Isolation of ESBL producing E. coli from Urine

By Acharya Tankeshwar •  Updated: 05/28/22 •  6 min read

Collection of  Urine Specimen:

  1. Instruct the patient to collect midstream urine.
  2.  Give the patient a sterile, dry, wide-necked leak-proof container and explain the importance of collecting a specimen with as little contamination as possible.
  3. Instruct the female patients to cleanse the area around the urethral opening with clean water, dry the area, and collect the urine with the labia held apart.
  4. Collect about 10-15 ml of urine. If the patient is in renal failure or a young child, it may not be possible to obtain more than a few milliliters of urine.

Labeling and rejection criteria: Label the specimen container appropriately with registration/collection number, patient name, type of specimen, date and time of collection, and the tests requested.


  1. As soon as possible, deliver the specimen with a request form to the laboratory.
  2. Keep the urine in refrigerator at 4ºC, if immediate delivery to the laboratory is not possible.
  3. Add boric acid (0.1g/10ml) to the urine, if a delay in delivery of more than 1 hour is anticipated.

Microbiologic methods for detection of ESBL E.coli

a.       General consideration

  1. Escherichia coli is the predominant cause of both community and nosocomial urinary tract infection (UTI) and also is the commonest cause of uncomplicated UTI in women and children.
  2. Extended-spectrum ß-lactamases (ESBL) are a rapidly evolving group of ß-lactamases that share the ability to hydrolyze third-generation cephalosporins and aztreonam but are inhibited by clavulanic acid. Increasing resistance to third-generation cephalosporins amongst E. coli is predominantly due to the production of ESBLs. These plasmid-mediated enzymes mostly evolved via point mutations of the classical TEM-1 and SHV-1  β-lactamase.
  3. Accurate laboratory detection is important to avoid inappropriate antimicrobial therapy and clinical failure.

Culture and identification of Escherichia coli from Urine 

The standard urine culture protocol used 1 μl (0.001 mL) of urine, spread quantitatively onto 5% sheep blood agar plate and MacConkey agars, and incubated aerobically at 35°C for 24 h.


  1. A loopful of well-mixed midstream urine sample is inoculated into the blood agar plate, MacConkey agar plate and CLED (Cysteine Lysine Electrolyte Deficient agar) or using the semi-quantitative method (i.e with the help of the calibrated loop of 4 mm size)
  2. A loopful of the urine is touched to the center of the plate from which the inoculum is spread in diameter across the plate. Without flaming or reentering the urine, a loop is drawn across the entire plate, crossing the first inoculum streak numerous times.
  3. The Blood Agar plate and CLED or Mac-Conkey agar plate both are incubated aerobically at 37°C for 24 hours.
  4. The following day the plates are observed for the growth of the organism, their characteristics on the plates, or any possible contamination.

Interpretation of the result:

  1. Less than 104 organisms/ml: not significant
  2. 104– 10organisms/ml: doubtful significance (suggest repeat collection)
  3. More than 105 organisms/ml: significant bacteriuria

Only samples showing significant growth are further processed.


In certain conditions, even the growth between 103-10is also considered significant. Those conditions are:

  • Patient being on diuretics
  • Patients being on antimicrobial

In certain conditions, even any count is considered significant. Those conditions are

  • Specimen obtained from catheter tubing
  • Suprapubic aspirate
  • Suspected hematogenously acquired  infection (eg. Staphylococcus  aureus)

ii) Colony morphology

  • Typical colonies on MacConkey agar will appear pink, shiny, and have a diameter of 0.5 – 1 mm after overnight incubation.
  • Colony appearance varies from grey to white, transparent to opaque, and raised convex to flat on blood agar plates.

 iii) Presumptive identification.

 Confirmatory tests for E. coli

 The suspected organism is subjected to various biochemical tests such as SIM, MR-VP, Urease, Citrate Utilization, and TSI tests.

IsolateTSISIMCitrateUreaseMethyl RedInference
Lactose fermenting (pink) on MacConeky Agar  or yellow colonies in CLEDAcid/Acid,
Gas +
H2S not produced Indole +ve, motileNegativeNegativePositiveE. coli

Note: However some E.coli belonging to the Alkaligens-Dispar (A-D) group may be non-lactose fermenting on Mac-Conkey agar. All the other characteristics are similar to E.coli.  

Antimicrobial susceptibility testing

  • Perform the susceptibility test by a disc-diffusion method using standard methods as described in the guidelines.
  • For detection of ESBL producing E.coli, the isolate screened should be multidrug-resistant exhibiting resistance to at least one of the third-generation cephalosporins.

Screening test for ESBL producing E.coli

According to the CLSI guidelines, isolates showing an inhibition zone size of ≤ 22 mm with ceftazidime (30 µg), ≤ 25 mm with ceftriaxone (30 µg), and ≤ 27 mm with cefotaxime (30 µg) are identified as potential ESBL producers and shortlisted for confirmation of ESBL production.

Confirmatory tests for ESBL

According to CLSI guidelines suspected ESBL producers can be confirmed by two phenotypic methods:

Combination disc method

This test requires the use of a third-generation cephalosporin antibiotic disc alone and in combination with clavulanic acid. In this study, a disk of either ceftazidime (30µg)/ cefotaxime(30µg) /cefpodoxime (30µg) alone and a disk of corresponding clavulanic acid either ceftazidime – clavulanic acid (30 µg/10 µg)/ cefotaxime – clavulanic acid (30/10 µg)/cefpodoxime – clavulanic acid (30/10 µg) is used. Both the disks are placed at least 25 mm apart, center to center, on a lawn culture of the test isolate on Mueller Hinton Agar (MHA) plate and incubated overnight at 37°C. The difference in zone diameters with and without clavulanic acid is measured.

Interpretation: An increase of ≥ 5 mm in inhibition zone diameter around the combination disk of cephalosporin- clavulanic acid versus the inhibition zone diameter around cephalosporin disk alone, confirms ESBL production.

Double disc synergy method

This test requires two discs of third-generation cephalosporin either, cefotaxime, ceftazidime, or cefpodoxime. A ceftazidime 30µg disc and an amoxicillin+ clavulanic acid 20+10 µg disc are then placed 25 – 30 mm apart, center-to-center on a lawn culture of the test isolate on Mueller Hinton Agar (MHA) plate. Incubate overnight in air at 37°C.

Interpretation: ESBL production is inferred when the zone of inhibition around the ceftazidime disc is expanded by the clavulanate in a cloverleaf fashion.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

2 responses to “Isolation of ESBL producing E. coli from Urine”

  1. EKILA IFINJI Richard says:

    I’m very happy for what I use to read in this blog. But I’d like to have more details about streptococcus identification tests and enterococcus. In our our lab, we use bacitracine for beta hemolytic strains, optochin test for alpha hemolytic strains and bile esculine test for no hemolytic strains. If you have some suggestions to tell me,
    Kind regards!

  2. V for Vendetta says:

    Please, I hope you reply to my question this time around. Thank you for your good work.
    Please, how EXACTLY does one count colonies when estimating for significant bacteriuria? Especially colonies that have matted together.
    Thank you.

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