A rapid paper-strip method for the detection of penicillinase production by penicillin resistant strains of Staphylococcus aureus.

The size and nature of the zone of inhibition round a benzylpenicillin disc have been widely used as guides to the sensitivity or resistance of a strain of Staphylococcus aureus. However, the diameter of the zone ofinhibition depends on many factors, including inoculum size, depth of medium, and potency of the antibiotic test disc. Inoculum size, in particular, may be difficult to standardise when sensitivity testing is carried out directly from clinical material. The demonstration of penicillinase production is a valuable guide to potential clinical resistance. Many methods for the detection of staphylococcal peni-cillinase have been described, but some are unsuitable for routine use because of their complexity. The principles of the method which we describe here are as follows: Strains of Staph. aureus that are able to produce penicillinase are induced to do so by growth in a subinhibitory concentration of methi-cillin. The induced Pi lactamase hydrolyses added penicillin to form penicilloic acid, resulting in a fall of pH. A suitable indicator is used to demonstrate this pH change. This acidimetric principle has been used previously in an agar overlay method (Wong and Soo-Hoo, 1976). In the present method, however, benzyl-penicillin and an indicator are incorporated into paper strips. When desiccated and stored under optimum conditions the test strips retain their sensitivity for many months. Tests are performed on culture plates similar to those used routinely for the determination of methicillin sensitivity of Staph. aureus isolates. Organisms capable of producing penicillinase do so in the area immediately surrounding the zone of inhibition associated with a methicillin disc. The highly buffered sensitivity test agars tend to obscure the fall in pH caused by the hydrolysis of added benzyl-penicillin; a poorly buffered agar (2% Columbia, BBL) was found to give consistent and satisfactory results. The pH of freshly poured columbia agar was found to be 7-2, but after surface inoculation with Staph. aureus and overnight incubation the pH rose to 8-2-8-6. An indicator which encompassed this latter range but which would register a fall of pH was required; cresol red (pH range 7 2-8 8) was found to be the most satisfactory. Soluble benzylpenicillin (unbuffered), 600 mg, is dissolved in 3 ml 0-1 % aqueous cresol red; 0 1 ml N/10 sodium hydroxide is added. Three to four filter papers (Whatman, No. 1, 9 cm diam) are saturated with this solution. The impregnated papers are placed in the lids of 9 …