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Mechanisms of
Antimicrobial Action
and Resistance
Alan L. Goldin, M.D./Ph.D.
Sections in Medical Microbiology
& Immunology
„
Chapter 10
… Mechanisms
… Pages
„
of action
69-84
Chapter 11
… Resistance
… Pages
„
85-93
Useful reference, but recommendations
change about drugs of choice
Information on Antibiotics
„
The Medical Letter
… Bi-weekly
publication
… Independent evaluation of new drugs
… 100 Main Street, New Rochelle, NY 10801
… (800) 211-2769
… http://www.medletter.com/
„
Choice of Antibacterial Drugs (annual issue)
… http://medlet-best.securesites.com/restrictedtg/t57.pdf
„
Handbook of Antimicrobial Therapy
… Every
other year (small handbook)
Mechanisms of Action
Antibacterial drugs can be classified in
many ways – mechanism of action will be
used in these lectures
„ Biochemical mechanism of action is
crucial to understanding the mechanism of
selective toxicity
„
Mechanisms of Action
Antimetabolites (sulfonamides)
„ Affect nucleic acids (quinolones, rifampin)
„ Inhibit cell wall synthesis (penicillin)
„ Act on ribosomes
- Reversible (tetracycline, chloramphenicol)
- Irreversible (aminoglycosides)
„ Disrupt cell walls (nystatin, polymyxin)
„
Pharmacology
Route of administration (iv, oral)
„ Route of elimination (kidney, liver)
„ Half-life, which is affected by diseases
(liver or kidney disease) and other drugs
„ Interactions with other drugs
„ Dosing schedule, particularly compliance
„ Side effects and idiosyncratic responses
„
Resistance
The most important problem in therapeutic
use of antibacterial drugs
„ Biochemical mechanisms of resistance
„ Genetics
„ Societal and physician behaviors
„ Approaches to retard the development of
resistance
„
Definitions
„
Antimicrobial
… Inhibits
„
Antibacterial
… Inhibits
„
growth of micro-organisms
growth of bacteria
Antibiotic
… Inhibits
growth of micro-organisms
… Made by other micro-organisms
… Usually extended to include synthetic
drugs
Bacteriostatic versus Bactericidal
„
Bacteriostatic
… Reversible
inhibition of growth
… When the antibiotic is removed, almost all of
the bacteria can replicate
„
Bactericidal
… Irreversible
inhibition of growth
… When the antibiotic is removed, almost none
of the bacteria (10-7 to 10-3) can replicate
Minimal Inhibitory Concentration
MIC
„ Lowest concentration of antibiotic that
prevents visible growth
„ Broth or tube dilution method
„
… Serial
2-fold dilutions of the antibiotic
… Accurate but time-consuming
„
Disk sensitivity test
… Rapid,
but must be related to results from the
tube dilution method
MIC
Tube Dilution Method for
Determination of MIC
128
64
32
16
8
4
2
μg Antibiotic per ml
1
0.5
Disk Sensitivity Test
0 Time
Disk Sensitivity Test
24 Hours
Zone of Inhibition
(mm in diameter)
Correlation of Distance from Disk
and Antibiotic Concentration
Concentration
(μg per ml)
128
64
32
16
8
4
2
1
0.5
Tetracycline
Amikacin
4 8 12 16 20 24 28 32
Distance from Disk (mm)
Minimal Bactericidal Concentration
„
„
„
MBC
Lowest concentration of antibiotic that reduces
the number of viable cells by at least 1000-fold
Performed in conjunction with MIC by the tube
dilution method
… Aliquots
from the tubes at and above the MIC are
plated onto agar media
… The antibiotic is diluted, so that the remaining
viable cells grow and form colonies
„
The MBC of a truly bactericidal agent is equal to
or just slightly above its MIC
Tube Dilution Method for
Determination of MBC
128
64
32
16
8
4
2
μg Antibiotic per ml
1
0.5
MIC
Tube Dilution Method for
Determination of MBC
128
64
32
16
8
4
2
μg Antibiotic per ml
1
0.5
MIC
Tube Dilution Method for
Determination of MBC
128
64
32
16
8
4
2
μg Antibiotic per ml
1
0.5
64
32
MIC
128
MBC
Tube Dilution Method for
Determination of MBC
16
8
4
2
μg Antibiotic per ml
1
0.5
Attainable Level of Antibiotic
Concentration that can be reached in the
target tissue without toxic side effects
„ If the attainable level of an antibiotic is
greater than the MIC for at least 90% of
the isolates, that species is considered
susceptible to that antibiotic
„ For serious infections, those odds may
provide inadequate guidance for treatment
„
Trough Levels of Antibiotics
Levels of antibiotics reach minimal levels
(troughs) at roughly predictable times after
administration
„ The troughs may be at or below the MIC
„ This may or may not be a problem
because of two mitigating factors
„
… Post
Antibiotic Effect, a prolonged period
before bacteria resume growth
… Synergism between host defenses and subMIC levels of antibiotics
Trough Levels of Antibiotics
„
Trough levels may increase the frequency
of drug-resistant bacteria
… Frequency
of developing resistance is greatly
increased at levels just above the MIC
… Development of resistance to ciprofloxacin is
10,000 times more frequent at 2 times the
MIC compared to 8 times the MIC
Choice of Drugs Starts with
Susceptibility
Susceptibility by itself does not assure
therapeutic success
„ Lack of susceptibility guarantees
therapeutic failure
„ There are many other considerations in
the choice of antibacterial drugs
„
… Toxicity
and side-effects
… Interactions with other drugs
… Pharmacology of the drug
Antimetabolites
Sulfonamides
Prontosil
NH2
O
H2N
N
N
S
NH2
-
O
„
„
„
„
A red dye that cured streptococcal and
staphylococcal infections in mice (1933)
Ineffective against bacteria in laboratory media
Confirmed the dogma that clinically effective
treatment could not be achieved with drugs
acting directly on bacteria
The first Sulfonamide
Sulfanilamide
O
H2N
S
NH2
O„
„
„
The active component of Prontosil
A product of cleavage at the diazo bond, which
occurs naturally in the body
Effective against bacteria in both patients and
laboratory media
Sulfonamides and PABA Are Analogs
O
H2N
S
O
NHR
H2N
C
O-
O-
Sulfonamides
„
„
PABA
Sulfonamide antagonizes para-Aminobenzoic acid
Competition for uptake by bacteria
… PABA
is 1,000-fold more effective
… Small amounts of PABA negate large amount of
sulfonamides
… This competition is not a clinical problem, because we
don’t get PABA in out diets, and it is rapidly excreted
Sulfonamides and PABA Are Analogs
O
H2N
S
O
NHR
H2N
C
O-
O-
Sulfonamides
„
„
„
PABA
Sulfonamides competitively inhibit the condensation
of PABA with dihydropteridine to form
dihydropteroic acid
This is the first step in the biosynthesis of
tetrahydrofolic acid
Metabolic competition is roughly equivalent
Site of Action of Sulfonamides
Dihydropteridine
+ para-Aminobenzoic acid
(PABA)
SULFONAMIDES
INHIBIT
Dihydropteroic acid
+ Glutamic acid
Dihydrofolic acid (DHF)
NADPH
NADP
Tetrahydrofolic acid (THF)
Selective Toxicity of Sulfonamides
„
We lack dihydropteroic acid synthase
… We
„
require folic acid in our diet
Bacteria must synthesize folic acid using
dihydropteroic acid synthase
… They
cannot use an external source
… Sulfonamides are still effective even when
folic acid is present
Consequences of Inhibition by
Sulfonamides
Sulfonamide block
Tetrahydrofolic acid deficit
Tetrahydrofolic acid cofactor deficits
Thymidine
Purines
Methionine
DNA
DNA
RNA
Protein
Effect of Sulfonamides Depends
on the Environment
„
Bactericidal in blood and urine
… Blood
and urine have large amounts of methionine
and purines, so protein and RNA synthesis continue
… Selectively blocking DNA synthesis is lethal
„
Bacteriostatic if protein and RNA synthesis are
also blocked
… Adding
„
a bacteriostatic antibiotic decreases efficacy
Ineffective in purulent lesions
… Rich
in methionine, purines & thymidine from cells
that have lysed, so synthesis of proteins, RNA and
DNA can continue
Sulfonamides Introduced the
Problem of Drug Resistance
„
Development of sulfonamide resistance was
rapid
… Sulfonamides
were introduced to treat bacillary
dysentery during World War II
… 4 years later, most isolates were resistant
… About 10% were resistant to 3 biochemically
unrelated antibiotics
… This pattern has been repeated with each new drug
„
Resistance to multiple drugs is more common
than to a single drug
…R
factors, transposons, and integrons
Dynamics of Drug Resistance
„
People who receive an antibiotic are more likely to
harbor bacteria resistant to that antibiotic and
biochemically unrelated antibiotics
„
People who frequent environments in which antibiotics
are used are more likely to harbor drug-resistant
bacteria, even if they have not received antibiotics. This
applies to patients as well as to staff.
„
The probability of harboring drug-resistant bacteria
returns to normal within a few weeks after antibiotic
therapy is discontinued or after absence from the
antibiotic-rich environments
„
The prevalence of drug-resistant bacteria in the
community is increasing due to increasing use of
antibiotics in the environment
„
Antibiotics, use them and lose them
Resistance to Sulfonamides
„
Reduced uptake (Antiporter)
… Transposons
„
Altered dihydropteroic acid synthase
… Reduced
„
sensitivity to sulfonamides
Transposons & plasmids
… Increased
„
„
& plasmids
levels of synthase or synthase activity
Mutation or plasmid
Increased synthesis of PABA (rare)
… Mutation
… Loss
of end-product inhibition
… Promoter up mutation
Impact of Sulfonamide Discovery
„
Shattered vitalist dogma on treatment of infection
… Proved
„
in vitro effects are relevant
Initiated successful searches for antibiotics
… Penicillin
„
and streptomycin
Launched huge search for metabolic analogs
… Produced
thousands of rat poisons
… A few anticancer agents
… An immunsuppressant
… One antibacterial drug (Trimethoprim)
Trimethoprim
Competitive inhibitor of dihydrofolic acid
reductase
„ The competitive substrate is dihydrofolic
acid
„ Trimethoprim blocks a step in the
biosynthesis of tetrahydrofolic acid
„
Site of Action of Trimethoprim
Dihydropteridine
+ PABA
Sulfonamides
Inhibit
Dihydropteroic acid
+ glutamic acid
dTMP
Dihydrofolic acid
Trimethoprim
Inhibits
NADPH
NADP
Tetrahydrofolic acid (THF)
dUMP
5,10-methylene THF
5-methyl THF
methionine & purines
Site of Action of Trimethoprim
Dihydropteridine
+ PABA
„
Sulfonamides
Inhibit
„
Dihydropteroic acid
+ glutamic acid
Dihydrofolic acid
dTMP
Trimethoprim
„
NADPH
Inhibits
NADP
Trimethoprim acts rapidly,
sulonamides act slowly
With trimethoprin, dUMP ⇒
dTMP rapidly depletes THF
by conversion to DHF, and
there is no DHF ⇒ THF
With sulfonamides, there is
no net synthesis of THF, but
DHF ⇒ THF proceeds
…
Tetrahydrofolic acid (THF)
dUMP
5,10-methylene THF
5-methyl THF
„
Depletion of THF pool takes 3-4
generations
Synthesis of pyrimidines &
purines does not deplete THF
methionine & purines
Site of Action of Trimethoprim
Dihydropteridine
+ PABA
Sulfonamides
Inhibit
„
…
Bactericidal in blood
… Ineffective in purulent lesions
Dihydropteroic acid
+ glutamic acid
„
Dihydrofolic acid
dTMP
Trimethoprim
NADPH
Inhibits
NADP
„
But trimethoprim is not
antagonized by PABA
Trimethoprim and
sulfonamides are synergistic
…
Inhibitors of sequential steps
are often synergistic
5,10-methylene H4F
… Sulfonamides reduce DHF
5-methyl H4F
which competes with
methionine & purines trimethoprim
Tetrahydrofolic acid (H4F)
dUMP
Trimethoprim is like
sulfonamides
Trimethoprim and Sulfonamides
are Synergistic
Sulfamethoxazole inhibits an early step in
the pathway and lowers the concentration
of dihydrofolic acid
„ Dihydrofolic acid and trimethoprim
compete for binding to dihydrofolic acid
dehydrogenase
„ Less trimethoprim is required for inhibition
of dihydrofolic acid reductase in the
presence of sulfamethoxazole
„
Trimethoprim and Sulfonamides
are Synergistic
The synergism permits use of smaller
doses than if either drug were used alone
„ The use of two drugs together reduces the
frequency of resistance
„ The two drugs are marketed as a
combination in the fixed ratio of 5 parts
sulfamethoxazole to 1 part trimethoprim
„ There are only a few indications for the
use of either drug alone
„
Selectivity of Trimethoprim
Both bacteria and humans have
dihydrofolate reductase
„ The human enzyme is 60,000-fold less
sensitive to trimethoprim
„ There is no toxicity due to the antibacterial
action of trimethoprim
„ Folic acid deficiency can occur in patients
with inadequate dietary consumption
„
… Normal
bacterial flora can no longer make
folic acid to compensate
Resistance to Trimethoprim
„
„
Dihydrofolate reductases with decreased
sensitivity to trimethoprim
… Reduced affinity for trimethoprim
… Located in the intervening sequences of
transposons
… On a plasmid, but may transpose to the
chromosome
… It is not a mutant form of the bacterial
enzyme, but a new gene
Mutation of bacterial dihydrofolate reductase is
only important in the lab
Resistance to TMP/Sulfa
Resistance to TMP makes the
combination ineffective
„ Resistance to Sulfonamide maintains
considerable potency
„
Drugs to Remember
„
TMP/Sulfonamide Combination
… Trade
name Bactrim
Drugs that Affect
Nucleic Acid
Synthesis
Quinolones
Quinolones
„
Nalidixic was the first quinolone
… Too
toxic for systemic use (newer quinolones
can be used systemically)
… Rapidly excreted in the urine
… Effectively used to treat urinary tract infections
„
Inhibits the A subunit of DNA gyrase
… Human
analog (topoisomerase II) is several
hundred fold less sensitive
… Rapidly inhibits DNA synthesis
„
Bactericidal unless growth is prevented
Quinolones
O
O
F
COOH
H3C
N
N
N
C2H5
Nalidixic Acid
COOH
N
R2N
R1
6-FluoroQuinolones
Ciprofloxacin
R1 =
, R2 = H:
Norfloxacin
R1 = —C2H5 , R2 = H:
Ofloxacin
R1 = —C 2H5 , R2 = CH3:
Resistance to Quinolones
„
„
„
Missense mutations in gyrA
Missense mutations in a gene for a membrane
protein, which reduces the uptake of
fluoroquinolones
Development of resistance to ciprofloxacin
among nosocomial pathogens
… Between
1989 and 1992, resistance among S. aureus
increased 123%
… By the end of 1992, More than ¼ of all S. aureus
strains were resistant to ciprofloxacin
… Ciprofloxacin resistance was 80% among methicillin
resistant S. aureus
Resistance to Quinolones
„
Most frequent among important nosocomial
pathogens such as S. aureus and P. aeruginosa
… These
species were not highly susceptible to the first
fluoroquinolones
… Resistance developed rapidly because the drugs
were used at levels close to the MIC
„
„
Ciprofloxacin resistant organisms are cross
resistant to other fluoroquinolones
Plasmid encoded resistance is not a problem
…A
single copy of the sensitive gyrA gene makes the
bacteria susceptible
… Errors in DNA synthesis and repair are lethal
Drugs to Remember
Ciprofloxacin (Cipro)
„ Levofloxacin
„
Drugs that
Inhibit Cell Wall
Synthesis
Penicillins
Cephalosporins
Vancomycin
Penicillins
Penicillin G was the first penicillin in 1942
„ Advantages compared to sulfonamides
„
… Much
greater potency
… Much less toxicity
… Effective against organisms that were
resistant to sulfonamides
… Effective in wounds and purulent lesions
6-Aminopenicillanic Acid
H
H
S
H2N
C
N
O
CH3
CH3
H
COOH
β-lactam ring Thiazolidine ring
Peptidoglycan Cross Linking
L Ala D Glu m Dap D Ala
D Ala
D Ala
Site of action of penicillins
TRANSPEPTIDASE
L Ala D Glu m Dap D Ala TRANSPEPTIDASE
TRANSPEPTIDASE
D Ala D Ala m Dap L Glu L Ala
glycan ( N acetyl glucosamine-N acetyl muramic acid)n
Peptidoglycan Cross Linking
CH3 O
CH3 O
Ala – Glu –DAP -
N
H
HO
O
CH3
C
C
H
N
H
O
CH3
C
C
H
C
H
N
H
C
N
H2
NH
N
C
H
C
- DAP – Glu - Ala
glycan ( N acetyl glucosamine-N acetyl muramic acid)n
free amino group of DAP (m-diaminopimelic acid)
cross link
NH2
OH
Substrate-Enzyme Intermediate
in the Cross Linking Reaction
CH 3 O
Ala – Glu –DAP -
N
H
C
H
C
CH3 O
O
N
H2
C
H
C
Transpeptidase
O
= Serine hydroxyl group in active center of transpeptidase
OH
β-lactam Inactivation of Transpeptidases
H
H
S
C
C
CH3
H2N
C
C
CH3
N
C
O
H
COOH
+ Transpeptidase
H
H
S
C
C
CH3
H2N
C
C
HN
C
O
Serine OH
of Transpeptidases
O
H
Transpeptidase
CH3
COOH
Inactivation of Transpeptidases
by β-lactams
H
H
S
C
C
CH3
H2N
C
C
C
O
O
CH3
HN
H
COOH
Transpeptidase
Serine OH of Transpeptidases
Transpeptidases (Penicillin
Binding Proteins)
MW
PBP
Activity
Function
91,000
87,000
1a Transpeptidases
1b
66,000
2
Transpeptidase?
Maintenance of rod shape
60,000
3
Transpeptidase
Peptidoglycan synthesis
Septum formation
49,000
4
42,000
40,000
5
6
Peptidoglycan synthesis
Cell wall elongation
D-alanine
Control extent of x links
carboxypeptidases
Selectivity & Side Effects of
β-lactams
„
Selective toxicity
targets of β-lactams are uniquely bacterial
… The corresponding structures do not occur in humans
… The
„
Side effects
… The
earliest penicillins are exceptionally benign
… Some of the later derivatives have side effects related
to their side chains
… A nonspecific side effect is superinfection, such as
overgrowth of the large intestine with Clostridium
difficile (pseudomembranous colitis)
… Hypersensitivity is a common and serious problem
Haptene Formation: Reaction of
β-lactams with Serum Proteins
O
R
C
H
N
H
C
H
C
S
CH 3
C HN
O
ε amino group
of a Lys residue
NH
CH 3
CO O H
Serum protein
Resistance to β-lactams
Resistance of Staphylococci to penicillin G
became a major problem within 10 years
„ Resistance has since appeared in several
additional bacterial species
„ Most group A (β hemolytic) Streptococci
are still highly sensitive
„ Resistance is due to β-lactamase
„
Resistance to β-lactams
Destruction by β-lactamase
H
H
S
C
C
CH3
H2N
C
C
HN
C
O
Serine OH
O
H
β-lactamase
+ H2O
CH3
COOH
Penicilloic acid
+
Free β-lactamase
β-lactamases of Staphylococci
„
„
Primarily penicillinases
Inducible & extracellular
… Inoculum
„
„
„
size has large effect on MIC
MIC for β-lactamase negative is < 0.5 μg/ml for 10 – 106 cells
MIC for β-lactamase positive is < 0.5 μg/ml for 10 – 103 cells
MIC for β-lactamase positive Staph is 1250 μg/ml for 106 cells
… Large
initial dose is important (kill before induction)
… Destruction of penicillin by a few bacteria can protect a
sensitive pathogen (secretion of β-lactamase)
„
One of the major limitations of the early penicillins
Limitations of Early Penicillins
Hypersensitivity by a significant proportion
of the population
„ Need to use parenteral routes of
administration (no oral administration)
„ Development of resistance among
important groups of pathogens
„ Narrow antibacterial spectrum
„
Oral Penicillin
Penicillin G is hydrolyzed by acid in the
stomach
„ Penicillin V is acid-stable
„ Made by adding phenoxyacetic acid to the
medium of the mold producing penicillin
„ Penicillin G is now so inexpensive that it
can be used orally by giving a larger dose
„
Natural Penicillins
O
C C N
H2
H
CH3
Acid labile
CH3
O
COOH
PENCILLIN G (benzylpenicillin)
O
O C C N
H2
H
CH3
Acid stable
CH3
O
COOH
PENICILLIN V (phenoxymethyl penicillin)
β-Lactamase Refractory Penicillin
Penicillin G is hydrolyzed by β-lactamase
„ Methicillin is refractory to β-lactamase
hydrolysis
„ Steric hindrance of the side chain prevents
the hydrolysis
„ Penicillin G forces the β-lactamase into its
active conformation, so use with methicillin
will decrease the effectiveness of methicillin
„ These drugs are made semi-synthetically
„
Preparation of Semisynthetic
Penicilins
S
H2N
CH3
N
CH3
COOH
6-AMINOPENICILLANIC ACID
O
+ Acid anhydrides
or
Acid chlorides
OCH3
O
C
O
S
N
N
OCH3
O
METHICILLIN
CH3
CH3
COOH
C
S
N
N
OC2H5 O
NAFCILLIN
CH3
CH3
COOH
Broad Spectrum Penicillin
Penicillin G cannot pass through the outer
membrane of gram negative bacteria
„ Ampicillin has a charged amino group that
allows it to pass through the outer
membrane
„ Ampicillin is also acid-stable
„ These drugs are semi-synthetic
„
Penicillin G and Ampicillin
O
C
H2
C
S
N
H
N
CH3
Narrow Spectrum
CH3
O
COOH
PENICILLIN G
(Benzyl penicillin)
O
H
C
C
S
N
H
NH2
N
O
AMPICILLIN
CH3
Broad Spectrum
CH3
COOH
Broad Spectrum β-Lactamase
Refractory Penicillin?
There are none
„ The large side chains that make methicillin
refractory to β-lactamase prevent it from
crossing the outer membrane
„ A partial solution is to combine a broad
spectrum penicillin with a β-lactamase
inhibitor
„
Active Site Directed Inhibitors of
β-Lactamases
O
O
N
O
O
S
C C H 2O H
H
COOH
C lavulanic A cid
N
O
CH3
CH3
COOH
Sulbactam
Inhibition of β-Lactamases by
Clavulanic Acid
O
CHCH2OH
N
O
I
O
O
II
COOH
β-lactamase
+ β-lactamase
CHCH2OH
HN
O
COOH
CH2CH2OH
HN
O
COOH
β-lactamase
Effect of Clavulanic Acid on
Ampicillin Resistance
Antibiotic
MIC (μg per ml)
E. coli
β-lactamase -
E. coli
β-lactamase +
Ampicillin alone
2
> 2,000
Ampicillin +
Clavulanic Acid
2
4
Intrinsic Resistance to β-Lactams
„
Methicillin resistant Staph. aureus (MRSA)
… Still
cannot hydrolyze methicillin
… Resistant by an intrinsic mechanism
Resistance developed rapidly (in 10 years
of methicillin use)
„ Resistance is carried on a transposon,
frequently with other resistance genes
„ Resistance is easily transmitted to other
bacteria
„
Pencillin Binding Proteins (PBP) of Methicillin
Susceptible & Resistant S. aureus
Susceptible
PBP
1
2
3
4
Resistant
2A
Genetics of Methicillin Resistance
„
„
„
mecA encodes PBP 2A
mecA is a fusion gene
mecA is on a transposon
… Transmitted
by a plasmid, but stability requires
transposition to the chromosome
… Production of PBP 2A by mecA is essential but not
sufficient for methicillin resistance
„
Host (S. aureus) functions are also required
… Depending
on host functions, resistance is often
heterogeneous, leading to incorrect sensitivity reports
„
The mecA transposon is an attractant for other
resistance genes
Drugs to Remember
Penicillin
„ Ampicillin
„ Nafcillin
„ Amoxicillin/Clavulanate Combination
„
… Augmentin
Other β Lactam Antibiotics
„ Cephalosporins
„ Carbapenems
„ Monobactams
Cephalosporins
„
„
„
About 20 currently in use
Tend to be substrates for β-lactamases less frequently
than penicillins
1st generation (Cefazolin)
… Antibacterial spectra & potency
„ 2nd generation (Cefoxitin)
like penicillins
… More potent & better against gram
„ 3rd generation (Cefotaxime)
…
„
negatives
Even more potent & highly effective against gram negatives but
at the expense of reduced potency for gram positives
4th generation (Ceftazidime)
…
Enhanced activity against gram negatives without loss of
potency for gram positives
Core Structures of Penicillins &
Cephalosporins
H
H
S
H 2N
CH3
C
N
O
H
CH3
H
CO O H
6-Aminopenicillanic Acid
H
S
H 2N
R
N
O
COOH
7-Aminocephalosporanic Acid
O
R = CH2
O
HC
CH3
Cross Hypersensitivity of
Cephalosporins with Penicillins
About 2% of population are hypersensitive
to cephalosporins
„ About 8% of people who are
hypersensitive to penicillins are also
hypersensitive to cephalosporins
„
Penicillins versus Cephalosporins
Haptene Formation
Penicillins + Serum protein Cephalosporins + Serum protein
Rare
Frequent
O
R
C
H
N
H
C
O
H
C
S
CH3
C HN
O
NH
CH3
COOH
Serum protein
Penicilloyl protein
R
C
N
H
if at all
H
H
C
C
S
C HN
O
R1
NH
COOH
Serum protein
Cephasporyl protein
Resistance to Cephalosporins
„
β-lactamases
… Penicillins
only
… Cephalosporins only
… Penicillins & Cephalosporins
„
„
„
Specificities of β-lactamases are not predictable
Some bacteria may have more than one
β-lactamase
Assumptions about sensitivity can lead to
unpleasant surprises
Carbapenems versus Penicillin
Carbapenems
H
Penicillins
H
R1
H
CH
S R2
N
O
COOH
H atoms are trans
C replaces
R1 attached directly
R1 N
H
O
H
S
C
N
CH3
CH3
COOH
H atoms are cis
S in fused ring
R1 attached via
amino group
Monobactams
H
R
H
NH
CH 3
N
O
SO3
_
Drugs to Remember
„
Cephalosporins
… Cefazolin
… Cefotaxime
… Ceftazidime
„
Carbapenems
… Imipenem
Vancomycin
Inhibits peptidoglycan synthesis
„ The mechanism is different from that used
by penicillin
„
… Binds
„
to the D Ala – D Ala substrate
Narrow spectrum of action
… Complex
glycopeptide
… Cannot cross the outer membrane
Resistant to β-lactamases
„ Antibiotic of last resort
„
Vancomycin Target (D Ala – D Ala)
CH3 O
CH3 O
Ala – Glu –DAP -
N
H
HO
O
CH3
C
C
H
N
H
O
CH3
C
C
H
C
H
N
H
C
N
H2
NH
N
C
H
C
- DAP – Glu - Ala
glycan ( N acetyl glucosamine-N acetyl muramic acid)n
free amino group of DAP (m-diaminopimelic acid)
cross link
NH2
OH
Vancomycin Resistance
„
A Depsipentapeptide instead of the normal
Pentapeptide
„
Pentapeptide
…L
Alanyl - D Glutamyl - m DAP - D Alanyl - D Alanine
… VanSens
„
Depsipentapeptide
…L
„
„
Alanyl - D Glutamyl - m DAP - D Alanyl - D Lactate
… VanRes
VanSens Vancomycin can bind to D Alanyl - D Alanine
VanRes Vancomycin cannot bind to D Alanyl - D Lactate
Vancomycin Resistance I
„ Synthesis of
Pyruvate + NADH
D Alanine + D Lactate
the Depsipentapeptide
vanH
vanA
D Lactate + NAD
D Alanyl - D Lactate
L Alanyl - D Glutamyl - m DAP + D Alanyl - D Lactate
van?
L Alanyl - D Glutamyl - m DAP - D Alanyl - D Lactate
(Depsipentapeptide)
Vancomycin Resistance II
„
Destruction of Existing Vancomycin
Binding Sites
D Alanyl - D Alanine
vanX
D Alanine + D Alanine
L Alanyl - D Glutamyl – m DAP - D Alanyl - D Alanine
vanY
L Alanyl - D Glutamyl – m DAP - D Alanine + D Alanine
Drugs to Remember
„
Vancomycin
Drugs that Act on
Ribosomes
Aminoglycosides
Chloramphenicol
Macrolides
Clindamycin
Tetracycline
Mechanisms of Action
„ Act
on subunits of the bacterial
ribosome to disrupt translation
„ Aminoglycosides affect the 30 S
subunit and are bactericidal
„ The others are bacteriostatic
…Tetracycline
affects the 30 S subunit
…Chlorampenicol, Macrolides and
Clindamycin affect the 50 S subunit
Gentamicin (Aminoglycoside)
Aminosugar
Aminocyclitol
R1
CH
NH
NHR2
O
2
NH2
OH
O
NH
2
O
OH
Gentamicin C1
Gentamicin C2
Gentamicin C1a
R1 = CH3 R2 = CH3
R1 = CH3 R2 = H
R1 = H
R2 = H
NHCH
CH
3
3
OH
Aminosugar
O
Selective Toxicity
„ Inhibits
30 S ribosomal subunit
…Difference
between inhibition of
eukaryotic and bacterial ribosomes is not
very large
…Inhibits mitochondrial ribosomes
„ Mammalian
cell and mitochondrial
membranes are barriers
Mechanisms of Resistance
„ Proteins
modify and inactivate the
compounds
…Resistance
is additive
…Proteins are encoded on plasmids
„ Resistant
…This
ribosomal proteins
occurs very rarely
…Resistance is very high
Kanamycin Sites of Inactivation
ACII
ACIII
ACI
AC
Types of Inactivation
CH2-NH2
NH2
O
HO
NH2
OH
OH
O
PI
CH2OH
OH
O
PII
(AC)
HO
O
NH2
OH
AD
AC
(AC)
AD
P
N-Acetyl transferases
O-Acetyl transferases
O-Adenyl transferases
O-Phosphatases
Blocked reaction
Chloramphenicol
I
II
III
O H C H 2O H O
O 2N
C
H
C
H
N
H
C
C H C l2
Chloramphenicol
„ Binds
to the 50 S ribosomal subunit
…Does
not inhibit mammalian 80 S subunit
…Does inhibit mitochondrial 70 S subunit
„ Aplastic
anemia is possible
…1
in 25,000 to 40,000 administrations
…Life-threatening
…Never a drug of first choice
„ Resistance
as for aminoglycosides
Erythromycin
CH3
H3C
N
HO
CH3
O
O
H3 C
CH3
CH3
O
HO
HO
OH
H3 C
CH3
CH2
H3CO
CH3
OH
O
O
CH3
H3C
O
O
CH3
Erythromycin
„ Macrolide
antibiotic
…Does
not inhibit mammalian 80 S subunit
…Does inhibit mitochondrial 70 S subunit
…Does not cross the mitochondrial
membrane
„ Resistance
by rRNA methylation
„ Often an alternative for penicillin to
treat allergic patients
Clindamycin
CH3
CH3
N
H 3C C C
H2 H2
O
Cl
CH
N
H
CH
O
HO
OH
SC H 3
OH
Clindamycin
„ Similar
spectrum as erythromycin
„ Binds to the 50 S subunit
„ Frequent association with bowel
superinfection
…Pseudomembranous
colitis
…Clostridium difficile infections
„ Used
to treat anaerobic infections
Tetracylcines
OH
O
OH
O
OH
O
NH2
7
6
5
OH
N(CH3)2
„
„
„
Bacteriostatic inhibitors with broad spectrum
Block the binding of aminoacyl-tRNAs to the A site
of the ribosome 30 S subunit
Resistance due to efflux and insensitive ribosomes
Tetracylcines
OH
O
OH O
OH
O
NH2
7
6
5
OH
N(CH3)2
Drug
Position
Chlortetracycline
6
CH3 ; OH
Tetracycline
CH3 ; OH
Doxycycline
Minocycline
5
OH
7
Cl
CH3
N(CH3)2
Drugs to Remember
Gentamicin
„ Erythromycin
„ Clindamycin
„ Tetracycline
„
Drugs that
Disrupt Cell Walls
Nystatin
Polymyxin
L-Leu
L-Phe
(α) L-Dab
(α) L-Dab
(α) L-Dab
L-Thr
(γ)
L-Dab
(α)
L-Thr
(α) L-Dab
6-Methyloctanoic
POLYMYXIN B1
L-Dab = L-α, γ-Diaminobutyric acid
(α) and (γ) indicate NH2 groups
L-Dab involved in peptide linkages
Polymyxins
Too toxic for systemic use
„ Effective against gram negative but not
gram positive bacteria
„ Bactericidal, disrupting the outer
membrane
„ Used in topical creams and ointments
„
Newer Antibiotics for
Use Against Antibiotic
Resistant Bacteria
Semisynthetic Streptogramins
Oxazolidinones
Lipopeptides
Glycylcylines
Ketolides
Newer Antibiotics for Use Against
Antibiotic Resistant Bacteria
„ Semisynthetic
streptogramins
… Quinupristin/dalfopristin
(Synercid) was
approved by the FDA in 1999
… Effective against Vancomycin Resistant Staph.
aureus (VRSA) and Enterococci (VRE)
„
Oxazolidinones
„ Lipopeptides
„ Glycylcylines
„ Ketolides
Streptogramins
N
O
N
HN
O
N
O
O
O
O
N
OH
O
H
N
N
N
O
NH
O
O
O
O
OH
N
O
N
O
P ristinom ycin Ia
Pristinom ycin IIa
Quinupristin/Dalfopristin
Act synergistically on the bacterial
ribosome to disrupt protein synthesis
„ Active against S. aureus and E. faecium
but not against E. faecalis
„ Must be administered intravenously
„ High incidence of adverse effects and drug
interactions
„ Wholesale cost for 10 day treatment is
about $3,000 plus hospitalization
„ No longer used very often
„
New Antibiotics for Use Against
Antibiotic Resistant Bacteria
„ Semisynthetic
„
streptogramins
Oxazolidinones
… Linezolid
(Zyvox) was approved by the FDA in
2000
… Effective against Vancomycin Resistant Staph.
aureus (VRSA) and Enterococci (VRE)
„ Lipopeptides
„ Glycylcylines
„ Ketolides
Oxazolidinones
O
R1
N
O
R2
Linezolid
Inhibits protein synthesis at the bacterial
ribosome
„ Bacteriostatic against staphylococci and
enterococci
„ Active against S. aureus, E. faecium and
E. faecalis
„ Administered intravenously or orally
„ Generally well-tolerated
„ Wholesale cost for 10 day treatment is
about $1,000
„
New Antibiotics for Use Against
Antibiotic Resistant Bacteria
„ Semisynthetic
„
streptogramins
Oxazolidinones
„ Lipopeptides
… Daptomycin
(Cubicin) was approved by the FDA
in 2003
… Effective against Vancomycin Resistant
Enterococci (VRE)
„ Glycylcylines
„ Ketolides
Daptomycin (Cubicin)
Daptomycin (Cubicin)
Binds to the cell membrane of grampositive bacteria and causes membrane
depolarization
„ Effective against Vancomycin Resistant
Staph. aureus (VRSA) and Enterococci
(VRE), including E. faecium and E.
faecalis
„ Administered intravenously
„ Approved for treatment of complicated
skin and skin structure infections
„
New Antibiotics for Use Against
Antibiotic Resistant Bacteria
„ Semisynthetic
„
streptogramins
Oxazolidinones
„ Lipopeptides
„ Glycylcylines
… 9-Aminotetracyclines
acylated with
N-dimethylglycine
… Tigecycline was approved by the FDA in 2005
„ Ketolides
Glycylcyclines
O
H3C
OH
H
N
OH O
OH
O
9
N
H3C
O
NH2
8
7
6
5
OH
N(CH3)2
„Glycylcyclines
are not substrates for the efflux
process and they block insensitive ribosomes
Tigecycline
H 3C
H 3C
H 3C
N
H
OH
H
N
O
O
OH
OH
O
O
9
NH2
8
7
6
5
OH
N (C H 3 ) 2
Tigecylcine (Tygacil)
Active against methicillin-resistant S.
aureus and probably VRE (in vitro)
„ Broad spectrum
„ Approved for complicated intra-abdominal
and skin and skin structure infections
„ Not a substrate for tetracycline antiporters
or ribosome protection proteins
„ Intravenous administration
„ Bacteriostatic
„
New Antibiotics for Use Against
Antibiotic Resistant Bacteria
„ Semisynthetic
„
streptogramins
Oxazolidinones
„ Lipopeptides
„ Glycylcylines
„ Ketolides
… Telithromycin
(Ketek) was approved by the FDA
in 2004
… Effective against multi-drug resistant
Streptococcus pneumoniae
Telithromycin (Ketek)
Telithromycin (Ketek)
Structurally related to the macrolides,
which include Erythromycin
„ Blocks protein synthesis by binding to 23S
rRNA of the 50S ribosomal subunit
„ Effective against
„
… gram-positive
S. aureus (MRSA, not VRA)
and S. pneumoniae (increasingly resistant to
penicillin and macrolides)
… gram negative Haemophilus influenzae
… Mycoplasma pneumoniae and Chlamydia
Telithromycin (Ketek)
Approved for treatment of bronchitis,
sinusitis and pneumonia
„ Alternative to a fluoroquinolone for
macrolide-resistant pneumococci
„ Cost is $114 for 10 day course
„
… Comparable
cost to fluoroquinolones and
newer macrolides such as Clarithromycin
… Erythromycin costs about $6
„
Use with caution because of reports of
serious hepatotoxicity
Drugs to Remember
Linezolid
„ Daptomycin
„ Tigecycline
„
Antibiotic
Resistance
Current Status of Resistance
Introduction of new antibiotics had been
keeping up with resistance
„ Declining investment in antibiotic
discovery during the 1980s altered the
balance
„ Accelerated investment in the 1990s is
beginning to yield new drugs
„ Avoidance of resistance to new drugs has
been a consistent but never achieved
design objective
„
The Problems in Avoiding
Resistance
Mobile genetic elements
„ Multiple resistance and association with
virulence markers
„ Increasing use of drugs is associated with
increasing frequency of resistance
„ Worst case scenarios are already here for
some nosocomial infections
(Staphylococci and Enterococci)
„
Antibiotic Resistance in the US
Sept. 2002 – ASM Meeting
„
Methicillin-Resistant Staph. aureus
… >50%
of nosocomial bloodstream infections
… 31% of Staph infections outside the hospital
… 71% of Staph infections in nursing homes
First case in US of vancomycin resistant
Staph. aureus (from Enterococcus)
„ Campylobacter jejuni and coli
„
… Most
common cause of diarrhea
… 50% are resistant to Ciprofloxacin (Cipro)
Retarding Emergence of Resistance
„
Maintenance of therapeutic levels
… Ensure
patient compliance
… Avoid the use of drugs when the MIC is at or
only slightly below the attainable level
… Prevent biofilms and treat them aggressively
Use combinations of antibiotics when
indicated (but not otherwise)
„ Avoid over and ill-advised use of
antibiotics
„
… Prescriptions
for infections that won’t respond
… Tendency to use hot new drugs
… Self medication
Antibiotic Resistance of Bacteria from
Sewers Serving Isolated Locations
Sewer
Serving
General
Hospital
Mental
Hospital
Residential
Area
Percent of Bacteria Resistant to
Streptomycin Chloramphenicol Tetracycline
34.7
0.7
32.0
6.5
0.3
0.4
0.7
0.007
0.1
Gentamicin Resistant
P. aeruginosa in Burn Patients
1965 - 90 % susceptible
„ 1968 - 636 kg (0.7 tons) of topical
gentamicin used
„ 1969 - 9 % susceptible
„ late 1969 - gentamicin discontinued
„ 1970 - 95 % susceptible
„
Antibiotic Treatment of Adults
with Sore Throat
1989-1999 (JAMA 2001, vol. 286:1181)
„ 6.7 million annual visits in the US
„ Antibiotics were prescribed in 73% of
cases
„
… Decreasing
use of penicillin and
erythromycin
… Increasing use of non-recommended,
extended-spectrum macrolides and
fluoroquinolones
Antibiotic Treatment of Adults
with Sore Throat
„
„
Most sore throats are due to viral upper
respiratory tract infections
Group A β-hemolytic Streptococci is the only
common cause warranting antibiotics
… Streptococci
„
„
cultured in 5-17% of cases
Penicillin and erythromycin are still
recommended in most cases
Other drugs increase likelihood of resistance
to those drugs and greatly increase the cost
(> 20-fold for quinolones versus penicillin)
Societal Contributors
„
„
Antibiotic additives in stock feed
Chlorine treatment of water
… Reduces
number of bacteria by > 100
… Survivors are resistant to antibiotics
„
Mercury and other contaminants in water
… Bacteria
resistant to mercury are also resistant to
antibiotics
„
Antibacterial soaps
… Any
inhibitor selects for resistance to other inhibitors,
including antibacterial drugs
… Criticized by the AMA and CDC, which agree that
regular soap and water is equally effective
Current Status of Antibiotic
Discovery
„
Empiricism
… At
first highly successful
… Now marginal
„
Rational approach
… Molecular
modeling is being used extensively
… Low yield so far, but promising
„
Novel agents from non-microbial biological
systems
New or Improved Antibiotics in
Development
„ Synthetic
Vancomycins
„ For resistance to Fluoroquinolones
New Antibiotics in Development
„ Synthetic
…A
„ For
Vancomycins
promising but unproven prospect
resistance to Fluoroquinolones
Synthetic Vancomycins
„
„
„
„
„
The sugar groups on the peptide backbone were
modified (Science 1999, vol. 284:508)
Completely synthetic drug
The modified drug was more efficient at killing
both vancomycin-sensitive and vancomycinresistant organisms
Mechanism of action is different, blocking
transglycosylation rather than transpeptidation
Additional modifications are being tried
New Antibiotics in Development
„ Synthetic
Vancomycins
„ For resistance to Fluoroquinolones
2-Pyridones
O
O
F
COOH
F
COOH
N
N
N
CH3
N
HN
NH2
2-Pyridone
„
„
Ciprofloxacin
Inhibits DNA gyrase A, like quinolones
May be more effective against gyrA mutants
Approaches to Identify New
Antibacterial Drugs
„
Peptides from higher organisms
… Magainin
from frogs, reached phase III
trials but never proceeded further
„
Steroids from higher organisms
… Squalamine
„
from sharks
Inhibitors of additional pathways
… Block
lipid A synthesis, which is an
essential component of the outer
membrane of gram negative bacteria
Functional Genomics
The genomes of more than 20 microbial
organisms have been sequenced
„ Sequence data are used to identify
essential targets by comparative
genomics
„ The targets are experimentally tested
„ Drugs are developed to block those
targets, based on structural predictions
„
The Future of Antibiotics
The best long-term solution is to
minimize the development of resistance
„ Doctors have a critical role in
accomplishing this goal
„