A characteristic vascular lesion has been described in human systemic infections caused by Pseudomonas aeruginosa (Forkner et al., 1958; Margaretten et al., 1961: Rabin et al., 1961). In an attempt to elucidate its pathogenesis, experimental animals were infected with P. aeruginosa to reproduce this vascular lesion. It was found that destruction of the arterial elastic lamina was frequently associated with the lesion, suggesting a probable role of the elastase of P. aeruginosa in the pathogenesis. Some of our studies on the elastase of P. aeruginosa are reported here. MATERIALS AND METHODS Source of Microorganism. A strain of P. aeruginosa, isolated from an infected burn wound of a patient and previously shown to have high elastolytic activity (Mull and Callahan, 1963)) was employed. Preparation of elastase extract. Elastase was prepared from P. aeruginosa cultured in nutrient broth (37°C for 3 days) by the following fractionation procedure carried out at 4°C. Slime and bacterial cells were removed by filtration and/or centrifugation and ammoni'G sulfate (400 gm/liter) was added to the supernatant. The precipitate that formed overnight was recovered by centrifugation, dissolved in a small volume of distilled water, and dialysed for 48 hours. After inert material was removed by precipitation with streptomycin sulfate (10 gm/liter), protein was reprecipitated from the supernatant by the addition of ammonium sulfate (300 gm/liter) after which the precipitate was redissolved in water, dialysed, and lyophilized. The resulting brown product which was completely water soluble was used in the following assays. Enzyme assay. Twenty mg of elastin-orcein (Worthington) and 3 mg of elastase extract dissolved in 3 ml tris buffer [ tris (hydroxy-methyl) aminomethane] 0.05 ICI pH 7.8 were incubated at 37°C with gentle shaking for 4 hours. Reactions were stopped by the addition of 2 ml Sorensen's phosphate buffer, 0.7 M pH 7.0 (Sacher et al., 1955). Since bacterial pigments present in the enzyme preparation had optical absorption properties similar to that of orcein, the extent of elastolysis was determined indirectly by estimating the remaining undigested elastin-orcein. The undigested

No abstract

technique, and 9 of 20 when an anaerobic jar was used). Thus, anaerobic fermentation of mannitol seems to be too exacting a criterion, but all methods using glucose fermentation classified the 20 coagulase-positive strains as staphylococci.The classification of other staphylococci into S. saprophyticus and S. lactis on their ability to produce acetoin from glucose (Shaw et al.) was not supported by the results of tests reported here. Some strains of each of these species were, in the newer tests, classified a's staphylococci and some as micrococci, but the different methods did not agree in every case. The best agreement was attained when anaerobic fermentation of glucose was tested by Baird-Parker's method, by peptone-water cultures in an anaerobic jar, and by Evans' method when a final pH of less than 5.0 was required.It is clear that those catalase-positive grampositive cocci that ferment glucose under anaerobic conditions can be regarded as staphylococci, and those that fail to do so (because they oxidize the sugar, do not attack it, or do not grow under anaerobic conditions) are not staphylococci and, by inference, are micrococci.At this stage, we recommend that Staphylococcus be divided into two species, S. aureus (coagulase-positive) and S. epidermidis (coagulasenegative). The species previously named S. afermentans was shown to consist of two organisms: one attacked carbohydrates by oxidation; the other did not seem to attack them at all.

No abstract

No abstract