Transcript Lecture 7 Bio3124 - University of Ottawa

Antimicrobial Chemotherapy
Lecture 7
Bio3124
Chemotherapeutic agents



Chemical agents used to treat infectious
diseases
Kill or inhibit the growth of pathogens
within the host
Antibiotics
 Natural microbial products/semi
synthetic/synthetic
The Development of
Chemotherapy


Paul Ehrlich (1904)

Concept of selective toxicity

identified dyes that effectively treated African
sleeping sickness
Sahachiro Hato (1910)


Paul Ehrlich
1854 – 1915
working with Ehrlich, identified arsenic compounds
(Salvarsan) that effectively treated syphilis
Gerhard Domagk, and Jacques and Therese
Trefouel (1935)

discovered sulfonamides and sulfa drugs
Penicillin

Ernest Duchesne (1896),
discovery lost

Alexander Fleming (1928)
 observed penicillin activity on a
contaminated plate
 did not follow up
•
Penicillium mold kills S.aureus
effectiveness demonstrated by Florey, Chain, and Heatley
(1939)
•
Fleming, Florey and Chain received Nobel Prize in 1945
Later discoveries

Selman Waksman (1944): Streptomycin, an
antibiotic active against tuberculosis


Nobel Prize in 1952 for this discovery
by 1953 chloramphenicol, terramycin,
neomycin, and tetracycline were isolated
Properties of an antibiotic

Selective toxicity (Paul Ehrlich)

ability of drug to kill or inhibit pathogen with a minimal
or no harm to host
For a good antibiotic:
 Therapeutic dose


Toxic dose


drug level required for clinical treatment; should be
low
drug level at which drug becomes too toxic for patient
(i.e., produces side effects); should be high
Therapeutic index

ratio of toxic dose to therapeutic dose; should be high
Determining Antimicrobial Activity

expressed in two ways

minimal inhibitory concentration (MIC)

lowest concentration of drug that inhibits the growth of a
pathogen

minimal lethal concentration (MLC) or minimal
bactericidal concentration (MBC)


lowest concentration of drug that kills a pathogen
two techniques used to determine MIC and MBC
Dilution Susceptibility tests
Tube dilution assay: tests for one
antibiotic at a time
MIC=16 ug/ml
• MIC: half dilution series of AB in Müller
Hinton medium prepared
• equal amount of bacterial broth added
• incubation at 37°C for 18 hours, visible
growth checked
• MBC: lowest AB concentration to kill
completely
• Agar dilution assay: a variation of tube
dilution assay done on agar containing
solid medium where visible colonies are
scored (multiple microorganisms vs one type
of antibiotic)
2n
128 64
32
16
8
4
2
0
Antibiotic concentration (ug/ml)
MBC=64 ug/ml
2n
128 64
32
16
8
4
2
Antibiotic free medium
0
Kirby-Bauer method: disc diffusion test
 Agar disc diffusion test: Bacterial culture
is uniformly inoculated onto the surface of
an agar plate
 antibiotic discs are placed onto the plate
AMP
 antibiotic diffuses out of discs to form a
Tb
Tet
concentration gradient
 after incubation the diameter of the zone
Gm
of growth inhibition is measured and
compared to standard tables
Suceptibility order: Tet>Tb>Gm>Amp
 the diameter of the zone of inhibition is
inversely related to MIC
Kirby-Bauer method



standardized method for carrying out disk diffusion
test
sensitivity and resistance determined using tables
that relate zone diameter to degree of microbial
resistance
table values plotted and used to determine if
concentration of drug reached in body will be
effective
Determining the susceptibility or resistance
• log2 MIC vs
diameter of
inhibition zone
is linear
512
128
64
(μg/ml)
• MIC values
are compared
to standard
value set by
international
committees
32
A
16
Resistant
Intermediate Susceptible
8
4
B
2
• effective
dosage
determined
0
6
10
14
18
Diameter if inhibition zone (mm)
22
Determining the Therapeutic Dose
• antibiotics decay in
vivo
• time required to keep
the serum concentration
above MIC
• without significant
toxicity to host
• effective dosage and
repetition of dose
determined
The E test





similar to disk diffusion method, but uses strip
instead of disk
convenient to use, more informative than discs
MIC value can be read directly
E-test strips contain a gradient
of an antibiotic
intersection of elliptical zone of
inhibition with strip indicates MIC
MIC
Classification of antibiotics




inhibitors of cell wall synthesis

β-lactams (penicillins and cephalosporins)

Vancomycin
protein synthesis inhibitors

Aminoglycosides (Kanamycin, Gentamicin etc.)

Tetracyclins

Macrolides

Chloramphenicol
metabolic antagonists

Trimethoprim

Sulfonamides
nucleic acid synthesis inhibitors

Quinolones
1. Inhibitors of Cell Wall Synthesis

Penicillins
 6-aminopenicillanic acid derivatives,
differ in side chain

Have b-lactam ring
 essential for bioactivity
 many penicillin resistant organisms
produce b-lactamase (penicillinase)
which hydrolyzes a bond in this ring
Mode of action

irreversible competitive binding to the active site of PBPs (transpeptidases),
normally catalyse the transpeptidation step in bacterial cell wall synthesis

prevents the synthesis of complete cell walls =>cell lysis

acts only on growing bacteria that are synthesizing new peptidoglycan
PBP locked in hemi-acyl reaction on antibiotic
PBP catalyzes transpeptidation reaction
Cephalosporins



structurally and functionally similar to penicillins
broad-spectrum antibiotics that can be used on patients that are
allergic to penicillin
grouped into four categories based on their spectrum of activity
From Cephalosporium acremonium
Fourth generation
Resistance mechanisms against B-lactams
• Resistance:
•
•
•
PBPs with lowered affinity to β-lactam
Overproduction of β –lactamases
acquisition of inducible β –lactamases
•
resistance to penicillins, including the semisynthetic
analogs, continues to be a problem
Vancomycin

glycopeptide antibiotics produced by Streptomyces
orientalis

Competitive binding to terminal D-Ala residues of
the peptidoglycan, interferes with transpeptidation
reaction of PBPs

vancomycin has been important for treatment of
antibiotic resistant staphylococcal and enterococcal
infections


considered “drug of last resort”

rise in resistance to vancomycin is of great
concern
Resistance:

Mutational change of D-ala-D-Ala to D-Ala-Lactate
in cell wall structure
2. Protein Synthesis Inhibitors

many antibiotics bind specifically to the prokaryotic
ribosomes


inhibit a step in protein synthesis





30S (small) or 50S (large) ribosomal subunits
aminoacyl-tRNA binding
peptide bond formation
mRNA reading
Ribosomal translocation
Antibiotics:

Aminoglycosides, Tetracyclines, Macrolides and
Chloramphenicol
Aminoglycosides
• Wide spectrum, fungal and bacterial
metabolic or synthetic products
• 1944: first aminoglycoside,
Streptomycin from Streptomyces griseus,
used to treat of tuberculosis
• more synthetic products
• Polycationic cyclohexane amino sugars with diverse structural
properties
• Three families: Kanamycin, Neomycin and Gentamicin
• Structural diversity: type of sugar and sugar ring substitutions, ie NH3, OH, H
• example shows derivatives in Kanamycin family
Mode of action and resistance mechanism
• Mode of action: bind to 16S rRNA of 30S ribosomal subunit
and interfere with protein synthesis
• ribosomal shifting, amino acid misincorporation, misfolded proteins
• Resistance mechanisms:
enzymatic modification of ABs that results
in loss of capacity to bind the target
Modifying enzymes:
• Aminoglycoside
neucleotidyle
transferases (ANTs): covalently bind
an NTP in sugar ring I or III
• Aminoglycoside acyl transferases (AACs)
AAC6’ transfer an acyl group to 6’ position
• Aminoglycoside phosphotransferase (APHs): e.g. APH3’ transfers a
phosphate group from ATP to the 3’ position
Tetracyclines





produced by Streptomyces or synthesized
are broad spectrum, bacteriostatic, eg. used in acne treatment
all have a four-ring structure to which a variety of side chains are
attached
Bind to 30S ribosomal subunit A site
 inhibits binding of aminoacyl-tRNA molecules to the A site of the
ribosome
Resistance: mutational target modification resulting in
loss of binding site
Macrolides

contain 12 to 22-carbon lactone
rings linked to one or more sugars

used on patients allergic to penicillin

e.g., erythromycin, Azithromycin

broad spectrum, bacteriostatic

binds to 23S rRNA of
50S ribosomal subunit


inhibits peptide chain elongation
Resistnace: mutational target modification
Chloramphenicol

First isolated from Streptomyces venezuelae

now is chemically synthesized

binds to 23S rRNA in 50S ribosomal subunit and inhibits peptidyl
transferase reaction during protein synthesis

Toxic, numerous side effects, carcinogenic

used in life-threatening situations

Resistance: enzymatic acetylation by acetyltransferases on –OH
groups
3. Metabolic Antagonists

act as antimetabolites


antagonize metabolic
pathways by competitively
inhibiting the use of
metabolites by key enzymes
are structural analogs


molecules that are structurally
similar to, and compete with,
naturally occurring metabolic
intermediates
Synergism: two antibiotics
working together better than
individually
Dihydrofolic acid analog
Sulfonamides or Sulfa Drugs


structurally related to a p-aminobenzoic acid (PABA)
PABA used for the synthesis of folic acid and is made by
many pathogens
 unlike humans, these pathogens cannot take up PABA
 selectively toxic, compete against PABA for the active site
of an enzyme involved in folic acid synthesis, resulting in a
decline in folic acid concentration
 pathogen dies because folic acid is a precursor to
purines and pyrimidines which are nucleic acid building
blocks
Trimethoprim



synthetic antibiotic that also interferes with folic acid
production
broad spectrum
can be combined with sulfa drugs to increase
efficacy of treatment



combination blocks two steps in folic acid pathway
has a variety of side effects including abdominal
pain and photosensitivity
Resistance:

Overproduction of PABA

Mutant enzymes that evade the binding of drugs but not
the natural substrates
4. Nucleic Acid Synthesis Inhibition

a variety of mechanisms


block DNA replication

inhibition of DNA polymerase

inhibition of DNA helicase
block transcription


inhibition of RNA polymerase
Must target DNA/RNA biosynthetic steps that are
different between prokaryotes and eukaryotes
Quinolones

broad-spectrum, synthetic, contain quinolone
ring

Nalidixic acid, first synthesized (1962)

Fluoroquinlones like Ciprofloxfacin,
norfloxacin are new drugs with a wider
antibacterial spectra

act by inhibiting bacterial DNA-gyrase
(Topoisomerase II)

disrupts DNA supercoiling and replication

selective toxicity: DNA gyrase is not present
in eukaryotic cells

Resistance:

mutations in quinolone binding cleft on
gyrase (gyrB subunit) that results in loss
of affinity

Reduced uptake due to mutations in
ompF gene that encodes for porins

Active efflux pumps
Antifungal Drugs


fewer effective agents because of similarity of
fungal cells and human cells
easier to treat superficial mycosis than
systemic infections
Antifungal Agents

Fungal infections difficult to treat

Eukaryotic cells similar to animal cells
 Fungi inactivate many drugs
Inhibitors:


Treatment of superficial mycosis
 Clotrimazole: inhibits sterol synthesis

Griseofulvin: disrupts mitotic spindle formation
Treating systemic infections
 Polyene produced by Streptomyces nodosus
 binds membrane ergosterol found in fungal membranes
 disrupts fungal membrane integrity
Antiviral Drugs


drug development has been slow because it
is difficult to specifically target viral cycle
drugs currently used inhibit virus-specific
enzymes and life cycle processes
Amantadine



used to prevent
influenza
infections
Inhibits M2 viral
envelop protein
blocks
penetration and
uncoating of
influenza virus
Tamiflu and Zanamivir




anti-influenza agents
neuraminidase inhibitors
prevent the release of virions from infected cells,
not a cure for influenza, has been shown to
shorten course of illness
A. Oseltamivir (Tamiflu)
inhibits herpes virus enzymes
involved in DNA and RNA
synthesis and function
inhibits herpes
virus and
cytomegalovirus
DNA polymerase
inhibits herpes
virus DNA
polymerase
Anti-HIV drugs

two main targets


HIV reverse transcriptase
HIV protease
Reverse transcriptase
inhibitors
Protease inhibitors
• Gag polyprotein is expressed from
a single mRNA
• cleaved by viral protease to viral coat
proteins p15, p17and p24
• Ritonavir , inhibitor of protease
mimics peptide bond
that is normally attacked
by the protease
Summary of Mechanisms of Drug Resistance


alteration of target enzyme or organelle
eg. PBPs
inactivation of drug


Drug exclusion: prevent entrance of drug




enzymatic modification of drug by pathogen (eg. AGs)
drug can’t bind to or penetrate pathogen
decreased permeability
Efflux pumps: pump drug out
use of alternative pathways or increased production
of target metabolite, eg. PABA
Drug Resistance



an increasing problem
resistance mutants arise spontaneously or by
mutagen induction and are then selected
once resistance establishes in a population it can be
transmitted to other bacteria horizontally by



Transduction: viruses transmit resistance genes
Transformation: genes picked up from environment
Conjugation: conjugative plasmids transmit the resistance
genes
After 100 years of golden antibiotic era we have a
problem on our hands: Emerging Multidrug
Resistance

Multidrug resistance: clinical and social problem
inherent to antibiotic era

Reasons?



Mutation rates in bacteria and viruses are
enormously high and cumulative
Selection of antibiotic resistant microorganisms is
unavoidable direct result of antibiotic use
Increased volume of antibiotic use, most probably
prone to improper practice and misuse. Statistical
data suggest a direct correlation (see examples..)
Antibiotics Consumption in Canada
Canada, Quebec, Ontario

Number of prescriptions per 1000 citizens per year
1000
900
800
700
600
500
400
300
200
100
0
Canada
Ontario
Quebec
1994
1995
1996
1997
1998
1999
2000
This excess is attributed to What?

1990 - 300 metric tons of antibiotics were used.

70% are not used appropriately

Incorrect usage.

Prescription for viral infections.

Prescription for bacterial infections which can
recover on their own.
S. pneumoniae resistance to Penicillin and
Macrolides in Canada
16%
% Resistant
14%
12%
Penicillin
Macrolides
10%
8%
6%
4%
2%
0%
1988
1994
1996
1998
Resistance of H. influenzae to
Penicillin in Canada
45%
40%
35%
30%
25%
20%
15%
10%
5%
0%
1977
1985
1993
1996
1998
Economical impact
 Bacterial infections and antibacterial
resistance constitute an ever
increasing problem
 this is reflected in leading market
share (62%) for anti-bacterial agents
 Fast growing antibiotic resistance
has negative impact on R&D in
pharmaceutical initiatives
 the least relative growth in
antibacterial sector is alarming
Measures to counter the Acquired Resistance

Public education.

Usages must be effectively controlled.

Eliminate the prophylactic administration in livestock.

Reduce the usage in the household products.

Reduce the prophylactic usage in humans.

Discover and synthesize new antibiotics.

Would this solve the problem?

What are the possible alternatives?

Probiotic approach?

Natural antimicrobial products? Phage therapy?

New antimicrobial targets? Vaccine development?