Transcript SET UP: Susceptibility Testing – KB, MIC and MBC

• KIRBY – BAUER
• MINIMUM INHIBITORY
CONCENTRATION
• MINIMUM BACTERIOCIDAL
CONCENTRATION
SUSCEPTIBILITY TESTING
• Antibiotic - Antimicrobial agent
produced by a bacterium or fungus
• Chemotherapeutic agent – can be
produced either naturally or
synthetically
SUSCEPTIBILITY TESTING
SUSCEPTIBILITY TESTING PERFORMED……WHY????
• Isolate is considered to be clinically significant
• Examples: Sterile site, wound, abscess, UTI, RTI, BSI
• Isolate susceptibility IS NOT predictable
• GAS (Streptococcus pyrogenes), causative agent of
‘Group A Strep’ still has a predictable susceptibility to
PENICILLIN (Still the drug of choice [if patient is not
allergic])
• Isolate must be able to grow rapidly on solid media
(within 24 hours)
SUSCEPTIBILITY TESTING
CRITERIA IS STANDARDIZED BY THE
‘CLINCAL LABORATORY STANDARDS
INSTITUTE’ (CLSI)
CRITERIA THAT IS STANDARDIZED:
• INOCULUM (0.5 McFarland = 1.5 x 108
cfu/mL) in LOG Phase
• MEDIUM
• ATMOSPHERE
• INCUBATION TIME
• INTERPRETATION
SUSCEPTIBILITY TESTING
STANDARDIZED SUSCEPTIBILITY
TESTING FOR:
• BACTERIA
• YEAST (Candida spp.)
• MYCOBACTERIA
• MOULDS
• VIRUSES
KIRBY – BAUER SUSCEPTIBILITY METHOD
• DISK DIFFUSION
• Paper impregnated with a certain concentration
of antibiotic/chemotherapeutic agent
• WHAT DO WE NEED?????
• PURE CULTURE - One type of microorganism
• STANDARDIZATION – 0.5 McFarland with a
turbidity meter
KIRBY – BAUER SUSCEPTIBILITY METHOD
• WHAT DO WE NEED?????
• STANDARD GROWTH MEDIUM – MUELLER – HINTON
(standard pH, vitamins, elements)
• TEMPERATURE – 35C
• ATMOSPHERE – AIR
• DURATION – 18 – 24 hours
• STANDARD DISKS IMPREGNATED WITH A CERTAIN
ANTIBIOTIC THAT CAN BE USED TO TREAT THE
PARTICULAR MICROORGANISM
KIRBY – BAUER SUSCEPTIBILITY METHOD
AFTER PREPARATION OF 0.5
MCFARLAND STANDARD SUSPENSION
IN STERILE SOLUTION (WATER,
SALINE, OR BROTH):
• INOCULATE MUELLER – HINTON
AGAR PLATE
• SET UP A PURITY PLATE
KIRBY – BAUER TESTING (KB)
RESULTS
DIAMETER= ZONE OF INHIBITION (MEASURED IN MILLIMETERS)
KIRBY – BAUER INTERPRETATIONS
INTERPRETATION STANDARDS FOR EACH
ANTIMICROBIAL AGENT ARE SET BY THE
CLSI (CLINICAL LABORATORY STANDARDS
INSTITUTE)
DIAMETER IS MEASURED IN MILLIMETERS.
THIS RELATES APPROXIMATELY LINEARLY
TO THE ANTIMICROBIAL’S LOG2 MIC
KIRBY – BAUER INTERPRETATIONS
ZONE SIZES PLACED INTO CATEGORIES:
SENSITIVE – Largest zone of inhibition; maybe the
appropriate drug; bacterial resistance is absent
INTERMEDIATE – Smaller zone of inhibition; may be
appropriate drug if a high concentration can be achieved;
may be susceptible, but possibly to a lesser degree than
against an isolate which is susceptible
RESISTANT – Smallest zone of inhibition; may not be an
appropriate choice; organism does not exhibit invitro serum
achievable levels of the drug; treatment success is doubtful
KIRBY – BAUER INTERPRETATIONS
ADVANTAGES:
•SIMPLE
•INEXPENSIVE
•STANDARDIZED
DISADVANTAGES:
•BACTERIOSTATIC
•DRUGS NEED TO BE SOLUBLE TO DISPURSE
THROUGH MEDIUM
•USED AS A GUIDE; DOES NOT DETERMINE THE
EXACT AMOUNT OF ANTIBIOTIC TO BE USED
MINIMUM INHIBITORY CONCENTRATION
(MIC)
• MORE ACCURATE WAY OF MEASURING THE
SUSCEPTIBILITY OF A MICROORGANISM
• MICRODILUTION IN 96 WELL PLATE WITH
DOUBLING DILUTIONS WITH INCREASING
CONCENTRATIONS OF A DRUG AGAINST A
STANDARDIZED INOCULUM OF MICROORGANISM
0.25
0.5
1.0
2.0
4.0
8.0
16.0
MINIMUM INHIBITORY CONCENTRATION
(MIC)
RESULTS
0.25
0.5
1
2
4
8
CLOUDINESS OR A PELLET = GROWTH
THE ‘MIC’ = THE FIRST WELL THAT HAS NO GROWTH
VISUALLY
MIC = __________________
MINIMUM INHIBITORY
CONCENTRATION (MIC)
ADVANTAGES:
• MIC IS A BETTER PREDICTOR OF APPROPRIATE DRUG
• MAY BE ABLE TO USE A LOWER DOSE OF ANTIBIOTIC
• MAY BE ABLE TO USE A LESS EXPENSIVE BUT STILL
EFFECTIVE ANTIBODY
• DECREASE CHANCE OF TOXIC EFFECTS ON THE
PATIENT’S SYSTEMS/ORGANS
• GET A ‘QUALITATIVE’ (INTERPRETATIONS) AND
‘QUANTITATIVE’ (MIC LEVEL)
MINIMUM INHIBITORY
CONCENTRATION (MIC)
DISADVANTAGES:
• NOT APPROPRIATE FOR FASTIDIOUS
MICROORGANISMS
• DRUGS MAY COME OUT OF SOLUTION AND
RENDER
THEM INEFFECTIVE
MINIMUM BACTERIOCIDAL
CONCENTRATION (MBC)
• Inoculate the negative (no growth)
wells from an MIC plate onto solid
media
• Incubate overnight
• Look for growth
• The first plate with NO GROWTH
is the ‘MBC’
MINIMUM Bacteriocidal
CONCENTRATION (MBC)
RESULTS
0.25
0.5
1
2
4
8
So, our MIC is 4 mcg/mL
• Plate the 4 and 8 well to solid media
• Incubate overnight
If we saw growth in 4 but not 8?MBC = _________
If we saw growth in both 4 and 8  ?MBC=_______
TM
GM
TM
YELLOW = GROWTH AREA
WHITE = ZONE OF INHIBITION
CENTER CIRCLE IS THE DISK
AK