Novel Method for Detection of β-Lactamases by Using a Chromogenic Cephalosporin Substrate

Abstract

A new cephalosporin with a highly reactive 3-lactam ring was found to give an immediate color change in the presence of 3-lactamases from many bacteria, including staphylococci, Bacillus species, Enterobacteriaceae, and Pseudomnonas. The reaction is confined to organisms producing ,B-lactamases, but it is sufficiently sensitive to indicate the presence of this enzyme in small amounts in strains previously considered not to produce it. The compound has an unusual ultraviolet spectrum, and the color change can be followed quantitatively by measuring changes in absorption which occur in the 380-to 500-nm region, where cephalosporins normally have no absorption. The development of color is thought to be a consequence of the 3-lactam ring being unusually highly conjugated with the 3-substituent. Although in the bacteria only 3-lactamases produce this color change, it was found that serum and tissues from experimental animals also rapidly produced the colored breakdown product, which was then excreted in the urine. The mechanism of the mammalian breakdown was considered to be different from that found in bacteria.There are several techniques available for the detection and estimation of ,3-lactamases (2). Some are normally used quantitatively, and others can also be applied as qualitative spot tests. None of these methods is entirely satisfactory and, for carrying out spot tests, there is a requirement for a new reliable and sensitive diagnostic agent. This report describes the properties of such a substance. In the course of a screening program, a new cephalosporin was made: 3-(2,4-dinitrostyryl)-(6R, 7R)-7-(2-thienylacetamido) -ceph-3-em-4-carboxylic acid, E-isomer. It will be referred to here under the code number 87/312, and has the structure shown in Fig. 1 Spectrophotometric method for determination of g-lactamase activity. The method used was adapted from one described previously using cephaloridine as a substrate (9), in which the rate of breakdown of this substrate in the presence of g-lactamase was determined by measuring the rate of change of the ultraviolet absorption associated with the ,3-lactam ring.In aqueous solution at pH 7, 87/312 has two main absorption peaks, one at 217 nm and one at 386 nm (Fig. 2). The peak at 217 nm is associated with the 7-acyl group, and no change in absorbance occurred after the enzymatic hydrolysis. (Fig. 2). There was also a change in spectrum and color when 87/312 was attacked by serum; in this case, the decrease at 386 nm was the same, but the new peak was formed at 510 nm instead of 482 nm. At this wavelength, the maximal change observed was from 0.12 to 1.74 for a solution of 10-4 M.The shift in wavelength is due to serum binding (Fig. 3).

A new method to study permeation of β‐lactam antibiotics into reconstituted vesicles from the outer membrane of Pseudomonas aeruginosa NCTC 10662

“…The product(s) had E~TcSrnOf 15 000 [15]. Hydrolysis of cefamandole was followed at 275 nm; an E27cSmOf 6800 was calculated.…”

Transcriptional analysis of the dd-peptidase/penicillin-binding protein-encoding dac gene of Streptomyces R61: Use of the promoter and signal sequences in a secretion vector

“…Do-carboxypeptidase-producing Streptomyces colonies were detected using the immunological test described in Duez et al (1987) except that the [125I]-labelled protein A was replaced with alkaline phosphatase coupled to goat antibodies directed against rabbit IgG (Bio-Rad immunoblot assay kit). /LLactamase activity in culture fluids was estimated with nitrocefin as substrate (O'Callaghan et al 1972) and/Llactamase-producing Streptomyces colonies were detected as described in Dehottay et al (1986). 115 Cellfractionation.…”

A novel isoquinoline alkaloid, DD-carboxypeptidase inhibitor, with antibacterial activity isolated from Streptomyces sp. 8812. Part I: Taxonomy, fermentation, isolation and biological activities

“…34 A mixture of b-lactamase and JS-1 in 0.1 M phosphate buffer, pH 7.0, was incubated for 30 min at 37 1C; thereafter, nitrocephin was added and the mixture was incubated for an additional 10 min at 37 1C. Subsequently, absorbance was measured at 482 nm.…”