Extended-Spectrum Extended-Spectrum ββ--LactamasesLactamases

(ESBLS)(ESBLS)Laboratory Guideline For The

Detection Of This Important Resistance Treat

Prof. Ellabib MSDept. of Medical Microbiology

Medical Collegeellabib@dmi.ly

Introduction to Introduction to ββ-Lactamases-Lactamases

What is β-Lactamases ?Enzymes produced by Bacteria Inactivate β-Lactam antibioticsChromosomal or plasmid-mediatedRare among gram positive pathogensExceptions being Staph. aureus and Enteroccocus faecalisCommon among gram negative bacteriaEnterobacteriaceaeInducible, constitutive and /or hyper productive strain Such as TEM-1 and SHV-1

Introduction to Introduction to ββ- Lactamases cont.- Lactamases cont.

What is Extended-Spectrum β- Lactamases?Mutation of TEM-1 AND SHV-1 leading to Enzymes hydrolyze β- Lactam antibioticsCause resistant to Oxyimino - cephalosporinsCeftazidimeAztreonamProduced by E. coli, Klebsiella pneumoniaeOther EnterobacteriaceaeP. aeruoginosaChromosomally mediated or plasmid- mediated

History to History to ββ-Lactamases -Lactamases

β-Lactamases in gram positive bacteria Represented by Penicillinase enzyme S. aureus, coagulase negative staphylococci

and Enteroccocus faecalis 80 to 90% resistant to penicillin'sBenzylpenicillinsAminopenicillins (ampicillin, amoxicillin)carboxypenicillins (ticarcillin, carbenicillin)Remain sensitive to stable penicillinMethicillin, oxacillin

ββ-Lactamases in gram positive bacteria cont.-Lactamases in gram positive bacteria cont.

Class A enzyme (plasmid mediated) Borderline oxacillin resistant Hyper producer strains of S. aureus Show resistant to oxacillinRetain sensitivity with clavulanic acidNot major problem in other G+ bacteriaMajor important resistant in S. aureus is

Methicillin resistant

ββ-Lactamases in gram negative -Lactamases in gram negative

Leading cause of resistantChromosomal or plasmidConstitutive or inducibleTEM-1 (E. coli, 1960) TemonieraPlasmidSHV-1Plasmid mediated in E. coliChromosomally in most K. pneumoniae

ββ-Lactamases in gram negative-Lactamases in gram negative

New β-Lactamases for every new β-LactamSelective pressureOver use of new antibioticsSelect new variant of β-Lactamases resistant SHV-2First enzyme hydrolysis newer β-Lactam Found in K. ozraenae in Germany Called ESBLSOVER 150 ESBLS were discoveredEnterobacteriaceae and Ps. aeruginosa

Characterization and Classification of ESBLSCharacterization and Classification of ESBLS

Most of them has serine at active site Class A enzymeHydrolysis penicillin'sSuch as TEM-1, SHV-1 and penicillinase

Bush, Jacoby and Medeiros Classification schemeUse biochemical enzyme propertiesMolecular structureNucleotide sequence genesPlace β-Lactamases into functional groups

Types of ESBLSTypes of ESBLS Most of them derived from TEM OR SHV Mostly found in E. coli and K. pneumoniae Also found in other Enterobacteriaceae

TEM typesTEM-1 E. coli90% amp Resistant H. influenzae and N. gonorrhoeae Amp and p resistant Hydrolyze penicillins and early cephalosporin's

TEM-2TEM-2

Derived from TEM-1 NO CHANGE IN SUBSTRATE PROFILE Tem-3 First TEM type displayed ESBLS Over 90 TEM derivatives described latter ESBLS and inhibitory resistant (IRT) Derivatives of TEM and SHV types Resistant to inhibition by clavulanic acid TEM-1 and TEM-2 Mainly found in E. coli Some strain of K. pneumoniae and K. oxytoca

ESBLS and inhibitory resistant (IRT) cont.ESBLS and inhibitory resistant (IRT) cont. Resistant to sulbactam Remain susceptible to tazobactam Sulphydryl variable (SHV) SHV-1 K. pneumoniae Plasmid mediated Amp resistant In many strains genes integrated in chromosomes Few derivatives of SHV-1 Serine residue critical for hydrolysis of ceftazidime Lysine for cefotaxime

Sulphydryl variable (SHV) cont.Sulphydryl variable (SHV) cont.

SHV-10 Have an IRT Majority found in K. pneumoniae Citrobacter diversus and E. coli P. aeruginosa CTX-M Plasmid mediated ESBLS Hydrolyze cefotaxime over ceftazidime Strains of Salmonella enteric serovar Typhimurium E. coli and other some species of enterobacter Better inhibited by Tazobactam

OXAOXAClass D group 2dConfer resistant to Amp and cephalothinHigh hydrolytic activity against OX and CLOXPoorly inhibited by clavulanic acidFound mainly in P. aeruginosa PER-1Plasmid encodingP. aeruginosa and Salmonella enteric serovar

Typhimurium Acinetobacter baumaniiCeftazidime resistant strains in A. baumanii

Laboratory detection of ESBLSLaboratory detection of ESBLS

Why ?Major leading cause of resistance to β - Lactam

antibiotics Increasing prevalence among

EnterobacreriaceaeDue to its fast Spread amongst various bacteriaVery Important by infection control committeeClinical and therapeutic implication

Methods Methods

1. Double disk diffusion Synergy test (DDST)2. Broth dilution 3. Etest ESBL strip

Double disk diffusion (DDST) Using Kirby-Bauer disc diffusion (NCCLS) Synergy between augmentin+3GC Ceftazidime, Cefpodoxime, Cefepime, cefotaxime, cefoxitin Increased zone with the addition of inhibitor

DOUBLE DISK TEST FOR ESBL CONFIRMATIONDOUBLE DISK TEST FOR ESBL CONFIRMATION

l. Introduction Cefpodoxime* (can be used alone) third generation cephalosporins Aztreonam are all extremely susceptible to ESBLs used as screening agents MIC disk diffusion E. coli, Klebsiella species or Proteus species confirmation of ESBL be determined by the

double disk test.

II. II. MaterialsMaterials

Mueller‑Hinton (MH) agar (90 0r 150) mm20/10 µg amoxicillin‑ clavulanate disc30 µ g ceftazidime disc30 µ g ceftriaxone or cefotaxime disc30 µ g aztreonam disc10 µ g cefpodoxime disc (optional)30 µ g cefoxitin disc30 µ g cefepime discQuality control strain: E. coli ATCC 51446

lll. Procedurelll. Procedure1. Prepare a bacterial suspension of the organism to be

tested that has a turbidity equivalent to that of a 0.5 McFarland standard.

2. Inoculate a Mueller‑Hinton agar plate with this suspension in accordance with CLSI M100‑S16 (M2) guidelines for disc diffusion testing.

3. Place the amoxicillin‑ clavulanic acid disc towards the centre of the plate.

4. Carefully measure 15 mm out from the edge of that disc at 90o angles marking the plate.

5. Place a ceftazidime disk on the plate so that its inner edge is 15 mm (the mark) from the amoxicillin‑ clavulanic acid disc (See Figure 1 KB-ESBL Template or Figure 2 IC ESBL Screen).

Procedure cont. Procedure cont.

6. Do the same with cefotaxime (or ceftriaxone), aztreonam and cefpodoxime discs so that they are spaced 90o apart and 15mm from the centre disc.

7. Place one each of cefoxitin, ceduroxime, cefepime and pipercillin/tazobactum discs in any available space remaining on the plate.

8. Incubate 35oC, in O2 x 18-24 hours and record the zone diameters for the all cephalosporins as per CLSI guidelines.

9. For E. coli, Klebsiella species and Proteus species, instead of using standard cut offs to determine S, I or R, screening test cut offs are used and interpretations as R and S are reported if zone size is < or > of these screening breakpoints.

Figure 1. KB-ESBL Template

FOX CPD

15 mm AMC

CRO 15 mm 15 mm CAZ 15 mm

ATM

TZP

CXM FEP

Placement of antibiotics for ESBLS testing

Figure 2.Figure 2. IC-ESBL TemplateIC-ESBL TemplateTo be used for Infection Control ESBL Screen isolatesTo be used for Infection Control ESBL Screen isolates

InterpretationInterpretation

Note: The following applies to cefpodoxime-nonsusceptible E. coli, Klebsiella

species and Proteus species only.1. After incubation, measure the diameters of the zone of complete

inhibition with calipers/ruler. Measure at the narrowest side of the zone.

2. Document zone size for all antibiotics into the LIS.3. Observe for potentiation of the inhibition zone (i.e. increase in the

inhibition zone) of any one of Cefpodoxime, Ceftazidime, ceftriaxone or aztreonam when combined with clavulanic acid (enter Yes or No to the “drug” named “Potentiation” in the LIS).

4. If a reduction of zone of inhibition of any one of Cefpodoxime, Ceftazidime, ceftriaxone or aztreonam when combined with clavulanic acid is observed (i.e. a D zone formation), enter Yes or No to the “drug” named “D zone” in the LIS. Recheck the identification of the isolate and repeat testing if the identification is questionable.

NCCLS INTERPRETATION FOR NCCLS INTERPRETATION FOR ESBLS ESBLSThe National Committee for Clinical Laboratory Standards (NCCLS) has

developed broth microdilution and disk diffusion screening tests using selected antimicrobial agents (1). Each Klebsiella pneumoniae, K. oxytoca, or Escherichia coli isolate should be considered a potential ESBL-producer if the test results are as follows:

Disk diffusion MICscefpodoxime < 22 mm cefpodoxime > 2 µg/ml ceftazidime < 22 mm ceftazidime > 2 µg/mlaztreonam < 27 mm aztreonam > 2 µg/mlcefotaxime < 27 mm cefotaxime > 2 µg/mlceftriaxone < 25 mm ceftriaxone > 2 µg/mlThe sensitivity of screening for ESBLs in enteric organisms can vary

depending on which antimicrobial agents are tested. The use of more than one of the five antimicrobial agents suggested for screening will improve the sensitivity of detection. Cefpodoxime and ceftazidime show the highest sensitivity for ESBL detection.

Class A ESBL presentClass A ESBL present

I. Potentiation of the inhibition zone of any one of Cefpodoxime, Ceftazidime, ceftriaxone or Aztreonam when combined with clavulanic acid (see below for examples of different patterns of potentiation that can be seen with organisms that contain Class A ESBLs)

II. Susceptibility to cefoxitin.III. Susceptibility or resistance to any one of

Ceftazidime, ceftriaxone or Aztreonam

ATM

CPD

CAZ

AMC

CRO

FOX

Detection of ESBLSDetection of ESBLS

Detection of ESBLSDetection of ESBLS

Class A and Class C ESBL presentClass A and Class C ESBL present

I. Potentiation of the inhibition zone of any one of Cefpodoxime, Ceftazidime, ceftriaxone or Aztreonam when combined with clavulanic acid

II. Resistant or Intermediate to cefoxitin.

III. Susceptibility or resistance to any one of Ceftazidime, ceftriaxone or Aztreonam

Class C‑ESBL presentClass C‑ESBL present

I. No potentiation with clavulanic acid

II. Resistance or Intermediate to cefoxitin

III. Resistance to any one of ceftazidime, ceftriaxone or Aztreonam.

Inducible Class C‑ESBL presentInducible Class C‑ESBL present

I. No potentiation with clavulanic acid

II. Resistance or Intermediate to cefoxitin

III. Susceptible, Intermediate or Resistance to any one of Ceftazidime, ceftriaxone or Aztreonam.

IV. D zone with clavulanic acid

ESBL not Class A or Class C presentESBL not Class A or Class C present

I. No potentiation with clavulanic acid

II. Susceptibility to cefoxitin

III. Resistance to any one of ceftazidime, ceftriaxone or Aztreonam

ESBL absentESBL absentI. No potentiation with clavulanic acid

II. Susceptibility or resistance to cefoxitin

III. Susceptibility to all of Ceftazidime, ceftriaxone or Aztreonam

Double disc diffusion and Etest to Double disc diffusion and Etest to detect ESBLSdetect ESBLS

• Double disc diffusion

• Etest

Technique type Test Advantages Disadvantages

Clinical microbiolo

gy

Standard NCCLS interpretive criteria

Easy to use, performed in every lab

ESBLs not always “resistant”

NCCLS ESBL confirmatory test

Easy to use and interpret Sensitivity depends on choice of oxyimino-cephalosporin

Double-disk approximation test

Easy to use, easy to interpret Distance of disk placement for optimal sensitivity not

standardized

Three-dimensional test

Sensitive, easy to interpret Not specific for ESBLS, labor intensive

Etest ESBL strips Easy to use Not always easy to interpret, not as sensitive as double-disk

test

Vitek ESBL test Easy to use, easy to interpret Reduced sensitivity

Molecular detection

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