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Antibiotic Sensitivity Testing

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					    Antibiotic
Sensitivity Testing

     Dr.T.V.Rao MD
Modern era in Antibiotics
 begins with Fleming.
History of Antibiotic Discovery
    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.

   Dr.T.V.Rao MD
       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
 3. Assay of the concentration of an administered
  drug in the blood or body fluid of patient
  required to control the schedule of dosage.
Dr.T.V.Rao MD
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.
 Helps the local pattern of antibiotic prescribing.
   Dr.T.V.Rao MD
          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.
   Dr.T.V.Rao MD
            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;
    Dr.T.V.Rao MD
               Testing Antibiotic
                 Susceptibility
 Antibiotic sensitivity test: A laboratory
  test which determines how effective
  antibiotic therapy is against a bacterial
  infections.
 Antibiotic sensitivity test: the in vitro
  testing of bacterial cultures with antibiotics
  to determine susceptibility of bacteria to
  antibiotic therapy. Bauer-Kirby test.
    Dr.T.V.Rao MD
        How results to be Reported
         after Sensitivity Testing
    One should follow guidelines in reporting the results and
     make matters simple with clear words as

    Organism A isolated and sensitive ( or resistant )
    to Antibiotic B

    Such a report is relevant to present clinical condition “
     that the minimum inhibitory concentration( MIC )
     of the antibiotic for it has been measured in some
     way and that, if the organism is reported as
     sensitive the MIC is less than a half or quarter of
     the concentration of antibiotics likely to be found
     in the infected tissues of a patient given the usual
     schedule of doses i.e. that the infection is
     treatable”
       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.
 Intermediate sensitivity means they show a
  unimodal distribution, but they can often
  be reclassified as sensitive or resistant if
  retested.
   Dr.T.V.Rao MD
     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

   Dr.T.V.Rao MD
    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 .
   Dr.T.V.Rao MD
Steps in Antibiotic sensitivity
           testing
        Streaking the Inoculum
   Kirby-Bauer (also Bauer-
    Kirby) disk diffusion
    antibiotic susceptibility
    testing applies a defined
    inoculum (compared to
    McFarland 0.5 OD
    standard streaked as a
    lawn onto a large
    Mueller-Hinton agar or
    Blood agar plate (in 3
    directions to ensure
    confluence).
   Dr.T.V.Rao MD
        Making proper inoculum
   Swab a Mueller-Hinton
    plate with each of the
    bacteria. Dip a sterile
    swab into the broth and
    express any excess
    moisture by pressing the
    swab against the side of
    the tube.


    Dr.T.V.Rao MD
Bacteria are inoculated as lawn
            culture
   Method of
    inoculation- Good
    results are obtained
    by placing a standard
    loopful of inoculum
    suspension on the
    plate and then
    spreading it with a
    dry sterile swab.
         Disk Diffusion Method
   After completely swabbing the plate, turn
    it 90 degrees and repeat the swabbing
    process. (It is not necessary to re-moisten
    the swab.) Run the swab around the
    circumference of the plate before
    discarding it in the discard bag.


   Dr.T.V.Rao MD
    Placing the Antibiotic disks
   Then, using a
    dispenser such as the
    one pictured,
    antibiotic-
    impregnated disks are
    placed onto the agar
    surface. As the
    bacteria on the lawn
    grow, they are
    inhibited to varying
    degrees by the
    antibiotic diffusing
    from the disk
   Dr.T.V.Rao MD
Zone sizes differ on sensitivity
           pattern
   It has been
    determined that
    zones of inhibition of
    a certain diameter
    (varies for antibiotic
    and to a lesser
    extent, bacterial
    species) correlate
    with sensitivity or
    resistance to the
    antibiotic tested
       Look at the Charts for
      establishing the zones of
              Sensitivity
   The zone sizes are looked up on a
    standardized chart to give a result of
    sensitivie, resistant, or intermediate. Many
    charts have a corresponding column that
    also gives the MIC (minimal inhibitory
    concentration) for that drug.
            Interpretation
 Place the metric ruler across the zone of
  inhibition, at the widest diameter, and measure
  from one edge of the zone to the other
  edge. HOLDING THE PLATE UP TO THE LIGHT
  MIGHT HELP.
 The disc diameter will actually be part of that
  number. If there is NO zone at all, report it as
  0---even though the disc itself is around 7
  mm.
 Zone diameter is reported in millimetres, looked
  up on the chart, and result reported as S
  (sensitive), R (resistant), or I (intermediate).
    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   traanmitted
             light
The zone of inhibition guides
the right choice of Antibiotic
        How results to be Reported
         after Sensitivity Testing
    One should follow guidelines in reporting the results and
     make matters simple with clear words as

    Organism A isolated and sensitive ( or resistant )
    to Antibiotic B

    Such a report is relevant to present clinical condition “
     that the minimum inhibitory concentration( MIC )
     of the antibiotic for it has been measured in some
     way and that, if the organism is reported as
     sensitive the MIC is less than a half or quarter of
     the concentration of antibiotics likely to be found
     in the infected tissues of a patient given the usual
     schedule of doses i.e. that the infection is
     treatable”
                    Stokes’ Method
   In original Stokes’
    method the inoculum of
    the control strain is
    evenly spread over the
    upper and lower thirds of
    a plate and that of the
    test strain over the
    central third;uninoculated
    gaps 2 – 3 mm wide are
    left to test from the
    control areas.

   Dr.T.V.Rao MD
             Control strains
           in Stokes’ Method
   The Microbiology laboratories should
    always practice to test the quality control
    of their work with the use of standard
    strains in Stokes method with following
    standard strains and results are compared
     1 Escherichia coli NCTC 10418
    2 Pseudomonas aeruginosa NCTC 10662
    3 Staphylococcus aureus NCTC 6571
    Stokes Method for Antibiotic
         Sensitivity testing
   In the Stokes controlled
    sensitivity test, a control
    organism is inoculated on part
    of a plate and the test
    organism is plated on the
    remainder. Disks are placed at
    the interface and the zones of
    inhibition are compared. The
    use of a sensitive control
    shows that the antibiotic is
    active, so that if the test
    organism grows up to the disk
    it may safely be assumed that
    the test organism is resistant
    to that drug.
   Dr.T.V.Rao MD
    The strips with multiple
Antibiotics can be tested in one
               go
  Other methods of Antibiotic
     susceptibility testing
 Othermethods 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

   Dr.T.V.Rao MD
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
MIC test results graphed
                    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 strips are impregnated with
    various concentration of
           Antibiotics
E = testing on various isolates
           Multiple drug resistant
                 organisms

  Multiple drug resistant organisms are resistant to
  treatment with several, often unrelated,
  antimicrobial agents as described above in
  Shigella. Some of the most important types of
  multiple drug resistant organisms that have
  been encountered.

Dr.T.V.Rao MD
Examples of Bacteria of Clinical
        Importance
   MRSA - methicillin/oxacillin-resistant
    Staphylococcus aureus
    VRE - vancomycin-resistant enterococci
    ESBLs - extended-spectrum beta-
    lactamases (which are resistant to
    cephalosporins and monobactams)
    PRSP - penicillin-resistant Streptococcus
    pneumoniae
   Dr.T.V.Rao MD
Methicillin resistance in
 Staphylococcu aureus
     Detection of MRSA made
    simple with Disk Diffusion
             Methods
 The MRSA among the Staphylococcus
  aureus needs detection in special
  condtions either by carrying out the test
  on medium containing 5 % Nacl with
  Methicillin disk with 5 or 10 micrograms
 Recently the detection was made simple
  with testing on Oxacillin or Cefotixin disks
      Newer Methods in MRSA
            detection
   Recently developed chromogenic media
    combine primary growth and selectivity
    with differentiation from coagulase
    negative staphylococci. These media show
    improved specificity when compared with
    traditional media. Sensitivity is also
    improved but requires 48hrs incubation to
    achieve >85%.
       Chromagar for detection of
                MRSA
   BBL™ CHROMagar™ MRSA
    is a selective and differential
    medium for the qualitative
    direct detection of nasal
    colonization by Methicillin
    resistant Staphylococcus
    aureus (MRSA) to aid in the
    prevention and control of
    MRSA infections in healthcare
    settings. The test is performed
    on anterior nares swab
    specimens from patients and
    healthcare workers to screen
    for MRSA colonization. BBL
    CHROMagar MRSA is not
    intended to diagnose MRSA
    infection nor to guide or
    monitor treatment for
    infections
Detection of ESBL strains in
    Enterobacteriaceae
    Methods to detect ESBL
         producers
 Various
 combination of
 Antibiotic disks
 are used for the
 detection of
 ESBL producers
 in Gram negative
 bacilli.
  Antibiotic disks helps in
detection of ESBL producers
                 Various Antibiotic
                  disks kept at specified
                  place helps in
                  detection of ESBL
                  producers
     Limitation of Disk Diffusion
              Methods
   Disk diffusion methods
    are not suitable for slow
    grwoing bacterial
    pathogens
   The great limitation being
    for testing on
    Mycobacterium
    tuberculosis which
    needs varied, technically
    demanding methods
                Antibiotic Assays
   The amount of antibiotic in the Blood or other body
    fluids of patients measured for following reasons
    1 To ensure adequate theraputic concentrations are
    reached – in serious infections with parental
    administration
    2 To avoid toxic concentration
    3 To study pharmacokinetics of Antibiotic
    4 To identify the clinical response with identified
    concentration
    At present Automated methods are popularly
    used as the testing protocols are technically
    demanding
Dr.T.V.Rao MD
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.
Various Methods of Antibiotic
sensitivity testing are used for
    quality control studies
Created for awareness to Medical
  and Paramedical staff in the
       Developing world
   Dr.T.V.Rao MD ‘s Basic learning series
                   Email
          doctortvrao@gmail.com

				
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Description: Antibiotic Sensitivity Testing