Rapid method for screening large numbers of Escherichia coli colonies for production of plasmid-mediated beta-lactamases.

A rapid, simple assay for screening large numbers of Escherichia 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 (Lancet ii:906, 1977), 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 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 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 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 beta-lactamases (OXA-2, OXA-3, and PSE-2) and also would miss intrinsic penicillin resistance.