Antibiotic Sensitivity Testing

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ANTIBIOTIC SENSITIVITY TESTINGSKILL BASED
LEARNING

• Dr.T.V.Rao MD

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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.

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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.

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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.

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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

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Introduction

• 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)

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Introduction

• 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

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What Does the Laboratory Need to Knowabout
Antimicrobial Susceptibility Testing (AST) ?

• Which organisms to test?
• What methods to use?
• What antibiotics to test?
• How to report results?

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Routine Susceptibility Tests

• Disk diffusion (Kirby Bauer)
• Broth micro-dilution MIC
• NCCLS reference method
• Etest

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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.

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Choosing the Appropriate Antibiotic

• 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

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Table 1 Basic sets of drugs for routine
susceptibility tests (http//w3.whosea.org/)
Set 1 Set 2
Staphylococcus Benzyl penicillinOxacillinErythromycinTetracyclineChloramphenicol GentamicinAmikacinCo-trimoxazoleClindamycin
Intestinal AmpicillinChloramphenicolCo-trimoxazoleNalidixic acidTetracycline Norfloxacin
Enterobacteriaceae Urinary SulfonamideTrimethoprimCo-trimoxazoleAmpicillinNitrofurantoinNalidixic acidTetracycline NorfloxacinChloramphenicolGentamicin
Blood and tissues AmpicillinChloramphenicolCotrimoxazoleTetracyclineGentamicin CefuroximeCeftriaxoneCiprofloxacinPiperacillinAmikacin
Pseudomonas aeruginosa PiperacillinGentamicinTobramycin Amikacin
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Antimicrobial Susceptibility Testing

• 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

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Susceptibility Testing Methods
Incubate plate 18-24 hr, 35 C Measure and record
zone of inhibition around each disk
Inoculate MH plate
Place disks on agar plate
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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

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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

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Examining purity of plateSelect the Colonies
from Pure Isolates
Transmitted light
Reflected light
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Disk Diffusion Test
Prepare inoculum suspension
Prepare inoculum suspension
Select colonies
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Prepare the Material for Inoculation
Standardize inoculum Suspension as per Mac
farland standard
Mix well
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Swab the plate with optimal sample
Remove sample
Swab plate
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Select the Disks and Apply
Select disks
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Incubate Overnight
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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 mm

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Read the Results with Precision
Transmitted Light
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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

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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.

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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

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Factors Affecting Size of Zone of Inhibition

• 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 lt 35 oC
• Ideal 16-18 hours less time does not give
  reliable results

• Inoculum density
• Timing of disc application
• Temperature of incubation
• Incubation time

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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

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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

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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

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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 )

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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 -20 oC
• Use Mueller-Hinton medium for antibiotic
  sensitivity determination
• Use appropriate control cultures
• Use standard methodology for the test

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Need for Modified Methods

• 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

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Dilution Method

• Minimum Inhibition Concentration (MIC)
• 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

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Antimicrobial susceptibilitytesting using
micro-broth dilutions
ug/ml 64 32 16 8 4 2
96 well microtiter plate
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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

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Creating Dilutions
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Broth Dilution Method
Day 1 Add 1 ml of test bacteria (1106 CFU/ml)
to tubes containing 1 ml broth and concentration
of antibiotic (mg/l)
Controls C1 No antibiotic, check viability on
agar plates immediately C2 No test bacteria
Bacterial conc. 5105 CFU/ml Incubate 35 oC, o/n
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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 gt 0.1 of 5105 CFU/ml MBC 32 mg/l
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Broth Dilution Method

• 100 of original bacterial conc.
• 5105 CFU/ml
• 0.1
• (5105)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
• 5000.01 5 CFU

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Broth Dilution Method are Technically Difficult

• Disadvantages
• Only one antibiotic one organism can be tested
  each time
• Time-consuming

• Solutions??
• Agar dilution method
• Disc diffusion method
• Micro broth dilution method

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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

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Agar Dilution Method

• Procedure
• 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
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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

MIC
32 ug/ml
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Minimal inhibitory concentration

• The lowest concentration of antimicrobial agent
  that inhibits the growth of a bacterium
• Interpret
• Susceptible
• Intermediate
• Resistant

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Clinical Conditions when MICs are Useful

• Endocarditis
• Meningitis
• Septicemia
• Osteomyelitis
• Immunosuppressed patients (HIV, cancer, etc.)
• Prosthetic devices
• Patients not responding despite S Reports

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Inoculum Preparation MIC Testing (NCCLS
Reference Method)

• Standardize inoculum suspension
• Final inoculum concentration
• 3 5 x 105 CFU/ml
• (3 5 x 104 CFU/well)

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Select Micro titration plate and prepare optimal
inoculum
Prepare inoculum suspension
Micro dilution MIC tray
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Dilute mix inoculumsuspension
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Pour inoculum into reservoir and inoculate MIC
tray
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Incubate overnightDo not forget to check the
purity of Inoculum
Inoculate purity plate
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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

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Read MICs
-

0.5
1
2
4
8
16
32
64
gt64
gt64
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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.

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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 particularly valuable in such
  situations, when on-scale MICs are needed for
  treatment decisions.

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Antimicrobial Gradient TestingE-test
Read plates after recommended Incubation
Read MIC where elipse intersects scale
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MIC of the Bacteria can be read Directly
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MIC on a strip abbiodisk.com
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Serum Susceptibility Tests

• To determine drug concentration in the patients
  serum MICSIT
• The Serum Inhibitory Titer (SIT)
• The highest dilution of patients serum that
  inhibit bacteria
• To determine the ability of drug in the patients
  serum to kill bacteria
• The Serum Bactericidal Level (SBL)
• The lowest dilution of patients serum that kills
  bacteria
• Technically Demanding

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Antibiotic Sensitivity testing can be done with
automation
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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.

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What is the Role of Microbiology Departments

• 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

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References

• 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.

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