DR.T.V.RAO MD 1

Dr.T.V.Rao MD

EXTENDED SPECTRUM

B-LACTAMASES(ESBL)

BASIS, DETECTION AND REPORTING

DR.T.V.RAO MD 2

Gram Negative Bacilli

V. choleraC. jejuni

Helicobacter pylori

EnterobacteriaceaePseudomonas

aeruginosa

Stenotrophomonas maltophilia

Acinetobacter spp.

Many other

(H. influenza, etc..)

DR.T.V.RAO MD 3

HISTORY OF RESISTANCE IN GRAM-NEGATIVE BACTERIA

1928

Fleming

1941

Penicillin use

1940 Penicillinase detected in

E. coli

1959

β -lactamase resistant penicillin's: Methicillin

1960s

Broad spectrum/ extended spectrum

penicillin's

1964

Cephalothin use

1965

Broad spectrum β –lactamases (TEM-1 in E. coli)

1983 Extended

spectrum β-lactamases

1950 1960 1970 1980 1990 2000

1985

Carbapenems (Imipenem)

Early 1980s

3rd generation ceph.

Carbapenemases

TEM-1 widespread

2005

Tigecycline

ESBL outbreaks in

France

1976

β –lactamases inhibitors

4

DR.T.V.RAO MD

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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COMMITTEES CONTROLLING RESISTANCE PATTERNS

BSACUnited Kingdom

CA-SFMFrance

CLSI (NCCLS) USA

DINGermany

SRGASweden

CRGNetherlands

NWGANorway

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STANDARDISATION• 1959 Ericsson & Steers – evaluation of methods

• 1961 WHO – standardisation

• 1964 Isenberg – comparison of methods in USA

• 1964 Truant – standardised tube dilution MICs

• 1966 Bauer-Kirby

• 1975 NCCLS CLSI• 1998 BSAC Standardised Method

• 2009 EUCAST Standardised Method

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GROWING INCIDENCE OF ESBL PRODUCERS

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Β-LACTAMASES CLASSIFICATION

• Molecular class:• A:

• TEM

• SHV

• other

• B: • Metalloenzymes

(carbapenemases)

• C: • Prototype: chromosomal

ampC

• D: • OXA (oxacillin

hydrolyzing enzymes)

• Enzyme type (by substrate profile):• Penicillinase

• Broad-spectrum

• Extended Spectrum

• Carbapenemase

• Genetic classification:• plasmids mediated

• Chromosomal

http://www.lahey.org/studies/webt.asp

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TYPES OF Β-LACTAMASES

• β-lactamases

• Penicillinase: gene blaZ , inducible, on transposon (can move between chromosome and plasmid).

• Broad spectrum β-lactamases • (plasmid encoded)

• TEM

• SHV

• OXA (mainly in pseudomonas)

• ESBLs

• TEM related

• SHV related

• OXA related

• CTX-M

• Other

• ampC β-lactamases

• Resistant to β-lactamase inhibitors

• chromosomal

• Carbapenemases

• Metallo- β-lactamases

• Serine carbapenemases

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EXTENDED-SPECTRUM -LACTAMASES ESBLS

• Mutations of TEM-1, TEM-2, SHV-1

• Inactivate -lactams with an oxyimino group (third-generation cephalosporins and aztreonam)

• Plasmid-mediated (often other R genes)

• 70 TEM and 15 SHV types

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GENETIC MECHANISM

Transformation

Penicillinase blaZ

Plasmidtransfer

Broad spectrum b-lactamase

(blaTEM)

&

Mutation

ESBL(TEM related)

&

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-LACTAMASE ACTIVITY

C C

C N

H H

R-CONH

S

COOH

CH3

CH3

O

Enzyme-Ser-OH

-lactam

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-LACTAMASE ACTIVITY

C C

C N

H H

R-CONH

S

COOH

CH3

CH3

O

HO

Ser

Enzyme

HOH

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L

L

L

LL

L

L

L Lb-lactamaseproduction

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• The term ESBLs is used to mean acquired, class A β-lactamases that hydrolyze and confer resistance to oxyimino- ‘2nd- and 3rd-generation’ cephalosporins, eg cefuroxime, cefotaxime, ceftazidime and ceftriaxone

WHAT ARE ESBL PRODUCING BACTERIA.

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ESBL• Confer resistance to 1st , 2nd, 3rd cephalosporins.

• Most are susceptible to β-lactamase inhibitors

• Most are susceptible to 4th cephalosporins

• All are susceptible to carbapenems

• Diversity of ESBL• SHV (widespread)

• TEM (>100 types)

• OXA • Predominantly in Pseudomonas

• less susceptible to β-lactamase inhibitors

• CTX-M• Probably independent evolution

• Highly resistant to 3rd generation cephalosporins

• initially in South America, Far East & Eastern Europe

• Probably most frequent worldwide

• Clonal spread has been documented

FURTHER COMPLICATING MATTERS:

• More than one gene of β-lactamase / ESBL / ampC / carbapenemase can be carried on the same plasmid.

• Genes of ESBL are carried on plasmids that usually carry additional resistant genes: frequently MDR

• Laboratory diagnosis confusing:

susceptibility profiles sometimes

misleading: “hidden resistance” ->

CLSI guidelines are changing.

• CTX-M clones appearing in the

community (Canada, Greece, Spain,

Italy).DR.T.V.RAO MD 17

DR.T.V.RAO MD 18

ESBLS INCLUDE:• Cephalosporin-hydrolysing mutants of TEM and SHV -

the common plasmid-mediated penicillinases of Enterobacteriaceae. Well over 100 such variants are known

• • CTX-M types. These evolved separately, at least some of them via the escape and mutation of chromosomal β-lactamases of Kluyvera species. Over 30 variants are known Obscure types, e.g. VEB and PER, not yet of concern in the UK; also OXA (Class D) ESBLs from Pseudomonas aeruginosa, in Turkey.

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• ESBLs are not the sole β-lactamases to confer resistance to 2nd and 3rd generation cephalosporins, but are the most important. They occur mostly in Enterobacteriaceae (e.g. E. coli, Klebsiella species and Enterobacter species) and rarely in non-fermenters (e.g. P. aeruginosa).

ESBL ARE IMPORTANT CAUSE OF RESISTANCE

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THEY SHOULD BE DISTINGUISHEDFROM OTHER IMPORTANT MODES OF RESISTANCE TO 2ND

AND 3RD GENERATION CEPHALOSPORINS, EG:

• Hyper produced chromosomal AmpC β-lactamases, especially in Enterobacter species.

• • Plasmid-mediated AmpC β-lactamases, in Klebsiella spp. and E. coli (rare)

• • Hyper produced K1 chromosomal β-lactamases in K. oxytoca not pneumoniae)

• • Efflux-mediated resistance in P. aeruginosa

• • Various ill-defined mechanisms in Acinetobacter species.

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LABORATORY DETECTION: SCREENING ANDCONFIRMATION

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• The basic strategy to detect ESBL producers is to use an indicator cephalosporin to screen for likely producers, then to seek cephalosporin/clavulanate synergy, which distinguishes ESBL producers from, for example, strains that hyper producer AmpC or K1 enzymes.

PRIMARY TESTING FOR ESBL DETECTION

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SCREENING• The ideal indicator cephalosporin is one to which all

ESBLs confer resistance, even when their production is scanty. Choice is predicated by the following general traits:

• TEM and SHV ESBLs – obvious resistance to ceftazidime, variable to cefotaxime

• CTX-M ESBLs – obvious resistance to cefotaxime: variable to ceftazidime

• All ESBLs – obvious resistance to cefpodoxime

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DETECTION STRATEGY: STEP 1

See http://www.hpa.org.uk

• Screen Enterobacteriaceae with :• Cefpodoxime- best general ESBL substrate

• Cefotaxime & ceftazidime- good substrates for CTX-M & TEM/SHV, respectively

Spread of CTX-M into community means screening must be wider than before

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DETECTION OF ESBLS: STEP 2

See http://www.hpa.org.uk

• Seek ceph/clav synergy in ceph R isolates

•Double disc•Combination disc•Etest

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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

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ESBL CONFIRMATION

Clavulanate Inhibition with

Cefotaxime - + +-

Ceftazidime - + - +

NOT ESBLreport results as they appear

ESBLreport cephalosporins, penicillins,

aztreonam as “R”

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CTXCAZ

CTX+CA CAZ+CA

CMZTest for E. coli K. pneumoniaeK. oxytoca

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ESBL Confirmatory TestPositive for ESBL

Ceftaz/CACefotax/CA

Ceftaz Cefotax

33

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ESBL CONFIRMATORY TEST NEGATIVE FOR ESBL

Ceftaz/CA Cefotaxime/CA

Ceftaz Cefotax

34

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ESBL EXAMPLEMIC (g/ml)

Cefotaxime >32

Ceftazidime 0.5

Cefotaxime/clavulanate 0.5

Ceftazidime/clavulanate 1.0

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HOW TO REPORTKLEBSIELLA PNEUMONIAE (ESBL)

Ampicillin R

Amoxicillin/clavulanate S

Cefazolin R

Cefotiam R

Cefmetazole S

Cefotaxime I R

Aztreonam R

, NCCLS not recommended

Cefepime S (?) R

Gentamicin R

Amikacin S

Imipenem S

Ciprofloxacin S

Flomoxef S ?

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• It follows that the logical indicator is either cefpodoxime or BOTH of cefotaxime and ceftazidime resistance.

• An alternative strategy has been proposed for community urines: testing cephalexin or cephradine as the indicator drug, then doing confirmatory ESBL tests on all isolates that are found resistant (these include e.g. all Enterobacter species. and some hyper producers of classical TEM, as well as the ESBL producers). This is not recommended, as some CTX-M-15 producers,

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AMPC

• Gene on chromosome

• Produced by virtually all GNBs

• Activity generally low

• NOT inhibited by -lactamase inhibitors

• Differ in E. coli/Klebsiella versus other GNBs

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AMPC -LACTAMASEE. COLI/KLEBSIELLA SPP.-TYPICAL

• Chromosomal

• Produced in minimal amount

• NOT inducible

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AMPC -LACTAMASEE. COLI/KLEBSIELLA SPP.- GENETIC

CHANGES

• AmpC genes mutation

• Low amount-destroy ampicillin, 1st cephalosporins

• High amount-destroy expanded spectrum -lactams

• Transfer of ampC to plasmid

• Hyper production

• Resistant to 3rd cephalosporins, cephamycins

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AMPC -LACTAMASEOTHER GNBS-TYPICAL

• Chromosomal

• Produced in small amount

• Inducible (hyper production)

• Reversible

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Etest for ESBLs

Cefotaxime

Cefotaxime+

clavulanate

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Etest for ESBLs

Cefotaxime

Cefotaxime+

clavulanate

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QUALITY CONTROL IN ESBL DETECTION

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• Positive controls should be used to ensure the performance of ESBL confirmatory tests. Three ESBL-positive E. coli strains are available from the NCTC: • CTX-M-15 (cefotaximase) NCTC 13353 TEM-3 (broad-spectrum) NCTC 13351 TEM-10 (ceftazidimase) NCTC 13352

CONTROLS FOR ESBL TESTS

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• The CLSI recommends K. pneumoniae ATCC 700603 as an ESBL-producing QC control, as does AB Biodisk (Etest). This strain may be sourced from the ATCC.

• Either E. coli NCTC 10418 or ATCC 25922 should also be used as a negative control in ESBL confirmation tests. Use of such controls is especially important when the cephalosporin and cephalosporin + clavulanate combination discs are from different batches, which may vary in original content or retained potency.

CLSI RECOMMENDS

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• Zones of the cephalosporin and cephalosporin + clavulanate discs for ESBL-negative E. coli should be equal or, at worst, within + 2 mm. Any greater difference implies malfunction or deterioration.

ZONE DETERMINATION GUIDES …

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• Briefly, revising breakpoints involves systematic review of microbiological, pharmacologic, and clinical data. Recognized experts, sponsors (pharmaceutical industry), and regulators participate in the process which includes discussions at public meetings of the CLSI Subcommittee on Antimicrobial Susceptibility Testing that take place twice a year. When establishing original breakpoints for new agents, controlled clinical trial data are required

ROLE OF CLSI IN REVISING BREAKPOINTS IN ANTIBIOTIC RESISTANCE

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FOLLOW THE NEW GUIDELINES CLSI 2010

• Guidelines for cephalospins for Enterobacteriaceae in accordance with the 2010 Clinical Laboratory Standards Institute (CLSI) recommendations. The following changes will be made to comply with the CLSI.

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WHY DO BREAKPOINTS SOMETIMES NEED

TO BE REVISED?

• Breakpoints need to be revised due to changing resistance mechanisms and bacterial population distributions, changing science leading to a better understanding of the pharmacologic determinants of clinical response, and adoption of “best practices” by clinicians.

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AgentCLSI 2009 CLSI 2010

S I R S I R

Cefazolin ≤8 16 ≥32 ≤1 2 ≥4

Cefotaxime ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftriaxone ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftazidime ≤8 16 ≥32 ≤4 8 ≥16

Aztreonam ≤8 16 ≥32 ≤4 8 ≥16

Cefipime ≤8 16 ≥32 ≤8 16 ≥32

ENTEROBACTERIACEAE: REVISED BREAKPOINTS FOR CEPHALOSPORINS

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• Agent Old (M100-S19) Revised (M100-S20)

S I R S I R

• Cefazolin ≥18 15-17 ≤14 NA NA NA

• Cefotaxime ≥23 15-22 ≤14 ≥26 23-25 ≤22

• Ceftizoxime ≥20 15-19 ≤14 ≥25 22-24 ≤21

• Ceftriaxone ≥21 14-20 ≤13 ≥23 20-22 ≤19

• Ceftazidime ≥18 15-17 ≤14 ≥21 18-20 ≤17

• Aztreonam ≥22 16-21 ≤15 ≥21 18-20 ≤17

• S – susceptible

• I – Intermediate

• R – Resistant.

DISK DIFFUSION BREAKPOINTS (MM):

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• Agent M100-S19 M100-S20

S I R S I R

• Cefuroxime ≤8 16 ≥32 ≤8 16 ≥32

• Cefepime ≤8 16 ≥32 ≤8 16 ≥32

• Cefotetan ≤16 32 ≥64 ≤16 32 ≥64

• Cefoxitin ≤8 16 ≥32 ≤8 16 ≥32

• S – susceptible

• I – Intermediate

• R – Resistant

FOLLOWING MIC BREAKPOINTS WERE REEVALUATED FOR ENTEROBACTERIACEAE BUT WERE NOT REVISED

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WHY WERE THE BREAKPOINTS FOR CEFEPIME AND CEFUROXIME (PARENTERAL) NOT REVISED?

• The cefepime breakpoints were not revised based upon clinical trial data and PK-PD evaluations. The clinical trial data showed cefepime efficacy for patients infected with isolates that tested cefepime susceptible (MIC ≤8 μg/ml), but produced an ESBL

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WHY ARE THERE NO DISK DIFFUSION BREAKPOINTS FOR CEFAZOLIN?

• Studies have not yet been completed to identify the zone diameter breakpoints that correlate with the revised MIC breakpoints for Cefazolin. Initial studies did not reveal clear zone diameter breakpoints and disk diffusion testing of Cefazolin may require a new disk with alternate disk content.

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BACTERIA NOT TO TEST FOR ESBL’S

• Acinetobacter• Acinetobacter often S to clavulanate alone

• S. maltophilia• You get +ve results via inhibition of L-2

chromosomal b-lactamase, which is ubiquitous in the species

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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

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MOLECULAR DETECTION: WHERE AND WHY ?

• In the Reference Laboratory

– confirmation of unusual resistance– surveillance of resistance mechanisms– monitoring spread of resistance genes / strains– identify strains likely to contain novel resistance

mechanisms

• In the clinical diagnostic laboratory

– rapid detection for patient management– infection control

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MOLECULAR DETECTION: DIFFERENT NEEDS

• In the Reference Laboratory,

– testing “pure” cultures– myriad assays and formats – numerous bug-drug combinations

• In the clinical diagnostic laboratory

– directly from specimens– need to target key species– format must be simple, rapid and cost-effective– problems with genes in commensals

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• Simple and multiplex PCR

• Real-time PCR• DNA sequencing• Hybridisation-

based techniques

MOLECULAR DETECTION

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MOLECULAR TESTS IN CLINICAL LABS

Black box approach:

molecular biology steps hidden

Simple end-product detection

Simple samplepreparation

• Must be rapid (TATs), inexpensive, reliable !• Platform must be sufficiently versatile to justify investment• Relatively hands-free, with scope for automation• On-going – e.g. <30 min test for ESBL detection

Chips with everything…going beyond AST

!Total profiling; more cost-effective than PCR

• species identification• resistance genes• virulence genes• epidemicity predictors• strain-specific markers

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MOLECULAR DETECTION: THE INHERENT PROBLEM

• Molecular methods only detect known mechanisms• only as good as available sequence data• resistant isolates with known genes identified

• & new variants, if sufficient homology• false-resistance (unexpressed / partial genes)

• Susceptibility must always be confirmed• can’t base treatment on a negative molecular result • can’t detect genuinely new resistance mechanisms• will never (?) replace cheap phenotypic methods

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HAND WASHING CAN REDUCE THE PREVALENCE AND SPREAD OF ESBL PRODUCERS

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