Antibiotic sensitivity testing   Skills in Microbiology                                       in
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Antibiotic sensitivity testing , Skills in Microbiology

Antibiotic sensitivity testing , Skills in Microbiology



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Antibiotic sensitivity testing   Skills in Microbiology                                       in Antibiotic sensitivity testing Skills in Microbiology in Presentation Transcript

    Dr.T.V.Rao MD
    Dr.T.V.Rao MD
  • 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
  • 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
    What is the goal of Antibiotic Sensitivity testing?
  • 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
  • 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
  • Dr.T.V.Rao MD
    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
    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
  • Which organisms to test?
    What methods to use?
    What antibiotics to test?
    How to report results?
    What Does the Laboratory Need to Knowabout Antimicrobial Susceptibility Testing (AST) ?
    Dr.T.V.Rao MD
  • Routine Susceptibility Tests
    Disk diffusion (Kirby Bauer)
    Broth micro-dilution MIC
    NCCLS reference method
    Dr.T.V.Rao MD
  • Dr.T.V.Rao MD
    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 (Spectrophotometry)
    Plate count: making dilution first
    Turbidity standard (McFarland) routinely performed.
  • Dr.T.V.Rao MD
    Choosing the Appropriate Antibiotic
    Drugs for routine susceptibility tests:
    Set 1:the drugs that are availablein 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 isresistantto the first-choice drugs
    or when other reasons (allergy to a drug, or its unavailability) make further testing justified
  • Dr.T.V.Rao MD
    Table 1: Basic sets of drugs for routine susceptibility tests(
  • 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
    Antimicrobial Susceptibility Testing
  • Susceptibility Testing Methods
    Incubate plate
    18-24 hr, 35 C
    Measure and record zone of inhibition around each disk
    MH plate
    Place disks
    on agar plate
  • Dr.T.V.Rao MD
    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
    Disc Diffusion Method
    Procedure(Modified Kirby-Bauer method: National Committee for Clinical Laboratory Standards. NCCLS)
    Prepareapproximately.108 CFU/ml bacterial inoculum in a saline or tryptic soy broth tube(TSB) or Mueller-Hinton broth (5ml)
    Pick 3-5 isolated colonies from plate
    Adjust the turbidity tothesame as the McFarland No. 0.5 standard.*
    Streak the swabon 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
    Examining purity of plateSelect the Colonies from Pure Isolates
    Transmitted light
    Reflected light
  • Disk Diffusion Test
    Prepare inoculum
    Prepare inoculum
    Select colonies
    Dr.T.V.Rao MD
  • Prepare the Material for Inoculation
    Standardize inoculum
    Suspension as per Mac farland standard
    Mix well
    Dr.T.V.Rao MD
  • Swab the plate with optimal sample
    Remove sample
    Swab plate
    Dr.T.V.Rao MD
  • Select the Disks and Apply
    Select disks
    Dr.T.V.Rao MD
  • Incubate Overnight
    Dr.T.V.Rao MD
  • Disc Diffusion Method
    • Place the appropriate drug-impregnated disc on the surface of the inoculated agar plate
    • Invert the plates and incubate them at35oC, o/n (18-24 h)
    • Measure the diameters of inhibition zone in mm
    Dr.T.V.Rao MD
  • Read the Results with Precision
    Dr.T.V.Rao MD
  • Dr.T.V.Rao MD
    Disc Diffusion Method
    • Measurement of the diameters of inhibition zone
    • 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 inhibition
    • 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
    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 drug.
  • Dr.T.V.Rao MD
    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
    Factors Affecting Size of Zone of Inhibition
    Inoculum density
    Timing of disc application
    Temperature of incubation
    Incubation time
    • Larger zones with light inoculum and vice versa
    • If after application of disc, the plate is kept for longer time at room temperature, small zones may form
    • Larger zones are seen with temperatures < 35oC
    • Ideal 16-18 hours; less time does not give reliable results
  • Dr.T.V.Rao MD
    Factors Affecting Size of Zone of Inhibition
    • Size of the plate
    • Depth of the agar medium(4 mm)
    • Proper spacing of the discs (2.5 cm)
    • Smaller plates accommodate less number of discs
    • Thin media yield excessively large inhibition zones and vice versa
    • Avoids overlapping of zones
  • Dr.T.V.Rao MD
    Factors Affecting Size of Zone of Inhibition
    • Deterioration in contents leads to reduced size
    • Affects rate of growth, diffusion of antibiotics and activity of antibiotics
    • Tetracycline, novobiocin, methicillin zones are larger
    • Aminoglycosides, erythromycin zones are larger
    • Subjective errors in determining the clear edge
    • Potency of antibiotic discs
    • Composition of medium
    • Acidic pH of medium
    • Alkaline pH of medium
    • Reading of zones
  • Dr.T.V.Rao MD
    Quality Assurance in Antibiotic Susceptibility Testing
    Visit - WHO-Regional Office for South East Asia website
    Medium:Mueller-Hinton agar plates
    Enterococcus faecalis (ATCC 29212 or 33l86) and a disc of co-trimoxazole 20 mm in diameter of the inhibition zone
    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
    Quality Assurance in Antibiotic Susceptibility Testing with Control strains
    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
    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 disc
    • Store supply of antimicrobial discs at -20oC
    • Use Mueller-Hinton medium for antibiotic sensitivity determination
    • Use appropriate control cultures
    • Use standard methodology for the test
  • Modified Methods in Disc diffusion for Antibiotic sensitivity testing to be used for detections of following bacterial isolates
    1 MRSA
    2 ESBL
    3 Enterobacteriaceae and Gram negative bacteria and Carbapenems resistant using Modified Hodge test
    Dr.T.V.Rao MD
    Need for Modified Methods
  • 35
    Dilution Method
    Minimum Inhibition Concentration (MIC)
    The lowest concentration of antimicrobial agent thatinhibitsbacterial 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 susceptibilitytesting using micro-broth dilutions
    64 32 16 8 4 2
    96 well microtiter plate
  • 37
    Broth Dilution Method
    Procedure Making dilutions (2-fold) of antibiotic in broth Mueller-Hinton, Tryptic Soy Broth
    Inoculation of bacterial inoculum, incubation, overnight
    Controls: no inoculum, no antibiotic
    Turbidity visualization  MIC
    Sub culturing of non-turbid tubes, overnight
    Growth (bacterial count)  MBC
  • Dr.T.V.Rao MD
    Creating Dilutions
  • 39
    Broth Dilution Method
    128 64 32 16 8 4 2 C1 C2
    64 32 16 8 4 2 1 C1 C2
    Day 1
    Add 1 ml of test bacteria (1*106 CFU/ml) to tubes containing 1 ml broth and concentration of antibiotic (mg/l)
    C1 = No antibiotic, check viability on agar plates immediately
    C2 = No test bacteria
    Bacterial conc.= 5*105 CFU/ml
    Incubate 35 oC, o/n
  • 40
    64 32 16 8 4 2 1 C1 C2
    64 32 16
    Broth Dilution Method
    Day 2
    Record visual turbidity
    Subculture non-turbid tubes to agar plates (use 0.01 ml standard loop)
    MIC = 16 mg/l
    0.01 ml (spread plate), Incubate 35 oC, o/n
    Day 3
    Determine CFU on plates:
    At 16 mg/ = 700 CFU/ml > 0.1% of 5*105 CFU/ml
    MBC = 32 mg/l
  • 41
    Broth Dilution Method
    100% of original bacterial conc.
    = 5*105 CFU/ml
    = [(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
  • Dr.T.V.Rao MD
    Broth Dilution Method are Technically Difficult
    Disadvantages :
    Only one antibiotic & one organism can be tested each time
    Agar dilution method
    Disc diffusion method
    Micro broth dilution method
  • 43
    Micro broth Dilution Method
    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
  • 44
    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
  • 45
    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
  • Dr.T.V.Rao MD
    Minimal inhibitory concentration
    The lowest concentration of antimicrobial agent that inhibits the growth of a bacterium
  • Clinical Conditions when MICs are Useful
    Immunosuppressed patients (HIV, cancer, etc.)
    Prosthetic devices
    Patients not responding despite “S” Reports
    Dr.T.V.Rao MD
  • Dr.T.V.Rao MD
    Inoculum PreparationMIC Testing (NCCLS Reference Method)
    Standardize inoculum suspension
    Final inoculum concentration
    3 – 5 x 105 CFU/ml
    (3 – 5 x 104 CFU/well)
  • Dr.T.V.Rao MD
    Select Micro titration plate and prepare optimal inoculum
    Prepare inoculum
    Micro dilution MIC tray
  • Dr.T.V.Rao MD
    Dilute & mix inoculumsuspension
  • Dr.T.V.Rao MD
    Pour inoculum into reservoir and inoculate MIC tray
  • Dr.T.V.Rao MD
    Incubate overnightDo not forget to check the purity of Inoculum
    purity plate
  • 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 colonies)
    Examine before reading MIC (usually at 16-20 h)
    Re-incubate if Antibiograms questionable
  • Read MICs
  • Dr.T.V.Rao MD
    The gradient technique, Etest®
    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 agents.
  • Dr.T.V.Rao MD
    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 decisions.
  • Antimicrobial Gradient TestingE-test®
    Read plates
    Read MIC
    where elipse
  • Dr.T.V.Rao MD
    MIC of the Bacteria can be read Directly
  • MIC on a strip
  • 5-Jan-06
    Chiang Mai University
    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 bacteria
    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
  • Antibiotic Sensitivity testing can be done with automation
    Dr.T.V.Rao MD
  • Dr.T.V.Rao MD
    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 resistance.
  • Each laboratory should have a staff member 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
    What is the Role of Microbiology Departments
  • 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, Pa.
    Dr.T.V.Rao MD
  • Dr.T.V.Rao MD
    For Articles of Interest on Antibiotics follow me on
  • Created by Dr.T.V.Rao MD for ‘e’ learning resources for Microbiologists in Developing World
    Dr.T.V.Rao MD