antimicrobial chemotherapy and antibiotic sensitivity testing
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Antimicrobial Chemotherapy and Antibiotic Sensitivity TestingTRANSCRIPT
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Antimicrobial Chemotherapy and
Antibiotic Sensitivity TestingDr.T.V.Rao MD
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Uses of Antimicrobial Agents
• Antimicrobial agents are widely employed
to cure bacterial diseases
• Definition of Antibiotic – Antibiotics are
substances that are derived from a various
species of microorganisms and are
capable of inhibiting the growth of other
microorganism even in small
concentrations.
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Beginning of Antibiotics with
Discovery of Pencillin
• The discovery of penicillin
has been attributed to
Scottish scientist
Alexander Fleming in
1928 and the
development of penicillin
for use as a medicine is
attributed to the
Australian Nobel
Laureate Howard Walter
Florey.
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Discovery of Pencillin
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Discovery of Pencillin
Awarded Nobel Prize
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Chemotherapeutic Agents
• Antimicrobial agents – that are
produced synthetically but
have action similar to that of
antibiotics and are defined as
chemotherapeutic agents
• Eg Sulphonamides,
Quinolones.11/1/2014 Dr.T.V.Rao MD 6
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Definition
• Bacteriostatic - Antimicrobial agents
that reversibly inhibit growth of
bacteria are called as bacteriostic (
Tetracyclnes, Chloramphenicol )
• Bactericical – Those with an
irreversible lethal action on bacteria
are known as bactericidal ( Pencillin,
Isoniazid )11/1/2014 Dr.T.V.Rao MD 7
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How Drugs Act
• Drugs differ on their
capabilities to act at different
sites on bacteria.
• Some drugs have more than
one site of action
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Major mechanisms of
antimicrobial drugs
• 1 Inhibition of cell wall synthesis
• 2 Inhibition of cell membrane function
• 3 Inhibition of protein synthesis ( inhibition
of translation and transcription of genetic
material)
• 4 Inhibition of nucleic acid synthesis.
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Pencillin
• Pencillin and cephalosporins act inhibiting
Trans peptidases, the enzyme catalyses the final
linking step in synthesis of peptidoglycan.
• Due to this reason Pencillin in bactericidal for
grwoing bacteria since new peptidoglycan is
synthesized at that stage only.
• In nongrwoing cells pencillin is inactive
• An intact beta – lactum is essential for
antibacterial activity of pencillins
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Classification of Pencillins
• Natural
Benzyl pencillin
Phenoxymethyl pencillin Pencillin v
Semi synthetic and pencillase resistant
1 Methicillin
2 Nafcillin
3 Cloxacillin
4 Oxacillin
5 Floxacillin
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Extended spectrum pencillins
Aminopencillins - Ampicillin, Amoxycillin
Carboxypencillins – Carbencillin, Ticarcillin
Ureidopencillin - Piperacillin
Resistance to pencillin is due to pencillinase commonly called as ßlactamase
The enzyme opens Betalactam ring hydrolytically and thus converts the antibiotic to inactive pencillonic acid.
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Inhibitors to Beta lactamase
• Clavulinic acid which is a product of
Strept.clavuligerus
• Acts against the Staphylococcal beta
ßlactamase.
• And plasmid mediated Betalactamase of
Gram negative bacteria.
• Salbactum – this is a semisyntetic sulfone
derivative with weak antibacterial activity
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• Tazobactam – A penicillonic acid sulfone
derivative acts well when combined with
piperacillin.
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Cephalosporins• Like pencillin acts similar
• Products of the molds of genus
Cephalosporium except cefoxilin
• Divided into 4 generation of
cephalosporins depending on the
spectrum of activity.
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Different Generations of
Cephalosporins
• Cephalosporins are grouped into
"generations" based on their
spectrum of antimicrobial activity. The
first cephalosporins were designated
first generation while later, more
extended spectrum cephalosporins
were classified as second generation
cephalosporins.
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Major generations of Cephalosporins
• Cephalosporins are divided into 3 generations:
• 1st generation: Cephelexin, cefadroxil,
cephradine
• 2nd generation: Cefuroxime, cefaclor
• 3rd generation: cefotaxime, Ceftazidime,
cefixime - these give the best CNS penetration
• 4th generation Cephalosporins are already
available
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Basis of generations in
Cephalosporins
• Cephalosporins are grouped into
"generations" based on their spectrum of
antimicrobial activity. The first
cephalosporins were designated first
generation while later, more extended
spectrum cephalosporins were classified
as second generation cephalosporins.
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Advantages with Newer
generations
• Each newer generation of cephalosporins
has significantly greater gram-negative
antimicrobial properties than the preceding
generation, in most cases with decreased
activity against gram-positive organisms.
Fourth generation cephalosporins,
however, have true broad spectrum
activity
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Other Beta-lactams include
• Other beta-lactams include:
• Aztreonam: a monocytic beta-lactam,
with an antibacterial spectrum which
is active only against Gram negative
aerobes, including Pseudomonas
aeruginosa, Neisseria meningitidis
and N. gonorrhoea.
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Other drugs
• Imipenem: a carbapenem with a
broader spectrum of activity
against Gram positive and
negative aerobes and anaerobes.
Needs to be given with cilastatin
to prevent inactivation by the
kidney.
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Quinolones
• Quinolones are the first wholly synthetic
antimicrobials. The commonly used
Quinolones.
• Act on the DNA gyrase which prevents
DNA polymerase from proceeding at the
replication fork and consequently stopping
synthesis.
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Aminoglycosides
• Aminoglycosides are group of antibiotics in
which amino sugars liked by glycoside bonds
• Eg Streptomycin,
• Act at the level of Ribosome's and inhibits
protein synthesis
• Other Aminoglycosides –
Gentamycin,
neomycins,paromomycins,tobramycins
Kanamycins and spectinomycins
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Tetracycline's
• Broad spectrum antibiotic produced by
Streptomyces species
• 1. Oxytetracycle, chlortetracycle and
tetracycline
• Tetracyclnes are bacteriostatic drugs inhibits
rapidly multiplying organisms
• Resistance develops slowly and attributed to
alterations in cell membrane permeability to
enzymatic inactivation of the drug
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Choramphenicol
• Choramphenicol is
bacteriostatic drug
• Can produce bone marrow
depression
• Chloramphenicol interferes
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Macrolides,Azalides,Ketolides
• Contain macro cyclic lactone ring
Erythromycin. Is popularly used drug
• Other drugs
Roxithromycin,Azithromycin
• Inhibits the protein synthesis.
• Used as alternative to pencillin allergy
patients.
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Other Antimicrobial agents
• Lincomycins
Clindamycin resembles Macrolides in
biting site and antimicrobial activity.
Streptogramins
Quinpristin / dalfopristin
useful in gram postive bacteria
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Antibiotics in Anaerobes
• Major anaerobes – Anaerobic cocci, clostridia and Bacteroides are susceptible to Benzyl pencillin
• Bact.fragilis as well as many other anaerobes are treatable with Erythromycin,Lincomycin, tetracycline and Chloramphenicol
• Clindamycin is effective against many strains of Bacteroides
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Metronidazole in Anaerobic
Infections• Since the discovery of Metronidazole in
1973 since then it was identified as
leading agent anaerobes.
• But also useful in treating parasitic
infections
Trichomonas, Amoebiasis and other
protozoan infections.
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Antiviral drugs
• Acyclovir
• Ribavirin
• Azidothymidine
• Amantidine
• Idoxuridine
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Antifungal Agents
• Grisofulvin
• Imidazole
• Polyenes Nystatin, Amphotericin B
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Antibiotic resistance
• Antibiotic resistance is the ability of a micro
organism to withstand the effects of antibiotics. It
is a specific type of drug resistance. Antibiotic
resistance evolves naturally via natural selection
acting upon random mutation, but it can also be
engineered by applying an evolutionary stress
on a population. Once such a gene is generated,
bacteria can then transfer the genetic
information in a horizontal fashion (between
individuals) by plasmid exchange.
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Detection of Antibiotic sensitivity and Resistance
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Routine Susceptibility Tests
• Disk diffusion
(Kirby Bauer)
• Broth micro-
dilution MIC
– NCCLS reference
method
• Etest
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• Diffusion method
– Put a filter disc, or a porous cup/a bottomless
cylinder containing measured quantity of
drugs on the a solid medium that has been
seeded with test bacteria
• Dilution method
– vary amount of antimicrobial substances
incorporated into liquid or solid media
– followed by inoculation of test bacteriaDr.T.V.Rao MD 35
Antimicrobial Susceptibility
Testing
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Susceptibility Testing
Methods
Innoculate
MH plate
Place disks
on agar plate
Incubate plate
18-24 hr, 35 C
Measure and
record zone of
inhibition around
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Antimicrobial susceptibility testsMinimum inhibitory concentration [MIC]
– The smallest concentration of antibiotic that
inhibits the growth of organism
Liquid media (dilution) allows MIC estimation
Solid media (diffusion)
– Disk diffusion (Kirby-Bauer)
– E-tests
– Allows MIC estimation
Beta lactamase production: quick screening
method11/1/2014 Dr.T.V.Rao MD 37
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Disk Diffusion
Susceptibility Testing
Improper agar & disk placement Mueller Hinton agar &
good disk placement
Use Mueller Hinton agar
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Antimicrobial susceptibility
testing using micro-broth
dilutions
•
•
•
•
•
•
•
• •
•
•
•
•
96 well microtiter plate
ug/ml
64 32 16 8 4 2
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Dilution in liquid broth
• Tubes containing increasing antibiotic
concentrations
• Incubation during 18 hr at 37°C
• Tedious
0 (Control) 0,25 0,50 1 2 4 8 mg/l
MICBacterial growth Inhibition
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Kirby-Bauer disc testingAntibiotic-impregnated discs placed on an agar plate at the
interface between test organism and susceptible control organism
Resulting zones of inhibition compared, use of controls
Susceptibility is inferred (standard tables)
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Antimicrobial Gradient
Testing
E-test®
Read plates
after
recommended
Incubation
Read MIC
where elipse
intersects
scale
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E test – MIC Reports are helpful in
Critical management decisions
• Quantitative MIC
data is a
prerequisite for
the management
of critical
infections,
including sepsis,
especially among
critical care
patients. Etest is
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E-testPlastic strips with a predefined
gradient of
– One antibiotic
– One antifungal
Only one manufacturer
One strip per antibiotic
Wide range of antibiotics
Easy to use
Storage at -20°C
Short shelf life, expensive
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Reading E-tests
Susceptible < 1
Resistant > 4 ug/ml
Ciprofloxacin for
Yersinia pestis
Intermediate 1-4 ug/ml
Upper reading
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Are we overusing Antibiotics
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Choose the Appropriate
Antibiotic
Think before
prescribing
Are we using
Right drug
for the Right
bug ?
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•Program created by Dr.T.V.Rao MD for Medical and Paramedical Students in
the Developing world
• Email.
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