Antimicrobial ChemotherapyAntimicrobial Chemotherapy

Lecture 7Bio3124

Chemotherapeutic agents

Chemical agents used to treat infectious diseases

Kill or inhibit the growth of pathogens within the host

AntibioticsAntibiotics Natural microbial products/semi

synthetic/synthetic

The Development of Chemotherapy

Paul Ehrlich (1904) Concept of selective toxicity identified dyes that effectively treated African

sleeping sickness

Sahachiro Hato (1910) working with Ehrlich, identified arsenic compounds

(Salvarsan) that effectively treated syphilis

Gerhard Domagk, and Jacques and Therese Trefouel (1935) discovered sulfonamides and sulfa drugs

Paul Ehrlich 1854 – 1915

Penicillin

Ernest Duchesne (1896), discovery lost

Alexander Fleming (1928) observed penicillin activity on a

contaminated plate did not follow up

• effectiveness demonstrated by Florey, Chain, and Heatley

(1939)

• Fleming, Florey and Chain received Nobel Prize in 1945

Penicillium mold kills S.aureus

Later discoveries

Selman Waksman (1944)Selman Waksman (1944): Streptomycin, an

antibiotic active against tuberculosis

Nobel Prize in 1952 for this discovery

by 1953 chloramphenicol, terramycin,

neomycin, and tetracycline were isolated

Properties of an antibiotic

Selective toxicity Selective toxicity (Paul Ehrlich) ability of drug to kill or inhibit pathogen with a minimal

or no harm to host

For a good antibiotic: Therapeutic doseTherapeutic dose

drug level required for clinical treatment; should be low

Toxic doseToxic dose drug level at which drug becomes too toxic for patient

(i.e., produces side effects); should be high Therapeutic indexTherapeutic index

ratio of toxic dose to therapeutic dose; should be high

Determining Antimicrobial Activity

expressed in two ways minimal inhibitory concentration (MIC)

lowest concentration of drug that inhibits the growth of a

pathogen

minimal lethal concentration (MLC) or minimal

bactericidal concentration (MBC)

lowest concentration of drug that kills a pathogen

two techniques used to determine MIC and MBC

Dilution Susceptibility testsDilution Susceptibility tests

MIC=16 ug/ml

Tube dilution assay:Tube dilution assay: tests for one

antibiotic at a time

• MIC: half dilution series of AB in Müller

Hinton medium prepared

• equal amount of bacterial broth added

• incubation at 37°C for 18 hours, visible

growth checked

• MBC: lowest AB concentration to kill

completely

• Agar dilution assayAgar dilution assay: a variation of tube

dilution assay done on agar containing

solid medium where visible colonies are

scored (multiple microorganisms vs one type

of antibiotic)

Antibiotic concentration (ug/ml)

2n 128 64 32 16 8 4 2 0

Antibiotic concentration (ug/ml)

2n 128 64 32 16 8 4 2 0

Antibiotic free medium 2n 128 64 32 16 8 4 2 0

MBC=64 ug/ml

Agar disc diffusion test:Agar disc diffusion test: Bacterial culture

is uniformly inoculated onto the surface of

an agar plate

antibiotic discs are placed onto the plate

antibiotic diffuses out of discs to form a

concentration gradient

after incubation the diameter of the zone

of growth inhibition is measured and

compared to standard tables

the diameter of the zone of inhibition is

inversely related to MIC

Tb

AMP

Tet

Gm

Suceptibility order: Tet>Tb>Gm>Amp

Kirby-Bauer method: disc diffusion test

Kirby-Bauer method

standardized method for carrying out disk diffusion test

sensitivity and resistance determined using tables that relate zone diameter to degree of microbial resistance

table values plotted and used to determine if concentration of drug reached in body will be effective

Determining the susceptibility or resistance

• MIC values are compared to standard value set by international committees

• log2 MIC vs diameter of inhibition zone is linear

• effective dosage determined

SusceptibleSusceptible

Diameter if inhibition zone (mm)

(μg/

ml)

6 10 14 18 22

MIC

0

4

16

64

512

A

B

ResistantResistant

2

8

32

128

IntermediateIntermediate

Determining the Therapeutic DoseDetermining the Therapeutic Dose

• antibiotics decay in vivo

• time required to keep the serum concentration above MIC• without significant toxicity to host

• effective dosage and repetition of dose determined

MIC

The E test

similar to disk diffusion method, but uses strip instead of disk

convenient to use, more informative than discs MIC value can be read directly E-test strips contain a gradient

of an antibiotic intersection of elliptical zone of

inhibition with strip indicates MIC

Classification of antibiotics

inhibitors of cell wall synthesis β-lactams (penicillins and cephalosporins) Vancomycin

protein synthesis inhibitors Aminoglycosides (Kanamycin, Gentamicin etc.)

Tetracyclins Macrolides Chloramphenicol

metabolic antagonists Trimethoprim Sulfonamides

nucleic acid synthesis inhibitors Quinolones

1. Inhibitors of Cell Wall Synthesis Penicillins

6-aminopenicillanic acid derivatives, differ in side chain

Have -lactam ring essential for bioactivity many penicillin resistant organisms

produce -lactamase (penicillinase) which hydrolyzes a bond in this ring

PBP locked in hemi-acyl reaction on antibiotic

Mode of action irreversible competitive binding to the active site of PBPs (transpeptidases),

normally catalyse the transpeptidation step in bacterial cell wall synthesis

prevents the synthesis of complete cell walls =>cell lysis

acts only on growing bacteria that are synthesizing new peptidoglycan

PBP catalyzes transpeptidation reaction

Cephalosporins structurally and functionally similar to penicillins broad-spectrum antibiotics that can be used on patients that are

allergic to penicillin grouped into four categories based on their spectrum of activity

Fourth generation

From Cephalosporium acremonium

Resistance mechanisms against B-lactams

• Resistance: • PBPs with lowered affinity to β-lactam• Overproduction of β –lactamases• acquisition of inducible β –lactamases

• resistance to penicillins, including the semisynthetic

analogs, continues to be a problem

Vancomycin

glycopeptide antibiotics produced by Streptomyces orientalis

Competitive binding to terminal D-Ala residues of the peptidoglycan, interferes with transpeptidation reaction of PBPs

vancomycin has been important for treatment of antibiotic resistant staphylococcal and enterococcal infections considered “drug of last resort” rise in resistance to vancomycin is of great

concern Resistance:

Mutational change of D-ala-D-Ala to D-Ala-Lactate

in cell wall structure

2. Protein Synthesis Inhibitors many antibiotics bind specifically to the prokaryotic

ribosomes 30S (small) or 50S (large) ribosomal subunits

inhibit a step in protein synthesis aminoacyl-tRNA binding peptide bond formation mRNA reading Ribosomal translocation

Antibiotics: Aminoglycosides, Tetracyclines, Macrolides and

Chloramphenicol

AminoglycosidesAminoglycosides

• Wide spectrum, fungal and bacterial metabolic or synthetic products

• 1944: first aminoglycoside, Streptomycin from Streptomyces griseus,used to treat of tuberculosis

• more synthetic products

• Polycationic cyclohexane amino sugars with diverse structural

properties

• Three families: Kanamycin, Neomycin and Gentamicin

• Structural diversity: type of sugar and sugar ring substitutions, ie NH3, OH, H

• example shows derivatives in Kanamycin family

Mode of action and resistance mechanismMode of action and resistance mechanism

• Mode of action:Mode of action: bind to 16S rRNA of 30S ribosomal subunit

and interfere with protein synthesis

• ribosomal shifting, amino acid misincorporation, misfolded proteins

• Resistance mechanisms: enzymatic modification of ABs that results in loss of capacity to bind the target

Modifying enzymes:• Aminoglycoside neucleotidyle transferases (ANTs): covalently bind an NTP in sugar ring I or III• Aminoglycoside acyl transferases (AACs)AAC6’ transfer an acyl group to 6’ position• Aminoglycoside phosphotransferase (APHs): e.g. APH3’ transfers a phosphate group from ATP to the 3’ position

Tetracyclines produced by Streptomyces or synthesized are broad spectrum, bacteriostatic, eg. used in acne treatment all have a four-ring structure to which a variety of side chains are

attached Bind to 30S ribosomal subunit A site

inhibits binding of aminoacyl-tRNA molecules to the A site of the ribosome

Resistance: mutational target modification resulting in loss of binding site

Macrolides

contain 12 to 22-carbon lactone

rings linked to one or more sugars

used on patients allergic to penicillin

e.g., erythromycin, Azithromycin

broad spectrum, bacteriostatic

binds to 23S rRNA of

50S ribosomal subunit

inhibits peptide chain elongation

Resistnace: mutational target modification

Chloramphenicol

First isolated from Streptomyces venezuelae

now is chemically synthesized

binds to 23S rRNA in 50S ribosomal subunit and inhibits peptidyl

transferase reaction during protein synthesis

Toxic, numerous side effects, carcinogenic

used in life-threatening situations

Resistance: enzymatic acetylation by acetyltransferases on –OH

groups

3. Metabolic Antagonists act as antimetabolites

antagonize metabolic pathways by competitively inhibiting the use of metabolites by key enzymes

are structural analogs molecules that are structurally

similar to, and compete with, naturally occurring metabolic intermediates

Synergism: two antibiotics working together better than individually

Dihydrofolic acid analog

Sulfonamides or Sulfa Drugs

structurally related to a p-aminobenzoic acid (PABA) PABA used for the synthesis of folic acid and is made by

many pathogens unlike humans, these pathogens cannot take up PABA selectively toxic, compete against PABA for the active site

of an enzyme involved in folic acid synthesis, resulting in a decline in folic acid concentration pathogen dies because folic acid is a precursor to

purines and pyrimidines which are nucleic acid building blocks

Trimethoprim

synthetic antibiotic that also interferes with folic acid production

broad spectrum can be combined with sulfa drugs to increase

efficacy of treatment combination blocks two steps in folic acid pathway

has a variety of side effects including abdominal pain and photosensitivity

Resistance: Overproduction of PABA Mutant enzymes that evade the binding of drugs but not

the natural substrates

4. Nucleic Acid Synthesis Inhibition

a variety of mechanisms block DNA replication

inhibition of DNA polymerase

inhibition of DNA helicase

block transcription

inhibition of RNA polymerase

Must target DNA/RNA biosynthetic steps that are

different between prokaryotes and eukaryotes

Quinolones broad-spectrum, synthetic, contain quinolone

ring Nalidixic acid, first synthesized (1962) Fluoroquinlones like Ciprofloxfacin,

norfloxacin are new drugs with a wider antibacterial spectra

act by inhibiting bacterial DNA-gyrase (Topoisomerase II) disrupts DNA supercoiling and replication

selective toxicity: DNA gyrase is not present in eukaryotic cells

Resistance: mutations in quinolone binding cleft on

gyrase (gyrB subunit) that results in loss of affinity

Reduced uptake due to mutations in ompF gene that encodes for porins

Active efflux pumps

Antifungal Drugs

fewer effective agents because of similarity of fungal cells and human cells

easier to treat superficial mycosis than systemic infections

Antifungal Agents Fungal infections difficult to treat

Eukaryotic cells similar to animal cells Fungi inactivate many drugs Inhibitors: Treatment of superficial mycosis Clotrimazole: inhibits sterol synthesis

Griseofulvin: disrupts mitotic spindle formation

Treating systemic infections

Polyene produced by Streptomyces nodosus binds membrane ergosterol found in fungal membranes disrupts fungal membrane integrity

Antiviral Drugs

drug development has been slow because it is difficult to specifically target viral cycle

drugs currently used inhibit virus-specific enzymes and life cycle processes

used to prevent influenza infections

Inhibits M2 viral envelop protein

blocks penetration and uncoating of influenza virus

Amantadine

Tamiflu and Zanamivir

anti-influenza agents neuraminidase inhibitors prevent the release of virions from infected cells,

not a cure for influenza, has been shown to

shorten course of illnessA. Oseltamivir (Tamiflu)

inhibits herpes virus enzymesinvolved in DNA and RNAsynthesis and function

inhibits herpesvirus DNA polymerase

inhibits herpesvirus andcytomegalovirusDNA polymerase

Anti-HIV drugs

two main targets HIV reverse transcriptase HIV protease

Reverse transcriptase inhibitors

Protease inhibitors

mimics peptide bondthat is normally attacked

by the protease

mimics peptide bondthat is normally attacked

by the protease

• Gag polyprotein is expressed from a single mRNA• cleaved by viral protease to viral coat proteins p15, p17and p24• Ritonavir , inhibitor of protease

Summary of Mechanisms of Drug ResistanceSummary of Mechanisms of Drug Resistance

alteration of target enzyme or organelleeg. PBPs

inactivation of drug enzymatic modification of drug by pathogen (eg. AGs)

Drug exclusion: prevent entrance of drug drug can’t bind to or penetrate pathogen decreased permeability

Efflux pumps: pump drug out use of alternative pathways or increased production

of target metabolite, eg. PABA

Drug Resistance

an increasing problem resistance mutants arise spontaneously or by

mutagen induction and are then selected once resistance establishes in a population it can be

transmitted to other bacteria horizontally by Transduction: viruses transmit resistance genes Transformation: genes picked up from environment Conjugation: conjugative plasmids transmit the resistance

genes

After 100 years of golden antibiotic era we have a problem on our hands: Emerging Multidrug Resistance

Multidrug resistance: clinical and social problem inherent to antibiotic era

Reasons? Mutation rates in bacteria and viruses are

enormously high and cumulative Selection of antibiotic resistant microorganisms is

unavoidable direct result of antibiotic use Increased volume of antibiotic use, most

probably prone to improper practice and misuse. Statistical data suggest a direct correlation (see examples..)

Antibiotics Consumption in Canada

0100200300400500600700800900

1000

1994 1995 1996 1997 1998 1999 2000

Canada

Ontario

Quebec

Canada, Quebec, Ontario Number of prescriptions per 1000 citizens per year Number of prescriptions per 1000 citizens per year

This excess is attributed to What?

1990 - 300 metric tons of antibiotics were used.

70% are not used appropriately

Incorrect usage.

Prescription for viral infections.

Prescription for bacterial infections which can recover on their own.

S. pneumoniae resistance to Penicillin and Macrolides in Canada

Resistance of H. influenzae to Penicillin in Canada

Economical impactEconomical impact

Bacterial infections and antibacterial

resistance constitute an ever

increasing problem

this is reflected in leading market

share (62%) for anti-bacterial agents

Fast growing antibiotic resistance

has negative impact on R&D in

pharmaceutical initiatives

the least relative growth in

antibacterial sector is alarming

Measures to counter the Acquired Resistance Public education.

Usages must be effectively controlled.

Eliminate the prophylactic administration in livestock.

Reduce the usage in the household products.

Reduce the prophylactic usage in humans.

Discover and synthesize new antibiotics.

Would this solve the problem?

What are the possible alternatives?

Probiotic approach?

Natural antimicrobial products? Phage therapy?

New antimicrobial targets? Vaccine development?