SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 Detection and Reporting of Beta-lactam Resistance in Enterobacteriaceae.

SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli
October 3, 2007 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 Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

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 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

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

Thomson KS et al. JCM 2007 Aug;45(8):2380-4.
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 Phoenix 73(96) 21(81) Phoenix* 75(99) 15(58) Vitek 2 69(91) 68(89) 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):

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 97/89 Etest 58/96 55/96 90/84 Disk Diffusion 71/96 42/96 97/87 Vitek Legacy 52/98 NA Vitek 2 48/96 MicroScan 84/98 74/96 100/89 Phoenix 61/98 81/96 Sensititre 42/98 29/96 Anderson KF et al. JCM 2007 Aug;45(8):

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
Penicillins Cephalosporins Cephamycins Carbapenems Monobactams Benzyl-penicillin Cephalothin 1st Cefoxitin Imipenem Aztreonam Methicillin Cefamandole 2nd Cefotetan Meropenem Ampicillin Cefuroxime 2nd Cefmetazole Ertapenem Carbenicillin Cefotaxime 3rd Mezlocillin Ceftazidime 3rd Ticarcillin Ceftriaxone 3rd Cefepime 4th

Penicillin nucleus S CH3 R 1 5 2 6 CH3 7 4 N 3 O COOH

Cephalosporin nucleus
1 S 7 R1 C O HN R2 O COOH

MODE OF ACTION OF BETA LACTAMS IN GRAM NEGATIVES
SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 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 Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

The Gram Negative Cell Wall
Efflux system Porin channels B-lactamases PBPs Adapted from Livermore and Woodford, Trends in Microbiol, 2006.

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

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)

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)

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?
SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 Why Test for β-lactamases? Correct therapy Breakpoints do not reliably detect new β-lactamases Infection control Identify drugs causing resistance Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

Detection of ESBLs: Two Approaches
Screening tests and confirmatory tests for positive screens Confirmatory tests

Detection of ESBLs: Screening Tests
Advantages Less work Cheaper Disadvantages Sensitivity less than 100% Delayed confirmation Reporting of positive screens?

CLSI Confirmatory Test – Klebsiella, E. coli, P. mirabilis
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

FDA-Approved Commercial Tests
BD Sensi Disks Etest MicroScan Phoenix Trek Vitek Vitek 2

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

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

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 (0/2) (1/3) (1/1) (CLSI breakpoint 8 g/ml) Kim YK, et al. AAC 46: , 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 ESBL

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 (CLSI breakpoint 8 g/ml) 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 5 x 107 256 32 >1024 (CLSI breakpoint 8 g/ml) 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 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 Sep;44:

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 0.4 8.9 P. mirabilis 5/359 1.4 3.1 E. aerogenes 2/189 1.1 0.6 3.3 Moland ES, et al. J Clin Microbiol Sep;44:

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 1.3 K. pneumoniae 1149 37 3.2 M. morganii 70 5.7 P. mirabilis 592 25 4.2 P. stuartii 16 12.5 Schreckenberger P, LUMC Antibiogram

P. 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

October 3, 2007 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 P A C E - Serratia - Providencia/P. aeruginosa - Aeromonas - Citrobacter freundii - Enterobacter, Hafnia Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

October 3, 2007 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 Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

AmpC Beta Lactamases Inducer Potential GOOD VARIABLE POOR Cefoxitin
SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 AmpC Beta Lactamases Inducer Potential GOOD VARIABLE POOR Cefoxitin Clavulanate Sulbactam Cefmetazole Desacetyl Cefotaxime Tazobactam Imipenem Cefamandole Aztreonam Ampicillin Cephalothin 3rd Gen Cephs Cefonicid 4th Gen Cephs Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

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

Induced AmpC -lactamase AmpD More recycling: AmpD overwhelmed
ampR ampC More recycling: AmpD overwhelmed Wall fragments convert AmpR to activator ampC (-lactamase gene) expressed

E. cloacae expressing Induced Chromosomal AmpC
But mutational derepression is the problem, not induction

Derepressed AmpC -lactamase++ ampD inactivated by mutation
ampR ampC ampD inactivated by mutation AmpR constantly converted to activator ampC hyper-expressed

E. cloacae derepressed mutant expressing AmpC

Class C AmpC Beta Lactamases
SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli Class C AmpC Beta Lactamases October 3, 2007 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 Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

AmpC Beta Lactamases Mutant Selection GOOD SELECTORS POOR SELECTORS
SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 AmpC Beta Lactamases Mutant Selection GOOD SELECTORS POOR SELECTORS 3rd Gen Cephs Imipenem 4th Gen Cephs Cephamycins Older Cephalosporins Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

MICs (mg/L) for E. cloacae AmpC mutants

Bush Group 1 or AmpC Inducible Beta Lactamases
SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 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.” Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

E. cloacae not expressing Chromosomal AmpC

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 R Cefazolin R Ceftazidime S Cefepime S Ceftriaxone S Gentamicin S Levofloxacin S

E. cloacae AmpC Derepressed Mutant

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 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

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 mediated AmpC

AmpC Disk Test Test Organism on disk Lawn culture: E. coli ATCC 25922
SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 Test Organism on disk Lawn culture: E. coli ATCC 25922 AmpC Disk Test Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

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

Chromosomal AmpC that is Suspicious for ESBL
Growth of: Enterobacter cloacae Amikacin S Ampicillin R Cefazolin R Ceftazidime I Cefepime S Ceftriaxone S Gentamicin S Levofloxacin S

E. cloacae with Chromosomal AmpC and ESBL

Chromosomal AmpC that is Suspicious for 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 not Suspicious for ESBL
Growth of: Enterobacter cloacae Amikacin S Ampicillin R Cefazolin R Ceftazidime R Cefepime S Ceftriaxone R Gentamicin S Levofloxacin S

E. cloacae with Chromosomal AmpC derepressed mutant and ESBL
11 mm 7 mm

Chromosomal AmpC that is not Suspicious for ESBL
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 R* See Comment

S. marcesens with Chromosomal AmpC and ESBL

Differences between ESBL and AmpC Beta Lactamases
SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 Differences between ESBL and AmpC Beta Lactamases Test Result ESBL AmpC Inhibited by clavulanate Yes No Hydrolyzes -1st, 2nd, 3rd, Cephalosporins Yes (R) -Cephamycins No (S) -Cefepime Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

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

K. oxytoca with K1 -lactamase

K. Oxytoca with K1 -lactamase

K-1 Beta Lactamase

K. pneumoniae with ESBL, AmpC, and Carbapenemase

Resistance to Carbapenems
SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 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 Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

Resistance to Carbapenems
SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 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 Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

Class A Carbapenemases
SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 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 Paul C. Schreckenberger, Ph.D., D(ABMM), Loyola University Medical Center

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: , 2005

Results of Testing of 62 KPC % Susceptible Imipenem MBD 105 MBD 104 5 44 Etest 2 Disk Meropenem Ertapenem Bratu, S. et al AAC 49: , 2005

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: ; Schreckenberger, P personal observation) Bratu, S. et al AAC 49: , 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): ; AAC. 2006; 50(8): ; AAC. 2007; 5(2): 763-5; 47th ICAAC. Abstract C ; 47th ICAAC. Abstract C ; 47th ICAAC. Abstract C

Courtesy of J. Patel, PhD., CDC
Widespread Sporadic Isolate(s) Geographical Distribution of KPC-Producers Courtesy of J. Patel, PhD., CDC

K. 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

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-β-lactamase production (negative test). Procedure described by Ellen Molan and Ken Thompson, Creighton University

Imipenem resistant K. pneumoniae expressing Class A carbapenemase
Imipenem resistant S. maltophilia expressing Class B carbapenemase

Anderson KF et al. JCM 2007 Aug;45(8):2723-5.
Modified Hodge Test Inoculate MH agar with a 1:10 dilution of a 0.5 McFarland suspension of E. coli ATCC 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):

*CLSI breakpoint for “S”; Courtesy of J. Patel, PhD., CDC
KPC Producer - Example imipenem ≤4 µg/ml* meropenem ≤4 µg/ml* ertapenem ≤2 µg/ml* *CLSI breakpoint for “S”; marked w/ arrow Courtesy of J. Patel, PhD., CDC

Ertapenem Resistant E. cloacae

E. cloacae: ertapenem resistance, meropenem susceptible

E. cloacae derepressed mutant expressing AmpC and porin mutation
KPC positive Control Patient Isolate

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 Interpretive reading: recognizing the unusual and inferring resistance mechanisms from resistance phenotypes. J Antimicrob Chemother. 48:S1, Web version (2004…with a few changes) available… Then to “Susceptibility Testing” link Then to “Guide to Susceptibility Testing” Then to “Chapter 11”

SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 Detection and Reporting of Beta-lactam Resistance in Enterobacteriaceae.

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SCACM Workshop Detection of Antibiotic Resistance in Gram-Negative Bacilli October 3, 2007 Detection and Reporting of Beta-lactam Resistance in Enterobacteriaceae.

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