4. within chromosome

Integron
large cassette region that contains antibiotic
resistance genes

Integrase
required for movement of integrin from one
genetic element to another\

Resistance Mechanisms
1. Genetic mutations in PBP coding sequence
2. Genetic Recombination
3. Overproduction of PBP
4. Acquiring new genetic coding sequence

** Acquired type of B-lactam resistance are commonly

found in Gram + bacteria
2.

Glycopeptides
Inhibit bacterial cell wall synthesis by binding to
end of peptidoglycan
Interferes with transpeptidation
Interferes with PBP enzymes that incorporate
precursors into growing cell wall

a.
b.
3.

Vancomycin
Levels should be monitored
Potential for toxicity
Teicoplanin
Approved
Not available in the US

Lipoglycopeptides
Semi-synthetic molecules
Glycopeptides that contain hydrophobic chemical
groups
Increase cell permeability
Cause depolarization of cell membrane potential
Inhibits transglycosylation
a. Oritavancin
b. Telavancin

Transglycosylation
-

Necessary for cell wall synthesis by complexing

with D-alanyl-D-alanine residues

Spectrum of Activity: VISA (Vancomycin Intermediate

Staphylococcus aureus.

B.

Inhibitors of Cell Membrane Function

1.

Lipopeptides
a. Daptomycin
Recently developed
Bind and disrupts cell membrane of Gram +
bacteria
Has potent activity against Gram + cocci,
MRSA,VRSA
b. Bacitracin
Inhibits recycling of certain metabolites required
for maintaining peptidoglycan synthesis
Only used as tropical antibacterial agent
Internal consumption is avoided

2.

Polymyxins
Cyclic polypeptide agents that disrupt cell
membranes
Acts as detergents (interacts with phospholipids
and increased permeability)
Disruption results to leakage of macromolecules
and ions
Not equally effective agains all bacteria and more
effective against Gram - and poor activity to
Gram +
Pose a risk for toxicity for humans have
membranes
Last resort microbial agents

Spectrum of
Acinetobacter

Major Side effects

1. Neurotoxicity
2. Nephrotoxicity

C.

Inhibitors of Protein Synthesis

1.

Aminoglycosides and Aminocyclitols

Activity:

Gram

-,

P. aeruginosa,

(Finals) MICROBIOLOGY / Principles of Antimicrobial Action and Resistance

a.
-

Aminoglycosides
Inhibit protein synthesis
Irreversible binding to protein receptors on 30S
ribosomal unit
Major toxicity:
1. Nephrotixicity
2. Auditory/Vestibular Toxicity
Interrupts the following:
o Initial formation of protein synthesis
complex
o Reading of mRNA code
o Formation of rRNA complex
Gentamycin
Commonly used aminoglycoside
1. Tobramycin
2. Amikacin
3. Streptomycin

Spectrum of Activity: Not effective against anaerobic

bacteria, Effective against aerobic gram and
Staphylococcus aureus.

2.

Macrolide-Lincosdamide-Sreptogramin
Inhibits protein synthesis by binding to 23s rRNA
on bacteria 50S ribosomal subunit
Disrupts pepide chain (blocks translocation
reaction
Bacteriostatic
Can be bactericidal if organism is low and if used
in high concentrations

Spectrum of Activity: Effective against Gram +,

mycoplasma, Treponemes, Rickettsiae & Not effective
against Gram

Quinupristin- dalfopristin
Dual streptogramin
Targets 2 sites of 50S ribosomal subunit
Toxicity is generally low
Lincosamides, Clindamycin, Lincomycin
Bind to 50S ribosomal subunit
Prevent elongation
Interfere with peptidyl transfer

Spectrum of Activity: Effective against Gram +

Spectrogramins
Naturally occurring cyclic peptides
a. Quinupristin-daltopristin
Enter cell through massive diffusion
Bind irreversibly to 50s subunit that induces
conformational change in the ribosomal structure
Alteration of ribosome
Interferes with peptide bond formation that
disrupts elongation
Toxicity:
1. Low toxicity
2. Localized phlebitis major complication of IV
infusion

Spectrum of Activity: Effective agains Gram + and

some Gram -

3.

Ketolides

Consist of:
1. Chemical derivatives of erithromycn A
2. Other macrolides
Bind to 23s rRNA of 50S ribosomal subunit
Inhibits protein synthesis

4.

Telithromycin
Maintain activity agains most macroled-resistant
gram +
Does not induce common macrolide resistance
mechanism
Effective against respiratory pathogens &
intracellular organisms
Low toxicity
Major side effects:
1. Gastrointestinal symptoms

Spectrum of Activity: Effective against gram +, some

gram -, mycoplasma, mycobacteria, chlamydia,
rickettsia, Francisella tularensis

5.

Oxazolidinones
Represented by linezolid
Linezolid
o Synthetic agent
o Inhibits protein synthesis by interactive
with 23s rRNA in 50S ribosomal subunit
o Interfere with binding of tRNA for
formylated-methionine
Low toxicity
Gastrointestinal symptoms

Spectrum of Activity: Effective against Gram +,

mycobacteria

6.

Chloramphenicol
Inhibits addition of amino acid to growing peptide
chain
Reversibly bind to 50S ribosomal subunit
Inhibits transpeptidation
Highly active
Uses has dwindled because of drug toxicity and
development of new effective and safer drugs
Bone marrow toxicity
Major side effect: Aplasia

Spectrum of Activity: Effective against Gram and

Gram +

7.

Tetracyclines
Broad spectrum bacteriostatic
Inhibit protein synthesis by binding reversibly to
30S ribosomal subunit
-

Toxicity:
o Upper gastrointestinal effects
o Cutaneous phototoxicity
o Photoallergic immune reaction

Spectrum of Activity: Effective against Gram and

Gram +, Intracellular pathogens (Rickettsia and
Chlamydia), Protozoa, N. gonorrhoeae, Mycoplasma,
Spirochetes

(Finals) MICROBIOLOGY / Principles of Antimicrobial Action and Resistance

8.

Glycylglycines
Semi-synthetic tetracycline derivatives
Tigecycline
o 12st agent of glycylglycines approved
for clinical use
o Inhibits protein synthesis by binding
reversibly to 30S ribosomal subunit
o Has advantage of being refractory to
most common tetracycline resistance
o Most common effects are:

Nausea

Vomiting

Diarrhea

D.

Inhibitors of DNA and RNA Synthesis

1.

Fluoroquinolones
Simply quinolones
Derivatives of Nalidixic acid
o Ciprofloxacin
o Ofloxacin
Bind and interfere with DNA gyrase enzymes
regulating DNA supercoiling
Also inhibit topoisomerase IV
Topoisomerase IV
o Unlinks DNA after replication
Potent bactericidal agents
Toxicity:
o Tendinitis
o Rupture of Achilles tendon

2.

3.

Spectrum of Activity: Effective against gram

(topoisomerase is targeted) and gram + (DNA gyrase
is targeted)
Metronidazole
Antibacterial activity is related to the presence of
nitro group in chemical structure
Nitro group
o Reduced by nitroreductase in bacterial
cytoplasm
o Generates:

Autotoxic compounds

Free radicals
Activation requirements
o Reduction under low redox potentials
(anaerobic)
Low toxicity
Side effects: Mild gastrointestinal symptoms
Spectrum of Activity: Effective against Anaerobic,
Microaerophilic
gram

and
Protozoans
(Trichomonas, Giardia, Entamoeba histolytica)
Rifamycin
Include Rifampin (Rifampicin)
Semisynthetic
Bind to the enzyme
o DNA dependent
o RNa polymerase
Inhibits RNA synthesis
Does not effectively penetrate oputer membrane
of gram (-)

Spontaneous mutation
Rifampin are usually used in combination with
other agents
Side effects:
o Gastrointestinal symptoms
o Hypersensitivity reactions

E.

Inhibitors of Other Metabolic Processes

1.

Sulfonamides
Disrupts folic acid pathway
Bind to dihydropteroate synthase
Moderate toxicity
Side effects:
o Vomiting
o Nausea
o Hypersensitivity reactions
Antagonistic for other medications

Spectrum of Activity: Effective against Gram and

Gram +

2.

Trimethoprim
Targets folic acid pathway
Inhibits dihydrofolate reductase
Usually
combined
with
sulfonamide
(sulfamethoxazole) to simultaneously attack 2
targets
Mild toxicity
Adverse side effects:
o Person with AIDS develop side effects
more often

3.

Nitrofurantoin
Consists of nitrogroup on heterocyclic ring
Mechanism is diverse and multifaceted
Used to treat uncomplicated UTI
Side effects:
o Gastrointestinal infections or symptoms

Diarrhea

Nausea

Vomiting
o Chronic pulmonary condition
o Irreversible pulmonary fibrosis

Spectrum of Activity: Effective against Gram and

Gram +

MECHANISMS OF ANTIBIOTIC RESISTANCE

Principles

Resistance requires: interruption or disturbance

of one or more steps
Result is:
o Partial loss of effectiveness
o Complete loss of effectiveness

Aspects
1.
2.
3.

Biologic vs. Clinical

Environmentally-mediated
Microorganism-mediated

(Finals) MICROBIOLOGY / Principles of Antimicrobial Action and Resistance

A.

Biologic vs. Clinical Resistance

Biologic resistance
Changes that result in observed
susceptibility to a particular agent

Clinical resistance
Most laboratory
resistance

methods

S. pneumoniae
Inhibited by penicillin

B.

Environmentally
Mediated
Resistance
Antimicrobial Resistance
1. Drug
2. Microorganisms
3. Environment

used

to

2 subcategories of Organism based resistance

a. Intrinsic/inherent
b. Acquired

Intrinsic Resistance
Results from the normal genetic, structural or
physiologic state of microorganism
Considered as natural inherited characteristic
Resistance pattern may be predictable
Intrinsic resistance profiles are also useful
markers to aid identification of certain bacteria

reduced

detect

Antimicrobial

Intrinsic Resistance
Anaerobic bact vs.
aminoglycosides
Enterococci vs.
aminoglycosides
Gram + vs. Aztreonam
Enterococci vs.
cephalosporin
Gram vs. Vancomycin
P. aeruginosa vs.
Sulfonamides
Klebsiella spp vs.
ampicillin
Stenotrophomonas
maltophilia vs. imipenem
Aerobic bact vs.
metronidazole
Lactobacilli and
Leuconostoc sp. vs.
vancomycin

**These three factors coexist

Environmentally mediated resistance

Resistance directly resulting from physical or
chemical changes of environment
Alters:
o Microbial agent
o Microorganisms normal physiological
response

Environmental factors
1. pH
2. anaerobic atmosphere
3. cation concentration
4. thymidine content

pH
Erythromycin & Aminoglycosides
o Low pH; Low effect of agent
Tetracycline
o High pH; Low effect of agent

Aminoglycoside mediated shutdown of bacterial protein

synthesis
-

Requires intracellular uptake

Driven by oxidative process
No oxygen, low uptake
Affected by Ca++ and Mg++
o P. aeruginosa has negative net charge
in cell membrane
o Cations compete with aminoglycoside
for negative charged binding sites
o High cations, low effect of agent

Antimicrobial resistance that results from

genetically encoded traits of the microorganisms

C.

Presence of Metabolites
Enterococci
o Uses thymine and exogenous folic acid
to circumvent activities of sulfonamides
and trimethoprim
o Absence
of
metabolites,
full
susceptibility to antibiotics
Microorganism Mediated Resistance

Lack of PBP

Lack of uptake

Production of enzymes

Inability to anaerobically
reduce to active form
Lack of appropriate cell
wall precursor target

Acquired Resistance
Results from altered cellular physiology and
structure caused by changes in microorganism
genetic makeup
Trait associated with specific strains of particular
organism
Unpredictable
May be acquired by:
o Genetic mutation
o Acquisition of genes
o Combination of mutational and gene
transfer events
COMMON PATHWAYS FOR ANTIMICROBIAL
RESISTANCE
1.
2.

Mechanism
Lack
of
ocidative
metabolism

3.
4.
5.
6.

Enzymatic degradation or modification of

antimicrobial agent
Decreased
uptake
or
accumulation
of
antimicrobial agent
Altered antimicrobial agent
Circumvention
of
the
consequences
of
antimicrobial action
Uncoupling
of
antimicrobial
agent-target
interactions and subsequent effects on bacterial
metabolism
Any combination of mechanisms 1 to 5

(Finals) MICROBIOLOGY / Principles of Antimicrobial Action and Resistance

Resistance mechanisms against:

1. B-lactams
a. Enzymatic destruction
- B-lactamase
b. Altered target
- Mutational changes in PBP
c. Decreased uptake
- Porin channels change in number
2. Glycopeptides
a. Altered target
- Alteration in molecular structure
b. Target overproduction
- Excess peptidoglycan
3. Aminoglycoside
a. Enzymatic modification
- Modifying enzymes
b. Decreased uptake
- Porin channels change in number
c. Altered target
- Mutational changes in ribosomal
binding site

4.

5.

Quinolones
a. Decreased uptake
- Alterations in the outer membrane
b. Altered target
- Changes in DNA gyrase subunits
Macrolides
a. Efflux
- Pumps drugs out of cell
b. Altered target
- Enzymatic alteration

Factors contributing to the emergence and

dissemination of antimicrobial resistance among
bacteria
1. Emergence of new genes
2. Spread of old genes to new hosts
3. Mutations of old genes resulting in more potent
resistance
4. Emergence of intrinsically resistant opportunistic
bacteria