Beta-Lactam Resistance in Gram Negative Bacilli Flashcards
Beta-Lactam Antibiotics: Mechanism of Action
• Penicillin binding proteins (PBPs) are responsible for assembly, maintenance, and regulation of peptidoglycan (cell wall) metabolism • Beta-lactams bind and inhibit PBPs • Disruption of peptidoglycan synthesis • Bactericidal
Beta-lactam antibiotics
• Penicillins – Benzylpenicillin (natural) – Ampicillin (extended spectrum) – Piperacillin (Ureidopenicillin) – Ticarcillin (Carboxypenicillin) • Beta-lactam/beta-lactamase inhibitors – Ampicillin/sulbactam (Unasyn) – Amoxicillin/clavulanate (Augmentin) – Ticarcillin/clavulanate (Timentin) – Piperacillin/Tazobactam (Zosyn)
B-lactam antibiotics : Cephalosporins
• First Generation cephalosporins – Cefazolin – Cephalothin – Cefadroxil • Second Generation oral antibiotics – Cefuroxime (many others) • Second Generation cephamycins – Cefoxitin – Cefotetan
Beta-lactam antibiotics : Cephalosporins continued
• Third generation cephalosporins – Cefotaxime – Ceftriaxone – Ceftazidime – Cefixime • Fourth generation cephalosporins – Cefepime • Monobactams – Aztreonam
Beta-lactam antibiotics : Carbapenems
• Carbapenems – Imipenem – Meropenem – Ertapenem – Doripenem
Resistance to Beta-Lactams
• Six “P’s” that can explain resistance to β-lactams:
– Penetration: poor access to intracellular bacteria
– Porins: prevent entry of β-lactams into bacterial cell
– Pumps: prevent accumulation of β-lactams in bacterial cell (efflux pumps)
– Penicillin-binding proteins: production of PBPs that do not bind β-lactams (or only bind certain types of beta-lactams)
– Peptidoglycan: some bacteria do not have a cell wall
– Penicillinases (β-lactamases): production of enzymes that hydrolyze β-lactams
Location of β -lactamases
• Most β -lactamases of gram-negative species are periplasmic in location.
Classification of β-lactamases
• Four ways based on: – Spectrum and preference of subsrates – Susceptibility to inhibitors – Chromosomal vs plasmid mediated – Molecular Sequence • Classification schemes include: – Jack and Richmond (1970) – Richmond and Sykes (1973) – Ambler (1980 based on molecular phylogeny) – Bush (1989, 1995) based on substrate preference
Molecular classification of β-lactamases
• Four molecular groupings: A,B,C, and D
– A,C, and D are all serine β-lactamases
– B is reserved for metallo β –lactamases (eg zinc requiring)
• There is good correlation between the Bush scheme and molecular taxonomy
Chromosomal β -lactamases:
Miscellaneous Gram-negative
• Pseudomonas aeruginosa
– Inducible, chromososmal Amp C
– Totally derepressed mutants occur at low frequency but can be selected by cephalosporin or ureido-penicillin therapy
• Stenotrophomonas maltophilia
– Produces 2 inducible, chromosomal enzymes both
regulated by the same induction system.
• One is a Class B metalloenzyme
• One is a Class A
– Between the two, the hit almost all β -lactams
Chromosomal β –lactamases–Klebsiella
• Primary Class A chromosomal enzymes
– K. pneumoniae = SHV-1
– K. oxytoca = KOXY
• All are constitutive but at levels to produce
resistance to ampicillin, amoxicillin, ticarcillin, etc
- Regardless of MIC, no unprotected penicillin should be used against Klebsiella
- Higher and broader levels of resistance in Klebsiella are usually caused by plasmid-mediated or hyperproduced B-lactamases
ESBL - Extended Spectrum Beta-Lactamase
• Class A, plasmid encoded enzymes • Derivatives (mutants) of original TEM-1 and SHV-1 -lactamases • Spreading throughout the Enterobacteriaceae • More than 200 described to date • Labile – 1st and 3rd generation cephalosporins – Ureidopenicillins • Stable – Cephamycins – Carbapenems – Cefepime** • Inhibited by Beta-lactamase inhibitors – Tazobactam – Sulbactam – Clavulanate
Extended Spectrum β-Lactamases
● Extended Spectrum β-Lactamases (ESBLs) are Ambler class A enzymes.
● Encoded on either the chromosome or mobile genetic elements.
● Inhibited by β-lactamase inhibitors e.g., clavulanic acid.
AmpC β-lactamases
• Can be encoded on chromosome (SPACE
organisms)
– Serratia, Providencia/Proteus
vulgaris/Pseudomonas, Acinetobacter,
Citrobacter freundii complex, Enterobacter
• Constitutive expression
• Penicillins, First- and Second- generation cephalosporins are hydrolyzed (and usually 3rd
-gen but R to 3rd gen not always detected in vitro)
• In Enterobacteriaceae, expression can be “upregulated” by exposure to beta-lactam antibiotics (esp carbapenems) and beta-lactamse inhibitors
• Cefepime and carbapenems are not hydrolyzed
AmpC β-lactamases
Continued
• Plasmid mediated AmpC
– Kleb, E. coli
– Clue=cefoxtin resistance in these organisms
• ESBL “skips” a generation—cephamycins are not
hydrolyzed, first and third generation cephalosporins
are usually resistant
AmpCs
● AmpCs are Ambler class C enzymes.
● Encoded on the chromosome (SPACE organisms) or mobile genetic elements.
● Expression of chromosomal-encoded enzymes can be induced.
● Inhibited by cloxacillin
ESBL vs. AmpC
• Plasmid (ESBL) vs. Chromosomal (AmpC)
• Inhibited by β-lactamase inhibitors (ESBL) vs. not
inhibited (AmpC)
• Cephamycin S (ESBL) vs. Cephamycin R (AmpC)
• Sometimes confers Cefepime R (ESBL) vs. does not confer Cefepime R (AmpC)
CRE
– Carbapenem Resistant Enterobacteriaceae
• Generic descriptor until mechanism of resistance has been established
– Exception: Proteus and Proteus-like (Proteus,
Morganella, Providencia) intrinsically have reduced susceptibility to Imipenem
CRE—CDC Definition
• Enterobacteriaceae that are non-susceptible to one of: doripenem, meropenem or
imipenem
AND
• Resistant to all of the third generation cephalosporins tested (ceftriaxone, cefotaxime, and ceftazidime)
CRE
• May be attributed to: – Acquisition of a Carbapenemase enzyme (eg. KPC, IMP, VIM, NDM-1, OXA-48) OR – ESBL and/or AmpC with a porin or efflux mutation
KPC
• Klebsiella pneumoniae Carbapenemase
• Molecular class A
– In vitro, inhibited by clavulanic acid but not by EDTA
• Confers resistance to ALL -LACTAM antibiotics in vivo
• Plasmid-encoded
– Transferable
– Can be found in all Enterobacteriaceae
– Typically other resistance genes transmitted on the same plasmid (aminoglycosides,
fluoroquinolones)
Klebsiella pneumoniae Carbapenemases
● Klebsiella pneumoniae (Kpne) carbapenemases (KPC) are Ambler class A enzymes •Can be found in all Enterobacteriaceae
KPC at Barnes
• First “arrived” at BJH in 2007
– Index patient: surgery in South America
• Approximately 150 unique patients with KPC positive isolates to date
• 2011/2012: Average 2 new patients/month
• Many of the isolates are susceptible to Gent (but
resistant to Tobra and Amikacin)
• Most isolates susceptible to colistin and tigecycline (and sometimes that is all)
• About ¼ of the isolates test susceptible to cefepime in vitro (but reported as R)
KPC at Barnes - continued
• Mostly isolated from urine and tracheal aspirate samples
• ~1% of Enterobacteriaceae
• Most patients have been in long term care facilities prior to transfer to BJH
• Small number of isolates from blood cultures, and one from CSF
• Many different species
– K. pneumoniae, K. oxytoca, E. coli, Serratia, Proteus, E.
cloacae, E. aerogenes, Citrobacter freundii complex
NDM-1 Beta-Lactamase
• New Dehli Metallo-beta-lactamase-1
• Metallo beta-lactamase: requires zinc for enzyme
function
– Similar to other metallos (IMP and VIM)
• Plasmid encoded
• Now seen world-wide
• Becoming widespread in Europe and Asia
• In the UK:
– Now the predominant mechanism of carbapenem
resistance
– Spread not clonal
NDM-1 Beta-Lactamase : Continued
• Although molecular class B, most isolates aztreonam resistant
• Typically resistant to other classes of antimicrobials
• Origin unknown (environmental source?)
• Reported in all Enterobacteriaceae, although E.
coli and Klebsiella spp. most common
• Most patients have an epi-link to India
• Many specimen types
• Both community and hospital-associated cases
Metallo-β-Lactamases
● Metallo--Lactamases (MBL) are Ambler class B enzymes (active site Zn2+ enzyme).
● Encoded on the chromosome or mobile genetic elements.
NDM-1: mobile genetic element, rare chromosomal insertions reported
● Inhibited by metal-chelating agents: e.g., dipicolinic acid and EDTA.
OXA-48
• OXA
– “Oxacillinase”
– Class D beta-lactamase
• Much variability of spectrum of Class D
• Not inhibited by beta-lactamase inhibitors
• Do not require divalent cations
• Can be chromosomal or on mobile genetic elements
OXA-48 - Continued
• Until recently, associated with Acinetobacter but not Enterobacteriaceae
• Until recently, not reported in US
– Middle East, North Africa, India, Europe
• 2013: First reports of OXA-48 in USA
– Klebsiella pneumoniae; patients with epi-links to
India and Saudi Arabia
Automated Susceptibility Testing Methods
• Vitek2
– Card with ability to test 19-25 agents (and might infer more based on expert rules)
• Growth curves are used to
determine MIC
• BD Phoenix
– 84 well system with approx. 20 antimicrobial agents
– Reading every 20 min with colorimetric growth detection (redox indicator in wells)
• Microscan
– Microbroth dilution method
Automated Systems and KPC…
• AST systems are IVDs regulated by FDA, can’t
change interpretive criteria until FDA does
• Most don’t currently have low enough dilutions to accommodate new breakpoints
• Labs that rely exclusively on automated systems for CRE detection are likely underreporting these organisms
BJH Laboratory Workflow
• No Modified Hodge!!! • Alert to isolates with reduced meropenem susceptibility – Lab developed KPC PCR assay • TaqMan • Smart Cycler – MDR panel set up
Case • Specimen: Blood • Organism: Klebsiella pneumoniae • Susceptibility profile: – Ampicillin: S – Cefazolin: S – Cefotetan: S – Ceftriaxone: S – Ceftazidime: S – Cefepime: S – Meropenem: S – Trimethoprim-Sulfamethoxazole: S
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Case
• Specimen: Blood • Organism: Klebsiella pneumoniae • Susceptibility profile: – Ampicillin: R – Cefazolin: S – Cefotetan: S – Ceftriaxone: S – Ceftazidime: S – Cefepime: S – Meropenem: S – Trimethoprim-Sulfamethoxazole: S
Case
• Specimen: Blood • Organism: Klebsiella pneumoniae • Susceptibility profile: – Ampicillin: R – Cefazolin: R – Cefotetan: S – Ceftriaxone: R – Ceftazidime: S – Cefepime: R – Meropenem: S – Trimethoprim-Sulfamethoxazole: S
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Case • Specimen: Blood • Organism: Klebsiella pneumoniae • Susceptibility profile: – Ampicillin: R – Cefazolin: R – Cefotetan: R – Ceftriaxone: R – Ceftazidime: S – Cefepime: S – Meropenem: S – Trimethoprim-Sulfamethoxazole: S
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Case • Specimen: Blood • Organism: Klebsiella pneumoniae • Susceptibility profile: – Ampicillin: R – Cefazolin: R – Cefotetan: R – Ceftriaxone: R – Ceftazidime: R – Cefepime: R – Meropenem: R – Trimethoprim-Sulfamethoxazole: S
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Case • Specimen: Urine • Organism: Enterobacter cloacae • Susceptibility profile: – Ampicillin: R – Cefazolin: R – Cefotetan: R – Ceftriaxone: R – Ceftazidime: R – Cefepime: S – Meropenem: S – Trimethoprim-Sulfamethoxazole: S
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Case • Specimen: Urine • Organism: Enterobacter cloacae • Susceptibility profile: – Ampicillin: R – Cefazolin: R – Cefotetan: R – Ceftriaxone: R – Ceftazidime: R – Cefepime: R – Meropenem: R – Trimethoprim-Sulfamethoxazole: S
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Case • Specimen: Urine • Organism: Enterobacter cloacae • Susceptibility profile: – Ampicillin: R – Cefazolin: R – Cefotetan: R – Ceftriaxone: R – Ceftazidime: R – Cefepime: S – Meropenem: R – Trimethoprim-Sulfamethoxazole: S
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Case • Specimen: Tracheal Aspirate • Organism: Stenotrophomonas maltophilia • Susceptibility profile: – Ampicillin: R – Cefazolin: R – Cefotetan: R – Ceftriaxone: R – Ceftazidime: R – Cefepime: S – Meropenem: S – Trimethoprim-Sulfamethoxazole: S
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Case • Specimen: Tracheal Aspirate • Organism: Stenotrophomonas maltophilia • Susceptibility profile: – Ampicillin: R – Cefazolin: R – Cefotetan: R – Ceftriaxone: R – Ceftazidime: R – Cefepime: R – Meropenem: R – Trimethoprim-Sulfamethoxazole: S
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Case • Specimen: Tracheal Aspirate • Organism: Stenotrophomonas maltophilia • Susceptibility profile: – Ampicillin: R – Cefazolin: R – Cefotetan: R – Ceftriaxone: R – Ceftazidime: R – Cefepime: R – Meropenem: R – Trimethoprim-Sulfamethoxazole: R
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