Mechanisms of Antimicrobial Action and ResistanceAlan L. Goldin, M.D./Ph.D.

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

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

MIC

Tube Dilution Method for Determination of MIC

0 Time

Disk Sensitivity Test

24 Hours

Disk Sensitivity Test

Zone of Inhibition(mm in diameter)

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

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

Tube Dilution Method for Determination of MBC

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

MIC

Tube Dilution Method for Determination of MBC

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

MIC

Tube Dilution Method for Determination of MBC

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

MIC

Tube Dilution Method for Determination of MBC

MB

C

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

Antimetabolites

Sulfonamides

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

Drugs to Remember

TMP/Sulfonamide CombinationTrade name Bactrim

Drugs that Affect Nucleic Acid SynthesisQuinolones

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

Drugs to Remember

Ciprofloxacin (Cipro)Levofloxacin

Drugs that Inhibit Cell Wall Synthesis

PenicillinsCephalosporinsVancomycin

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

6-Aminopenicillanic Acid

NC

S CH3

CH3

COOHO

H H

H2N

H

β-lactam ring Thiazolidine ring

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

Drugs to Remember

PenicillinAmpicillinNafcillinAmoxicillin/Clavulanate Combination

Augmentin

Other β

Lactam Antibiotics

CephalosporinsCarbapenemsMonobactams

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

N

NH CH

HH

R

O SO3_

3

Monobactams

Drugs to Remember

CephalosporinsCefazolinCefotaximeCeftazidime

CarbapenemsImipenem

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

Drugs to Remember

Vancomycin

Drugs that Act on Ribosomes

AminoglycosidesChloramphenicolMacrolidesClindamycinTetracycline

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

Chloramphenicol

CH

CH

NH

C CHCl2O 2N

O H CH2O H O

I 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 administrationsLife-threateningNever a drug of first choice

Resistance as for aminoglycosides

Erythromycin

O

CH3

O

H3C

HO

OH

H3C

CH2

O

CH3

CH3

CH3

HO

O

O

H3C

O

OH

CH3

H3CO CH3

O CH3

HO

NCH3H3C

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

Clindamycin

O

CH

CH

CH3

Cl

NH

N

CH2

CH2

H3C O

O H

SCH3

H OO H

CH3

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

Drugs to Remember

GentamicinErythromycinClindamycinTetracycline

Drugs that Disrupt Cell Walls

NystatinPolymyxin

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

Oxazolidinones

ON

O

R1

R2

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)

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

Tigecycline

OH O OH O

NH 2

O

OH

OH

N(CH 3)2

NH

HN

7 6 58

9

O

H 3CH 3C

H 3C

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)

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

Drugs to Remember

LinezolidDaptomycinTigecycline

Antibiotic Resistance

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 or Improved Antibiotics in DevelopmentSynthetic VancomycinsFor resistance to Fluoroquinolones

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

New Antibiotics in Development

Synthetic VancomycinsFor 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 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

The Future of Antibiotics

The best long-term solution is to minimize the development of resistanceDoctors have a critical role in accomplishing this goal