Carbapenamases

in
Antibiotic
Resistance
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. Carbapenems
antibiotics were originally
developed from thienamycin, a
naturally-derived product of
Streptomyces cattleya
 Carbapenems - Structure

 Carbapenems are
structurally very
similar to the
pencillins, but the
sulfur atom in
position 1 of the
structure has
been replaced
with a carbon
atom, and hence
the name of the
group, the
 Drugs belong to the
carbapenem class:
 Imipenem
 Meropenem

Ertapenem

Doripenem
Panipenem/ betamipron
 Background of

Carbapenamases
 Carbapenem-resistant
Enterobacteriaceae (CRE) are
usually resistant to all β-lactam
agents as well as most other
classes of antimicrobial agents. The
treatment options for patients
infected with CRE are very limited.
Healthcare-associated outbreaks of
CRE have been reported
 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
 Carbapenemases are produced by
several commonly infecting Gram
Negative Bacteria

 Carbapenemases are known to exist in
several different species of gram-
negative bacilli including species of
Enterobacteriaceae and Pseudomonas
aeruginosa. However,
carbapenemases are more common in
lactose-fermenting species of
Enterobacteriaceae (e.g., K.
pneumoniae and E. coli) than in non-
lactose fermenting Enterobacteriaceae
(e.g. Serratia marcescens and some
Enterobactericae spp.) and P.
aeruginosa.
 How Carbapenamase
resistance is initiated

 Carbapenem resistance in
Enterobacteriaceae occurs when an
isolate acquires a carbapenemase
or when an isolate produces an
extended-spectrum
cephalosporinase, such as an AmpC-
type β-lactamase, in combination
with porin loss. In the United States,
the most common mechanism of
carbapenem resistance is the
Klebsiella pneumoniae
 Carbapenamases a Global
Problem
 The resistance to
Carbapenems has
emerged worldwide
and the
predominant
mechanism of
resistance is
attributed by the
production of
various
Carbapenems-
hydrolyzing β-
 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
 Carbapenem Resistance:
Mechanisms
Enterobacteriaceae Cephalosporinase + porin loss

Carbapenemase

P. aeruginosa Porin loss

Up-regulated efflux

Carbapenemase

Acinetobacter spp. Cephalosporinase + porin loss

Carbapenemase
 Transposons and Integrons
contribute for spread of resistance,

 The genes of these

MBL enzymes are
often plasmid
borne and are
associated with
mobile genetic
elements
(transposons and
integrons),
making them
 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-β -lactamse) GIM, SPM Enterobacteriacea
Acinetobacter spp.

Class D OXA Acinetobacter spp.

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
 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,
 Klebsiella pneumoniae
Carbapenamases
 KPC-producing organisms continues
to evolve. Although most KPCs are
detected in isolates of Klebsiella
and Escherichia coli, KPCs have
been extensively reported in other
genera of the Enterobacteriaceae
family, such as Proteus,
Serratia, Salmonella, and
Citrobacter.
 K leb siella p n eu m on iae
 C arb ap en am ases
 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
 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
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.

Pseudomonas aeruginosa – Columbia & Puerto Rico

 Pseudomonas aeruginosa
Carbapenamases
 KPC resistance has
been reported in
inherently
resistant
organisms such
as
Pseudomon
as from Trinidad,
an isolate of
multidrug-
resistant
Pseudomonas
aeruginosa that
 Special antibiotic sensitivity testing is
emerging need in Microbiology
laboratories
 Supplemental
testing, in
addition to the
routine
susceptibility
tests of isolates,
has become
necessary in
order to detect
the deluge of
beta-lactamases
and
carbapenemases
 When to Suspect a KPC-
Producers
 Enterobacteriaceae – especially
Klebsiella pneumoniae that are
resistant to extended-spectrum
cephalosporins:


 MIC range for 151 KPC-producing

isolates
 Ceftazidime 32 to >64 µ g/ml
 Ceftriaxone ≥ 64 µ g/ml
 Cefotaxime ≥ 64 µ g/ml


 Variable susceptibility to cefoxitin and


Modified Hodge
Test for
Carbapenemase
Detection in
Enterobacteriacea
 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.
 CLSI Recommends
 CLSI Recommends doing Modified
Hodge test before reporting
Carbapenam susceptibility results
if results are elevated but
susceptible to Carbapenam by
Minimum inhibitory concentration.
 The results of intermediate or
resistance to Carbapenems need
not be tested with MHT
 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
 The Carapnemase is detected by
antibiotic sensitivity patterns

 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.
The result is a
characteristic
cloverleaf-like
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
 K.pneumonia showing
Carbapenem resistance
 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 35OC ± 2OC in
ambient air for 16–24 hours

 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
 MHT detection (photo courtesy of CDC)

 The MHT performed

on a 100 mm
MHA plate. (1) K.
pneumoniaeATCC
BAA 1705,
positive result K.
pneumoniaeATCC
BAA 1706,
negative result;
and a clinical
isolate, positive
result312
A Positive HMT test

 MHT Positive test
has a clover leaf-
like indentation
of the E.coli
25922 growing
along the test
organism growth
streak within the
disk diffusion
zone.
 A positive MHT
indicates that
this isolate is
producing a
 A negative HMT Test

 MHT Negative test has no growth
of the E.coli 25922 along the
test organism growth streak
within the disc diffusion.
 A negative MHT indicates
that this isolate is not
producing a
carbapenemase
 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

 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
 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
 New CLSI
guidelines
Nonsusceptible
”Interpretive
Category”
 “Nonsusceptible”
Interpretive Category
 “…. used for organisms that have
only a susceptible interpretive
category, but not intermediate
or resistant interpretive
categories (ie, susceptible-only
interpretive category). A
susceptible-only interpretive
category may be applied to new
antimicrobial agents for which
no resistant isolates have been
encountered at the time the
initial interpretive criteria are
 Nonsusceptible”
Interpretive Category
 The isolates that test with an MIC
above the susceptible interpretive
breakpoint are designated as
nonsusceptible. A designation of
nonsusceptible does not
necessarily mean that a
resistance mechanism exists in
the isolate. The MIC of the isolate
in the nonsusceptible range may
be within the previously
recognized wild-type distribution
of susceptibility results; however,
 Interpretation/Results
Conveyed to Infection Control Departments
 Report all cultures
that are positive
for CRE or
carbapenemase-
producing
Enterobacteriace
ae to the
appropriate
infection control
personnel.
 Patients infected will be dealt with
caution to prevent spread

 patients colonized with carbapenem-
resistant or Carbapenamases-
producing Enterobacteriaceae in
the intestinal tract and the Patients
who grow these organisms should
be placed on Contact Precautions
to prevent transmission of the
resistant bacteria
 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
 Follow contact Precautions

 Contact
Precautions
should be
implemented for
all patients with
positive cultures
for CRE or
carbapenemase-
producing
Enterobacteriace
ae

Hand washing can save
several patients
 Care of the patients colonized with
Carapnemase resisistant
Enterobactericae
 Patients colonized with CRE are
thought to be a source of
transmission in the healthcare
setting . Identifying patients who
are colonized with CRE and placing
these patients in isolation
precautions may be an important
step in preventing transmission
Detection of Antibiotic resistance
patterns is more than past
 Resistance to our beta-lactam and
carbapenem antibiotics is becoming
daunting for antimicrobial therapy
for infections involving the
Enterobacteriaceae. Similarly,
laboratory testing to detect these
resistance mechanisms is becoming
more complex and perplexing for
microbiology laboratories.
 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
 Detection of Drug resistance helps to
control the spread of Hospital acquired
infections
 In addition to the risk
of compromised
care of the patient,
when pathogens
with these
enzymes go
undetected,
necessary
infection control
measures are
precluded, thereby
allowing the risk of
these resistant
organisms
  Created by Dr.T.V.Rao MD for
“e” learning in
Microbiology


 Email
 doctortvrao@gmail.com
