Chapter 12

Drugs, Microbes, Host – The Elements of

Chemotherapy

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2

12.1 Principles of Antimicrobial Therapy

• Administer a drug to an infected person that

destroys the infective agent without harming the

host’s cells

• Antimicrobial drugs are produced naturally or

synthetically

3

The Ideal Antimicrobial Drug

4

The Origins of Antimicrobial Drugs

• Antibiotics are common metabolic products of aerobic

bacteria and fungi

– Bacteria in genera Streptomyces and Bacillus

– Molds in genera Penicillium and Cephalosporium

• By inhibiting the other microbes in the same habitat, antibiotic producers have less competition for nutrients and space

Insight 12.1 Sir Alexander

Fleming

5

Interactions Between Drugs and Microbe

• Antimicrobial drugs should be selectively toxic

– drugs should kill or inhibit microbial cells

without simultaneously damaging host tissues

• As the characteristics of the infectious agent

become more similar to the vertebrate host cell,

complete selective toxicity becomes more

difficult to achieve and more side effects are

seen

6

Mechanisms of Drug Action

1. Cell wall inhibitors

Block synthesis and repair

Penicillins

Cephalosporins

Vancomycin

Bacitracin

Monobactams/carbapenems

Fosfomycin

Cycloserine

Isoniazid

2. Cell membrane

Cause loss of selective permeability

Polymyxins

3. DNA/RNA

DNA mRNA

Inhibit replication and transcription

Inhibit gyrase (unwinding enzyme)

Quinolones (ciprofloxacin)

Inhibit RNA polymerase

Rifampin

4. Protein synthesis inhibitors acting

on ribosomes

Site of action

50S subunit

Chloramphenicol

Erythromycin

Clindamycin

Streptogramin (Synercid)

Site of action

30S subunit

Aminoglycosides

Gentamicin

Streptomycin

Tetracyclines

Both 30S

and 50S

Blocks initiation of protein

Synthesis

Linezolid (Zyvox)

5. Metabolic pathways and products

Substrate

Ribosome

Enzyme

Product

Block pathways and inhibit

Metabolism

Sulfonamides (sulfa drugs)

Trimethoprim

Figure 12.2 Major targets of drugs in bacteria

7

The Spectrum of an Antimicrobic Drug

• Spectrum – range of activity of a drug

– Narrow-spectrum: effective on a small

range of microbes

• Target a specific cell component that is found

only in certain microbes

– Broad-spectrum: greatest range of activity

• Target cell components common to most

pathogens (ribosomes)

8

12.2 Survey of Major Antimicrobial Drug Groups

Antimicrobial Drugs That Affect the Bacterial Cell Wall

• Most bacterial cell walls contain peptidoglycan

• Penicillins and cephalosporins block synthesis of peptidoglycan, causing the cell wall to lyse

• Active on young, growing cells

• Penicillins that do not penetrate the outer membrane and are less effective against gram-negative bacteria

• Broad spectrum penicillins and cephalosporins can cross the cell walls of gram-negative bacteria

9

Figure 12.3 Activity of Penicillin

Peptidoglycan

(cell wall)

Exposure to cephalosporin

or penicillin

(a) Normal untreated cells. Left and center views

are Staphylococcus cells; right view is the molecular

structure of peptidoglycan, with chains of NAM

(N-acetyl muramic acid) and NAG (N-acetyl

glucosamine) linked together by cross bridges

(green).

Membrane bulges

out as water diffuse

into cell.

Membrane breaks.

Cell lyses.

Weak points lacking

peptidoglycan

(b) Effects of treatment with penicillin. Left side depicts

the stages in the breakdown of a Staphylococcus cell

exposed to penicillin. Center view shows actual cells

undergoing lysis and cell death. Right is a molecular view

of how penicillin blocks the formation of the cross bridges,

which removes the support that holds the cell wall together.

10,000

Janice Carr/CDC

10,000

© CNRI/Photo Researchers, Inc.

Antibacterial Drugs that Act on the Cell Wall

• Beta-lactam antimicrobials - all contain a highly reactive 3 carbon, 1 nitrogen ring

• Primary mode of action is to interfere with cell wall synthesis

• Greater than ½ of all antimicrobic drugs are beta-lactams

• Penicillins and cephalosporins most prominent beta-lactams

10

Figure 12.7 Penicillin and Its Relatives

• Large diverse group of compounds

• Could be synthesized in the laboratory

• More economical to obtain natural penicillin through microbial fermentation and modify it to semi-synthetic forms

• All consist of 3 parts:

– Thiazolidine ring

– Beta-lactam ring

– Variable side chain dictating microbial activity (R)

Nafcillin

Beta-

lactam

Thiazolidine

CO

R Group

CH3

O COOH

CH3

N

N

S

CH3

O COOH

CH3

N

N

S

CH3

O COOH

CH3

N

N

S

CH3

O COOH

CH3

N

N

S

Nucleus

(Aminopenicillanic acid)

Ticarcillin

CO CH

COONa

Cloxacillin

CO

CH3

Cl

Carbenicillin

CO CH

COONa

S

O N

12

Subgroups and Uses of Penicillins

• Penicillins G and V most important natural forms

• Penicillin is the drug of choice for gram-positive cocci (streptococci) and some gram-negative bacteria (meningococci and syphilis spirochete)

• Semisynthetic penicillins – ampicillin, carbenicillin, and amoxicillin have broader spectra – Gram-negative infections

• Penicillinase-resistant – methicillin, nafcillin, cloxacillin

• Primary problems – allergies and resistant strains of bacteria

13

Cephalosporins

• Account for one-third of all antibiotics administered

• Synthetically altered beta-lactam structure

• Relatively broad-spectrum, resistant to most penicillinases, and cause fewer allergic reactions

• Some are given orally; many must be administered parenterally

• Generic names have root – cef, ceph, or kef

Basic Nucleus

COOH CH2

6 5

4

3 2

CH2

S or O

OC

O

CH2

OCH3

Cefotiam

(second generation)

Cephalothin

(first generation*)

Moxalactam

(third generation)

Cefepime

(fourth generation)

O

C

O

NH2

NH2

N CH2

C CH

COONa

OH

O

N

N

CH2

N

N S CH2 N

CH3

CH3

S CH2

N

N N

N

O

N

S

N

CH3

CH3 CH2

R Group 1 R Group 2

N1 S

S

*New improved versions of drugs are referred to as new “generations.”

N

2nd

3rd

4th

7

Figure 12.8 The structure of

cephalosporins

14

Non Beta-lactam Cell Wall Inhibitors

• Vancomycin – narrow-spectrum, most effective in treatment of Staphylococcal infections in cases of penicillin and methicillin resistance or if patient is allergic to penicillin; toxic and hard to administer; restricted use

• Bacitracin – narrow-spectrum produced by a strain of Bacillus subtilis; used topically in ointment

• Isoniazid (INH) – works by interfering with mycolic acid synthesis; used to treat infections with Mycobacterium tuberculosis

15

Antimicrobial Drugs That Disrupt Cell Membrane Function

• A cell with a damaged membrane dies from disruption in metabolism or lysis

• These drugs have specificity for a particular microbial group, based on differences in types of lipids in their cell membranes

• Polymyxins interact with phospholipids and cause leakage, particularly in gram-negative bacteria

• Amphotericin B and nystatin form complexes with sterols on fungal membranes which causes leakage

16

Figure 12.4 Activity of Polymyxins

Cell

membrane

Polymyxin B

A detergent that

causes openings in the

cell membrane

Antibiotics That Damage Bacterial Cell Membranes

• Polymixins, narrow-spectrum peptide

antibiotics with a unique fatty acid component

– Treat drug resistant Pseudomonas

aeruginosa and severe UTI

17

18

Drugs That Act on DNA or RNA

• May block synthesis of nucleotides, inhibit replication, or

stop transcription

• Chloroquine binds and cross-links the double helix;

quinolones inhibit DNA helicases

• Antiviral drugs that are analogs of purines and

pyrimidines insert in viral nucleic acid, preventing

replication

Drugs that Act on DNA or RNA

• Fluoroquinolones – work by binding to DNA

gyrase and topoisomerase IV

– Broad spectrum effectiveness

• Concerns have arisen regarding the overuse

of quinoline drugs

– CDC is recommending careful monitoring

of their use to prevent ciprofloxacin-

resistant bacteria

19

20

Drugs That Interfere with Protein Synthesis

• Ribosomes of eukaryotes differ in size and structure

from prokaryotes; antimicrobics usually have a selective

action against prokaryotes; can also damage the

eukaryotic mitochondria

• Aminoglycosides (streptomycin, gentamycin) insert on

sites on the 30S subunit and cause misreading of mRNA

• Tetracyclines block attachment of tRNA on the A

acceptor site and stop further synthesis

21

Figure 12.5 Targets on the 70S Ribosome

mRNA

mRNA

Aminoglycosides

50S

30S

aa aa mRNA is

misread,

protein is

incorrect

Chloramphenicol

mRNA

aa aa Formation

of peptide

bonds is

blocked

Erythromycin

mRNA

aa aa 50S

30S

Ribosome

is prevented

from

translocating

Tetracyclines

50S

30S

tRNA is

blocked,

no protein is

synthesized

Oxazolidinones

Prevent

initiation and

block ribosome

assembly

50S

30S

22

Drugs That Interfere with Protein Synthesis

• Aminoglycosides – composed of one or more amino sugars and an aminocyclitol (6C) ring; binds ribosomal subunit

-

NH2

OH

OH

O OH

NH C

NH

NH NH2 C

NH

CHO

O

CH3

O

OH

CH2OH O

OH

OH

6-carbon ring

2 sugars

CH3NH

Figure 12.9 The

structure of an

aminoglycoside

23

Drugs That Interfere with Protein Synthesis

• Aminoglycosides –

• Products of various species of soil actinomycetes in genera Streptomyces and Micromonospora

• Broad-spectrum, inhibit protein synthesis, especially useful against aerobic gram-negative rods and certain gram-positive bacteria – Streptomycin: bubonic plague, tularemia, TB

– Gentamicin: less toxic, used against gram-negative rods

– Newer – tobramycin and amikacin gram-negative bacteria

24

Tetracycline Antibiotics

• Broad-spectrum, block protein synthesis by binding ribosomes

• Treatment for STDs, Rocky Mountain spotted fever, Lyme disease, typhus, acne, and protozoa

• Generic tetracycline is low in cost but limited by its side effects

OH

R

O OH

R R R N

CH3 CH3

OH

OH

COCH2

O

(a) Tetracyclines

Figure 12.10 Structure

of a broad spectrum

antibiotic

25

Chloramphenicol • Potent broad-spectrum drug with unique nitrobenzene

structure

• Blocks peptide bond formation and protein synthesis

• Entirely synthesized through chemical processes

• Very toxic, restricted uses, can cause irreversible damage to bone marrow

• Typhoid fever, brain abscesses, rickettsial, and chlamydial infections

O

Cl2

C NO2

H

OH

C

NH

H

CH2 OH

C CH

(b) Chloramphenicol

Figure 12.10

Structure of a broad

spectrum antibiotic

26

Macrolides and Related Antibiotics

• Erythromycin – large lactone ring with sugars; attaches to ribosomal 50s subunit

• Broad-spectrum, fairly low toxicity

• Taken orally for Mycoplasma pneumonia, legionellosis, Chlamydia, pertussis, diphtheria and as a prophylactic prior to intestinal surgery

• For penicillin-resistant – gonococci, syphilis, acne

• Newer semi-synthetic macrolides – clarithromycin, azithromycin

OH

O O

CH3

CH3

OH

(c) Erythromycin

CH3

CH2

CH3 CH3 OH

CH3

O

O

OH

CH3

O

N(CH3)2

Sugar

O

Sugar

CH3

OH CH3 CH3O

Lactone ring

O Figure 12.10

Structure of a

broad spectrum

antibiotic

27

Drugs that Block Metabolic Pathways

• Sulfonamides and trimethoprim block enzymes required for tetrahydrofolate synthesis needed for DNA and RNA synthesis

Sulfonamides

inhibit enzyme

Only certain bacteria

perform this step

PABA Dihydropteroic

acid

Dihydrofolic

acid

Trimethoprim

inhibits enzyme

Folic acid is coenzyme

required to synthesize

Tetrahydrofolic

acid (folic acid) Pyrimidines

Purines

Amino acids

(a) The metabolic pathway needed to synthesize

tetrahydrofolic acid (THFA) contains two enzymes

that are chemotherapeutic targets. Under normal

circumstances, PABA acts as a substrate for the

enzyme pteridine synthetase, the product of which

is needed for the eventual production of folic acid. Enzyme Enzyme Enzyme

PABA

(substrate)

Sulfa drug

(inhibitor) Sulfa drug

(inhibitor)

Higher levels of

sulfa drug

more likely to

bind to enzyme

Figure 12.6 Competitive

inhibition as a mode of action

28

Drugs that Block Metabolic Pathways

• Competitive inhibition – drug competes with normal substrate for enzyme’s active site

• Synergistic effect – the effects of a combination of antibiotics are greater than the sum of the effects of the individual antibiotics

N

N

H

H H

H

O S O

N H

O HO

H

C

(b)

Sulfanilamide PABA

Structural differences

Figure 12.6 Competitive

inhibition as a mode of

action

29

• Most are synthetic; most important are sulfonamides, or sulfa drugs - first antimicrobic drugs

• Narrow-spectrum; block the synthesis of folic acid by bacteria – Sulfisoxazole: shigellosis, UTI,

protozoan infections

– Silver sulfadiazine: burns, eye infections

– Trimethoprim: given in combination with sulfamethoxazole: UTI, PCP(Pneumocystis carinii pneumonia)

Nucleus R Group

CH3

O

C

CH3

O

CH3

N

N

N

NH2 SO2 NH

(a)

(b)

(c)

Drugs That Block Metabolic Pathways

30

12.3 Drugs to Treat Fungal, Parasitic and Viral Infections

• Fungal cells are eukaryotic; a drug that is toxic to fungal cells also toxic to human cells

• Five antifungal drug groups: – Macrolide polyene

• Amphotericin B – mimic lipids, most versatile and effective, topical and systemic treatments

• Nystatin – topical treatment

(a)

OH

OH O

OH OH

OH

OH OH

OH

O

O

Figure 12.12 an

antifungal drug

31

Antifungal Drugs • Five antifungal drug groups:

– Griseofulvin: stubborn cases of dermatophyte infections, nephrotoxic

– Synthetic azoles: broad-spectrum; ketoconazole, clotrimazole, miconazole

– Flucytosine: analog of cytosine; cutaneous mycoses or in combination with amphotericin B for systemic mycoses

– Echinocandins: damage cell walls; capsofungin

(b) (c) NH2

O

N F

N

H Cl

C

N

N

Figure 12.12 b.

clotrimazole,

inhibits fungal

cell membrane

Figure 12.12 c.

flucytosine,

inhibits DNA and

protein synthesis

32

Antiparasitic Chemotherapy

• Antimalarial drugs: quinine, chloroquinine, primaquine, mefloquine

• Antiprotozoan drugs: metronidazole (Flagyl), quinicrine, sulfonamides, tetracyclines

• Antihelminthic drugs: immobilize, disintegrate, or inhibit metabolism – Mebendazole, thiabendazole: broad-spectrum,

inhibit function of microtubules, interferes with glucose utilization and disables them

– Pyrantel, piperazine: paralyze muscles

– Niclosamide: destroys scolex

33

Antiviral Chemotherapeutic Agents

• Selective toxicity is almost impossible due to obligate intracellular parasitic nature of viruses

• Block penetration into host cell

• Block replication, transcription, or translation of viral genetic material

– Nucleotide analogs • Acyclovir – herpesviruses

• Ribavirin – a guanine analog – RSV, hemorrhagic fevers

• AZT – thymine analog – HIV

• Prevent maturation of viral particles – Protease inhibitors : HIV

34

Drugs for Treating Influenza

• Amantadine, rimantidine – restricted almost exclusively

to influenza A viral infections; prevent fusion of virus with

cell membrane

• Relenza and tamiflu – slightly broader spectrum; blocks

neuraminidase in influenza A and B

Fuzeon

Prevents binding of viral GP-41 receptors to cell

receptor which blocks fusion of virus with cell

Maravoc

Covers up the cell receptors;

HIV cannot adhere and is inert

Amantidine and relatives

Block entry of influenza virus by interfering with

fusion of virus with cell membrane (also release)

Tamiflu, Relenza

Stop the actions of influenza neuraminidase,

required for entry of virus into cell

HIV

virus

Host cell membrane

Drug No Fusion/no entry

Cell

receptors

Influenza

virus Amantidine, Tamiflu

Thought to interfere with

influenza virus assembly or

budding/release

Nucleus

I. Inhibition of Virus Entry or Release

1

2

3

4

1

2

3

4

4

Table 12.6 Antiviral Drugs

35

Antiherpes Drugs

• Many antiviral agents mimic the structure of nucleotides

and compete for sites on replicating DNA

– Acyclovir (Zovirax), Valacyclovir (Valtrex),

Famiciclovir (Famvir), Peniciclovir (Denavir)

• Oral and topical treatments for oral and genital herpes,

chickenpox, and shingles

DNA

Polymerase

Virus DNA

Herpesvirus

Drug

HIV

virus

HIV RNA

RT

no reverse transcription

No HIV

DNA

No Viral DNA

replication

Drug

Acyclovir, other “cyclovirs

Terminates DNA replication in herpesviruses

Nucleoside analog reverse transcriptase (RT) inhibitors

(zidovudine - retrovir)

Stop the action of reverse transcriptase in HIV,

blocking viral DNA production

Non-nucleoside reverse transcriptase inhibitors

(nevirapine)

Attach to HIV RT binding site, stopping its

action

II. Inhibition of Nucleic Acid Synthesis

5

6

7 6

7

5

Table 12.6 Antiviral Drugs

36

Drugs for Treating HIV Infections and AIDS

• Retrovirus offers 2 targets for chemotherapy: – Interference with viral DNA synthesis from viral RNA

using nucleoside reverse transcriptase inhibitors (nucleotide analogs)

– Interference with synthesis of DNA using nonnucleoside reverse transcriptase inhibitors

Azidothymidine (AZT) – first drug aimed at treating AIDS, thymine analog

37

12.4 Interactions between Microbes and Drugs: The Acquisition of Drug

Resistance

• Adaptive response in which microorganisms begin

to tolerate an amount of drug that would ordinarily

be inhibitory; due to genetic versatility or variation;

intrinsic and acquired

38

How Does Drug Resistance Develop?

• Acquired resistance:

– Spontaneous mutations in critical chromosomal genes

– Acquisition of new genes or sets of genes via transfer from another species

• Originates from resistance factors (plasmids) encoded with drug resistance, transposons

Resistance gene

Virus

Plasmid

Resistance

gene

Plasmid donor

DNA fragment

Gene goes to

plasmid or to

chromosome

Transduction

Transfer

by viral

delivery

Transformation

Transfer of

free DNA

Conjugation

Plasmid

transfer

Bacterium

Receiving

Resistance

genes

Dead

bacterium

Figure 12.13 Transfer of drug resistance

39

Specific Mechanisms of Drug Resistance

N

S R

O COOH Penicillinase N

H

R

O C

OH COOH

S CH3

CH3

D C

Cell surface

of microbe Cell surface

of microbe

Cell surface

of microbe Cell surface

of microbe

Drug

Drug

B A C D Product

Drug acts

Active

Drug

pump

Inactive

Drug

pump

Normal

receptor

Different

receptor

X

1. Drug inactivation Result

Active penicillin Inactive penicillin

2. Decreased permeability

3. Activation of drug pumps

4. Change in drug binding site

5. Use of alternate metabolic pathway

1 Inactivation of a drug like penicillin by

penicillinase, an enzyme that cleaves

a portion of the molecule and renders

it inactive.

1.

2

4

3

5

The receptor that transports the drug

is altered, so that the drug cannot

enter the cell.

2.

Specialized membrane proteins are

activated and continually pump the

drug out of the cell.

3.

Binding site on target (ribosome) is

altered so drug has no effect 4.

The drug has blocked the usual

metabolic pathway (green), so the

microbe circumvents it by using an

alternate, unblocked pathway that

achieves the required outcome (red) .

5.

Figure 12.14

Examples of

mechanisms

of drug

resistance

40

12.5 Interactions Between Drugs and Hosts

• Estimate that 5% of all persons taking antimicrobials will

experience a serious adverse reaction to the drug – side

effects

• Major side effects:

– Direct damage to tissue due to toxicity of drug

– Allergic reactions

– Disruption in the balance of normal flora-

superinfections possible

© Kenneth E. Greer/Visuals Unlimited

Figure 12.16

Drug induced

side effects

41

Figure 12.17 Superinfections

(a) (b) (c)

Circulating

drug

Intestine Intestine Intestine

Superinfection Drug

Infection

Pathogen

overgrows

Drug destroys beneficial

flora

Potential pathogen

resistant to drug

but held in check

by other microbes

Normal flora important

to maintain intestinal

balance

(a) A primary

infection in the

throat is treated

with an oral

antibiotic

(b) The drug is

carried to the

intestine and

is absorbed

into the

circulation.

(c) The primary

infection is cured, but

drug-resistant

pathogens have

survived and create

an intestinal

superinfection.

42

12.6 Considerations in Selecting an Antimicrobial Drug

• Identify the microorganism causing the

infection

• Test the microorganism’s susceptibility

(sensitivity) to various drugs in vitro when

indicated

• The overall medical condition of the patient

43

Testing for the Drug Susceptibility of Microorganisms

• Essential for groups of bacteria commonly showing resistance – Kirby-Bauer disk diffusion test

Oxytetracycline 30 µg

(R < 17 mm; S ≥ 22 mm)

Enrofloxacin 5 µg

(R < 17 mm; S ≥ 22 mm) Gentamicin 10 µg

(R < 17 mm; S ≥ 21 mm)

Ampicillin 10 µg

(R < 14 mm; S ≥ 22 mm) Cefotaxime 30 µg

(R < 14 mm; S ≥ 23 mm) Chloramphenicol 30 µg

(R < 21 mm; S ≥ 21 mm)

= Zone of

Inhibition

= Region of

bacterial growth

Disk DifusionT est (schematic).

Example and evaluation of a sensitivity test, agar diffusion methoid

R = resistant, I = intermediate, S = sensitive

OT

30 GN

10

AMP

10 CTX

30

C

30

1 2 3 4 0mm

ENR

5

Kirby-Bauer Disk Diffusion Test*

= Antibiotic carrier (disc) imprinted with abbreviation and concentration

ENR

5

© Prof. Patrice Courvalin, MD, FRCR, Unite desAgentsAntibactcriens, Institut Pasteur, Paris, France

CTX AMC

(a) If the test bacterium is sensitive to a drug, a zone of inhibition develops around

its disk. The larger the size of this zone, the greater is the bacterium’s

sensitivity to the drug. The diameter of each zone is measured in millimeters

and evaluated for susceptibility or resistance by means of a comparative

standard (R, I, or S).

(b) Result for gram negative Klebsiella pneumoniae, with zones

of inhibition indicating sensitivity to cefotaxime (CTX) and

ampicillin and clavulanic acid (AMC). Observe the expanded

zone between these two drugs that indicates their enhanced

action, or synergy, when combined.

(c) Result for gram positive Staphylococcus aureus using

gentamicin (GM, left) and aztreonam (ATM, right), Although the

bacteria are sensitive to both drugs, the GM has a few colonies

growing in the zone of inhibition, indicating some cells that

have developed resistance to that concentration of the drug.

© Kathy Park Talaro

Figure 12.18 Techniques

for preparation and

interpretation of disc

diffusion tests.