Download - Lecture 7 Bio3124
Antimicrobial ChemotherapyAntimicrobial Chemotherapy
Lecture 7Bio3124
Chemotherapeutic agents
chemical agents used to treat infectious diseases
Kill or inhibit the growth of pathogens within the host
Antibiotics 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): 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 (Paul Ehrlich) ability of drug to kill or inhibit pathogen with a minimal
or no harm to host
For a good antibiotic: Therapeutic dose
drug level required for clinical treatment; should be low
Toxic dose drug level at which drug becomes too toxic for patient
(i.e., produces side effects); should be high Therapeutic 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 therapeutic dose
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 dose
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
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 reactionPBP locked in hemi-acyl reaction on antibiotic
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
• 1940: 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 actively 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• Ritanovir , 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?