Dr.T.V.

Rao MD

CARBAPENAMS
Uses and Resistance
DR.T.V.RAO MD

1
 Dr.T.V.Rao MD

CONVENTIONAL ANTIBIOTICS
 Nitrofurantoin, metronidazole, clindamycin,
Penicillins
 vancomycin,
Cephalosporinsteicoplanin, cotrimoxazole, fusidic
 acid, etc
Carbapenems

 Isoniazid,
Quinolones pyrazinamide, ethambutol, rifampin,
cycloserine, etc
 Amino glycosides
 Macrolides
 Tetracyclines

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 Dr.T.V.Rao MD

CARBAPENEMS X PENICILLINS
 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

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 Dr.T.V.Rao MD

ANTIBIOTIC FORMULATION CONTINUE TO GROW

CONTINUE TO CONFUSE

 50 penicillins  9 macrolides
 71 Cephalosporins  2 streptogramins
 12 Tetracyclines  3 dihydrofolate
 8 amino glycosides reductase
 1 monobactam inhibitors
 >3 carbapenems
 1 oxazolidinone
 5.5 quinolones

4
 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
5
 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 Streptomyces cattleya.

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 Dr.T.V.Rao MD

CARBAPENEMS COMMON USES

 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

7

HOW ARE CARBAPENEMS USED?
Uses by Clinical Syndrome
 Bacterial meningitis
 Hospital-associated
sinusitis
 Sepsis of unknown origin
 Hospital-associated
pneumonia
Reference: Sanford Guide
Dr.T.V.Rao MD
Use by Clinical Isolate
 Acinetobacter spp.
 Pseudomonas aeruginosa
 Alcaligenes spp.
 Enterobacteriaceae
 Mogenella spp.
 Serratia spp.
 Enterobacter spp.
 Citrobacter spp.
 ESBL or AmpC + E. coli
and Klebsiella spp.
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 Dr.T.V.Rao MD

ENTEROBACTERIACEAE ARE REAL PROBLAMATIC MICROBES

 The rapid and disturbing

spread of:
 extended-spectrum ß-
lactamases
 AmpC enzymes
 carbapenem
resistance
 metallo-β-lactamases
 KPC and OXA-48 β-
lactamases
 Quinolones resistance
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 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)
 .
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 Dr.T.V.Rao MD

CARBAPENEMS ARE BROAD SPECTRUM

ANTIBIOTIC
 These agents have the broadest antibacterial
spectrum compared to other beta-lactam classes
such as penicillins and cephalosporins. Additionally
they are generally resistant to the typical bacterial
beta-lactamase enzymes which are one of the
principal resistance mechanisms of bacteria. They
are active against both Gram positive and gram
negative bacteria, with the exception of intracellular
bacteria, such as the Chlamydia

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 Dr.T.V.Rao MD

DRUGS BELONG TO THE CARBAPENEM

CLASS:
 Imipenem
 Meropenem

Ertapenem

Doripenem
Panipenem/
betamipron
Biapenem
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 Dr.T.V.Rao MD

BROADEST ANTIBACTERIAL SPECTRUM

 These agents have the broadest antibacterial
spectrum compared to other beta-lactam classes
such as penicillins and cephalosporins. Additionally
they are generally resistant to the typical bacterial
beta-lactamase enzymes which are one of the
principal resistance mechanisms of bacteria. They
are active against both Gram positive and gram
negative bacteria, with the exception of intracellular
bacteria, such as the Chlamydia.

13
 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 14
 SPECTRUM OF ACTIVITY
Strep spp. & Entero- Non-
Drug Anaerobes
MSSA bacteriaeae fermentors

Imipenem + + + +

Meropenem + + + +

Limited
Ertapenem + + activity +

Doripenem + + + +

Dr.T.V.Rao MD 15
 Dr.T.V.Rao MD

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 penicillins,

extended spectrum cephalosporins, and
carbapenems
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 Dr.T.V.Rao MD

MECHANISMS OF CARBAPENEM
RESISTANCE
 Carbapenemase hydrolyzing enzymes

 Porin loss “OprD”

 ESBL or AmpC + porin loss

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 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 18
 Dr.T.V.Rao MD

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

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 Dr.T.V.Rao MD

CARBAPENEMASES
 Ability to hydrolyze penicillins, cephalosporins,
monobactams, and carbapenems
 Resilient against inhibition by all commercially viable ß-
lactamase inhibitors
 Subgroup 2df: OXA (23 and 48) carbapenemases
 Subgroup 2f : serine carbapenemases from molecular class A:
GES and KPC
 Subgroup 3b contains a smaller group of MBLs that
preferentially hydrolyze carbapenems
 IMP and VIM enzymes that have appeared globally, most frequently in
non-fermentative bacteria but also in Enterobacteriaceae

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 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.

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 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 to
meropenem or Imipenem.
 Modified Hodge test is confirmatory. Discussed
in manual. PCR is gold standard.

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 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 includes
cephalosporins and
carbapenems
23
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.

Pseudomonas aeruginosa – Columbia & Puerto

Dr.T.V.Rao MD Rico 24
 Dr.T.V.Rao MD

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.

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 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
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 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
 Dr.T.V.Rao MD

RESISTANCE TO CARBAPENEMS
 Carbapenems = ertapenem, imipenem, meropenem
 Intrinsically less susceptible organisms – Acinetobacter, P.
aeruginosa
 Other organisms may acquire resistance – K. pneumoniae, other
Enterobacteriaceae
 Know mechanisms of carbapenem resistance:
 Class A carbapenemases (KPC, SME,…)
 Class B metallo-β-lactamases (IMP, VIM, SPM…)
 Class D oxa 23, -40, -51, -58
 Organisms that acquire these resistance mechanisms will be
resistant to all carbapenems but may test susceptible to
imipenem

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 CARBAPENEMASE CLASSIFICATION
Molecular A B D
Class
Functional 2f 3 2d
Group
Aztreonam + - -
Hydrolysis
EDTA - + -
Inhibition
Clavulanate + - 
Inhibition

Dr.T.V.Rao MD 29
 Dr.T.V.Rao MD

EMERGING METALLO-Β-LACTAMASES
WITH MOBILE GENETICS
(SENTRY PROGRAM 2001-2005)

Genetic group Geographic origin Characterized enzymes

imp Japan IMP-1 through IMI-13a
IMP-14, and IMP-16a
vim Italy VIM-1 through VIM-7a
spm Brazil SPM-1a
gim Germany GIM-1a
vim USA VIM-7a,b
sim Korea SIM-1

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 Dr.T.V.Rao MD

RESISTANCE TO CARBAPENEMS
 Can also have
carbapenem resistance
due to
 Class A ESBL’s (CTX-M)
+ reduced permeability
 Class C High AmpC +
reduced permeability
 These hydrolyze
ertapenem more than
meropenem or imipenem

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 Dr.T.V.Rao MD

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)
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 Dr.T.V.Rao MD

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
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 CARBAPENEMASE-PRODUCING
Dr.T.V.Rao MD

KLEBSEILLA PNEUMONIA (KPC)

 KPC-3 is the most recently reported enzyme in
that group
 KPC-3 is closely related to its predecessors,
differing by only 1 amino acid from KPC-2 and
by 2 amino acids from KPC-1
 It has been recovered from isolates of K.
pneumoniae, E. coli, and E. cloacae

34
 CARBAPENEM RESISTANCE: MECHANISMS

Enterobacteriaceae Cephalosporinase + porin loss

Carbapenemase
P. aeruginosa Porin loss
Up-regulated efflux
Carbapenemase
Acinetobacter spp. Cephalosporinase + porin loss
Carbapenemase

Dr.T.V.Rao MD 35
 CARBAPENEMASES
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 36
 Dr.T.V.Rao MD

KLEBSIELLA PNEUMONIAE AND

CARBAPENEMASE
 KPC is a class A b-lactamase
 Confers resistance to all b-lactams including extended-spectrum
cephalosporins and carbapenems

 Occurs in Enterobacteriaceae
 Most commonly in Klebsiella pneumoniae
 Also reported in: K. oxytoca, Citrobacter freundii, Enterobacter
spp., Escherichia coli, Salmonella spp., Serratia spp.,

 Also reported in Pseudomonas aeruginosa (Columbia)

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 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

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 Dr.T.V.Rao MD

KPC ENZYMES
 Molecular class A and functional group 2f
 Inhibited by clavulanic acid but not by EDTA
 Confers resistance to ALL -LACTAM
antibiotics
 Plasmid-encoded
 Associated with other resistant genes

(aminoglycosides, fluoroquinolones)
 Transferable

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 Dr.T.V.Rao MD

KPC EPIDEMIOLOGY
 Predominantly in K. pneumoniae (KP)
 Reported in Enterobacter spp., Salmonella
spp., E. coli, P. aeruginosa, and Citrobacter
spp.
 First identified in KP clinical isolate from
North Carolina in 1996 (KPC-1)
 KPC-2, -3, and -4 have been reported.
 Mostly identified on the East cost

40
 Dr.T.V.Rao MD

KPC EPIDEMIOLOGY
 KPC producers have been identified outside USA
 France
 Brazil
 Columbia
 China
 Not detected at the University of Nebraska Medical
Center
 45 ESBL-like isolates collected-6 had elevated
carbapenem MICs-none contained KPC

41
 Dr.T.V.Rao MD

K. PNEUMONIAE WITH CARBAPENEMASE-

PRODUCING CLONES

42
Norman P et al. LID 2009
 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 to
meropenem or Imipenem.
 Modified Hodge test is confirmatory. Discussed
in manual. PCR is gold standard.

43
Major families of β-lactamases of clinical importance
Dr.T.V.Rao MD

Bush K and Jacboy G AAC 2010 44

 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
 Ceftazidime 32 to >64 mg/ml
 Ceftriaxone ≥ 64 mg/ml
 Cefotaxime ≥ 64 mg/ml
 Variable susceptibility to cefoxitin and cefepime

45
 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."
 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
 GENETIC ORIGIN OF THE NDM-1
 An intact ISCR1 element was shown to be downstream from
the qac/sul genes. The third region consisted of a new MBL
gene, designated bla(NDM-1), flanked on one side by K.
pneumoniae DNA and a truncated IS26 element on its other
side. The last two regions lie adjacent to one another, and all
three regions are found on a 180-kb region that is easily
transferable to recipient strains and that confers resistance to
all antibiotics except fluoroquinolones and colistin. NDM-1
shares very little identity with other MBLs, with the most
similar MBLs being VIM-1/VIM-2, with which it has only
32.4% identity.
 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
 BLANDM-1 IS EXPRESSED IN...
 Isolates, which include an
Escherichia coli, Klebsiella
pneumoniae, and
Enterobacter cloacae, carry
blaNDM-1, which confers
resistance to all beta-lactam
agents except aztreonam (a
monobactam antimicrobial) ;
all three isolates were
aztreonam resistant,
presumably by a different
mechanisms.
 Dr.T.V.Rao MD

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.

53
 Dr.T.V.Rao MD

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

54
 Dr.T.V.Rao MD

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. 55

 Dr.T.V.Rao MD

MODIFIED HODGE TEST

 Preliminary results suggest that any of the three

carbapenem disks work in the Modified Hodge Test
56
Dr.T.V.Rao MD

57
 Dr.T.V.Rao MD

INDIAN DATA ON CARBAPENEM RESISTANCE

 Incidence of Meropenem resistance higher

than that of Imipenem/cilastatin across
clinically significant nosocomial pathogens

Carbapenem Overall Incidence of

Resistance
Meropenem 22.16%
Imipenem/cilastatin 17.32%

Gupta E et al, Indian J Med Res 2006 July; 124: 95-98 58

 Dr.T.V.Rao MD

INDIAN DATA ON CARBAPENEM RESISTANCE

Organism MEM resistance IMI resistance

Pseudomonas 37.6% 30.0%
spp.
Acinetobacter 34.7% 27.2%
spp.
E. coli 3.5% 2.1%
Klebsiella spp. 6.9% 4.3%
 Overall Imipenem/cilastatin showed better activity than
Meropenem

Gupta E et al, Indian J Med Res 2006 July; 124: 95-98 59

 Dr.T.V.Rao MD

MECHANISMS OF CARBAPENEM
RESISTANCE
 Carbapenemase hydrolyzing enzymes

 Porin loss “OprD”

 ESBL or AmpC + porin loss

60
 Dr.T.V.Rao MD

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
 Can submit initial isolate to CDC via NJ State Lab
for confirmation by blaKPC PCR if KPC-producers
not previously identified in hospital’s isolate
population
 Alert clinician and infection control practitioner to
possibility of mobile carbapenemase in isolate
61
 Dr.T.V.Rao MD

CARBAPENEMASES 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)
62
 Dr.T.V.Rao MD

KPC EPIDEMIOLOGY
 Predominantly in K. pneumoniae (KP)
 Reported in Enterobacter spp., Salmonella
spp., E. coli, P. aeruginosa, and Citrobacter
spp.
 First identified in KP clinical isolate from
North Carolina in 1996 (KPC-1)
 KPC-2, -3, and -4 have been reported.
 Mostly identified on the East cost

63
 Dr.T.V.Rao MD

WHEN TO SUSPECT A KPC PRODUCER

 Enterobacteriaceae
 Resistance to extended spectrum
Cephalosporins (cefotaxime, ceftazidime, and
ceftriaxone)
 Variable susceptibility to cephamycins
(cefoxitin, cefotetan)
 Carbapenem MICs  2 g/ml

64
 Dr.T.V.Rao MD

HOW TO DETECT A KPC PRODUCER

 Antimicrobial susceptibility tests (ASTs)
 MIC

 Carbapenem MIC  2 g/ml

 Disk diffusion

 Carbapenem: “I” or “R”

 Among carbapenems, ertapenem:

 Most sensitive

 less specific

Anderson et al. 2007. JCM 45 (8): 2723 65

 DEFINITIVE IDENTIFICATION OF A KPC
PRODUCER
 Modified Hodge test
 100% sensitivity to detect KPC
1. Swab E. coli ATCC 25922
onto plate to create lawn
pos pos
Place imipenem disk in
center.
pos
2. Streak test isolates from edge
of disk to end of plate.
3. Incubate overnight. neg neg
4. Look for growth of E. coli
around test isolate streak -
indicates carbapenem-
neg
hydrolyzing enzyme.
meropenem ertapenem imipenem
Dr.T.V.Rao MD 66
Janet Hindler, What’s New in the 2008 CLSI Standards for (AST)?
Dr.T.V.Rao MD
K. PNEUMONIAE WITH KPC-2

67
 Dr.T.V.Rao MD

TRIS/EDTA DISK TEST

 Tris/EDTA disks used in
combination with a
carbapenem disk provides a
sensitive test for class A
carbapenem-hydrolyzing
enzymes
 Imipenem disks most
sensitive carbapenem disks to
use with this method, but
ertapenem and meropenem
also work well

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 Dr.T.V.Rao MD

TRIS/EDTA DISK TEST

 KPC-2 producing K.
pneumoniae is both the lawn
culture and inoculated onto
Tris/EDTA disk placed beside
imipenem disk.
 Indentation indicates
production of carbapenem-
hydrolyzing enzyme (positive
test).
 Second Tris/EDTA disk (not
inoculated with test organism)
is placed further away from
imipenem disk to test for
metallo-β-lactamase
production (negative test).

Procedure described by Ellen Molan and Ken Thompson, Creighton

University 69
 IMIPENEM RESISTANT K. PNEUMONIAEDr.T.V.Rao MD
EXPRESSING CLASS A
CARBAPENEMASE AND
IMIPENEM RESISTANT S. MALTOPHILIA EXPRESSING CLASS B
CARBAPENEMASE

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 Dr.T.V.Rao MD

MODIFIED HODGE TEST

 Inoculate MH agar with a 1:10
dilution of a 0.5 McFarland
suspension of E. coli ATCC
25922 and streak for confluent
growth using a swab.
 Place 10-µg imipenem disk in
center
 Streak each test isolate from
disk to edge of plate
 Isolate A is a KPC producer
and positive by the modified
Hodge test.
Anderson KF et al. JCM 2007
Aug;45(8):2723-5.

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 Dr.T.V.Rao MD

KPC PRODUCER - EXAMPLE

imipenem meropenem ertapenem

≤4 µg/ml* ≤4 µg/ml* ≤2 µg/ml*

*CLSI breakpoint for “S”;

marked w/ arrow Courtesy of J. Patel, PhD., CDC
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E. cloacae: ertapenem resistance,
Dr.T.V.Rao MD

meropenem susceptible

73
E. cloacae derepressed mutant
Dr.T.V.Rao MD

expressing AmpC and porin

mutation

KPC positive Control

Patient Isolate

74
 ACTIVITY AGAINST
ACINETOBACTER SPP.
 Carbapenems are considered the drugs of choice for treating
serious infections caused by Acinetobacter baumanii
 Progressive antimicrobial resistance in Acinetobacter is a cause
of concern
 Imipenem/cilastatin demonstrates lower MICs and lower
resistance rates than Meropenem against Acinetobacter
baumanii

Carbapenem Resistance rate against

Acinetobacter baumanii
Imipenem / cilastatin 70%
Meropenem 80%
Dr.T.V.Rao MD 75
Canduela MJ et al, J Antimocrob Chemother 2006; 57: 1220-1222
 Dr.T.V.Rao MD

ALTERNATIVE TREATMENT FOR A KPC

PRODUCER
 Tigecycline (100.0% effective)

 Colistin (88.1% effective)

 SENTRY report. AAC. 2008. Feb;52(2):570-3

 Minocycline
 A strategy for susceptibility testing is needed

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 CARBAPENEMASE-PRODUCING
Dr.T.V.Rao MD

KLEBSEILLA PNEUMONIA (KPC)

 Conclusions:
 Correct inoculum's of any organism undergoing
identification and susceptibility testing should
be assured
 K. pneumoniae intermediate or resistant to
ertapenem or meropenem should be considered
resistant to all carbapenems, regardless of the
other susceptibility results
 Inoculum effect with imipenem has also been
observed in KPC-possessing Enterobacter spp.

Bratu, S. et al AAC 49:3018-3020, 2005

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 Dr.T.V.Rao MD

CLSI GUIDELINES TO BE
FOLLOWED IN DETECTION
 ESBL detection—CLSI guidelines present
 Need to have guidelines to detect ESBLs present in
other species besides E. coli, K. pneumoniae, K.
oxytoca, and P. mirabilis.
 AmpC detection-No guidelines available
 KPC detection-Not widespread, need to have
lower concentrations of carbapenems on panels.

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 Dr.T.V.Rao MD

PURCHASE QC STRAINS
 ATCC BAA-1708-
mupA S. aureus isolate,
ATCC BAA-1705 and
BAA-1706. Positive and
Negative modified Hodge
test isolates, respectively.
 New ampicillin,
piperacillin and ticarcillin
QC tests for E. coli
ATCC35218.
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 Dr.T.V.Rao MD

DEFINITIVE IDENTIFICATION OF A KPC

PRODUCER
 PCR
 The method
of choice to
confirm
KPC
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 Dr.T.V.Rao MD

AUTOMATED SYSTEMS CANNOT DETECT

ALL TYPES OF ANTIBIOTIC RESISTANCE
 Limitations of Automated Systems in detecting
emerging resistance in Gram-Negative Bacilli
 Unable to detect ESBLs in organisms other than E.
coli and Klebsiella
 Unable to detect Inducible AmpC
 Unable to detect ESBLs in AmpC positive strains
 Unable to detect imipenem resistance in strains
producing KPC carbapenemases

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 Dr.T.V.Rao MD

CARBAPENEMS: MYTHS AND REALITY

 Inspite of expensiveness of Carbapenems,
pharmacoeconomic studies have demonstrated the
advantages of these drugs over a number of cheaper
conventional antibiotics

 Since inadequate empirical antimicrobial therapy is

associated with significantly higher mortality in serious
infections, Carbapenems are no longer considered second-
line antimicrobials

 The main ways to improve use of Carbapenems is:

- De escalation therapy
- Optimal dosing of Carbapenems

Bereznyakov IG, Kharkov Medical Academy of Post Graduate Education, Kharkov, Ukraine
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 Dr.T.V.Rao MD

TESTING OTHER DRUGS FOR CLINICAL USE

IN CARBAPENEM RESISTANT STRAINS
 Polymixin 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 microdilution – reference labs
 Breakpoints - none
 MIC ≤ 2 mg/ml, normal MIC range
 MIC ≥ 4 mg/ml indicates increased resistance

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 Dr.T.V.Rao MD

TESTING OTHER DRUGS TO TREAT PATIENTS WITH RESISTANT

CARBAPENEM ISOLATES

 Tigecycline:
 Test by Etest if possible –
disk diffusion tends to
overcall resistance

 No CLSI breakpoint, but

there are FDA breakpoint
 Susceptible ≤ 2 mg/ml
 Intermediate = 4 mg/ml
 Resistant ≥ 8 mg/ml

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 Dr.T.V.Rao MD

HOW TO IMPROVE OUR

MICROBIOLOGY DEPARTMENTS
 Each laboratory should have a staff member with
the time, interest, and expertise to provide
leadership in antibiotic testing and resistance. This
person would read relevant publications, network
with other laboratories, and evaluate potentially
useful tests to detect new forms of resistance
before new CLSI-recommended tests
become available”
 Ken Thomson, Emerging Infect. Dis., 2001

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 Dr.T.V.Rao MD

INTERPRETATION/RESULTS
CONVEYED TO INFECTION CONTROL DEPARTMENTS

 Report all cultures

that are positive for
CRE or
carbapenemase-
producing
Enterobacteriaceae to
the appropriate
infection control
personnel.
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 Dr.T.V.Rao MD
INSPITE OF SEVERAL ADVANCES
SIMEPLE HAND WASH
CAN SAVE SEVERAL LIVES

87
 Dr.T.V.Rao MD

THE PROGRAMME DESIGNED WITH

REFERENCES
 Livermore et. al. 2001. Interpretive reading: recognizing the unusual and inferring
resistance mechanisms from resistance phenotypes. J Antimicrob Chemother. 48:S1, 87-
102.
 Paul C. Schreckenberger, Ph.D., D(ABMM) Professor of Pathology
Director, Clinical Microbiology Laboratory Loyola University Medical Center
Ken Thomson, Emerging Infect. Dis., 2001
Gupta E et al, Indian J Med Res 2006 July; 124: 95-98
Bratu S et al AAC 49:776-778; Schreckenberger, P personal observation
Canduela MJ et al, J Antimocrob Chemother 2006; 57: 1220-1222
Lee et al. CMI, 7, 88-102. 2001.

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 Dr.T.V.Rao MD

Programme created by Dr.T.V.Rao.MD

for ‘e’ learning by Medical
Professionals
Email
doctortvrao@gmail.com

89