Multipoint microbiological assay for detecting f-lactamase

A multipoint microbiological assay for determining f-lactamase production by clinical isolates of bacteria was evaluated. With strains of Haemophilus influenzae, Neisseria gonorrhoeae, and Branhamella catarrhalis there was excellent correlation between results obtained using this method and those obtained using the chromogenic cephalosporin reference method. The multipoint method is an inexpensive yet reliable adjunct to conventional susceptibility testing methods. Department of Bacteriology, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF K J Thickett T G Winstanley Accepted for publication 21 November 1990 The susceptibility testing of micro-organisms such as Branhamella catarrhalis, Haemophilus influenzae, and Neisseria gonorrhoeae using the agar-dilution method has been described.' One drawback of this method is that traditional indicators of,-lactamase activityreduced zone size and increased colony size at zone edges around antibiotic discs-are absent. We believe B catarrhalis poses a particular problem (unpublished observations). Although around 50% of strains are resistant to ampicillin as indicated by ,B-lactamase production,2 many appear susceptible using the accepted ampicillin "break-point" of 1 mg/I.' With B catarrhalis, therefore, it is imperative to determine whether ,B-lactamase is produced if an agar dilution susceptibility testing method is to be used. Detection of f-lactamase production in H influenzae and N gonorrhoeae also has important clinical implications. Diagnostic laboratories use four methods for ,B-lactamase detection. Hydrolysis of the filactam bond yields a dibasic acid which can be detected by a change in pH-the acidometric method4 or by the ability to reduce iodinethe iodometric method.5 With the chromogenic cephalosporin,6 an electron shift in the cephalosporin molecule yields a coloured product. Loss of antibacterial activity may also be detected by microbiological assays which use susceptible bacteria to detect breakdown of fl-lactam antibiotics by organisms which are applied by streaking, or on membranes or filter paper discs.7 The origins of such assays lie in clinical observations of "indirect pathogenicity".8 Of these methods, the microbiological assay has proved the easiest to adapt to multipoint methodology. Methods Diagnostic Sensitivity Test agar (DST:Oxoid) was supplemented with lysed horse blood (7%) and nicotinamide adenine dinucleotide (Boehringer Mannheim; 20 mg/l) to facilitate growth of fastidious organisms. An antiswarming agent (1-4-Nitrophenl-glycerol: 55 mg/l) and penicillin (0-02 mg/l) were added and the plate was surface seeded with Staphylococcus aureus (NCTC 6571: IO' cfu/ml). This plate was inoculated with the bacteria under test after the conventional antibiotic "break-point" set had been inoculated. Overnight incubation in 7% carbon dioxide showed a "satellite" of growth around organisms producing ,B-lactamase (figure). To assess the reliability of the multipoint method we compared it with the chromogenic 435 group.bmj.com on June 23, 2017 Published by http://jcp.bmj.com/ Downloaded from