SCI 8008SEF Medical Microbiology & Virology II

SCI 8008SEF Medical Microbiology & Virology II
Lecture 10
Advanced laboratory technologies in Clinical Microbiology

– Carbapenemase-producing Enterobacteriaceae
By Dr. Andy YY CHEUNG

cheungyy@hkmu.edu.hk
After this lecture, you will be able to:
• List current CLSI recommendations for antimicrobial
susceptibility testing (AST) and reporting of Carbapenem-
resistant Enterobacteriaceae (CRE)
• Describe the differences between CRE and Carbapenemase-
producing Enterobacteriaceae (CPE)
• List and compare old and current phenotypic detection methods
for CPE
• Explain the significance of CRE and CPE from clinical and
epidemiological perspectives
Acronyms
• CRE = Carbapenem-Resistant Enterobacterales /
Enterobacteriaceae
• CPE = Carbapenemase-Producing Enterobacterales
/ Enterobacteriaceae
• GNR = Gram-negative rod
As of 2021,
• the order Enterobacterales contains 7 validly published families
(Budviciaceae, Enterobacteriaceae, Erwiniaceae, Hafniaceae, Morgan
ellaceae, Pectobacteriaceae and Yersiniaceae)
β-Lactams
• Beta-lactams are antibiotics that have a beta-lactam ring nucleus
• Subclasses include
• Carbapenems
• Cephalosporins and cephamycins (cephems)
• Clavams
• Monobactams
• Penicillins (Penicillin)
• All β-lactams contain a 4-membered β-lactam ring that is essential

for activity
• All beta-lactams bind to and inactivate enzymes required for bacterial
cell wall synthesis
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
Different β-Lactams
Penicillins Cephalosporins Cephamycins
Carbapenems Monobactam
β-Lactams against Gram-Negative Rods (GNR)
Inhibitor
Penicillins Cephalosporins Cephamycin Monobactam Carbapenems
Combinations
Amoxicillin-
Amoxicillin Cefazolin (1) Cefoxitin Aztreonam Doripenem
clavulanic acid
Ampicillin-
Ampicillin Cefuroxime (2) Cefotetan Ertapenem
sulbactam
Piperacillin-
Piperacillin Cefotaxime (3) Imipenem
tazobactam
Ticarcillin-
Ticarcillin Ceftazidime (3) Meropenem
clavulanic acid
Ceftolozane-
Ceftriaxone (3)
tazobactam
Ceftazidime-
Cefepime (4)
avibactam
ß-Lactams: Mechanisms of Action
• β-Lactam antibiotics are bactericidal agents that interrupt bacterial
cell-wall formation as a result of covalent binding to essential
penicillin-binding proteins (PBPs), enzymes that are involved in the
terminal steps of peptidoglycan cross-linking in both Gram-negative
and Gram-positive bacteria
ß-Lactams: Mechanisms of Resistance
• β-Lactamases
• Loss of porin channel or decreased expression of porin
• Efflux pump altered
• Altered PBPs
ß-Lactams: Mechanisms of Action and
Resistance
Intro to Bacteria & Antibiotics: Beta Lactams Pt 1
• https://www.youtube.com/watch?v=oBmVHRuhm3A
ß-Lactams: Mechanisms of Action and Resistance
• https://www.youtube.com/watch?v=qBdYnRhdWcQ
Mechanisms of Resistance in Gram negative Bacteria to Beta Lactam
Antibiotics
https://www.youtube.com/watch?v=xAqceL9A-Bs
Penicillin and Beta lactam Antibiotics | Mechanism of Action and
Resistance | Antibacterial spectrum
• https://www.youtube.com/watch?v=rmWR6xEIsQI
β-Lactamases Produced by Gram-negatives
• Enzymes that hydrolyze the β-lactam ring, inactivating the β-lactam
• Hundreds of different types including:
• Penicillinases
• ESBLs
• Carbapenemases
• AmpCs
• Selectively inactivate various β-lactam antimicrobial agents
Phenotypic detection of β-lactamases
Which? Where?
ESBL
AmpC Enterobacteriaceae, A. baumannii, P. aeruginosa
Carbapenemases
Why? Legislation: mandatory reporting

Definition of "multidrug-resistance"
In-house surveillance
Outbreak investigations
Epidemiology national/international
Clinical relevance and therapeutic options

Pfeifer,
Yvonne Y. (2014, September 24). Interpretation of ESBL and carbapenemase
Pfeifer phenotypes
EURL-AR Training Course [Powerpoint slides].
2014, Copenhagen 12
Extended-Spectrum β-Lactamases (ESBL )
1960 Usage penicillins; cephalosporins 1st/2nd generation

ampicillin, mezlocillin, cefotiam
→ 1965: Detection of β-lactamases (penicillinases)
TEM-1 und SHV-1 in E. coli and K. pneumoniae
1980 Usage “new” cephalosporins 3rd generation

cefotaxime, ceftazidime, cefpodoxime
→ 1983: Detection of cephalosporinases
Extended-Spectrum Beta-Lactamases
TEM-ESBL n > 100 variants
SHV-ESBL n > 60 variants “classic” ESBL in
CTX-M-ESBL n > 130 variants Enterobacteriaceae
PER-ESBL
GES-ESBL rare in Enterobacteriaceae A. baumannii,
VEB-ESBL P. aeruginosa
OXA-ESBL
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 13
β-lactamases class C = AmpC
Chromosomal-encoded Plasmid-encoded
AmpC β-lactamases AmpC β-lactamases
● E. cloacae CMY FOX

ACC LAT
● Citrobacter freundii ACT MOX
● Hafnia alvei
● Morganella morganii • Horizontal gene transfer of formerly
chromosomal ampC
● Aeromonas hydrophila • Acquired by E. coli, K. pneumoniae,
● E. coli Proteus mirabilis
Modifications in regulatory mechanisms AmpC overexpression due to a promotor of an

result in ampC overexpression insertion sequence upstream of the gene
→ Resistance to penicillins and → Resistance to penicillins und

cephalosporins cephalosporins
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 14
Diagnostics: ESBL and AmpC β-lactamases
Phenotype: Resistance to penicillins (ampicillin)
1st, 3rd, 4th generation cephalosporins (cefotaxime,
cefpodoxime, ceftazidime, cefepime)
Resistance to monobactams (aztreonam)
Susceptible to cephamycins (cefoxitin) and carbapenems
(imipenem, meropenem, ertapenem)
ESBL inhibitors: clavulanic acid, sulbactam, tazobactam
Phenotype: Resistance to penicillins, 1st, 3rd gen. cephalosporins and

cephamycins (cefoxitin, cefotetan)
AmpC Susceptible to carbapenems
Stable to ESBL inhibitors (clavulanic acid)
AmpC inhibitor: cloxacillin
Screening: Chromogenic agar for selection of "ESBL-producing" bacteria:

with cefotaxime (1mg/L) or cefpodoxime (4mg/L)
+/- cloxacillin
Brilliance ESBL Agar (Oxoid); ChromID ESBL (Biomerieux),
Chromagar ESBL (MAST)
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 15
Diagnostics: ESBL and AmpC β-lactamases
• AST by automated systems:
• "ESBL-positive” → cefotaxime- and/or ceftazidime-resistant and inhibition by an ESBL

inhibitor (clavulanic acid)
• "AmpC-positive" → "high-level-cephalosporinase" or "cephamycinase"

• cefotaxime- and/or ceftazidime- and/or cefoxitin-resistant
• No inhibition by an ESBL inhibitor (clavulanic acid)
ESBL/AmpC confirmation tests

Etest ESBL → cefotaxime/ceftazidime/cefepime + clavulanic acid
Etest AmpC → cefotetan/cefoxitin + cloxacillin
Disk-tests ESBL/AMPC → Combined-Disk-Tests (CDT)
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 16
Etest: ESBL confirmation
CT cefotaxime
CTL cefotaxime + clavulanic acid
TZ ceftazidime
TZL ceftazidime + clavulanic acid
K. pneumoniae ESBL-type CTX-M-15
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 17
Combined Disk Test: ESBL+AmpC confirmation
A cefpodoxime C. cefpodoxime + AmpC inhibitor
B cefpodoxime + ESBL inhibitor D. cefpodoxime + ESBL- and AmpC inhibitor
ESBL-negative ESBL-positive AmpC-positive ESBL-positive

AmpC-negative AmpC-positive
Limitations: Low -lactamase production → false-negative results

Production of other -lactamases than ESBL/AmpC:
→ false positive/no results
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 18
Limitations CDT: ESBL + AmpC confirmation
ESBL or AmpC or…?
resistant subpopulation? “multidrug-resistant”?
K. pneumoniae CTX-M-15 + OXA-48 K. pneumoniae KPC-2

Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 19
Mechanism of Action of Carbapenemase
Carbapenemases
Hydrolyzed
Carbapenem β-Lactam
(Carbapenem)
Mechanisms of carbapenem resistance in GNR
Efflux-pumps Inner membrane
Periplasmic space
Outer membrane
Active transport of antibiotics outwards;

common in Pseudomonas aeruginosa
Loss of porins
Porins = outer menbrane proteins (OMPs)
Mutations in different porin genes lead to loss of porins → loss of
permeability of the cell wall
Porin loss + ESBL/AmpC production → carbapenem resistance (ETP, MPM)
Common in Enterobacter aerogenes, K. pneumoniae
Carbapenemase production Beta-lactamase
Hydrolysis of carbapenems
by specific beta-lactamases
= carbapenemases
Outer membrane Periplasmic space
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 21
Concepts Related to AST
• When an enzyme produced by a bacterium hydrolyzes an
antimicrobial agent, the enzyme inactivates that agent
• Low level activity of an enzyme implies that the MICs are reduced to a
lesser extent than occurs with enzymes that exhibit higher level
activity
• MICs may be in the resistant, intermediate or even the high end of
the susceptible range
• Carbapenemases can exhibit low level or higher-level activity
Carbapenem-Resistant Enterobacteriaceae
• Two mechanisms may result in carbapenem MICs or zone diameters
in the “I” or “R” range among Enterobacteriaceae
• Carbapenemase production
• Cephalosporinase or ESBL together with porin loss
• Some AmpC -lactamases and ESBLs have low-level carbapenem-
hydrolyzing activity
• Porin loss limits entry of the carbapenem into the cell
GNR β-Lactamases (Non-carbapenemase)
Class Examples Produced by: Notes
Most inhibited by β-lactamase inhibitors

K. pneumoniae and
ESBLs [TEM, SHV,
other Usually plasmid-mediated; Can confer
A CTX-M]
Enterobacteriaceae carbapenem resistance if other “R”
mechanisms are present (e.g., porin
modification)
B --- --- ---

Inducible in some genera
Enterobacteriaceae and some (SPACE/SPICE organisms); Not
C AmpC
non-fermenters inhibited by clavulanic acid
D --- --- ---

“SPICE” often stands for the following bacterial species: Serratia spp, Providencia spp, indole-positive Proteae (e.g. Proteus
spp. [not mirabilis], Morganella spp., Providencia spp.), Citrobacter spp., and Enterobacter spp. Some have also included Pseudomonas
spp (“P”).
GNR Carbapenemases
Class Examples Produced by: Notes
K. pneumoniae and other

Serine carbapenemases: Enterobacteriaceae Usually plasmid-
A
KPC, SME, IMI, NMC, GES mediated (not SME)
S. marcescens
P. aeruginosa Inhibited by EDTA

MBL carbapenemases:
Enterobacteriaceae
B e.g. NDM, VIM, IMP, GIM,
Acinetobacter Do not hydrolyze
SPM
S. maltophilia aztreonam
C --- --- ---

Acinetobacter baumannii
Weakly hydrolyze
D OXA carbapenemases Pseudomonas
carbapenems
Enterobacteriaceae
Most Common Carbapenemases- KPC
KPC (Klebsiella pneumoniae Carbapenemase)
• Most common carbapenemase in USA
• First report 1996 from North Carolina
• Usually, a high level of enzyme can be produced
• Mostly K. pneumoniae, also K. oxytoca, E. coli, C. freundii,
Enterobacter spp., Salmonella, Serratia spp., P. aeruginosa and other
GNRs
• Plasmid with KPC gene generally has other resistant genes including
genes for ESBLs
Most Common Carbapenemases- MBL
• Metallo β-Lactamase (MBL) Carbapenemase
• NDM (New Delhi MBL) is the most common MBL worldwide;
frequently encountered in India and Pakistan
• First report 2008 in a Swedish patient who was hospitalized in India
• Called MBL because zinc is required for activity
• Mostly K. pneumoniae and E. coli
• blaNDM gene is highly mobile
• Also includes blaIMP, blaVIM
OXA Carbapenemase
• First described in Acinetobacter baumannii in 1985
• OXA-48 Commonly found in Europe and Africa; relatively rare in USA
• First reported in 2008 in Turkey
• Mostly K. pneumoniae, E. coli
• Many OXA-48-like variants described to date (OXA-181, OXA-232)
• Weakly hydrolyze carbapenems and cephalosporins (OXA-48 has
greater hydrolytic properties than some other OXAs against
carbapenems)
Carbapenemases
KPC → "Klebsiella pneumoniae Carbapenemase" mainly in K. pneumoniae
Common in „endemic areas“ (e.g. Greece, Israel, Italy)
OXA-48 → in Enterobacteriaceae; common in Turkey, North Africa, India
VIM → "Verona Integron-borne Metallo-beta-lactamase" in
Enterobacteriaceae and P. aeruginosa
common in Mediterranean countries (Italy, Greece)
NDM → "New-Delhi Metallo-Beta-Lactamase" in Enterobacteriaceae
and A. baumannii from India, North Africa, Balkan states
IMP → rare in E. cloacae, K. pneumoniae; more common in P. aeruginosa
metallo-beta-
GIM → "German Imipenemase" single cases in E. cloacae, lactamases
S. marcescens, P. aeruginosa, A. pittii (MBL)
AIM → "Adelaide Imipenemase" single cases in P. aeruginosa

FIM → "Florence Imipenemase" single cases in P. aeruginosa
DIM → "Dutch Imipenemase" single cases in Pseudomonas spp.
SIM → "Seoul Imipenemase" single cases in A. baumannii
SPM → "São Paulo metallo-β-lactamase" single cases in P. aeruginosa
OXA-58/23/24 → common in A. baumannii
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 29
Number of carbapenemase-
producing Enterobacteriaceae confirmed at the Public
Health Laboratory Services Branch, CHP, 2009 to 2016
• A HK wide surveillance was implemented since the last quarter of 2010
https://impact.chp.gov.hk/chapters_1_5.php
Longitudinal Genomic Characterization of Carbapenemase-
producing Enterobacteriaceae Reveals Changing Pattern of CPE
Isolated in Hong Kong Hospitals
Zhu, C., Li, C., Lai, C. K., Ng, R., Chau, K. Y., Wong, K. T., ... & Margaret, I. P. (2021). Longitudinal genomic
characterization of carbapenemase-producing Enterobacteriaceae (CPE) reveals changing pattern of CPE Isolated
in Hong Kong Hospitals. International Journal of Antimicrobial Agents, 58(5), 106430.
Longitudinal Genomic Characterization of Carbapenemase-
producing Enterobacteriaceae Reveals Changing Pattern of CPE
Isolated in Hong Kong Hospitals
Zhu, C., Li, C., Lai, C. K., Ng, R., Chau, K. Y., Wong, K. T., ... & Margaret, I. P. (2021). Longitudinal genomic
characterization of carbapenemase-producing Enterobacteriaceae (CPE) reveals changing pattern of CPE Isolated
in Hong Kong Hospitals. International Journal of Antimicrobial Agents, 58(5), 106430.
Where can I find information about
Carbapenem breakpoint in Enterobacteriaceae?
CLSI. Performance Standards for Antimicrobial Susceptibility

Testing, 33rd Edition. CLSI supplementary M100. Clinical and
Laboratory Institute; 2023
https://clsi.org/all-free-resources/
3
Interpreting Carbapenem Results
- Enterobacteriaceae
CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 33rd Edition. CLSI
supplementary M100. Clinical and Laboratory Institute; 2023
CLSI Carbapenem Dosage Comment
• “Because of limited treatment options for infections caused by
organisms with carbapenem MICs or zone diameters in the
intermediate range, clinicians may wish to design carbapenem
dosage regimens that use maximum recommended doses and
possibly prolonged intravenous infusion regimens as has been
reported in the literature.”
• Maintain higher antimicrobial agent levels in patients for longer
periods of time.
CLSI- testing and reporting tier
Testing for the Enzymes- Carbapenemase
Do we need to determine if a
carbapenemase is present in CRE?
⚫ Patient care
⚫ No, when using the “current” CLSI breakpoints
⚫ Infection control
⚫ Yes, if outbreak suspected; possibly in other settings
⚫ Epidemiology / research
⚫ Yes, to better understand emerging resistance and
⚫ plan for “challenges”
⚫ Some say MUST identify resistance mechanism in all CRE as carbapenemase-

producers are more worrisome than noncarbapenemase-producing CRE
Referral Form for Carbapenemase-Producing
Enterobacteriaceae Isolates (except Proteus,
Providencia and Morganella spp.)
Diagnostics: Carbapenemases
Phenotype: Resistance to penicillins, 1st-4th gen. cephalosporins and
Carbapenemase carbapenems
producer
Metallo-beta-lactamases (MBL) → susceptible to aztreonam
OXA-48 carbapenemases → susceptible to 3rd gen. cephalosporins
Stable to ESBL inhibitors (clavulanic acid)
Carbapenemase inhibitors: EDTA (MBL); boric acid (KPC)
Screening:
Chromogenic agar for selection of „Carbapenemase-producing“ bacteria:
with Meropenem oder Ertapenem (Girlich et al., 2013, Diagn. Microbiol. Infect. Dis.)
Brilliance CRE Agar (Oxoid)
ChromID Carba (Biomerieux)
Chromagar KPC (MAST)
SUPERCARBA medium (Nordmann P.,et al., JCM, 2012)
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 40
Diagnostics: Carbapenemases
AST by automated systems:
„Carbapenemase producer (KPC or MBL) or loss of permeability + ESBL/high-
level-cephalosporinase“
→ resistant/intermediate-resistant to imipenem/meropemen/
ertapenem
→ Manual confirmation tests is needed!
Carbapenemase confirmation tests:
Carbapenemase production
→ mod. Hodge Test,
→ imipenem/meropenem-hydrolysis (Carba NP, MALDI)
Etest MBL → imipenem + imipenem/EDTA
Disk-Tests → CDT with carbapenemase inhibitors
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 41
chromID® CARBA SMART
Study: Screening (rectal swabs) of patients
with KPC-producer colonisation
KPC OXA-48
KPC/MBL OXA-48
selective selective
Red = KPC-2 E. coli

Blue = KPC-2 K. pneumoniae
Grey = KPC-2 C. freundii (outbreak strain)
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 42
Tests for Carbapenemases in
Enterobacteriaceae (Old methods)
Combined disc test method / Etest:
Modified Hodge test
Double Disk Synergy Test Metallo-beta-lactamases (MBL)
Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae

Use P. aeruginosa P. aeruginosa
Acinetobacter Acinetobacter
Strengths Simple Simple Simple
Some false positive results Special “fresh” boric acid and EDTA MBL producer only
(e.g., ESBL/AmpC + porin) reagents
Some false negative Some false positive results due to
Limitations
results (e.g., NDM) EDTA
Negative result for OXA-type
Enterobacteriaceae only
carbapenemase
Modified Hodge Test for Carbapenemases
M100S 26th ed. Table 3B.

Modified Hodge Test for Carbapenemases
• https://www.youtube.com/watch?v=YCNHLCYihJg&t=315s
• https://www.youtube.com/watch?v=HBG6_sobcQQ
• https://www.youtube.com/watch?v=EleJS6pq5U4
Modified Hodge Test
Mod. Hodge-Test
Disks:
Imipenem
Meropenem
Ertapenem
K. pneumoniae
K. pneumoniae carbapenem-susceptible
carbapenemase
producer
E. coli – reference strain
carbapenem-susceptible
K. pneumoniae
carbapenem-resistant but no
carbapenemase production
- Confirmation of any carbapenamase production (OXA-48, KPC, MBL)

- reliable for outbreak investigations
- low specifity - false positive results (e.g. AmpC producer) are common
Girlich, D., Poirel, L., Nordmann, P., 2012. Value of the modified Hodge test for detection of emerging
carbapenemases in Enterobacteriacae. J. Clin. Microbiol. 50, 477-479.
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 46
Modified Hodge Test - Examples
Neg Control Pos Control NDM OXA 232 SME

KPC False neg Positive Positive
Modified Hodge Test False Positive
Pos Control
AmpC + porin loss

Neg Control
Neg Control
Combined Disk Test:
Carbapenemase confirmation
MRP Meropenem MR+BO Meropenem + KPC inhibitor
MR+DP Meropenem + MBL inhibitor MR+CL Meropenem + AmpC inhibitor
KPC-positive MBL-positive (e.g. VIM, NDM)

Boric acid derivate = KPC inhibitor EDTA derivate = MBL inhibitor
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 49
Limitations CDT: Carbapenemase confirmation
MRP Meropenem MR+BO Meropenem + KPC inhibitor
MR+DP Meropenem + MBL inhibitor MR+CL Meropenem + AmpC inhibitor
Carbapenemase or no carbapenemase?
P. aeruginosa carbapenem resistant K. pneumoniae carbapenem resistant
Mod. Hodge-Test positive
P. aeruginosa without carbapenemase K. pneumoniae CTX-M-15 + OXA-48

Pfeifer, Y. (2014, September 24). Interpretation of ESBL and carbapenemase phenotypes [Powerpoint slides].
Double Disk Synergy Test
Rao, M. J., Harle, S. A., Ravi, J., Padmavathy, M., Umapathy, B. L., & Navaneeth, B. V. (2014). Metallo beta lactamase mediated resistance in carbapenem resistant gram-negative bacilli: A cause
for concern. International Journal of Medical Research & Health Sciences, 3(2), 263-268.
Problem of EDTA
Lee, K., Lim, Y. S., Yong, D., Yum, J. H., & Chong, Y. (2003). Evaluation of the Hodge test and the imipenem-EDTA double-disk synergy test for differentiating
metallo-β-lactamase-producing isolates of Pseudomonas spp. and Acinetobacter spp. Journal of clinical microbiology, 41(10), 4623-4629.
Etest: Metallo-beta-lactamases (MBL)
K. pneumoniae VIM-1 E. coli NDM-1
Imipenem
Deformation of ellipse/phantom zone
Imipenem + EDTA
(MBL inhibitor)
Metallo-β-lactamase-positive
Pfeifer,
Pfeifer phenotypes
2014, Copenhagen 53
Limitations: Etest MBL and P. aeruginosa
A) B) C)
Resistant
subpopulation
Phantom zone
P. aeruginosa P. aeruginosa P. aeruginosa

MBL-suspicious MBL-suspicious MBL-positive
Limitations: Etest MBL and Nonfermenter
IP Imipenem
IPI Imipenem+EDTA
General growth inhibition due to EDTA
independently from MBL-production
The imipenem/EDTA inhibition zone is a

"narrow ellipse"
⚫ common in carbapenem-resistant
P. aeruginosa or A. baumannii
narrow ellipse producing OXA carbapenemases
⚫ false-positive MBL-test
P. aeruginosa without carbapenemase
Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen

Testing for the Enzymes- Carbapenemase
(current-2023)
Carba NP Test for Carbapenemase Production
⚫ Isolated colonies (lyse)

⚫ Hydrolysis of imipenem
⚫ Detected by change in pH of indicator
(red changes to yellow/orange) Neg Pos
⚫ Rapid <2h
⚫ Microtube method
Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase-producing Enterobacteriaceae.

Emerg Infect Dis. 2012 ; 18:1503-7.
“To rapidly identify carbapenemase producers in

Enterobacteriaceae, we developed the Carba NP test.
The test uses isolated bacterial colonies and is based
on in vitro hydrolysis of a carbapenem, imipenem.“
"It was 100% sensitive and specific compared

with molecular-based techniques. “
“This rapid (<2 hours), inexpensive technique

may be implemented in any laboratory."
Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase-producing Enterobacteriaceae.

Emerg Infect Dis. 2012 ; 18:1503-7.
Carba NP Test - Examples
Blank Neg KPC OXA48 OXA181 NDM IMP VIM SME

Pos Invalid Neg Pos Pos Pos Pos
• https://www.youtube.com/watch?v=SjpfzB2bO3U
Blue Carba Test
Advantages:
• direct use of colonies (instead of bacterial extracts
that need the extraction buffer (B-PER II);
• reduced cost per reaction due to use of Tienam
( 10× cheaper than an imipenem monohydrate formula)
J. Pires, Â. Novais, and L. Peixe Blue-Carba, an Easy Biochemical Test for Detection of Diverse Carbapenemase Producers Directly from Bacterial
Cultures J Clin Microbiol. Dec 2013; 51(12): 4281–4283
Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 61

Blue Carba Test
1. The Carba NP method relies on the detection in a bacterial extract of hydrolysis of the carbapenem β-
lactam ring through the acidification of a phenol red solution used as color indicator
2. In the Blue-Carba test variant, bromothymol blue was selected as the indicator, since it includes the
optimal pH range (6.0 to 7.6) for most β-lactamases (pH = 6.8), which was a key factor for a direct colony
approach
3. A commercially and widely available imipenem (Tienam 500; Merck Sharp & Dohme, France) was used as
the substrate for carbapenemases
4. The test solution consisted of an aqueous solution of bromothymol blue at 0.04% (Merck Millipore,
Germany) adjusted to pH 6.0, 0.1 mmol/liter ZnSO4, and 3 mg/ml of imipenem, with a final pH of 7.0
5. A negative-control solution (0.04% bromothymol blue solution, pH 7.0) was prepared to control the
influence of bacterial components or products in the pH of the solution
6. A loop (approximately 5 μl) of a pure bacterial culture recovered from Mueller-Hinton agar was directly
suspended in 100 μl of both test and negative-control solutions in a 96-well microtiter plate and
incubated at 37°C with agitation (150 rpm) for 2 h
7. Carbapenemase activity was revealed when the test and negative-control solutions, respectively, were (i)
yellow versus blue, (ii) yellow versus green, or (iii) green versus blue
8. Noncarbapenemase producers remained blue or green on both solutions
J. Pires, Â. Novais, and L. Peixe Blue-Carba, an Easy Biochemical Test for Detection of Diverse Carbapenemase Producers Directly from Bacterial
Cultures J Clin Microbiol. Dec 2013; 51(12): 4281–4283
Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 62

CIM Test – for Carbapenemase
(Carbapenem Inactivation Method)
⚫ Principle:
• A suspension of bacteria is incubated
with a standard meropenem disk
• If the organism produces carbapenemase, it
will inactivate the meropenem in the disk
• Following 2-hour incubation, the meropenem
disk is removed from the suspension and placed
on a lawn of E. coli ATCC 25922 #1 #2
• Following overnight incubation,
carbapenemase activity is demonstrated by a
loss of meropenem activity in the disk.
#1 carbapenemase positive
#2 carbapenemase negative
van der Zwaluw K, et al. 2015. PLoS One. 10(3):e0123690

Modified CIM Test – for Carbapenemase
• https://www.youtube.com/watch?v=pPBZ62n3sqY
EDTA-Modified Carbapenem Inactivation Method
A phenotypic method for
Detecting metallo-β-lactamase-producing Enterobacteriaceae
1. A 1-µl loopful of organism was resuspended in a 2-ml tube

of tryptic soy broth (TSB)
2. Another 1-µl loopful of organism was resuspended in a 2-ml
tube of TSB supplemented with EDTA at a final
concentration of 5 mM EDTA
3. A meropenem disk was placed in each tube (disks were
submerged using a 10-µl inoculation loop), and the tubes
were incubated at 35°C in ambient air without agitation for
4 h ± 15 min
4. Subsequently, the disks were removed using a 10-µl
inoculation loop and applied to Mueller-Hinton agar plates
(BD) freshly plated with a 0.5 McFarland suspension of a
carbapenem-susceptible strain (Escherichia coli ATCC 25922)
Sfeir, M. M., Hayden, J. A., Fauntleroy, K. A., Mazur, C., Johnson, J. K., Simner, P. J., ... & Westblade, L. F. (2019). EDTA-modified carbapenem inactivation method: a
phenotypic method for detecting metallo-β-lactamase-producing Enterobacteriaceae. Journal of clinical microbiology, 57(5), e01757-18.
EDTA-Modified Carbapenem Inactivation Method: a Phenotypic Method
for Detecting Metallo-β-Lactamase-Producing Enterobacteriaceae (cont’)
• An eCIM result is only recorded if the isolate is positive for

carbapenemase production (i.e., mCIM positive)
• A test isolate is positive for MBL production when the zone size
increases by ≥5 mm compared to the zone size observed for the
mCIM and is considered negative for an MBL if the increase in zone
size is ≤4 mm
Sfeir, M. M., Hayden, J. A., Fauntleroy, K. A., Mazur, C., Johnson, J. K., Simner, P. J., ... & Westblade, L. F. (2019). EDTA-modified carbapenem inactivation method: a
phenotypic method for detecting metallo-β-lactamase-producing Enterobacteriaceae. Journal of clinical microbiology, 57(5), e01757-18.
Molecular Tests for Carbapenemases
➢ Biofire F
– KPC, NDM, OXA, IMP, VIM
➢ Nanosphere F
➢ Cepheid (Xpert® Carba-R) F
– KPC, NDM, OXA-48, IMP-1, VIM
➢ BD MAX Check-Points CPO assay F
F FDA-cleared
Carbapenem hydrolysis + confirmation with
UV-Spectrophotometry
• Measuring imipenem hydrolysis by UV spectrophotometry requires technically challenging and

time-consuming incubation and protein extraction. (Kruse et al. 2013)
Carbapenem hydrolysis + confirmation with
MALDI-TOF
Burckhardt, I., & Zimmermann, S. (2011). Using matrix-assisted laser

desorption ionization-time of flight mass spectrometry to detect
carbapenem resistance within 1 to 2.5 hours. Journal of clinical
microbiology, 49(9), 3321-3324.
Hrabák, J., Walková, R., Študentová, V., Chudáčková, E., & Bergerová, T. (2011). Carbapenemase
activity detection by matrix-assisted laser desorption ionization-time of flight mass
spectrometry. Journal of clinical microbiology, 49(9), 3222-3227.
NG-Test® CARBA-5 (immunochromatographic assay)
NG-Test® CARBA-5 is a visual

multiplex immunochromatographic
(lateral flow) qualitative assay for the
detection and differentiation of the
five most common carbapenemase
families (KPC, OXA-48-like, VIM, IMP
and NDM) from carbapenem non-
susceptible pure bacterial colonies
of Enterobacterales and Pseudomon
as aeruginosa
https://hardydiagnostics.com/industry_content/ngtestcarba5/

SCI 8008SEF Medical Microbiology & Virology II - Lecture 10 - OLE

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SCI 8008SEF Medical Microbiology & Virology II - Lecture 10 - OLE

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Advanced laboratory technologies in Clinical Microbiology

By Dr. Andy YY CHEUNG

• All β-lactams contain a 4-membered β-lactam ring that is essential

Penicillins Cephalosporins Cephamycins

Why? Legislation: mandatory reporting

Clinical relevance and therapeutic options

1960 Usage penicillins; cephalosporins 1st/2nd generation

1980 Usage “new” cephalosporins 3rd generation

● E. cloacae CMY FOX

Modifications in regulatory mechanisms AmpC overexpression due to a promotor of an

→ Resistance to penicillins and → Resistance to penicillins und

Phenotype: Resistance to penicillins, 1st, 3rd gen. cephalosporins and

Screening: Chromogenic agar for selection of "ESBL-producing" bacteria:

• "ESBL-positive” → cefotaxime- and/or ceftazidime-resistant and inhibition by an ESBL

• "AmpC-positive" → "high-level-cephalosporinase" or "cephamycinase"

ESBL/AmpC confirmation tests

ESBL-negative ESBL-positive AmpC-positive ESBL-positive

Limitations: Low -lactamase production → false-negative results

K. pneumoniae CTX-M-15 + OXA-48 K. pneumoniae KPC-2

Active transport of antibiotics outwards;

Carbapenemase production Beta-lactamase

Most inhibited by β-lactamase inhibitors

B --- --- ---

D --- --- ---

K. pneumoniae and other

P. aeruginosa Inhibited by EDTA

C --- --- ---

AIM → "Adelaide Imipenemase" single cases in P. aeruginosa

CLSI. Performance Standards for Antimicrobial Susceptibility

⚫ Some say MUST identify resistance mechanism in all CRE as carbapenemase-

Carbapenemase inhibitors: EDTA (MBL); boric acid (KPC)

Carbapenemase confirmation tests:

Etest MBL → imipenem + imipenem/EDTA

Disk-Tests → CDT with carbapenemase inhibitors

Red = KPC-2 E. coli

Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae

M100S 26th ed. Table 3B.

- Confirmation of any carbapenamase production (OXA-48, KPC, MBL)

Neg Control Pos Control NDM OXA 232 SME

AmpC + porin loss

KPC-positive MBL-positive (e.g. VIM, NDM)

P. aeruginosa without carbapenemase K. pneumoniae CTX-M-15 + OXA-48

Deformation of ellipse/phantom zone

P. aeruginosa P. aeruginosa P. aeruginosa

The imipenem/EDTA inhibition zone is a

P. aeruginosa without carbapenemase

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen

⚫ Isolated colonies (lyse)

Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase-producing Enterobacteriaceae.

“To rapidly identify carbapenemase producers in

"It was 100% sensitive and specific compared

“This rapid (<2 hours), inexpensive technique

Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase-producing Enterobacteriaceae.

Blank Neg KPC OXA48 OXA181 NDM IMP VIM SME

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 61

Yvonne Pfeifer EURL-AR Training Course 2014, Copenhagen 62

van der Zwaluw K, et al. 2015. PLoS One. 10(3):e0123690

1. A 1-µl loopful of organism was resuspended in a 2-ml tube

• An eCIM result is only recorded if the isolate is positive for

• Measuring imipenem hydrolysis by UV spectrophotometry requires technically challenging and

Burckhardt, I., & Zimmermann, S. (2011). Using matrix-assisted laser

NG-Test® CARBA-5 is a visual