Extended spectrum Betalactamases

8/7/2019 Extended spectrum Betalactamases

1/63

ESBLDETECTION

METHODSDR.T.V.RAO MD

DR.T.V.RAO MD 1


2/63

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


3/63

3

SURVIVAL OF THE FITTEST

Resistant bacteria survive, susceptible ones die

Mutant emerges

slowly

Sensitive cells

killed by antibiotic

Mutants progeny

overrun


4/63

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


5/63

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


6/63

-LACTAM ANTIBIOTICS Penicillin's

Ampicillin

Piperacillin

Beta-lactam/beta-lactamase inhibitors

Ampicillin/sulbactam

Amoxicillin/clavulanate

Ticarcillin/clavulanate

Piperacillin/TazobactamDR.T.V.RAO MD 6


7/63

-LACTAM ANTIBIOTICS

First Generation cephalosporins Cefazolin

Cephalothin

Second Generation oral antibiotics

Cefuroxime (many others)

Second Generation cephamycins Cefoxitin

CefotetanDR.T.V.RAO MD 7


8/63

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.


9/63

-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)

DR.T.V.RAO MD 9


10/63

-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


11/63

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


12/63

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


13/63


14/63

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


15/63

15

AMBLER CLASSIFICATION OF -LACTAMASES

Active site

Nucleotide

sequence

Four evolutionarily distinct molecular classes

A C D

Serine-enzymes

B

Zinc-enzymes

-lactamases

DR.T.V.RAO MD


16/63

16

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

DR.T.V.RAO MD


17/63

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

DR.T.V.RAO MD 17


18/63

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


19/63

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


20/63

20

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


21/63

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. 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


22/63

22

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


23/63

23

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

DR.T.V.RAO MD


24/63

24

TYPES OF ESBLS TEM

SHV

CTX-M

OXA

Mutations

ESBL PhenotypePlasmid-mediated

DR.T.V.RAO MD


25/63

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

DR.T.V.RAO MD 25


26/63

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


27/63

LABORATORY DETECTION OF ESBL

Phenotypic Methods

Screening methods

Confirmatory Methods

Genotypic Methods


28/63

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


29/63

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


30/63

WHY DETECT ESBL PRODUCERS?

ESBL producers may:Appear Sensitive to some cephalosporins s

in vitro Show major inoculum effects

Fail in therapy, despite appearingsusceptible

DR.T.V.RAO MD 30


31/63

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


32/63

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


33/63

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


34/63

Klebsiella pneumoniae

Escherichia coli

Proteus mirabilis

Enterobacter cloacae Non-typhoidal Salmonella

(in some countries)

COMMON ESBL PRODUCERS

DR.T.V.RAO MD 34


35/63

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


36/63

DETECTION STRATEGY: STEP 1

Screen Enterobacteriaceae with : Cefpodoxime- best general ESBL substrate

Cefotaxime & ceftazidime- good substratesfor CTX-M & TEM/SHV, respectively

DR.T.V.RAO MD 36


37/63

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


38/63

COMBINATION DISK METHODCARTER MW ET AL: J CLIN MICROBIOL 2000; 38: 4228 -

4232

Difference > 5 mm


39/63

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


40/63

DR.T.V.RAO MD 40


41/63

DR.T.V.RAO MD 41


42/63

42

ESBLS DETECTION METHODS:

INHIBITION BY CLAVULANIC ACID

Co-amoxiclav disc surrounded by cefotaxime, ceftriaxone, ceftazidime and

aztreonam discs (30 mcg each)


43/63

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

DR.T.V.RAO MD 43


44/63

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

DR.T.V.RAO MD 44


45/63

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


46/63

Etest for ESBLsCefotaxime

Cefotaxime+

clavulanate

DR.T.V.RAO MD 46

ESBL C fi t T t


47/63

ESBL Confirmatory Test

Positive for ESBL

Ceftaz/CA Cefotax/CA

Ceftaz Cefotax

47DR.T.V.RAO MD 47

ESBL CONFIRMATORY TEST NEGATIVE FOR


48/63

48

ESBL CONFIRMATORY TEST NEGATIVE FOR

ESBL

Ceftaz/CA Cefotaxime/CA

Ceftaz Cefotax

48DR.T.V.RAO MD


49/63

ESBL CONFIRMATORY TEST

Ceftaz/CA CeftazEtest

49DR.T.V.RAO MD 49

S S C O


50/63

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

DR.T.V.RAO MD 50


51/63

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


52/63

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


53/63

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

DR.T.V.RAO MD 53


54/63

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


55/63

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


56/63

PCR and sequencing

The gold standard

Can detect all variants

Easy to perform Labor intensive

MOLECULAR DETECTION OF ESBLS

ESBL DETECTION: AUTOMATED


57/63

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


58/63

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


59/63

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


60/63

60

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


61/63

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


62/63

HAND WASHING STILL CAN REDUCE THE ESBL

SPREAD

DR.T.V.RAO MD 62


63/63

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

[email protected]

Documents

Extended spectrum Betalactamases