Antimicrobial Susceptibility Tests Clinical Microbiology 2: Antimicrobial Susceptibility Tests • Basic principle • Antimicrobial Susceptibility Tests (AST) Nuntra Suwantarat, MD, D(ABMM) - Phenotypic test °µµ¦¥r¡¥r®· ´¦µ »ª´µ¦´r - Genotypic test Chulabhorn International College of Medicine • Clinical correlation and clinical application Division of Infectious Diseases • Summary Thammasat University Hospital Thammasat University 13 March 2018 • I have no conflict interest to be disclosure IDAT Week 2018
Clinical Microbiology Laboratory Antimicrobial Susceptibility Tests
• Identification (ID) • Standardized, reproducible methods for assessing Causative pathogens antibiotic activity • Routine tests (manual and automated methods) • Antimicrobial susceptibility tests (AST)* Phenotypic >>>> Genotypic Phenotypic vs Genotypic • Guideline for performing the tests and breakpoints interpretation (CLSI/ EUCAST) • Antimicrobial resistance (AMR) • Specialized tests for specific applications Chromosomal vs plasmid resistance ESBL, CRE confirmation tests Intrinsic vs acquired resistance Methicillin resistance in Staphylococcus (mecAtest) Basic and conventional biochemical methods Advance and molecular identification methods Inducible clindamycin resistance (D test) Kirby-Bauer Method 1. Disk Diffusion Antibiotic susceptibility testing in which disks containing various antibiotics are placed on a plate swabbed with the organism. Zones of inhibition are measured to determine whether the organism is susceptible, intermediate or resistant. (Based on interpretation guideline, CLSI, EUCAST)
Manual of antimicrobial susceptibility testing, JHU microbiology laboratory ASM, 2005 Disk Diffusion Test Disk Diffusion Test
Manual of antimicrobial Manual of antimicrobial susceptibility testing, susceptibility testing, ASM, 2005 ASM, 2005
Disk Diffusion Test Disk Diffusion Test
GOOD TO rule-out mix infection (not pure colonies)
Manual of antimicrobial susceptibility testing, Manual of antimicrobial ASM, 2005 susceptibility testing, ASM, 2005 Disk Diffusion Test Disk Diffusion Test
Manual of antimicrobial Manual of antimicrobial susceptibility testing, susceptibility testing, ASM, 2005 ASM, 2005
Disk Diffusion Test 2. Tube Dilution Method The first tube in which there is no visible growth is the MIC level Variable factors of the antibiotic for the organism tested.
• Media composition • Media pH • Agar depth • Concentration of inoculum • Inoculation procedure • Antimicrobial concentration in disk
• Disk storage MIC
Antibiotic Concentration Low ------High JHU microbiology laboratory Illustration of the difference between MIC test MIC, minimum inhibitory concentration, and MBC, minimum bactericidal concentration, of an antibiotic.
MBC MIC *MIC= 1st tube with no visible growth Routinely use, CLSI and EUCAST Interpretation Use for guiding treatment* Endocarditis (Enterococcus, S. bovis) meningitis (S. pneumoniae) MDR bacteria; MBC = 99% bacterial growth inhibition MRSA (vancomycin MIC) (Currently use only for research, ESBL/CRE previously use for endocarditis treatment) Broth microdilution: manual or commercial system ie MicroScan, TREK panels Phoenix system, VITEK2 system --- Give MIC interpretation
MIC Test MIC test
Æ MIC 4. E-test Method 3. Agar Dilution Method Each strip is impregnated with increasing concentrations of a different antibiotic ; Formerly used at JHH, this method measures MICs of antibiotics by comparing strips are placed on a plate swabbed with the organism to be tested, and incubated growth of colonies on plates of increasing antibiotic concentrations. overnight. The MIC level for each antibiotic is at the line crossed on the strip where Labor intensive/ rarely use in routine labs the organism is inhibited from growing. For research: Standard for Anaerobic bacteria AST, N. gonorrhea AST
Note E-test/ Gradient concentration may have variable of diffusion E= elliptical shape as disk diffusion JHU microbiology laboratory JHU microbiology laboratory
New Jan 2018 (CLSI: M100S28) Automated biochemical identification and susceptibility method What do you need from AST? MicroScan system (Walkaway system) VITEK 2 system • Accurate results BD Phoenix system (QA/QC, interpretation Æ identification) Sensititer • Short/ Appropriate TAT • Clinical applicable • Clinical impact for patient care • Cost effectiveness
MicroScan Identification and AST cards (pH changes)
Routine ID method at TUH since September 2015 Nonfermenters Æ Elizabethkingae, Ekinella, Chryseobacterium, Salphingomonas, Roseomonas, Methylobacterium etc Beta-hemolytic streptococci Æ GAS: S. pyogenes, GBS: S. agalactiae, Group D strep Æ S. gallolyticus, Enterococcus spp.
Calculated MIC (TAT 6-8 hrs) Pro: Faster for common bacteria, size of machine Con: Indeterminate for special pathogens (not good for yeast), need to refrigerate ID/AST cards
Fluorescent detection + Semi-calculated MIC (TAT 6-8hrs) Pro: Faster(common bacteria), confirmation tests (CPE?), accurate MIC Con: Indeterminate for special bacteria size of machine (better with M50)/ package?
Mechanism of GN resistance Phenotypic resistance - Enzymatic resistance vs Non-enzymatic resistance Pro - Acquired (plasmid/transferable resistance Genotypic resistance Good for Intrinsic (chromosomal) resistance Yeast Enzymatic (Candida AST) (hydrolytic enz) OprD = “+” betalactamase (several bla genes) MTB/NTM OprE/F = “no charge” Non-enzymatic Enterobactericae Æ Plasmid transfer (mainly) Colistin AST? -Efflux pump Æ need isolation -Porin change - Nonfermenters Æ non-plasmid transfer (oprD gene) (mainly) Æ +/- isolation Con -Decreased Not automated membrane permeability (omp gene) system -Aminoglycoside (aac gene) (target site changed) -Quinolone resistance (gyrA, gyrB, parC, qnr genes)
AAC 1999;43(2):424-7 NEJM 2008;358:1271-81
CRE vs CPE (CP-CRE) CRE vs CPE (CP-CRE) Carbapenem resistant Enterobactericeae • Definition
• CRE = Carbapenem Resistance Enterobacteriaceae CDC 2015 definition Resistance to imipenem, meropenem, doripenem or ertapenem KPC OXA KPC VIM OR documentation that the isolate produce carbapenemase NDM-1 MCR-1 NDM-1 • CP-CRE = Carbapenem-Producing Enterobacteriaceae CRE/CP-CRE in Thailand Æ plasmid transferable gene (carbapenemase) = NDM-1, OXA ÆÆInfection control implementation needed KPC
CDC; Healthcare associate infection
Variable in geographic distribution (Genotypic resistance) CRE confirmation test No need to perform if use new CLSI breakpoints (2010) JCM, Sep 2013
The CIM (Carbapenemase inactivation method) PCR Results (Manual in house test) a new phenotypic Test to assess Carbapenemase activity
NDM 561 bp
OXA 438 bp
IMP 232 bp
van der Zwaluw K, de Haan A, Pluister GN, et al. The Carbapenem Inactivation Method (CIM), a simple and low-cost alternative for the Carba NP Test to assess phenotypic carbapenemase activity in Gram-Negative rods. PLoS ONE 2015; 10(3): e0123690. Cepheid GeneXpert
CRE stool Screening
KPC NDM
ASM 2012
Gene target gyrB hsp60 sodA gyrB tuf gene gyrB hsp60 hsp60
gene hsp60 hsp60
Verigene: BC- GP test Limitation (Nanosphere) FDA cleared blood c/s Aerobic bottle multiplex PCR, only microarray: Mixed TAT: 2.5 hrs infection Mixed c/s • Biofire Filmarray
Positive Blood panel (Multiplex PCR) (1 h result)
Caution! Gram-negative bacteria resistance mechanism = genotypic (enzymatic) and non genotypic (non-enzymatic) Antimicrobial Susceptibility Tests
• Basic principle • Antimicrobial Susceptibility Tests Phenotypic test Genotypic test • Clinical correlation • Summary
MIC vs Disk Diffusion Test MIC vs Disk Diffusion Test
Interpretative criteria/guideline: CLSI, EUCAST Patient dose not response with Gentamicin. Why? Treatment guideline: research data, patient care
Note: MIC Tobramycin *** *** is lower than Gentamicin (all susceptible) In vitro data Tobramycin is a better choice. New Jan 2018 (CLSI: M100S28)
New Jan 2017 (CLSI: M100S27)
Caution! No breakpoint for Burkholderia pseudomallei in CLSI (Meliodosis) See CDC recommendation or reference articles
Non-Enterobacteriacae (not Acinetobacter, Pseudomonas): No disk diffusion breakpoint Identification Automated system: Burkholderia pseudomallei misidentification is not uncommon.
AST: No CLSI interpretative criteria Disk diffusion: overcall TMP-SMX resistance MIC test is recommended.
New Jan 2017 (CLSI: M100S27)
MRSA: Vancomycin test = MIC only No disk diffusion breakpoint Using CLSI 2010 breakpoint: no need for ESBL/ CRE confirmation test mecA gene VanA Vancomycin MIC creeping (MIC > 1) Æ more treatment failure Æ Vancomycin should not be avoided. NEJM-review, IDSA Guideline for MRSA treatment, Endocarditis treatment MSSA vs MRSA Use: Linezolid/ Daptomycin/ Ceftaroline Definition, Mechanism of resistance, CA-MRSA vs HA-MRSA (USA type, Scc type) Endocarditis, Severe Pneumonia, Severe Skin infection, Osteomyelitis Need to monitor Vancomycin MIC and vancomycin trough level (drug level)
Sanford Guideline (Infectious Disease Treatment GL)
Viridans strep, S. bovis (S. gallolyticus) endocarditis • Pen G MIC <0.12 mcg/mL = Susceptible Æ Pen G or Ceftriaxone x 4 weeks Æ shorten duration = Pen G or Ceftriaxone PLUS gentamicin x 2 wks • Pen G MIC > 0.12 to < 0.5 = Intermediate resistance Æ Pen G or Ceftriaxone x 4 weeks PLUS gentamicin x 2 weeks Æ Vancomycin x 4 weeks • Pen G MIC > 0.5 = Resistance Æ Pen G (or Ampicillin) PLUS gentamicin x 4-6 weeks Æ Vancomycin PLUS gentamicin x 4-6 weeks
D- test: Micro labs report should be…. Other indication for MIC monitoring/use Erythromycin-Resistance *Enterococcus Endocarditis, S. pneumoniae meningitis Clindamycin-Resistance Osteomyelitis, Endovascular infection, treatment failure etc. **MDR pathogen: MRSA (Macrolide-inducible clindamycin resistance) ESBL/CRE ESBLs treatment • Severe infection/ bacteremia • Recommended to use Carbapenems • Fosfomycin? • Aminoglycoside (source of infection) • UTIs, de-escalation therapy • Depend on susceptibility profiles ID consultation • Pip/tazo (if low MIC)? • Newer version quinolones? (if no bacteremia) Combination therapy with • Fosfomycin Colistin/ polymixin B/ Aminoglycoside/ Tigecycline etc. • Aminoglycoside etc Carbapenem high dose/ prolong infusion • Isolation the patient if possible (or cohorting the patient) (need to check MIC**) • In Thailand, may not need for isolation (high prevalence) Tamma PD et al. Carbapenem therapy is associated with improved survival compared with Room Isolation (Plasmid transferable) piperacillin-tazobactam for patients with extended-spectrum ɴ-lactamase bacteremia. Clin Infect Dis. 2015;60(9):1319-25. Perez F, Bonomo RA Editorial Commentary: Bloodstream Infection Caused by Extended-Spectrum ɴ-Lactamase–Producing Gram-Negative Bacteria: How to Define the Best Treatment Regimen? Clin Infect Dis. 2015;60(9):1326-29
A.baumannii/ P.aeruginosa Update CLSI 2017 Antimicrobial agents Inhibition MIC • Colistin susceptibility test matter? zone (mm) (μg/mL) No disk diffusion test interpretation S R S* R Colistin --<2>4
*loading dose, maximum recommended dose, and combination with other agents
Nuntra/Dr. Surapee IDAT Oct 2017, CLSI Update Dr. Surapee IDAT Oct 2017, CLSI Update A.baumannii/ P.aeruginosa Enterobacteriaceae: Colistin
Antimicrobial agents Inhibition MIC Organization MIC zone (mm) (μg/mL) (μg/mL) S R S* R WT NWT --<2>4 Colistin CLSI 2017 <2 >4 (ECV)
S R *loading dose, maximum recommended dose, EUCAST 2017 and combination with other agents (Clinical <2 > 2 breakpoint)
Comparative methods for colistin Susceptibility tests
1. Broth microdilution (BMD) 2. Agar dilution (AD) 3. Agar gradient (Etest) 4. Disk diffusion (DD) Rapid polymyxin NP Colistin susceptibility test
TAT 2 h • BMD: gold standard Sensitivity 99.3% Specificity 95.4% • AD: essential agreement with BMD
From 135 colistin R isolates • DD and Etest: poor agreement with and 65 cosistin S isolates BMD
• Need clinical breakpoints : MIC-PK/PD-Clinical outcomes
Dr. Surapee IDAT Oct 2017, CLSI Update