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Detection of Carbapenemases From a Technologist’s Perspective

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Presentation on theme: "Detection of Carbapenemases From a Technologist’s Perspective"— Presentation transcript:

1 Detection of Carbapenemases From a Technologist’s Perspective
Courtney Fraser MLS (ASCP) Microbiology Supervisor ACL Laboratories

2 Disclosure I will discuss specific brand names in this presentation. I do not have stock in, nor receive honoraria for any commercial product mentioned in this presentation.

3 Introduction Automation is not available for carbapenemase confirmatory testing. Resistance mechanisms are not typically discussed in the classroom for most MLS students. Seasoned Techs currently in the field are not used to resistance in organisms and are unfamiliar with the mechanisms related to resistance. Education needed on antibiotic classifications.

4 Purpose of Presentation
Define types of carbapenemases and understand classifications When to test How to test How to incorporate instrumentation tools

5 What is a Carbapenemase?
First, need to understand what are the carbapenems: Ertapenem Imipenem Meropenem Doripenem Second, the suffix –ase indicates an enzyme A carbapenemase is an enzyme produced by bacteria that hydrolyze (deactivate) carbapenems


7 Carbapenems Large groups
Meropenem All share a common β-lactam ring, β-lactamases target the β-lactam ring) differences are in ring stabilization from hydrolysis protection of ring from β-lactamase by addition of large groups blocking the substrate site Imipenem Ertapenem Large groups

8 Why Important? Studies have shown that rapid detection of carbapenemases have attributed to positive patient treatment outcomes. Infection Control emergency – These mechanisms can be plasma-mediated allowing for easy transmission to other organisms. Detection of these resistance mechanisms should initiate contact precautions for patients , thus reducing the spread of nosocomial related infections. Deter inappropriate antibiotic use thus prolonging the efficacy of available antibiotics

9 The Rise of Carbapenemases

10 ACL Laboratories Overview
ACL Laboratories is defined as an operating management agreement between Advocate Health Care and Aurora Health Care. Two Central Labs (Rosemont, IL., West Allis, WI.) Total of 27 hospitals Rosemont

11 Wisconsin vs. Illinois 2011

12 KPC Isolates 2009 and 2011 - Chicago

13 Types of β-Lactamases/Carbapenemases
Type A (Ambler) – KPC – 1st described 1998, endemic in NYC since Moving west. 1st isolates in Chicago area in 2/08 Type B – Metallo ß-lactamases Found in Stenotrophomonas maltophilia Enterobacteriaceae and P. aeruginosa - Reported sporadically in U.S. Watch out for NDM-1 which has spread to Europe (Sweden and England) from India Type D – Oxa-40 – Endemic in Acinetobacter baumannii in the Chicago area since 2002 Hyper AmpC producers Mutations in AmpC promoter and attenuator/promoter regions Poorly inhibited by lactam—lactamase inhibitor combinations E. coli produces AmpC (poorly) and is unlike other bacteria with AmpC the gene is not inducible No ampR regulatory gene All the above can give a positive Hodge Test! 13 13

14 Types of Carbapenemases
Enzyme Type Ambler Class Activity Spectrum Organism(s) KPC (1-10) (plasmid) A All β-lactams Enterobacteriaceae Ps. aeruginosa SME Carbapenems and aztreonam, but not 3rd/4th Gen cephalosporins S. marcescens , not plasmid Associated. NMC–A, IMI MNC = Not metallo carbapenemase IMI = IMI hydrolyzing Β-lactamase Same as for SME Enterobacter spp. GES GES= Guiana extended spectrum (plasmid) Imipenem and 3rd/4th cephalosporins Ps. Aeruginosa and Enterobacteriaceae IMI, VIM, NDM-1 VIM = Verona Integron encoded MBL) NDM-1 = New Delhi metallo β lactamase B (metallo-β-lactamases) All β-lactams; can test susceptible to aztreonam (NDM-1 variable AZT resistance) Pseudomonas spp. Acinetobacter spp. Enterobacteriaceae OXA (Oxacillin hydrolyzing) D Weakly active against carbapenems A. baumanii, P. Aeruginosa, and rare Enterobacteriaceae S. marcescens enzyme Pediatr Infect Dis J. 2010;29(1):68-70. 

15 Recommendations for Screening
Any Enterobacteriaceae At least one carbapenem with an increased MIC (ertapenem is most sensitive, but not specific) and at least one resistant 3rd or 4th generation cephalosporins: Ceftriaxone Ceftazidime Cefotaxime Ceftizoxime Cefepime Screen for KPC, MBL, and AmpC ESBL testing is performed by automated instrumentations.

16 KPC Klebsiella pneumoniae Carbapenemase
1st reported on the East Coast of the United States in the late 1990’s Currently found worldwide. Can be associated with other Enterobacteriaceae. KPC gene is plasmid-mediated which has contributed to the rapid dissemination across the globe. Hydrolyze all beta-lactam antibiotics. Can be detected by Modified Hodge Test Boronic Acid inhibition

17 Modified Hodge Test (MHT)
Used for the detection of KPC producing isolates Carbapenem disk: meropenem – gold standard – most specific Increased MIC for imipenem seen is Proteus, Providencia, and Morganella Indentation of the inhibition zone indicates that the test strain is hydrolyzing the carbapenem. Not specific for KPC AmpC enzymes can give weak false positive results that can be accentuated by porin loss

18 Modified Hodge Test (MHT)

19 Alternative Test Rosco kit for carbapenemase detection
4 disks all on one plate: Meropenem (MRP10) Meropenem + Boronic Acid (MRPBO) *KPC Meropenem + Cloxacillin (MR+CX) *AmpC Meropenem + DPA Dipicolinic acid (MR+DP) *MBL Easier to read compared to the MHT Reduced the risk of false positives for KPC Detects true hyperproduction of AmpC by utilizing a carbapenem instead of a cephamycin

20 Rosco Kit

21 Positive for KPC

22 Metallo Beta-Lactamase (MBL)
Uses a zinc cation for the hydrolysis of the beta-lactam ring Activity is inhibited by EDTA (similar to clavulanic acid and ESBL) Hydrolyze all beta-lactam antibiotics except aztreonam. Chromosomal presence found in Stenotrophomonas, Aeromonas, and Chryseobacterium. Clinically significant MBLs have transmitted to other bacterial pathogens.

23 Detection of MBL Ratio MP/MPI >8 (or 3 fold)

24 AmpC Chromosomal = MY SPACE bugs
(Morgenella, Y. enterocolitica, Serratia, Providencia, Aeromonas, Citrobacter, Enterobacter) Inducible = Any MYSPACE or Enterobacteriaceae containing AmpC plasmid Organism may develop resistance during prolonged therapy with 3rd generation cephalosporins. Identified in organisms exhibiting the following: Resistant to cephamycins Cefoxitin Cefotetan Sensitive to Cefepime Hyperproduction = Any Enterobacteriaceae Caused by a mutation in the AmpC gene leading to permanent hyperproduction or derepression.

25 The AmpC gene is now found on a plasmid
We can no longer predict which bacteria are AmpC positive Amp C is upregulated by treatment with β-lactam drugs Amount of enzyme produced is dependant on the selection of stable mutants with upregulated genes.

26 AmpC + Porin Loss = “Carbapenemase”

27 AmpC Many different AmpC enzymes
C. freundii cluster CMY-2 Enterobacter cluster MIR-1, ACT-1 M. morganii cluster** DHA-1 H. alvei cluster ACC-1 Aeromonas cluster CMY-1 and FOX-1 Degree of carbapenemase resistance is dependant on type of AmpC in a porin deficient isolate

28 Detection of AmpC Hyperproducer

29 Education and Training
In the past, technologists did not need to use antibiotic classification in everyday use. Education may be needed to help technologists understand the importance of classifications. Tools are available in automated instrumentation that can help technologists on the bench.

30 MicroScan Report

31 Vitek Observa Report

32 Tools in Instrumentation
Custom comments/alerts can be printed on the patient report generated from the instrument. Activation of these tools can significantly increase the technologist’s awareness on when to appropriately screen for carbapenemases. Can also be used for many other difficulties in AST reporting.

33 Creating a MicroScan Comment

34 MicroScan Alert Rules

35 MicroScan Patient Report

36 How to Build a BIOART Rule


38 Vitek Patient Report

39 References Bush, Karen, Jacoby, George, Antimicrobial Agents and Chemotherapy,Minireview Updated Functional Classification of Beta-Lactamases, Mar. 2010, Vol.54, No. 3, p Fernando, Pasteran, Tania Mendez, Melina Rapoport, Leonor Guerriero, and Alejandra Corso, Controlling False Positive Results Obtained with the Hodge…J. Clin. Microbiol. 2010, 48(4):1323 (2010). Gazin, Muriel, Fabienne Paasch, Herman Goossens and Surbhi Malhotra-Kumar, Current Trends in Culture-Based and Molecular Detection of ESBL Harboring and Carbapenem Resistant Enterobacteriaceae, J. Clin. Microbiol. 2012, 50(4). Rosco Diagnotica Siemens Microscan Biomerieux Vitek 2 Observa

40 THANK YOU!!! A big THANK YOU to the ACL Sensitivity Training Team!!! Your passion and dedication to AST testing has served both patients and your fellow coworkers. Questions?

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