esbl extended spectrum beta lactamases dynamics and detection l.
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survival of the fittest
Resistant bacteria survive, susceptible ones dieSurvival of the fittest

Mutant emerges

slowly

Sensitive cells

killed by antibiotic

Mutant’s progeny

overrun

Dr.T.V.Rao MD

slide3

PENICILLIN

BETA LACTAM RING

BETA LACTAMASES enzymes that inactivate the beta-lactam ring

CEPHALOSPORIN

BETA LACTAM RING

Dr.T.V.Rao MD

action of a b lactamase
Action of a b-lactamase

S

H2O

N

Inactive penicilloate

O

COOH

S

Active penicillin

HN

O

OH

COOH

Dr.T.V.Rao MD

basis of betalactamse activity
Basis of Betalactamse Activity
  • Time-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

Dr.T.V.Rao MD

b lactam antibiotics
b-lactam antibiotics
  • Penicillin's
    • Ampicillin
    • Piperacillin
  • Beta-lactam/beta-lactamase inhibitors
    • Ampicillin/sulbactam
    • Amoxicillin/clavulanate
    • Ticarcillin/clavulanate
    • Piperacillin/Tazobactam

Dr.T.V.Rao MD

penicillins
Penicillins
  • 1st 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

slide8

Some beta-lactamases only inactivate a small number of antibiotics e.g. penicillin

  • Others have extendedspectrum to all the penicillins and cephalosporins e.g. cefuroxime, ceftriaxone (ESBLs)
  • In addition may also carry resistance to other antibiotics e.g. ciprofloxacin.

Dr.T.V.Rao MD

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

Definition of ESBL

Dr.T.V.Rao MD

esbl evolution
ESBL Evolution
  • Mid 1980s
  • Variants of TEM and SHV
  • Breakdown 3rd generation cephalosporins
  • Mainly in hospital Klebsiella
  • Spread world wide

Dr.T.V.Rao MD

what are extended spectrum lactamases
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).

Dr.T.V.Rao MD

ambler classification of lactamases
Ambler Classification of β-Lactamases

β-lactamases

Active site

Serine-enzymes

Zinc-enzymes

Nucleotide sequence

C

D

B

A

Four evolutionarily distinct molecular classes

Dr.T.V.Rao MD

what is a beta lactam
Abx

Penicillin

Cephalosporin

Monobactam

Carbapenem

Bacteriocidal

What is a beta-lactam?

Google Images

Dr.T.V.Rao MD

cephalosporins
Cephalosporins

4th

3rd

2nd

1st

Willey, et al., 2008

Dr.T.V.Rao MD

cephalosporins uses
Cephalosporins-uses
  • 1st 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

Dr.T.V.Rao MD

others
Others
  • Monobactams
  • Monobactams very active against G- including E.coli Ex. Aztreonam
  • Carbapenems
  • Carbapenems have an extremely broad spectrum. Cross-reactivity with penicillins or cephalosporins Ex. Imipenam

Dr.T.V.Rao MD

the fight goes on
The Fight goes on ..
  • Beta-lactam
  • Beta-lactamase
  • Beta-lactamase inhibitor
  • ESBL

Google Images

Dr.T.V.Rao MD

common esbl producers
Common ESBL producers

Klebsiella pneumoniae

Escherichia coli

Proteus mirabilis

Enterobacter cloacae

Non-typhoidal Salmonella (in some countries)

Dr.T.V.Rao MD

the fight beta lactam
The FightBeta-Lactam

PG

cell

N

O

LYSIS

Dr.T.V.Rao MD

the fight beta lactamase
The FightBeta-lactamase

PG

beta-lactamase

cell

N

O

Dr.T.V.Rao MD

the fight beta lactamase21
The FightBeta-lactamase

PG

O

NH

OH

cell

Dr.T.V.Rao MD

the fight beta lactamase inhibitor
The FightBeta-lactamase inhibitor

PG

beta-lactamase

cell

Inhibitor

N

O

Dr.T.V.Rao MD

the fight beta lactamase inhibitor23
The FightBeta-lactamase inhibitor

PG

beta-lactamase

Inhibitor

cell

N

O

LYSIS

Dr.T.V.Rao MD

esbls
ESBLs
  • Enterobacteriaceae
  • Resistance to oxyimino-cephalosporins and Monobactams but not cephamycins and carbapenem
    • Susceptible to beta-lactamase inhibitors
    • Genes
  • SHV
  • TEM
  • CTX-M
  • OXA
  • AmpC

Oteo, et al., 2010

Dr.T.V.Rao MD

slide25

Evolution of -Lactamases

Plasmid-mediated TEM and SHV -lactamases

Extended-spectrum

Cephalosporins

Ampicillin

1983

1965

1970s

1988

2000

1963

Look and you will find ESBL

TEM-1

E.coli

S.paratyphi

TEM-1

Reported in

28 Gm(-) sp

ESBL in

Europe

ESBL

in USA

> 130 ESBLs

Worldwide

Dr.T.V.Rao MD

classification of 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

Classification of β lactamases

Dr.T.V.Rao MD

classification
Classification
  • Ambler Classification
    • Molecular class A – D
      • A
  • Bush-Jacoby-Medeiros Classification
    • Functional group 1 – 4
      • 2
      • 2b
      • 2be

Paterson and Bonomo, 2005

Dr.T.V.Rao MD

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

Beta-lactamase inhibitors

Dr.T.V.Rao MD

resistance and genetics
Resistance and genetics

Know the species

Dr.T.V.Rao MD

slide30

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

types of esbls
TEM

SHV

CTX-M

OXA

Mutations

Plasmid-mediated

ESBL Phenotype

Types of ESBLs

Dr.T.V.Rao MD

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

Choice of Indicator Cephalosporin

Livermore D and Woodford N HPA Guidance 2004

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, VitekNeo tabs & others

Current Modern Methods

Slide

detection of esbls
Detection of ESBLs
  • Seek ceph/clav synergy in ceph R isolates
      • Double disc
      • Combination disc
      • Etest

Dr.T.V.Rao MD

challenges for the diagnostic lab
Challenges for the diagnostic lab
  • Detection…. Hemophilus, Neisseriaetc.
  • Predicting b-lactamase types. Have GNB got ?:
    • ESBL,
      • AmpC
        • Metallo types, VIM, IMP etc…
  • Spotting unusual patterns; knowing what to refer ???
detection strategy step 1
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

combination disk method carter mw et al j clin microbiol 2000 38 4228 4232
Combination disk methodCarter MW et al: J Clin Microbiol 2000; 38: 4228 - 4232

Difference > 5 mm

Dr.T.V.Rao MD

klebsiella pneumonia producing a higher activity esbl
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).

Dr.T.V.Rao MD

slide41

Double disc antagonism for inducible AmpC

Cefoxitin

Ceftazidime

Dr.T.V.Rao MD

ampc inducibility when to look
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

esbls detection methods inhibition by clavulanic acid
ESBLs Detection Methods: Inhibition by Clavulanic Acid

Co-amoxiclav disc surrounded by cefotaxime, ceftriaxone, ceftazidime and aztreonam discs (30 mcg each)

Dr.T.V.Rao MD

esbl detection
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

Dr.T.V.Rao MD

slide45

ESBL Confirmatory Test

Positive for ESBL

Cefotax/CA

Ceftaz/CA

Ceftaz

Cefotax

45

Dr.T.V.Rao MD

esbl confirmatory test negative for esbl
ESBL Confirmatory Test Negative for ESBL

Ceftaz/CA

Cefotaxime/CA

Ceftaz

Cefotax

46

Dr.T.V.Rao MD

esbl confirmatory test
ESBL Confirmatory Test

Ceftaz/CA

Ceftaz

Etest

47

Dr.T.V.Rao MD

esbls times a changing with ctx m
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

Dr.T.V.Rao MD

esbl confirmatory tests
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

synergy tests with 4 gen cephalosporins
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

Dr.T.V.Rao MD

pitfalls in esbl detection
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

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

Risk Factors for ESBL Infection

Dr.T.V.Rao MD

bacteria not to test for esbl s
Bacteria not to test for ESBL’s
  • Acinetobacter
    • Often S to clavulanate alone
  • S. maltophilia
    • +vet result by inhibition of L-2 chromosomal b-lactamase, ubiquitous in the species

Dr.T.V.Rao MD

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

ESBL Reporting Rule

55

Dr.T.V.Rao MD

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

ESBLsoccur in species other than E. coli, Klebsiella spp., and Proteus mirabilis which CLSI does not currently address

Will CLSI confirmatory test detect ALL ESBL-producing GNB?

56

Dr.T.V.Rao MD

esbl detection automated systems as
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)

Dr.T.V.Rao MD

the resistance becoming complex
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

Dr.T.V.Rao MD

antibiotic policy changes practised
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

Dr.T.V.Rao MD

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.

Microbiology Laboratories and ESBL’s

Dr.T.V.Rao MD

slide62

The programme created by Dr.T.V.Rao MD for basic understanding by Medical Microbiologists in the Developing World

  • Email
  • doctortvrao@gmail.com

Dr.T.V.Rao MD