Detection and Reporting of Beta-lactam Resistance in Enterobacteriaceae Paul C. Schreckenberger,...

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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

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?

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

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

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

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.

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

Penicillin nucleus

Cephalosporin nucleus

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

Adapted from Livermore and Woodford, Trends in Microbiol, 2006.

The Gram Negative Cell Wall

Porin channels

Efflux system

B-lactamases

Definition of beta lactamases

• Beta lactamases are enzymes produced by some gram-positive and gram-negative bacteria that hydrolyze beta lactam antibiotics

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

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)

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)

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

Why Test for β-lactamases?

•Correct therapy

•Breakpoints do not reliably detect new

β-lactamases

• Infection control

• Identify drugs causing resistance

Detection of ESBLs: Two Approaches

1. Screening tests and confirmatory tests for positive screens

2. Confirmatory tests

Detection of ESBLs: Screening Tests

•Advantages Less work Cheaper

•Disadvantages Sensitivity less than 100% Delayed confirmation Reporting of positive screens?

• 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

• 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

• 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

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)

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

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

• 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

E. coli with CTX-M ESBLE. coli with CTX-M ESBL

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

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

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 $$$$$!

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

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

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

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

P. mirabilis with ESBLP. mirabilis with ESBL

• 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

AmpC Beta Lactamases

- 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:

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

Inducer Potential

GOOD VARIABLE POOR

Cefoxitin Clavulanate Sulbactam

Cefmetazole Desacetyl Cefotaxime

Tazobactam

Imipenem Cefamandole Aztreonam

Ampicillin Cephalothin 3rd Gen Cephs

Cefonicid 4th Gen Cephs

Uninduced AmpC

•Wall fragments recycled by AmpD•AmpR in repressor conformation• ampC (-lactamase gene) NOT expressed

ampCampRampD

AmpD AmpR

Induced AmpC

ampCampR

• More recycling: AmpD overwhelmed• Wall fragments convert AmpR to activator • ampC (-lactamase gene) expressed

AmpD-lactamase

But mutational derepression is the problem, not induction

E. cloacae expressing Induced E. cloacae expressing Induced Chromosomal AmpCChromosomal AmpC

Derepressed AmpC

ampCampR

• ampD inactivated by mutation• AmpR constantly converted to activator • ampC hyper-expressed

-lactamase++

E. cloacae derepressed mutant E. cloacae derepressed mutant expressing AmpCexpressing AmpC

• 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

Mutant Selection

GOOD SELECTORS POOR SELECTORS

3rd Gen Cephs Imipenem

4th Gen Cephs Cephamycins

Older Cephalosporins

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

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

E. cloacae not expressing E. cloacae not expressing Chromosomal AmpCChromosomal AmpC

• 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

E. cloacae AmpC E. cloacae AmpC Derepressed MutantDerepressed Mutant

• 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

• 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

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

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

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

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

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)

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

• 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

• 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

How to Determine if AmpC and ESBL Both Present

•Double Disk Diffusion Test Look for AmpC type pattern plus clavulanic

effect

Amikacin S Ampicillin R Cefazolin R Ceftazidime I Cefepime S Ceftriaxone S Gentamicin S Levofloxacin S

Chromosomal AmpC that is Suspicious for ESBL

E. cloacae with Chromosomal E. cloacae with Chromosomal AmpC and ESBLAmpC and ESBL

• 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

Chromosomal AmpC that is not Suspicious for ESBL

11 mm11 mm

7 mm7 mm

E. cloacae with Chromosomal E. cloacae with Chromosomal AmpC derepressed mutant and AmpC derepressed mutant and

ESBLESBL

• Growth of: Enterobacter cloacae - this organism possess an ESBL. Contact Isolation is required

Chromosomal AmpC that is not Suspicious for ESBL

R* See Comment

S. marcesens with Chromosomal AmpC S. marcesens with Chromosomal AmpC and ESBLand ESBL

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)

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

K. oxytoca with K1 K. oxytoca with K1 -lactamase-lactamase

K. Oxytoca with K1 K. Oxytoca with K1 -lactamase-lactamase

K-1 Beta LactamaseK-1 Beta Lactamase

K. pneumoniae with ESBL, K. pneumoniae with ESBL, AmpC, and CarbapenemaseAmpC, and Carbapenemase

• 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

• 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

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

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

• 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

• 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

Results of Testing of 62 KPC % Susceptible

Imipenem MBD 105

MBD 104

Etest 2

Disk 2

Meropenem MBD 105

MBD 104

Etest 5

Disk 2

Ertapenem MBD 105 2

MBD 104 2

Etest 0

Disk 0

• 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)

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

Widespread

Sporadic Isolate(s)

Geographical Distribution of KPC-Producers

Courtesy of J. Patel, PhD., CDC

K. Pneumoniae with KPC-2K. Pneumoniae with KPC-2

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

• 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

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

Ertapenem Resistant E. cloacaeErtapenem Resistant E. cloacae

E. cloacae: ertapenem resistance, E. cloacae: ertapenem resistance, meropenem susceptiblemeropenem susceptible

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

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

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”

Detection and Reporting of Beta-lactam Resistance in Enterobacteriaceae Paul C. Schreckenberger,...

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