Phenotypic Detection of β-Lactamase Resistance in Gram-Negative

Bacilli: Testing and Interpretation Guide (Rev. 2-21-12)

Paul C. Schreckenberger, Ph.D., Violeta Rekasius, MT(ASCP)

INTRODUCTION:
To detect isolates with AmpC, K1, and KPC enzymes, as well as those with ESBLs, it is desirable
to test all Enterobacteriaceae with a combination of antibiotics that will allow detection of
resistant mechanisms. In our laboratory we have expanded the ESBL confirmatory disk test to
include 12 antibiotic disks and have applied the test to all members of the Enterobacteriaceae that
have a susceptibility pattern that is suspicious for the presence of an ESBL, AmpC, K1 or KPC
type resistance gene. Through the application of this test we have been able to successfully detect
antibiotic resistance in many species of Enterobacteriaceae that would have not been detected
using our automated susceptibility testing system.

WHEN TO PERFORM 12-DISK TEST:

Set up 12-disk test to confirm presence of ESBL in following instances:
1. Any E. coli or Klebsiella when phenotype does not agree with ESBL confirmation test on
Vitek, MicroScan, or other automated susceptibility system (e.g. ceftazidime is I or R but
ESBL confirmatory test is Neg)
2. Any Enterobacteriaceae when one of the 3rd gen. cephalosporins tests I or R and no ESBL
confirmatory test result available
3. Any Enterobacteriaceae when atypical or multi-drug resistant pattern exists (e.g. P.
mirabilis resistant to multiple drugs)
4. Any Enterobacteriaceae resistant to all drugs except imipenem
5. Any Enterobacteriaceae resistant to ertapenem, imipenem or meropenem

SPECIMEN:
The specimen consists of a pure isolate of the Enterobacteriaceae, which has a susceptibility
result that is consistent with an ESBL or an AMPC pattern, ie.; ceftazidime is I/R, cefrtriaxone is
I/R, aztreonam is I/R and requires confirmation by a disk method.

MATERIALS:
1. Antibiotic disks are placed in 12 cartridge dispenser, kept in fridge (2-8C), until use:
Aztreonam (30)
Ceftazidime (30)
Ceftazidime + clavulante (30/10)
Cefotaxime (30)
Cefotaxime + clavulante (30/10)
Cefoxitin (30)
Cefotetan (30)
Ceftriaxone (30)
Cefepime (30)
Ertapenem (10)
Imipenem(10)
Meropenem (10)
2. Mueller Hinton (MH) agar plate, 150 mm , kept in fridge (2-8C), until use

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3. sterile saline or tryptic soy broth (TSB)
4. sterile swabs
5. 0.5 McFarland barium turbidity standard / photometer (colorimeter)

METHOD:
1. Allow the MH agar plate and disk dispenser to come to room temperature before use.
2. Prepare a 0.5 McFarland standard of the organism to be tested in sterile saline or TSB.
Standardize the inoculum using the colorimeter.
3. Streak the bacterial suspension evenly in 3 planes onto the surface of the MH agar plate,
using a cotton swab. Rim the edge of the plate.
4. Place the disk dispenser over the MH agar plate and depress the knob. This will allow the
antibiotic disks to dispense and automatically “tamp” the disk into place.
5. All of the disks must be placed on the same MH agar plate in a specified order (See Figure
1)
6. Incubate the MH agar plate overnight in a non-CO2 incubator at 35C.
7. The following day read and record all zones of inhibition.
8.
RESULTS:

1. Detection of ESBLs (ceftazidime and cefotaxime disks with and without clavulanic
acid are used to detect ESBLs)
A. If the zone size increases 5 mm or more when clavulanate is added compared to the
drug alone the isolate is considered an ESBL. Only one antibiotic must be "reversed"
by the clavulanate to be an ESBL. Note: For MYSPACE organisms if the zone size
increases 3 mm or more with clavulanic-containing disk compared to the drug alone
then the isolate is considered an ESBL. See reference 14.

Combination Disk (CLISI) Method – E.coli with ESBL

22 mm

CAZ/CLA – 22 mm
CAZ – 11 mm
1 1 mm 22 –11 = > 5mm
= ESBL

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 B. If an “enhancement” or extension of the zone of inhibition is seen between any of the
cephalosporin antibiotics and the clavulanate containing disks, the presence of an
ESBL can be predicted. This phenomenon is often referred to as the “KEYHOLE”
effect, or “CLAVULANIC” effect and is indicative of ESBL production.
Double Disk Potentiation Method – P. mirabilis with ESBL

Keyhole Formation
Around Clavulanic
Containing Disk
= ESBL

2. Detection of AmpC beta lactamases (cefepime and cefoxitin disks are used to detect
ampC beta lactamases)

a. AmpC strains are resistant to the cephamycins (ie; cefoxitin and cefotetan).
b. AmpC strains are susceptible to cefepime.
c. High level AmpC producers causes resistance to all 1st, 2nd and 3rd generation
cephalosporins, the beta lactam-inhibitor drugs and the monobactams (ie;
aztreonam).

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 Double Disk Potentiation Method - E. coli with plasmid mediated AmpC, ESBL Negative

No Clavulanic Effect

Cefepime (FEP) – S

Cefoxitin (FOX) – R

= AmpC

Double Disk Potentiation Method - E. cloacae with chromosomal AmpC, ESBL Positive

Keyhole Formation
between cefepime and
clavulanic containing
disk
= ESBL

Cefepime (FEP) – S
Cefoxitin (FOX) – R
= AmpC

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3. Detection of K1 beta lactamases (aztreonam, ceftazidime, cefotaxime and ceftriaxone
disks are used to detect K1 beta lactamases)

Double Disk Potentiation Method – K. oxytoca with chromosomal K1 beta lactamase,

ESBL Negative, AmpC Negative

Cefoxitin S = Neg AmpC

No Clavulanic Effect =
Neg ESBL

Aztreonam - R
Ceftazidime - S

Cefotaxime S: Ceftriaxone R

4. Detection of Carbapenemase (ertapenem and imipenem disks are used to screen for
carbapenemase resistance)

Double Disk Potentiation Method – K. pneumoniae with KPC beta lactamase

i. Imipenem - S
ii.
Ertapenem - R
iii.
iv. Suggests possible KPC
or metallo-beta-
lactamase. Confirm
with Hodge test and
MBL Etest or send to
reference lab for
confirmation

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5. Metallo-Beta-Lactamases are resistant to ALL antibiotics. May be susceptible to Aztreonam,
Colistin And/or Tigecycline

Suggests possible metallo-beta-lactamase. Confirm with MBL Etest or EDTA Disks (See below)
or send to reference lab for confirmation

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5. Metallo-Beta-lactamase (MBL) Etest

POSITIVE FOR MBL – MIC of

Imipenem (IP) is reduced by at least three
doubling dilutions in presence of EDTA
(IPI)

NEGATIVE FOR MBL - MIC of

Imipenem (IP) is NOT reduced by at least
three doubling dilutions in presence of
EDTA (IPI)

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6. SME – Chromosomal carbapenemase

SME showing imipenem

resistance and susceptibility to
all 3rd and 4th generation
cephalosporins

SME are resistant to Imipenem by a chromosomal mechanism. Contact isolation is NOT

necessary.

REPORTING RESULTS:
1. Record all disk diffusion mm zone size readings in the culture work up and on recording
form.
2. Reporting of detected resistance mechanisms:

ESBL:
• If ESBL is detected, change/override any previous susceptibility result to resistant, for all
penicillins, cephalosporins, and monobactams, regardless of how the drug tests,
following CLSI interpretive guidelines for ESBL. Refer to current CLSI M100-S21
document. Report beta lactam inhibitor drugs as they test.
• If ESBL is not detected, report drugs as tested. For example if organism is shown to be
an ampC or K1, report drugs as they test, do not override and make resistant
• If ESBL is present along with AmpC or K1, apply the ESBL reporting rules and report
all penicillins, cephalosporins and monobactams as resistant.

AmpC:
• Chromosomal AmpC detected in MYSPACE organisms, do NOT change any
antibiotics, report as they test.
• Plasmid-mediated AmpC detected in E. coli, Klebsiella species, Proteus, do NOT
change any antibiotics, report as they test with comment “Plasmid-mediated

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 transmissible resistance mechanism detected (AmpC). Patient requires
contact isolation.”
K1:
• Report all drugs as they test. Do NOT apply ESBL reporting rules.

KPC:
• Report confirmed positive Modified Hodge Test as: “Carbapenem resistant
Enterobacteriaceae (CRE) detected (plasmid mediated KPC type). Patient
requires contact isolation.”

MBL
• Report confirmed MBLs as: “Carbapenem resistant Enterobacteriaceae
(CRE) detected (plasmid mediated metallo-beta-lactamase MBL type).
Patient requires contact isolation.”

SME
• Report Serratia marcescens SME as: “Imipenem resistance due to
carbapenemase production (chromosomal-type). The effectiveness of other
beta-lactams (that test susceptible) in treating infections due to
carbapenemase-producing Serratia marcescens has not been established.”

QUALITY CONTROL:

Disk diffusion testing is performed weekly with ATCC# 700603 Klebsiella pneumoniae and E.
coli ATCC 25922 following CLSI guidelines. If correct quality control results are not obtained,
the test is invalid and patient results cannot be reported.

REFERENCE:

Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial

Susceptibility Testing, Twenty-second Informational Supplement. CLSI document M100-S22.
Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,
Pennsylvania 19087-1898 USA, 2012.

General Review Articles

1. Livermore DM, Winstanley TB, Shannon KP. Interpretative reading: recognizing the
unusual and inferring resistance mechanisms from resistance phenotypes.
J Antimicrob Chemother. 2001 Jul;48 Suppl 1:87-102
2. Thomson KS, Moland ES. Version 2000: the new β-lactamases of Gram-negative bacteria
at the dawn of the new millennium (Review). Microbes and Infection, 2000;2:1225-1235

Review of Phenotypic Detection Methods

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 3. Drieux L, Brossier F, Sougakoff W, Jarlier V. Phenotypic detection of extended-spectrum
β-lactamase production in Enterobacteriaceae: review and bench guide. Clin Microbiol
Infect 2008; 14 (Suppl. 1) 90-103.
4. Livermore DM, Brown DF. Detection of beta-lactamase-mediated resistance. J
Antimicrob Chemother. 2001 Jul;48 Suppl 1:59-64.

ESBL Review Articles

5. Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization,
epidemiology, and detection of this important resistance threat. Clin Microbiol Rev. 2001
Oct;14(4):933-51, table of contents. Review.
6. Jacoby GA, Munoz-Price LS: The new beta-lactamases.
N Engl J Med. 2005 Jan 27;352(4):380-91. Review
7. Paterson DL, Bonomo RA: Extended-spectrum beta-lactamases: a clinical update.
Clin Microbiol Rev. 2005 Oct;18(4):657-86. Review.
8. Schreckenberger, P.C. Conundrums in the Laboratory Detection of Antimicrobial
Resistant Gram-Negative Bacteria. Reviews in Medical Microbiology; 15:45-49, April
2004.
9. Thomson KS: Controversies about extended-spectrum and AmpC beta-lactamases. Emerg
Infect Dis. 2001 Mar-Apr;7(2):333-6. Review.

AmpC B-Lactamases Review Articles

10. Jacoby GA. AmpC β-Lactamases. Clin Microbiol Rev; 2009 Jan; 22(1):161-182.

Double-Disk Potentiation Method

11. Coudron PE, Moland ES, Sanders CC: Occurrence and detection of extended-spectrum
beta-lactamases in members of the family Enterobacteriaceae at a veterans medical center:
seek and you may find. J Clin Microbiol. 1997 Oct;35(10):2593-7.
12. Jarlier V, Nicolas MH, Fournier G, Philippon A: Extended broad-spectrum beta-
lactamases conferring transferable resistance to newer beta-lactam agents in
Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis. 1988
Jul-Aug;10(4):867-78.
13. Pitout JD, Reisbig MD, Venter EC, Church DL, Hanson ND. Modification of the double-
disk test for detection of enterobacteriaceae producing extended-spectrum and AmpC
beta-lactamases. J Clin Microbiol. 2003 Aug;41(8):3933-5.
14. Towne TG, Lewis JS 2nd, Herrera M, Wickes B, Jorgensen JH: Detection of SHV-type
extended-spectrum beta-lactamase in Enterobacter isolates. J Clin Microbiol. 2010
Jan;48(1):298-9.
15. Tzelepi E, Giakkoupi P, Sofianou D, Loukova V, Kemeroglou A, Tsakris A: Detection of
extended-spectrum beta-lactamases in clinical isolates of Enterobacter cloacae and
Enterobacter aerogenes. J Clin Microbiol. 2000 Feb;38(2):542-6.
16. Yan JJ, Hsueh PR, Lu JJ, Chang FY, Shyr JM, Wan JH, Liu YC, Chuang YC, Yang YC,
Tsao SM, Wu HH, Wang LS, Lin TP, Wu HM, Chen HM, Wu JJ: Extended-spectrum
beta-lactamases and plasmid-mediated AmpC enzymes among clinical isolates of
Escherichia coli and Klebsiella pneumoniae from seven medical centers in Taiwan.
Antimicrob Agents Chemother. 2006 May;50(5):1861-4.
17. Yan JJ, Ko WC, Wu HM, Tsai SH, Chuang CL, Wu JJ. Complexity of Klebsiella
pneumoniae isolates resistant to both cephamycins and extended-spectrum cephalosporins
at a teaching hospital in Taiwan. J Clin Microbiol. 2004 Nov;42(11):5337-40.

Loyola University Medical Center Page 10 5/7/2012

Hodge Test
18. Anderson KF, Lonsway DR, Rasheed JK, Biddle J, Jensen B, McDougal LK, Carey RB,
Thompson A, Stocker S, Limbago B, Patel JB. Evaluation of methods to identify the
Klebsiella pneumoniae carbapenemase in Enterobacteriaceae. J Clin Microbiol. 2007
Aug;45(8):2723-5.
19. Lee K, Chong Y, Shin HB, Kim YA, Yong D, Yum JH. Modified Hodge and EDTA-disk
synergy tests to screen metallo-beta-lactamase-producing strains of Pseudomonas and
Acinetobacter species. Clin Microbiol Infect. 2001 Feb;7(2):88-91.
20. Lee K, Lim YS, Yong D, Yum JH, Chong Y. Evaluation of the Hodge test and the
imipenem-EDTA double-disk synergy test for differentiating metallo-beta-lactamase-
producing isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol. 2003
Oct;41(10):4623-9

Carbapenemase-Producing Klebsiella pneumoniae (KPC)

21. Alba J, Ishii Y, Thomson K, Moland ES, Yamaguchi K. Kinetics study of KPC-3, a
plasmid-encoded class A carbapenem-hydrolyzing beta-lactamase.
Antimicrob Agents Chemother. 2005 Nov;49(11):4760-2.
22. Bradford PA, Bratu S, Urban C, Visalli M, Mariano N, Landman D, Rahal JJ, Brooks S,
Cebular S, Quale J. Emergence of carbapenem-resistant Klebsiella species possessing the
class A carbapenem-hydrolyzing KPC-2 and inhibitor-resistant TEM-30 beta-lactamases
in New York City. Clin Infect Dis. 2004 Jul 1;39(1):55-60.
23. Bratu S, Mooty M, Nichani S, Landman D, Gullans C, Pettinato B, Karumudi U, Tolaney
P, Quale J. Emergence of KPC-possessing Klebsiella pneumoniae in Brooklyn, New
York: epidemiology and recommendations for detection. Antimicrob Agents Chemother.
2005 Jul;49(7):3018-20.
24. Woodford N, Tierno PM Jr, Young K, Tysall L, Palepou MF, Ward E, Painter RE, Suber
DF, Shungu D, Silver LL, Inglima K, Kornblum J, Livermore DM. Outbreak of Klebsiella
pneumoniae producing a new carbapenem-hydrolyzing class A beta-lactamase, KPC-3, in
a New York Medical Center. Antimicrob Agents Chemother. 2004 Dec;48(12):4793-9.

New Dehli Metallo beta-lactamase (NDM-1)

25. Centers for Disease Control and Prevention (CDC). Detection of Enterobacteriaceae
isolates carrying metallo-beta-lactamase - United States, 2010. MMWR Morb Mortal
Wkly Rep. 2010 Jun 25;59(24):750.
26. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R,
Chaudhary U, Doumith M, Giske CG, Irfan S, Krishnan P, Kumar AV, Maharjan S,
Mushtaq S, Noorie T, Paterson DL, Pearson A, Perry C, Pike R, Rao B, Ray U, Sarma JB,
Sharma M, Sheridan E, Thirunarayan MA, Turton J, Upadhyay S, Warner M, Welfare W,
Livermore DM, Woodford N. Emergence of a new antibiotic resistance mechanism in
India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet
Infect Dis. 2010 Sep;10(9):597-602.
27. Mochon AB, Garner OB, Hindler JA, Krogstad P, Ward KW, Lewinski MA, Rasheed JK,
Anderson KF, Limbago BM, Humphries RM. New Delhi Metallo-{beta}-lactamase
(NDM-1) Producing Klebsiella pneumoniae: a case report and laboratory detection
strategies. J Clin Microbiol. 2011 Feb 16. [Epub ahead of print]

Loyola University Medical Center Page 11 5/7/2012

 28. Mulvey MR, Grant JM, Plewes K, Roscoe D, Boyd DA. New Delhi metallo-β-lactamase
in Klebsiella pneumoniae and Escherichia coli, Canada. Emerg Infect Dis. 2011
Jan;17(1):103-6.
29. Sidjabat H, Nimmo GR, Walsh TR, Binotto E, Htin A, Hayashi Y, Li J, Nation RL,
George N, Paterson DL. Carbapenem resistance in Klebsiella pneumoniae due to the New
Delhi Metallo-β-lactamase. Clin Infect Dis. 2011 Feb;52(4):481-4.

SME Carbapenemases
30. Walther-Rasmussen J, Høiby N. Class A carbapenemases. J Antimicrob Chemother. 2007
Sep;60(3):470-82.
31. Queenan AM, Shang W, Schreckenberger P, Lolans K, Bush K, Quinn J. SME-3, a novel
member of the Serratia marcescens SME family of carbapenem-hydrolyzing beta-
lactamases. Antimicrob Agents Chemother. 2006 Oct;50(10):3485-7.

NMCA Carbapenemases
32. Pottumarthy S, Moland ES, Juretschko S, Swanzy SR, Thomson KS, Fritsche TR. NmcA
carbapenem-hydrolyzing enzyme in Enterobacter cloacae in North America. Emerg Infect
Dis. 2003 Aug;9(8):999-1002. Erratum in: Emerg Infect Dis. 2003 Sep;9(9):1188.

OXA-type Carbapenemases
33. Walther-Rasmussen J, Høiby N. OXA-type carbapenemases. J Antimicrob Chemother.
2006 Mar;57(3):373-83. Review.

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 Fig 1. Template for Disk Potentiation Method for Detecting
ESBL and ampC beta-lactamases

2
9 CTX-CLA

10 ATM
IMP

8 3
CTX 11

ERT 12 CAZ-CLA FEP

7
CAZ 4

MEM CRO
6 5

Abbreviation KEY CTT FOX

1 cefotaxime-
clavulanate
2 aztreonam
3 cefepime
4 ceftriaxone
5 cefoxitin
6 cefotetan
7 meropenem
8 ertapenem
9 imipenem
10 cefotaxime
11 ceftazidime-
clavulanate
12 ceftazidime

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