Extend Spectrum Beta-Lactamase (ESBL) strain of Escherichia coli Isolation: Introduction, Isolation Methods and Result Interpreation

Extend Spectrum Beta-Lactamase (ESBL) strain of Escherichia coli Isolation- Introduction, Isolation Methods and Result Interpreation

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

Screening Method

Antibiotics      Zone of Inhibition (ZOI)

Ceftazidime(30µg)  ≤ 22 mm

Cefotaxime (CTX ) ≤ 27 mm

Cefpodoxime (CPD) ≤ 17 mm

Confirmatory method

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

  1. Collect and label the urine sample from OPD or indoor patients requested for culture/sensitivity.
  2. Subject the sample to culture on MacConckey Agar plate and incubate at 37° C for 24 hours.
  3. Identify E.coli by different biochemical tests or serological tests.
  4. 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.
  5.  Measure the diameter of the zone of inhibition.
  6.  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.
  7. Isolates may be either sensitive or resistant to Nalidixic Acid, Nitrofurantoin, Gentamicin, or Ciprofloxacillin.
  8. Confirm the ESBL strain by using Cefpodoxime, Cefpodoxime with Clavulanate, Carbapenems (Meropenem).
  9. Compare the zone of inhibition of the Cepfodxime disc to the zone of inhibition of cefpodoxime with the Clavulanate disc.
  10. 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).
  11. 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

  1. 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.
  2. For uniformity of the test use the antibiotic discs from Oxoid or MAST.
  3. Control strains are  K. pneumoniae ATCC 7000603 (ESBL positive control strain) and  E.coli ATCC 25922 (ESBL negative control strain).

Further Reading

  1. https://clsi.org/media/3481/m100ed30_sample.pdf
  2. Ahmed I, Salam A. Extended-spectrum beta-lactamases and bacterial resistance. PAKISTAN JOURNAL OF MEDICAL SCIENCES. 2002;18(2):151-5.
  3. Knothe H, Shah PDP, Krcmery V, Antal M, Mitsuhashi S. Transferable resistance to cefotaxime, cefoxitin, cefamandole, and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection. 1983;11(6):315-7.
  4. Jacoby GA, Medeiros AA. More extended-spectrum beta-lactamases. Antimicrobial Agents and Chemotherapy. 1991;35(9):1697
  5. Du Bois S, Marriott M, Amyes S. TEM-and SHV-derived extended-spectrum β-lactamases: relationship between selection, structure, and function. Journal of Antimicrobial Chemotherapy. 1995;35(1):7-22
  6. Rupp ME, Fey PD. Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. Drugs. 2003;63(4):353-65.
  7. Paterson DL, Ko W-C, Von Gottberg A, Mohapatra S, Casellas JM, Goossens H, et al. Antibiotic therapy for Klebsiella pneumoniae bacteremia: implications of production of extended-spectrum β-lactamases. Clinical Infectious Diseases. 2004;39(1):31-7.
  8. Paterson DL, Ko W-C, Von Gottberg A, Casellas JM, Mulazimoglu L, Klugman KP, et al. Outcome of cephalosporin treatment for serious infections due to apparently susceptible organisms producing extended-spectrum β-lactamases: implications for the clinical microbiology laboratory. Journal of clinical microbiology. 2001;39(6):2206-12.
  9. Gold HS, Moellering Jr RC. Antimicrobial-drug resistance. New England Journal of Medicine. 1996;335(19):1445-53.
  10. Ahmed AA, Osman H, Mansour AM, Musa HA, Ahmed AB, Karrar Z, et al. Antimicrobial agent resistance in bacterial isolates from patients with diarrhea and urinary tract infection in Sudan. The American journal of tropical medicine and hygiene. 2000;63(5):259-63.
  11. Medeiros AA. Nosocomial outbreaks of multiresistant bacteria: extended-spectrum beta-lactamases have arrived in North America. Annals of internal medicine. 1993;119(5):428-30.
  12. Merie Queenan A, Bush K. carbapenemases: the versatile B-lactamases. Clin Microbiol Rev. 2007;20(3):440-58
  13. Forbes BA, Sahm DF, Weissfeld AS, Trevino E. Bailey & Scott’s diagnostic microbiology, Mosby. Inc, St Louis. 2002.
  14. Bradford PA. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clinical microbiology reviews. 2001;14(4):933-51.
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