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									Dr.T.V.Rao MD

   Dr.T.V.Rao MD   1
  Uses of Antibiotic Sensitivity
                    test: A laboratory
Antibiotic sensitivity
 test which determines how effective
 antibiotic therapy is against a bacterial
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
                   Dr.T.V.Rao MD               2
  What is the goal of Antibiotic
      Sensitivity testing?
 The goal of antimicrobial susceptibility testing is to
  predict the in vivo success or failure of antibiotic
  therapy. Tests are performed in vitro, and measure
  the growth response of an isolated organism to a
  particular drug or drugs. The tests are performed
  under standardized conditions so that the results are
  reproducible. The test results should be used to
  guide antibiotic choice. The results of antimicrobial
  susceptibility testing should be combined with
  clinical information and experience when selecting
  the most appropriate antibiotic for our patients.

                         Dr.T.V.Rao MD                     3
    Components of Antibiotic
      Sensitivity Testing
1.The identification of relevant pathogens in
 exudates and body fluids collected from
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
                      Dr.T.V.Rao MD               4
 Why Need Continues for Testing
     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.
 Antibiotic sensitivity
  testing is essential part of
  Medical Care
                           Dr.T.V.Rao MD   5
 Susceptibility test, main purposes:
   As a guide for treatment
     Sensitivity of a given m.o. to known conc. of drugs
     Its concentration in body fluids or tissues

   As an epidemiological tool
     The emergence of resistant strains of major
       pathogens (e. g. Shigella, Salmonella typhi)
     Continued surveillance of the susceptibility pattern
       of the prevalent strains (e. g. Staphylococci, Gram-
       negative bacilli)

                         Dr.T.V.Rao MD                        6
 Methods for antimicrobial susceptibility testing
    Indirect method
      cultured plate from pure culture

    Direct method
      Pathological specimen
      e.g. urine, a positive blood culture, or a swab of pus

                           Dr.T.V.Rao MD                        7
    What Does the Laboratory Need to Know
about Antimicrobial Susceptibility Testing (AST) ?
 Which organisms to test?
 What methods to use?
 What antibiotics to test?
 How to report results?

                     Dr.T.V.Rao MD                   8
Routine Susceptibility Tests
Disk diffusion
 (Kirby Bauer)
Broth micro-
 dilution MIC
  NCCLS reference

                     Dr.T.V.Rao MD   9
  Preparing for Testing
 Inoculum preparation
 - Number of test organisms can be determined using
  different methods:

   Direct count (Microscopic examination)
   The optical density (OD) at 600 nm
   Plate count: making dilution first
   Turbidity standard (McFarland) routinely
                      Dr.T.V.Rao MD                   10
   Choosing the Appropriate
 Drugs for routine susceptibility tests:
    Set 1: the drugs that are available in most hospitals
     and for which routine testing should be carried out for
     every strain

    Set 2: the drugs that are tested only:
       at the special request of the physician
       or when the causative organism is resistant to the first-
        choice drugs
       or when other reasons (allergy to a drug, or its
        unavailability) make further testing justified

                            Dr.T.V.Rao MD                           11
    Table 1: Basic sets of drugs for routine susceptibility
                         tests (
                                Set 1                Set 2
Staphylococcus                  Benzyl penicillin    Gentamicin
                                Oxacillin            Amikacin
                                Erythromycin         Co-trimoxazole
                                Tetracycline         Clindamycin

Intestinal                      Ampicillin           Norfloxacin
                                Nalidixic acid

Enterobacteriaceae              Sulfonamide          Norfloxacin
Urinary                         Trimethoprim         Chloramphenicol
                                Co-trimoxazole       Gentamicin
                                Nalidixic acid

Blood and tissues               Ampicillin           Cefuroxime
                                Chloramphenicol      Ceftriaxone
                                Cotrimoxazole        Ciprofloxacin
                                Tetracycline         Piperacillin
                                Gentamicin           Amikacin

Pseudomonas aeruginosa          Piperacillin         Amikacin

                                     Dr.T.V.Rao MD                     12
Antimicrobial Susceptibility
 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 bacteria

                         Dr.T.V.Rao MD                         13
    Susceptibility Testing Methods

                               Incubate plate
Inoculate      Place disks
                               18-24 hr, 35 C
MH plate       on agar plate   Measure and
                               record zone of
                               inhibition around
                               each disk
        Diffusion Method
 Disc diffusion method : The Kirby-Bauer test
   Antibiotic-impregnated filter disc*
   Susceptibility test against more than one
    antibiotics by measuring size of “inhibition zone
   1949: Bondi and colleagues paper disks
   1966: Kirby, Bauer, Sherris, and Tuck  filter
    paper disks
     Demonstrated that the qualitative results of
       filter disk diffusion assay correlated well with
       quantitative results from MIC tests
                       Dr.T.V.Rao MD                      15
   Disc Diffusion Method
Procedure (Modified Kirby-Bauer method: National
  Committee for Clinical Laboratory Standards. NCCLS)
   Prepare approximately. 108 CFU/ml bacterial inoculum in
    a saline or tryptic soy broth tube (TSB) or Mueller-Hinton
    broth (5 ml)
     Pick 3-5 isolated colonies from plate
     Adjust the turbidity to the same as the McFarland No.
       0.5 standard.*
   Streak the swab on the surface of the Mueller-Hinton agar (3
    times in 3 quadrants)
   Leave 5-10 min to dry the surface of agar

                          Dr.T.V.Rao MD                            16
      Examining purity of plate
Select the Colonies from Pure Isolates


           Reflect               Transmitted light
           ed light

                 Dr.T.V.Rao MD                       17
     Disk Diffusion
                                Prepare inoculum
   Prepare inoculum
Select colonies

                Dr.T.V.Rao MD                      18
          Prepare the Material for
Standardize inoculum
Suspension as per Mac farland              Mix well

                                Dr.T.V.Rao MD         19
 Swab the plate with optimal

Remove sample           Swab plate

                Dr.T.V.Rao MD        20
 Select the Disks and Apply

Select disks

               Dr.T.V.Rao MD   21
Incubate Overnight

       Dr.T.V.Rao MD   22
     Disc Diffusion Method
   Place the
    appropriate drug-
    impregnated disc on
    the surface of the
    inoculated agar plate
   Invert the plates and
    incubate them at 35 oC, o/n
    (18-24 h)
   Measure the
    diameters of
    inhibition zone in

                            Dr.T.V.Rao MD   23
Read the Results with

                  Dr.T.V.Rao MD   24
   Disc Diffusion Method
 Measurement of the diameters of inhibition
     Measure from the edge where the growth stats,
      BUT there are three exceptions
          With sulfonamides and co-trimoxazole, ignore slight
           growth within the zone
          Certain Proteus spp. may swarm into the area of
          When beta-lactamase producing Streptococci are tested,
           zone of inhibition are produced with a heaped-up,
           clearly defined edge, regardless of the size of the
           inhibition zone, they should be reported as resistant

                           Dr.T.V.Rao MD                            25
 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
  sensitive, resistant, or
  intermediate. Many
  charts have a
  corresponding column
  that also gives the MIC
  (minimal inhibitory
  concentration) for that
                         Dr.T.V.Rao MD   26
            Disc Diffusion Method
            Reporting the Results
 Interpretation of results
    By comparing with the diameters with
     “standard tables”
   Susceptible
   Intermediate susceptible
          Low toxic antibiotics: Moderate susceptible
          High toxic antibiotics: buffer zone btw resistant
           and susceptible
     Resistant
                         Dr.T.V.Rao MD                     27
  Factors Affecting Size of Zone
           of Inhibition
                                  Larger zones with light
 Inoculum density                    inoculum and vice versa

                                  If after application of disc, the
 Timing of disc application          plate is kept for longer time at
                                      room temperature, small zones
                                      may form

 Temperature of incubation       Larger zones are seen with
                                      temperatures < 35 oC
 Incubation time
                                  Ideal 16-18 hours; less time
                                      does not give reliable results
                          Dr.T.V.Rao MD                                  28
 Factors Affecting Size of Zone of

 Size of the plate
                          Smaller plates
                                  accommodate less
                                  number of discs
 Depth of the agar
  medium (4 mm)                Thin media yield
                                  excessively large
                                  inhibition zones and vice

 Proper spacing of            Avoids overlapping of
  the discs (2.5 cm)              zones

                       Dr.T.V.Rao MD                          29
Factors Affecting Size of Zone of
 Potency of antibiotic           Deterioration in contents leads
  discs                              to reduced size

 Composition of                  Affects rate of growth,
  medium                             diffusion of antibiotics and
                                     activity of antibiotics

 Acidic pH of medium             Tetracycline, novobiocin,
                                     methicillin zones are larger

                                  Aminoglycosides,
 Alkaline pH of                     erythromycin zones are larger
                                  Subjective errors in
 Reading of zones                   determining the clear edge

                          Dr.T.V.Rao MD                              30
   Quality Assurance in Antibiotic
       Susceptibility Testing
 Visit - WHO-Regional Office for South East Asia
   Medium: Mueller-Hinton agar plates
      Enterococcus faecalis (ATCC 29212 or 33l86) and a disc of
       co-trimoxazole 20 mm in diameter of the inhibition
   Procedure: Modified Kirby-Bauer method
    recommended by National Committee on Clinical
    Laboratory Services (NCCLS)
   Susceptibility test with quality control strains

                          Dr.T.V.Rao MD                            31
     Quality Assurance in Antibiotic
    Susceptibility Testing with Control
 Susceptibility test with
   quality control strains
for every new batch of
   Mueller-Hinton agar
    Staphylococcus
     aureus (ATCC 25923)
    Escherichia coli
     (ATCC 25922)
    Pseudomonas
     aeruginosa (ATCC
     2785 )

                             Dr.T.V.Rao MD   32
  Quality Assurance in Antibiotic
        Susceptibility Test
 Salient features of quality control
     Use antibiotic discs of 6 mm diameter
     Use correct content of antimicrobial agent per
     Store supply of antimicrobial discs at -20 oC
     Use Mueller-Hinton medium for antibiotic
      sensitivity determination
     Use appropriate control cultures
     Use standard methodology for the test

                      Dr.T.V.Rao MD                    33
 Need for Modified Methods
Modified Methods in Disc diffusion for
 Antibiotic sensitivity testing to be used for
 detections of following bacterial isolates
3 Enterobacteriaceae and Gram negative
 bacteria and Carbapenems resistant using
 Modified Hodge test
                   Dr.T.V.Rao MD                 34
         Dilution Method
                Concentration (MIC)
Minimum Inhibition
   The lowest concentration of antimicrobial agent that
    inhibits bacterial growth/ multiplication

 Minimum Bactericidal Concentration (MBC) or
  Minimum Lethal Concentration (MLC)
  The lowest concentration of
   antimicrobial agent that allows less than
   0.1% of the original inoculum to survive
           Antimicrobial susceptibility
            testing using micro-broth
                                           64 32 16 8 4   2

                  •                •
•      •                       •
•      •                               •
•     •
    96 well microtiter plate
  Broth Dilution Method
 Making dilutions (2-fold) of antibiotic in broth
 Mueller-Hinton, Tryptic Soy Broth
  Inoculation of bacterial inoculum, incubation,
    Controls: no inoculum, no antibiotic
  Turbidity visualization  MIC
  Sub culturing of non-turbid tubes, overnight
  Growth (bacterial count)  MBC

Creating Dilutions

       Dr.T.V.Rao MD   38
     Broth Dilution Method
                                              Day 1

128 64     32   16   8   4       2 C1 C2
                                               Add 1 ml of test bacteria
                                               (1*106 CFU/ml) to tubes
                                               containing 1 ml broth and
                                               concentration of
                                               antibiotic (mg/l)

64   32   16    8    4   2   1 C1 C2
                                           C1 = No antibiotic, check
     Bacterial conc.= 5*105 CFU/ml         viability on agar plates
           Incubate 35 oC, o/n
                                           C2 = No test bacteria
            Broth Dilution Method

                                              Day 2
 64   32   16    8      4   2   1 C1 C2
                                              Record visual turbidity
                                              Subculture non-turbid tubes
                                              to agar plates (use 0.01 ml
                                              standard loop)
0.01 ml (spread plate), Incubate 35 oC, o/n
                                              MIC = 16 mg/l

                                              Day 3
                                              Determine CFU on plates:
                                              At 16 mg/ = 700 CFU/ml >
                                              0.1% of 5*105 CFU/ml
      64           32           16
                                              MBC = 32 mg/l

    Broth Dilution Method
 100% of original bacterial conc.
    = 5*105 CFU/ml

 0.1%
    = [(5*105)*0.1]/100 CFU/ml
    = 500 CFU/ml

 The bacteria count should be less than 5 CFU on agar plate
  subcultured with 0.01 ml
    500*0.01 = 5 CFU

   Broth Dilution Method are
      Technically Difficult
Disadvantages :        Solutions??
 Only one                       Agar dilution
  antibiotic & one                method
  organism can be                Disc diffusion
  tested each time
                                 Micro broth dilution
 Time-consuming                  method

                Dr.T.V.Rao MD                            42
    Micro broth Dilution
 Micro dilution plates:
   “Micro dilution/ Micro broth dilutions”
  96 wells/ plate: simultaneously performed
    with many tests organisms/ specimens, less
    reagent required

 Manually prepared
 Commercially prepared
   Frozen or Dried/ lyophilized
   Consistent performance but high cost
   May suffer from degradation of antibiotic during
    shipping and storage

  Agar Dilution Method
   Making dilutions of antimicrobial agent in melted
    media and pouring plates
     One concentration of antibiotic/ plate
     Possible for several different strains/plate

        64 uGu/ml       32 ug/ml        16 ug/ml

  Agar Dilution Method
 Procedure                  
     Inoculation of bacterial inoculum (McFarland
      No. 0.5)
          Using a replicating inoculator device called “A Steers-
           Foltz replicator”
          Delivers 0.001 ml of bacterial inoculum
     Incubation
     Spot of growth

                                               32 ug/ml          45
 Minimal inhibitory concentration

The lowest
 concentration of
 antimicrobial agent
 that inhibits the
 growth of a
   Susceptible
   Intermediate
   Resistant
                   Dr.T.V.Rao MD    46
Clinical Conditions when MICs are
  Immunosuppressed patients (HIV, cancer,
  Prosthetic devices
  Patients not responding despite “S” Reports
                    Dr.T.V.Rao MD                47
    Inoculum Preparation
              MIC Testing
         (NCCLS Reference Method)
Final inoculum
 3 – 5 x 105 CFU/ml
 (3 – 5 x 104
                  Dr.T.V.Rao MD     48
Select Micro titration plate and
  prepare optimal inoculum
                          Prepare inoculum
     Micro dilution
     MIC tray

                      Dr.T.V.Rao MD          49
Dilute & mix inoculum

        Dr.T.V.Rao MD   50
Pour inoculum into reservoir and
       inoculate MIC tray

             Dr.T.V.Rao MD         51
               Incubate overnight
    Do not forget to check the purity of Inoculum

purity plate

                       Dr.T.V.Rao MD                52
   Optimal Use of Purity Plates
Sub final test suspension to non-selective medium
 (after inoculating MIC test)
Streak for isolation (avoid several specimens per
 plate - may not reveal contaminants if no isolated
Examine before reading MIC (usually at 16-20 h)
Re-incubate if Antibiograms questionable
MICs         - +

         >6        >6
         4         4
The gradient technique,
Etest is a well established
  AST method in
  microbiology laboratories
  around the world. The Etest
  technique comprises a
  predefined gradient of
  antibiotic concentrations on
  a plastic strip, and can be
  used to determine the
  Minimum Inhibitory
  Concentration (MIC) of
  antibiotics, antifungal agents
  and antimycobacterial

                            Dr.T.V.Rao MD   55
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
                            particularly valuable in
                            such situations, when
                            on-scale MICs are
                            needed for treatment
              Dr.T.V.Rao MD                             56
     Antimicrobial Gradient Testing
 Read plates
                                Read MIC
                               where elipse
MIC of the Bacteria can be read

             Dr.T.V.Rao MD        58
MIC on a strip
    Serum Susceptibility Tests
     To determine drug concentration in the patient’s
      serum = MIC*SIT
            The Serum Inhibitory Titer (SIT)
              The highest dilution of patient’s serum that inhibit

     To determine the ability of drug in the patient’s
      serum to kill bacteria
            The Serum Bactericidal Level (SBL)
              The lowest dilution of patient’s serum that kills bacteria
             Technically Demanding
5-Jan-06                         Chiang Mai University                      60
Antibiotic Sensitivity testing
can be done with automation

            Dr.T.V.Rao MD        61
  VITEK 2 Automates Reporting
         of Resistance
 Integrated in the VITEK 2
  system is the Advanced
  Expert System (AES™), a
  software which validates
  and interprets susceptibility
  test results, and detects
  antibiotic resistance
  mechanisms. The AES
  Expert System is the most
  developed software system
  in this field, and is capable
  of identifying even
  emerging and low-level

                            Dr.T.V.Rao MD   62
     What is the Role of
  Microbiology Departments
                       a staff member
 Each laboratory should have
  with the time, interest, and expertise to provide
  leadership in antibiotic testing and resistance.
  This person would read relevant publications,
  network with other laboratories, and evaluate
  potentially useful tests to detect new forms of
  resistance before new CLSI-recommended
  tests become available”
 - Ken Thomson, Emerging Infect. Dis., 2001

                      Dr.T.V.Rao MD                   63
1Usanee Anukool (Ph.D.) Clinical Microbiology,AMS,
Chiang Mai University
2National Committee For Clinical Laboratory Standards. 1998.
NCCLS document M100 - S8 . Performance Standards for
Antimicrobial Susceptibility Testing. 8th edition, NCCLS, Waynae,

                            Dr.T.V.Rao MD                           64
Created by Dr.T.V.Rao MD for ‘e’
learning resources for Microbiologists
        in Developing World
                Email

               Dr.T.V.Rao MD             65

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