Detection and Reporting of Beta-lactam Resistance in Enterobacteriaceae

Paul C. Schreckenberger, Ph.D., D(ABMM) Professor of Pathology Director, Clinical Microbiology Laboratory Loyola University Medical Center [email protected]

Objectives

• Participants will be able to:  Set up and interpret the double disk diffusion method for detecting ESBLs and ampCs.

 Describe methods for detection of carbapenamases, including the Hodge test and Tris EDTA double disk test  Modify susceptibility reports based on characterization of resistance Genotypes.

2

Detecting Antibiotic Resistance Is there a Problem?

Automated Systems

• Poor performance by automated systems in detecting resistance has necessitated use of off line screening/confirmatory tests  Oxacillin screening plates for MRSA  Vancomycin screening plates for MRSA and VRE  D-Zone Test for detection of inducible clinidamycin resistance 4

Automated Systems

• Limitations of Automated Systems in detecting emerging resistance in 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.

6

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.

7

Role of the Microbiology Lab

• “Each laboratory should have a staff member with the time, interest, and expertise to provide leadership in antibiotic testing and resistance. This person would read relevant publications, network with other laboratories, and evaluate potentially useful tests to detect new forms of resistance before new CLSI-recommended tests become available” • - Ken Thomson, Emerging Infect. Dis., 2001 8

The β-lactam family of antibiotics

Penicillins Cephalosporins Cephamycins Carbapenems 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

11

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.

13

Definition of beta lactamases

• Beta lactamases are enzymes produced by some gram-positive and gram-negative bacteria that hydrolyze beta lactam antibiotics 14

β-Lactamase Classes

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

16

ESBLs

Extended spectrum β-lactamases

• Primarily found in: 

Klebsiella, E. coli

•

Also found in:



Proteus, Serratia



Enterobacter, Salmonella



Morganella, etc.

• Most are inhibited well by clavulanic acid and tazobactam (less so by sulbactam) 17

Beta-lactamase inhibitors

• • • Resemble β-lactam antibiotic structure Bind to β-lactamase and protect the antibiotic from destruction • Most successful when they bind the 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?

22

CLSI Confirmatory Test –

Klebsiella, E. coli, P. mirabilis

• MIC Test  cefotaxime and ceftazidime +/ 4 μg/ml clavulanate:  > 3 doubling dilution decrease with either drug e.g.

ceftazidime 8 μg/ml • 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

34

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

35

• •

Enterobacteriaceae



-Lactam Breakpoints and ESBL Issues

CLSI is re-evaluating  -lactam breakpoints for Enterobacteriaceae  Example: cefotaxime   Current – Susceptible at  8  g/ml Proposed – Susceptible at  1 or  2  g/ml   Substantial data needed Goal is to more accurately detect all  -lactamase and other  lactam resistance mechanisms with revised breakpoints Changing breakpoints – commercial systems project it will take 3 years … much $$$$$!

36

ESBLs in organisms other than

E. coli and Klebsiella spp.

• • Most labs do not attempt to detect ESBLs in organism other than

E. coli

and

Klebsiella

Two Indications for ESBL Testing in Other Organisms  ESBLs detected in

E. coli or Klebsiella

 Suspicious phenotype • How to test?

 Use specific (confirmatory) test  Perform Double Disk Diffusion 37

Prevalence of ESBLs

• • Aim of study was to detect ESBL prevalence in all GNB in US medical centers 6,421 consecutive non-duplicate GNB screened for reduced susceptibility to cephems and aztreonam or potentiation of cefepime by clavulanate  Patients were from 42 ICU and 21 non-ICU sites throughout the US, 9/00 to 9/02 • Screen positive isolates were then investigated in a central lab for ESBL status Moland ES, et al. J Clin Microbiol. 2006 Sep;44:3318-24 38

Prevalence of ESBLs

Organism

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

59

• 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