Chapter 28 Antimicrobial Susceptibility Testing Wang Hui COMMON IN VITRO ANTIMICROBIAL SUSCEPTIBILITY TESTS • Frequently used in vitro susceptibility tests • Infrequently used in vitro susceptibility tests Frequently used in vitro susceptibility tests • Dilution tests Broth microdilution Agar Semiautomated • Diffusion tests Disk (Kirby- Bauer) Fixed gradient (Etest or epsilometer) Spot tests (e.g.β-lactamase) Infrequently used in vitro susceptibility tests • • • • • • • Minimum bactericidal concentration (MBC) Serum bactericidal test (SBT,”Schlichter test”) Time-kill kinetic assay Drug synergy test Disk approximation “Checkerboard” Time-kill kinetic assay Broth Microdilution Minimum Inhibitory Concentration Test The broth microdilution minimum inhibitory concentration (MIC) test provides a quantitative measurement (in μg/ml) of the lowest concentration of an antimicrobial agent that inhibit the growth of a bacterium. Broth Microdilution Minimum Inhibitory Concentration Test • • • • • • • Selected antimicrobics Growth Media Bacteria for Inoculation Incubated of Inoculated Panels Reading Panels Specific Control Strains Interpretation of MIC results Specific Control Strains (1) (2) (3) (4) (5) (6) Enterococcus faecalis, ATCC 29212; S.aureus, (ATCC) 29213(β-lactamase positive); Escherichia coli, ATCC 25922; and Pseudomonas aeruginosa, ATCC 27853. Escherichia coli, ATCC 35218 H.influenzae, ATCC 49247 or 49766 when testing an H.influzae isolate; and (7) Neisseria gonorrhoeae, ATCC 49226 when testing an isolate of N. gonorrhoeae. Interpretation of MIC results • Susceptible, • Intermediately susceptible, or • Resistant. Broth Microdilution Disk diffusion Method(1) This method, commonly referred to as the Kirby-Bauer test, provides a qualitative measure of the ability of an antimicrobic to inhibit the growth of a rapidly growing bacterium. Disk diffusion Method(2) Disks containing a given concentration of an antimicrobic are placed on a confluently inoculated agar plate and incubated for 16 to 24 hours. At the end of the incubation period, zones of growth inhibition are measured across the disk diameter and recorded to the nearest millimeter. Disk Diffusion Method(3) Procedure of the Kirby-Bauer test 1、Growth Media 2、Bacteria for Inoculation 3、Filter Paper Disks Containing of an Antimicrobic 4、Zone Diameter of Inhibition Advantages and Disadvantages of Tests Advantages : 1.easy to substitute one disk for another 2. dependent on a commercial provider for the drug profiles available 3. easier to spot contamination and low-level resistance Disadvantages: 1. use only with rapidly growing organism 2. MBC can not be done using agar diffusion techniques E Test (1) A new test method has been developed recently called the E Test, which is a modification of the disk diffusion test but provides an MIC result. E Test(2) The MIC is read at the point where the zone intersects the MIC scale on the strip. Studies show this method to give greater than 95% agreement with the standardized broth microdilution method E test(3) Susceptibility Testing Of Anaerobic Bacteria The recommended control strains are: • Bacteroides fragilis, ATCC 25285; • Bacteroides thetaiotaomicron, ATCC 29741; • Clostridiun perfringens, ATCC 13124; • Eubacterium lentum , ATCC 430555 The β-lactamase Test •The Purpose of β-lactamase Test •Methods of β-lactamase •Organisms of routinely testing for βlactamase •D Test Methods of β-lactamase 1. Acidometric Method 2. Chromogenic Cephalosporin Method 3. Iodometric Method D Test At the time of inoculation, a disk containing a low concentration of an inducing antimicrobic is placed just outside the zone of inhibition normally obtained with an enzyme-susceptible antimicrobic . if induced β-lactamase is produced in the presence of the inducing agent, the zone will become flattened at the side adjacent to the inducing disk. Resistance Screening Plates Agar plates containing a specified concentration of a single antimicrobial agent are frequently used to screen selected organisms for resistance to the agent. S.aureus :oxacillin E.faecium and E.fecalis :vancomycin S.pneumoniae :penicillin Detection Of Extended-Spectrum β-Lactamase-Producing Isolates Certain enteric Gram-negative bacilli, primarily E.coli and Klebsiella spp., demonstrating resistance to various extendedspectrum cephalosporins. ESBLs(1) If reduced activity is detected by either diskdiffusion or MIC, a confirmation test is to be performed that involves testing the isolate’s relative susceptibility to both ceftazidime and cefotaxime and without the addition of clavulanic acid. If either the zone of inhibition increases by >5mm or the MIC decreases <3 two-fold dilutions when clavulanic acid is added, the isolate is declared an ESBL-producing strain and it is reported as resistant to all cephalosporins and monobactams. ESBLs(2) ESBLs(3) ESBLs(4) ESBLs(5) ESBLs(6) ESBLs(7) ESBLs(8) Other Tests To Detect Drug-Inactivating Enzymes H. influenzae produce choramphenicol acetyltransferase Semi-automated Susceptibility Testing • Systems Composed of Major Components • Testing Errors • Advantages and Disadvantages of Test INFREQUENTLY USED ANTIMICROBIC SUSCEPTIBILITY TEST METHODS • • • • The Minimum Bactericidal Concentration Test The Serum Bactericidal Test Time-Kill Kinetics Test Checkerboard Microdilution Test The Minimum Bactericidal Concentration Test Certain organisms may be inhibited by low concentration of an antimicrobic but require much higher concentrations to be killed. This can be determined by the minimum bactericidal concentration (MBC) The Serum Bactericidal Test The serum bactericidal test(SBT) measures the level of cidal antimicrobial activity in patient serum directed against the patient`s isolate .The result is expressed as the highest serum dilution that retains the ability to kill 99.9% of the inoculum . Time-Kill Kinetics Test The time-kill kinetic assay measures the rate of killing while being exposed to a single drug concentration, usually one that is physiologically achievable at standard dosage. The time-kill kinetic assay is currently thought to be the best method to study in vitro drug synergy . Checkerboard Microdilution Test A standard 96-well microtiter tray is loaded horizontally with increasing concentrations of drug A, and vertically with increasing concentrations of drug B so that as one scans the plate from lower left to upper right, both drugs in each well are present in increasing concentrations that span the therapeutic range from easily achievable (susceptible) to unachievable (resistant). REFERENCES • 1.Snydman DR, Cuchural GJ Jr, McDermont L, et al. Correlation of various in vitro testing methods with clinical outcomes in patients with Bacteroides fragilis group infections treated with cefoxitin:a retrospective analysis. Antimicrob Agents Chemother 1992; 36: 504-544. • 2.Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Susceptibility Tests.9th ed. Standard M2-A9. Villanova, PA: National Committee for Clinical Laboratory Standards, 2006. • 3.National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically.2nd ed. Standard M6-A2. Villanova, PA: National Committee for Clinical Laboratory Standards, 1990. • 4.Hindler JA, Swenson JM. Susceptibility testing of fastidious bacteria. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, and Yollken RH.eds. Manual of Clinical Microbiology.7th ed. Washington D.C.: American Society for Microbiology, 1999: 1544-1554. REFERENCES • 5.Berke I, Tierno PM. Comparison of efficacy and cost-effectiveness of BIOMIC VIDEO and vitek antimicrobial susceptibility test system for use in the clinical microbiology laboratory. J Clin Microbiol 1996; 34: 19801984. • 6.Livermore DM.β-lactamases in laboratory and clinical resistance. Clin Microbiol Rev, 1995; 8:557-584. • 7. Blaser J. Interactions of antimicrobial combinations in vitro: the relativity of synergism. Scand J Infect Dis 1991; 74(suppl):71-79. 8.Van der Auwera P. Synergistic, additive, and antagonistic activity of antimirobial agents: how to measure? What are the clinical consequences? Applications to the new fluoroquinolones. Infect Dis Newsl 1992; 11:49-54.