This document discusses carbapenamases, which are beta-lactamase enzymes that can hydrolyze carbapenem antibiotics, rendering them ineffective. It notes that carbapenamases are an emerging problem and now represent one of the most versatile beta-lactamase families. The document summarizes the main types of carbapenamases, including KPC, NDM, VIM, and OXA. It discusses the increasing spread of resistant bacteria producing these enzymes worldwide and outlines challenges for detection and treatment. Laboratory tests for detecting carbapenamase activity like the modified Hodge test are also summarized.

1. CARBAPENAMASES
Facts, Controversies, Detection
T.V.Rao MD
Sri Dev Raj Urs Medical College.
Kolar, Karnataka

2. A Changing Landscape for
Numbers of Approved Antibacterial Agents
We have more resistant Microbes
Bars represent number of new antimicrobial agents approved by the FDA during the period listed.
0
0
2
4
6
8
10
12
14
16
18
Numberofagentsapproved
1983-87 1988-92 1993-97 1998-02 2003-05 2008
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
Resistance
2Dr.T.V.Rao MD

3. 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
3Dr.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 Streptomyces cattleya.
4Dr.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
6Dr.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)
– . 7Dr.T.V.Rao MD

8. Spectrum of Activity
Drug
Strep spp. &
MSSA
Entero-
bacteriaeae
Non-
fermentors
Anaerobes
Imipenem + + + +
Meropenem + + + +
Ertapenem + +
Limited
activity
+
Doripenem + + + +
8Dr.T.V.Rao MD

9. Carbapenems
Drug
Route of
Administration
FDA Status
Imipenem IV Cleared
Meropenem IV Cleared
Ertapenem IM, IV Cleared
Doripenem IV Application Submitted
9Dr.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
10Dr.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 β-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
Classification Enzyme Most Common Bacteria
Class A KPC, SME,
IMI, NMC,
GES
Enterobacteriaceae
(rare reports in P. aeruginosa)
Class B
(metallo-β-lactamse)
IMP, VIM,
GIM, SPM
P. aeruginosa
Enterobacteriacea
Acinetobacter spp.
Class D OXA Acinetobacter spp.
15Dr.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.
16Dr.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 µg/ml to
ertapenem or an MIC of 2-4 µg/ml to meropenem
or Imipenem.
• Modified Hodge test is confirmatory..
PCR is gold standard.
17Dr.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 includes
cephalosporins and
carbapenems
18Dr.T.V.Rao MD

19. KPC’s in Enterobacteriaceae
Species Comments
Klebsiella spp. K. pneumoniae-cause of outbreaks
K. oxytoca-sporadic occurrence
Enterobacter spp.
Sporadic occurrence
Escherichia coli
Salmonella spp.
Citrobacter freundii
Serratia spp.
19Dr.T.V.Rao MD

20. 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

21. 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.
21Dr.T.V.Rao MD

22. Enzyme Ambler
Class
Country Spectrum of activity Organisms
GES
Guiana Extended
spectrum
A French
Guiana
Imipenem & extended
spectrum cephalosporins
Ps.
SME
Serratia
marcesance
enzyme
A USA Carbapenem, aztreonam
but not 3rd gen
cephalosporins
Serratia
marcescens
NMC – A,
IMI
Non metallo
carbapenamse
A Europe Carbapenem, aztreonam
but not 3rd gen
cephalosporins
Enterobacter spp
KPC
Kleb pn.
carbapenamase
A USA All β lactams Kleb. pneumoniae
OXA
Oxacillin
hydrolysing
D Scotland Carbapenems (weak) Acinetobacter, Ps.
Serine β lactamases:

23. Enzyme Ambler
Class
Country Spectrum of activity Organisms
IMP (18)
Imipenem
Japan
B Japan All β lactams Ps., Acinetobacter
VIM (12)
Verona
B Italy Pan R, may be S to
aztreonam
Ps. , Acinetobacter
SPM
Sao Paulo
B Brazil Pan R Ps
GIM German B Germany Pan R Ps.
SIM
Souel
B South
Korea
Pan R Acinetobacter, Ps.
NDM
New Delhi
B India,
UK
Pan R Kleb pneu, E.
coli
Metallo β lactamases (Zn at active site)

25. Carbapenemase Class A
• First identified 1982 in UK
• Four major families
• Chromosomally encoded
– Serratia marcescens enzyme (SME)
– Not metalloenzyme carbapenemases (NMC)
– Imipenem-hydrolyzing β-lactamases (IMI)
• Plasmid encoded
– Klebsiella pneumoniae carabapenemases (KPC)
– Guiana Extended-Spectrum (GES)
25Dr.T.V.Rao MD

26. Lancet Infect Dis 2010; 10: 597–602 Published Online August 11, 2010
1985 1986 1990 1995 2000 2008 2010
Imipenem
launched
Chromosomal
R
in Ps
IMP in
Japan
VIM in
Verona
KPC in
USA
March of Carbapenemases
NDM 1
Clinical Microbiology and Infection, Volume 16 Number 12, December 2010
Castanheira M et al: Anti Agents Chem 2010
SENTRY Program
Out of 39 strains collected from India in 2006
15 strains had NDM 1
10 strains carried OXA 48 variant
2 strains carried VIM 6
Multiple PFGE patterns

27. 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
27Dr.T.V.Rao MD

28. Enterobacteriaceae: Breakpoints revised so need
for other newer drugs, may be carbapenms ?
Agent
CLSI 2009 CLSI 2010
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
28Dr.T.V.Rao MD

29. 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
< 1
2-4 8
2
>16
>4
>16
>23
NA 14-15
20-22
<13
<19
MEM < 1
< 1
2-4 8
2
>16
>4
>22
>23
16-21 14-15
20-22
<13
<19
ERT < 1
< 0.25
2 4
0.5
>8
>1
> 22
>23
19-21 16-18
20-22
<15
<19

30. 30
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

31. 31
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

32. • Located on plasmids; conjugative and
nonconjugative
• blaKPC is usually flanked by transposon sequences
• blaKPC reported on plasmids with:
– Normal spectrum β-lactamases
– Extended spectrum β-lactamases
– Aminoglycoside resistance
Dr.T.V.Rao MD 32
KPC Enzymes

33. 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 µg/ml
• Ceftriaxone ≥ 64 µg/ml
• Cefotaxime ≥ 64 µg/ml
– Variable susceptibility to cefoxitin and cefepime
33Dr.T.V.Rao MD

34. Modified Hodge Test for
Carbapenemase Detection in
Enterobacteriaceae

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

36. 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.

37. 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.

38. 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

39. 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.

40. 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.

41. Protocols in Modified Hodge Test

42. 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

43. 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
Carbapenem Disk
Susceptible
E. coli
Test Isolate

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

45. 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.

46. 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

47. 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

48. What Labs Should Do Now
• Look for isolates of Enterobacteriaceae (especially
K. pneumoniae), with carbapenem MIC ≥ 2 µg/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

49. 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."

50. • 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
NDM-1

51. 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

52. • 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
New Delhi metallo-beta-lactamase 1

53. 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.

54. 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."

55. 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

56. 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."

57. 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

58. 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.

59. 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

60. 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

61. 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

62. CHROMagar
ESBL & KPC

63. 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

64. 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 µg/ml, normal MIC range
• MIC ≥ 4 µg/ml indicates increased resistance

65. 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

66. 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.

67. 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 67

68. Thank Q