Antibacterials Lecture

7/31/2019 Antibacterials Lecture

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Mechanisms ofAntimicrobial Actionand Resistance

Alan L. Goldin, M.D./Ph.D.



Sections in Medical Microbiology& Immunology

Chapter 10Mechanisms of action

Pages 69-84Chapter 11

ResistancePages 85-93

Useful reference, but recommendationschange 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 Therapy

Every other year (small handbook)

http://www.medletter.com/

http://medlet-best.securesites.com/restrictedtg/t57.pdf

http://medlet-best.securesites.com/restrictedtg/t57.pdf

http://www.medletter.com/



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



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 therapeuticuse of antibacterial drugsBiochemical mechanisms of resistanceGeneticsSocietal and physician behaviorsApproaches to retard the development ofresistance



DefinitionsAntimicrobial

Inhibits growth of micro-organismsAntibacterial

Inhibits growth of bacteria

AntibioticInhibits 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 replicateBactericidal

Irreversible inhibition of growthWhen the antibiotic is removed, almost noneof the bacteria (10 -7 to 10 -3) can replicate



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



μg Antibiotic per ml128 64 32 16 8 4 2 1 0.5

M I C

Tube Dilution Method forDetermination of MIC



0 Time

Disk Sensitivity Test



24 Hours

Disk Sensitivity Test

Zone of Inhibition(mm in diameter)



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



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




Tube Dilution Method forDetermination of MBC




M I C





M I C





M I C


M B C



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



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



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



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



Antimetabolites

Sulfonamides



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



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



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



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



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 usingdihydropteroic acid synthase

They cannot use an external source

Sulfonamides are still effective even whenfolic acid is present



Sulfonamide block

Tetrahydrofolic acid deficit

Tetrahydrofolic acid cofactor deficits

DNA

Thymidine Purines Methionine

DNA

RNA

Protein

Consequences of Inhibition bySulfonamides



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



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



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



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



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)



Trimethoprim

Competitive inhibitor of dihydrofolic acidreductaseThe competitive substrate is dihydrofolicacidTrimethoprim blocks a step in thebiosynthesis of tetrahydrofolic acid



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



Dihydrofolic acid

Tetrahydrofolic acid (THF)

NADPH

NADP

Trimethoprim

Inhibits



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



Dihydrofolic acid

Tetrahydrofolic acid (H 4F)

NADPH

NADP

Trimethoprim

Inhibits



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



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



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



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



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



Resistance to TMP/Sulfa

Resistance to TMP makes thecombination ineffectiveResistance to Sulfonamide maintainsconsiderable potency



Drugs to Remember

TMP/Sulfonamide CombinationTrade name Bactrim



Drugs that AffectNucleic AcidSynthesis

Quinolones



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



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



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



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



Drugs to Remember

Ciprofloxacin (Cipro)Levofloxacin



Drugs thatInhibit Cell WallSynthesis

PenicillinsCephalosporins

Vancomycin



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



6-Aminopenicillanic Acid

NC

S CH 3

CH 3

COOHO

HH

H 2N

H

-lactam ring Thiazolidine ring

Peptidoglycan Cross Linking



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 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



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



-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



Inactivation of Transpeptidasesby -lactams

C

HNC

C

SC

CO

CH 3

CH 3

COOHH

HH

H 2N

O

Transpeptidase

Serine OH of Transpeptidases



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)



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



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



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



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



-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



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



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



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



-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



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



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



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



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



Active Site Directed Inhibitors of-Lactamases

N

SOO

CH 3

CH 3

COOHON

O

O COOH

CH CH 2O H

Clavulanic Acid Sulbactam



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



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



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



Susceptible

PBP 1

23

4

2A

Pencillin Binding Proteins (PBP) of Methicillin

Susceptible & Resistant S. aureus

Resistant



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



Drugs to RememberPenicillinAmpicillinNafcillinAmoxicillin/Clavulanate Combination

Augmentin



Other Lactam Antibiotics

CephalosporinsCarbapenems

Monobactams



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



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



Cross Hypersensitivity ofCephalosporins with Penicillins

About 2% of population are hypersensitiveto cephalosporinsAbout 8% of people who arehypersensitive to penicillins are alsohypersensitive to cephalosporins



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



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



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



N

NH CH

HH

R

O SO3

_

3

Monobactams



Drugs to RememberCephalosporins

CefazolinCefotaximeCeftazidime

Carbapenems

Imipenem



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)



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



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



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)



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



Drugs to RememberVancomycin



Drugs that Act onRibosomes

AminoglycosidesChloramphenicolMacrolides

ClindamycinTetracycline



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 )



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



Selective ToxicityInhibits 30 S ribosomal subunit

Difference between inhibition ofeukaryotic and bacterial ribosomes is notvery largeInhibits mitochondrial ribosomes

Mammalian cell and mitochondrialmembranes are barriers



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



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



Chloramphenicol

CH

CH

NH

C C H C l 2O 2 N

O H C H 2 O H OI II III



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



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



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



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



ClindamycinSimilar spectrum as erythromycin

Binds to the 50 S subunitFrequent association with bowel

superinfectionPseudomembranous colitis

Clostridium difficile infectionsUsed to treat anaerobic infections

Tetracylcines



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



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



Drugs to RememberGentamicinErythromycinClindamycinTetracycline



Drugs thatDisrupt Cell Walls

NystatinPolymyxin

L ( ) D b



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



PolymyxinsToo toxic for systemic useEffective against gram negative but notgram positive bacteriaBactericidal, disrupting the outermembrane

Used in topical creams and ointments



Newer Antibiotics forUse Against Antibiotic

Resistant BacteriaSemisynthetic Streptogramins

OxazolidinonesLipopeptides

GlycylcylinesKetolides

Newer Antibiotics for Use Against



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



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



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



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



Oxazolidinones

ON

O

R 1

R 2

Linezolid



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






Daptomycin (Cubicin) was approved by the FDAin 2003Effective against Vancomycin Resistant

Enterococci (VRE)GlycylcylinesKetolides

Daptomycin (Cubicin)







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






Glycylcylines9-Aminotetracyclines acylated withN-dimethylglycineTigecycline was approved by the FDA in 2005

Ketolides



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



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)



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




Antibiotic Resistant BacteriaSemisynthetic streptogramins


GlycylcylinesKetolidesTelithromycin (Ketek) was approved by the FDAin 2004Effective against multi-drug resistantStreptococcus pneumoniae

Telithromycin (Ketek)







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)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



Drugs to RememberLinezolidDaptomycinTigecycline



AntibioticResistance

Current Status of Resistance



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



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 )



Retarding Emergence of Resistance



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



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



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



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



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



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



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



DevelopmentSynthetic Vancomycins

For resistance to Fluoroquinolones

New Antibiotics in Development




Synthetic Vancomycins

A promising but unproven prospectFor resistance to Fluoroquinolones



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





Synthetic Vancomycins

For resistance to Fluoroquinolones



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 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



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



The Future of Antibiotics

The best long-term solution is tominimize the development of resistanceDoctors have a critical role inaccomplishing this goal

Documents

Antibacterials Lecture