Detection and Reporting of Beta-lactam Resistance in Enterobacteriaceae

1. Detection and Reporting of Beta-lactam Resistance in Enterobacteriaceae Paul C. Schreckenberger, Ph.D., D(ABMM) Professor of Pathology Director, Clinical Microbiology Laboratory Loyola University Medical Center pschrecken@lumc.edu

2. Objectives • Participants will be able to: • Set up and interpret the double disk diffusion method for detecting ESBLs and ampCs. • Describe methods for detection of carbapenamases, including the Hodge test and Tris EDTA double disk test • Modify susceptibility reports based on characterization of resistance Genotypes.

3. Detecting Antibiotic Resistance Is there a Problem?

4. Automated Systems • Poor performance by automated systems in detecting resistance has necessitated use of off line screening/confirmatory tests • Oxacillin screening plates for MRSA • Vancomycin screening plates for MRSA and VRE • D-Zone Test for detection of inducible clinidamycin resistance

5. Automated Systems • Limitations of Automated Systems in detecting emerging resistance in Gram-Negative Bacilli • Unable to detect ESBLs in organisms other than E. coli and Klebsiella • Unable to detect Inducible AmpC • Unable to detect ESBLs in AmpC positive strains • Unable to detect imipenem resistance in strains producing KPC carbapenemases

6. Comparison of Phoenix & Vitek 2 for Detecting ESBLs in E.coli and Klebsiella *Phoenix results after activation of two normally inactive Phoenix expert rules (rules 325 and 1437) intended to enhance ESBL detection based on susceptibility results Thomson KS et al. JCM 2007 Aug;45(8):2380-4.

7. Evaluation of Methods to Identify KPC in Enterobacteriaceae Anderson KF et al. JCM 2007 Aug;45(8):2723-5.

8. Role of the Microbiology Lab • “Each laboratory should have a staff member with the time, interest, and expertise to provide leadership in antibiotic testing and resistance. This person would read relevant publications, network with other laboratories, and evaluate potentially useful tests to detect new forms of resistance before new CLSI-recommended tests become available” • - Ken Thomson, Emerging Infect. Dis., 2001

9. The β-lactam family of antibiotics Penicillins Cephalosporins Cephamycins Carbapenems Monobactams Benzyl-penicillin Cephalothin 1st Cefoxitin Imipenem Aztreonam Methicillin Cefamandole 2nd Meropenem Cefotetan Ampicillin Cefuroxime 2nd Ertapenem Cefmetazole Carbenicillin Cefotaxime 3rd Mezlocillin Ceftazidime 3rd Ticarcillin Ceftriaxone 3rd Cefepime 4th

10. Penicillin nucleus S CH3 R 1 5 2 6 CH3 7 4 3 N O COOH

11. Cephalosporin nucleus 1 S 7 R1 C O HN R2 O COOH

12. MODE OF ACTION OF BETA LACTAMS IN GRAM NEGATIVES SUSCEPTIBLERESISTANT -Lactam Antibiotic  Diffusion through  Porin Blocks Entry Outer Membrane  Efflux Pump  Diffusion through  Beta-Lactamase Peptidoglycan Hydolyzes Beta-Lactam  Penicillin Binding Proteins  Changes in PBP results in  Failure to Bind to -Lactam Cell Death

13. The Gram Negative Cell Wall Efflux system Porin channels B-lactamases PBPs Adapted from Livermore and Woodford, Trends in Microbiol, 2006.

14. Definition of beta lactamases • Beta lactamases are enzymes produced by some gram-positive and gram-negative bacteria that hydrolyze beta lactam antibiotics

15. β-Lactamase Classes

16. ESBLsExtended-spectrum β-lactamases • >180 enzymes described (119 TEM, 45 SHV) • All mutations of older TEM and SHV plasmid-mediated β -lactamases • TEM-3, TEM-4, etc. • SHV-2, SHV-3, etc. • CTX-M-1,2, etc. and Toho-type • OXA-type • PER-1 and 2 • Resistance conferred to extended-spectrum penicillins, 3rd and 4th generation cephalosporins and aztreonam (not imipenem or cephamycins) www.lahey.org/studies/webt.htm

17. ESBLsExtended-spectrum β-lactamases • Primarily found in: • Klebsiella, E. coli • Also found in: • Proteus, Serratia • Enterobacter, Salmonella • Morganella, etc. • Most are inhibited well by clavulanic acid and tazobactam (less so by sulbactam)

18. Beta-lactamase inhibitors • Resemble β-lactam antibiotic structure • Bind to β-lactamase and protect the antibiotic from destruction • Most successful when they bind the β-lactamase irreversibly • Three important in medicine • Clavulanic acid • Sulbactam • Tazobactam

20. Why Test for β-lactamases? • Correct therapy • Breakpoints do not reliably detect new β-lactamases • Infection control • Identify drugs causing resistance

21. Detection of ESBLs: Two Approaches • Screening tests and confirmatory tests for positive screens • Confirmatory tests

22. Detection of ESBLs: Screening Tests • Advantages • Less work • Cheaper • Disadvantages • Sensitivity less than 100% • Delayed confirmation • Reporting of positive screens?

23. CLSI Confirmatory Test – Klebsiella, E. coli, P. mirabilis • MIC Test • cefotaxime and ceftazidime +/- 4 μg/ml clavulanate: • > 3 doubling dilution decrease with either drug • Disk Test • cefotaxime and ceftazidime +/- 10 μg clavulanate • > 5 mm zone increase e.g. ceftazidime 8 μg/ml ceftazidime + clavulanate 1 μg/ml

24. FDA-Approved Commercial Tests • BD Sensi Disks • Etest • MicroScan • Phoenix • Trek • Vitek • Vitek 2

25. Combination Disk Method CLSI Approved Method

26. Double Disk Method Not CLSI Approved

27. CLSI Reporting Recommendation • ESBL confirmed:E. coli, Klebsiella,P. mirabilis • Report resistant for all penicillins, cephalosporins and aztreonam (except cephamycins, e.g., cefoxitin and cefotetan) regardless of in vitro status

28. Treatment of ESBL Positive Organisms with Cephalosporins MICFAILURE DEATH 8 100% (6/6) 33% (2/6) 4 67% (2/3) 0% (0/3) 2 33% (1/3) 0% (0/3) ≤1 27% (3/11) 18% (2/11) (CLSI breakpoint 8 g/ml) Paterson, DL, et al. JCM 39: 2206 – 2212, 2001

29. ESBL Blood Stream Infections Clinical Outcome FATALITY RATE: ESBL Producers = 26.7% (12/45) Non-ESBL Producers = 5.7% (5/87) MICFavorable Outcome pts given only Suscep. 3rd gen ceph 8 0 (0/2) 4 33 (1/3) 2 100 (1/1) (CLSI breakpoint 8 g/ml) Kim YK, et al. AAC 46:1481-1491, 2002

30. Pitfalls of ESBL Testing • CTX-M type -lactamases - novel group of Class A plasmid-encoded cephalosporinases • CTX abbreviation for cefotaximase. Includes CTX-M-type (17 to date), Toho-1, Toho-2, MEN-1 • Rapidly hydrolyze cefotaxime but not ceftazidime (some MICs  4) • Inhibited better by tazobactam than by sulbactam and clavulanate

31. Pitfalls of ESBL Testing • CTX-M-type found in Salmonella sp., E. coli, K. pneumoniae, C. freundii, P. mirabilis, S. marcescens • More common in S. America than N. America, also common in Europe and Asia • Have decreased susceptibility to inhibitor drugs therefore may not be confirmed with CLSI confirmatory test

33. E. coli with CTX-M ESBL

34. Pitfalls of ESBL TestingEffects of Inoculum (CLSI breakpoint 8 g/ml) KS Thomson and ES Moland, Creighton University

35. Pitfalls of ESBL TestingEffects of Inoculum (CLSI breakpoint 8 g/ml) KS Thomson and ES Moland, Creighton University

36. Enterobacteriaceae -Lactam Breakpoints and ESBL Issues • CLSI is re-evaluating -lactam breakpoints for Enterobacteriaceae • Example: cefotaxime • Current – Susceptible at  8 g/ml • Proposed – Susceptible at  1 or  2 g/ml • Substantial data needed • Goal is to more accurately detect all -lactamase and other -lactam resistance mechanisms with revised breakpoints • Changing breakpoints – commercial systems project it will take 3 years …much $$$$$!

37. ESBLs in organisms other than E. coli and Klebsiella spp. • Most labs do not attempt to detect ESBLs in organism other than E. coli and Klebsiella • Two Indications for ESBL Testing in Other Organisms • ESBLs detected in E. coli or Klebsiella • Suspicious phenotype • How to test? • Use specific (confirmatory) test • Perform Double Disk Diffusion

38. Prevalence of ESBLs • Aim of study was to detect ESBL prevalence in all GNB in US medical centers • 6,421 consecutive non-duplicate GNB screened for reduced susceptibility to cephems and aztreonam or potentiation of cefepime by clavulanate • Patients were from 42 ICU and 21 non-ICU sites throughout the US, 9/00 to 9/02 • Screen positive isolates were then investigated in a central lab for ESBL status Moland ES, et al. J Clin Microbiol. 2006 Sep;44:3318-24

39. Prevalence of ESBLs Moland ES, et al. J Clin Microbiol. 2006 Sep;44:3318-24

40. Prevalence of ESBLs at LUMC2006 and 2007 (Jan-Sept) Schreckenberger P, LUMC Antibiogram 2006-07

42. P. mirabilis with ESBL

43. Pitfalls of ESBL Testing • Recommendation (not CLSI endorsed): Extend CLSI reporting recommendations to all ESBL-producing organisms • Report all ESBL-producing organisms the same way: resistant to all penicillins, cephalosporins, and aztreonam

44. AmpC Beta Lactamases • Cephalosporinases, hydrolyze all beta lactam antibiotics except carbapenems and cefepime • Not Inhibited by clavulanate and sulbactam • Some inducible • Characteristic of certain genera: S P A C E - Serratia - Providencia/P. aeruginosa - Aeromonas - Citrobacter freundii - Enterobacter, Hafnia

45. AmpC Beta Lactamases • High level production of enzyme can be inducible or constitutive • With inducibleproduction, enzyme produced at low level unless organism exposed to inducing agents • Induction is a reversible mechanism

46. AmpC Beta Lactamases

47. AmpD AmpR ampD ampR ampC Uninduced AmpC • Wall fragments recycled by AmpD • AmpRin repressor conformation • ampC (-lactamase gene) NOT expressed

48. Induced AmpC -lactamase AmpD ampD ampR ampC • More recycling: AmpD overwhelmed • Wall fragments convert AmpR to activator • ampC (-lactamase gene) expressed

49. But mutational derepression is the problem, not induction E. cloacae expressing Induced Chromosomal AmpC

50. Derepressed AmpC -lactamase++ ampD ampR ampC • ampD inactivated by mutation • AmpR constantly converted to activator • ampC hyper-expressed