SCI 8008SEF Medical Microbiology & Virology II - Lecture 10 - OLE
SCI 8008SEF Medical Microbiology & Virology II - Lecture 10 - OLE
Lecture 10
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
Different β-Lactams
Carbapenems Monobactam
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
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
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
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
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
Phenotypic detection of β-lactamases
Which? Where?
ESBL
AmpC Enterobacteriaceae, A. baumannii, P. aeruginosa
Carbapenemases
In-house surveillance
Outbreak investigations
Epidemiology national/international
Pfeifer,
Yvonne Y. (2014, September 24). Interpretation of ESBL and carbapenemase
Pfeifer phenotypes
EURL-AR Training Course [Powerpoint slides].
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
Pfeifer,
Yvonne Y. (2014, September 24). Interpretation of ESBL and carbapenemase
Pfeifer phenotypes
EURL-AR Training Course [Powerpoint slides].
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,
Yvonne Y. (2014, September 24). Interpretation of ESBL and carbapenemase
Pfeifer phenotypes
EURL-AR Training Course [Powerpoint slides].
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
Carbapenemases
Hydrolyzed
Carbapenem β-Lactam
(Carbapenem)
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
Mechanisms of carbapenem resistance in GNR
Efflux-pumps Inner membrane
Periplasmic space
Outer membrane
Hydrolysis of carbapenems
by specific beta-lactamases
= carbapenemases
Outer membrane Periplasmic space
Pfeifer,
Yvonne Y. (2014, September 24). Interpretation of ESBL and carbapenemase
Pfeifer phenotypes
EURL-AR Training Course [Powerpoint slides].
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
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
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
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
GNR β-Lactamases (Non-carbapenemase)
Class Examples Produced by: Notes
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
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
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
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
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
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)
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
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)
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?
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. Performance Standards for Antimicrobial Susceptibility Testing, 33rd Edition. CLSI
supplementary M100. Clinical and Laboratory Institute; 2023
CLSI- testing and reporting tier
CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 33rd Edition. CLSI
supplementary M100. Clinical and Laboratory Institute; 2023
Testing for the Enzymes- Carbapenemase
CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 33rd Edition. CLSI
supplementary M100. Clinical and Laboratory Institute; 2023
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”
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
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)
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,
Yvonne Y. (2014, September 24). Interpretation of ESBL and carbapenemase
Pfeifer phenotypes
EURL-AR Training Course [Powerpoint slides].
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 production
→ mod. Hodge Test,
→ imipenem/meropenem-hydrolysis (Carba NP, MALDI)
Pfeifer,
Yvonne Y. (2014, September 24). Interpretation of ESBL and carbapenemase
Pfeifer phenotypes
EURL-AR Training Course [Powerpoint slides].
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
K. pneumoniae
K. pneumoniae carbapenem-susceptible
carbapenemase
producer
E. coli – reference strain
carbapenem-susceptible
K. pneumoniae
carbapenem-resistant but no
carbapenemase production
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
Modified Hodge Test False Positive
Pos Control
Neg Control
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
Combined Disk Test:
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
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
Imipenem + EDTA
(MBL inhibitor)
Metallo-β-lactamase-positive
Pfeifer,
Yvonne Y. (2014, September 24). Interpretation of ESBL and carbapenemase
Pfeifer phenotypes
EURL-AR Training Course [Powerpoint slides].
2014, Copenhagen 53
Limitations: Etest MBL and P. aeruginosa
A) B) C)
Resistant
subpopulation
Phantom zone
CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 33rd Edition. CLSI
supplementary M100. Clinical and Laboratory Institute; 2023
Carba NP Test for Carbapenemase Production
CLSI Outreach Working Group (Spring 2016). Laboratory Detection and Reporting of Carbapenem-Resistant Enterobacteriaceae (CRE) [Powerpoint slides].
Carba NP Test for Carbapenemase Production
• 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
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
• https://www.youtube.com/watch?v=pPBZ62n3sqY
EDTA-Modified Carbapenem Inactivation Method
A phenotypic method for
Detecting metallo-β-lactamase-producing Enterobacteriaceae
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’)
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
– KPC, NDM, OXA, IMP, VIM
➢ Cepheid (Xpert® Carba-R) F
– KPC, NDM, OXA-48, IMP-1, VIM
➢ BD MAX Check-Points CPO assay F
– KPC, NDM, OXA, IMP, VIM
F FDA-cleared
Carbapenem hydrolysis + confirmation with
UV-Spectrophotometry
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)
https://hardydiagnostics.com/industry_content/ngtestcarba5/