Carbapenamases in Antibiotic Resstance

1. Carbapenamases in Antibiotic Resistance Dr.T.V.Rao MD

2. 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. Carbapenems antibiotics were originally developed from thienamycin, a naturally-derived product of Streptomyces cattleya

3. Carbapenems - Structure • Carbapenems are structurally very similar to the pencillins, but the sulfur atom in position 1 of the structure has been replaced with a carbon atom, and hence the name of the group, the carbapenems.

4. Drugs belong to the carbapenem class: • Imipenem • Meropenem Ertapenem Doripenem Panipenem/ betamipron Biapenem

5. Background of Carbapenamases • Carbapenem-resistant Enterobacteriaceae (CRE) are usually resistant to all β-lactam agents as well as most other classes of antimicrobial agents. The treatment options for patients infected with CRE are very limited. Healthcare-associated outbreaks of CRE have been reported

6. 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.

7. Carbapenemases are produced by several commonly infecting Gram Negative Bacteria • Carbapenemases are known to exist in several different species of gram-negative bacilli including species of Enterobacteriaceae and Pseudomonas aeruginosa. However, carbapenemases are more common in lactose-fermenting species of Enterobacteriaceae (e.g., K. pneumoniae and E. coli) than in non-lactose fermenting Enterobacteriaceae (e.g. Serratia marcescens and some Enterobactericae spp.) and P. aeruginosa.

8. How Carbapenamase resistance is initiated • Carbapenem resistance in Enterobacteriaceae occurs when an isolate acquires a carbapenemase or when an isolate produces an extended-spectrum cephalosporinase, such as an AmpC-type β-lactamase, in combination with porin loss. In the United States, the most common mechanism of carbapenem resistance is the Klebsiella pneumoniae carbapenemase (KPC).

9. Carbapenamases a Global Problem • The resistance to Carbapenems has emerged worldwide and the predominant mechanism of resistance is attributed by the production of various Carbapenems-hydrolyzing β-lactamases referred as Carbapenamases

10. 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

11. Carbapenem Resistance: Mechanisms

12. Transposons and Integrons contribute for spread of resistance, • The genes of these MBL enzymes are often plasmid borne and are associated with mobile genetic elements (transposons and integrons), making them readily transferable among various species

13. Carbapenamases

14. Carbapenamases are complex in Mechanisims • Carbapenamases constitute the most versatile family of β-lactamases belonging to molecular classes A, B and D and are capable of hydrolyzing almost all β-lactams. Given their zinc dependent hydrolytic activity, Carbapenamases of class B is designated as metallo-ß-lactamases (MBL) that include, for example, IMP, GIM, SIM, SPM, and VIM carbapenemases, and these MBL enzymes have been reported in P.aeruginosa and other multidrug resistant pathogens

15. 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.

16. Klebsiella pneumoniae Carbapenamases • KPC-producing organisms continues to evolve. Although most KPCs are detected in isolates of Klebsiella and Escherichia coli, KPCs have been extensively reported in other genera of the Enterobacteriaceae family, such as Proteus,Serratia, Salmonella, and Citrobacter.

17. Klebsiella pneumoniae Carbapenamases • 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

18. 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

19. KPC’s in Enterobacteriaceae Pseudomonas aeruginosa – Columbia & Puerto Rico

20. 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.

21. Special antibiotic sensitivity testing is emerging need in Microbiology laboratories • Supplemental testing, in addition to the routine susceptibility tests of isolates, has become necessary in order to detect the deluge of beta-lactamases and carbapenemases that clinical laboratories face today

22. When to Suspect a KPC-Producers • 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

23. Modified Hodge Test for Carbapenemase Detection in Enterobacteriaceae

24. 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.

25. CLSI Recommends • CLSI Recommends doing Modified Hodge test before reporting Carbapenam susceptibility results if results are elevated but susceptible to Carbapenam by Minimum inhibitory concentration. • The results of intermediate or resistance to Carbapenems need not be tested with MHT

26. 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

27. The Carapnemase is detected by antibiotic sensitivity patterns • 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. The result is a characteristic cloverleaf-like indentation

28. 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.

29. 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.

30. Protocols in Modified Hodge Test

31. K.pneumonia showing Carbapenem resistance

32. 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 35OC ± 2OC in ambient air for 16–24 hours

33. 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.

34. 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.

35. MHT detection (photo courtesy of CDC) • The MHT performed on a 100 mm MHA plate. (1) K. pneumoniaeATCC BAA 1705, positive result K. pneumoniaeATCC BAA 1706, negative result; and a clinical isolate, positive result312

36. A Positive HMT test • MHT Positive test has a clover leaf-like indentation of the E.coli 25922 growing along the test organism growth streak within the disk diffusion zone. • A positive MHT indicates that this isolate is producing a carbapenemase

37. A negative HMT Test • MHT Negative test has no growth of the E.coli 25922 along the test organism growth streak within the disc diffusion. • A negative MHT indicates that this isolate is not producing a carbapenemase

38. 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

39. CLSI guidelines for Carbapenamases detection • CLSI has published guidelines for detection of isolates producing carbapenemases (CLSI document M100) . For isolates that test susceptible to a carbapenem but demonstrate reduced susceptibility either by disk diffusion or MIC testing, performing a phenotypic test for carbapenemase activity, the Modified Hodge Test (MHT), is recommended

40. 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

41. New CLSI guidelines Nonsusceptible ”Interpretive Category”

42. “Nonsusceptible”Interpretive Category • “…. used for organisms that have only a susceptible interpretive category, but not intermediate or resistant interpretive categories (ie, susceptible-only interpretive category). A susceptible-only interpretive category may be applied to new antimicrobial agents for which no resistant isolates have been encountered at the time the initial interpretive criteria are determined.

43. Nonsusceptible”Interpretive Category • The isolates that test with an MIC above the susceptible interpretive breakpoint are designated as nonsusceptible. A designation of nonsusceptible does not necessarily mean that a resistance mechanism exists in the isolate. The MIC of the isolate in the nonsusceptible range may be within the previously recognized wild-type distribution of susceptibility results; however, there is limited clinical experience with these isolates in clinical trials.”

44. Interpretation/ResultsConveyed to Infection Control Departments • Report all cultures that are positive for CRE or carbapenemase-producing Enterobacteriaceae to the appropriate infection control personnel.

45. Patients infected will be dealt with caution to prevent spread • patients colonized with carbapenem-resistant or Carbapenamases-producing Enterobacteriaceae in the intestinal tract and the Patients who grow these organisms should be placed on Contact Precautions to prevent transmission of the resistant bacteria

46. Laboratories should create protocols for detection of CRE • The exact procedure for confirmation of CRE or carbapenemase-production should be laboratory-specific and chosen based upon laboratory workflow and the types of isolates causing clinical infections in the patient population served. It may be helpful to refer to the CLSI guidelines for identification of carbapenemase production in isolates that test susceptible to Carbapenems

47. Follow contact Precautions • Contact Precautions should be implemented for all patients with positive cultures for CRE or carbapenemase-producing Enterobacteriaceae

48. Hand washing can save several patients

49. Care of the patients colonized with Carapnemase resisistant Enterobactericae • Patients colonized with CRE are thought to be a source of transmission in the healthcare setting . Identifying patients who are colonized with CRE and placing these patients in isolation precautions may be an important step in preventing transmission

50. Detection of Antibiotic resistance patterns is more than past • Resistance to our beta-lactam and carbapenem antibiotics is becoming daunting for antimicrobial therapy for infections involving the Enterobacteriaceae. Similarly, laboratory testing to detect these resistance mechanisms is becoming more complex and perplexing for microbiology laboratories.