Rapid Method for Screening Large Numbers of Escherichia coli Colonies for Production of Plasmid-Mediated β-Lactamases

Abstract

A rapid simple assay for screening large numbers of E. coli colonies for production of certain plasmid-mediated .beta.-lactamases (including TEM-1, TEM-2, HMS-1, SHV-1, OXA-1, PSE-1, PSE-4 and CEP-2) is described. The technique, a modification of the method of Slack et al. for detection of .beta.-lactamase in limited numbers of Haemophilus influenzae clinical isolates, uses filter paper impregnated with benzylpenicillin and a pH indicator dye (bromocresol purple) that changes color in the presence of .beta.-lactamase activity. The test paper is briefly applied to an agar surface containing bacterial colonies which are subsequently scored individually on the paper by color; yellow indicates the presence of .beta.-lactamase, dark green, its absence, and variegation (yellow and dark green), a mixed population. Concordance of results of this assay with those of replica plating for antibiotic resistance was > 99%. Hundreds of colonies per plate can be scored quickly and remain viable for further evaluation. The assay is useful for studies of the stability of plasmids encoding .beta.-lactamases and in cloning with vectors such as pBR322 in which insertion of DNA fragments can be detected by inactivation of the .beta.-lactamase gene. Whenever the assay is used, results should be confirmed initially by another method, such as replica plating, because the test paper assay does not detect 3 .beta.-lactamases (OXA-2, OXA-3 and PSE-2) and misses intrinsic penicillin resistance.