SOP for processing of urine specimen for laboratory diagnosis of Extended spectrum Beta lactamase producing E.coli

 Collection of 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.

Labelling 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.

Transport

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 1hr 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 infections  in women and children.
2. Extended-spectrum ß-lactamases (ESBL) are a rapidly evolving group of ß-lactamases which 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 extended-spectrum b-lactamases (ESBLs). These plasmid mediate enzymes mostly evolved via point mutations of the classical TEM-1 and SHV-1 b-lactamases.
3. Accurate laboratory detection is important to avoid  inappropriate antimicrobial therapy and clinical failure.

Culture and identification.

i)   Culture

1. A loopful of well mixed mid stream urine sample is inoculated into the Blood Agar plate and CLED(Cysteine Lysine Electrolyte Deficient agar) or Mac-Conkey Agar plate 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 a diameter across the plate. Without flaming or reentering the urine, 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 37⁰C 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- 10⁵ organisms/ml- Doubtful significance (suggest repeat collection)
3. More than 10⁵ organisms/ml- Significant bacteriuria

Only samples showing significant growth are further processed.

NOTE: In certain conditions even the growth between 10³-10⁵ is also considered significant. Those conditions are:

a) Patient being on diuretics

b) Patients being on antimicrobial

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

a) Specimen obtained from catheter tubing

b) Suprapubic aspirate

c) Suspected heamtogenously acquired  infection (eg.Staphylococcus  aureus )

ii)  Colony morphology

• Typical colonies on Mac -Conkey agar will appear pink,shiny have a diameter  of 0.5 - 1 mm after over night incubation.
• Colony appearance varies from grey to white, transparent to opaque and raised convex to  flat on Blood agar plates.

 iii) Presumptive identification.

• Perform Gram staining  of suspected colonies (Gram negative bacilli).
• Perform oxidase test and catalase test.

Note:   Colonies should be picked with a wooden tooth pick or platinum wire. Use of a chrome wire may cause a false positive oxidase test.

9.3  Confirmatory tests

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

Isolate	TSI	SIM	Citrate	Urease	Methyl Red	Inference
Lactose fermenting (pink)on MA or yellow colonies in CLED	Acid/Acid,Gas +	H2S not produced Indole + Motile	Negative	Negative	Positive	E. 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.

9.4   Antimicrobial susceptibility testing

• Perform the susceptibility test by 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.

9.5  Screening test for ESBL producing E.coli
According to the CLSI guidelines, isolates showing 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.

9.6       Confirmatory tests for ESBL

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

i) 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)/ Ceftazidime - 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. Difference in zone diameters with and without clavulanic acid is measured.

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

ii) Double disc synergy method : This test requires two discs of third generation cephalosporin either,cefotaxime,ceftazidime or cefpodoxime. A ceftazidime 30 mg disc and an amoxicillin+ clavulanic acid 20+10 mg disc are then placed 25 - 30 mm apart, centre-to-centre.on a lawn culture of the test isolate on Mueller Hinton Agar (MHA) plate. Incubate overnight in air at 37⁰C.

 Interpretation: ESBLproduction is inferred when the zone of inhibition around the ceftazidime disc is expanded by the clavulanate in a clover leaf fashion