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.
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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 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 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 Test 73(96) 73(96) 69(91) Expert System 73(96) 75(99) 68(89) 26 ESBL-neg strains ESBL Test 21(81) 21(81) 22(85) Expert System 21(81) 15(58) 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.
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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 Etest 94/98 58/96 94/93 55/96 97/89 90/84 Disk Diffusion Vitek Legacy Vitek 2 MicroScan Phoenix Sensititre 71/96 52/98 48/96 84/98 61/98 42/98 42/96 55/96 71/96 74/96 81/96 29/96 97/87 NA 94/93 100/89 NA NA
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 8
The β-lactam family of antibiotics
Penicillins Cephalosporins Cephamycins Carbapenems Monobactams
Benzyl penicillin Methicillin Ampicillin Cephalothin 1 st Cefoxitin Imipenem Cefamandole 2 nd Cefotetan Cefuroxime 2 nd Cefmetazole Meropenem Ertapenem Carbenicillin Cefotaxime 3 rd Mezlocillin Ticarcillin Ceftazidime 3 rd Ceftriaxone 3 rd Cefepime 4 th Aztreonam
Penicillin nucleus
R O 6 7 5 N 4
1
S 3 2 CH 3 CH 3 COOH
10
Cephalosporin nucleus
1 S R 1 C O HN 7 O COOH R 2
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MODE OF ACTION OF BETA LACTAMS IN GRAM NEGATIVES SUSCEPTIBLE -Lactam Antibiotic Diffusion through Outer Membrane Diffusion through RESISTANT Porin Blocks Entry Efflux Pump Beta-Lactamase Peptidoglycan Hydolyzes Beta-Lactam Penicillin Binding Proteins Cell Death 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.
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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
Chromosomal Plasmid A B C D
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, 3 rd and 4 th generation cephalosporins and aztreonam (not imipenem or cephamycins) www.lahey.org/studies/webt.htm
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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 β lactamase 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 less than 100% Delayed confirmation Reporting of positive screens?
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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 • Disk Test ceftazidime + clavulanate 1 μg/ml 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 8 4 2 ≤1 FAILURE DEATH 100% (6/6) 33% (2/6) 67% (2/3) 33% (1/3) 27% (3/11) 0% (0/3) 0% (0/3) 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 = 26.7% (12/45) = 5.7% (5/87) MIC 8 4 2 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, MEN 1 • 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.
CFU/ml 5 x 10 5 Cefotaxime 2 Ceftazidime 1 Aztreonam 0.5
Cefepime 0.5
(CLSI breakpoint
8
g/ml) KS Thomson and ES Moland, Creighton University
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Pitfalls of ESBL Testing
Effects of Inoculum
MICs in g/ml: SHV-3 producing
Citrobacter freundii
Inocul.
CFU/ml 5 x 10 5 Cefotaxime 2 Ceftazidime 1 Aztreonam 0.5
Cefepime 0.5
5 x 10 7 256 32 32 >1024
(CLSI breakpoint
8
g/ml) KS Thomson and ES Moland, Creighton University
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• •
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 $$$$$!
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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
K. oxytoca K. pneumoniae E. cloacae E. coli S. marcescens P. mirabilis E. aerogenes
# Pos/Total tested
18/137 96/853 25/453 42/1616 5/306 5/359 2/189
% Overall % in ICUs % in Non ICUs
13.1
9.2
23 11.3
5.5
2.6
1.6
1.4
1.1
13.7
4.3
3.6
0.4
3.1
0.6
4.7
14.3
1.6
8.9
0 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
C. freundii complex C. koserii E. aerogenes E. cloacae E. coli K. oxytoca K. pneumoniae M. morganii P. mirabilis P. stuartii
Total tested
165 110 197 387 5131 151 1149 70 592 16
ESBL Pos
4 6 2 20 96 2 37 4 25 2
% ESBL
2.4
5.5
1.0
5.2
1.9
1.3
3.2
5.7
4.2
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 ESBL producing 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 A
- Providencia/P. aeruginosa - Aeromonas
C E
- Citrobacter freundii - 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
GOOD Cefoxitin Cefmetazole Imipenem Ampicillin Inducer Potential VARIABLE Clavulanate Desacetyl Cefotaxime Cefamandole Cephalothin Cefonicid POOR Sulbactam Tazobactam Aztreonam 3 rd Gen Cephs 4 th Gen Cephs 46
AmpD
Uninduced AmpC
AmpR
amp
D
amp
R
amp
C • •Wall fragments recycled by AmpD •AmpR in repressor conformation
amp
C ( -lactamase gene) NOT expressed 47
Induced AmpC
-lactamase AmpD
amp
D
amp amp
C R • • More recycling: AmpD overwhelmed • Wall fragments convert AmpR to activator
amp
C ( -lactamase gene) expressed 48
E. cloacae expressing Induced Chromosomal AmpC But mutational derepression is the problem, not induction 49
Derepressed AmpC
-lactamase++
amp
D
amp amp
C R • •
amp
D inactivated by mutation • AmpR constantly converted to activator
amp
C 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
GOOD SELECTORS 3 rd Gen Cephs Mutant Selection POOR SELECTORS Imipenem 4 th Gen Cephs Cephamycins Older Cephalosporins 53
MICs (mg/L) for E. cloacae
AmpC mutants
Ampicillin Cephalothin Piperacillin Cefotaxime Ceftazidime Aztreonam Imipenem Meropenem
Inducible Derepressed Basal
512 2048 4 256 4 1024 128 16 1 0.5 0.25 0.06 0.25 0.06 256 256 16 0.25 0.12 0.06 0.25 0.06 0.06 0.015 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 S
Ampicillin
Cefazolin
Ceftazidime
Cefepime
Ceftriaxone
Gentamicin
Levofloxacin S S S S R R 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 S
Ampicillin
Cefazolin
Ceftazidime
Cefepime
Ceftriaxone
Gentamicin
Levofloxacin S R S S R R R
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• Chromosomal AmpC that is Expressing High Level Resistance
Growth of: Enterobacter cloacae
Amikacin
Ampicillin
Cefazolin
Ceftazidime
Cefepime
Ceftriaxone
Gentamicin
Levofloxacin S R S S S R R R
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. 1/3 of U.S. laboratories 3.3 – 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 screens: isolates positive in any of following 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 Test Organism on disk AmpC Disk Test
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
AmpC plasmids and
K. pneumoniae
that acquire 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 S S S S R I R
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 S S S R R R
75
E. cloacae with Chromosomal AmpC derepressed mutant and ESBL
7 mm 11 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 S S R* See Comment
77
78
S. marcesens with Chromosomal AmpC and ESBL 79
Differences between ESBL and AmpC Beta Lactamases
Test Result Inhibited by clavulanate Hydrolyzes -1 st , 2 nd , 3 rd , Cephalosporins -Cephamycins -Cefepime ESBL Yes Yes (R) No (S) Yes (R) AmpC No Yes (R) 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 –
P. aeruginosa Acinetobacter,
Other organisms may acquire resistance
pneumoniae,
other Enterobacteriaceae
– K.
• 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 . p neumoniae
c arbapenemase carbapenem-hydrolyzing enzymes most common on East Coast of U.S.
(
KPC-type) possess 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 found to possess
bla K. pneumoniae
collected in Brooklyn, NY, 62 (24%) were KPC 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, 2005
96
Carbapenemase-Producing Klebsiella pneumonia (KPC)
Results of Testing of 62 KPC
% Susceptible Imipenem MBD 10 5 5 MBD 10 4 Etest 44 2 Meropenem Ertapenem Disk MBD 10 5 MBD 10 4 Etest Disk MBD 10 5 MBD 10 4 Etest Disk 2 2 2 0 0 2 2 5 5
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 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 (negative test).
β lactamase production
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 3 rd 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