esbl - dynamics and detection
DESCRIPTION
ESBL - Dynamics and DetectionTRANSCRIPT
Dr.T.V.Rao MD 1
ESBLEXTENDED SPECTRUM BETA
LACTAMASESDYNAMICS AND DETECTION
DR.T.V.RAO
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Dr.T.V.Rao MD
SURVIVAL OF THE FITTESTResistant bacteria survive, susceptible
ones die
Mutant emergesslowly
Sensitive cellskilled by antibiotic
Mutant’s progenyoverrun
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BETA LACTAM RING
PENICILLIN
BETA LACTAM RING
CEPHALOSPORIN
BETA LACTAMASES enzymes that inactivate the beta-lactam ring
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ACTION OF A B-LACTAMASE
NO
COOH
S
HN
OCOOH
S
OH
Active penicillin
Inactive penicilloateH2O
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BASIS OF BETALACTAMSE ACTIVITYTime-delayed Growth. Beta-lactamase (red) is produced by the central colony, promoting growth of nearby, non-resistant colonies as it deactivates ampicillin (blue). Diffusion of beta lactamase through agar leads to time-delayed growth of non-resistant colonies
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B-LACTAM ANTIBIOTICSPenicillin's
•Ampicillin•Piperacillin
Beta-lactam/beta-lactamase inhibitors•Ampicillin/sulbactam•Amoxicillin/clavulanate•Ticarcillin/clavulanate•Piperacillin/Tazobactam
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PENICILLINS1st gen – strep infection (G+) Ex. Penicillin, Cloxacillin
Extended-spectrum – have broader spectrum against G- including E.Coli Ex. Amoxicillin With inhibitor would protect against some beta-lactamase producers Ex. Amoxicillin/Clav
Broad spectrum – many Enterobacteriaceae Ex Piperacillin/Tazobactam
Dr.T.V.Rao MD
Some beta-lactamases only inactivate a small number of antibiotics e.g. penicillin
Others have extended spectrum to all the penicillins and cephalosporins e.g. cefuroxime, ceftriaxone (ESBLs)
In addition may also carry resistance to other antibiotics e.g. ciprofloxacin. 8
Dr.T.V.Rao MD 9
DEFINITION OF ESBL :
Class A by Ambler or Group 2be by Bush classifications
Typically, enzymes are plasmid-mediated derived from older ß-lactamases of TEM and SHV
In early 2000s, CTX-M derived ß-lactamases are included
Dr.T.V.Rao MD
ESBL EVOLUTION
Mid 1980s
Variants of TEM and SHV
Breakdown 3rd generation cephalosporins
Mainly in hospital Klebsiella
Spread world wide
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WHAT ARE EXTENDED-SPECTRUM Β-
LACTAMASES?
ESBLs are enzymes that mediate resistance to extended-spectrum (third generation) cephalosporins (e.g., ceftazidime, cefotaxime, and ceftriaxone) and monobactams (e.g., aztreonam) but do not affect cephamycins (e.g., cefoxitin and Cefotetan) or carbapenems (e.g., meropenem or imipenem).
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AMBLER CLASSIFICATION OF Β-LACTAMASES
Active site
Nucleotide sequence
Four evolutionarily distinct molecular classes
A C D
Serine-enzymes
B
Zinc-enzymes
β-lactamases
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WHAT IS A BETA-LACTAM?
Abx• Penicillin• Cephalosporin• Monobactam• Carbapenem
Bacteriocidal
Google Images
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CEPHALOSPORINS
Willey, et al., 2008
1st
2nd
3rd
4th
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CEPHALOSPORINS-USES1st gen: strep, staph, G- including E.coli Ex. Cefazolin
2nd gen: greater spectrum against G- Ex. Cefoxitin
3rd gen: even greater activity, combat narrow-spectrum beta-lactamase producers ESBLs emerged Ex. Ceftazidime
4th gen: effective against G- bacilli expressing Xm AmpC resistant to 3rd gen Ex. Cefepime
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OTHERSMonobactams Monobactams very active against G- including E.coli Ex. Aztreonam
CarbapenemsCarbapenems have an extremely broad spectrum. Cross-reactivity with penicillins or cephalosporins Ex. Imipenam
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THE FIGHT GOES ON ..
Beta-lactam
Beta-lactamase
Beta-lactamase inhibitor
ESBLGoogle Images
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Klebsiella pneumoniae
Escherichia coli
Proteus mirabilis
Enterobacter cloacae
Non-typhoidal Salmonella (in some countries)
COMMON ESBL PRODUCERS
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THE FIGHTBETA-LACTAM
cell
PG
NO
LYSIS
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THE FIGHTBETA-LACTAMASE
cell
PG
NO
beta-lactamase
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THE FIGHTBETA-LACTAMASE
cell
PG
NHO
OH
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THE FIGHTBETA-LACTAMASE INHIBITOR
cell
PG
NO
beta-lactamase
Inhibitor
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THE FIGHTBETA-LACTAMASE INHIBITOR
cell
PG
NO
beta-lactamase
Inhibitor
LYSIS
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ESBLSEnterobacteriaceae Resistance to oxyimino-cephalosporins and Monobactams but not cephamycins and carbapenem
• Susceptible to beta-lactamase inhibitors
•GenesSHV
TEM
CTX-M
OXA
AmpCOteo, et al., 2010
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Plasmid-mediated TEM and SHV -lactamases
Ampicillin
1965
TEM-1E.coliS.paratyphi
1970s
TEM-1Reported in 28 Gm(-) sp
1983
ESBL in Europe
1988
ESBL in USA
2000
> 130 ESBLsWorldwide
Extended-spectrumCephalosporins
1963
Evolution of -Lactamases
Look and you will find ESBL
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CLASSIFICATION OF Β LACTAMASES
Richards and Sykes (1971)• substrate
Ambler (1969)• structure
Bush, Jacoby, Medeiros (1995)• Substrate; correlation with molecular structure
• 150 TEM; • 88 SHV; • 88 OXA, • 53 CTX-M; • 22 IMP; • 12 VIM + smaller number of other enzymes (http://www.lahey.o
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CLASSIFICATIONAmbler Classification
•Molecular class A – D• A
Bush-Jacoby-Medeiros Classification
•Functional group 1 – 4• 2• 2b• 2be
Paterson and Bonomo, 2005
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BETA-LACTAMASE INHIBITORSResemble β-lactam antibiotic structure
Bind to β-lactamase and protect the antibiotic from destruction
Most successful when they bind the β-lactamase irreversibly
Three important in medicine•Clavulanic acid•Sulbactam•Tazobactam
RESISTANCE AND GENETICS
AmpCHi-level
TEM ESBL
CTX-M K1
Ceftazidime R R v S
Cefotaxime R v R S
Cefoxitin R S S S
Aztreonam R v v R
Synergy + clav No +++ +++ No
Know the speciesDr.T.V.Rao MD 29
Why Test for β-lactamases ?
Improve clinical outcome Inappropriate treatment leads to poor outcome Each 1 hour delay increases mortality by 7.6% in septic
shock1
Encourage antimicrobial stewardship Spare carbapenems.. Reduce C. difficile / antibiotic associated diarhoea
Enhanced surveillance Identify emerging resistance problems Develop structures to prevent dissemination
Infection Control ‘Search and Destroy’ analogous to MRSA ?
Laboratory Detection is not always easy… OR Rapid
1Kumar, Crit Care Med, 2006
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TYPES OF ESBLS
TEM
SHV
CTX-M
OXA
Mutations
ESBL PhenotypePlasmid-mediated
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CHOICE OF INDICATOR CEPHALOSPORIN
TEM & SHV – obvious resistance to ceftazidime, variable to cefotaxime
CTX-M – obvious resistance to cefotaxime, variable to ceftazidime
All ESBLs – obvious resistance to cefpodoxime
Cefuroxime, cephalexin and cephradine are unreliable indicators
Livermore D and Woodford N HPA Guidance 2004
Slide 33
CURRENT MODERN METHODS
CLSI – Clinical Laboratory and Standards Institute
ARMRL - Antibiotic Resistance Monitoring and Reference Laboratory, Health Protection Agency Centre for Infections, London
EUCAST- European Society of Clinical Microbiology & Infectious Diseases
Commercial methods – Etest, BD Phoenix, Vitek Neo tabs & others
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DETECTION OF ESBLS
Seek ceph/clav synergy in ceph R isolates
•Double disc•Combination disc•Etest
CHALLENGES FOR THE DIAGNOSTIC LAB
Detection…. Hemophilus, Neisseria etc.
Predicting b-lactamase types. Have GNB got ?:
ESBL,AmpC
Metallo types, VIM, IMP etc…
Spotting unusual patterns; knowing what to refer ???
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DETECTION STRATEGY: STEP 1
Screen Enterobacteriaceae with :
• Cefpodoxime- best general ESBL substrate
• Cefotaxime & ceftazidime- good substrates for CTX-M & TEM/SHV, respectively
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COMBINATION DISK METHODCARTER MW ET AL: J CLIN MICROBIOL 2000; 38: 4228 - 4232
Difference > 5 mm
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KLEBSIELLA PNEUMONIA PRODUCING A HIGHER ACTIVITY ESBL
The higher level of ESBL production is indicated by the inhibition of the β‑lactamase by clavulanic acid and the resulting elliptical inhibitory zone between cefotaxime (CTX 5) and Augmentin (AMC 60).
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Double disc antagonism for inducible AmpC
Cefoxitin Ceftazidime
AMPC INDUCIBILITY- WHEN TO LOOK
Rarely!!!!!Risk is mutation, not inducibility per se
Best to identify & predict risk from species
Biggest risk Enterobacter & C freundii
Avoid cephalosporins against them
Identify means identify TO SPECIES LEVEL all Enterobacteriaceae (‘coliforms’) ex serious infections
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ESBLS DETECTION METHODS: INHIBITION BY CLAVULANIC ACID
Co-amoxiclav disc surrounded by cefotaxime, ceftriaxone, ceftazidime and aztreonam discs (30 mcg each)
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ESBL DETECTION
–Screen cefpodoxime ; cefotaxime & ceftazidime
–Synergy test with ceph/clav
Combination discs are most cost effective synergy tests; Etests a good alternative.. or automate
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ESBL Confirmatory TestPositive for ESBL
Ceftaz/CACefotax/CA
Ceftaz Cefotax
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ESBL CONFIRMATORY TEST NEGATIVE FOR ESBL
Ceftaz/CA Cefotaxime/CA
Ceftaz Cefotax
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ESBL CONFIRMATORY TEST
Ceftaz/CA CeftazEtest
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ESBLS: TIMES A’ CHANGING WITH CTX-M
Old advice- test ceftazidime; ESBL test if R
New advice- test ceftazidime & cefotaxime; ESBL test if R to either
• Alternative- test cefpodoxime; ESBL test if R
• Still true- Only testing cefuroxime is inadequate
COMPARING DISK DIFFUSION WITH MINIMUM INHIBITORY CONCENTRATIONS
Disk diffusion MICs
cefpodoxime < 22 mm cefpodoxime > 2 µg/ml
ceftazidime < 22 mm ceftazidime > 2 µg/ml
aztreonam < 27 mm aztreonam > 2 µg/ml
cefotaxime < 27 mm cefotaxime > 2 µg/ml
ceftriaxone < 25 mm ceftriaxone > 2 µg/ml
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ESBL CONFIRMATORY TESTS Double-disk synergy (DDS) test• CAZ and CAZ/CA disks• CTX and CTX\CA disks• Confirmatory testing requires using both CAZ and CTX alone and with CA
• 5 mm enhancement of the inhibition zone of antibiotic/CA combination vs antibiotic tested alone = ESBL
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SYNERGY TESTS WITH 4-GEN CEPHALOSPORINS
Cefepime/clav (Mast & AB Biodisk)
Cefpirome clav (Oxoid)
• Devt. driven by spread of clonal E. aerogenes with TEM-24 in Belgium & France
• Sensitivity for weak ESBLs remains to be proven
• Cefpirome & cefepime products need comparison
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PITFALLS IN ESBL DETECTION
•Methods optimised for E. coli & Klebsiella
•More difficult with Enterobacter
–clavulanate induces AmpC; hides ESBL
•Best advice is to do synergy test (NOT SCREEN) with 4th gen ceph
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RISK FACTORS FOR ESBL INFECTION
Length of hospital stay
Severity of illness
Time in the ICU
Intubation and mechanical ventilation
Urinary or arterial catheterization
Previous exposure to antibiotics
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BACTERIA NOT TO TEST FOR ESBL’S
Acinetobacter– Often S to
clavulanate aloneS. maltophilia
– +vet result by inhibition of L-2 chromosomal b-lactamase, ubiquitous in the species
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ESBL REPORTING RULE
The rule (CLSI =NCCLS) M100-S15)… • “Strains of Klebsiella spp. E. coli, and Proteus mirabilis
that produce ESBLs may be clinically resistant to therapy with penicillin's, cephalosporins, or aztreonam, despite apparent in vitro susceptibility to some of these agents.”
The message…• Report “confirmed” ESBL-producing strains as R to all
penicillin's, cephalosporins, and aztreonam
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WILL CLSI CONFIRMATORY TEST DETECT ALL ESBL-PRODUCING GNB?
No - some isolates have ESBLs plus other resistance mechanisms that mask ESBL detection in the confirmatory test, e.g.,
• > 1 ESBL
• ESBL + AmpC
• ESBL + porin mutation
ESBLs occur in species other than E. coli, Klebsiella spp., and Proteus mirabilis which CLSI does not currently address
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144 putative of ESBL producers
ESBL detection:
•AS: Microscan, Vitek2, Phoenix•Phenotypic tests: Etest, DDS•Molecular tests: PCR, IsoElectric Focusing (IEF)
Molecular identification: the reference method
ESBL DETECTION: AUTOMATED SYSTEMS (AS)
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THE RESISTANCE BECOMING COMPLEX
Beta-lactamases are getting more complex
Full I/D needs complex molecular methods
Much can be inferred from simple tests.
Needs I/D
Testing wide panels of antibiotics; synergy tests
Knowledge of what’s unusual
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ANTIBIOTIC POLICY CHANGES PRACTISED
Nitrofurantion substituted for quinolones in UTIs
Imipenem substituted for quinolones in serious sepsis
Ertapenem introduced for ESBL sepsis
Gentamicin substituted for cephalosporins in surgical prophylaxis
Return to amoxycillin in respiratory tract infections
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MICROBIOLOGY LABORATORIES AND ESBL’S
Unfortunately, many clinical laboratories lack of understanding regarding ESBLs and Ampc ß-lactamase and their detection .This has been documented in a study in Connecticut USA, where it was found that 21% of laboratories failed to detect extended –spectrum cephalosporins and Aztreonam in ESBLs and Ampc. The true prevalence of ESBLs is not known and is probably underestimated because of difficulties encounter in their detection. However, it is clear that ESBLs –producing organisms are distributed worldwide and their prevalence is increasing.
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HAND WASHING STILL CAN REDUCE THE ESBL SPREAD
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The programme created by Dr.T.V.Rao MD for basic understanding by Medical
Microbiologists in the Developing World Email