Isolation of Legionella spp. from environmental samples in the Okayama area

Abstract

During the period from October 1981 to March 1982, 24 water samples collected from coolingtowers and 32 environmental samples from Okayama prefecture, were cultured for isolation for Legionella spp. Environmental samples included 3 groups;(1) Seven samples of tap water, nebulizers, portable shower and baths, in Kawasaki Medical Hospital. (2) Nine samples of tap water, circulatingwater of air-conditioning systems and well water, from three private houses. (3) Seventeen variedsamples of pond soil mud and creek water, around Kawasaki Medical Hospital in autumn and winter.The isolation was carried out by guinea pig inoculation and direct plating on E-F agar after Low-p Htreatment of samples. Isolated strains were identfied as Legionella sp. by 19 ordinary biochemical tests, gaschromatography and direct fluorescent antibody techniques (DFA) useing FITC labeled antiserumwhich were prepared by injection of L. pneumophila serogroup 1, 2, 3 and 4 into rabbits. Theseserogroups were also used in biochemical tests and gaschromatography as controls.Sixteen strains (66.7%) of L. pneumophila were isolated from 24 water samples collected fromcooling towers, 4 strains of L. pneumophila and one strain of Legionella Like Organisms (L.L.O.) wereisolated from 6 samples of group 3 collected in autumn, while no Legionella sp. was isolated from othersamples in group 1, 2, and 3 collected in winter. From these result, it was very likely that amount of L.pneumophila decreased in winter season, and these organisms could not grow up in circulating water at ahigh temperature (50-75°) in air-conditioning systems set up in the private houses.Of 20 strains of L. pneumophila, only 10 stratins showed positive reaction in the Kovac oxidase test.However, several of these strains were negative when they were re-examined by the same test. Itseemed, therefore, that L. pneumophila was unstable in oxidase activity. In other biochemical tests, isolates showed identical results with those of standard strains. All isolates of L. pneumophila grew wellon B-CYE agar at 25 and 41°, indicating that B-CYE agar was more appropriate for the L. pneumophilacultivation than other media reported previously.The analysis of cellular fatty acid composition clearly demonstrated that differances existed between L. pneumophila and L.L.O., showing the usefulness of gaschromatography for classification ofthese organisms.DFA demonstrated that 18 isolates were identified as serogroup 1, one isolate serogroup 4, and theother one was seemed to be serogroup 5, 6 or nontypable.It is emphasized, that L. pneumophila is widely distributed in water in cooling towers and environmentsin Okayama prefecture.