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ESBLDETECTION
METHODSDR.T.V.RAO MD
DR.T.V.RAO MD 1
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ESBLs are enzymes that
mediate resistance toextended-spectrum (third
generation) cephalosporins
(e.g., ceftazidime, cefotaxime,
and ceftriaxone) andmonobactams (e.g.,
aztreonam) but do not affect
cephamycins (e.g., cefoxitin
and Cefotetan) or
carbapenems (e.g.,
meropenem or imipenem).
WHAT ARE EXTENDED-SPECTRUM
-LACTAMASES?
DR.T.V.RAO MD 2
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3
SURVIVAL OF THE FITTEST
Resistant bacteria survive, susceptible ones die
Mutant emerges
slowly
Sensitive cells
killed by antibiotic
Mutants progeny
overrun
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WHY SHOULD CLINICAL LABORATORY PERSONNEL BECONCERNED 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 isused. 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 minimuminhibitory concentrations (MICs) of 256 g/ml, but have poor activity
on cefotaxime, producing MICs of only 4 g/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
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SLIDE 5
DEFINITION OF ESBLBL:
Class A by Ambler or Group 2be by Bushclassifications
Typically, enzymes are plasmid-mediatedderived from older -lactamases of TEM andSHV
In early 2000s, CTX-M derived -lactamasesare included
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-LACTAM ANTIBIOTICS Penicillin's
Ampicillin
Piperacillin
Beta-lactam/beta-lactamase inhibitors
Ampicillin/sulbactam
Amoxicillin/clavulanate
Ticarcillin/clavulanate
Piperacillin/TazobactamDR.T.V.RAO MD 6
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-LACTAM ANTIBIOTICS
First Generation cephalosporins Cefazolin
Cephalothin
Second Generation oral antibiotics
Cefuroxime (many others)
Second Generation cephamycins Cefoxitin
CefotetanDR.T.V.RAO MD 7
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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.
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-LACTAM RESISTANCE IN GRAM NEGATIVE
BACTERIA Ampicillin resistance in Enterobacteriaceae
Acquisition of TEM-1 -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 -lactamases
More TEM and SHV variants and emergence of CTX-M, VEB, PER
Resistance to 3rd and 4th generation cephalosporins (ceftazidime,
cefotaxime)
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-LACTAM RESISTANCE IN GRAM NEGATIVEBACTERIA
AmpC -lactamases Natural -lactamases able to hydrolyze cephalosporins at low
level
Mutations in regulatory genes leave to derepression andoverexpression 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
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THERE ARE MORE THAN 200 BETA-LACTAMASE TYPES IN GRAMNEGATIVE 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
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Plasmid-mediated TEM and SHV -lactamases
Ampicillin
1965
TEM-1E.coli
S.paratyphi
1970s
TEM-1Reported in
28 Gm(-) sp
1983
ESBL inEurope
1988
ESBLin USA
2000
> 130 ESBLsWorldwide
Extended-spectrum
Cephalosporins
1963
Evolution of-Lactamases
Look and you will find ESBL
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Plasmid-mediated TEM and SHV -lactamases
Ampicilli
n
1965
TEM-1E.coli
S.paratyphi
1970s
TEM-1Reported in
28 Gm(-) sp
1983
ESBL inEurope
1988
ESBLin USA
2000
> 130 ESBLsWorldwide
Extended-spectrum
Cephalosporins
1963
Evolution of-Lactamases
14DR.T.V.RAO MD 14
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15
AMBLER CLASSIFICATION OF -LACTAMASES
Active site
Nucleotide
sequence
Four evolutionarily distinct molecular classes
A C D
Serine-enzymes
B
Zinc-enzymes
-lactamases
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MODIFIED BUSHJACOBYMEDEIROS
CCLASSIFICATION OF LACTAMASES
Functional Substrate profileGroup
MolecularClass
Inhibitor Example
1 Cephalosporinase C Oxa AmpC, MIR-1
2a Penicillinase A Clav. S.aureus
2b Broad spectrum A Clav. TEM-1/2, SHV-12be Extended spectrum A Clav. TEM 3-29, TEM46-104 SHV2-
28, CTX-M types2br Inhibition resistant A - TEM 30-41 (IRT1-12)
2c Carbenicillinase A Clav. PSE-1
2d Oxacillinase D (Clav.) OXA-1 (OXA-2 &-10 derivedESBL)
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.maltophilia4 Penicillinase - - B.cepacia
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AMPC -LACTAMASES
Chromosomally encoded-cell wall turnover
Enterobactersp., Citrobactersp., Serratia sp.,Morganella sp. Even E. coli.
Third-generation cephalosporins are not good
inducers of AmpC -lactamase Third-generation cephalosporin resistant strains
are derepressedmeaning that the AmpC -
lactamase is not inducible anymore. AmpC mutants are cephamycin resistant
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AMPC -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
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AMPC -LACTAMASES
Expression of the chromosomal ampC is
generally low Inducible in response to certain -lactams
Factors involved in ampC induction:
-lactam interaction with PBPs
Byproducts of cell wall synthesis
Gene products
AmpR
AmpD
AmpG
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CTX-M Fast growing important group Preferentially hydrolyse, and confer resistance to cefotaxime
Escape of chromosomal -lactamase genes from Kluyvera spp (a bug
of no clinical importance!)
Having migrated to mobile DNA, CTX-M -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 ofK. 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
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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. Coliisolates tested by HPAwere multi-resistant to aminoglycosides, fluoroquinolones andtrimethoprim as well as all -lactams, exceptcarbapenems
and temocillin
HPA Report September 2005 www.hpa.org.uk/publications
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INCREASING NUMBERS OF ESBLS
0
10
20
30
40
50
60
70
80
2000 2001 2002 2003 2004 2005 2006 2007
#ofESBLsperyear
Lewis, et al. AAC 51:4015, 2007
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BETA-LACTAMASE INHIBITORS
Resemble -lactam antibiotic structure
Bind to -lactamase and protect the antibiotic fromdestruction
Most successful when they bind the -lactamase
irreversibly Three important in medicine
Clavulanic acid
Sulbactam
Tazobactam
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TYPES OF ESBLS TEM
SHV
CTX-M
OXA
Mutations
ESBL PhenotypePlasmid-mediated
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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 inhibitorssuch as clavulanic acid
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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
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LABORATORY DETECTION OF ESBL
Phenotypic Methods
Screening methods
Confirmatory Methods
Genotypic Methods
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Why Test for -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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SLIDE 29
LABORATORY DETECTION
1996
Etest with ceftazidime and clavulanate was recommended (CormicanMG et al JCM)
1996
>50% ESBL E. coliand 29% of ESBL K. pneumoniae were resistant tocefoxitin and 10% of non-ESBL E.coliand K. pneumoniae alsoresistant to cefoxitin Jacoby GA & Han P JCM)
2001
Cefpodoxime recommended for screening Clin Microbiol Rev2001
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WHY DETECT ESBL PRODUCERS?
ESBL producers may:Appear Sensitive to some cephalosporins s
in vitro Show major inoculum effects
Fail in therapy, despite appearingsusceptible
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APPEAR SUSCEPTIBLE TO
CEPHALOSPORINS
0
10
20
30
40
50
60
70
80
=32
(R)
Cefotax.
Ceftriax.Ceftaz.
Enterobacteriaceae are
traditionally reported assusceptible to ceftazidime,
cefotaxime, ceftriaxone,
aztreonam, and cefepimewhen MIC
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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
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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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Klebsiella pneumoniae
Escherichia coli
Proteus mirabilis
Enterobacter cloacae Non-typhoidal Salmonella
(in some countries)
COMMON ESBL PRODUCERS
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DIVERSITY OF ESBLS
Confer resistance to 1st , 2nd, 3rd cef.
Most are susceptible to -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 -lactamase inhibitors CTX-M
Probably independent evolution
Highly resistant to 3rd generation cephalosporines
initially in South America, Far East & Eastern Europe
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DETECTION STRATEGY: STEP 1
Screen Enterobacteriaceae with : Cefpodoxime- best general ESBL substrate
Cefotaxime & ceftazidime- good substratesfor CTX-M & TEM/SHV, respectively
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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 METHOD
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COMBINATION DISK METHODCARTER MW ET AL: J CLIN MICROBIOL 2000; 38: 4228 -
4232
Difference > 5 mm
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ESBL CONFIRMATORY TESTSDouble-disk synergy (DDS) test
CAZ and CAZ/CA disks CTX and CTX\CA disks
Confirmatory testing
requires using both CAZand 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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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 : COMBINATION DISCS: +VE RESULT,
ZONE ENLARGED 50%
D iscs (30+10 g) % Detected (n =100)
Ceftazidime +/- clav 88
Cefotaxime +/- clav 66
Both 93
MZali et al. 2000,JAC, 45, 881
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ESBL DETECTION
2 Steps:
Screen cefpodoxime ; cefotaxime & ceftazidime
Synergy test with ceph/clav
Combination discs are most cost effective synergy tests; Etestsa good alternative.. or automate
Guidelines on http//www.hpa.org.uk- type ESBL in search facility
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COMPARING DISK DIFFUSION WITH MINIMUM
INHIBITORY CONCENTRATIONS
Disk diffusion MICs
cefpodoxime < 22 mm cefpodoxime > 2 g/ml
ceftazidime < 22 mm ceftazidime > 2 g/ml
aztreonam < 27 mm aztreonam > 2 g/ml
cefotaxime < 27 mm cefotaxime > 2 g/mlDR.T.V.RAO MD 45
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Etest for ESBLsCefotaxime
Cefotaxime+
clavulanate
DR.T.V.RAO MD 46
ESBL C fi t T t
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ESBL Confirmatory Test
Positive for ESBL
Ceftaz/CA Cefotax/CA
Ceftaz Cefotax
47DR.T.V.RAO MD 47
ESBL CONFIRMATORY TEST NEGATIVE FOR
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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
49DR.T.V.RAO MD 49
S S C O
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PITFALLS IN ESBL DETECTION
Methods optimised forE. 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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SYNERGY TESTS WITH 4-GEN CEPHS
Cefepime/clav (Mast & AB Biodisk)
Cefpirome clav (Oxoid)
Devt. driven by spread of clonalE. 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
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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 -gen cephs
Carbapenemase
Metallo or not
R includes imipenem & / or meropenem
DR.T.V.RAO MD 52
BACTERIA NOT TO TEST FOR ESBLS
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BACTERIA NOT TO TEST FOR ESBLS
Acinetobacter Often S to clavulanate alone
S. maltophilia
+vet result by inhibition of L-2 chromosomal -
lactamase, ubiquitous in the species
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ESBL REPORTING RULE The rule (CLSI =NCCLS) M100-S15)
Strains ofKlebsiella 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
54DR.T.V.RAO MD 54
WILL CLSI CONFIRMATORY TEST DETECT ALL
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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., andProteus mirabilis which CLSI does not currently address
55
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PCR and sequencing
The gold standard
Can detect all variants
Easy to perform Labor intensive
MOLECULAR DETECTION OF ESBLS
ESBL DETECTION: AUTOMATED
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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
JCM. Apr. 2007, p.1167-1174
SYSTEMS (AS)
ESBL DETECTION: AUTOMATED
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O O
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
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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
MICROBIOLOGY LABORATORIES
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AND ESBLS
Unfortunately ,many clinical laboratories lack of
understanding regarding ESBLs and Ampc -lactamase andtheir detection .This has been documented in a study inConnecticut USA, where it was found that 21% of laboratoriesfailed to detect extendedspectrum cephalosporins and
Aztreonam in ESBLs andAmpc. 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 producingorganisms are distributed worldwide and their prevalence isincreasing.
CARBAPENEMS TREATMENT OF CHOICE FOR
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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 DMID2006; Zanetti AAC 2003]
HAND WASHING STILL CAN REDUCE THE ESBL
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HAND WASHING STILL CAN REDUCE THE ESBL
SPREAD
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DR.T.V.RAO MD 63
Created by Dr.T.V.Rao MD for e-learning resourceson implication of misuse of Antibiotics and
consequences for Medical and Paramedical students in
Developing WorldEmail