Laboratory Methods and Strategies for Antimicrobial Susceptibility Testing PDF

Laboratory Methods and Strategies for Antimicrobial Susceptibility Testing Chapter 11 Bailey & Scott’s Diagnostic Microbiology Patricia M. Tille Fourteenth Edition Copyright © 2017 Elsevier Inc. All Rights Reserved Objectives • Determine standardized measures and general conditions required in...

Laboratory Methods and Strategies for Antimicrobial Susceptibility Testing Chapter 11 Bailey & Scott’s Diagnostic Microbiology Patricia M. Tille Fourteenth Edition Copyright © 2017 Elsevier Inc. All Rights Reserved Objectives • Determine standardized measures and general conditions required in antimicrobial susceptibility testing • Outline procedures for conducting and evaluating antimicrobial susceptibility testing: Broth dilution Agar dilution Disk Diffusion E-test Automated antimicrobial susceptibility testing systems D-test Predictor Antimicrobial Agents β-Lactamase detection & Chloramphenicol acetyltransferase Time-kill Studies Serum Bactericidal (Schlichter Test) • Decide on when to perform a susceptibility tests and how to evaluate the accuracy of the used strategy Antimicrobial Susceptibility Testing Methods Three general methods are available to detect and evaluate antimicrobial susceptibility: 1) Methods that directly measure the activity of antimicrobial agents against a bacterial isolate 2) Methods that directly detect the presence of a specific resistance mechanism in a bacterial isolate 3) Special methods that measure complex antimicrobial-organism interactions Antimicrobial Susceptibility Testing Methods 1) METHODS THAT DIRECTLY MEASURE ANTIMICROBIAL ACTIVITY: -Involves bringing the antimicrobial agents and the infecting bacterium together in the same in vitro environment to determine the impact of the drug on bacterial growth or viability -The level of effect on bacterial growth is measured, and the organism’s resistance or susceptibility is reported -Direct measures of antimicrobial activity are accomplished using: A. Conventional susceptibility testing methods (such as Broth dilution, Agar dilution, and Disk diffusion) B. Commercial susceptibility testing systems C. Special screens and indicator tests A. Conventional Testing Methods: Broth Dilution -Liquid environment -Range of concentrations of antimicrobial agent (µg of active drug/ml of broth) Medium: -Two general categories: microdilution and macrodilution * Same test principle, different broth volume -For microdilution testing: the broth volume is 0.05 to 0.1 mL (microtiter tray) -For macrodilution testing: the broth volume is 1 mL or greater (Rarely used in clinical labs) Inoculation: -Standardized bacterial suspensions that match the turbidity of the 0.5 McFarland standard (1.5 × 108 CFU/mL) usually serve as the starting point for dilutions *final standard bacterial concentration of 5 x 105 CFU/mL in each microtiter well -Standard inoculum is prepared from a fresh, overnight, & pure culture -Use of manual or automated multi prong calibrated inoculator Incubation time and conditions: -To optimize bacterial growth without interfering with antimicrobial activity -Reading and Interpretation of results: -Includes internal controls *Growth control: No antimicrobial agent added-Detect positive growth (Positive control) *Sterility Control: No inoculation-No growth (Negative control) -Provide data on: *Growth profiles *MIC (quantitative data) *Translated into an interpretive category (qualitative): -Susceptible (S), intermediate (I), resistant (R) -Recently added categories: Non-susceptible (NS) and Susceptible-dose dependent (SDD) (Broth) (CAMHB) (for meth-R) (CAMHB + 2% NaCl) (CAMHB + 2%-5% LHB) (CAMHB + 2.5%-5% LHB) 16-20 hr Conventional Testing Methods: Agar Dilution -Agar based medium -Series of agar plates with doubling dilution of an antimicrobial agent + positive growth control plate without antibiotic -Inoculated with 1 x 104 CFU/ml -Provide data on: *Growth *MBC/MIC and/or interpretive categories -Labor intensive -Examination of growth of one or more bacterial isolate per plate -Determining MICs for N. gonorrhoeae which does not grow sufficiently in broth Conventional Testing Methods: Disk Diffusion (Kirby-Bauer) -Antibiotics-impregnated filter paper disks (antibiotic disks) (of known concentration) on the surface of an agar plate seeded with a lawn of bacteria *Antibiotic begins to immediately diffuse into the agar and establish a concentration gradient around the paper disk (Highest concentration is closest to the disk) -Upon incubation, bacteria will grow except where the antibiotic concentration is sufficiently high to inhibit growth -Thickness of the agar affects the antimicrobial drug concentration gradient -Disks must be placed flat on the surface and firmly applied -Disks should be stored at proper temperature with desiccants -Disks should not be expired Generally not an acceptable method for testing slowgrowing organisms such as mycobacteria and anaerobes -After incubation, the diameter of the zone of inhibition around each disk is measured in millimeters -Inhibition zone sizes are correlated with MICs * Determines if susceptible, intermediate, resistant *The Larger the zone of inhibition the greater the antimicrobial activity/greater diffusion * As MICs increase (more resistant bacterial strains), the corresponding inhibition zone sizes (diameters) decrease *No zone of inhibition: resistance -Size of the zone of inhibition depends on the antibiotic disk concentration -Convenience and user friendly -Up to 12 antimicrobial agents can be tested against one bacterial isolate -Generally accurate -The major disadvantage is the lack of interpretive criteria for organisms not previously referenced Some tips to consider -Inoculum from overnight culture -Lawn of growth must be confluent -Tiny colonies at the zone edge are ignored -Swarming is ignored (Ex: Proteus) -Obvious colonies with clear zone should not be ignored (contamination, mixed culture, resistance subpopulation) Hazy Zone: *Hetero-resistant population *Motile bacterial swarming Clear Zone: *Susceptible *Bacterial grew for a while before antibiotic inhibited growth B. Commercial Testing Methods: -Broth Microdilution Methods -Agar Dilution Derivations -Diffusion in Agar Derivations -Automated Antimicrobial Susceptibility Test Systems Diffusion in Agar Derivations -Example: E-test -Combines the convenience of disk diffusion with the ability to generate MIC data -Agar plates are inoculated in the same way as in the disk diffusion method and strips are placed on the inoculum lawn -After overnight incubation, the plate is examined for elliptical zone of inhibition and the number present at the point where the border of growth inhibition intersects the E-strip is the MIC Automated Antimicrobial Susceptibility Test Systems -Vitek Legacy and Vitek 2 systems -MicroScan WalkAway system -Phoenix system These different systems vary in: -Extent of automated inoculum preparation and inoculation -Methods used to detect growth -Algorithms used to interpret and assign MIC values and categorical findings ( susceptible, intermediate, resistant) Vitek 2 AST -Inoculum is automatically introduced by a filling tube into a miniaturized, plastic, 64-well, closed card containing specified concentrations of antibiotics -Cards are incubated in a temperature-controlled compartment -Optical readings every 15 minutes to measure the amount of light transmitted through each well (including a growth control well) -Algorithmic analysis of the growth kinetics by the system’s software to derive the MIC data -The MIC results are validated with the Advanced Expert System (AES) software, category interpretation is assigned, and organism’s antimicrobial resistance patterns are reported -6 to 8 hours to detect antimicrobial resistance mechanism for most clinically relevant bacteria MicroScan WalkAway system -Use broth microdilution panel format manually inoculated with a multi pronged device -Inoculated panels are placed in an incubator-reader unit for the required time -Bacterial growth patterns may be detected using spectrophotometry or fluorometry *Spectrophotometric: overnight incubation, read manually as described for routine microdilution testing *Fluorometric: Based on the degradation of fluorogenic substrates by viable bacteria (provides susceptibility results in 3.5 to 5.5 hours) Phoenix system -Manual inoculation process -Growth is monitored based on a redox indicator system -Supplemental testing (ex: confirmatory extended spectrum beta-lactamase test for E. coli) -Effective Interpretation of results *Rules based data management expert system -Results in 8 to 12 hours C. Special Screens and Indicator Tests Alternative Approaches for Enhancing Resistance Detection Oxacillin Resistance -Certain strains of staphylococci (MRSA) are resistance to oxacillin and related drugs (methicillin & nafcillin…) -Resistant strains to one of these agents are usually resistant to all other beta-lactam antibiotics *Oxacillin disk screens *Growth on agar screen: presence of oxacillin (or methicillin) resistance No growth: Isolate is susceptible -Use of 30μg cefoxitin disks to improve the detection of mecA-oxacillin resistant staphylococci *Inhibitory zones ≤ 21 mm: oxacillin resistance in S. aureus & S. lugdunensis ≤ to 24 mm indicate oxacillin resistance in coagulase negative staphylococci -Detection of oxacillin resistance in S.aureus & S. lugdunensis can be also performed by the broth dilution method (growth in 4µg/ml cefoxitin indicates oxacillin resistance) N.B. -Heterogeneous Staph resistance population -Resistance can be detected in other ways: *Mec-A gene detection by PCR *Rapid latex agglutination and immunochromatogenic qualitative assays (Detection of PBP2a) Vancomycin Resistance -Vancomycin agar screens -For the detection of Staph or Enterococci -Non-motile and non-pigmented Enterococcal species: acquired resistance by van A or van B gene transfer -Motile and pigmented species: van C gene responsible for intrinsic resistance Aminoglycoside Resistance -Aminoglycoside acquired high-level resistance in Enterococci -Screens using high concentrations of Aminoglycoside Macrolide-Lincosamide-StreptograminB Resistance -Some Isolates produce a profile of resistance to a macrolide (ex: erythromycin) and susceptibility to lincosamide (clindamycin) *Two different mechanisms of resistance: -efflux (msrA gene): isolate susceptible to clindamycin -iMLSB mechanism (erythromycin ribosomal methylase gene (erm) that alters ribosomal targets): clindamycin resistant -Perform D-test to distinguish between these two resistant mechanisms *Positive D test: Presence of macrolide-inducible resistance to clindamycin produced by an inducible methylase that alters the common ribosomal binding site for macrolides, clindamycin, and the group B streptogrammins *The cross-resistance is called the MLSB phenotype *The methylase is encoded by a plasmid-borne gene erm Resistance of gram-negative rods to extended Beta lactams and carbapenems -Interpretive criteria for Cefpodoxime, Ceftazidime, Cefotaxime, Ceftriaxone & Aztreonam -Carbapenemase production in species belonging to Enterobacteriaceae, P. aeruginosa, and Acinetobacter  Ex: Carba NP test in detecting Klebsiella pneumoniae carbapenemase (KPC) Predictor Antimicrobial Agents -Staphylococcal resistance to oxacillin: resistance to all currently available beta-lactams -Enterococcal high-level gentamicin resistance: resistance to nearly all other currently available aminoglycosides -Enterococcal resistance to ampicillin: resistance to all penicillin derivatives Antimicrobial Susceptibility Testing Methods Three general methods are available to detect and evaluate antimicrobial susceptibility: 1) Methods that directly measure the activity of antimicrobial agents against a bacterial isolate 2) Methods that directly detect the presence of a specific resistance mechanism in a bacterial isolate 3) Special methods that measure complex antimicrobial-organism interactions 2. METHODS THAT DIRECTLY DETECT SPECIFIC RESISTANCE MECHANISMS Phenotypic methods *Test for the presence of β-lactamase enzymes (most common) *Test for chloramphenicol-modifying enzyme: chloramphenicol acetyltransferase (less common) Beta Lactamase Detection -Rapid test -Does not require overnight incubation like disc diffusion -Most useful assay is the chromogenic cephalosporinase test (ex: commercially available Cefinase disk) -The disk incorporates chromogenic cephalosporin (nitrocefin) as the substrate - Intact β-lactam ring: yellow color (negative) - cleaved β-lactam ring: red color (positive) Chloramphenicol acetyltransferase (CAT) -If CAT is positive: Chloramphenicol resistance -If CAT is negative: Does not rule out resistance mediated by other mechanisms (ex: decrease uptake of drug) Genotypic Methods: -Confirm results obtained by phenotypic methods -Molecular methods: nucleic acid hybridization and amplification to detect of antimicrobial resistance genes *Examples: Detection of mecA gene, van genes -Disadvantages: * Resistance mediated by divergent genes * Resistance may be due to a culmination of processes (enzymatic modification, decrease uptake, altered affinity of the drug’s target, combination of mechanisms) * The presence of a gene encoding resistance does not provide information about the status of the control genes (silent, nonfunctional…) Antimicrobial Susceptibility Testing Methods Three general methods are available to detect and evaluate antimicrobial susceptibility: 1) Methods that directly measure the activity of antimicrobial agents against a bacterial isolate 2) Methods that directly detect the presence of a specific resistance mechanism in a bacterial isolate 3) Special methods that measure complex antimicrobial-organism interactions 3. SPECIAL METHODS FOR COMPLEX ANTIMICROBIAL/ORGANIMS INTERACTIONS Bactericidal Tests: -Minimal Bactericidal concentration (MBC) testing -Time-kill Studies -Serum-bactericidal testing (SBT)/Schlichter Test Time-Kill Studies -Detect bactericidal activity of antimicrobial agent -Expose a bacterial isolate to a concentration of antibiotic in a broth medium and measure the rate of killing over a specified time period -Each time-sample is plated on agar plates after incubation -CFU counts are performed -Number of viable bacteria from each sample is plotted over time to determine the rate of killing Generally, a 1000-fold decrease in the number of viable bacteria in the antibiotic-containing broth after a 24-hour period, compared with the number of bacteria in the original inoculum, is interpreted as bactericidal activity Quality Control of Antimicrobial Susceptibility Tests  Standardized methods  CLSI guidelines for interpretations  Reference strains -Defined susceptibility/resistance patterns  Consecutive testing  Periodic testing *Specific combos (MRSA with hetero-resistance, Ampicillin-resistant E. cloacae)  Antibiograms: Overall antimicrobial susceptibility profile of an organism