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Antibiotic Sensitivity Testing 2020 Update



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Antibiotic Sensitivity Testing 2020 Update

  1. 1. Antibiotic Sensitivity Testing 2020 Update Margie A. Morgan, PhD, MT(ASCP), D(ABMM)
  2. 2. Antibiotic Classes • Beta Lactams –contain a beta lactam ring in molecular structure of antibiotic • Penicillins • Penicillin • Amoxicillin • Ampicillin • Amp/Clavulanate • Amp/Sulbactam • Anti-Pseudomonal Penicillins • Piperacillin/Tazobactam • Ticarcillin/Clavulanate • Anti- Staphylococcal Penicillins • Nafcillin • Oxacillin • Cloxacillin • Dicloxacillin
  3. 3. Antibiotic Classes (2) • Also beta lactam antibiotics Carbapenems • Imipenem • Meropenem • Ertapenem • Doripenem • Monobactam • Aztreonam • Cephalosporins • First generation • Cefazolin • Second generation • Cefotetan • Cefoxitin Cefuroxime • Third generation • Ceftriaxone • Ceftazidime Cefpodoxime • Fourth generation • Cefepime Ceftaroline
  4. 4. Antibiotic Classes (3) • Fluoroquinolones • Ciprofloxacin • Levofloxacin • Moxifloxacin • Aminoglycosides • Gentamicin • Tobramycin • Amikacin • Sulfonamide • Trimethoprim/ sulfamethozaxole • Macrolides • Azithromycin • Clarithromycin • Erythromycin • Tetracyclines • Tetracycline • Minocycline • Doxycycline • Lincosamide • Chloramphenicol • Nitrofurans • Nitrofurantoin
  5. 5. Four Major mechanisms of antibiotic resistance •Enzymatic cleavage leads to inactivation of an antibiotic • Active Beta lactamases and amino-glycoside modifying enzymes cleave and destroy antibiotic •Altered receptors and binding proteins prevent attachment of antibiotics to the bacterial surface • Penicillin binding proteins (PBPs) • Mechanism for Strep pneumoniae resistance to penicillin and MRSA resistance to methicillin •Altered permeability/influx and efflux pumps stop passage of antibiotic through cell membrane porins – gram negative bacilli • Pseudomonas aeruginosa resistance to amino-glycosides •Bypass of a metabolic block/metabolic block imposed by antibiotic • Enterococcus resistance to TMP/SXT
  6. 6. How are rules established for Susceptibility Testing? CLSI – Clinical Laboratory Standards Institute publish approved standards for the testing & reporting of susceptibility results 1. Prepare documents with appropriate antibiotics to test for each organism 2. Charts outlining interpretation of susceptibility results 3. Quality control standards and proper testing procedures
  7. 7. Methods/Bacteria in Review Susceptibility testing methods 1. Kirby Bauer disk diffusion (KB) 2. E Test Strip Minimum inhibitory concentration (MIC ) 3. Broth dilution Minimum inhibitory concentration (MIC) Resistant Bacteria of Importance •MRSA methicillin resistant Staphylococcus aureus •VRE vancomycin resistant enterococcus •ESBL Extended Spectrum Beta Lactamase •CRE Carbapenamase Resistant Enteric Gram negative rods / also Carbapenamase resistant organisms (CRO) •Streptococcus pneumoniae •Neisseria gonorrhoeae
  8. 8. Preparation of Bacteria for all Susceptibility Methods Pure culture of one organism/ never test a mixed culture •Log phase growth of bacteria - 16-24 hours old •Susceptibility methods test for stasis not killing of bacteria •Standardized suspension of bacteria needed for testing. This is accomplished by: • O.5 McFarland Standard – Barium sulfate solution prepared in an acid pH that equals the turbidity of 10 8 bacteria/ml • Alternative method – use spectrophotometer •Incubation at 35 °C in room air (or CO²) for 18- 24 hours •If inoculum anount is NOT controlled, false resistance or susceptible results could occur
  9. 9. Quality Control Verification: Before testing patient organisms: Must correctly test multiple QC strains for 20 consecutive days. This is to assure you are able to perform the tests correctly. Must use ATCC strains of bacteria (American Type Culture Collection) with known activity for testing QC. If QC strains are tested and within appropriate values - you can then do weekly quality control on all lots of antibiotic cards, disks, plates. • Data must be recorded and reviewed monthly by supervisor •If weekly QC results are out of control: • Immediately repeat/ inform supervisor • If repeat is OK and you know the source of your error – continue routine testing • If repeat is NOT OK or if you do not know why you had a failure – must investigate/document/ repeat 5 times to start routine testing. All repeats must be in control • Must not report results of out of control antibiotic until back in control
  10. 10. Agar Disk Diffusion (Kirby Bauer) Qualitative Susceptibility Method •Mueller Hinton agar –with or without blood • 150 mm plate diameter • 4mm in depth • Agar specifically balanced in Ca+ and Mg+, • if the ions are too high % amino-glycosides test falsely resistant, • if the ions too low % falsely susceptible amino-glycoside results •Streak bacteria on plate with cotton tipped swab •Apply 6mm paper disks that contain a single antibiotic •Incubate for 16-24 hrs at 35*C •Measure zone of diameter of inhibition of growth (mm) – CLSI charts used to interpret results
  11. 11. Kirby Bauer disk Dispenser Each cartridge contains a separate antibiotic Growth inside a zone is considered resistance Measure the diameter of the zone of inhibition Watch out for double Zones /contaminated? Proteus will swarm Into a zone
  12. 12. E Test Quantitative MIC Susceptibility •Calibrated plastic strips impregnated with concentration gradient (mcg/ml) of one antibiotic •Diffusion gradient created as antibiotic diffuses into agar in an elliptical shape •MIC (minimum inhibitory concentration) is where the ellipse ends on the plastic strip •Good method for slower growing fastidious organisms that do not give consistent results on Kirby Bauer test .
  13. 13. Broth Dilution minimum inhibitory concentration/MIC Quantitative Susceptibility Method •Bacteria inoculum: adjusted to 0.5 McFarland standard then further diluted to 5x105 organisms /ml in saline •Suspension is inoculated into micro titer trays containing broth growth medium and known 2 fold dilutions (mcg/ml) of antibiotics •Each horizontal row is a unique antibiotic • Growth/resistance leads to turbidity in the broth •Lowest concentration of antibiotic with No Growth (clear well)= MIC value.
  14. 14. Broth Dilution Definitions •MIC = lowest concentration of antibiotic inhibiting growth •MBC = lowest concentration of antibiotic killing 99.9% •Antibiotic tolerance – Ratio of MBC/MIC (>=32) • MBC = 16 MIC= 8 16/8= 2 No Tolerance • MBC = 128 MIC = 8128/2 = 64 Tolerance* • *This means bacteria will tolerate the antibiotic and not be killed
  15. 15. Becton-Coulter Microscan walkaway AST system BD Phoenix AST systemBiomerieux Vitek2 AST Automated Identification and Susceptibility Testing Systems (AST) – use broth dilution MIC type testing
  16. 16. Methicillin Resistant Staph aureus (MRSA) •OLD WAY/ Test for oxacillin (OX) resistance to detect methicillin resistance - it is more stable for testing in the laboratory •If OX is resistant - S. aureus is reported as a MRSA •NEW and better way – If Cefoxitin is resistant report as a MRSA Cefoxitin is more reliable and a preferred way to confirm MRSA •All cephalosporin antibiotics are also reported resistant when a MRSA is detected and should never be used for therapy •Resistance mechanism for MRSA is penicillin binding proteins (PBPs) • PBPs bind penicillin and related antibiotics • The binding prevents disruption of the peptidoglycan synthesis in the Staph aureus cell wall • PBPs are produced by the mecA gene • New emerging strain of MRSA produced by mecC gene
  17. 17. Picture: Oxacillin KB disk = resistant / MRSA Cefoxitin KB disk Newer method –more sensitive screen for MRSA Picture: Cefoxitin (FOX) KB = a methicillin sensitive S. aureus Methods to Detect MRSA Oxacillin Testing is no longer the preferred method for detection
  18. 18. Clindamycin Induction Test – The D test •The D test determines if Staph aureus, including MRSA, is susceptible to Clindamycin more reliably than testing Clindamycin resistance by KB or MIC methods •During antibiotic therapy, Staph aureus isolates resistant to Erythromycin possess enzymes that can be induced to make the S. aureus also resistant to Clindamycin •Clindamycin is often used to treat serious soft tissue infections with MRSA – so reliable testing necessary
  19. 19. D test Negative- round Clindamycin zone Kirby Bauer zone around Clindamycin will be blunted to form a D if Clindamycin can be induced by Erythromycin to be resistant – so called INDUCIBLE RESISTANCE. Clindamycin should be reported as “resistant by clindamycin induction” and not used for therapy The D Test
  20. 20. Enterococcus •All Enterococcus are intrinsically resistant to: • Cephalosporins • Clindamycin • Trimethoprim/sulfamethoxazole •Synergistic antibiotic therapy can be important for the treatment of Enterococcus • Ampicillin plus Gentamicin is synergistic = increased bacteria killing potential with combination of two antibiotics that kill by different mechanisms • Particularly important for endocarditis therapy •Acquired resistance to vancomycin – • Plasmid mediated vanA associated with E. faecium • Plasmid mediated vanB associated with E. faecalis
  21. 21. Extended Spectrum Beta Lactamase [ESBL] •Enzymes produced by Enteric Gram negative bacilli • Confer resistance to Cephalosporins, Penicillins and Monobactam (Aztreonam) antibiotics by opening the beta lactam ring of the antibiotic and inactivating the antibiotic • ESBLs do not attack Cephamycin (cefoxitin, cefotetan) or the Carbapenem antibiotic classes •Plasmid mediated CTX-M beta lactamases are the most common ESBL enzymes in the US currently, but many more ESBL types can be found worldwide •Therapy for ESBL producing gram negative rods: • Carbapenems: Imipenem, Meropenem, Doripenem, and Ertapenem
  22. 22. Detecting ESBL in the laboratory •Standard of Practice: • Recently, CLSI established new Cephalosporin MIC values and KB zone breakpoints to safely detect ESBL activity • New MIC breakpoints are one to three doubling dilutions lower than previously used • New KB zones for susceptible are larger than before •Molecular testing needed for confirmation of actual enzyme present (CTX-M)– this is beyond the scope of most clinical laboratories
  23. 23. Why all the fuss about ESBLs? • GNRs with ESBL phenotypes >=10% in US and the numbers are increasing • Limited treatment options • Carbapenems: meropenem, imipenem, ertapenem • Risk factors: • Long hospital stay – particularly in the ICU • Central lines • Issues with the intestine • Long term care facility • Ventilator assistance
  24. 24. Carbapenemases – CRE and CRO •CRE = Carbapenamase resistant Enteric / CRO= Carbapenamase resistant organism •Carbapenem antibiotics currently have the highest spectrum of activity against multi- drug resistant GNRs •But the worst scenario has come true – appearance of carbapenem-hydrolyzing-beta- lactamases which confer resistance to a broad spectrum of beta lactam antibiotics including carbapenems – making GNRs resistant to most if not all antibiotics •Two CREs are getting the most attention: • KPC – “Klebsiella pneumoniae carbapenemase” most common in the US • NDM-1 – New Delhi metallo-beta-lactamase. Resistance determinants are numerous and great concern about its spread. •Infections with CRE/CRO producing GNRs can produce a 50% fatality rate
  25. 25. Most sensitive way to detect a CRE is to perform an Ertapenem MIC test. However, all Carbapenem antibiotics (meropenem, imipenem) usually have MICs in the resistance range for CREs. CROs can be identified by detecting elevated/resistant MICs to meropenem or imipenem Treatment of CRE: Polymyxins (Colistin and Polymyxin B) Newer combination antibiotics preferred: Ceftazidime/avibactam Ceftolozane/tazobactam Meropenem/Vabomere Carbapenemase Resistant CRE Laboratory Testing
  26. 26. Streptococcus pneumoniae and resistance to Penicillin (PEN) • Perform a Penicillin Minimum Inhibitory Concentration (MIC) by E Test or broth microdilution •KB method cannot be used to test Penicillin against Strep pneumoniae because it under predicts resistance to Penicillin (PEN) •High level PEN resistance is <=10% in US • If PEN resistant - antibiotics of choice becomes a 3rd gen Cephalosporin, vancomycin or quinolone
  27. 27. Neisseria gonorrhoeae (GC) • Increasing resistance of GC over last two decades • 1980’s beta lactamase producing strains with Penicillin resistance • By 2000, the quinolones were resistant due to the acquisition of mutations by GC that altered the binding sites, preventing Quinolone activity • Currently Cephalosporins (Ceftriaxone & Cefixime) are the mainstay for therapy in the US – however resistance to these antibiotics are becoming common in Asia. • This resistance due to Penicillin Binding Proteins (PBPs) and over production of efflux pump mechanism • Currently susceptibility testing of GC is not performed in most labs • Detection of resistant strains in the USA could be problematic due to our reliance on amplification testing for STD diagnosis that only test for the genetic presence of GC and not for resistance markers