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Cefinase™ discs are intended for use in rapid testing of isolated colonies of Neisseria gonorrhoeae, Staphylococcus species, Haemophilus influenzae, enterococci and anaerobic bacteria for the production of β-lactamase.

The ability of certain bacteria to produce enzymes that inactivate β-lactam antibiotics, i.e., penicillins and cephalosporins, has long been recognized. Abraham and Chain in 1940 first recognized an enzymatic activity in extracts of Escherichia coli that inactivated penicillin.¹ Since then a large number of similar enzymes have been isolated from a number of bacterial species with somewhat different substrate specificities. Some selectively hydrolyze penicillin-class antimicrobics (i.e., penicillin G, ampicillin, carbenicillin) and have been described as penicillinases. Others selectively hydrolyze the caphalosporin-class antimicrobics (i.e., cephalothin, cephalexin, cephradine) and have been described as cephalosporinases. Still other enzymes hydrolyze both cephalosporins and penicillins.²

A large number of β-lactamase-resistant penicillin and cephalosporin class antimicrobics have been developed by various pharmaceutical companies. One group includes the semisynthetic penicillins; methicillin, oxacillin, nafcillin and others, which are resistant to the penicillinase enzymes produced by staphylococci.³ A large number of cephalosporins have also been developed which have varying degrees of resistance to β-lactamases. These include the second-generation cephalosporins (cefoxitin, cefamandole and cefuroxime) and third-generation cephalosporins (cefotaxime, moxalactam, cefoperazone and others).⁴

Several clinical tests have been developed for the detection of β-lactamases. These tests provide rapid information predictive of the development of resistance. Interpretation of β-lactamase test results must consider: the sensitivity of the test for different classes of β-lactamase enzymes, the types of β-lactamases produced by different taxonomic groups of organisms and the substrate specificities of the different β-lactamases.

The most commonly used clinical procedures include the iodometric method, the acidometric method, and a variety of chromogenic substrates.⁵ 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 (Calbiochem-Behring) has been found effective in detecting most of the known β-lactamases except for some of the penicillinases produced by staphylococci, and some β-lactamases produced by anaerobic bacteria.⁶ Another chromogenic cephalosporin, nitrocefin (Glaxo Research), has been found effective in detecting all known β-lactamases including the staphylococcal penicillinases.^7-9

In other bacteria, for example, penicillin-resistant Neisseria gonorrhoeae,¹¹ Staphylococcus aureus,^12,13 Moraxella catarrhalis,¹⁴ and ampicillin-resistant Haemophilus influenzae, ^5,9,15 only one class of enzyme is produced by resistant strains. The β-lactamase test performed with these organisms enables a prediction of resistance to be made immediately after primary isolation, 18-24 h prior to the time that growth-dependent susceptibility results would be available.

While the prevalence of β-lacatmase-producing enterococci appears to be small, a low inoculum may result in strains going undetected by susceptibility-testing procedures, and routine screening by the nitrocefin disc procedure is recommended.¹⁶

With anaerobic bacteria, the relationship between the production of β-lactamase and resistance to β-lactam antimicrobics is complicated and somewhat similar to Enterobacteriaceae. β-lactamases are most commonly found within the Bacteroides species,¹⁷ however, β-lactamase-producing strains of Clostridium butyricum, C. perfringens and Fusobacterium sp. have been reported.^18,19 Among the Bacteroides group, a variety of enzymes may be produced with different substrate specificities. The β-lactamases frequently found in strains of Prevotella melaninogenica and P. oralis are usually specific for penicillins (penicillinase),²⁰ whereas the β-lactamases frequently found in the B. fragilis group are cephalosporinases.^21,22 A variety of cephalosporinases have been reported in the B. fragilis group and they include some very active enzymes which can hydrolyze some of the reportedly β-lactamase-resistant cephalosporins such as cefotaxime.^23,24 Rare strains have been reported which hydrolyze at high rates all known β-lactams including cefoxitin.^24,25

Even though the β-lactamases produced by the B. fragilis group are most active against cephalosporins, most strains are found to be resistant to penicillin, carbenicillin and ampicillin in growth-dependent susceptibility tests.^17,26 This finding suggests that the B. fragilis group may be intrinsically resistant to penicillins through factors such as permeability barriers,²² or that the β-lactamase is produced in quantities sufficient to overcome the relatively slow hydrolysis rate of the enzyme with penicillins. Evidence which tends to support a contributory role for β-lactamase in the resistance to penicillins is found in reports that the combination of clavulanic acid (a β-lactamase inhibitor) and penicillins is many times more active against B. fragilis than is the penicillin alone.²⁷

Whatever the cause or causes of penicillin resistance in B. fragilis, all strains should probably be considered resistant.²⁸ The other gram-negative anaerobic strains are probably susceptible to penicillin so long as they are β-lactamase negative.²⁸

Control reference cultures should be run with each group of unknowns. The following organisms are recommended for use as test strains.

Quality control requirements must be performed in accordance with applicable local, state and/or federal regulations or accreditation requirements and your laboratory's standard Quality Control procedures. It is recommended that the user refer to pertinent NCCLS guidance and CLIA regulations for appropriate Quality Control practices.

Cefinase discs impregnated with nitrocefin.

For in vitro Diagnostic Use.

These discs are not for use in susceptibility testing.

Observe aseptic techniques and established precautions against microbiological hazards throughout all procedures. After use, prepared plates and other contaminated materials must be sterilized by autoclaving before discarding.

Nitrocefin induces mutations in certain strains of bacteria (Ames test) and may be sensitizing. Ingestion, inhalation or contact with the skin or eyes should be avoided.