Laboratory Diagnostic Of Ctx-m Eschericia Coli

CTX-M βlactamase in (Detection in Laboratory)

INTRODUCTION 1.E.coli are the most frequently associated with ESBL. 2.In the Class A ESBLs based on Ambler Classification. 3.ESBLs are enzmes that mediate resistance to extended-spectrum (third generation) cephalosporins and monobactams.

INTRODUCTION 4. ESBLs can be difficult to detect because they have different levels of activity against various cephalosporins. 5. Thus, the choice of which antimicrobial agents to test is critical. 6. Any decrease in the zone sizes or MIC less than 2 mg/L for third generation cephalosporins should be

METHOD DETECTION

Antimicrobial Susceptibility Standard Disc Diffusion Method 1. In-vitro sensitivity testing using established NCCLS (National Committe for Clinical Laboratory Standards) procedure is carried out with : •Ceftazidime (30 µg) •Cefotaxime (30 µg) •Ceftriaxone (30 µg) •Aztreonam (30 µg) •Cefpodoxime (10 µg)

Antimicrobial Susceptibility Standard Disc Diffusion Method 2. Zone diameters are read using the revised NCCLS document where any zone within “grey zone” are considered as a probable ESBL producing strain which requiring phenotypic confirmatory testing

Antimicrobial Susceptibility Standard Disc Diffusion Method

Antimicrobial Susceptibility Standard Disc Diffusion Method

Double Disk Synergy / Disk Approximation Method 1. This method uses multiple target disc with clavulanic acid disc or a single cefpodoxime disc with clavulanic acid disc. 2. Mueller – Hinton agar plate is inoculated with a suspension (0.5 McFarland) made from an overnight agar plate of the test strain

Double Disk Synergy / Disk Approximation Method 3. Disc containing the standard, • Ceftazidime (30 µg) • Ceftriaxone (30 µg) • Aztreonam (30 µg) • Cefpodoxime (10 µg) 4. Plates are then incubated overnight at 35°C. 5. Enhancement of zone inhibition is indicative of presence of an ESBL

Double Disk Synergy / Disk Approximation Method Zone Enhanceme nt

Disc Diffusion Method 1. Ceftazidime (30 µg) versus ceftazidime / clavulanic (30 / 10 µg) and cefotaxime (30 µg) versus (cefotaxime / clavulanic acid (30 / 10 µg) are placed onto a Muller – Hinton agar plate lawned with the test organism and incubated at 35°C.

Disc Diffusion Method 2. Result : Clavulnic acid restores activity of cefotaxime or ceftazidime or both 3. QC : E.Coli ATCC 25922

Disc Diffusion Method 2. E-test combination strips, example ceftazidime / ceftazidime – clavulanic acid and cefotaxime / cefotaxime – clavulanate are employed to perform the phenotypic confirmatory testing 3. Strips are inoculated on the surface of the agar plate and incubated overnight 4. Any reduction of ≥ 3 log 2

Disc Diffusion Method

Ceftaz/ CA

Cefta z

Others Method •

Molecular detection •

PCR and sequencing • • • •

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The gold standard Can detect all variants Easy to perform Labor intensive

Isoelectric focusing (IEF)

Automated System •

Microscan, Vitek2 & Phoenix