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7/31/2019 Antibacterials Lecture
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Mechanisms ofAntimicrobial Actionand Resistance
Alan L. Goldin, M.D./Ph.D.
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Sections in Medical Microbiology& Immunology
Chapter 10Mechanisms of action
Pages 69-84Chapter 11
ResistancePages 85-93
Useful reference, but recommendationschange about drugs of choice
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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 Therapy
Every other year (small handbook)
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Mechanisms of Action
Antibacterial drugs can be classified inmany ways – mechanism of action will beused in these lecturesBiochemical mechanism of action iscrucial to understanding the mechanism of
selective toxicity
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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)
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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
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ResistanceThe most important problem in therapeuticuse of antibacterial drugsBiochemical mechanisms of resistanceGeneticsSocietal and physician behaviorsApproaches to retard the development ofresistance
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DefinitionsAntimicrobial
Inhibits growth of micro-organismsAntibacterial
Inhibits growth of bacteria
AntibioticInhibits growth of micro-organismsMade by other micro-organismsUsually extended to include synthetic
drugs
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Bacteriostatic versus Bactericidal
BacteriostaticReversible inhibition of growthWhen the antibiotic is removed, almost all of
the bacteria can replicateBactericidal
Irreversible inhibition of growthWhen the antibiotic is removed, almost noneof the bacteria (10 -7 to 10 -3) can replicate
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Minimal Inhibitory ConcentrationMICLowest concentration of antibiotic thatprevents visible growth
Broth or tube dilution methodSerial 2-fold dilutions of the antibioticAccurate but time-consuming
Disk sensitivity testRapid, but must be related to results from the
tube dilution method
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μg Antibiotic per ml128 64 32 16 8 4 2 1 0.5
M I C
Tube Dilution Method forDetermination of MIC
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0 Time
Disk Sensitivity Test
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24 Hours
Disk Sensitivity Test
Zone of Inhibition(mm in diameter)
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1286432
16842
10.5
Distance from Disk (mm)
4 8 12 16 20 24 28 32
Concentration(μ g per ml) Tetracycline
Correlation of Distance from Diskand Antibiotic Concentration
Amikacin
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Minimal Bactericidal Concentration
MBC
Lowest concentration of antibiotic that reducesthe number of viable cells by at least 1000-foldPerformed in conjunction with MIC by the tube
dilution methodAliquots from the tubes at and above the MIC areplated onto agar media
The antibiotic is diluted, so that the remainingviable cells grow and form coloniesThe MBC of a truly bactericidal agent is equal to
or just slightly above its MIC
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μg Antibiotic per ml128 64 32 16 8 4 2 1 0.5
Tube Dilution Method forDetermination of MBC
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μg Antibiotic per ml128 64 32 16 8 4 2 1 0.5
M I C
Tube Dilution Method forDetermination of MBC
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μg Antibiotic per ml128 64 32 16 8 4 2 1 0.5
M I C
Tube Dilution Method forDetermination of MBC
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μg Antibiotic per ml128 64 32 16 8 4 2 1 0.5
M I C
Tube Dilution Method forDetermination of MBC
M B C
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Attainable Level of Antibiotic
Concentration that can be reached in thetarget tissue without toxic side effectsIf the attainable level of an antibiotic isgreater than the MIC for at least 90% ofthe isolates, that species is considered
susceptible to that antibioticFor serious infections, those odds may
provide inadequate guidance for treatment
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Trough Levels of AntibioticsLevels of antibiotics reach minimal levels(troughs) at roughly predictable times afteradministration
The troughs may be at or below the MICThis may or may not be a problembecause of two mitigating factors
Post Antibiotic Effect, a prolonged periodbefore bacteria resume growthSynergism between host defenses and sub-MIC levels of antibiotics
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Trough Levels of Antibiotics
Trough levels may increase the frequencyof drug-resistant bacteriaFrequency of developing resistance is greatly
increased at levels just above the MICDevelopment of resistance to ciprofloxacin is10,000 times more frequent at 2 times the
MIC compared to 8 times the MIC
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Choice of Drugs Starts withSusceptibility
Susceptibility by itself does not assuretherapeutic successLack of susceptibility guaranteestherapeutic failureThere are many other considerations in
the choice of antibacterial drugsToxicity and side-effectsInteractions with other drugs
Pharmacology of the drug
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Antimetabolites
Sulfonamides
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Prontosil
H2N
NH 2
N N S NH 2
O
O-
A red dye that cured streptococcal andstaphylococcal infections in mice (1933)Ineffective against bacteria in laboratory mediaConfirmed the dogma that clinically effectivetreatment could not be achieved with drugsacting directly on bacteriaThe first Sulfonamide
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Sulfanilamide
H2N S NH 2
O
O-
The active component of Prontosil
A product of cleavage at the diazo bond, whichoccurs naturally in the bodyEffective against bacteria in both patients and
laboratory media
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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 ofsulfonamides
This competition is not a clinical problem, because wedon’t get PABA in out diets, and it is rapidly excreted
Sulfonamides PABA
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Sulfonamides and PABA Are Analogs
H2N S NHR
O
O-
H2N C
O
O-
Sulfonamides competitively inhibit the condensationof PABA with dihydropteridine to form
dihydropteroic acidThis is the first step in the biosynthesis oftetrahydrofolic acidMetabolic competition is roughly equivalent
Sulfonamides PABA
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Dihydropteridine + para-Aminobenzoic acid(PABA)
SULFONAMIDESINHIBIT
Dihydropteroic acid+ Glutamic acid
Dihydrofolic acid (DHF)
Tetrahydrofolic acid (THF)
NADPH
NADP
Site of Action of Sulfonamides
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Selective Toxicity of Sulfonamides
We lack dihydropteroic acid synthaseWe require folic acid in our diet
Bacteria must synthesize folic acid usingdihydropteroic acid synthase
They cannot use an external source
Sulfonamides are still effective even whenfolic acid is present
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Sulfonamide block
Tetrahydrofolic acid deficit
Tetrahydrofolic acid cofactor deficits
DNA
Thymidine Purines Methionine
DNA
RNA
Protein
Consequences of Inhibition bySulfonamides
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Effect of Sulfonamides Dependson the Environment
Bactericidal in blood and urineBlood and urine have large amounts of methionineand purines, so protein and RNA synthesis continueSelectively blocking DNA synthesis is lethal
Bacteriostatic if protein and RNA synthesis arealso blocked
Adding a bacteriostatic antibiotic decreases efficacyIneffective in purulent lesions
Rich in methionine, purines & thymidine from cellsthat have lysed, so synthesis of proteins, RNA andDNA can continue
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Sulfonamides Introduced theProblem of Drug Resistance
Development of sulfonamide resistance wasrapid
Sulfonamides were introduced to treat bacillarydysentery during World War II4 years later, most isolates were resistantAbout 10% were resistant to 3 biochemicallyunrelated antibiotics
This pattern has been repeated with each new drugResistance to multiple drugs is more commonthan to a single drug
R factors, transposons, and integrons
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Dynamics of Drug ResistancePeople who receive an antibiotic are more likely toharbor bacteria resistant to that antibiotic andbiochemically unrelated antibiotics
People who frequent environments in which antibioticsare used are more likely to harbor drug-resistantbacteria, even if they have not received antibiotics. Thisapplies to patients as well as to staff.The probability of harboring drug-resistant bacteriareturns to normal within a few weeks after antibiotictherapy is discontinued or after absence from theantibiotic-rich environmentsThe prevalence of drug-resistant bacteria in thecommunity is increasing due to increasing use ofantibiotics in the environmentAntibiotics, use them and lose them
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Resistance to SulfonamidesReduced uptake (Antiporter)
Transposons & plasmidsAltered dihydropteroic acid synthase
Reduced sensitivity to sulfonamidesTransposons & plasmidsIncreased levels of synthase or synthase activity
Mutation or plasmid
Increased synthesis of PABA (rare)MutationLoss of end-product inhibitionPromoter up mutation
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Impact of Sulfonamide DiscoveryShattered vitalist dogma on treatment of infection
Proved in vitro effects are relevantInitiated successful searches for antibiotics
Penicillin and streptomycin
Launched huge search for metabolic analogsProduced thousands of rat poisonsA few anticancer agentsAn immunsuppressantOne antibacterial drug (Trimethoprim)
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Trimethoprim
Competitive inhibitor of dihydrofolic acidreductaseThe competitive substrate is dihydrofolicacidTrimethoprim blocks a step in thebiosynthesis of tetrahydrofolic acid
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Dihydrofolic acid
Tetrahydrofolic acid
dTMP
dUMP
(THF)
NADPH
NADP
TrimethoprimInhibits
THF
THF
methionine & purines
Dihydropteridine + PABA
Dihydropteroic acid
SulfonamidesInhibit
+ glutamic acid
5-methyl5,10-methylene
Site of Action of Trimethoprim
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Dihydrofolic acid
Tetrahydrofolic acid (THF)
NADPH
NADP
Trimethoprim
Inhibits
methionine & purines
Dihydropteridine + PABA
Dihydropteroic acid
SulfonamidesInhibit
+ glutamic acid
5,10-methylene THF
5-methyl THF
dTMP
dUMP
Site of Action of TrimethoprimTrimethoprim acts rapidly,sulonamides act slowlyWith trimethoprin, dUMP ⇒
dTMP rapidly depletes THFby conversion to DHF, andthere is no DHF ⇒ THF
With sulfonamides, there isno net synthesis of THF, butDHF ⇒ THF proceeds
Depletion of THF pool takes 3-4generations
Synthesis of pyrimidines &purines does not deplete THF
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Dihydrofolic acid
Tetrahydrofolic acid (H 4F)
NADPH
NADP
Trimethoprim
Inhibits
methionine & purines
Dihydropteridine + PABA
Dihydropteroic acid
SulfonamidesInhibit
+ glutamic acid
5,10-methylene H 4F
5-methyl H 4F
dTMP
dUMP
Site of Action of Trimethoprim
Trimethoprim is likesulfonamides
Bactericidal in bloodIneffective in purulent lesions
But trimethoprim is not
antagonized by PABATrimethoprim andsulfonamides are synergistic
Inhibitors of sequential stepsare often synergisticSulfonamides reduce DHFwhich competes with
trimethoprim
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Trimethoprim and Sulfonamidesare Synergistic
Sulfamethoxazole inhibits an early step inthe pathway and lowers the concentrationof dihydrofolic acid
Dihydrofolic acid and trimethoprimcompete for binding to dihydrofolic aciddehydrogenaseLess trimethoprim is required for inhibitionof dihydrofolic acid reductase in the
presence of sulfamethoxazole
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Trimethoprim and Sulfonamidesare Synergistic
The synergism permits use of smallerdoses than if either drug were used aloneThe use of two drugs together reduces the
frequency of resistanceThe two drugs are marketed as acombination in the fixed ratio of 5 partssulfamethoxazole to 1 part trimethoprimThere are only a few indications for the
use of either drug alone
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Selectivity of TrimethoprimBoth bacteria and humans have
dihydrofolate reductaseThe human enzyme is 60,000-fold lesssensitive to trimethoprimThere is no toxicity due to the antibacterialaction of trimethoprim
Folic acid deficiency can occur in patientswith inadequate dietary consumptionNormal bacterial flora can no longer makefolic acid to compensate
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Resistance to TrimethoprimDihydrofolate reductases with decreasedsensitivity to trimethoprim
Reduced affinity for trimethoprimLocated in the intervening sequences oftransposonsOn a plasmid, but may transpose to thechromosome
It is not a mutant form of the bacterialenzyme, but a new geneMutation of bacterial dihydrofolate reductase isonly important in the lab
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Resistance to TMP/Sulfa
Resistance to TMP makes thecombination ineffectiveResistance to Sulfonamide maintainsconsiderable potency
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Drugs to Remember
TMP/Sulfonamide CombinationTrade name Bactrim
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Drugs that AffectNucleic AcidSynthesis
Quinolones
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QuinolonesNalidixic was the first quinolone
Too toxic for systemic use (newer quinolonescan be used systemically)Rapidly excreted in the urine
Effectively used to treat urinary tract infectionsInhibits the A subunit of DNA gyrase
Human analog (topoisomerase II) is severalhundred fold less sensitiveRapidly inhibits DNA synthesis
Bactericidal unless growth is prevented
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Quinolones
N NH3C
O
COOH
C2H5
NN
O
COOH
R1R2N
F
Nalidixic Acid 6-FluoroQuinolones
= ,R1 R2 = H:
R1 = —C 2H5 , R 2 = H:
R1 = —C 2H5 , R 2 = CH 3:
Ciprofloxacin
Norfloxacin
Ofloxacin
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Resistance to QuinolonesMissense mutations in gyrAMissense mutations in a gene for a membraneprotein, which reduces the uptake offluoroquinolonesDevelopment of resistance to ciprofloxacinamong 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 methicillinresistant S. aureus
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Resistance to QuinolonesMost frequent among important nosocomialpathogens such as S. aureus and P. aeruginosa
These species were not highly susceptible to the firstfluoroquinolonesResistance developed rapidly because the drugs
were used at levels close to the MICCiprofloxacin resistant organisms are crossresistant to other fluoroquinolonesPlasmid encoded resistance is not a problem
A single copy of the sensitive gyrA gene makes thebacteria susceptibleErrors in DNA synthesis and repair are lethal
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Drugs to Remember
Ciprofloxacin (Cipro)Levofloxacin
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Drugs thatInhibit Cell WallSynthesis
PenicillinsCephalosporins
Vancomycin
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Penicillins
Penicillin G was the first penicillin in 1942Advantages compared to sulfonamidesMuch greater potencyMuch less toxicityEffective against organisms that were
resistant to sulfonamidesEffective in wounds and purulent lesions
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6-Aminopenicillanic Acid
NC
S CH 3
CH 3
COOHO
HH
H 2N
H
-lactam ring Thiazolidine ring
Peptidoglycan Cross Linking
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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
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HO C CH NH C CH NH
O CH 3 O CH 3
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
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O = Serine hydroxyl group in active center of transpeptidase
Substrate-Enzyme Intermediatein the Cross Linking Reaction
N CH
C
OCH3
H
Transpeptidase
OAla – Glu –DAP -
NH2
CH
C OH
OCH3
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-lactam Inactivation of Transpeptidases
C
N C
C
SC
CO
CH 3
CH 3
COOHH
HH
H 2N
C
HNC
C
SC
C
O
CH 3
CH 3
COOHH
HH
H 2N
O
Transpeptidase
+ Transpeptidase
Serine OHof Transpeptidases
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Inactivation of Transpeptidasesby -lactams
C
HNC
C
SC
CO
CH 3
CH 3
COOHH
HH
H 2N
O
Transpeptidase
Serine OH of Transpeptidases
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MW
91,00087,000
66,000
60,000
49,000
42,00040,000
1a
2
3
456
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)
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Selectivity & Side Effects of-lactamsSelective toxicity
The 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 relatedto their side chains
A nonspecific side effect is superinfection, such asovergrowth of the large intestine with Clostridium difficile (pseudomembranous colitis)
Hypersensitivity is a common and serious problem
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Haptene Formation: Reaction of
-lactams with Serum Proteins
C
HN
S CH 3
CH 3
COOH
CNH
CR
O
C
NHO
Serum protein
HH
amino groupof a Lys residue
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Resistance to -lactamsResistance of Staphylococci to penicillin Gbecame a major problem within 10 yearsResistance has since appeared in several
additional bacterial speciesMost group A ( β hemolytic) Streptococci
are still highly sensitiveResistance is due to β-lactamase
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Resistance to -lactams
Destruction by -lactamase
C
HNC
C
SC
C
O
CH 3
CH 3
COOHH
HH
H2N
O
β-lactamase
+ H 2O
Penicilloic acid+
Free β-lactamaseSerine OH
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-lactamases of StaphylococciPrimarily penicillinases
Inducible & extracellularInoculum size has large effect on MICMIC for β-lactamase negative is < 0.5 μg/ml for 10 – 10 6 cells
MIC for β-lactamase positive is < 0.5 μg/ml for 10 – 103
cellsMIC for β-lactamase positive Staph is 1250 μg/ml for 10 6 cells
Large initial dose is important (kill before induction)
Destruction of penicillin by a few bacteria can protect asensitive pathogen (secretion of β-lactamase)
One of the major limitations of the early penicillins
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Limitations of Early Penicillins
Hypersensitivity by a significant proportionof the populationNeed to use parenteral routes ofadministration (no oral administration)Development of resistance among
important groups of pathogensNarrow antibacterial spectrum
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Oral Penicillin
Penicillin G is hydrolyzed by acid in thestomachPenicillin V is acid-stableMade by adding phenoxyacetic acid to themedium of the mold producing penicillin
Penicillin G is now so inexpensive that itcan be used orally by giving a larger dose
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Natural PenicillinsNH
CCH2
CH 3
CH 3
COOHO
O
NH
CCH2
O
O COOH
CH 3
CH 3
O
PENCILLIN G (benzylpenicillin)
PENICILLIN V (phenoxymethyl penicillin)
Acid labile
Acid stable
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-Lactamase Refractory Penicillin
Penicillin G is hydrolyzed by β-lactamase
Methicillin is refractory to β-lactamasehydrolysisSteric hindrance of the side chain preventsthe hydrolysisPenicillin G forces the β-lactamase into its
active conformation, so use with methicillinwill decrease the effectiveness of methicillinThese drugs are made semi-synthetically
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Preparation of SemisyntheticPenicilins
N
S CH 3
CH 3
COOHO
H 2N
N
S CH 3
CH 3
COOHO
NC
O
OC 2H 5
N
S CH 3
CH 3
COOHO
NC
OOCH 3
OCH 3
6-AMINOPENICILLANIC ACID
METHICILLIN NAFCILLIN
+ Acid anhydridesor
Acid chlorides
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Broad Spectrum PenicillinPenicillin G cannot pass through the outermembrane of gram negative bacteriaAmpicillin has a charged amino group thatallows it to pass through the outermembrane
Ampicillin is also acid-stableThese drugs are semi-synthetic
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Penicillin G and Ampicillin
N
S CH3
CH3O
NHCCH2
O
N
S CH 3
CH 3O
NH
CHC
O
NH2
COOH
COOHPENICILLIN G(Benzyl penicillin)
AMPICILLIN
Narrow Spectrum
Broad Spectrum
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Broad Spectrum -LactamaseRefractory Penicillin?
There are noneThe large side chains that make methicillinrefractory to β-lactamase prevent it fromcrossing the outer membraneA partial solution is to combine a broad
spectrum penicillin with a β-lactamaseinhibitor
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Active Site Directed Inhibitors of-Lactamases
N
SOO
CH 3
CH 3
COOHON
O
O COOH
CH CH 2O H
Clavulanic Acid Sulbactam
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Inhibition of -Lactamases byClavulanic Acid
HN
O
O
CHCH 2OH
COOH
β-lactamase
I
II
+ β -lactamase
N
O
O
CHCH 2OH
COOH
HN
O
O
CH2CH2OH
COOH
β-lactamase
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Effect of Clavulanic Acid onAmpicillin Resistance
Antibiotic MIC (μg per ml)
E. coli β-lactamase - E. coli β-lactamase +Ampicillin alone 2 > 2,000
Ampicillin +Clavulanic Acid 2 4
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Intrinsic Resistance to -LactamsMethicillin resistant Staph. aureus (MRSA)
Still cannot hydrolyze methicillinResistant by an intrinsic mechanism
Resistance developed rapidly (in 10 yearsof methicillin use)Resistance is carried on a transposon,frequently with other resistance genesResistance is easily transmitted to other
bacteria
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Susceptible
PBP 1
23
4
2A
Pencillin Binding Proteins (PBP) of Methicillin
Susceptible & Resistant S. aureus
Resistant
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Genetics of Methicillin ResistancemecA encodes PBP 2AmecA is a fusion gene
mecA is on a transposonTransmitted by a plasmid, but stability requirestransposition to the chromosome
Production of PBP 2A by mecA is essential but notsufficient for methicillin resistanceHost ( S. aureus ) functions are also required
Depending on host functions, resistance is oftenheterogeneous, leading to incorrect sensitivity reportsThe mecA transposon is an attractant for otherresistance genes
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Drugs to RememberPenicillinAmpicillinNafcillinAmoxicillin/Clavulanate Combination
Augmentin
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Other Lactam Antibiotics
CephalosporinsCarbapenems
Monobactams
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CephalosporinsAbout 20 currently in useTend to be substrates for β-lactamases less frequentlythan penicillins1st generation (Cefazolin)
Antibacterial spectra & potency like penicillins
2nd generation (Cefoxitin)More potent & better against gram negatives3rd generation (Cefotaxime)
Even more potent & highly effective against gram negatives butat the expense of reduced potency for gram positives
4 th generation (Ceftazidime)Enhanced activity against gram negatives without loss of
potency for gram positives
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Core Structures of Penicillins &Cephalosporins
N
C
SCH 3
CH 3COOH
O
H H
H 2N
HNO
H H
H 2 N
S
R
COOH
R =
6-Aminopenicillanic Acid 7-Aminocephalosporanic Acid
CH 2 O HC
O
CH3
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Cross Hypersensitivity ofCephalosporins with Penicillins
About 2% of population are hypersensitiveto cephalosporinsAbout 8% of people who arehypersensitive to penicillins are alsohypersensitive to cephalosporins
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Penicillins + Serum protein
Frequent
Cephalosporins + Serum proteinRare if at all
Penicilloyl protein Cephasporyl protein
Penicillins versus CephalosporinsHaptene Formation
H HC
HN
S
COOH
R 1
CNH
CRO
C
HN
S CH 3
CH 3
COOH
CNH
CR
O
C
NHO
Serum protein
HH
O
C
NH
Serum protein
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Resistance to Cephalosporinsβ-lactamases
Penicillins onlyCephalosporins only
Penicillins & CephalosporinsSpecificities of β-lactamases are not predictableSome bacteria may have more than oneβ-lactamaseAssumptions about sensitivity can lead to
unpleasant surprises
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Carbapenems versus Penicillin
NC
SC H 3
C O O HO
H H
N
HNC H
S
COOHO
H H
C H 3
R 2 R 1R 1
H atoms are trans H atoms are cisC replaces S in fused ringR 1 attached directly R 1 attached via
amino group
Carbapenems Penicillins
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N
NH CH
HH
R
O SO3
_
3
Monobactams
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Drugs to RememberCephalosporins
CefazolinCefotaximeCeftazidime
Carbapenems
Imipenem
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VancomycinInhibits peptidoglycan synthesisThe mechanism is different from that usedby penicillin
Binds to the D Ala – D Ala substrateNarrow spectrum of actionComplex glycopeptideCannot cross the outer membrane
Resistant to β-lactamases
Antibiotic of last resort
Vancomycin Target (D Ala – D Ala)
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HO C CH NH C CH NH
O CH 3 O CH 3
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
Va co yc a get ( )
Ala – Glu –DAP -
- DAP – Glu - Ala
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Vancomycin ResistanceA Depsipentapeptide instead of the normal
PentapeptidePentapeptideL Alanyl - D Glutamyl - m DAP - D Alanyl - D Alanine
Van Sens
DepsipentapeptideL Alanyl - D Glutamyl - m DAP - D Alanyl - D LactateVan Res
Van Sens Vancomycin can bind to D Alanyl - D AlanineVan Res Vancomycin cannot bind to D Alanyl - D Lactate
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Vancomycin Resistance ISynthesis of the Depsipentapeptide
Pyruvate + 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)
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Vancomycin Resistance IIDestruction of Existing VancomycinBinding 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
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Drugs to RememberVancomycin
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Drugs that Act onRibosomes
AminoglycosidesChloramphenicolMacrolides
ClindamycinTetracycline
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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 andClindamycin affect the 50 S subunit
G i i (A i l id )
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Gentamicin (Aminoglycoside)
O
N H 2
R 1
C H N H R 2
N 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 C 1 R1 = CH 3 R2 = CH 3Gentamicin C 2 R1 = CH 3 R2 = HGentamicin C 1a R1 = H R 2 = H
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Selective ToxicityInhibits 30 S ribosomal subunit
Difference between inhibition ofeukaryotic and bacterial ribosomes is notvery largeInhibits mitochondrial ribosomes
Mammalian cell and mitochondrialmembranes are barriers
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Mechanisms of ResistanceProteins modify and inactivate the
compoundsResistance is additive
Proteins are encoded on plasmidsResistant ribosomal proteins
This occurs very rarelyResistance is very high
Kanamycin Sites of Inactivation
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Kanamycin Sites of Inactivation
Types of InactivationAC N-Acetyl transferases(AC) O-Acetyl transferases
AD O-Adenyl transferasesP O-PhosphatasesBlocked reaction
AC II
AC IIIAC IAC
O
O
OO
HO
CH 2-NH 2 NH 2
NH 2
CH 2OH
HONH 2
OH
OH
OH
OH
PI
PII
(AC)
AD
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Chloramphenicol
CH
CH
NH
C C H C l 2O 2 N
O H C H 2 O H OI II III
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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 administrations
Life-threateningNever a drug of first choice
Resistance as for aminoglycosides
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Erythromycin
O
CH 3
O
H3C
HO
OH
H3C
CH 2
O
CH 3
CH 3
CH 3
HO
O
O
H3C
O
OH
CH 3
H3CO CH 3
O CH 3
HO
N
CH 3H3C
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ErythromycinMacrolide antibiotic
Does not inhibit mammalian 80 S subunitDoes inhibit mitochondrial 70 S subunit
Does not cross the mitochondrialmembrane
Resistance by rRNA methylationOften an alternative for penicillin totreat allergic patients
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Clindamycin
O
C H
C H
C H 3
C l
NH
N
CH 2
CH 2
H 3 C O
O H
SC H 3
H O
O H
C H 3
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ClindamycinSimilar spectrum as erythromycin
Binds to the 50 S subunitFrequent association with bowel
superinfectionPseudomembranous colitis
Clostridium difficile infectionsUsed to treat anaerobic infections
Tetracylcines
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OH O OH O
NH 2
O
OH
OH
N(CH3)2
7 6 5
Tetracylcines
Bacteriostatic inhibitors with broad spectrum
Block the binding of aminoacyl-tRNAs to the A siteof the ribosome 30 S subunitResistance due to efflux and insensitive ribosomes
Tetracylcines
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5 6 7Chlortetracycline CH 3; OH Cl
Tetracycline CH 3; OH
Doxycycline OH CH 3
Minocycline N(CH 3)2
PositionDrug
OH O OH O
NH 2
O
OH
OH
N(CH 3)2
7 6 5
Tetracylcines
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Drugs to RememberGentamicinErythromycinClindamycinTetracycline
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Drugs thatDisrupt Cell Walls
NystatinPolymyxin
L ( ) D b
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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 NH groupsL-Dab involved in peptide linkages
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PolymyxinsToo toxic for systemic useEffective against gram negative but notgram positive bacteriaBactericidal, disrupting the outermembrane
Used in topical creams and ointments
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Newer Antibiotics forUse Against Antibiotic
Resistant BacteriaSemisynthetic Streptogramins
OxazolidinonesLipopeptides
GlycylcylinesKetolides
Newer Antibiotics for Use Against
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Newer Antibiotics for Use AgainstAntibiotic Resistant BacteriaSemisynthetic streptogramins
Quinupristin/dalfopristin (Synercid) wasapproved by the FDA in 1999Effective against Vancomycin Resistant Staph.
aureus (VRSA) and Enterococci (VRE)Oxazolidinones
LipopeptidesGlycylcylinesKetolides
St t g i
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Streptogramins
N
NH
HN
O
NN
NHN
O
O
O
N
O
OO
OH
O N
N
O
NO
O
O
O
O H
O
Pristinom ycin Ia Pristinom ycin IIa
Q i i ti /D lf i ti
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Quinupristin/DalfopristinAct synergistically on the bacterialribosome to disrupt protein synthesisActive against S. aureus and E. faecium but not against E. faecalis
Must be administered intravenouslyHigh incidence of adverse effects and druginteractionsWholesale cost for 10 day treatment isabout $3,000 plus hospitalization
No longer used very often
New Antibiotics for Use Against
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New Antibiotics for Use AgainstAntibiotic Resistant BacteriaSemisynthetic streptogramins
OxazolidinonesLinezolid (Zyvox) was approved by the FDA in2000Effective against Vancomycin Resistant Staph.aureus (VRSA) and Enterococci (VRE)
LipopeptidesGlycylcylinesKetolides
O a olidinones
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Oxazolidinones
ON
O
R 1
R 2
Linezolid
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LinezolidInhibits protein synthesis at the bacterialribosomeBacteriostatic against staphylococci andenterococci
Active against S. aureus , E. faecium andE. faecalis Administered intravenously or orallyGenerally well-toleratedWholesale cost for 10 day treatment is
about $1,000
New Antibiotics for Use Against
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New Antibiotics for Use AgainstAntibiotic Resistant BacteriaSemisynthetic streptogramins
OxazolidinonesLipopeptides
Daptomycin (Cubicin) was approved by the FDAin 2003Effective against Vancomycin Resistant
Enterococci (VRE)GlycylcylinesKetolides
Daptomycin (Cubicin)
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Daptomycin (Cubicin)
Daptomycin (Cubicin)
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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.faecalis Administered intravenouslyApproved for treatment of complicatedskin and skin structure infections
New Antibiotics for Use Against
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New Antibiotics for Use AgainstAntibiotic Resistant BacteriaSemisynthetic streptogramins
OxazolidinonesLipopeptides
Glycylcylines9-Aminotetracyclines acylated withN-dimethylglycineTigecycline was approved by the FDA in 2005
Ketolides
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Glycylcyclines
OH O OH O
NH 2
O
OH
OH
N(CH 3)2
N
HN
OH 3C
H3C
7 6 58
9
Glycylcyclines are not substrates for the effluxprocess and they block insensitive ribosomes
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Tigecycline
O H O O H O
N H 2
O
O H
O H
N(CH3
)2
NH
HN
7 6 58
9
O
H 3 C
H 3 C
H 3C
Tigecylcine (Tygacil)
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g y ( yg )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 antiporters
or ribosome protection proteinsIntravenous administrationBacteriostatic
New Antibiotics for Use Against
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Antibiotic Resistant BacteriaSemisynthetic streptogramins
OxazolidinonesLipopeptides
GlycylcylinesKetolidesTelithromycin (Ketek) was approved by the FDAin 2004Effective against multi-drug resistantStreptococcus pneumoniae
Telithromycin (Ketek)
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Telithromycin (Ketek)
Telithromycin (Ketek)
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Telithromycin (Ketek)Structurally related to the macrolides,which include ErythromycinBlocks protein synthesis by binding to 23SrRNA of the 50S ribosomal subunit
Effective againstgram-positive S. aureus (MRSA, not VRA)
and S. pneumoniae (increasingly resistant topenicillin and macrolides)gram negative Haemophilus influenzae
Mycoplasma pneumoniae and Chlamydia
Telithromycin (Ketek)
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Telithromycin (Ketek)Approved for treatment of bronchitis,sinusitis and pneumoniaAlternative to a fluoroquinolone formacrolide-resistant pneumococci
Cost is $114 for 10 day courseComparable cost to fluoroquinolones and
newer macrolides such as ClarithromycinErythromycin costs about $6
Use with caution because of reports ofserious hepatotoxicity
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Drugs to RememberLinezolidDaptomycinTigecycline
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AntibioticResistance
Current Status of Resistance
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Current Status of ResistanceIntroduction of new antibiotics had beenkeeping up with resistanceDeclining investment in antibioticdiscovery during the 1980s altered the
balanceAccelerated investment in the 1990s isbeginning to yield new drugsAvoidance of resistance to new drugs hasbeen a consistent but never achieved
design objective
The Problems in Avoiding
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gResistance
Mobile genetic elements
Multiple resistance and association withvirulence markers
Increasing use of drugs is associated withincreasing frequency of resistance
Worst case scenarios are already here forsome nosocomial infections(Staphylococci and Enterococci )
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Retarding Emergence of Resistance
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Maintenance of therapeutic levelsEnsure patient complianceAvoid the use of drugs when the MIC is at oronly slightly below the attainable levelPrevent biofilms and treat them aggressively
Use combinations of antibiotics whenindicated (but not otherwise)Avoid over and ill-advised use ofantibiotics
Prescriptions for infections that won’t respondTendency to use hot new drugsSelf medication
Antibiotic Resistance of Bacteria from
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Sewers Serving Isolated Locations
SewerServing
Percent of Bacteria Resistant to
Streptomycin Chloramphenicol Tetracycline
GeneralHospital 34.7 0.7 32.0
MentalHospital 6.5 0.3 0.4Residential
Area0.7 0.007 0.1
Gentamicin Resistant
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P. aeruginosa in Burn Patients
1965 - 90 % susceptible1968 - 636 kg (0.7 tons) of topicalgentamicin used1969 - 9 % susceptiblelate 1969 - gentamicin discontinued1970 - 95 % susceptible
Antibiotic Treatment of Adults
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with Sore Throat1989-1999 (JAMA 2001, vol. 286:1181)
6.7 million annual visits in the USAntibiotics were prescribed in 73% of
casesDecreasing use of penicillin anderythromycinIncreasing use of non-recommended,extended-spectrum macrolides and
fluoroquinolones
Antibiotic Treatment of Adults
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with Sore ThroatMost sore throats are due to viral upper
respiratory tract infectionsGroup A β-hemolytic Streptococci is the onlycommon cause warranting antibiotics
Streptococci cultured in 5-17% of casesPenicillin and erythromycin are still
recommended in most casesOther drugs increase likelihood of resistanceto those drugs and greatly increase the cost
(> 20-fold for quinolones versus penicillin)
Societal Contributors
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Antibiotic additives in stock feedChlorine treatment of water
Reduces number of bacteria by > 100Survivors are resistant to antibiotics
Mercury and other contaminants in water
Bacteria resistant to mercury are also resistant toantibioticsAntibacterial soaps
Any inhibitor selects for resistance to other inhibitors,including antibacterial drugsCriticized by the AMA and CDC, which agree thatregular soap and water is equally effective
Current Status of Antibiotic
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DiscoveryEmpiricism
At first highly successfulNow marginal
Rational approachMolecular modeling is being used extensively
Low yield so far, but promisingNovel agents from non-microbial biologicalsystems
New or Improved Antibiotics in
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DevelopmentSynthetic Vancomycins
For resistance to Fluoroquinolones
New Antibiotics in Development
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New Antibiotics in Development
Synthetic Vancomycins
A promising but unproven prospectFor resistance to Fluoroquinolones
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Synthetic VancomycinsThe sugar groups on the peptide backbone weremodified (Science 1999, vol. 284:508)Completely synthetic drug
The modified drug was more efficient at killingboth vancomycin-sensitive and vancomycin-resistant organisms
Mechanism of action is different, blockingtransglycosylation rather than transpeptidationAdditional modifications are being tried
New Antibiotics in Development
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New Antibiotics in Development
Synthetic Vancomycins
For resistance to Fluoroquinolones
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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
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Antibacterial DrugsPeptides from higher organisms
Magainin from frogs, reached phase IIItrials but never proceeded further
Steroids from higher organismsSqualamine from sharks
Inhibitors of additional pathwaysBlock lipid A synthesis, which is anessential component of the outer
membrane of gram negative bacteria
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Functional GenomicsThe genomes of more than 20 microbial
organisms have been sequencedSequence data are used to identify
essential targets by comparativegenomicsThe targets are experimentally testedDrugs are developed to block thosetargets, based on structural predictions