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Beta-Lactamase 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 8/29/2019 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. Page 1 of 3 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. Page 2 of 3 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 Page 3 of 3 .
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