Transcript Document

Detection and Reporting of
Beta-lactam Resistance in
Enterobacteriaceae
Paul C. Schreckenberger, Ph.D., D(ABMM)
Professor of Pathology
Director, Clinical Microbiology Laboratory
Loyola University Medical Center
[email protected]
Objectives
• Participants will be able to:

Set up and interpret the double disk diffusion
method for detecting ESBLs and ampCs.

Describe methods for detection of
carbapenamases, including the Hodge test and
Tris EDTA double disk test

Modify susceptibility reports based on
characterization of resistance Genotypes.
2
Detecting Antibiotic
Resistance
Is there a Problem?
Automated Systems
• Poor performance by automated systems in
detecting resistance has necessitated use of
off line screening/confirmatory tests

Oxacillin screening plates for MRSA

Vancomycin screening plates for MRSA and VRE

D-Zone Test for detection of inducible clinidamycin
resistance
4
Automated Systems
• Limitations of Automated Systems in
detecting emerging resistance in GramNegative 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
5
Comparison of Phoenix & Vitek 2 for
Detecting ESBLs in E.coli and Klebsiella
No. (%) of tests that were correct
Phoenix
Phoenix*
Vitek 2
76 ESBL-pos strains
ESBL
Expert
Test
System
73(96)
73(96)
73(96)
75(99)
69(91)
68(89)
26 ESBL-neg strains
ESBL
Expert
Test
System
21(81)
21(81)
21(81)
15(58)
22(85)
22(85)
*Phoenix results after activation of two normally inactive Phoenix expert
rules (rules 325 and 1437) intended to enhance ESBL detection based
on susceptibility results
Thomson KS et al. JCM 2007 Aug;45(8):2380-4.
6
Evaluation of Methods to Identify
KPC in Enterobacteriaceae
Sensitivity/Specificity of Methods for Detecting KPCmediated resistance (31 KPC-pos; 45 KPC-neg)
Meropenem Imipenem Ertapenem
Reference BMD
94/98
94/93
97/89
Etest
58/96
55/96
90/84
Disk Diffusion
71/96
42/96
97/87
Vitek Legacy
52/98
55/96
NA
Vitek 2
48/96
71/96
94/93
MicroScan
84/98
74/96
100/89
Phoenix
61/98
81/96
NA
Sensititre
42/98
29/96
NA
Anderson KF et al. JCM 2007 Aug;45(8):2723-5.
7
Role of the Microbiology Lab
• “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
8
The β-lactam family of antibiotics
Penicillins
Cephalosporins Cephamycins Carbapenems
Benzylpenicillin
Cephalothin 1st
Cefoxitin
Imipenem
Methicillin
Cefamandole 2nd
Cefotetan
Meropenem
Ampicillin
Cefuroxime 2nd
Cefmetazole
Ertapenem
Carbenicillin
Cefotaxime 3rd
Mezlocillin
Ceftazidime 3rd
Ticarcillin
Ceftriaxone 3rd
Cefepime 4th
Monobactams
Aztreonam
Penicillin nucleus
S
R
6
7
O
CH3
1
5
2
CH3
N
4
3
COOH
10
Cephalosporin nucleus
1
R1
7
C
O
S
HN
R2
O
COOH
11
MODE OF ACTION OF BETA
LACTAMS IN GRAM NEGATIVES
SUSCEPTIBLE
-Lactam Antibiotic

Diffusion through
Outer Membrane

Diffusion through
Peptidoglycan

Penicillin Binding Proteins

Cell Death
RESISTANT
 Porin Blocks Entry
 Efflux Pump
 Beta-Lactamase
Hydolyzes Beta-Lactam
 Changes in PBP results in
Failure to Bind to -Lactam
The Gram Negative Cell Wall
Efflux system
Porin
channels
B-lactamases
PBPs
Adapted from Livermore and Woodford, Trends in Microbiol, 2006.
13
Definition of beta lactamases
• Beta lactamases
are enzymes
produced by some
gram-positive and
gram-negative
bacteria that
hydrolyze beta
lactam antibiotics
14
β-Lactamase Classes
A
B
C
D
Chromosomal
Plasmid
Bacteroides,
Klebsiella,
P. vulgaris
S. maltophilia,
flavobacteria
Most
enterobacteria
Aeromonas
Staph pen’ase
TEM, SHV
KPC
IMP, VIM
CMY, LAT, FOX
OXA
15
ESBLs
Extended-spectrum β-lactamases
• >180 enzymes described (119 TEM, 45 SHV)
• All mutations of older TEM and SHV plasmid-
mediated β -lactamases
 TEM-3, TEM-4, etc.
 SHV-2, SHV-3, etc.
 CTX-M-1,2, etc. and Toho-type
 OXA-type
 PER-1 and 2
• Resistance conferred to extended-spectrum
penicillins, 3rd and 4th generation cephalosporins and
aztreonam (not imipenem or cephamycins)
www.lahey.org/studies/webt.htm
16
ESBLs
Extended-spectrum β-lactamases
• Primarily found in:

Klebsiella, E. coli
• Also found in:

Proteus, Serratia

Enterobacter, Salmonella

Morganella, etc.
• Most are inhibited well by clavulanic acid and
tazobactam (less so by sulbactam)
17
Beta-lactamase inhibitors
• Resemble β-lactam antibiotic structure
• Bind to β-lactamase and protect the antibiotic
from destruction
• Most successful when they bind the βlactamase irreversibly
• Three important in medicine
Clavulanic acid
 Sulbactam
 Tazobactam

18
Why Test for β-lactamases?
• Correct therapy
• Breakpoints do not reliably detect new
β-lactamases
• Infection control
• Identify drugs causing resistance
20
Detection of ESBLs:
Two Approaches
1. Screening tests and confirmatory tests
for positive screens
2. Confirmatory tests
21
Detection of ESBLs:
Screening Tests
• Advantages
 Less
work
 Cheaper
• Disadvantages
 Sensitivity
 Delayed
less than 100%
confirmation
 Reporting
of positive screens?
22
CLSI Confirmatory Test –
Klebsiella, E. coli, P. mirabilis
• MIC Test

cefotaxime and ceftazidime +/- 4 μg/ml clavulanate:

> 3 doubling dilution decrease with either drug
e.g.
ceftazidime
8 μg/ml
ceftazidime + clavulanate 1 μg/ml
• Disk Test

cefotaxime and ceftazidime +/- 10 μg clavulanate

> 5 mm zone increase
23
FDA-Approved Commercial Tests
• BD Sensi Disks
• Etest
• MicroScan
• Phoenix
• Trek
• Vitek
• Vitek 2
24
Combination Disk Method
CLSI Approved Method
Double Disk Method
Not CLSI Approved
CLSI Reporting Recommendation
• ESBL confirmed: E. coli, Klebsiella, P. mirabilis
• Report resistant for all penicillins,
cephalosporins and aztreonam (except
cephamycins, e.g., cefoxitin and cefotetan)
regardless of in vitro status
27
Treatment of ESBL Positive
Organisms with Cephalosporins
MIC
FAILURE DEATH
8
100% (6/6)
33% (2/6)
4
67% (2/3)
0% (0/3)
2
33% (1/3)
0% (0/3)
≤1
27% (3/11)
18% (2/11)
(CLSI breakpoint 8 g/ml)
Paterson, DL, et al. JCM 39: 2206 – 2212, 2001
28
ESBL Blood Stream Infections
Clinical Outcome
FATALITY RATE:
ESBL Producers
Non-ESBL Producers
MIC
8
4
2
= 26.7% (12/45)
= 5.7% (5/87)
Favorable Outcome
pts given only Suscep. 3rd gen ceph
0 (0/2)
33 (1/3)
100 (1/1)
(CLSI breakpoint 8 g/ml)
Kim YK, et al. AAC 46:1481-1491, 2002
29
Pitfalls of ESBL Testing
• CTX-M type -lactamases - novel group of
Class A plasmid-encoded cephalosporinases
• CTX abbreviation for cefotaximase. Includes
CTX-M-type (17 to date), Toho-1, Toho-2, MEN1
• Rapidly hydrolyze cefotaxime but not
ceftazidime (some MICs  4)
• Inhibited better by tazobactam than by
sulbactam and clavulanate
30
Pitfalls of ESBL Testing
• CTX-M-type found in Salmonella sp., E. coli, K.
pneumoniae, C. freundii, P. mirabilis, S.
marcescens
• More common in S. America than N. America,
also common in Europe and Asia
• Have decreased susceptibility to inhibitor
drugs therefore may not be confirmed with
CLSI confirmatory test
31
32
E. coli with CTX-M ESBL
33
Pitfalls of ESBL Testing
Effects of Inoculum
MICs in g/ml: SHV-3 producing Citrobacter freundii
Inocul.
Cefotaxime
Ceftazidime
Aztreonam
Cefepime
2
1
0.5
0.5
CFU/ml
5 x 105
(CLSI breakpoint 8 g/ml)
KS Thomson and ES Moland, Creighton University
34
Pitfalls of ESBL Testing
Effects of Inoculum
MICs in g/ml: SHV-3 producing Citrobacter freundii
Inocul.
Cefotaxime
Ceftazidime
Aztreonam
Cefepime
5 x 105
2
1
0.5
0.5
5 x 107
256
32
32
>1024
CFU/ml
(CLSI breakpoint 8 g/ml)
KS Thomson and ES Moland, Creighton University
35
Enterobacteriaceae -Lactam
Breakpoints and ESBL Issues
•
CLSI is re-evaluating -lactam breakpoints for
Enterobacteriaceae

Example: cefotaxime
 Current – Susceptible at  8 g/ml
 Proposed – Susceptible at  1 or  2 g/ml


•
Substantial data needed
Goal is to more accurately detect all -lactamase and other lactam resistance mechanisms with revised breakpoints
Changing breakpoints – commercial systems project it
will take 3 years …much $$$$$!
36
ESBLs in organisms other than
E. coli and Klebsiella spp.
• Most labs do not attempt to detect ESBLs in
organism other than E. coli and Klebsiella
• Two Indications for ESBL Testing in Other
Organisms
ESBLs detected in E. coli or Klebsiella
 Suspicious phenotype

• How to test?
Use specific (confirmatory) test
 Perform Double Disk Diffusion

37
Prevalence of ESBLs
• Aim of study was to detect ESBL prevalence in all
GNB in US medical centers
• 6,421 consecutive non-duplicate GNB screened for
reduced susceptibility to cephems and aztreonam or
potentiation of cefepime by clavulanate

Patients were from 42 ICU and 21 non-ICU sites
throughout the US, 9/00 to 9/02
• Screen positive isolates were then investigated in a
central lab for ESBL status
Moland ES, et al. J Clin Microbiol. 2006 Sep;44:3318-24
38
Prevalence of ESBLs
Organism
# Pos/Total
tested
% Overall % in ICUs % in NonICUs
K. oxytoca
18/137
13.1
9.2
23
K. pneumoniae
96/853
11.3
13.7
4.7
E. cloacae
25/453
5.5
4.3
14.3
E. coli
42/1616
2.6
3.6
1.6
S. marcescens
5/306
1.6
0.4
8.9
P. mirabilis
5/359
1.4
3.1
0
E. aerogenes
2/189
1.1
0.6
3.3
Moland ES, et al. J Clin Microbiol. 2006 Sep;44:3318-24
39
Prevalence of ESBLs at LUMC
2006 and 2007 (Jan-Sept)
Organism
Total tested
ESBL Pos
% ESBL
C. freundii complex
165
4
2.4
C. koserii
110
6
5.5
E. aerogenes
197
2
1.0
E. cloacae
387
20
5.2
E. coli
5131
96
1.9
K. oxytoca
151
2
1.3
K. pneumoniae
1149
37
3.2
M. morganii
70
4
5.7
P. mirabilis
592
25
4.2
P. stuartii
16
2
12.5
Schreckenberger P, LUMC Antibiogram 2006-07
40
P. mirabilis with ESBL
42
Pitfalls of ESBL Testing
• Recommendation (not CLSI endorsed): Extend
CLSI reporting recommendations to all ESBLproducing organisms
• Report all ESBL-producing organisms the
same way: resistant to all penicillins,
cephalosporins, and aztreonam
43
AmpC Beta Lactamases
• Cephalosporinases, hydrolyze all beta lactam
antibiotics except carbapenems and cefepime
• Not Inhibited by clavulanate and sulbactam
• Some inducible
• Characteristic of certain genera:
S - Serratia
P - Providencia/P. aeruginosa
A - Aeromonas
C - Citrobacter freundii
E - Enterobacter, Hafnia
44
AmpC Beta Lactamases
• High level production of enzyme can be
inducible or constitutive
• With inducible production, enzyme
produced at low level unless organism
exposed to inducing agents
• Induction is a reversible mechanism
45
AmpC Beta Lactamases
Inducer Potential
GOOD
Cefoxitin
Cefmetazole
Imipenem
Ampicillin
VARIABLE
Clavulanate
Desacetyl
Cefotaxime
Cefamandole
Cephalothin
Cefonicid
POOR
Sulbactam
Tazobactam
Aztreonam
3rd Gen Cephs
4th Gen Cephs
46
Uninduced AmpC
AmpD
ampD
AmpR
ampR
ampC
•Wall fragments recycled by AmpD
•AmpR in repressor conformation
• ampC (-lactamase gene) NOT expressed
47
Induced AmpC
-lactamase
AmpD
ampD
amp
R
ampC
• More recycling: AmpD overwhelmed
• Wall fragments convert AmpR to activator
• ampC (-lactamase gene) expressed
48
E. cloacae expressing Induced
Chromosomal AmpC
But mutational
derepression is
the problem,
not induction
49
Derepressed AmpC
-lactamase++
ampD
amp
R
ampC
• ampD inactivated by mutation
• AmpR constantly converted to activator
• ampC hyper-expressed
50
E. cloacae derepressed mutant
expressing AmpC
51
Class C
AmpC Beta Lactamases
• With constitutive production - mutant
strains arise spontaneously at frequencies
of about 10-6 to 10-9
• Cephalosporinase produced constitutively
at high levels
• Not reversible
• Antibiotics that are poor inducers tend to
be good selectors of mutants
52
AmpC Beta Lactamases
Mutant Selection
GOOD SELECTORS
POOR SELECTORS
3rd Gen Cephs
Imipenem
4th Gen Cephs
Cephamycins
Older Cephalosporins
53
MICs (mg/L) for E. cloacae
AmpC mutants
Inducible Derepressed Basal
512
2048
4
Ampicillin
256
1024
16
Cephalothin
4
128
1
Piperacillin
0.5
256
0.06
Cefotaxime
0.25
256
0.25
Ceftazidime
0.06
16
0.06
Aztreonam
0.25
0.25
0.06
Imipenem
0.06
0.12
0.015
Meropenem
54
Bush Group 1 or AmpC
Inducible Beta Lactamases
Recommendation for Laboratory
• Laboratories should flag all organisms known
to posses inducible ß-lactamases (S/IB)
• Sample footnote:
“This organism is known to possess inducible
ß-lactamases. Isolates may become resistant
to all cephalosporins after initiation of therapy.
Avoid ß-lactam-inhibitor drugs.”
55
E. cloacae not expressing
Chromosomal AmpC
56
•
Chromosomal AmpC that is not
Expressing High Level Resistance
Growth of: Enterobacter cloacae - This organism is
known to possess inducible ß-lactamases. Isolates
may become resistant to all cephalosporins after
initiation of therapy. Avoid ß-lactam-inhibitor drugs








Amikacin
Ampicillin
Cefazolin
Ceftazidime
Cefepime
Ceftriaxone
Gentamicin
Levofloxacin
S
R
R
S
S
S
S
S
57
E. cloacae AmpC
Derepressed Mutant
58
•
Chromosomal AmpC that is
Expressing High Level Resistance
Growth of: Enterobacter cloacae - This organism is
known to possess inducible ß-lactamases. Isolates
may become resistant to all cephalosporins after
initiation of therapy. Avoid ß-lactam-inhibitor drugs








Amikacin
Ampicillin
Cefazolin
Ceftazidime
Cefepime
Ceftriaxone
Gentamicin
Levofloxacin
S
R
R
R
S
R
S
S
59
•
Chromosomal AmpC that is
Expressing High Level Resistance
Growth of: Enterobacter cloacae








Amikacin
Ampicillin
Cefazolin
Ceftazidime
Cefepime
Ceftriaxone
Gentamicin
Levofloxacin
S
R
R
R
S
R
S
S
60
Plasmid-Mediated AmpCs
• B-lactamases derrived from chromosomally
encoded clavulanate-resistant AmpC
cephalosporinases of Citrobacter,
Enterobacter & Morganella spp.
• Genes are typically encoded on large
plasmids and carry additional resistance
genes
61
Plasmid-Mediated AmpCs
• Reported in Klebsiella, E. coli, Salmonella, P.
mirabilis
• Many enzymes, CMY, BIL, ACT, MOX etc.,
some inducible
• Prevalence low but increasing
 Approx.
 3.3
1/3 of U.S. laboratories
– 8.5% K. pneumoniae in USA
62
AmpCs in E. coli
• E. coli possess a chromosomal gene that
encodes for AmpC -lactamase
• Usually low amounts of -lactamase
produced because AmpC gene regulated by
a weak promoter and strong attenuator
• These strains are cefoxitin susceptible
63
AmpCs in E. coli
• Some strains have promoter or attenuator
mutations that result in the upregulation of
AmpC -lactamase production resulting in
cefoxitin-resistant strains.These are referred
to as AmpC hyperproducers
• Some strains acquire plasmid-mediated
AmpC -lactamase e.g. CMY-2. These are
also cefoxitin-resistant
64
When to Suspect AmpC Plasmid
• Disk tests for AmpC β-lactamases should be
performed on E. coli, Klebsiella spp, P. mirabilis,
Salmonella isolates positive in any of following
screens:

Cefoxitin-nonsusceptible (i.e. I or R)

ESBL screen-positive but ESBL confirmatory test
negative

Ceftazidime and cefoxitin intermediate or resistant (i.e.
MIC > 16 µg/ml for both drugs) and ESBL confirmatory
test negative (this screen may have good specificity)
65
66
E. coli with plasmid
mediated AmpC
67
Lawn culture:
E. coli ATCC 25922
AmpC Disk Test
Test Organism
on disk
68
Pitfalls of ESBL Testing
• High level expression of AmpC may prevent
recognition of an ESBL
• Problem in species that produce chromosomally
encoded inducible AmpC beta-lactamase (eg.
Enterobacter, Serratia, Providencia, etc.)
• Problem in E. coli and K. pneumoniae that acquire
AmpC plasmids
• Clavulanate may act as an inducer of high level
AmpC and increase resistance to screening drugs
giving false negative ESBL confirmatory test
69
Pitfalls of ESBL Testing
• Approaches to detecting ESBL in AmpC
producing strains
Use tazobactam or sulbactam in place of clavulanate
in ESBL confirmatory test because these are less
likely to induce AmpC production
 Include cefepime as screening agent because high
level AmpC expression has minimal effect on the
activity of cefepime
 Include cephamycins (cefoxitin) as screening agent
because cephamycins are hydrolyzed by AmpCs but
not by ESBLs
 Add boronic acid as AmpC inhibitor to CLSI ESBL
confirmatory disks

70
How to Determine if AmpC and
ESBL Both Present
• Double Disk Diffusion Test
 Look
for AmpC type pattern plus clavulanic
effect
71
•
Chromosomal AmpC that is
Suspicious for ESBL
Growth of: Enterobacter cloacae








Amikacin
Ampicillin
Cefazolin
Ceftazidime
Cefepime
Ceftriaxone
Gentamicin
Levofloxacin
S
R
R
I
S
S
S
S
72
E. cloacae with Chromosomal
AmpC and ESBL
73
•
Chromosomal AmpC that is
Suspicious for ESBL
Growth of: Enterobacter cloacae - This organism
possess an ESBL. Contact Isolation is required.








Amikacin
Cefazolin
Ampicillin
Ceftazidime
Cefepime
Ceftriaxone
Gentamicin
Levofloxacin
S
R
R
I R
S R
S R
S
S
74
•
Chromosomal AmpC that is not
Suspicious for ESBL
Growth of: Enterobacter cloacae








Amikacin
Ampicillin
Cefazolin
Ceftazidime
Cefepime
Ceftriaxone
Gentamicin
Levofloxacin
S
R
R
R
S
R
S
S
75
E. cloacae with Chromosomal
AmpC derepressed mutant and
ESBL
11 mm
7 mm
76
•
Chromosomal AmpC that is not
Suspicious for ESBL
Growth of: Enterobacter cloacae - this organism
possess an ESBL. Contact Isolation is required
Amikacin
 Ampicillin
 Cefazolin
 Ceftazidime
 Cefepime
 Ceftriaxone
 Gentamicin
 Levofloxacin

S
R
R
R
S R* See Comment
R
S
S
77
78
S. marcesens with Chromosomal AmpC
and ESBL
79
Differences between ESBL and
AmpC Beta Lactamases
Test Result
ESBL
AmpC
Inhibited by
clavulanate
Hydrolyzes
Yes
No
-1st, 2nd, 3rd,
Cephalosporins
-Cephamycins
Yes (R)
Yes (R)
No (S)
Yes (R)
-Cefepime
Yes (R)
No (S)
80
Pitfalls of ESBL Testing
K1 -lactamase of K. oxytoca
• Predominantly penicillinase, can also significantly
hydrolyze aztreonam, cefuroxime and ceftriazone
• Weak activity against cefotaxime or ceftazidime
• Low-level production causes resistance to penicillins
• Hyperproduction causes resistance to aztreonam and
labile cephalosporins
• Distintinctive features of hyperproducers of K1

Greater activity against ceftriaxone than cefotaxime

Greater activity against aztreonam than ceftazidime
81
82
K. oxytoca with K1 -lactamase
83
K. Oxytoca with K1 -lactamase
84
85
K-1 Beta Lactamase
86
87
88
89
K. pneumoniae with ESBL,
AmpC, and Carbapenemase
90
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
91
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
92
Class A Carbapenemases
• Rare – Enterobacteriaceae
• 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
93
Carbapenemase-Producing
Klebsiella 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
94
Carbapenemase-Producing
Klebsiella pneumonia (KPC)
• Identifying isolates possessing KPC type
resistance may be difficult using current
methods of susceptibility testing
• The presence of KPC in K. pneumoniae may
increase the MIC of imipenem, but not to the
level of frank resistance
• Therefore, strains carrying this enzyme may
only be recognized as ESBL-producing
isolates
95
Carbapenemase-Producing
Klebsiella pneumonia (KPC)
• Among 257 isolates of K. pneumoniae
collected in Brooklyn, NY, 62 (24%) were
found to possess blaKPC
• Clinical microbiology laboratories that used
automated broth microdilution systems (All
MicroScan Users) reported 15% of KPCproducing isolates as susceptible to
imipenem
• Imipenem MIC was found to be markedly
affected by inoculum
Bratu, S. et al AAC 49:3018-3020, 2005
96
Carbapenemase-Producing
Klebsiella pneumonia (KPC)
Results of Testing of 62 KPC
Imipenem
Meropenem
Ertapenem
% Susceptible
MBD 105
5
MBD 104
44
Etest
2
Disk
2
MBD 105
2
MBD 104
5
Etest
5
Disk
2
MBD 105
2
MBD 104
2
Etest
0
Disk
0
Bratu, S. et al AAC 49:3018-3020, 2005
97
Carbapenemase-Producing
Klebsiella 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 49:776-778; Schreckenberger, P personal
observation)
Bratu, S. et al AAC 49:3018-3020, 2005
98
Extent of Problem
• Highly endemic in greater NY area

Endemic in ICUs at Columbia, Cornell, St. Vincent’s,
Mount Sinai, SUNY Downstate (Brooklyn), ………
Officially a reportable disease in New York State
• Still relatively uncommon, now being reported from
multiple other regions of U.S.: AZ, NJ, DE, NC, NM, FL,
PA, DE, GA, MD, MI, MO, MA, CA, AK, OH, VA……

• Reports from other parts of world: Scotland, Israel,
Colombia, China, Brazil, France, Turkey, Greece,
Singapore, Korea, Puerto Rico……
AAC. 2005; 49(10): 4423-4; AAC. 2006; 50(8): 2880-2 ; AAC. 2007;
5(2): 763-5; 47th ICAAC. Abstract C2-1929.2007; 47th ICAAC. Abstract
C2-2063. 2007; 47th ICAAC. Abstract C2-1933. 2007
Geographical Distribution of KPC-Producers
Widespread
Sporadic Isolate(s)
Courtesy of J. Patel, PhD., CDC
100
K. Pneumoniae with KPC-2
101
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
102
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 carbapenemhydrolyzing enzyme
(positive test).
Second Tris/EDTA disk (not
inoculated with test
organism) is placed further
away from imipenem disk
to test for metallo-βlactamase production
(negative test).
Procedure described by Ellen Molan and Ken
Thompson, Creighton University
103
Imipenem resistant K. pneumoniae
expressing Class A carbapenemase
Imipenem resistant S. maltophilia
expressing Class B carbapenemase
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.
105
KPC Producer - Example
imipenem
≤4 µg/ml*
meropenem
≤4 µg/ml*
*CLSI breakpoint for “S”;
marked w/ arrow
ertapenem
≤2 µg/ml*
Courtesy of J. Patel, PhD., CDC
Ertapenem Resistant E. cloacae
107
E. cloacae: ertapenem resistance,
meropenem susceptible
108
E. cloacae derepressed mutant
expressing AmpC and porin
mutation
KPC positive Control
Patient Isolate
109
When to Perform the Double Disk
Test
• Any E. coli and Klebsiella when phenotype
does not agree with ESBL confirmation test
on Vitek or other commercial system
• Any Enterobacteriaceae when one of the 3rd
gen. cephalosporins tests I or R
• Any Enterobacteriaceae when atypical
pattern exists (e.g. P. mirabilis resistant to
multiple drugs)
• Any Enterobacteriaceae resistant to all drugs
except imipenem
110
Good resource for understanding specific
natural and acquired resistance…….
• Livermore et. al. 2001. Interpretive reading:
recognizing the unusual and inferring resistance
mechanisms from resistance phenotypes. J
Antimicrob Chemother. 48:S1, 87-102.
• Web version (2004…with a few changes) available…
http://www.bsac.org.uk
 Then to “Susceptibility Testing” link
 Then to “Guide to Susceptibility Testing”
 Then to “Chapter 11”

111