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Sections in Medical Microbiology & Immunology
Chapter 10Mechanisms of actionPages 69-84
Chapter 11ResistancePages 85-93
Useful reference, but recommendations change about drugs of choice
Information on AntibioticsThe Medical Letter
Bi-weekly publicationIndependent evaluation of new drugs100 Main Street, New Rochelle, NY 10801(800) 211-2769http://www.medletter.com/
Choice of Antibacterial Drugs (annual issue)http://medlet-best.securesites.com/restrictedtg/t57.pdf
Handbook of Antimicrobial TherapyEvery other year (small handbook)
Mechanisms of Action
Antibacterial drugs can be classified in many ways – mechanism of action will be used in these lecturesBiochemical mechanism of action is crucial to understanding the mechanism of selective toxicity
Mechanisms of ActionAntimetabolites (sulfonamides)Affect nucleic acids (quinolones, rifampin)Inhibit cell wall synthesis (penicillin)Act on ribosomes- Reversible (tetracycline, chloramphenicol)- Irreversible (aminoglycosides)Disrupt cell walls (nystatin, polymyxin)
PharmacologyRoute of administration (iv, oral)Route of elimination (kidney, liver)Half-life, which is affected by diseases (liver or kidney disease) and other drugsInteractions with other drugsDosing schedule, particularly complianceSide effects and idiosyncratic responses
ResistanceThe most important problem in therapeutic use of antibacterial drugsBiochemical mechanisms of resistanceGeneticsSocietal and physician behaviorsApproaches to retard the development of resistance
DefinitionsAntimicrobial
Inhibits growth of micro-organismsAntibacterial
Inhibits growth of bacteriaAntibiotic
Inhibits growth of micro-organismsMade by other micro-organismsUsually extended to include synthetic drugs
Bacteriostatic versus Bactericidal
BacteriostaticReversible inhibition of growthWhen the antibiotic is removed, almost all of the bacteria can replicate
BactericidalIrreversible inhibition of growthWhen the antibiotic is removed, almost none of the bacteria (10-7 to 10-3) can replicate
Minimal Inhibitory Concentration
MICLowest concentration of antibiotic that prevents visible growthBroth or tube dilution method
Serial 2-fold dilutions of the antibioticAccurate but time-consuming
Disk sensitivity testRapid, but must be related to results from the tube dilution method
12864321684210.5
Distance from Disk (mm)
4 8 12 16 20 24 28 32
Concentration(μg per ml) Tetracycline
Correlation of Distance from Disk and Antibiotic Concentration
Amikacin
Minimal Bactericidal Concentration
MBCLowest concentration of antibiotic that reduces the number of viable cells by at least 1000-foldPerformed in conjunction with MIC by the tube dilution method
Aliquots from the tubes at and above the MIC are plated onto agar mediaThe 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
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 antibioticFor serious infections, those odds may provide inadequate guidance for treatment
Trough Levels of AntibioticsLevels of antibiotics reach minimal levels (troughs) at roughly predictable times after administrationThe troughs may be at or below the MICThis may or may not be a problem because of two mitigating factors
Post Antibiotic Effect, a prolonged period before bacteria resume growthSynergism between host defenses and sub-MIC 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 MICDevelopment 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 successLack of susceptibility guarantees therapeutic failureThere are many other considerations in the choice of antibacterial drugs
Toxicity and side-effectsInteractions with other drugsPharmacology of the drug
Prontosil
H2N
NH2
N N S NH2
O
O-
A red dye that cured streptococcal and staphylococcal infections in mice (1933)Ineffective against bacteria in laboratory mediaConfirmed the dogma that clinically effective treatment could not be achieved with drugs acting directly on bacteriaThe first Sulfonamide
Sulfanilamide
H2N S NH2
O
O-
The active component of ProntosilA product of cleavage at the diazo bond, which occurs naturally in the bodyEffective against bacteria in both patients and laboratory media
Sulfonamides and PABA Are Analogs
H2N S NHR
O
O-
H2N C
O
O-
Sulfonamide antagonizes para-Aminobenzoic acidCompetition for uptake by bacteria
PABA is 1,000-fold more effectiveSmall amounts of PABA negate large amount of sulfonamidesThis competition is not a clinical problem, because we don’t get PABA in out diets, and it is rapidly excreted
Sulfonamides PABA
Sulfonamides and PABA Are Analogs
H2N S NHR
O
O-
H2N C
O
O-
Sulfonamides competitively inhibit the condensation of PABA with dihydropteridine to form dihydropteroic acidThis is the first step in the biosynthesis of tetrahydrofolic acid Metabolic competition is roughly equivalent
Sulfonamides PABA
Dihydropteridine + para-Aminobenzoic acid(PABA)
SULFONAMIDESINHIBIT
Dihydropteroic acid+ Glutamic acid
Dihydrofolic acid (DHF)
Tetrahydrofolic acid (THF)
NADPH
NADP
Site of Action of Sulfonamides
Selective Toxicity of Sulfonamides
We lack dihydropteroic acid synthaseWe require folic acid in our diet
Bacteria must synthesize folic acid using dihydropteroic acid synthase
They cannot use an external sourceSulfonamides are still effective even when folic acid is present
Sulfonamide block
Tetrahydrofolic acid deficit
Tetrahydrofolic acid cofactor deficits
DNA
Thymidine Purines Methionine
DNARNA
Protein
Consequences of Inhibition by Sulfonamides
Effect of Sulfonamides Depends on the Environment
Bactericidal in blood and urineBlood and urine have large amounts of methionine and purines, so protein and RNA synthesis continueSelectively blocking DNA synthesis is lethal
Bacteriostatic if protein and RNA synthesis are also blocked
Adding a bacteriostatic antibiotic decreases efficacyIneffective 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 II4 years later, most isolates were resistantAbout 10% were resistant to 3 biochemically unrelated antibioticsThis 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 ResistancePeople 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 environmentsThe prevalence of drug-resistant bacteria in the community is increasing due to increasing use of antibiotics in the environmentAntibiotics, use them and lose them
Resistance to Sulfonamides
Reduced uptake (Antiporter)Transposons & plasmids
Altered dihydropteroic acid synthaseReduced sensitivity to sulfonamides
Transposons & plasmidsIncreased levels of synthase or synthase activity
Mutation or plasmid
Increased synthesis of PABA (rare)MutationLoss of end-product inhibitionPromoter up mutation
Impact of Sulfonamide DiscoveryShattered vitalist dogma on treatment of infection
Proved in vitro effects are relevantInitiated successful searches for antibiotics
Penicillin and streptomycinLaunched huge search for metabolic analogs
Produced thousands of rat poisonsA few anticancer agentsAn immunsuppressantOne antibacterial drug (Trimethoprim)
Trimethoprim
Competitive inhibitor of dihydrofolic acid reductaseThe competitive substrate is dihydrofolic acidTrimethoprim blocks a step in the biosynthesis of tetrahydrofolic acid
Dihydrofolic acid
Tetrahydrofolic acid
dTMP
dUMP
(THF)
NADPH
NADP
Trimethoprim Inhibits
THFTHF
methionine & purines
Dihydropteridine + PABA
Dihydropteroic acid
Sulfonamides Inhibit
+ glutamic acid
5-methyl5,10-methylene
Site of Action of Trimethoprim
Dihydrofolic acid
Tetrahydrofolic acid (THF)
NADPH
NADP
TrimethoprimInhibits
methionine & purines
Dihydropteridine + PABA
Dihydropteroic acid
Sulfonamides Inhibit
+ glutamic acid
5,10-methylene THF5-methyl THF
dTMP
dUMP
Site of Action of TrimethoprimTrimethoprim acts rapidly, sulonamides act slowlyWith trimethoprin, dUMP ⇒dTMP rapidly depletes THF by conversion to DHF, and there is no DHF ⇒ THFWith sulfonamides, there is no net synthesis of THF, but DHF ⇒ THF proceeds
Depletion of THF pool takes 3-4 generations
Synthesis of pyrimidines & purines does not deplete THF
Dihydrofolic acid
Tetrahydrofolic acid (H4 F)
NADPH
NADP
TrimethoprimInhibits
methionine & purines
Dihydropteridine + PABA
Dihydropteroic acid
SulfonamidesInhibit
+ glutamic acid
5,10-methylene H4 F5-methyl H4 F
dTMP
dUMP
Site of Action of Trimethoprim
Trimethoprim is like sulfonamides
Bactericidal in bloodIneffective in purulent lesions
But trimethoprim is not antagonized by PABATrimethoprim and sulfonamides are synergistic
Inhibitors of sequential steps are often synergisticSulfonamides reduce DHF which competes with trimethoprim
Trimethoprim and Sulfonamides are Synergistic
Sulfamethoxazole inhibits an early step in the pathway and lowers the concentration of dihydrofolic acidDihydrofolic acid and trimethoprimcompete 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 aloneThe use of two drugs together reduces the frequency of resistanceThe two drugs are marketed as a combination in the fixed ratio of 5 parts sulfamethoxazole to 1 part trimethoprimThere are only a few indications for the use of either drug alone
Selectivity of TrimethoprimBoth bacteria and humans have dihydrofolate reductaseThe human enzyme is 60,000-fold less sensitive to trimethoprimThere is no toxicity due to the antibacterial action of trimethoprimFolic acid deficiency can occur in patients with inadequate dietary consumption
Normal bacterial flora can no longer make folic acid to compensate
Resistance to TrimethoprimDihydrofolate reductases with decreased sensitivity to trimethoprim
Reduced affinity for trimethoprimLocated in the intervening sequences of transposonsOn a plasmid, but may transpose to the chromosomeIt 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 ineffectiveResistance to Sulfonamide maintains considerable potency
QuinolonesNalidixic was the first quinolone
Too toxic for systemic use (newer quinolonescan be used systemically)Rapidly excreted in the urineEffectively used to treat urinary tract infections
Inhibits the A subunit of DNA gyraseHuman analog (topoisomerase II) is several hundred fold less sensitiveRapidly inhibits DNA synthesis
Bactericidal unless growth is prevented
Quinolones
N NH3C
O
COOH
C2H5
NN
O
COOH
R1R2N
F
Nalidixic Acid 6-FluoroQuinolones
= , R1 R2 = H:
R1 = —C2H5 , R2 = H:
R1 = —C2H5 , R2 = CH3:
Ciprofloxacin
Norfloxacin
Ofloxacin
Resistance to QuinolonesMissense mutations in gyrAMissense mutations in a gene for a membrane protein, which reduces the uptake of fluoroquinolonesDevelopment of resistance to ciprofloxacin among nosocomial pathogens
Between 1989 and 1992, resistance among S. aureusincreased 123%By the end of 1992, More than ¼ of all S. aureusstrains were resistant to ciprofloxacinCiprofloxacin resistance was 80% among methicillinresistant S. aureus
Resistance to QuinolonesMost frequent among important nosocomial pathogens such as S. aureus and P. aeruginosa
These species were not highly susceptible to the first fluoroquinolonesResistance developed rapidly because the drugs were used at levels close to the MIC
Ciprofloxacin resistant organisms are cross resistant to other fluoroquinolonesPlasmid encoded resistance is not a problem
A single copy of the sensitive gyrA gene makes the bacteria susceptibleErrors in DNA synthesis and repair are lethal
Penicillins
Penicillin G was the first penicillin in 1942Advantages compared to sulfonamides
Much greater potencyMuch less toxicityEffective against organisms that were resistant to sulfonamidesEffective in wounds and purulent lesions
L Ala D Glu m Dap D Ala D Ala
D Ala D Ala m Dap L Glu L Ala
L Ala D Glu m Dap D Ala TRANSPEPTIDASE
TRANSPEPTIDASE
D Ala
TRANSPEPTIDASE
glycan ( N acetyl glucosamine-N acetyl muramic acid)n
Site of action of penicillins
Peptidoglycan Cross Linking
HO C CH
NH
C CH
NH
O CH3 O CH3
N CH
C
O
NH2
CH
C OH
OCH3CH3
H
NH N
glycan ( N acetyl glucosamine-N acetyl muramic acid)n
NH2free amino group of DAP (m-diaminopimelic acid)
cross link
Peptidoglycan Cross LinkingAla – Glu –DAP -
- DAP – Glu - Ala
O = Serine hydroxyl group in active center of transpeptidase
Substrate-Enzyme Intermediate in the Cross Linking Reaction
N CH
C
OCH3
H
Transpeptidase
OAla – Glu –DAP -
NH2
CH
C OH
OCH3
β-lactam Inactivation of Transpeptidases
C
NC
C
SC
CO
CH3
CH3
COOHH
HH
H2N
C
HNC
C
SC
CO
CH3
CH3
COOHH
HH
H2N
O
Transpeptidase
+ Transpeptidase
Serine OH of Transpeptidases
Inactivation of Transpeptidases by β-lactams
C
HNC
C
SC
CO
CH3
CH3
COOHH
HH
H2N
O
Transpeptidase
Serine OH of Transpeptidases
MW
91,00087,000
66,000
60,000
49,00042,00040,000
1a
2
3
4
56
1b Transpeptidases
Activity
Transpeptidase?
Transpeptidase
D-alaninecarboxypeptidases
Peptidoglycan synthesisCell wall elongation
Maintenance of rod shape
Peptidoglycan synthesisSeptum formation
Control extent of x links
PBP Function
Transpeptidases (Penicillin Binding Proteins)
Selectivity & Side Effects of β-lactams
Selective toxicityThe targets of β-lactams are uniquely bacterialThe corresponding structures do not occur in humans
Side effectsThe earliest penicillins are exceptionally benignSome of the later derivatives have side effects related to their side chainsA 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
C
HN
S CH 3
CH 3
CO OH
CNH
CR
O
C
NHO
Serum protein
HH
ε
amino groupof a Lys residue
Resistance to β-lactams
Resistance of Staphylococci to penicillin G became a major problem within 10 yearsResistance has since appeared in several additional bacterial speciesMost group A (β hemolytic) Streptococciare still highly sensitiveResistance is due to β-lactamase
Resistance to β-lactams Destruction by β-lactamase
C
HNC
C
SC
CO
CH3
CH3
COOHH
HH
H2N
O
β-lactamase
+ H2O
Penicilloic acid+
Free β-lactamaseSerine OH
β-lactamases of StaphylococciPrimarily penicillinasesInducible & extracellular
Inoculum size has large effect on MICMIC for β-lactamase negative is < 0.5 μg/ml for 10 – 106 cellsMIC for β-lactamase positive is < 0.5 μg/ml for 10 – 103 cellsMIC 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 populationNeed to use parenteral routes of administration (no oral administration) Development of resistance among important groups of pathogensNarrow antibacterial spectrum
Oral Penicillin
Penicillin G is hydrolyzed by acid in the stomachPenicillin V is acid-stableMade by adding phenoxyacetic acid to the medium of the mold producing penicillinPenicillin G is now so inexpensive that it can be used orally by giving a larger dose
Natural Penicillins
NH
CCH2
CH3
CH3
COOHO
O
NH
CCH2
O
O COOH
CH3
CH3
O
PENCILLIN G (benzylpenicillin)
PENICILLIN V (phenoxymethyl penicillin)
Acid labile
Acid stable
β-Lactamase Refractory Penicillin
Penicillin G is hydrolyzed by β-lactamaseMethicillin is refractory to β-lactamase hydrolysisSteric hindrance of the side chain prevents the hydrolysisPenicillin G forces the β-lactamase into its active conformation, so use with methicillinwill decrease the effectiveness of methicillinThese drugs are made semi-synthetically
Preparation of Semisynthetic Penicilins
N
S CH3
CH3
COOHO
H2N
N
S CH3
CH3
COOHO
NC
O
OC2H5N
S CH3
CH3
COOHO
NC
OOCH3
OCH3
6-AMINOPENICILLANIC ACID
METHICILLIN NAFCILLIN
+ Acid anhydrides or Acid chlorides
Broad Spectrum Penicillin
Penicillin G cannot pass through the outer membrane of gram negative bacteriaAmpicillin has a charged amino group that allows it to pass through the outer membraneAmpicillin is also acid-stableThese drugs are semi-synthetic
Penicillin G and Ampicillin
N
S CH3
CH3O
NH
CCH2
O
N
S CH3
CH3O
NH
CHC
O
NH2
COOH
COOHPENICILLIN G(Benzyl penicillin)
AMPICILLIN
Narrow Spectrum
Broad Spectrum
Broad Spectrum β-Lactamase Refractory Penicillin?
There are noneThe large side chains that make methicillinrefractory to β-lactamase prevent it from crossing the outer membraneA partial solution is to combine a broad spectrum penicillin with a β-lactamase inhibitor
Active Site Directed Inhibitors of β-Lactamases
N
SOO
CH 3
CH 3
COOHON
O
O COOH
CH
CH 2O H
Clavulanic Acid Sulbactam
Inhibition of β-Lactamases by Clavulanic Acid
HN
O
O
CHCH2OH
COOHβ-lactamase
I
II
+ β-lactamase
N
O
O
CHCH2OH
COOH
HN
O
O
CH2CH2OH
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 methicillinResistant by an intrinsic mechanism
Resistance developed rapidly (in 10 years of methicillin use)Resistance is carried on a transposon, frequently with other resistance genesResistance is easily transmitted to other bacteria
Susceptible
PBP 123
4
2A
Pencillin Binding Proteins (PBP) of Methicillin Susceptible & Resistant S. aureus
Resistant
Genetics of Methicillin ResistancemecA encodes PBP 2AmecA is a fusion genemecA is on a transposon
Transmitted by a plasmid, but stability requires transposition to the chromosomeProduction of PBP 2A by mecA is essential but not sufficient for methicillin resistance
Host (S. aureus) functions are also requiredDepending on host functions, resistance is often heterogeneous, leading to incorrect sensitivity reports
The mecA transposon is an attractant for other resistance genes
CephalosporinsAbout 20 currently in useTend to be substrates for β-lactamases less frequently than penicillins1st generation (Cefazolin)
Antibacterial spectra & potency like penicillins2nd generation (Cefoxitin)
More potent & better against gram negatives3rd generation (Cefotaxime)
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
NC
S CH 3
CH 3
CO OHO
H H
H 2N
H
NO
H H
H 2N
S
R
CO OH
R =
6-Aminopenicillanic Acid 7-Aminocephalosporanic Acid
CH2 O HC
O
CH3
Cross Hypersensitivity of Cephalosporins with Penicillins
About 2% of population are hypersensitive to cephalosporinsAbout 8% of people who are hypersensitive to penicillins are also hypersensitive to cephalosporins
Penicillins + Serum protein
Frequent
Cephalosporins + Serum proteinRare if at all
Penicilloyl protein Cephasporyl protein
Penicillins versus Cephalosporins Haptene Formation
H H
C
HN
S
COOH
R1
CNH
CR
O
C
HN
S CH3
CH3
COOH
CNH
CR
O
C
NHO
Serum protein
HH
OC
NH
Serum protein
Resistance to Cephalosporins
β-lactamasesPenicillins onlyCephalosporins onlyPenicillins & Cephalosporins
Specificities of β-lactamases are not predictableSome bacteria may have more than one β-lactamaseAssumptions about sensitivity can lead to unpleasant surprises
Carbapenems versus Penicillin
NC
S CH 3
CO OHO
H H
NHN
CHS
CO OHO
H H
CH 3
R 2 R1R1
H atoms are trans H atoms are cisC replaces S in fused ringR1 attached directly R1 attached via
amino group
Carbapenems Penicillins
VancomycinInhibits peptidoglycan synthesisThe mechanism is different from that used by penicillin
Binds to the D Ala – D Ala substrateNarrow spectrum of action
Complex glycopeptideCannot cross the outer membrane
Resistant to β-lactamasesAntibiotic of last resort
HO C CH
NH
C CH
NH
O CH3 O CH3
N CH
C
OCH3
H
NH N
NH2
CH
C OH
OCH3
glycan ( N acetyl glucosamine-N acetyl muramic acid)n
NH2free amino group of DAP (m-diaminopimelic acid)
cross link
Vancomycin Target (D Ala – D Ala)
Ala – Glu –DAP -
- DAP – Glu - Ala
Vancomycin ResistanceA Depsipentapeptide instead of the normal PentapeptidePentapeptide
L Alanyl - D Glutamyl - m DAP - D Alanyl - D AlanineVanSens
DepsipentapeptideL Alanyl - D Glutamyl - m DAP - D Alanyl - D LactateVanRes
VanSens Vancomycin can bind to D Alanyl - D AlanineVanRes Vancomycin cannot bind to D Alanyl - D Lactate
Vancomycin Resistance I
Synthesis of the DepsipentapeptidePyruvate + NADH D Lactate + NADvanH
D Alanine + D Lactate D Alanyl - D LactatevanA
L Alanyl - D Glutamyl - m DAP + D Alanyl - D Lactate
L Alanyl - D Glutamyl - m DAP - D Alanyl - D Lactate
van?
(Depsipentapeptide)
Vancomycin Resistance II
Destruction of Existing Vancomycin Binding Sites
D Alanyl - D Alanine D Alanine + D Alanine
L Alanyl - D Glutamyl – m DAP - D Alanyl - D Alanine
L Alanyl - D Glutamyl – m DAP - D Alanine + D Alanine
vanX
vanY
Mechanisms of ActionAct on subunits of the bacterial ribosome to disrupt translationAminoglycosides affect the 30 S subunit and are bactericidalThe others are bacteriostatic
Tetracycline affects the 30 S subunitChlorampenicol, Macrolides and Clindamycin affect the 50 S subunit
Gentamicin (Aminoglycoside)
O
N H 2
R 1
C H N H R 2N H 2
O H
N H 2
O
OO H
C H 3
O H
N H C H 3
Aminocyclitol
AminosugarAminosugar
AminosugarAminosugar
O
Gentamicin C1 R1 = CH3 R2 = CH3Gentamicin C2 R1 = CH3 R2 = HGentamicin C1a R1 = H R2 = H
Selective ToxicityInhibits 30 S ribosomal subunit
Difference between inhibition of eukaryotic and bacterial ribosomes is not very largeInhibits mitochondrial ribosomes
Mammalian cell and mitochondrial membranes are barriers
Mechanisms of ResistanceProteins modify and inactivate the compounds
Resistance is additiveProteins are encoded on plasmids
Resistant ribosomal proteinsThis occurs very rarelyResistance is very high
Kanamycin Sites of Inactivation
Types of InactivationAC N-Acetyl transferases(AC) O-Acetyl transferasesAD O-Adenyl transferasesP O-Phosphatases
Blocked reaction
ACII
ACIIIACIAC
O
O
OO
HO
CH2-NH2 NH2
NH2
CH2OH
HONH2
OH
OH
OH
OH
PI
PII
(AC)
AD
ChloramphenicolBinds to the 50 S ribosomal subunit
Does not inhibit mammalian 80 S subunitDoes inhibit mitochondrial 70 S subunit
Aplastic anemia is possible1 in 25,000 to 40,000 administrationsLife-threateningNever a drug of first choice
Resistance as for aminoglycosides
ErythromycinMacrolide antibiotic
Does not inhibit mammalian 80 S subunitDoes inhibit mitochondrial 70 S subunitDoes not cross the mitochondrial membrane
Resistance by rRNA methylationOften an alternative for penicillin to treat allergic patients
ClindamycinSimilar spectrum as erythromycinBinds to the 50 S subunitFrequent association with bowel superinfection
Pseudomembranous colitisClostridium difficile infections
Used to treat anaerobic infections
OH O OH O
NH2
O
OH
OH
N(CH3)2
7 6 5
Tetracylcines
Bacteriostatic inhibitors with broad spectrum Block the binding of aminoacyl-tRNAs to the A site of the ribosome 30 S subunitResistance due to efflux and insensitive ribosomes
5 6 7Chlortetracycline CH3; OH Cl
Tetracycline CH3; OH
Doxycycline OH CH3
Minocycline N(CH3)2
PositionDrug
OH O OH O
NH2
O
OH
OH
N(CH3)2
7 6 5
Tetracylcines
L-Leu (α) L-Dab
(α) L-Dab
L-Thr
(γ)L-Dab
L-Phe
(α) L-Dab
(α)
L-Thr
(α) L-Dab
6-Methyloctanoic
POLYMYXIN B 1
L-Dab = L-α, γ-Diaminobutyric acid
(α) and (γ) indicate NH2 groupsL-Dab involved in peptide linkages
Polymyxins
Too toxic for systemic useEffective against gram negative but not gram positive bacteriaBactericidal, disrupting the outer membraneUsed in topical creams and ointments
Newer Antibiotics for Use Against Antibiotic Resistant Bacteria
Semisynthetic StreptograminsOxazolidinonesLipopeptidesGlycylcylinesKetolides
Newer Antibiotics for Use Against Antibiotic Resistant BacteriaSemisynthetic streptogramins
Quinupristin/dalfopristin (Synercid) was approved by the FDA in 1999Effective against Vancomycin Resistant Staph. aureus (VRSA) and Enterococci (VRE)
OxazolidinonesLipopeptidesGlycylcylinesKetolides
Streptogramins
N
NH
HN
O
NN
NHN
O
O
O
N
O
OO
OH
O N
N
ON
OO
O
O
O H
O
Pristinom ycin Ia Pristinom ycin IIa
Quinupristin/DalfopristinAct synergistically on the bacterial ribosome to disrupt protein synthesisActive against S. aureus and E. faeciumbut not against E. faecalisMust be administered intravenouslyHigh incidence of adverse effects and drug interactionsWholesale cost for 10 day treatment is about $3,000 plus hospitalizationNo longer used very often
New Antibiotics for Use Against Antibiotic Resistant Bacteria Semisynthetic streptograminsOxazolidinones
Linezolid (Zyvox) was approved by the FDA in 2000Effective against Vancomycin Resistant Staph. aureus (VRSA) and Enterococci (VRE)
LipopeptidesGlycylcylinesKetolides
LinezolidInhibits protein synthesis at the bacterial ribosomeBacteriostatic against staphylococci and enterococciActive against S. aureus, E. faecium and E. faecalisAdministered intravenously or orallyGenerally well-toleratedWholesale cost for 10 day treatment is about $1,000
New Antibiotics for Use Against Antibiotic Resistant Bacteria Semisynthetic streptograminsOxazolidinonesLipopeptides
Daptomycin (Cubicin) was approved by the FDA in 2003Effective against Vancomycin Resistant Enterococci (VRE)
GlycylcylinesKetolides
Daptomycin (Cubicin)Binds to the cell membrane of gram-positive bacteria and causes membrane depolarizationEffective against Vancomycin Resistant Staph. aureus (VRSA) and Enterococci(VRE), including E. faecium and E. faecalisAdministered intravenouslyApproved for treatment of complicated skin and skin structure infections
New Antibiotics for Use Against Antibiotic Resistant Bacteria Semisynthetic streptograminsOxazolidinonesLipopeptidesGlycylcylines
9-Aminotetracyclines acylated with N-dimethylglycineTigecycline was approved by the FDA in 2005
Ketolides
Glycylcyclines
OH O OH O
NH2
O
OH
OH
N(CH3)2
N
HN
OH3C
H3C
7 6 58
9
Glycylcyclines are not substrates for the efflux process and they block insensitive ribosomes
Tigecylcine (Tygacil)Active against methicillin-resistant S. aureus and probably VRE (in vitro)Broad spectrumApproved for complicated intra-abdominal and skin and skin structure infectionsNot a substrate for tetracycline antiportersor ribosome protection proteinsIntravenous administrationBacteriostatic
New Antibiotics for Use Against Antibiotic Resistant Bacteria Semisynthetic streptograminsOxazolidinonesLipopeptidesGlycylcylinesKetolides
Telithromycin (Ketek) was approved by the FDA in 2004Effective against multi-drug resistant Streptococcus pneumoniae
Telithromycin (Ketek)Structurally related to the macrolides, which include ErythromycinBlocks protein synthesis by binding to 23S rRNA of the 50S ribosomal subunitEffective against
gram-positive S. aureus (MRSA, not VRA) and S. pneumoniae (increasingly resistant to penicillin and macrolides)gram negative Haemophilus influenzaeMycoplasma pneumoniae and Chlamydia
Telithromycin (Ketek)Approved for treatment of bronchitis, sinusitis and pneumoniaAlternative to a fluoroquinolone for macrolide-resistant pneumococciCost is $114 for 10 day course
Comparable cost to fluoroquinolones and newer macrolides such as ClarithromycinErythromycin costs about $6
Use with caution because of reports of serious hepatotoxicity
Current Status of ResistanceIntroduction of new antibiotics had been keeping up with resistanceDeclining investment in antibiotic discovery during the 1980s altered the balanceAccelerated investment in the 1990s is beginning to yield new drugsAvoidance of resistance to new drugs has been a consistent but never achieved design objective
The Problems in Avoiding Resistance
Mobile genetic elementsMultiple resistance and association with virulence markersIncreasing use of drugs is associated with increasing frequency of resistanceWorst 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 infections31% of Staph infections outside the hospital71% 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 diarrhea50% are resistant to Ciprofloxacin (Cipro)
Retarding Emergence of ResistanceMaintenance of therapeutic levels
Ensure patient complianceAvoid the use of drugs when the MIC is at or only slightly below the attainable levelPrevent 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 respondTendency to use hot new drugsSelf medication
Antibiotic Resistance of Bacteria from Sewers Serving Isolated Locations
Sewer Serving
Percent of Bacteria Resistant to
Streptomycin Chloramphenicol Tetracycline
General Hospital 34.7 0.7 32.0
Mental Hospital 6.5 0.3 0.4
Residential Area 0.7 0.007 0.1
Gentamicin Resistant P. aeruginosa in Burn Patients
1965 - 90 % susceptible1968 - 636 kg (0.7 tons) of topical gentamicin used1969 - 9 % susceptiblelate 1969 - gentamicin discontinued1970 - 95 % susceptible
Antibiotic Treatment of Adults with Sore Throat
1989-1999 (JAMA 2001, vol. 286:1181)6.7 million annual visits in the USAntibiotics were prescribed in 73% of cases
Decreasing use of penicillin and erythromycinIncreasing 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 infectionsGroup A β-hemolytic Streptococci is the only common cause warranting antibiotics
Streptococci cultured in 5-17% of casesPenicillin and erythromycin are still recommended in most casesOther drugs increase likelihood of resistance to those drugs and greatly increase the cost (> 20-fold for quinolones versus penicillin)
Societal ContributorsAntibiotic additives in stock feedChlorine treatment of water
Reduces number of bacteria by > 100Survivors are resistant to antibiotics
Mercury and other contaminants in waterBacteria resistant to mercury are also resistant to antibiotics
Antibacterial soapsAny inhibitor selects for resistance to other inhibitors, including antibacterial drugsCriticized by the AMA and CDC, which agree that regular soap and water is equally effective
Current Status of Antibiotic Discovery
EmpiricismAt first highly successfulNow marginal
Rational approachMolecular modeling is being used extensivelyLow yield so far, but promising
Novel agents from non-microbial biological systems
New Antibiotics in Development
Synthetic VancomycinsA promising but unproven prospect
For resistance to Fluoroquinolones
Synthetic Vancomycins
The sugar groups on the peptide backbone were modified (Science 1999, vol. 284:508)Completely synthetic drugThe modified drug was more efficient at killing both vancomycin-sensitive and vancomycin-resistant organismsMechanism of action is different, blocking transglycosylation rather than transpeptidationAdditional modifications are being tried
2-Pyridones
NN
O
COOH
HN
FN
N
O
COOHF
CH3
NH2
Inhibits DNA gyrase A, like quinolonesMay be more effective against gyrA mutants
2-Pyridone Ciprofloxacin
Approaches to Identify New Antibacterial Drugs
Peptides from higher organismsMagainin from frogs, reached phase III trials but never proceeded further
Steroids from higher organismsSqualamine from sharks
Inhibitors of additional pathwaysBlock lipid A synthesis, which is an essential component of the outer membrane of gram negative bacteria
Functional GenomicsThe genomes of more than 20 microbial organisms have been sequencedSequence data are used to identify essential targets by comparative genomicsThe targets are experimentally testedDrugs are developed to block those targets, based on structural predictions