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Carbapenamases Facts and Diagnosis

Carbapenamases Facts and Diagnosis

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Carbapenamases Facts and Diagnosis

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  1. CARBAPENAMASESFacts, Controversies, Detection Dr.T.V.Rao MD

  2. A Changing Landscape forNumbers of Approved Antibacterial AgentsWe have more resistant Microbes 18 16 14 12 10 Number of agents approved 8 6 4 0 2 0 Resistance 1983-87 1988-92 1993-97 1998-02 2003-05 2008 Bars represent number of new antimicrobial agents approved by the FDA during the period listed. Infectious Diseases Society of America. Bad Bugs, No Drugs. July 2004; Spellberg B et al. Clin Infect Dis. 2004;38:1279-1286; New antimicrobial agents. Antimicrob Agents Chemother. 2006;50:1912 Dr.T.V.Rao MD

  3. Carbapenems xPenicillin • The carbapenems are structurally very similar to the penicillins, but the sulphur atom in position 1of the structure has been replaced with a carbon atom, and hence the name of the group, the carbapenem Dr.T.V.Rao MD

  4. What are carbapenems • Carbapenems are a class of beta-lactam antibiotics with a broad spectrum of antibacterial activity. They have a structure that renders them highly resistant to beta-lactamases. Carbapenem antibiotics were originally developed from thienamycin, a naturally-derived product of Streptomycescattleya. Dr.T.V.Rao MD

  5. Spectrum of activity • Broad spectrum activity • GPC & GNB • Aerobic & Anaerobic bacteria • Active against MDR isolates • Active against ESBL +ve GNB • Active against Ps aeruginosa & Acinetobacter spp. • Not active against • MRSA • Enterococcus spp. • Stenotrophomonas maltophilia

  6. Carbapenems in Common Use • Imipenem • Broad spectrum, covers Gram-positive, Gram-negative (including ESBL-producing strains), Pseudomonas and anaerobes • Meropenem • Less seizure-inducing potential, can be used to treat CNS infections • Ertapenem • Lacks activity vs. Acinetobacter and Pseudomonas • Has limited activity against penicillin-resistant pneumococci Dr.T.V.Rao MD

  7. Carbapenems effective on several common isolates • Staph(not MRSA), Strep (highly resistant), Neisseria, Haemophilus, Proteus, Pseudomonas, Klebseilla, Bacteroides, anaerobes (excluding C. dif) • . Dr.T.V.Rao MD

  8. Spectrum of Activity Dr.T.V.Rao MD

  9. Carbapenems Dr.T.V.Rao MD

  10. Enterobacteriaceae are real problamatic microbes • The rapid and disturbing spread of: • ESBL extended-spectrum ß-lactamases • AmpC enzymes • carbapenem resistance • metallo-β-lactamases • KPC and OXA-48 β-lactamases • Quinolones resistance Dr.T.V.Rao MD

  11. Bush 2010 : Distribution of β lactamases according to function Most Carbapenemases can Hydrolyze ALL Beta lactam antibiotics

  12. Discovery of Carbapenamases • In 1996, the first isolate of KPC-producing bacteria was discovered in a clinical specimen of K pneumoniae from a hospital in North Carolina involved in the Intensive Care Antimicrobial Resistance Epidemiology (ICARE) surveillance program. KPCs were infrequently isolated until 2001, when KPC-producing Enterobacteriaceae were reported in several extended outbreaks in metropolitan hospitals of New York and New Jersey.

  13. Carbapenems used as important life saving option • Carbapenems are often used as antibiotics of last resort for treating infections due to multidrug-resistant gram-negative bacilli, because they are stable even in response to extended-spectrum and AmpC β-lactamases. However, gram-negative bacilli producing the acquired metallo-β-lactamases (MBLs) IMP and VIM have been increasingly reported in Asia and Europe and more recently, they have been detected in Canada and the United States

  14. Carbapenemases • The most versatile family of -lactamases • Two major groups based on the hydrolytic mechanism at the active site • Serine at the active site: class A and D • Zinc at the active site : class B • All carbapenemases hydrolyze penicillin's, extended spectrum cephalosporins, and carbapenems

  15. Carbapenamases Dr.T.V.Rao MD

  16. Carbapenamases are spreading faster • A new class of bacterial enzymes capable of inactivating Carbapenems, known as Klebsiella pneumoniae Carbapenamases (KPCs), has rapidly spread in the United States and continues to be extensively reported elsewhere in the world. KPCs are class A Carbapenamases that reside on transferable plasmids and can hydrolyze all pencillins, cephalosporins, and Carbapenems. Dr.T.V.Rao MD

  17. Carbapenemases within the Enterobacteriaceae • KPC carbapenemase Difficult to detect using current MIC breakpoints. • Isolates that have an MIC of 2 mg/ml to ertapenem or an MIC of 2-4 mg/ml tomeropenem or Imipenem. • Modified Hodge test is confirmatory.. PCR is gold standard. Dr.T.V.Rao MD

  18. KPC(K. pneumoniae carbapenemase) • KPCs are the most prevalent of this group of enzymes, found mostly on transferable plasmids in K.pneumoniae • Substrate hydrolysis spectrum includescephalosporins and carbapenems Dr.T.V.Rao MD

  19. KPC’s in Enterobacteriaceae Dr.T.V.Rao MD

  20. Mechanism of Resistance to Carbapenems 1. Cephalosporinase : Amp C & CTX- M + Porin mutation = low level resistance 2. Carbapenemase: βlactamases that can hydrolyze carbapenems Amber Class A: 9 families KPC, SME, NMC-A, IMI, PER, GES, SFO, SFC, IBC Amber Class B: 6 families VIM, GIM, SIM, NDM, IMP, SPM Amber Class D: 2 families OXA, PSE

  21. Pseudomonas aeruginosaCarbapenamases • KPC resistance has been reported in inherently resistant organisms such as Pseudomonasfrom Trinidad, an isolate of multidrug-resistant Pseudomonas aeruginosa that harboured a novel KPC-6 gene was detected. Dr.T.V.Rao MD

  22. Serine βlactamases:

  23. Metallo β lactamases (Zn at active site)

  24. Carbapenemase Class A • First identified 1982 in UK • Four major families • Chromosomally encoded • Serratia marcescens enzyme (SME) • Not metalloenzyme carbapenemases (NMC) • Imipenem-hydrolyzing -lactamases (IMI) • Plasmid encoded • Klebsiella pneumoniae carabapenemases (KPC) • Guiana Extended-Spectrum (GES) Dr.T.V.Rao MD

  25. Emerging Carbapenem Resistance in Gram-Negative Bacilli • Significantly limits treatment options for life-threatening infections • No new drugs for gram-negative bacilli • Emerging resistance mechanisms, carbapenemases are mobile, • Detection of carbapenemases and implementation of infection control practices are necessary to limit spread Dr.T.V.Rao MD

  26. Enterobacteriaceae: Breakpoints revised so need for other newer drugs, may be carbapenms ? Dr.T.V.Rao MD

  27. Laboratory Detection Clinical and Laboratory Standards Institute breakpoints: 2009 & 2010 Revised Break Points 2010

  28. Class ACarbapenemases • K. pneumoniae carbapenemase (KPC-type) possess carbapenem-hydrolyzing enzymes most common on East Coast of U.S. • Enzymes are capable of efficiently hydrolyzing penicillins, Cephalosporins, aztreonam, and carbapenems and are inhibited by clavulanic acid and tazobactam • To date 4 KPC enzymes have been identified: KPC-1, KPC-2, KPC-3, KPC-4 – E. coli, K. pneumoniae, K. oxytoca, E. cloacae Dr.T.V.Rao MD

  29. Class ACarbapenemases • K. pneumoniae carbapenemase (KPC-type) possess carbapenem-hydrolyzing enzymes most common on East Coast of U.S. • Enzymes are capable of efficiently hydrolyzing penicillins, Cephalosporins, aztreonam, and carbapenems and are inhibited by clavulanic acid and tazobactam • To date 4 KPC enzymes have been identified: KPC-1, KPC-2, KPC-3, KPC-4 – E. coli, K. pneumoniae, K. oxytoca, E. cloacae Dr.T.V.Rao MD

  30. KPC Enzymes • Located on plasmids; conjugative and nonconjugative • blaKPC is usually flanked by transposon sequences • blaKPC reported on plasmids with: • Normal spectrum b-lactamases • Extended spectrum b-lactamases • Aminoglycoside resistance Dr.T.V.Rao MD

  31. When to Suspect a KPC-Producer • Enterobacteriaceae – especially Klebsiella pneumoniae that are resistant to extended-spectrum cephalosporins: • MIC range for 151 KPC-producing isolates • Ceftazidime 32 to >64 mg/ml • Ceftriaxone ≥ 64 mg/ml • Cefotaxime ≥ 64 mg/ml • Variable susceptibility to cefoxitin and cefepime Dr.T.V.Rao MD

  32. Modified Hodge Test for Carbapenemase Detection in Enterobacteriaceae

  33. Laboratory Detection of KPC-Producers Problems: 1) Some isolates demonstrate low-level carbapenem resistance 2) Some automated systems fail to detect low-level resistance

  34. The Modified Hodge Test The Modified Hodge Test is a phenotypic confirmatory test for “Carapnemase” activity and is indicated when there is a positive screening test and resistance to one or more agents in cephalosporin subclass III (i.e., cefoperazone, cefotaxime, ceftazidime, ceftizoxime, and ceftriaxone) Be aware that imipenem disk tests perform poorly as a screen for carbapenemases.

  35. Phenotypic Tests for Carbapenemase Activity • Modified Hodge Test • 100% sensitivity in detecting KPC; also positive when other carbapenemases are present • 100% specificity Procedure described by Lee et al. CMI, 7, 88-102. 2001.

  36. The Modified Hodge Test (MHT) • The Modified Hodge Test (MHT) detects carbapenemase production in isolates of Enterobacteriaceae • Carbapenemase production is detected by the MHT when the test isolate produces the enzyme and allows growth of a carbapenem susceptible strain (E.coli ATCC 25922) towards a carbapenem disk

  37. Step 1 and 2 of MHT • Prepare a 0.5 McFarland dilution of the E.coli ATCC 25922 in 5 ml of broth or saline. • Dilute 1:10 by adding 0.5 ml of the 0.5 McFarland to 4.5 ml of MHB or saline.

  38. Step 3 and 4 of MHT • Streak a lawn of the 1:10 dilution of E.coli ATCC 25922 to a Mueller Hinton agar plate and allow to dry 3–5 minutes. • Place a 10 μg meropenem or ertapenem susceptibility disk in the center of the test area.

  39. Protocols in Modified Hodge Test

  40. Step 5 and 6 of MHT • In a straight line, streak test organism from the edge of the disk to the edge of the plate. Up to four organisms can be tested on the same plate with one drug. • Incubate overnight at 35C ± 2OC in ambient air for 16–24 hours

  41. Test for Carbapenemase DetectionAnderson KF et al. Evaluation of methods to identify KPC in enterobacteriaceae. JCM 2007; 45: 2723 – 2725. Susceptible E. coli Carbapenem Disk Test Isolate Modified Hodge Test (MHT) Carbapenem Inactivation Assay

  42. Modified Hodge Test Lawn of E. coli ATCC 25922 1:10 dilution of a 0.5 McFarland suspension Test isolates Imipenem disk Described by Lee et al. CMI, 7, 88-102. 2001.

  43. Observation for Carbapenamases detection by HMT • After 16–24 hours of incubation, examine the plate for a clover leaf-type indentation at the intersection of the test organism and the E. coli 25922, within the zone of inhibition of the carbapenem susceptibility disk.

  44. Quality control strains in Modified Hodge test • Perform quality control of the Carbapenems disks according to CLSI guidelines. • Perform quality control with each run. • MHT Positive Klebsiella pneumoniae ATCC BAA-1705 • MHT Negative Klebsiella pneumoniae ATCC BAA-1706

  45. Why Testing with Ertapenem or Meropenem • The procedure described by Landman et al. describes using a 10-μg imipenem disk for step 1. However, there are species of Enterobacteriaceae which have intrinsic mechanisms of resistance to imipenem other than a carbapenemase (See CLSI document M100, Appendix G). Therefore, ertapenem or meropenem may provide more specific selection for acquired carbapenem resistance in Enterobacteriaceae

  46. What Labs Should Do Now • Look for isolates of Enterobacteriaceae (especially K. pneumoniae), with carbapenem MIC ≥ 2 mg/ml or nonsusceptible to Ertapenem by disk diffusion • Consider confirmation by Modified Hodge Test • Alert clinician and infection control practitioner to possibility of mobile carbapenemase in isolate

  47. Newer Carbapenemases • As of June 2010, there were three reported cases of Enterobacteriaceae isolates bearing this newly described resistance mechanism in the US, the CDC stated that "All three U.S. isolates were from patients who received recent medical care in India."

  48. NDM-1 • K. pneumoniae containing NDM-1 was first discovered in 2008. By 2009, a study in Mumbai revealed 24 carbapenem-resistant Enterobacteriaceae, 22 of which were NDM-1 producers. Of these 22 organisms, 10 were klebsiella species, 9 were Escherichia coli, 2 were enterobacter species, and 1 was Morganella morganii — illustrating the ability of the plasmid to spread rapidly among strains of Enterobacteriaceae

  49. CDC reports the new genetic mechanisms • The isolate, Klebseilla pneumoniae 05-506, was shown to possess a metallo-beta-lactamase (MBL) but was negative for previously known MBL genes. Gene libraries and amplification of class 1 integrons revealed three resistance-conferring regions; the first contained bla(CMY-4) flanked by ISEcP1 and blc. The second region of 4.8 kb contained a complex class 1 integron with the gene cassettes arr-2, a new erythromycin esterase gene; ereC; aadA1; and cmlA7

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