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Controlling Microbial

Growth in the Body:


Antimicrobials
Question about Frontline
Video
At least two million people are infected with
drug-resistant superbugs in the U.S.. How
many of them die from those infections?
A.300
B.6,000
C.16,000
D.23,000
E.230,000
History of Antimicrobial Agents

Paul Ehrlich Alexander Fleming Selman Waksman


Magic bullets Antibiotics
Origin of Antibiotics
Most are natural
compounds produced by
other microorganisms

Semisynthetic:
naturally produced
antibiotics that have Streptomyces coelicolor

been chemically altered

Synthetic: fully synthesized in the laboratory


Features of Antimicrobial Drugs
Bactericidal: level of antibiotic that kills
the bacterium

Bacteriostatic: level of antibiotic that


inhibits the growth (ie reproduction) of
bacteria but doesnt otherwise kill them

depends on host immune system to


eliminate the pathogens
Antimicrobial Spectrum of Activity
Range of activity of an antibiotic against
bacteria

Broad spectrum antibiotics: effective against


a wide range of bacteria (ie tetracyclines)

Narrow spectrum antibiotics: effective


against a limited range of bacteria (ie
polymyxin)
Antimicrobial Spectrum of Activity

Broad spectrum
Narrow spectrum
Question
Which of the following is likely NOT
associated with broad spectrum antibiotics?

A. Can kill bacteria that make up the normal


microbiota
B. Is effective in treating illnesses in which
the identity of the pathogen is unknown
C. Can lead to opportunistic infections
D. Used to treat chronic infectious diseases
that have a definitive diagnosis
Features of Antimicrobial Drugs
Selective Toxicity
Effective antimicrobial agents must be
more toxic to the pathogen than to
the pathogens host
This requires the targeting of features
of the pathogen that differ from the
hosts cells
The majority of antimicrobials are
effective against bacteria (antibiotics)
Antiviral Drugs
Viruses are especially difficult to target with
antimicrobial drugs:
1. Viruses are intracellular parasites
The drug must effectively enter host cells

2. Viruses can be latent


Latent viruses are not actively replicating
Theres very little a drug can do to combat
a provirus
Antiviral Drugs

3. Viruses mutate very rapidly


Thus their molecular targets can change
rapidly

4. Viruses use host cell metabolic processes


and ribosomes
Its difficult to target viral processes
without harming the host
Antiviral Drug Targets

Block an aspect of the viral replication cycle


Some specific targets may include
Virus-specific enzymes (ie reverse
transcriptase from HIV
Viral Release
Viral nucleic acid synthesis
Viral entry
Viral uncoating
Main Targets of Antiviral Drugs
Virus
Entry Eukaryotic host
Uncoating cell
Nucleic acid synthesis
(viral enzyme directed)
Viral particle
production

Exit

Nucleic acid synthesis


Nucleoside analogs
Non-nucleoside polymerase inhibitors
Non-nucleoside reverse transcriptase
Drugs inhibiting
inhibitors (HIV) these processes
may harm host
cell also
Nucleoside, Nucleotide Analogs
Structure similar to nucleosides (sugar
+base) or nucleotides (sugar + base +
phosphates)

Antiviral drug
Nucleoside, Nucleotide Analogs
Incorporated into DNA or RNA
Can stop nucleotides from being added or alter
base-pairing properties

~100 times more likely to be used by viral


polymerase than host polymerase
o More damage done to rapidly replicating
viral genome
o But only effective against replicating virus
Main Targets of Antiviral Drugs
Virus
Entry
Eukaryotic host
Uncoating
Nucleic acid synthesiscell
(viral enzyme directed)
Viral particle Integrase (HIV
production
enzyme)
Exit
inhibitors
Assembly and release
of viral particles
Virus-specific Effective against HIV: Protease
enzymes inhibitors
Effective against influenza
viruses: Neuraminidase inhibitors
Main Targets of Antiviral Drugs
Entry inhibitors
Effective against HIV
Entry
Eukaryotic host
Uncoating
Nucleic acid synthesiscell
(viral enzyme directed)
Viral particle
production

Exit

Viral uncoating
Effective against
influenza A virus:
Amantadine
Rimantadine
Antifungal Drugs
Cell walls made of chitin
Fungal membranes have ergosterol as
opposed to cholesterol

Both of these are major targets of


antifungal drugs
Antiparasitic Drugs

Relatively little research and


development

Most drugs interfere with biosynthetic


pathways of protozoan parasites or
neuromuscular function of worms

Eukaryotic cellular targets harm to


human host cells
Antibacterial Drugs (Antibiotics)

(Folic acid synthesis)


Beta-Lactam Antibiotics
The first, most diverse, and most
commonly used class of antibiotics

Include penicillins, cephalosporins, and


carbapenems

Inhibit cell wall synthesis


Cell Wall Function
prevent against osmotic lysis of cell
confers rigidity; shape to cell

If cell wall is weak, cells lyse due to


osmotic pressure
Bacterial Cell Wall Synthesis

Normal bacterial
cell wall
Growth

As bacteria grow, they must synthesize


more peptidoglycan
Bacterial Cell Wall Synthesis
NAM
Crossbridge
NAG
between
NAG-NAM NAM
chain and NAM

Penicillin binding
protein (PBP),
called
transpeptidase
Bacterial Cell Wall Synthesis

New NAG and NAM


subunits
Beta Lactam Mechanism of Action

Irreversibly inhibits enzyme responsible


for the final step in the synthesis of the
peptidoglycan layer of bacterial cell walls
o Binds and inhibits transpeptidase
that cross links NAM subunits
o Transpeptidase is also called
penicillin binding protein (PBP)
Beta Lactam Blocks
Transpeptidase (PBP)
New cross-links
inhibited by
beta-lactam
Previously
formed
crossbridge
Cell bursts from osmotic pressure

Integrity of
peptidoglycan is
compromised
Question

Would it be advisable to administer an


antibiotic that inhibits cell growth at the
same time that the patient is receiving
penicillin? Why or why not?
Cell Wall Inhibitors

Prevent bacteria from increasing the


amount of peptidoglycan

Have no effect on existing peptidoglycan


layer

Effective only for growing cells


Beta lactamase
Penicillin
Bacteria resistant to O S CH3
beta lactam drugs R C NH CH CH C
CH3
produce beta C N CH COOH
lactamase that breaks O
-lactam ring
the critical beta lactam
ring

The beta lactam rings differ in the chemical


groups that are attached
Therefore antibiotics differ in their
sensitivity to different beta lactamases
Penicillins

Share basic structure


Side chain modified to
create derivatives
Cephalosporin
Penicillin
Structure makes R
O

C NH CH CH
S
C
CH3

resistant to some - CH3

C N
lactamases O
CH COOH

-lactam ring
(a)

Cephalosporin
O S

R C NH CH CH CH2

C N C
O R
-lactam ring COOH
(b)
Carbapenems
Resist most -lactamases
Carbapenems effective against a wide range
Question about Frontline
Video
KPC stands for Klebsiella pneumonia
carbapenemase. What does carbapenemase
do?
A. Cleaves all beta lactam rings
B. Cleaves beta lactam ring of just carbapenem
C. Confers resistance to all antibiotics
D. None of the above
Non beta-lactam Cell Wall Inhibitors:
Vancomycin

Is a glycopeptide
Binds to side chain of NAM molecules to
block the addition of the new NAM
subunits to a pre-existing chain (ie chain
elongation)
Weakens cell wall, causes lysis
Mechanism of vancomycin action
NAM NAG

V crosslink

D-ala-D-ala Peptide side


chain
Vancomycin- drug of last resort
Important for treating Gram-positives
resistant to -lactam antibiotics
Often antibiotic of last resort
Does not cross outer membrane of Gram-
negatives
Often administered via IV
Cell Membrane Inhibitors
Polymyxin B
Cell
membrane

Polymyxin B binds to phosophlipids and


lipopolysaccharides in outer membranes and
compromises membrane integrity
Could these damage human cell
membranes?

topical applications
Nucleic Acid Synthesis Inhibitors

Fluoroquinolones
Inhibit prokaryotic topoisomerases
o One type of topoisomerase, DNA
gyrase, is a common target

May have some toxicity


Protein Synthesis inhibitors
ie Erythromycin

Ie Clindamycin

Ie Streptomycin
Lincosamides and macrolides
Bind to the 50S subunit
Block proper mRNA movement through
ribosome
Clindamycin is an example of a lincosamide

Drug
50S

mRNA
Aminoglycosides
Ie streptomycin
Bind to 30S subunit
mRNA is misread
Causes premature release of the ribosome
from the mRNA
Incorrect
amino acids
50S Ribosome

mRNA
Aminoglycoside
30S
drug
Tetracyclines
Bind to 30S subunit
Block docking site of tRNA

Tetracycline
drug
50S
mRNA
Would protein synthesis inhibitors
have side effects?
Prokaryotes have 70S ribosomes
Eukaryotes have 80S ribosomes

Mitochondria also have 70S ribosomes


May account for some toxicity of
these antibiotics
Metabolic inhibitors
Sulfonamides- analogs of essential
metabolites used in folic acid synthesis
Sulfonamides
Sulfonamides
inhibit
Enzyme Enzyme Enzyme
Precursor X#1 Precursor #2
Dihydrofolic #3 Tetrahydrofolic
#1 #2 acid acid (folic acid)
PABA

Purines Pyrimidines Amino


acids
folic acid is required
to synthesize
Humans derive folic acid from their diet
and so do not possess this enzyme
system
Sulfa Drugs- Competitive Inhibitors
Compete with PABA for binding to the active
site of an enzyme used in folic acid synthesis
Sulfa drug
PABA (inhibitor)
(substrate)
Sulfa drug
(inhibitor)

Enzyme Enzyme Enzyme


Higher levels of sulfa drug
more likely to bind to enzyme
Determining the Susceptibility of a
Bacterial Strain to an Antibiotic

Minimum inhibitory concentration test


Minimum inhibitory concentration:
lowest concentration of a specific
antimicrobial drug needed to prevent
the growth of a given bacterial strain in
vitro

Diffusion test (Kirby Bauer disc test)


Minimum Inhibitory Concentration
(MIC) Test
Same inoculum size of test bacteria added

0 0.2 0.4 0.8 1.6 3.2 6.4 12.8


Negative mg/ml
control Increasing concentration of drug
Growth No growth
Kirby Bauer Disc Diffusion Test
Kirby Bauer Disc Diffusion Test
Potential Side Effects

Direct damage to tissue due to toxicity


of drug
Allergic reactions
Disruption of normal microbiota
After killing much of the normal
microbiota that confer protection
against pathogens, the patient could be
susceptible to a secondary infection
called an opportunistic infection
Disruption of the Normal Microbiota by
Broad Spectrum Antibiotics
Infection Drug Opportunistic
infection
Circulating
drug

Normal microbiota
important Intestine Intestine
to maintain
intestinal
balance

Potential pathogen Drug destroys


resistant to drug but held in beneficial Pathogen
check by other microbes microbiota overgrows
Drug Resistance
Innate Acquired
Naturally lack the drug Spontaneous
target; ie Mycoplasma mutations
lacks cell wall, resist Horizontal gene
penicillin etc. transfer
Outer membrane of
Gram-negatives blocks
many medications
Acquisition of Drug Resistance
DNA fragment Plasmid Plasmid
donor
Resistance
gene
Transformation Conjugation
(Transfer of (Plasmid transfer via
free DNA) direct contact)
Gene goes to plasmid
Dead Gene goes to
bacterium chromosome Phage
Resistance
gene
Transduction

Mutation
Bacterium receiving
Resistance genes
Evolution of Drug Resistance
Mutations and horizontal gene transfer
provide diversity

Fittest organisms for an environment are


selected by natural selection
Antibiotics Are a
Selective Pressure
Antibiotic

Sensitive organisms die

Resistant survivors can


multiply without
competition
Natural Selection and Drug Resistance
Just a single base-pair change in gene
encoding a ribosomal protein yields
resistance to streptomycin

In a population of 109 cells, at least one


likely has that mutation

if streptomycin is added, only that cell and


progeny will replicate, yielding resistant
population
Natural Selection and Drug Resistance
Non-drug-resistant cell
Drug-resistant cell Cells dying

Exposure
To drug Continued
Exposure
To drug
(b) Most sensitive
(a) Microbial (c) Most new cells are
cells are eliminated
cells resistant, infection will
by drug early :
involved in no longer respond to
resistant cells survive
an infection and grow drug
Frontline video Question
Why are antibiotic resistant infections often
hospital-acquired?
A. Hospitals house large numbers of people whose
immune systems are often in a weakened state.
B. Increased use of outpatient treatment means that
patients in the hospital are more vulnerable.
C. Medical staff move from patient to patient, providing a
way for pathogens to spread.
D. Many medical procedures bypass the body's natural
protective barriers.
E. All of the above
Mechanisms of Resistance
Drug Inactivation
Antibiotic-Inactivating Enzymes

Result
R S R S CH3
CH3
N Penicillinase O C N
O COOH H COOH
OH

Active penicillin Inactive penicillin


Membrane Modification
Antibiotics normally enter bacteria via porin
channels in the membrane

Decreased Uptake: alteration of the normal


receptor for the drug or the porins

Increased elimination: activation of or


increased production of efflux drug pumps
Decreased Uptake
New porin
Original porin

Outer membrane
Efflux Drug Pumps
Efflux pumps remove compounds from cell
Increased production of pumps allows
faster removal
Drug
Inactive drug Active
pump drug
pump
Cell surface
of microbe Cell surface
of microbe
Target Modification
Minor structural changes can prevent binding
For example, penicillin binding proteins (-lactam
antibiotics) and ribosomal subunit (macrolides)
Use of Alternate Metabolic Pathway

Drug inhibits
A B C D Product
X

.
C D
Question

How are most antibiotics being used in the


U.S.?
A. To treat hospitalized patients
B. To treat patients in the community (non
hospitalized)
C. To treat farm animals raised for food
D. To treat domesticated pets
Antibiotic Use in Animals
Antimicrobial drugs used in animal feeds at low
levels to enhance growth; selects for drug-
resistant microbes
ie Resistant Salmonella strains linked to animals
Antibiotic Overuse and Misuse

Avoid ordering costly and time-consuming


tests to pinpoint the patients illness
Patient Pressure
Patients self diagnose and take antibiotics
leftover from a previous prescription
Patients dont complete the full course of their
prescription
Antibiotic Overuse and Misuse
Antibiotics widely available
without prescription in some
countries, may allow
improper use

Avoid ordering costly and


time-consuming tests to
pinpoint the patients illness

Antibiotics for Sale


Example of Emerging Antibiotic
Resistance
Carbapenem-resistant Klebsiella
#1 Still not feeling well?
You must be immune to
the antibiotic. #2 I always stop
taking the antibiotic
as soon as I start
feeling better
because I dont want
to be taking drugs for
longer than
necessary

#3 When I take an antibiotic, the bacteria


will mutate in order to survive and thats #4 It doesnt matter
how I get drug resistance, right if my normal gut
bacteria become
resistant since they
dont typically cause
disease. Its only the
antibiotic resistant
pathogens that
matter
New Antibiotics are Needed

Modified from: Spellberg et al, 2009. Trends in Antimicrobial Drug Development: Implications for the
Future, Clinical Infectious Diseases 9(38): 1279-1286.
Strategies to Limit Drug Resistance
Speed development of new antibiotics

Track resistance data nationwide

Restrict antibiotic use- Only prescribe suitable


antimicrobials if appropriate

Use more narrow spectrum antibiotics

Use antimicrobial combination therapy

Educate patients about proper use of drugs

Directly observed therapy (used in TB treatment)

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