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 10 8 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 LOG 2 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

MINIMUM INHIBITORY CONCENTRATION (MIC) RESULTS 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 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 AK YELLOW = GROWTH AREA WHITE = ZONE OF INHIBITION CENTER CIRCLE IS THE DISK