LECTURES SERIES ESBLs

OVERVIEW OF EXTENDED

SPECTRUM BETA-LACTAMASES

(ESBLs)

DEPARTMENT OF PHARMACEUTICS AND

PHARMACEUTICAL MICROBIOLOGY

USMANU DANFODIYO UNIVERSITY, SOKOTO

NUHU, Tanko

30th June, 2016

Presentation Outline

Introduction

Beta-lactam antibiotics

Antimicrobial resistance

Beta-lactamases

Other definition of ESBLs

Methods of detection of ESBL production

July 16 2Overview of ESBLs Nuhu Tanko

Introduction

• The bactericidal effect of beta-lactam antibiotics

involves inhibition of cell wall synthesis.

• This effect occurs through covalent attachment to

PBP, which is a peptidoglycan transpeptidase

enzyme that catalyzes the final steps in cell wall

formation.

• The spectrum and effects of the different beta-

lactams are determined by the PBPs to which

these antibiotics bind.


Brief background of Beta-lactam (BL)

antibiotics• The 1st semi-synthetic cephalosporins were

introduced in the mid 1960s, and they showed asomewhat limited Gram-ve effect.

• In 2GC, coverage was expanded to include Gram-vebacteria in addition to the Gram+ve effect.

• The 3-GC (oxyimino-beta-lactams) offered extendedcoverage of Gram-ve bacteria and even better BLstability.

• In part, these cephalosporins were developedbecause of the discovery of narrow-spectrum beta-lactamases (e.g., TEM-1), and some of them alsohad good oral bioavailability.


• The Gram-ve effect was extended even further in

the 4-GC.

• A better Gram+ve effect was gained in the 5GC,

and this even applied to MRSA, and hence the

5GC are also called MRSA-active

cephalosporins.

• The cephamycins were developed in the 1970s.

• They proved to have the same antimicrobial

effect as the 2GC cephalosporins but were stable

against class A ESBLs.


• The carbapenems, were found to be highly

resistant to enzymatic hydrolysis.

• The 1st carbapenem (imipenem), which had to be

combined with cilastatin to protect it against renal

dehydropeptidase.

• The 2G of carbapenem, are resistant to renal

dehydropeptidase.

• The carbapenems are known to be the ONLY

antibiotics that have some degree of post-

antibiotic effect on infections with Gram-ve.


• The first monocyclic bacterially produced beta-

lactam antibiotics were named monobactams

(e.g., aztreonam).

• Monobactams have a good Gram-ve effect but

no useful Gram+ve effect, and they are stable

towards several beta-lactamases.


The menace called Antimicrobial

Resistance (AMR)

• Bacteria/microbes are remarkably resilient and

have developed ways to resist antibiotics/other

AM drugs.

• AMR is a resistance of microorganism to an AM

that was originally effective for treatment of

infections caused by it.


What are the Mechanism of

Resistance?

Destruction or inactivation of the antibiotic

Alteration or protection of the target site.

Reduction in cell surface permeability.

Metabolic by-pass.


• AMR can be:

Intrinsic resistance, or

Acquired resistance

• Factors promoting AMR:

Exposure to sub-optimal level of AM.

Exposure to broad spectrum AM.

Exposure to microbes carrying resistant genes.

Lack of hygiene in clinical environment.

Use of antibiotics in food and agriculture.


The Enzymes called Beta-lactamases (BLs)

• The BLs are the collective name of enzymes that

open the BL ring.

• Identified as a penicillinase or cephalosporinase on

the basis of the substrate hydrolysed.

• A water molecule is added to the common BL bond,

and this inactivates the BL antibiotic from penicillin

to carbapenems.

• Clinical effect of such hydrolyzation was not noted

until the beginning of the 1950s, when the first BL-

resistant S. aureus isolates appeared in hospitals.


What are these ESBLs?

• In 1983, Knothe found a single nucleotide mutation

in an SHV that represented the first plasmid-encoded

BL that could hydrolyze the extended-spectrum

cephalosporins in an isolate of K. ozaenae, and this

type was named SHV-2.


• ESBLs are defined as BLs that have the following

characteristics: they are transferable; they can

hydrolyze penicillins, 1st, 2nd, and 3rd GC, and

aztreonam (but not the cephamycins and

carbapenems); they can be blocked in vitro by

BLs inhibitors such as clavulanic acid.

• ESBL production is originally observed in E. coli and

Klebsiella spp., and has now been documented in

other Gram-ve bacilli, including Enterobacter spp.,

Proteus mirabilis and Providencia stuarti.


• Most ESBLs can be divided into three groups, which

are designated the TEM (approx. 200 variants), SHV

(over 140 variants), and CTX-M (approx. 130).

• In the beginning of the ESBL era, the clinical isolates

consisted of the TEMs and SHVs (mainly SHV-2 and

SHV-5).


• Members of the CTX-M group are now the most

common ESBLs worldwide.

• In Germany in 1989, an E. coli isolate that was

resistant to cefotaxime and produced a non-

TEM–non-SHV enzyme, was named CTX-M-1

due to its elevated activity against cefotaxime.


• The CTX-M enzymes can be classified into five

major groups, which are designated CTX-M-1, CTX-

M-2, CTX-M-8, CTX-M-9, and CTX-M-25.

• Each of these includes several plasmid-mediated

enzymes.

• For example, the CTX-M-1 group comprises CTX-

M-15 and several other types.


How can we detect ESBL production

in the laboratory?

• Can be Phenotypic or Genotypic.

• Increasing resistance to 3GC is predominantly

due to the production of ESBLs.

• Accurate laboratory detection is important to

avoid clinical failure due to inappropriate

antimicrobial therapy.


• As a general rule, laboratories should test all isolates

using both ceftazidime (the best indicator for

TEM and SHV-derived ESBLs) and cefotaxime

(the best indicator for CTX-M types).

• Alternative, they can test with cefpodoxime, as a

good indicator for all ESBL types.

• Earlier advice to screen only with ceftazidime is no

longer adequate in view of the emergence of CTX-M

types.


• Any organism showing reduced susceptibility to

cefotaxime, ceftazidime or cefpodoxime should

be investigated for ESBL production.

• Many different techniques exist for confirming

ESBL production but those utilising similar

methodology to standard susceptibility tests are

the most convenient for the routine diagnostic

laboratory.

• These all depend on detecting synergy between

clavulanic acid and the indicator

cephalosporin(s) used in the primary screening.


Each isolate should be considered a potential

ESBL-producer if the test results are as follows:

• Disk diffusion

Cefpodoxime < 22 mm

Ceftazidime < 22 mm

Cefotaxime < 27 mm

Ceftriaxone < 25 mm

Aztreonam < 27 mm

• MICs

Cefpodoxime > 2 µg/ml

Ceftazidime > 2 µg/ml

Cefotaxime > 2 µg/ml

Ceftriaxone > 2 µg/ml

Aztreonam > 2 µg/ml

July 16 Overview of ESBLs Nuhu Tanko 20

Double disc method

• Here it examines for the expansion of the

cephalosporin’s inhibition zone adjacent to a disc

containing Co-amoxiclav 20 + 10 mg.

• The agar is inoculated with the test organism to

give a semi-confluent growth.

• A ceftazidime 30 mg disc and an Co-amoxiclav

20+10 mg disc are then placed 25 - 30 mm apart,

centre-to-centre.

• This follow an overnight incubation in air at

37°C.


ESBL production is inferred when the zone of inhibition around the ceftazidime disc

is expanded by the clavulanate.

Additional extended-spectrum cephalosporins may be tested concurrently eg

cefotaxime (30 mg), aztreonam (30 mg) and ceftriaxone (30 mg), providing the Co-

amoxiclav disc is placed in the centre of the plate and the distance of 25 - 30 mm

between the cephalosporin and clavulanate-containing discs is observed.

The use of multiple cephalosporins may be helpful as ESBLs other than common

TEM and SHV mutants begin to spread.


Combination disc methods

• The zone of inhibition around a ‘combination’

disc containing the cephalosporin combined with

clavulanic acid is compared to the zone around a

disc containing the cephalosporin alone.

• An expansion of >5 mm or 50% (according to the

particular product and manufacturer’s guideline)

indicate ESBL production.

• Plates of test organisms are inoculated.July 16 23Overview of ESBLs Nuhu Tanko

• Pairs of discs containing an extended-spectrumcephalosporin (cefotaxime, ceftazidime orcefpodoxime) with and without clavulanic acidare placed on opposite sides of the sameinoculated plate.

• Zones of inhibition are measured followingovernight incubation in air at 37°C.

• The test organism is regarded as an ESBLproducer if the zone of inhibition around thecombination disc is at least 5 mm larger than thatof the cephalosporin alone, or if the zonediameter is expanded by 50% in the presence ofthe clavulanic acid.


CZC

CZ

CTC

CT

Based on CLSI recommendation, an increase in zone size > 5 mm than the zone when

tested alone will be accepted as confirmation of ESBL production.


ESBL Etest

• The detection of ESBLs by Etest is based on a

similar principle to that of the combination disc

method.

• Double-ended strips containing gradients of

cefotaxime or ceftazidime at one end and

cefotaxime or ceftazidime plus clavulanic acid at

the other end are tested in parallel.

• A decrease in MIC of 3 doubling dilutions in the

presence of clavulanate indicates ESBL

production.


Note

• Clinical isolates found to produce ESBLs should

be assumed to be resistant to all extended

spectrum cephalosporins irrespective of the

results of susceptibility testing.

• Not all resistance to 3GC due to ESBL

production - other potent BLs such as AmpC and

K1 enzymes may be responsible.


In Conclusion

• The presence of an ESBL-producing organism in aclinical infection can result in treatment failure if oneof the above classes of drugs is used.

• ESBLs can be difficult to detect because they havedifferent levels of activity against variouscephalosporins.

• Thus, the choice of which antimicrobial agents totest is critical. For example, one enzyme mayactively hydrolyze ceftazidime, resulting inceftazidime minimum inhibitory concentrations(MICs) of 256 µg/ml, but have poor activity oncefotaxime, producing MICs of only 4 µg/ml.


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LECTURES SERIES ESBLs

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