ESBL: From petri dish to patient

The First Choice Diagnostic Laboratory

Extended Spectrum Beta Lactamases (ESBL): From Petri Dish to Patient

Dr. Ashok Rattan,Super Religare Laboratories,

India


Sir Alexander Fleming

Ernest Boris Chain Sir Howard Florey


Beta lactams & β lactamases

Oxyimino aminothiazolyl caphalosporinCefuroxime, cefotaxime, ceftriaxone,

Ceftazidime, cefepime, cefpirome


TEM & SHV β lactamases


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


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


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)


β lactamases

• Penicillinases, Cephalosporinases• ESBL• Metallo β lactamases• Amp C• Carbapenemase


Redefining ESBLBalancing science and clinical need

• ESBL A

• ESBL M

– ESBL M-C

– ESBL M-D

• ESBL CARBA


Laboratory Detection of ESBL

• Phenotypic Methods– Screening methods– Confirmatory Methods

• Genotypic Methods


Double disk synergy test Jarlier V et al: Rev Infect Dis 1988;10: 867 - 878


Ceftaz/CA Ceftaz

>8 fold reduction in MICin presence of CA= ESBL

E test

Cefotaxime/CACefepime/CA

CefotaximeCefepime


Combination disk methodCarter MW et al: J Clin Microbiol 2000; 38: 4228 - 4232

Difference > 5 mm


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


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


Microscan ESBL PanelPanels contain ceftazidime alone and in combination with CAAnd cefotaxime alone and in combination with CA


Routine “research” laboratory in a teaching hospital ?


Amp C


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


• 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


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


A. TEM, B. SHV


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


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.


• 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


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


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.


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..


• 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)


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


Available lac + lac inhibitors

• Amoxacillin + Clavulonic acid

• Ampicillin + Sulbactum

• Ticarcillin + Clavulonic Acid

• Cefoperazone + Sulbactum

• Piperacillin + Tazobactum

• Cefotaxime + Sulbactum (India only)


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


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


• 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


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


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


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



• 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


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


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



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



CarbapenemCarbapenem


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


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


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


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


Infection control

• Transmitted through contact– ICU is hot spot– Hands of HCW, thermometer, ultrasound gel,

• Tag records• Education • Contact precautions• Transfer between wards & hospitals


Take home message

• Detect and report ESBL+ bacteria• Use the most appropriate antibiotic• Implement effective infection control

measures

ESBL: From petri dish to patient

Health & Medicine

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