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The First Choice Diagnostic Laboratory
Extended Spectrum Beta Lactamases (ESBL): From Petri Dish to Patient
Dr. Ashok Rattan,Super Religare Laboratories,
India
The First Choice Diagnostic Laboratory
Sir Alexander Fleming
Ernest Boris Chain Sir Howard Florey
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Beta lactams & β lactamases
Oxyimino aminothiazolyl caphalosporinCefuroxime, cefotaxime, ceftriaxone,
Ceftazidime, cefepime, cefpirome
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TEM & SHV β lactamases
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ESBL
• An ESBL is any β lactamase, ordinarily acquired and not inherent to a species, that can rapidly hydrolyse or confer resistance to oxyimino cephalosporins
– David Liverpool 2008
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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.org)
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β lactamases
• Penicillinases, Cephalosporinases• ESBL• Metallo β lactamases• Amp C• Carbapenemase
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Redefining ESBLBalancing science and clinical need
• ESBL A
• ESBL M
– ESBL M-C
– ESBL M-D
• ESBL CARBA
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Laboratory Detection of ESBL
• Phenotypic Methods– Screening methods– Confirmatory Methods
• Genotypic Methods
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Double disk synergy test Jarlier V et al: Rev Infect Dis 1988;10: 867 - 878
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Ceftaz/CA Ceftaz
>8 fold reduction in MICin presence of CA= ESBL
E test
Cefotaxime/CACefepime/CA
CefotaximeCefepime
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Combination disk methodCarter MW et al: J Clin Microbiol 2000; 38: 4228 - 4232
Difference > 5 mm
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Vitek ESBL confirmatory testVitek 2 Advanced Expert
Simultaneous assessment of antibacterial activity of cefepime, cefotaxime & ceftazidime either alone or+ CA interpretation through advanced expert system
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Phoenix ESBL test (BD)Uses growth response in 5 wells containing to
cefpodoxime, ceftazidime, Ceftazidime + CA, cefotaxime + CAceftriaxone + CA
Results are interpreted through a computer system
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Microscan ESBL PanelPanels contain ceftazidime alone and in combination with CAAnd cefotaxime alone and in combination with CA
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Routine “research” laboratory in a teaching hospital ?
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Amp C
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CefoxitinCefoxitin
• Broader spectrum of activity than most first generation Broader spectrum of activity than most first generation cephalosporinscephalosporins
• Greater resistance to Greater resistance to -lactamase enzymes-lactamase enzymes• The 7-methoxy group may act as a steric shieldThe 7-methoxy group may act as a steric shield• The urethane group is stable to metabolism compared to the The urethane group is stable to metabolism compared to the
esterester• Introducing a methoxy group to the equivalent position of Introducing a methoxy group to the equivalent position of
penicillins (position 6) eliminates activity.penicillins (position 6) eliminates activity.
N
O
HOMeHN S
CO2H
OO
CNH2
O
S
7
3
CephamycinsCephamycins
Second Generation CephalosporinsSecond Generation Cephalosporins
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• Aminothiazole ring enhances penetration of cephalosporins Aminothiazole ring enhances penetration of cephalosporins across the outer membrane of Gram -ve bacteriaacross the outer membrane of Gram -ve bacteria
• May also increase affinity for the transpeptidase enzymeMay also increase affinity for the transpeptidase enzyme• Good activity against Gram -ve bacteriaGood activity against Gram -ve bacteria• Variable activity against Gram +ve cocciVariable activity against Gram +ve cocci• Lack activity vs MRSALack activity vs MRSA• Generally reserved for troublesome infectionsGenerally reserved for troublesome infections
Third Generation CephalosporinsThird Generation Cephalosporins OximinocephalosporinsOximinocephalosporins
N
O
HHHN S
CO2H
R
C
O
N
S
NO
Me
H2N
CH2OCOMeH
CefotaximeCeftizoxime
N
NNCH2S
O
OH
Me
Ceftriaxone
RR
AminothiazoleAminothiazole ringring
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Test Advantages Disadvantages DNA probes Specific for gene family (e.g., TEM or
SHV) Labor intensive, cannot distinguish between ESBLs and non-ESBLs, cannot distinguish between variants of TEM or SHV
PCR Easy to perform, specific for gene family (e.g., TEM or SHV)
Cannot distinguish between ESBLs and non-ESBLs, cannot distinguish between variants of TEM or SHV
Oligotyping Detects specific TEM variants Requires specific oligonucleotide probes, labor intensive, cannot detect new variants
PCR-RFLP Easy to perform, can detect specific nucleotide changes
Nucleotide changes must result in altered restriction site for detection
PCR-SSCP Can distinguish between a number of SHV variants
Requires special electrophoresis conditions
LCR Can distinguish between a number of SHV variants
Requires a large number of oligonucleotide primers
Nucleotide sequencing
The gold standard, can detect all variants
Labor intensive, can be technically challenging, can be difficult to interpret manual methods
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A. TEM, B. SHV
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Clinical implications
• It is important to identify ESBL producing isolates ?
– No ! – particularly if there is no indication of such
organisms in the hospital in question
K Bush: Eur J Clin Microbiol Infect Dis 1996, 15: 361 – 364K Bush: Eur J Clin Microbiol Infect Dis 1996, 15: 361 – 364
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Emery CL & Weymouth LAJCM 1997; 35: 2061 – 2067
Detection & clinical significance of ESBL in a tertiary care medical center (Virginia, US)
• Prevalence of ESBL unknown, national guidelines for testing & reporting not developed– 750 bedded hospital, 6M, in vitro, MIC (if > 1 ug/ml)
-> ESBL by DDS & response from records– 50 isolates from 23 pts (1.2%)– Prevalence of ESBL is low– Respond favorably to antibiotic therapy based on
MIC– No clinical failure– May not be clinically necessary or cost effective to
institute additional testing to detect ESBL production on a routine basis.
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• 12 hospitals in USA, Taiwan, Australia, South Africa, Turkey, Belgium & Argentina Jan ’96 – Dec ’97
• Blood culture positive for K.pneumoniae
• Monitored for 1 M, 188 observational prospective study
In vitro susceptibility of isolates (%)
Antibiotics <1 2 4 8 16 32 >64
g / mlCefotaxime 5.6 18.1 5.6 19.4 13.9 15.3 22.2
Ceftriaxone 4.2 5.6 15.3 11.1 16.7 15.3 31.9
Ceftazidime 4.2 4.2 5.6 5.6 8.3 5.6 66.5
Cefepime 23.6 22.2 23.6 9.7 4.2 9.7 6.9
Cefoxitin 0 2.8 59.7 18.1 9.7 4.2 5.6
Cefotetan 65.3 19.4 8.3 1.4 1.4 2.8 1.4
Paterson et.al. 2001. JCM.39:2206-12Paterson et.al. 2001. JCM.39:2206-12
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Outcome of Serious ESBL Infections When Treated with 3rd gen Cephalosporin
MIC (µg/ml)
3rd gen cef
Failure Died within 14 d of bacteremia
8 S 100% (6/6) 33% (2/6)
4 S 67% (2/3) 0 (0/3)
2 S 33% (1/3) 0 (0/3)
≤1 S 27% (3/11) 18% (2/11)
Total 54% (15/28)
Paterson et.al. 2001. JCM.39:2206-12Paterson et.al. 2001. JCM.39:2206-12
4545
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Fatality rates for episodes of bloodstream infections
Immune status No. of fatal episodes (%)
ESBL group Non ESBL Total
Shock -> + - + -
Compromised 3 / 4 6 / 31 0 / 8 4 / 53 13 / 96
Competent 2 / 2 1 / 8 1 / 1 0 / 25 4 / 36
Subtotal 5 / 6 7 / 39 1 / 9 4 / 78
Kim et. al. 2002. AAC.46:1481 – 91.
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Molecular correlation for the treatment outcomes in bloodstream infections caused by E. coli &
K. pneumoniae with reduced susceptibility to Ceftazidime
• UCLA, 12 years retrospective study
• 23 E. coli, 13 K. pneumoniae ; CTZ MIC > 2 g/ml
• CTZ treatment was associated with failure of therapy in all patients
Wong Beringer et. al. 2002. CID.34:135 - 46Wong Beringer et. al. 2002. CID.34:135 - 46..
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• Conclusion: Do not use 3rd or 4th generation cephalosporins if infection is caused by ESBL +ve bacteria, even if sensitive in vitro
• Message to the lab: Always test for ESBL in E.coli & K.pneumoniae (Screen & confirm)
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Therapeutic options in infections caused by ESBL producing E.coli, K.pneumoniae
• Cephalosporins (3rd or 4th generation)• Cephamycins• lac + lac inhibitor combinations• Aminoglycosides• Fluoroquinolones• Carbapenems
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Available lac + lac inhibitors
• Amoxacillin + Clavulonic acid
• Ampicillin + Sulbactum
• Ticarcillin + Clavulonic Acid
• Cefoperazone + Sulbactum
• Piperacillin + Tazobactum
• Cefotaxime + Sulbactum (India only)
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MIC (g/ml) of Ceftazidime alone and in combination (4 +1) with Pot. Clavulonic Acid
MIC
E.coli
16
E.coli
435
E.coli
387
E.coli
324
kleb. p
neum
.7006
03
Kleb. p
neum
.WHO1
K.aeru
ginos
a KL 13
9
Klebse
illa s
pp. 53
Proteu
s spp.
Proteu
s spp. P
23
Proteu
s spp. P
18
Proteu
s spp. P
10
Pseudom
onas
210
Pseudom
onas
204
Pseudom
onas
35
Serra
tia 19
6
Serra
tia 63
H
S.aure
us 240
S.aure
us 292
13
Ceftazidime Cefta.+ Pot. Clav
32
8
16
0
64
128
256
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In vivoEscherichia coli lethal mouse model
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
MIC
E
Control CTZ 100mg/kg aloneCTZ 100mg + CA 50 mg/kg CTZ 100 mg + CA 25 mg/kgCTZ 100mg+ CA 12.5 mg/kg CTX 100 mg+ CA 6.25 mg?kg
0
6
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• Literature Conclusion: – Can be overwhelmed by lactamases– Inoculum effect present– Selection of porin less mutants
• Personal data• Recommendations: Serious infections with
ESBL producing organisms may not respond
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Association of ampicillin resistance & ESBL production with resistance to non lactam
antibiotics in invasive E. coli
Ampicillin ESBL
S (%) R (%) - +
Ciprofloxacin 7.3 24 17.2 63.3
Cotrimoxazole 9 44 32.9 77.3
Gentamicin 2.5 9.9 6.3 16.7
Oteo et. al. 2002. AAC.50:945 - 52Oteo et. al. 2002. AAC.50:945 - 52
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Potency & spectrum against ESBL+ve E.coli phenotype & all strains in European regions
(1997 – 2000) (n 189/1310)
MIC 90 (g/ml)
ESBL + All
Cefepime 8 1
Piper+ tazo 64 16
Gentamicin 128 8
Tobramycin 128 8
Ciprofloxacin 16 8
Jones et. al. 2003. CMI .9 : 708 - 12Jones et. al. 2003. CMI .9 : 708 - 12
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Potency & spectrum against ESBL+ve K. pneumoniae phenotype & all strains in European
regions (1997 – 2000) (n 306/934)
MIC 90 (g/ml)
ESBL + All
Cefepime 32 16
Piper+ tazo >128 64
Gentamicin >128 64
Tobramycin 128 64
Ciprofloxacin 16 4
Jones et. al. 2003. CMI .9 : 708 - 12Jones et. al. 2003. CMI .9 : 708 - 12
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• Ciprofloxacin was active in vitro against 21 of 28 isolates, only 21 analyzed– 2 of 7 had partial response– 5 of 7 cases, treatment failed– Isolates had MIC (0.38 ug/ml) close to susceptibility
breakpoint, treatment failure ascribed to the inability of the drug to reach therapeutic concentration at infected sites.
Endimiani et. al. 2004. CID.38: 243 - 51
Bacteremia due to K.pneumoniae isolates producing the TEM 52 ESBL : treatment outcome of patients receiving
Imipenem or ciprofloxacin
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Therapeutic options in infections caused by ESBL producing E.coli, K.pneumoniae
• Cephalosporins (3rd or 4th generation)• Cephamycins• lac + lac inhibitor combinations• Aminoglycosides• Fluoroquinolones• Carbapenems
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Potency & spectrum against ESBL+ve E.coli phenotype & all strains in European regions
(1997 – 2000) (n 189/1310)
MIC 90 (g/ml)
ESBL + All
Cefepime 8 1
Piper+ tazo 64 16
Gentamicin 128 8
Tobramycin 128 8
Ciprofloxacin 16 8
Meropenem 0.25 0.12
Jones et. al. 2003. CMI .9 : 708 - 12Jones et. al. 2003. CMI .9 : 708 - 12
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Potency & spectrum against ESBL+ve K. pneumoniae phenotype & all strains in European
regions (1997 – 2000) (n 306/934)
MIC 90 (g/ml)
ESBL + All
Cefepime 32 16
Piper+ tazo >128 64
Gentamicin >128 64
Tobramycin 128 64
Ciprofloxacin 16 4
Meropenem 1 0.12
Jones et. al. 2003. CMI .9 : 708 - 12Jones et. al. 2003. CMI .9 : 708 - 12
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CarbapenemCarbapenem
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Spectrum of activity of carbapenem
Susceptible Resistant
E. coli including ESBL Stenotrophomonas maltophilia
K.pneumoniae ESBL
Ps. aeruginosa
Acinetobacter spp
Proteus spp
Haemophilus influenza
Staph. aureus (MSSA) MRSA
Staph. epidermidis MRSE
Strept. pneumoniae Enterococcus faecalis
Imipenem better against GPC, active against GNB including Pseudo, anaerobes
Meropenem 2x to 4x better against GNB
Ertapenem 2x to 4x less active, not very active against GNB
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Breakpoints
Species Imipenem Meropenem Ertapenem
S I R Enterobact <4 8 >16 <4 8 >16 <2 4 >8
& staph
Anaerobes <4 8 >16 <4 8 >16 <4 8 >16
S.pneumoniae <0.12 >1 <0.25 >1 <1 >4
H. influenzae <4 <0.5 <0.5
S I RS I R S I RS I R
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Pharmacokinetics
Imipenem Meropenem Ertapenem
Dose 250 mg – 1 G 500 mg – 1 G 1 G
Frequency 6 – 8 hours 8 24
Route IV IV / IM IV / IM
T ½ 1 hour 1 4
Cmax 41 – 83 ug/ml 49 155
Protein binding 30% 2 95
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Carbapenem in clinical practice
• Suitable for Initial empirical &/or definitive mono-therapy of– Febrile neutropenia – ICU infections– Intra abdominal infections– Serious lower respiratory tract infections– Pediatric meningitis
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Infection control
• Transmitted through contact– ICU is hot spot– Hands of HCW, thermometer, ultrasound gel,
• Tag records• Education • Contact precautions• Transfer between wards & hospitals
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Take home message
• Detect and report ESBL+ bacteria• Use the most appropriate antibiotic• Implement effective infection control
measures
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