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ANTI MICROBIAL
CHEMOTHERAPY

Dr. Ashish Jawarkar
M.D.
Consultant Pathologist
Parul Sevashram Hospital
Modern era in Antibiotics begins
with Fleming.
Definition - Antibiotic







Anti – against
Bio – life
Originally extracted from compounds produced by
living organisms – aminoglycosides
Semi synthetic – cephalosporins, carbapenems
Synthetic – sulphonamides, quinolones,
oxazolidinediones
History of Antibiotic Discovery
Mechanisms of antibacterial action


1. Inhibition of cell wall synthesis




2. Inhibition of protein synthesis




Aminoglycosides, tetracyclines, chloramphenicol,
macrolides

3. Alteration of cell membranes




Penicillin, vancomycin, bacitracin

Polymyxin B, Bacitracin

4. Inhibition of nucleic acid synthesis
DNA – quinolones, Metronidazole
 RNA – Rifampicin, Bacitracin




5. Antimetabolite activity


Sulfonamides
Antimicrobial Resistance






How bacteria survive doses of antibiotics that
would otherwise have been lethal
Problem – treated with a higher dose, or newer
antibiotic
Side effects, Newer may not be available
antimicrobial chemotherapy
Types of resistance


Intrinsic




Bacteria may not make the protein that antibiotic
targets

Extrinsic


Due to acquired mutation in DNA or RNA
Causes of Resistance



Overuse/misuse of antibiotics
Example is sore throat – Viral , still antibiotics are
prescribed
Failure to take entire course
 Failure to take at correct intervals – two times a day
instead of three times

Antibiotic Susceptibility Testing




Pathogenic bacteria show large variations in
susceptibility to antibiotics
Particularly marked in case of Staphylococcus and
gram negative bacteria
Types



Diffusion tests
Dilution tests
Uses of Antibiotic Sensitivity
Testing






Antibiotic sensitivity test: A laboratory test
which determines how effective antibiotic therapy
is against a bacterial infections.
Antibiotic sensitivity testing will control the use of
Antibiotics in clinical practice
Testing will assist the clinicians in the choice of
drugs for the treatment of infections.
Components of Antibiotic
Sensitivity Testing



1.The identification of relevant pathogens in
exudates and body fluids collected from patients
2. Sensitivity tests done to determine the degree of
sensitivity or resistance of pathogens isolated from
patient to an appropriate range of antimicrobial
drugs
Antibiotic Sensitivity Testing Is
Essential of selection of
Antibiotics
Isolation and Identification of Bacteria
precedes the selection of Antibiotic
Testing Methods
Uses of Antibiotic Sensitivity
Testing






Helps to guide the Physician in choosing
Antibiotics
The accumulated results on different pathogens
their sensitivity will guide the physician in
choosing empirical treatment in serious patients
before the individual’s laboratory results are
analyzed in the Microbiology laboratory.
Reveals the changing trends in the local isolates.
Why Need continues for testing for
Antibiotic Sensitivity


Bacteria have the ability to
develop resistance following
repeated or subclinical
(insufficient) doses, so more
advanced antibiotics and
synthetic antimicrobials are
continually required to
overcome them.
Testing for Antibiotic sensitivity






The method includes several steps including
obtaining a bacterial sample;
identifying the type of bacteria in the bacterial
sample; selecting a set of antibiotics based on the
identity of the bacteria in the bacterial sample;
obtaining a control sample from the bacterial
sample;
What is Resistance in Antibiotic
Sensitivity Testing


Resistance implies that the infection is not
treatable with the tested Antibiotic because its
MIC exceeds achievable safe tissue or urine
levels.
Kirby-Bauer methods
A commonly used method in basic
laboratories


Kirby-Bauer antibiotic testing (KB testing or
disk diffusion antibiotic sensitivity testing) is a
test which uses antibiotic-impregnated wafers to
test whether particular bacteria are susceptible to
specific antibiotics
How to perform Kirby- Bauer
testing


The basics are easy: The bacterium is swabbed on
the agar and the antibiotic discs are placed on top.
The antibiotic diffuses from the disc into the agar
in decreasing amounts the further it is away from
the disc. If the organism is killed or inhibited by
the concentration of the antibiotic, there will be
NO growth in the immediate area around the disc:
This is called the zone of inhibition .
Steps in Antibiotic sensitivity testing
Bacteria are inoculated as
lawn culture


Method of inoculationGood results are
obtained by placing a
standard loopful of
inoculum suspension on
the plate and then
spreading it with a dry
sterile swab.
The area of Inhibition is measured
with a Scale
 Record

the
results for
everyone on
your table in the
table below.
The disk diffusion methods are
commonly used for routine testing
Read the plates in
transmitted light
The zone of inhibition guides
the right choice of Antibiotic
The strips with multiple
Antibiotics can be tested in one go
Other methods of Antibiotic
susceptibility testing


Other methods to test antimicrobial susceptibility
include the Stokes method, E-test (also based on
antibiotic diffusion). Agar and Broth dilution
methods for Minimum Inhibitory Concentration
determination.
Testing Minimum Inhibitory
Concentration


In alternative measure of susceptibility is to
determine the Minimum Inhibitory
Concentration (MIC) and the Minimum
Bactericidal Concentration (MBC) of a drug. A
series of broths are mixed with serially diluted
antibiotic solutions and a standard inoculum is
applied. After incubation, the MIC is the first
broth in which growth of the organism has been
What is Minimum Inhibitory
concentration


Minimum inhibitory concentration (MIC), in
microbiology, is the lowest concentration of an
antimicrobial that will inhibit the visible growth of
a micro organism after overnight incubation.
Minimum inhibitory concentrations are important
in diagnostic laboratories to confirm resistance of
micro organisms to an antimicrobial agent and
also to monitor the activity of new antimicrobial
agents.[
The Antibiotics are diluted to various
dilution to test the minimum
inhibitory concentration
What is E Test


Etest is an antimicrobial gradient technique in
which 15 reference MIC dilutions of an antibiotic
have been repackaged with innovative dry
chemistry technology onto a plastic strip. The
predefined gradient provides precise and accurate
assessment of antimicrobial activity against both
fastidious and non-fastidious microorganisms.



Dr.T.V.Rao MD
The s t ri ps are
im
pregnat ed w t h vari ous
i
c onc ent rat i on of
Ant i bi ot i c s
E = testing on various
isolates
Limitation of Disk Diffusion
Methods




Disk diffusion methods are
not suitable for slow
growing bacterial pathogens
The great limitation being
for testing on
Mycobacterium
tuberculosis which needs
varied, technically
demanding methods
Antibiotic Sensitivity testing can
be done with automation
There is a growing need for
Automation in Antibiotic sensitivity
testing
Limitation of Antibiotic Sensitivity
usage
Both Microbiologists and Clinician should however
bear in mind that the response therapy in vivo may
not always reflect the results of testing the
sensitivity of patient's pathogen in vitro.
Thank You

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antimicrobial chemotherapy

  • 1. ANTI MICROBIAL CHEMOTHERAPY Dr. Ashish Jawarkar M.D. Consultant Pathologist Parul Sevashram Hospital
  • 2. Modern era in Antibiotics begins with Fleming.
  • 3. Definition - Antibiotic      Anti – against Bio – life Originally extracted from compounds produced by living organisms – aminoglycosides Semi synthetic – cephalosporins, carbapenems Synthetic – sulphonamides, quinolones, oxazolidinediones
  • 6.  1. Inhibition of cell wall synthesis   2. Inhibition of protein synthesis   Aminoglycosides, tetracyclines, chloramphenicol, macrolides 3. Alteration of cell membranes   Penicillin, vancomycin, bacitracin Polymyxin B, Bacitracin 4. Inhibition of nucleic acid synthesis DNA – quinolones, Metronidazole  RNA – Rifampicin, Bacitracin   5. Antimetabolite activity  Sulfonamides
  • 7. Antimicrobial Resistance    How bacteria survive doses of antibiotics that would otherwise have been lethal Problem – treated with a higher dose, or newer antibiotic Side effects, Newer may not be available
  • 9. Types of resistance  Intrinsic   Bacteria may not make the protein that antibiotic targets Extrinsic  Due to acquired mutation in DNA or RNA
  • 10. Causes of Resistance   Overuse/misuse of antibiotics Example is sore throat – Viral , still antibiotics are prescribed Failure to take entire course  Failure to take at correct intervals – two times a day instead of three times 
  • 11. Antibiotic Susceptibility Testing   Pathogenic bacteria show large variations in susceptibility to antibiotics Particularly marked in case of Staphylococcus and gram negative bacteria
  • 13. Uses of Antibiotic Sensitivity Testing    Antibiotic sensitivity test: A laboratory test which determines how effective antibiotic therapy is against a bacterial infections. Antibiotic sensitivity testing will control the use of Antibiotics in clinical practice Testing will assist the clinicians in the choice of drugs for the treatment of infections.
  • 14. Components of Antibiotic Sensitivity Testing   1.The identification of relevant pathogens in exudates and body fluids collected from patients 2. Sensitivity tests done to determine the degree of sensitivity or resistance of pathogens isolated from patient to an appropriate range of antimicrobial drugs
  • 15. Antibiotic Sensitivity Testing Is Essential of selection of Antibiotics
  • 16. Isolation and Identification of Bacteria precedes the selection of Antibiotic Testing Methods
  • 17. Uses of Antibiotic Sensitivity Testing    Helps to guide the Physician in choosing Antibiotics The accumulated results on different pathogens their sensitivity will guide the physician in choosing empirical treatment in serious patients before the individual’s laboratory results are analyzed in the Microbiology laboratory. Reveals the changing trends in the local isolates.
  • 18. Why Need continues for testing for Antibiotic Sensitivity  Bacteria have the ability to develop resistance following repeated or subclinical (insufficient) doses, so more advanced antibiotics and synthetic antimicrobials are continually required to overcome them.
  • 19. Testing for Antibiotic sensitivity    The method includes several steps including obtaining a bacterial sample; identifying the type of bacteria in the bacterial sample; selecting a set of antibiotics based on the identity of the bacteria in the bacterial sample; obtaining a control sample from the bacterial sample;
  • 20. What is Resistance in Antibiotic Sensitivity Testing  Resistance implies that the infection is not treatable with the tested Antibiotic because its MIC exceeds achievable safe tissue or urine levels.
  • 21. Kirby-Bauer methods A commonly used method in basic laboratories  Kirby-Bauer antibiotic testing (KB testing or disk diffusion antibiotic sensitivity testing) is a test which uses antibiotic-impregnated wafers to test whether particular bacteria are susceptible to specific antibiotics
  • 22. How to perform Kirby- Bauer testing  The basics are easy: The bacterium is swabbed on the agar and the antibiotic discs are placed on top. The antibiotic diffuses from the disc into the agar in decreasing amounts the further it is away from the disc. If the organism is killed or inhibited by the concentration of the antibiotic, there will be NO growth in the immediate area around the disc: This is called the zone of inhibition .
  • 23. Steps in Antibiotic sensitivity testing
  • 24. Bacteria are inoculated as lawn culture  Method of inoculationGood results are obtained by placing a standard loopful of inoculum suspension on the plate and then spreading it with a dry sterile swab.
  • 25. The area of Inhibition is measured with a Scale  Record the results for everyone on your table in the table below.
  • 26. The disk diffusion methods are commonly used for routine testing
  • 27. Read the plates in transmitted light
  • 28. The zone of inhibition guides the right choice of Antibiotic
  • 29. The strips with multiple Antibiotics can be tested in one go
  • 30. Other methods of Antibiotic susceptibility testing  Other methods to test antimicrobial susceptibility include the Stokes method, E-test (also based on antibiotic diffusion). Agar and Broth dilution methods for Minimum Inhibitory Concentration determination.
  • 31. Testing Minimum Inhibitory Concentration  In alternative measure of susceptibility is to determine the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of a drug. A series of broths are mixed with serially diluted antibiotic solutions and a standard inoculum is applied. After incubation, the MIC is the first broth in which growth of the organism has been
  • 32. What is Minimum Inhibitory concentration  Minimum inhibitory concentration (MIC), in microbiology, is the lowest concentration of an antimicrobial that will inhibit the visible growth of a micro organism after overnight incubation. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of micro organisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents.[
  • 33. The Antibiotics are diluted to various dilution to test the minimum inhibitory concentration
  • 34. What is E Test  Etest is an antimicrobial gradient technique in which 15 reference MIC dilutions of an antibiotic have been repackaged with innovative dry chemistry technology onto a plastic strip. The predefined gradient provides precise and accurate assessment of antimicrobial activity against both fastidious and non-fastidious microorganisms.  Dr.T.V.Rao MD
  • 35. The s t ri ps are im pregnat ed w t h vari ous i c onc ent rat i on of Ant i bi ot i c s
  • 36. E = testing on various isolates
  • 37. Limitation of Disk Diffusion Methods   Disk diffusion methods are not suitable for slow growing bacterial pathogens The great limitation being for testing on Mycobacterium tuberculosis which needs varied, technically demanding methods
  • 38. Antibiotic Sensitivity testing can be done with automation
  • 39. There is a growing need for Automation in Antibiotic sensitivity testing
  • 40. Limitation of Antibiotic Sensitivity usage Both Microbiologists and Clinician should however bear in mind that the response therapy in vivo may not always reflect the results of testing the sensitivity of patient's pathogen in vitro.