Rapid Method for Screening Large Numbers ofEscherichia coliColonies for Production of Plasmid-Mediated β-Lactamases

Abstract

A rapid, simple assay for screening large numbers ofEscherichia colicolonies for production of certain plasmid-mediated β-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 β-lactamase in limited numbers ofHaemophilus influenzaeclinical isolates (Lancet ii:906, 1977), uses filter paper impregnated with benzylpenicillin and a pH indicator dye (bromocresol purple) that changes color in the presence of β-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 β-lactamase, dark green its absence, and variegation (yellow and dark green) a mixed population. Concordance of the results of this assay with those of replica plating for antibiotic resistance was over 99%. Hundreds of colonies per plate can be scored quickly and remain viable for further evaluation. The assay appears to be useful for studies of the stability of plasmids encoding β-lactamases and in cloning with vectors such as pBR322 in which insertion of DNA fragments can be detected by inactivation of the β-lactamase gene. Whenever the assay is to be used, results should always be confirmed initially by another method, such as replica plating, because the test paper assay does not detect three β-lactamases (OXA-2, OXA-3, and PSE-2) and also would miss intrinsic penicillin resistance.