CARBAPENAMASES

Facts, Controversies, Detection

Dr.T.V.Rao MD
 A Changing Landscape for
Numbers of Approved Antibacterial Agents
We have more resistant Microbes
18

16
Number of agents approved

14

12

Resistance
10

2
0
0
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 2
 Carbapenems x Penicillin
• The carbapenems are
structurally very similar
to the penicillins, but the
sulphur atom in position
1 of the structure has
been replaced with a
carbon atom, and hence
the name of the group,
the carbapenem

Dr.T.V.Rao MD 3
 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 Streptomyces cattleya.
Dr.T.V.Rao MD 4
 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 6
 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 7
 Spectrum of Activity
Strep spp. & Entero- Non-
Drug Anaerobes
MSSA bacteriaeae fermentors

Imipenem + + + +

Meropenem + + + +

Limited
Ertapenem + + activity +

Doripenem + + + +

Dr.T.V.Rao MD 8
 Carbapenems
Route of
Drug FDA Status
Administration
Imipenem IV Cleared

Meropenem IV Cleared

Ertapenem IM, IV Cleared

Doripenem IV Application
Submitted

Dr.T.V.Rao MD 9
 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 10
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
Classification Enzyme Most Common Bacteria
Class A KPC, SME, Enterobacteriaceae
IMI, NMC, (rare reports in P. aeruginosa)
GES

Class B IMP, VIM, P. aeruginosa

(metallo-b-lactamse) GIM, SPM Enterobacteriacea
Acinetobacter spp.

Class D OXA Acinetobacter spp.

Dr.T.V.Rao MD 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.
Dr.T.V.Rao MD 16
 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 to
meropenem or Imipenem.
• Modified Hodge test is confirmatory..
PCR is gold standard.

Dr.T.V.Rao MD 17
 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 includes
cephalosporins and
carbapenems
Dr.T.V.Rao MD 18
 KPC’s in Enterobacteriaceae
Species Comments
Klebsiella spp. K. pneumoniae-cause of outbreaks
K. oxytoca-sporadic occurrence
Enterobacter spp.
Escherichia coli
Salmonella spp. Sporadic occurrence
Citrobacter freundii
Serratia spp.

Dr.T.V.Rao MD 19
Mechanism of Resistance to Carbapenems
1. Cephalosporinase : Amp C & CTX- M
+ Porin mutation = low level resistance
2. Carbapenemase: β lactamases 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 aeruginosa Carbapenamases

• KPC resistance has been

reported in inherently
resistant organisms such
as Pseudomonas
from Trinidad, an isolate
of multidrug-resistant
Pseudomonas
aeruginosa that
harboured a novel KPC-6
gene was detected.
Dr.T.V.Rao MD 21
 Serine β lactamases:

Enzyme Ambler Country Spectrum of activity Organisms

Class
GES A French Imipenem & extended Ps.
Guiana Guiana spectrum cephalosporins
Extended
spectrum

SME A USA Carbapenem, aztreonam Serratia

Serratia but not 3rd gen marcescens
marcesance
enzyme cephalosporins

NMC – A, A Europe Carbapenem, aztreonam Enterobacter spp

IMI but not 3rd gen
Non metallo cephalosporins
carbapenamse

KPC A USA All β lactams Kleb.

Kleb pn. pneumoniae
carbapenamase

OXA D Scotland Carbapenems (weak) Acinetobacter,

Oxacillin Ps.
hydrolysing
 Metallo β lactamases (Zn at active site)
Enzyme Ambler Country Spectrum of activity Organisms
Class

IMP (18) B Japan All β lactams Ps.,

Imipenem Acinetobacter
Japan

VIM (12) B Italy Pan R, may be S to Ps. ,

Verona aztreonam Acinetobacter
SPM B Brazil Pan R Ps
Sao Paulo

GIM B Germany Pan R Ps.

German

SIM B South Pan R Acinetobacter,

Souel Korea Ps.
NDM B India, Pan R Kleb pneu,
New Delhi
UK E. coli
 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 ?
CLSI 2009 CLSI 2010
Agent
S I R S I R

Cefazolin ≤8 16 ≥32 ≤1 2 ≥4

Cefotaxime ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftriaxone ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftazidime ≤8 16 ≥32 ≤4 8 ≥16

Aztreonam ≤8 16 ≥32 ≤4 8 ≥16

Cefipime ≤8 16 ≥32 ≤8 16 ≥32

Dr.T.V.Rao MD 27
 Laboratory Detection
Clinical and Laboratory Standards Institute breakpoints: 2009 & 2010
Revised Break Points 2010

Agent MIC breakpoint (ug/ml) DD breakpoints (mm)

S MHT I R S MHT I R

IPM <1 2-4 8 >16 >16 NA 14-15 <13

<1 2 >4 >23 20-22 <19

MEM <1 2-4 8 >16 >22 16-21 14-15 <13

<1 2 >4 >23 20-22 <19

ERT <1 2 4 >8 > 22 19-21 16-18 <15

< 0.25 0.5 >1 >23 20-22 <19
 Class A Carbapenemases
• 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 A Carbapenemases
• 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
 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 Detection
Anderson KF et al. Evaluation of methods to identify KPC in enterobacteriaceae. JCM
2007; 45: 2723 – 2725.

Modified Hodge Test (MHT)

Carbapenem Inactivation Assay

Susceptible
E. coli
Carbapenem Disk

Test Isolate
 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."
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
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
CHROMagar
ESBL & 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 66
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