Pyocine typing of Pseudomonas aeruginosa. I. Investigation of culture conditions in the Gillies-Govan method

Abstract

Eighteen Pseudomonas aeruginosa strains of various pyocine types according to the Gillies-Govan method were selected from 126 isolates in Nagasaki area, using human blood as substitute for horse blood in the medium for typing. Standard strains of Types 1 and 16 supplied directly from Dr. R. R. Gillies were added to the mentioned 18 strains and used for the most part of this study. Indicator strains used for typing were also those presented as subcultures from him.First, the activity of pyocine produced by cultivating the test-strains at 32°C for 14 hours on Tryptosoy Agar (Eiken) plates containing horse, bovine, human or rabbit blood were studied in parallel and the results obtained were compared each other as for blood kinds. Thirteen out of 20 strains showed the same growth-inhibition patterns on the different media, and, as shown in Table 1 tabulating the results of remaining 7 strains including the standard strain Type 16, there was no difference between the media with horse and bovine blood respectively in every strain. Accordingly, bovine blood was used in further typing in place of horse blood used in the original method.The reproducibility of typing results of the 20 strains was discussed on the strength of growth-inhibition patterns shown in Table 2 which had been obtained by the tests repeated 6 times together with the last experiment. In 8 out of 20 strains the results were judged reproducible enough, but in 5 not so; and the remaining 7 strains were considered to correspond to “variable type” reported by Zabransky and Day. In conclusion, what is necessary in determination of pyocine type in wild strains is to repeat the typingtwice or more.Growth-inhibition patterns of 8 wild and 2 standard strains under various cultural conditions combining temperatures for cultivation (27°, 32° and 37°C) and incubation periods (14 and 24 hours) werefairly complicated (Table 3). It can safely be said that, from the respect of reproducibility, incubation of plates at 32°C for 14 hours is fitting for the pyocine typing, as adopted already by Gillies and Govan. “Self digestion” of pyocine once produced appears negligible, when the typing is carried out under these conditions at higher temperatures or/and longer period.Following results were obtained from the first typing of 143 isolates including 18 strains mentioned above, using bovine blood and by incubation at 32°C for 14 hours: 47 strains of Type 1, 24 Type 10, 18 Type 3, 7 Type 6, 5 Type 33, 4 Type 29, 3 Type 35, each two Types 5, 11 and 22, each one Types 4, 9, 16 and 20, 7 of non-producer, 8 of Type 1/10, and 10 unclassifiable strains (Table 4).