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Detection and Reporting of Beta-lactam Resistance in Enterobacteriaceae
Paul C. Schreckenberger, Ph.D., D(ABMM)Professor of PathologyDirector, Clinical Microbiology LaboratoryLoyola University Medical Centerpschrecken@lumc.edu
2
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.
Detecting Antibiotic ResistanceIs there a Problem?
4
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
5
Automated Systems
• 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
6
Comparison of Phoenix & Vitek 2 for Detecting ESBLs in E.coli and Klebsiella
No. (%) of tests that were correct
76 ESBL-pos strains 26 ESBL-neg strains
ESBL Test
Expert System
ESBL Test
Expert System
Phoenix 73(96) 73(96) 21(81) 21(81)
Phoenix* 73(96) 75(99) 21(81) 15(58)
Vitek 2 69(91) 68(89) 22(85) 22(85)
Thomson KS et al. JCM 2007 Aug;45(8):2380-4.Thomson KS et al. JCM 2007 Aug;45(8):2380-4.
*Phoenix results after activation of two normally inactive Phoenix expert rules (rules 325 and 1437) intended to enhance ESBL detection based on susceptibility results
7
Evaluation of Methods to Identify KPC in Enterobacteriaceae
Sensitivity/Specificity of Methods for Detecting KPC-mediated resistance (31 KPC-pos; 45 KPC-neg)
Meropenem Imipenem Ertapenem
Reference BMD 94/98 94/93 97/89Etest 58/96 55/96 90/84Disk Diffusion 71/96 42/96 97/87Vitek Legacy 52/98 55/96 NAVitek 2 48/96 71/96 94/93MicroScan 84/98 74/96 100/89Phoenix 61/98 81/96 NASensititre 42/98 29/96 NA
Anderson KF et al. JCM 2007 Aug;45(8):2723-5. Anderson KF et al. JCM 2007 Aug;45(8):2723-5.
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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
The β-lactam family of antibiotics
Ceftriaxone 3rdTicarcillin
Ceftazidime 3rdMezlocillin
Cefotaxime 3rdCarbenicillin
ErtapenemCefmetazoleCefuroxime 2ndAmpicillin
MeropenemCefotetanCefamandole 2ndMethicillin
AztreonamImipenemCefoxitinCephalothin 1stBenzyl-penicillin
MonobactamsCarbapenemsCephamycinsCephalosporinsPenicillins
Cefepime 4th
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Penicillin nucleus
COOHO
RS
CH3
CH3
N
1
2
34
56
7
11
Cephalosporin nucleus
C
O
HN
1
SR1
R2O
COOH
7
MODE OF ACTION OF BETA LACTAMS IN GRAM NEGATIVES
SUSCEPTIBLE RESISTANT
-Lactam Antibiotic
Diffusion through Porin Blocks Entry
Outer Membrane Efflux Pump
Diffusion through Beta-Lactamase
Peptidoglycan Hydolyzes Beta-Lactam
Penicillin Binding Proteins Changes in PBP results in
Failure to Bind to -Lactam Cell Death
13
Adapted from Livermore and Woodford, Trends in Microbiol, 2006.
The Gram Negative Cell Wall
Porin channels
Efflux system
PBPs
B-lactamases
14
Definition of beta lactamases
• Beta lactamases are enzymes produced by some gram-positive and gram-negative bacteria that hydrolyze beta lactam antibiotics
15
β-Lactamase Classes Chromosomal Plasmid
A Bacteroides, Klebsiella, P. vulgaris
Staph pen’ase TEM, SHV KPC
B S. maltophilia, flavobacteria
IMP, VIM
C Most enterobacteria
CMY, LAT, FOX
D Aeromonas OXA
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ESBLs
Extended-spectrum β-lactamases
www.lahey.org/studies/webt.htm
• >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)
17
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)
18
Beta-lactamase inhibitors• Resemble β-lactam antibiotic structure
• Bind to β-lactamase and protect the antibiotic from destruction
• Most successful when they bind the β-lactamase irreversibly
• Three important in medicine Clavulanic acid Sulbactam Tazobactam
20
Why Test for β-lactamases?
•Correct therapy
•Breakpoints do not reliably detect new
β-lactamases
• Infection control
• Identify drugs causing resistance
21
Detection of ESBLs: Two Approaches
1. Screening tests and confirmatory tests for positive screens
2. Confirmatory tests
22
Detection of ESBLs: Screening Tests
•Advantages Less work Cheaper
•Disadvantages Sensitivity less than 100% Delayed confirmation Reporting of positive screens?
23
• MIC Test cefotaxime and ceftazidime +/- 4 μg/ml clavulanate: > 3 doubling dilution decrease with either drug
• Disk Test cefotaxime and ceftazidime +/- 10 μg clavulanate > 5 mm zone increase
e.g. ceftazidime 8 μg/ml
ceftazidime + clavulanate 1 μg/ml
CLSI Confirmatory Test – Klebsiella, E. coli, P. mirabilis
24
• BD Sensi Disks
• Etest
• MicroScan
• Phoenix
• Trek
• Vitek
• Vitek 2
FDA-Approved Commercial Tests
Combination Disk MethodCombination Disk Method
CLSI Approved MethodCLSI Approved Method
Double Disk MethodDouble Disk Method
Not CLSI ApprovedNot CLSI Approved
27
• 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
CLSI Reporting Recommendation
28
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)
Paterson, DL, et al. JCM 39: 2206 – 2212, 2001
(CLSI breakpoint 8 g/ml)
29
ESBL Blood Stream Infections Clinical Outcome
FATALITY RATE:
ESBL Producers = 26.7% (12/45)
Non-ESBL Producers = 5.7% (5/87)
MIC Favorable Outcome pts given only Suscep. 3rd gen ceph
8 0 (0/2)
4 33 (1/3) 2 100 (1/1)
Kim YK, et al. AAC 46:1481-1491, 2002
(CLSI breakpoint 8 g/ml)
30
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, MEN-1
• Rapidly hydrolyze cefotaxime but not ceftazidime (some MICs 4)
• Inhibited better by tazobactam than by sulbactam and clavulanate
31
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
32
33
E. coli with CTX-M ESBLE. coli with CTX-M ESBL
34
MICs in g/ml: SHV-3 producing Citrobacter freundii
Inocul.
CFU/ml
Cefotaxime Ceftazidime Aztreonam Cefepime
5 x 105 2 1 0.5 0.5
KS Thomson and ES Moland, Creighton University
Pitfalls of ESBL Testing Effects of Inoculum
(CLSI breakpoint 8 g/ml)
35
MICs in g/ml: SHV-3 producing Citrobacter freundii
Inocul.
CFU/ml
Cefotaxime Ceftazidime Aztreonam Cefepime
5 x 105 2 1 0.5 0.5
5 x 107 256 32 32 >1024
Pitfalls of ESBL Testing Effects of Inoculum
KS Thomson and ES Moland, Creighton University
(CLSI breakpoint 8 g/ml)
36
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 $$$$$!
37
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
38
Prevalence of ESBLs
Moland ES, et al. J Clin Microbiol. 2006 Sep;44:3318-24Moland ES, et al. J Clin Microbiol. 2006 Sep;44:3318-24
• 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
39
Prevalence of ESBLs
Organism # Pos/Total tested
% Overall % in ICUs % in Non-ICUs
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;44:3318-24Moland ES, et al. J Clin Microbiol. 2006 Sep;44:3318-24
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Prevalence of ESBLs at LUMC2006 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 2006-07Schreckenberger P, LUMC Antibiogram 2006-07
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P. mirabilis with ESBLP. mirabilis with ESBL
43
Pitfalls of ESBL Testing
• Recommendation (not CLSI endorsed): Extend CLSI reporting recommendations to all ESBL-producing organisms
• Report all ESBL-producing organisms the same way: resistant to all penicillins, cephalosporins, and aztreonam
44
AmpC Beta Lactamases
SPACE
- Serratia- Providencia/P. aeruginosa- Aeromonas- Citrobacter freundii- Enterobacter, Hafnia
• Cephalosporinases, hydrolyze all beta lactam antibiotics except carbapenems and cefepime
• Not Inhibited by clavulanate and sulbactam• Some inducibleSome inducible• Characteristic of certain genera:Characteristic of certain genera:
45
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
46
Inducer Potential
GOOD VARIABLE POOR
Cefoxitin Clavulanate Sulbactam
Cefmetazole Desacetyl Cefotaxime
Tazobactam
Imipenem Cefamandole Aztreonam
Ampicillin Cephalothin 3rd Gen Cephs
Cefonicid 4th Gen Cephs
AmpC Beta Lactamases
47
Uninduced AmpC
•Wall fragments recycled by AmpD•AmpR in repressor conformation• ampC (-lactamase gene) NOT expressed
ampCampRampD
AmpD AmpR
48
Induced AmpC
ampCampR
ampD
• More recycling: AmpD overwhelmed• Wall fragments convert AmpR to activator • ampC (-lactamase gene) expressed
AmpD-lactamase
49
But mutational derepression is the problem, not induction
E. cloacae expressing Induced E. cloacae expressing Induced Chromosomal AmpCChromosomal AmpC
50
Derepressed AmpC
ampCampR
ampD
• ampD inactivated by mutation• AmpR constantly converted to activator • ampC hyper-expressed
-lactamase++
51
E. cloacae derepressed mutant E. cloacae derepressed mutant expressing AmpCexpressing AmpC
52
• 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
Class C
AmpC Beta Lactamases
53
Mutant Selection
GOOD SELECTORS POOR SELECTORS
3rd Gen Cephs Imipenem
4th Gen Cephs Cephamycins
Older Cephalosporins
AmpC Beta Lactamases
54
Inducible Derepressed Basal Ampicillin 512 2048 4 Cephalothin 256 1024 16 Piperacillin 4 128 1 Cefotaxime 0.5 256 0.06 Ceftazidime 0.25 256 0.25 Aztreonam 0.06 16 0.06 Imipenem 0.25 0.25 0.06 Meropenem 0.06 0.12 0.015
MICs (mg/L) for E. cloacaeAmpC mutants
55
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.”
Bush Group 1 or AmpC
Inducible Beta Lactamases
56
E. cloacae not expressing E. cloacae not expressing Chromosomal AmpCChromosomal AmpC
57
• 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 S Ampicillin R Cefazolin R Ceftazidime S Cefepime S Ceftriaxone S Gentamicin S Levofloxacin S
Chromosomal AmpC that is not Expressing High Level Resistance
58
E. cloacae AmpC E. cloacae AmpC Derepressed MutantDerepressed Mutant
59
• 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 S Ampicillin R Cefazolin R Ceftazidime R Cefepime S Ceftriaxone R Gentamicin S Levofloxacin S
Chromosomal AmpC that is Expressing High Level Resistance
60
• Growth of: Enterobacter cloacae
Amikacin S Ampicillin R Cefazolin R Ceftazidime R Cefepime S Ceftriaxone R Gentamicin S Levofloxacin S
Chromosomal AmpC that is Expressing High Level Resistance
61
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
62
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. 1/3 of U.S. laboratories 3.3 – 8.5% K. pneumoniae in USA
63
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
64
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
65
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)
66
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E. coli with plasmid E. coli with plasmid mediated AmpCmediated AmpC
68AmpC Disk TestAmpC Disk Test
Lawn culture:Lawn culture: E. coli E. coli ATCC 25922ATCC 25922
Test Organism Test Organism on diskon disk
69
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
70
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
71
How to Determine if AmpC and ESBL Both Present
•Double Disk Diffusion Test Look for AmpC type pattern plus clavulanic
effect
72
• Growth of: Enterobacter cloacae
Amikacin S Ampicillin R Cefazolin R Ceftazidime I Cefepime S Ceftriaxone S Gentamicin S Levofloxacin S
Chromosomal AmpC that is Suspicious for ESBL
73
E. cloacae with Chromosomal E. cloacae with Chromosomal AmpC and ESBLAmpC and ESBL
74
• Growth of: Enterobacter cloacae - This organism possess an ESBL. Contact Isolation is required.
Amikacin S Cefazolin R Ampicillin R Ceftazidime I R Cefepime S R Ceftriaxone S R Gentamicin S Levofloxacin S
Chromosomal AmpC that is Suspicious for ESBL
75
• Growth of: Enterobacter cloacae
Amikacin S Ampicillin R Cefazolin R Ceftazidime R Cefepime S Ceftriaxone R Gentamicin S Levofloxacin S
Chromosomal AmpC that is not Suspicious for ESBL
76
11 mm11 mm
7 mm7 mm
E. cloacae with Chromosomal E. cloacae with Chromosomal AmpC derepressed mutant and AmpC derepressed mutant and
ESBLESBL
77
• Growth of: Enterobacter cloacae - this organism possess an ESBL. Contact Isolation is required
Amikacin S Ampicillin R Cefazolin R Ceftazidime R Cefepime S Ceftriaxone R Gentamicin S Levofloxacin S
Chromosomal AmpC that is not Suspicious for ESBL
R* See Comment
78
79
S. marcesens with Chromosomal AmpC S. marcesens with Chromosomal AmpC and ESBLand ESBL
80
Differences between ESBL and AmpC Beta Lactamases
Test Result ESBL AmpC
Inhibited by clavulanate Yes No
Hydrolyzes
-1st, 2nd, 3rd, Cephalosporins
Yes (R) Yes (R)
-Cephamycins No (S) Yes (R)
-Cefepime Yes (R) No (S)
81
Pitfalls of ESBL TestingK1 -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
82
83
K. oxytoca with K1 K. oxytoca with K1 -lactamase-lactamase
84
K. Oxytoca with K1 K. Oxytoca with K1 -lactamase-lactamase
85
86
K-1 Beta LactamaseK-1 Beta Lactamase
87
88
89
90
K. pneumoniae with ESBL, K. pneumoniae with ESBL, AmpC, and CarbapenemaseAmpC, and Carbapenemase
91
• 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
Resistance to Carbapenems
92
• 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
Resistance to Carbapenems
93
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
94
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
95
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
96
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 KPC-producing isolates as susceptible to imipenem
• Imipenem MIC was found to be markedly affected by inoculum
Bratu, S. et al AAC 49:3018-3020, 2005Bratu, S. et al AAC 49:3018-3020, 2005
97
Carbapenemase-Producing Klebsiella pneumonia (KPC)
Bratu, S. et al AAC 49:3018-3020, 2005Bratu, S. et al AAC 49:3018-3020, 2005
Results of Testing of 62 KPC % Susceptible
Imipenem MBD 105
MBD 104
5
44
Etest 2
Disk 2
Meropenem MBD 105
MBD 104
2
5
Etest 5
Disk 2
Ertapenem MBD 105 2
MBD 104 2
Etest 0
Disk 0
98
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, 2005Bratu, S. et al AAC 49:3018-3020, 2005
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
100
Widespread
Sporadic Isolate(s)
Geographical Distribution of KPC-Producers
Courtesy of J. Patel, PhD., CDC
101
K. Pneumoniae with KPC-2K. Pneumoniae with KPC-2
102
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
103
• 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).
Tris/EDTA Disk Test
Procedure described by Ellen Molan and Ken Thompson, Creighton University
Imipenem resistant K. pneumoniae Imipenem resistant K. pneumoniae expressing Class A carbapenemaseexpressing Class A carbapenemase
Imipenem resistant S. maltophilia Imipenem resistant S. maltophilia expressing Class B carbapenemaseexpressing Class B carbapenemase
105
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. Anderson KF et al. JCM 2007 Aug;45(8):2723-5.
KPC Producer - Example
meropenem ≤4 µg/ml*
imipenem ≤4 µg/ml*
ertapenem ≤2 µg/ml*
Courtesy of J. Patel, PhD., CDC*CLSI breakpoint for “S”;
marked w/ arrow
107
Ertapenem Resistant E. cloacaeErtapenem Resistant E. cloacae
108
E. cloacae: ertapenem resistance, E. cloacae: ertapenem resistance, meropenem susceptiblemeropenem susceptible
109
KPC positive ControlKPC positive Control
Patient IsolatePatient Isolate
E. cloacae derepressed mutant E. cloacae derepressed mutant expressing AmpC and porin expressing AmpC and porin
mutationmutation
110
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
111
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”
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