Introduction of Extend spectrum beta-lactamase (ESBL) strain of Escherichia coli Isolation
On the last refresher training workshop of Antimicrobial Resistance (AMR) Surveillance, it has been decided to expand the scope of AMR surveillance by including the surveillance of Extended-spectrum β- lactamase ( ESBL) strain of E. coli from urine samples of OPD/Indoor patients Iactam antimicrobial agents in Gram-negative bacilli including E. coli. The term extended-spectrum β- lactamase is generally applied to plasmid-encoded β- lactamases that are capable of inactivating extended-spectrum cephalosporins and are inhabited by β lactamase inhibitors, such as clavulanic acid. In modern medical practice, newer antimicrobials drugs have been used extensively resulting in the emergence and rapid dissemination of resistant bacterial strains. Since one of the mechanisms of bacterial resistance to a β-lactam antibiotic is the production of β- lactamase enzyme that breaks down the structural β- lactam ring of penicillin and its synthetic derivatives. The property of stability to many bacterial β- lactamase was increased with the later generations of cephalosporins. However, the persistent exposure of bacterial strains to a multitude of β- lactams have induced a dynamic and continuous production and mutation of lactamase in many bacteria, expanding their activity even against the third and fourth-generation cephalosporins. These new β- lactamases are called ESBLs which were first reported in Germany.
ESBL Detection Method
Antibiotics Zone of Inhibition (ZOI)
Ceftazidime(30µg) ≤ 22 mm
Cefotaxime (CTX ) ≤ 27 mm
Cefpodoxime (CPD) ≤ 17 mm
Put ceftazidime (30 µg) and combination of ceftazidime (30 µg) and clavulanic acid ( 10 µg) disks with a distance of 20 mm from disk to disk on Muller-Hinton agar as shown above picture.
After overnight incubation, ZOI of ceftazidime clavulanic acid (CAC) should be equal to and greater than 5 mm that of ceftazidime.
The above isolate is showing the ESBL producer.
Requirements for Extend spectrum beta-lactamase (ESBL) strain of Escherichia coli Isolation
Proposed Method for Extend spectrum beta-lactamase (ESBL) strain of Escherichia coli Isolation
- Collect and label the urine sample from OPD or indoor patients requested for culture/sensitivity.
- Subject the sample to culture on MacConckey Agar plate and incubate at 37° C for 24 hours.
- Identify E.coli by different biochemical tests or serological tests.
- Perform antibodies susceptibility test of isolated E. coli using discs Ampicillin, Nalidixic Acid, Nitrofurantoin, Gentamycin, Ciprofloxacin, Cefotaxime (30 μg), Ceftazidime ( 30 μg), Cefpodoxime (10 μg) on Meuller Hinton Agar.
- Measure the diameter of the zone of inhibition.
- If the isolated strain of E. coli is resistant to Ampicillin and all cephalosporin groups (Cefotaxime, Ceftazidime, and Cefpodoxime), label the organism as suspected to be ESBL strain.
- Isolates may be either sensitive or resistant to Nalidixic Acid, Nitrofurantoin, Gentamicin, or Ciprofloxacillin.
- Confirm the ESBL strain by using Cefpodoxime, Cefpodoxime with Clavulanate, Carbapenems (Meropenem).
- Compare the zone of inhibition of the Cepfodxime disc to the zone of inhibition of cefpodoxime with the Clavulanate disc.
- If the difference between the two is (Cepfodxime disc and cefpodoxime/clavulanic acid disc) greater than or equal to 5mm (i.e >5mm), and strain is also sensitive to Carbapenems, report the isolate as ESBL E. coli (Clavulanic acid is the inhibitor of the β-lactamases).
- Preserve such ESBL E.coli isolate send to national reference laboratory of the country.
Zone of Inhibition for screening discs in Extend spectrum beta-lactamase (ESBL) strain of Escherichia coli Isolation
Antibiotics Screening zone size
- Cefotaxime (30 μg) <27mm
- Ceftazidime ( 30 μg) <22mm
- Cefpodoxime (10 μg) <17mm
Keynotes on Extend spectrum beta-lactamase (ESBL) strain of Escherichia coli Isolation
- The use of cefpodoxime is recommended in preference to cefotaxime or ceftazidime as per (CLSI documents) because cefpodoxime appears to be the most sensitive single substrate for the detection of ESBLs.
- For uniformity of the test use the antibiotic discs from Oxoid or MAST.
- Control strains are K. pneumoniae ATCC 7000603 (ESBL positive control strain) and E.coli ATCC 25922 (ESBL negative control strain).
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