β-lactamase detectionand typing tests-Nitrocefin test

β-lactamase detection and typing tests

β-Lactamases are the leading cause of bacterial resistance topenicillins and cephalosporins. Simple β-lactamase detection and typing tests can be valuable in the clinical laboratory. These include: (i) direct tests for β-lactamase activity infastidious Gram-negative species; (ii) tests for extended-spectrum β-lactamases (ESBLs); and (iii) tests for induci-bility of chromosomal β-lactamases. Indeed, direct β-lactamase tests are mainly used for Haemophilus influenzae, Moraxella catarrhalis and Neisseria spp., wherefew enzyme types occur, and where enzyme production hasclear implications for therapy.

An increasing array of PCR methods for specific detection or typing of β-lactamases is available but these are rarely used for routine purposes. The most commonly used routine test is the nitrocefin test, which is commercially available in various formats.

Nitrocefin test

Nitrocefin, a β-lactam molecule, is a chromogenic cephalosporin substrate used to detect the presence of beta-lactamase enzymes, an essential mediator of bacterial antibiotic resistance. While other detection methods exist, such as PCR, nitrocefin allows rapid detection using few materials and inexpensive equipment. It is sensitive to hydrolysis by all lactamases produced by gram-positive and gram-negative bacteria. This agent undergoes a colour change (from yellow to red on hydrolysis) as its amide bond is hydrolyzed by beta-lactamase, making it useful as a reagent in beta-lactamase activity studies.

The color change is often rapid but can take up to 60 min. The nitrocefin test works well with H. influenzae, Moraxella catarrhalis, N. gonorrhoeae, N. meningitidis and Enterococcus faecalis (β-lactamase production in the last two is rare). The method is less reliable with Staph. aureus, where induction with penicillin or oxacillin may be required, and the nitrocefin test should not be used for Staph because of false-positive reactions. saprophyticus. Alternative methods are the ‘clover leaf’ test, the acidometric method and the iodometric method[1].

Nitrocefinis is a pure powder from Becton Dickinson(Oxford, UK). The powder can also be obtained in commercial preparations， where it is supplied with a vial of diluent and mixed with materials that facilitate solubilization (e.g., Oxoid). Various commercial devices based on nitrocefin are also available (e.g., Oxoid and Becton Dickinson). The method described below is for pure powder; users of other preparations and devices should follow the instructions. Another chromogenic cephalosporin, PADAC,4 exists butis not readily available.

A 0.5 mM solution is prepared by dissolving 2.58 mg of nitrocefin powder in 0.5 mL of dimethylsulphoxide (DMSO), then diluting with 9.5 mL of 0.1 M phosphate buffer, pH 7.0. This solution is stable for 10 days at 4C in a foil-wrapped bottle. Glass containers should be used since DMSO degrades plastics. Colonies of the test isolates are scraped from nutrient agar plates and are suspended in20 uL volumes of 0.1 M phosphate buffer, pH 7.0, to pro-duce a dense suspension on a glass slide, and 20 uL amounts of the nitrocefin solution are added.3-Lactamaseactivity is indicated by a red colour within 1-2 min. Weak activities may take longer to appear, but reactions taking>10 min should be treated with scepticism, as they may reflect the secondary 3-lactamase activity of those penicillin-binding proteins that form unstable acyl complexes.

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