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CARBAPENAMASESFacts, Controversies, Detection

Dr.T.V.Rao MD


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


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


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


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


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


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


Spectrum of Activity

Dr.T.V.Rao MD


Carbapenems

Dr.T.V.Rao MD


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


Bush 2010 : Distribution of β lactamases according to function

Most Carbapenemases can Hydrolyze

ALL

Beta lactam antibiotics


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.


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


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


Carbapenamases

Dr.T.V.Rao MD


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


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


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


KPC’s in Enterobacteriaceae

Dr.T.V.Rao MD


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


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


Serine βlactamases:


Metallo β lactamases (Zn at active site)


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


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


Enterobacteriaceae: Breakpoints revised so need for other newer drugs, may be carbapenms ?

Dr.T.V.Rao MD


Laboratory Detection

Clinical and Laboratory Standards Institute breakpoints: 2009 & 2010

Revised Break Points 2010


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


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


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


When to Suspect a KPC-Producer

  • Enterobacteriaceae – especially Klebsiella pneumoniae that are resistant to extended-spectrum cephalosporins:

    • MIC range for 151 KPC-producing isolates

      • Ceftazidime32 to >64 mg/ml

      • Ceftriaxone≥ 64 mg/ml

      • Cefotaxime≥ 64 mg/ml

    • Variable susceptibility to cefoxitin and cefepime

Dr.T.V.Rao MD


Modified Hodge Test for Carbapenemase Detection in Enterobacteriaceae


Laboratory Detection of KPC-Producers

Problems:

1)Some isolates demonstrate low-level carbapenem resistance

2) Some automated systems fail to detect low-level resistance


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.


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.


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


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.


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.


Protocols in Modified Hodge Test


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


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


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.


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.


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


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


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


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."


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


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


New Delhi metallo-beta-lactamase 1

  • NDM-1, which stands for New Delhi metallo-beta-lactamase 1 and actually refers not to a single bacterial species but to a transmissible genetic element encoding multiple resistance genes that was initially isolated from a strain of Klebsiella obtained from a patient who acquired the organism in New Delhi, India

  • Subsequently, organisms in the Enterobacteriaceae family containing this genetic element (or variants thereof) have been found widely throughout India, Pakistan, and Bangladesh


NDM-1

  • NDM-1 symptoms are reported to be associated with the bacteria it attaches to. The currently known bacteria's hosting this gene are E.Coli and Klebsiella pneumoniae. The majority of the patients treated to date who are positive for NDM-1 were those with urinary tract infections, bacteraemia, or pneumonia NDM-1 is the gene responsible for the newest superbug. NDM-1 genes can live inside different bacteria and is resistant to currently available antibiotics.


Naming the strain as New Delhi creates Controversy

  • The gene was named after New Delhi, the capital city of India, as it was first described by Yong et al. in 2009 in a Swedish national who fell ill with an antibiotic-resistant bacterial infection that he acquired in India . The infection was unsuccessfully treated in a New Delhi hospital and after the patient's repatriation to Sweden, a carbapenem-resistant Klebsiella pneumoniae strain bearing the novel gene was identified. The authors concluded that the new resistance mechanism "clearly arose in India, but there are few data arising from India to suggest how widespread it is."


CDC reports

Three Enterobacteriaceae isolates carrying a newly described resistance mechanism, the New Delhi metallo-beta-lactamase (NDM-1) , were identified from three U.S. states at the CDC antimicrobial susceptibility laboratory. This is the first report of NDM-1 in the United States, and the first report of metallo-beta-lactamase carriage among Enterobacteriaceae in the United States


Blame on India Is it justified ?

  • 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."


CDCreports 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


Molecular configuration of NDM-1

  • NDM-1 also has an additional insert between positions 162 and 166 not present in other MBLs. NDM-1 has a molecular mass of 28 kDa, is monomeric, and can hydrolyze all beta-lactams except aztreonam. Compared to VIM-2, NDM-1 displays tighter binding to most Cephalosporins.


NDM genetic coding differs from other recent isolates

  • Compared to VIM-2, NDM-1 displays tighter binding to most cephalosporins, in particular, cefuroxime, cefotaxime, and cephalothin (cefalotin), and also to the penicillins. NDM-1 does not bind to the carbapenems as tightly as IMP-1 or VIM-2 and turns over the carbapenems at a rate similar to that of VIM-2. In addition to K. pneumoniae 05-506, bla(NDM-1) was found on a 140-kb plasmid in an Escherichia coli strain isolated from the patient's feces, inferring the possibility of in vivo conjugation


CLSI guidelines for assessing the antibiograms pattern

  • All patients colonized or infected with CRE or carbapenemase-producing Enterobacteriaceae should be placed on contact precautions. Acute care facilities should establish a protocol, in conjunction with CLSI guidelines, to detect nonsusceptibility and carbapenemase production in Enterobacteriaceae, particularly Klebseilla spp. and Escherichia coli, and immediately alert epidemiology and infection control staff members if identified


Phenotypic detection with Hodge test a Minimal requirement

  • Carbapenem resistance and carbapenemase production conferred by blaNDM-1 is detected reliably with phenotypic testing methods currently recommended by the Clinical and Laboratory Standards Institute , including disk diffusion testing and the modified Hodge test


CHROMagarESBL & KPC


Why is CRE a public health emergency ?

  • Significantly limits treatment options for life threatening infections

  • No new drug for GNB in the pipeline

  • Resistant mechanism easily transferable as it in now on a transposon

  • Rapid Detection & effective infection control measures essential to control spread


Testing Other Drugs

  • Polymyxin B or Colistin

    • Could test either, but colistin used clinically

    • Disk diffusion test does not work – don’t use!

    • Etest – works well, but not FDA cleared

    • Broth micro dilution – reference labs

    • Breakpoints- none

      • MIC ≤ 2 mg/ml, normal MIC range

      • MIC ≥ 4 mg/ml indicates increased resistance


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


Automation has limited use in Carbapenamases detection

  • Automated testing alone will not detect all of the resistance patterns that occur via beta-lactamases and carbapenemases. Failure to detect organisms with these enzymes can result in erroneous reports that would indicate an isolate is susceptible to beta-lactam and/or carbapenem antibiotics.


Become a Member of Alliance for the Prudent Use of Antibiotics (APUA) www.apua.org

An international organization dedicated to curbing antibiotic resistance

Chapters exist currently in several Asian countries: Australia, China, India, Nepal, Pakistan, Philippines, South Korea, Taiwan, Vietnam

Dr.T.V.Rao MD


Hand Washing Can Reduce the Spread of Microbes


Created by Dr.T.V.Rao MD for ‘ e ‘ learning resources for the Medical Professionals in the Developing World

email.

doctortvrao@gmail.com


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