Detection and Reporting of Betalactam Resistance in Enterobacteriaceae

1
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
• pschrecken_at_lumc.edu

2
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.

3
Detecting Antibiotic Resistance

• Is there a Problem?

4
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

5
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

6
Comparison of Phoenix Vitek 2 for Detecting
ESBLs in E.coli and Klebsiella
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 Aug45(8)2380-4.
7
Evaluation of Methods to Identify KPC in
Enterobacteriaceae
Anderson KF et al. JCM 2007 Aug45(8)2723-5.
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Role of the Microbiology Lab

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

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The ß-lactam family of antibiotics
Monobactams
Carbapenems
Cephamycins
Cephalosporins
Penicillins
Benzyl-penicillin
Aztreonam
Imipenem
Cefoxitin
Cephalothin 1st
Methicillin
Cefamandole 2nd
Meropenem
Cefotetan
Ampicillin
Cefuroxime 2nd
Ertapenem
Cefmetazole
Carbenicillin
Cefotaxime 3rd
Mezlocillin
Ceftazidime 3rd
Ceftriaxone 3rd
Ticarcillin
Cefepime 4th
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Penicillin nucleus
S
CH3
R
1
2
5
6
CH3
4
7
3
N
O
COOH
11
Cephalosporin nucleus
1 S
7
R1
C O
HN
R2
O
COOH
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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

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The Gram Negative Cell Wall
Efflux system
Porin channels
B-lactamases
PBPs
Adapted from Livermore and Woodford, Trends in
Microbiol, 2006.
14
Definition of beta lactamases

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

15
ß-Lactamase Classes
16
ESBLsExtended-spectrum ß-lactamases

• gt180 enzymes described (119 TEM, 45 SHV)
• All mutations of older TEM and SHV
  plasmid-mediated ß -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)

www.lahey.org/studies/webt.htm
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ESBLsExtended-spectrum ß-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)

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Beta-lactamase inhibitors

• Resemble ß-lactam antibiotic structure
• Bind to ß-lactamase and protect the antibiotic
  from destruction
• Most successful when they bind the ß-lactamase
  irreversibly
• Three important in medicine
• Clavulanic acid
• Sulbactam
• Tazobactam

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Why Test for ß-lactamases?

• Correct therapy
• Breakpoints do not reliably detect new
• ß-lactamases
• Infection control
• Identify drugs causing resistance

21
Detection of ESBLs Two Approaches

• Screening tests and confirmatory tests for
  positive screens
• Confirmatory tests

22
Detection of ESBLs Screening Tests

• Advantages
• Less work
• Cheaper
• Disadvantages
• Sensitivity less than 100
• Delayed confirmation
• Reporting of positive screens?

23
CLSI Confirmatory Test Klebsiella, E. coli, P.
mirabilis

• MIC Test
• cefotaxime and ceftazidime /- 4 µg/ml
  clavulanate
• gt 3 doubling dilution decrease with either drug
• Disk Test
• cefotaxime and ceftazidime /- 10 µg clavulanate
• gt 5 mm zone increase

e.g. ceftazidime 8 µg/ml ceftazidime
clavulanate 1 µg/ml
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FDA-Approved Commercial Tests

• BD Sensi Disks
• Etest
• MicroScan
• Phoenix
• Trek
• Vitek
• Vitek 2

25
Combination Disk Method CLSI Approved Method
26
Double Disk Method Not CLSI Approved
27
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

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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
29
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)

(CLSI breakpoint ?8 ?g/ml)
Kim YK, et al. AAC 461481-1491, 2002
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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

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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

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E. coli with CTX-M ESBL
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Pitfalls of ESBL Testing Effects of Inoculum
(CLSI breakpoint ?8 ?g/ml)
KS Thomson and ES Moland, Creighton University
35
Pitfalls of ESBL Testing Effects of Inoculum
(CLSI breakpoint ?8 ?g/ml)
KS Thomson and ES Moland, Creighton University
36
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 !

37
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

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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
Sep443318-24
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Prevalence of ESBLs
Moland ES, et al. J Clin Microbiol. 2006
Sep443318-24
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Prevalence of ESBLs at LUMC2006 and 2007
(Jan-Sept)
Schreckenberger P, LUMC Antibiogram 2006-07
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P. mirabilis with ESBL
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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

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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
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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

46
AmpC Beta Lactamases
47
Uninduced AmpC

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

48
Induced AmpC
?-lactamase
AmpD
ampC
ampR
ampD

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

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But mutational derepression is the problem, not
induction
E. cloacae expressing Induced Chromosomal AmpC
50
Derepressed AmpC
?-lactamase
ampC
ampR
ampD

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

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E. cloacae derepressed mutant expressing AmpC
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Class CAmpC 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

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AmpC Beta Lactamases
54
MICs (mg/L) for E. cloacaeAmpC mutants
55
Bush Group 1 or AmpCInducible 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.

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E. cloacae not expressing Chromosomal AmpC
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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

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E. cloacae AmpC Derepressed Mutant
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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

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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

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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

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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

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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

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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

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When to Suspect AmpC Plasmid

• Disk tests for AmpC ß-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 gt 16 µg/ml for both drugs)
  and ESBL confirmatory test negative (this screen
  may have good specificity)

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E. coli with plasmid mediated AmpC
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Test Organism on disk
Lawn culture E. coli ATCC 25922
AmpC Disk Test
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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 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

70
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

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How to Determine if AmpC and ESBL Both Present

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

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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

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E. cloacae with Chromosomal AmpC and ESBL
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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

75
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

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E. cloacae with Chromosomal AmpC derepressed
mutant and ESBL
11 mm
7 mm
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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
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S. marcesens with Chromosomal AmpC and ESBL
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Differences between ESBL and AmpC Beta Lactamases
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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

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K. oxytoca with K1 ?-lactamase
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K. Oxytoca with K1 ?-lactamase
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K-1 Beta Lactamase
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K. pneumoniae with ESBL, AmpC, and Carbapenemase
91
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-ß-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

92
Resistance to Carbapenems

• Can also have carbapenem resistance due to
• Class A ESBLs (CTX-M) reduced permeability
• Class C High AmpC reduced permeability
• These hydrolyze ertapenem more than meropenem or
  imipenem

93
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

94
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

95
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

96
Carbapenemase-Producing Klebsiella pneumonia (KPC)

• 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 493018-3020, 2005
97
Carbapenemase-Producing Klebsiella pneumonia (KPC)
Bratu, S. et al AAC 493018-3020, 2005
98
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 49776-778 Schreckenberger, P
  personal observation)

Bratu, S. et al AAC 493018-3020, 2005
99
Extent of Problem

• Highly endemic in greater NY area
• Endemic in ICUs at Columbia, Cornell, St.
  Vincents, 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
100
Geographical Distribution of KPC-Producers
Courtesy of J. Patel, PhD., CDC
101
K. Pneumoniae with KPC-2
102
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

103
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-ß-lactamase production
  (negative test).

Procedure described by Ellen Molan and Ken
Thompson, Creighton University
104
Imipenem resistant K. pneumoniae expressing Class
A carbapenemase
Imipenem resistant S. maltophilia expressing
Class B carbapenemase
105
Modified Hodge Test

• Inoculate MH agar with a 110 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 Aug45(8)2723-5.
106
KPC Producer - Example
meropenem 4 µg/ml
imipenem 4 µg/ml
ertapenem 2 µg/ml
CLSI breakpoint for S marked w/ arrow
Courtesy of J. Patel, PhD., CDC
107
Ertapenem Resistant E. cloacae
108
E. cloacae ertapenem resistance, meropenem
susceptible
109
E. cloacae derepressed mutant expressing AmpC
and porin mutation
KPC positive Control
Patient Isolate
110
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

111
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. 48S1, 87-102.
• Web version (2004with a few changes) available
• http//www.bsac.org.uk
• Then to Susceptibility Testing link
• Then to Guide to Susceptibility Testing
• Then to Chapter 11