Lab-Policies-Beta-Lactamase-Testing-by-Chromogenic-Cephalosporin-Lab-3340

Standard Operating Procedure

Subject Beta-Lactamase Testing by Chromogenic Cephalosporin
Index Number Lab-3340
Section Laboratory
Subsection Microbiology
Category Departmental
Contact Sarah Stoner
Last Revised 7/13/2017

References

Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers
for Medicare and Medicaid Services (CMS) and / or COLA.

Applicable To

Employees of Gundersen Health System laboratory, Gundersen St. Joseph’s Hospital laboratory,
Gundersen Palmer Lutheran Hospital laboratory, Gundersen Boscobel Area Hospital laboratory, and
Gundersen Tri-County Memorial laboratories.

Detail

This document establishes guidelines for performing cefinase testing for detection of beta lactamase in
unknown isolates.

PRINCIPLE:
The ability of certain bacteria to produce enzymes which inactivate beta lactam antibiotics, i.e.,
penicillins and cephalosporins, has long been identified. The most commonly used clinical procedures
include the iodometric method, the acidometric method, and a variety of chromogenic substrates. The
iodometric and acidometric tests are generally performed using penicillin as a substrate and therefore
can only detect enzymes which hydrolyze penicillin. One of the chromogenic cephalosporins, PADAC
(Calciochem-Behring) has been found effective in detecting most of the known beta-lactamases except
for some of the penicillinases produced by staphylococci, and some beta-lactamases produced by
anaerobic bacteria. Another chromogenic cephalosporin, Nitrocefin (Glaxo Research), has been
found effective in detecting all known beta-lactamases including the staphylococcal penicillinases.

The Cefinase disc is impregnated with the chromogenic cephalosporin, Nitrocefin. This compound
exhibits a very rapid color change from yellow to red as the amide bond in the beta lactam ring is
hydrolyzed by a beta-lactamase. When a bacterium produces this enzyme in significant quantities,
the yellow-colored disc turns red in the area where the isolate is smeared. Although other penicillin and
cephalosporins may be used as substrates for specific enzymes, Nitrocefin has the wide spectrum of
susceptibility and sensitivity of the commercially available beta lactams. It is not known to react with
other microbial enzymes. Each disc is used to test one bacterial strain for the presence of beta-
lactamase.

CLINICAL SIGNIFICANCE:
Bacteria possessing the enzyme beta-lactamase have the ability to break down beta lactam drugs such
as penicillins and cephalosporins. Detection of beta-lactamase producing strains allows the physician to
choose appropriate antibiotic therapy.

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Standard Operating Procedure

SPECIMEN:
Isolated colony to be tested.
REAGENTS / MATERIALS:
Cefinase disc impregnated with Nitrocefin. Store unopened at -20o to +8o C.
After use, the Cefinase cartridge should be stored in a sealed zip-lock bag containing desiccant and
stored at -20o to +8o C. Discard any discs 60 days after opening package. Do not use the cartridge if the
discs appear orange or red in color.
Sterile distilled water
Sterile loop or sticks
EQUIPMENT / INSTRUMENTATION: N/A
CALIBRATION: N/A
CALCULATIONS: N/A
QUALITY CONTROL:
Refer to Lab-3255 Quality Control - Microbiology Department.
Positive and negative controls are done with each new lot number or shipment of Cefinase discs.
Staphylococcus aureus ATCC 29213 Positive
Haemophilus influenzae ATCC 49247 Negative

Implementation

1. Using a disc dispenser, dispense required number of discs into an empty petri dish.
2. Moisten each disc with 1 drop of sterile water.
3. With sterile loop or applicator stick, remove several well-isolated similar colonies and smear

onto the disc surface.
4. Observe for color change.
Inducable Beta-lactamase
Organisms grown in the presence of oxacillin may exhibit an inducible beta-lactamase. Any beta-
lactamase negative staphylococcus should be rechecked after being exposed to penicillin overnight.
1. Streak the organism to be tested to a BAP.
2. The Vitek purity plate can be used for this purpose by dropping a penicillin disk onto the

inoculated purity plate.
3. Incubate at 35oC overnight.
4. Perform test using colonies surrounding the penicillin disk.
INTERPRETATION:
1. A positive reaction will show a yellow to red change on the area where culture was applied.
2. A negative result will show no color change on disc.

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Standard Operating Procedure

3. Haemophilus influenzae approximate reaction time is 1 minute. Resistant to ampicillin for a
positive reaction.

4. Neisseria gonorrhoeae and Moraxella catarrhalis approximate reaction time is 1 minute.
Resistant to penicillin for a positive reaction.

5. Staphlococcus aureus approximate reaction time is 1 hour. Resistant to penicillin, ampicillin,
carbenicillin and ticarcillin. Probably susceptible to cephalothin, methicillin, oxacillin, nofcillin
and other penicillinase-resistant penicillins for a positive reaction.

6. Enterococcus faecalis approximate reaction time is 5 minutes. Resistant to penicillin and
ampicillin for a positive reaction.

7. Anaerobic bacteria approximate reaction time 30 minutes. Probable identification if Bacteroides
species. Probable resistant to penicillin and may be resistant to cephalosporins including
cefotaxime and rarely cefoxitin.

8. If procedure results are questionable, other technologists or department medical director
should be consulted. The tests should be repeated or a new culture or stain requested if
deemed necessary.

LIMITATIONS OF THE PROCEDURE:
The efficacy of this test in predicting the beta-lactam resistance of microorganisms other than Neisseria
gonorrhoeae, Haemophilus influenzae, Moraxella catarrhalis, staphylococci, and certain anaerobic
bacteria is unproven. Resistance to beta-lactam antibiotics has been on rare occasions reported in some
of the above organisms without the production of beta-lactamases. In these cases, resistance
mechanisms such as permeability barriers have been postulated. Therefore, the beta-lactamase test
should be used as a rapid supplement and not a replacement for conventional susceptibility testing. For
some strains of staphylococci particularly Staphlococcus epidermidis, an inducible beta-lactamase has
been described which might result in a false negative beta-lactamase reaction with a strain which is
resistant to penicillin or ampicillin.
REVIEW AND CHANGES:
This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as
the maximum review date. Review will be done by the Technical Leader, Supervisor, Manager, Medical
Director or designee. Changes require retyping the document or form and review by the Medical
Director.
REFERENCES:

1. BD BBL Cefinase Disk Product Insert, April 2015.
2. Essential Procedure for Clinical Microbiology, Isenberg, Henry D., ASM, Washington D.C., 7th

Ed., 1998, pp. 224-226.
3. CAP Checklist 9/27/2007 MIC.21632

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