Extended spectrum Beta Lactamases
-
Upload
tumalapalli-venkateswara-rao -
Category
Health & Medicine
-
view
13.406 -
download
2
description
Transcript of Extended spectrum Beta Lactamases
ESBL
DETECTION
METHODSDR.T.V.RAO MD
DR.T.V.RAO MD 1
• 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).
WHAT ARE EXTENDED-SPECTRUM
Β-LACTAMASES?
DR.T.V.RAO MD 2
3
SURVIVAL OF THE FITTEST
• Resistant bacteria survive, susceptible ones die
Mutant emerges
slowly
Sensitive cells
killed by antibiotic
Mutant’s progeny
overrun
WHY SHOULD CLINICAL LABORATORY PERSONNEL BE
CONCERNED ABOUT DETECTING THESE ENZYMES?
DR.T.V.RAO MD 4
• The presence of an ESBL-producing organism in a clinical infection
can result in treatment failure if one of the above classes of drugs is
used. ESBLs can be difficult to detect because they have different
levels of activity against various cephalosporins. Thus, the choice of
which antimicrobial agents to test is critical. For example, one enzyme
may actively hydrolyze ceftazidime, resulting in ceftazidime minimum
inhibitory concentrations (MICs) of 256 µg/ml, but have poor activity
on cefotaxime, producing MICs of only 4 µg/ml. If an ESBL is
detected, all penicillin's, cephalosporins, and aztreonam should be
reported as resistant, even if in vitro test results indicate susceptibility
SLIDE 5
DEFINITION OF ESBL
BL:
• 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
ΒΒΒΒ-LACTAM ANTIBIOTICS• Penicillin's
• Ampicillin
• Piperacillin
• Beta-lactam/beta-lactamase inhibitors
• Ampicillin/sulbactam
• Amoxicillin/clavulanate
• Ticarcillin/clavulanate
• Piperacillin/TazobactamDR.T.V.RAO MD 6
Β-LACTAM ANTIBIOTICS
• First Generation cephalosporins
• Cefazolin
• Cephalothin
• Second Generation oral antibiotics
• Cefuroxime (many others)
• Second Generation cephamycins
• Cefoxitin
• CefotetanDR.T.V.RAO MD 7
8
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
Bradford PA. Clin Microbiol Rev. 2001;14:933-951.
Β-LACTAM RESISTANCE IN GRAM NEGATIVE
BACTERIA• Ampicillin resistance in Enterobacteriaceae
• Acquisition of TEM-1 β-lactamase in E. coli, SHV-1 in K. pneumonia
• Cephalosporin resistance developed by mutation of TEM and SHV
• Point mutations in TEM and SHV change structure of the enzyme
• Enables hydrolysis of cefuroxime, cephalexin, cefadroxil, cephalothin
etc…..
• Extended spectrum β-lactamases
• More TEM and SHV variants and emergence of CTX-M, VEB, PER
• Resistance to 3rd and 4th generation cephalosporins (ceftazidime,
cefotaxime)
DR.T.V.RAO MD 9
Β-LACTAM RESISTANCE IN GRAM NEGATIVE
BACTERIA
• AmpC β-lactamases
• Natural β-lactamases able to hydrolyze cephalosporins at low
level
• Mutations in regulatory genes leave to ‘derepression’ and
overexpression in Enterobacter, Serratia, Morganella spp
• Carbapenemases – resistance to cephalosporins and
carbapenems
• Acquired KPC in K. pneumoniae, Enterobacter, E.coli
• Zn dependent mettallo-enzymes (IMP, VIM) in P. aeruginosa, A.
baumanniiDR.T.V.RAO MD 10
THERE ARE MORE THAN 200 BETA-LACTAMASE TYPES IN GRAM
NEGATIVE BACILLI
• Class A: TEM-1,2; SHV-1; ESBLs, KPC
• Class B: MBLs
• Class C: AmpC
• Class D: OXA
DR.T.V.RAO MD 11
Plasmid-mediated TEM and SHV ββββ-lactamases
Ampicillin
1965
TEM-1
E.coli
S.paratyphi
1970s
TEM-1
Reported in
28 Gm(-) sp
1983
ESBL in
Europe
1988
ESBL
in USA
2000
> 130 ESBLs
Worldwide
Extended-spectrum
Cephalosporins
1963
Evolution of ββββ-Lactamases
Look and you will find ESBL
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
Plasmid-mediated TEM and SHV ββββ-lactamases
Ampicilli
n
1965
TEM-1
E.coli
S.paratyphi
1970s
TEM-1
Reported in
28 Gm(-) sp
1983
ESBL in
Europe
1988
ESBL
in USA
2000
> 130 ESBLs
Worldwide
Extended-spectrum
Cephalosporins
1963
Evolution of ββββ-Lactamases
14DR.T.V.RAO MD 14
15
AMBLER CLASSIFICATION OF Β-LACTAMASES
Active site
Nucleotide
sequence
Four evolutionarily distinct molecular classes
A C D
Serine-enzymes
B
Zinc-enzymes
β-lactamases
DR.T.V.RAO MD
16
MODIFIED BUSH–JACOBY–MEDEIROS
CCLASSIFICATION OF Β–LACTAMASES
Functional Substrate profile Group
Molecular Class
Inhibitor
Example
1 Cephalosporinase C Oxa AmpC, MIR-1
2a Penicillinase A Clav. S.aureus
2b Broad spectrum A Clav. TEM-1/2, SHV-1
2be Extended spectrum A Clav. TEM 3-29, TEM46-104 SHV2-28, CTX-M types
2br Inhibition resistant A - TEM 30-41 (IRT1-12)
2c Carbenicillinase A Clav. PSE-1
2d Oxacillinase D (Clav.) OXA-1 (OXA-2 &-10 derived ESBL)
2e Cephalosporinase A Clav. FPM-1 P. vulgaris, CepA B. fragilis.
2f Carbapenemase A Clav. IMI-1, NmcA, Sme 1-3
3 Metallo-enzyme B - S.maltophilia
4 Penicillinase - - B.cepacia
DR.T.V.RAO MD
AMPC ΒΒΒΒ-LACTAMASES
• Chromosomally encoded-cell wall turnover
• Enterobacter sp., Citrobacter sp., Serratia sp., Morganella sp. Even E. coli.
• Third-generation cephalosporins are not good inducers of AmpC β-lactamase
• Third-generation cephalosporin resistant strains are derepressed—meaning that the AmpC β-lactamase is not inducible anymore.
• AmpC mutants are cephamycin resistant
DR.T.V.RAO MD 17
AMPC ββββ-LACTAMASES
• Molecular class C, functional group 1
• Not inhibited by CA
• Confers resistance to penicillins, cephalosporins, monobactam, and cephamycin
• Chromosomally- or plasmid-mediated
• Many genera in Enterobacteriaceae encode chromosomal inducible AmpC
• Serratia marcescens
• Enterobacter cloacae
• Citrobacter freundii
• Morganella morganii
• Hafnia alvei
• Yersenia enterocolitica
• Pseudomonas aeruginosa
AMPC ββββ-LACTAMASES
• Expression of the chromosomal ampC is generally low
• Inducible in response to certain ββββ-lactams
• Factors involved in ampC induction:
• ββββ-lactam interaction with PBPs
• Byproducts of cell wall synthesis
• Gene products
• AmpR
• AmpD
• AmpG
20
CTX-M• Fast growing – important group
• Preferentially hydrolyse, and confer resistance to cefotaxime
• Escape of chromosomal ß-lactamase genes from Kluyvera spp (a bug
of no clinical importance!)
• Having migrated to mobile DNA, CTX-M ß-lactamases genes may
evolve further – undergoing mutations that increase activity against
ceftazidime
• The first CTX-M ESBL in the UK was found as recently as 2000, in a
solitary isolate of K. oxytoca
• First outbreak, caused by K. pneumoniae producing the new enzyme
CTX-M-26, was recorded in Birmingham in 2001Livermore D and Hawkey P. J Antimicrob Chemother 2005; 56: 451-454
HPA Report September 2005 www.hpa.org.uk/publications
21
CTX-M• Has supplanted TEM and SHV types as the predominant
ESBLs in the UK
• CTX-M-15 enzyme most common in UK
• 28/105 cases resulted in death in one UK PCT
• Most CTX-M-15 producing E. Coli isolates tested by HPA were multi-resistant to aminoglycosides, fluoroquinolones and trimethoprim as well as all ß-lactams, except carbapenems and temocillin
HPA Report September 2005 www.hpa.org.uk/publications
22
INCREASING NUMBERS OF ESBLS
0
10
20
30
40
50
60
70
80
2000 2001 2002 2003 2004 2005 2006 2007
# of ESBLs per year
Lewis, et al. AAC 51:4015, 2007
23
BETA-LACTAMASE INHIBITORS
• Resemble 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
DR.T.V.RAO MD
24
TYPES OF ESBLS
• TEM
• SHV
• CTX-M
• OXA
Mutations
ESBL PhenotypePlasmid-mediated
DR.T.V.RAO MD
ESBLS ARE BETA-LACTAMASES WHICH:
• Hydrolyse third generation cephalosporins
(and aztreonam, penicillins and many other
cephalosporins)
• Do not appreciably hydrolyse cephamycins
(cefoxitin or Cefotetan) or carbapenems
• Are inhibited by beta-lactamase inhibitors
such as clavulanic acid
DR.T.V.RAO MD 25
SLIDE 26
HISTORICAL PERSPECTIVES
LABORATORY DETECTION (V-1)1988
Jarlier effect – CTX with Augmentin (Jarlier V et al Rev Infect Dis 1988)
1990
NCCLS– ceftazidime zone <15mm Kirby Bauer Method for screening
1994
Synergy testing with ceftazidime (Sader HS et al Diagn Microbiol Infect Dis 1994)
LABORATORY DETECTION OF ESBL
• Phenotypic Methods
•Screening methods
•Confirmatory Methods
• Genotypic Methods
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
SLIDE 29
LABORATORY DETECTION
1996
Etest with ceftazidime and clavulanate was recommended (Cormican MG et al JCM)
1996
>50% ESBL E. coli and 29% of ESBL K. pneumoniae were resistant to cefoxitin and 10% of non-ESBL E.coli and K. pneumoniae also resistant to cefoxitin Jacoby GA & Han P JCM )
2001
Cefpodoxime recommended for screening Clin Microbiol Rev 2001
WHY DETECT ESBL PRODUCERS?
• ESBL producers may:
• Appear Sensitive to some cephalosporins s in vitro
• Show major inoculum effects
• Fail in therapy, despite appearing susceptible
DR.T.V.RAO MD 30
ESBL PRODUCERS FREQUENTLY
APPEAR SUSCEPTIBLE TO
CEPHALOSPORINS
0
10
20
30
40
50
60
70
80
<=8
(S)
>=32
(R)
Cefotax.
Ceftriax.
Ceftaz.
• Enterobacteriaceae are
traditionally reported as
susceptible to ceftazidime,
cefotaxime, ceftriaxone,
aztreonam, and cefepime
when MIC <= 8 µg/mL
32
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
• Klebsiella pneumoniae
• Escherichia coli
• Proteus mirabilis
• Enterobacter cloacae
• Non-typhoidal Salmonella
(in some countries)
COMMON ESBL PRODUCERS
DR.T.V.RAO MD 34
DIVERSITY OF ESBL’S
• Confer resistance to 1st , 2nd, 3rd cef.
• Most are susceptible to β-lactamase inhibitors
• Most are susceptible to 4th cef.
• All are susceptible to carbapenems
• Diversity of ESBL
• SHV (widespread)
• TEM (>100 types)
• OXA
• Predominantly in Pseudomonas
• less susceptible to β-lactamase inhibitors
• CTX-M
• Probably independent evolution
• Highly resistant to 3rd generation cephalosporines
• initially in South America, Far East & Eastern Europe
• Probably most frequent worldwide
DETECTION STRATEGY: STEP 1
• Screen Enterobacteriaceae with :
• Cefpodoxime- best general ESBL substrate
• Cefotaxime & ceftazidime- good substrates
for CTX-M & TEM/SHV, respectively
DR.T.V.RAO MD 36
DETECTION OF ESBLS: STEP 2
• Seek ceph/clav synergy in ceph R isolates
•Double disc
•Combination disc
•Etest
DR.T.V.RAO MD 37
COMBINATION DISK METHODCARTER MW ET AL: J CLIN MICROBIOL 2000; 38: 4228 -
4232
Difference > 5 mm
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
DR.T.V.RAO MD 40
DR.T.V.RAO MD 41
42
ESBLS DETECTION METHODS:
INHIBITION BY CLAVULANIC ACID
Co-amoxiclav disc surrounded by cefotaxime, ceftriaxone, ceftazidime and
aztreonam discs (30 mcg each)
ESBL DETECTION : COMBINATION DISCS: +VE RESULT, ZONE ENLARGED 50%
Discs (30+10 µµµµg) % Detected (n =100)
Ceftazidime +/- clav 88
Cefotaxime +/- clav 66
Both 93
M’Zali et al. 2000, JAC, 45, 881
DR.T.V.RAO MD 43
ESBL DETECTION
•2 Steps:
– Screen cefpodoxime ; cefotaxime & ceftazidime
– Synergy test with ceph/clav
•Combination discs are most cost effective synergy tests; Etests a good alternative.. or automate
•Guidelines on http//www.hpa.org.uk- type ESBL in search facility
DR.T.V.RAO MD 44
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/mlDR.T.V.RAO MD 45
Etest for ESBLs
Cefotaxime
Cefotaxime
+
clavulanateDR.T.V.RAO MD 46
ESBL Confirmatory Test
Positive for ESBL
Ceftaz/CACefotax/CA
Ceftaz Cefotax
47DR.T.V.RAO MD 47
48
ESBL CONFIRMATORY TEST NEGATIVE FOR
ESBL
Ceftaz/CA Cefotaxime/CA
Ceftaz Cefotax
48DR.T.V.RAO MD
ESBL CONFIRMATORY TEST
Ceftaz/CA CeftazEtest
49DR.T.V.RAO MD 49
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
DR.T.V.RAO MD 50
SYNERGY TESTS WITH 4-GEN CEPHS
•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
DR.T.V.RAO MD 51
CEPH R BUT SYNERGY –VE…
AmpC- plasmid or
chromosomal
S to 4 gen cephs
K1 hyperproducer
K. oxytoca
R cefuroxime, aztreonam, cefpodoxime
S ceftazidime, I to cefotaxime
May give false +ve ESBL test
Impermeable E.
coli, Kleb
R cefoxitin & cefuroxime; not ¾-gen cephs
Carbapenemase
Metallo or not
R includes imipenem & / or meropenem
DR.T.V.RAO MD 52
BACTERIA NOT TO TEST FOR ESBLS
•Acinetobacter
– Often S to clavulanate alone
•S. maltophilia
– +vet result by inhibition of L-2 chromosomal β-
lactamase, ubiquitous in the species
DR.T.V.RAO MD 53
ESBL REPORTING RULE
• The rule (CLSI =NCCLS) M100-S15)H• “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 messageH• Report “confirmed” ESBL-producing strains as R to all penicillin's,
cephalosporins, and aztreonam
54DR.T.V.RAO MD 54
WILL CLSI CONFIRMATORY TEST DETECT ALL
ESBL-PRODUCING GNR?
• No - some isolates have ESBLs plus other resistance mechanismsthat 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
55
• PCR and sequencing
• The gold standard
• Can detect all variants
• Easy to perform
• Labor intensive
MOLECULAR DETECTION OF ESBLS
• 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
• JCM. Apr. 2007, p.1167-1174
ESBL DETECTION: AUTOMATED
SYSTEMS (AS)
ESBL DETECTION: AUTOMATED
SYSTEMS
Detection
Method
Sensitivity
%
Specificity
%
PPV
%
NPV
%
MicroScan 83.5 72.9 81.6 75.4
Phoenix 98.8 52.2 75 96.6
Vitek2 85.9 78 84.9 79.3
DDS 92.9 96.6 97.5 90.5
Etest 94.1 84.7 89.9 90.9
JCM. Apr. 2007, p.1167-1174
PROBLEMATIC ORGANISMS….• ESBLA organisms with AmpC (Enterobacter, Citrobacter, Serratia)
• AmpC is induced by calvulanate
• Use cefipime in synergy tests
• ESBLCARBA
• Mettallocarbapenemases (Pseudomonas, Acinetobacter)
• Synergy with EDTA
• Hodge test
• ESBLM
• Difficult !
• Boronic acid for plasmidic AmpC
• Numerous commercial disc systems
• AmpC and ESBL inhibitors
60
MICROBIOLOGY LABORATORIES
AND ESBLS
• 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.
CARBAPENEMS - TREATMENT OF CHOICE FOR
SERIOUS INFECTIONS WITH ESBL PRODUCERS
• Carbapenems are not hydrolyzed by ESBLs to any great
extent
• Success rates with carbapenems for ESBL producers
consistently exceed 80%, and in no study has the outcome
with carbapenems been surpassed [Paterson CID 2004; Bhavnani DMID 2006; Zanetti AAC 2003]
HAND WASHING STILL CAN REDUCE THE ESBL
SPREAD
DR.T.V.RAO MD 62
DR.T.V.RAO MD 63
Created by Dr.T.V.Rao MD for ‘ ‘e-learning’ resources
on implication of misuse of Antibiotics and
consequences for Medical and Paramedical students in
Developing World