Seven bacteriophages active on Yersinia enterocolitica (YE) were isolated from surface water samples collected in Granada, Spain. A comparison of the respective host ranges of these new phages and of reference phages used for YE phage typing showed that YE strains belonging to various phage types, grown at either 37 or 25°C, expressed susceptibility to reference sewage water phages whereas susceptibility to new waterborne phages, as well as to reference phages from lysogenic YE, was only

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... rated in YE strains grown at 25°C. A YE strain isolated by stool culture from a pig was lysogenic for a bacteriophage which behaved like waterbome phages and reference phages from lysogenic YE strains. The possibility that the isolation of waterborne bacteriophages might, in certain circumstances, reflect the presence of lysogenic YE was raised. Yersinia enterocolitica (YE) is, among Yersinia, the single species in which lysogeny has been shown. Bacteriophages isolated from lysogenic strains of YE and active on other strains are used for classification of YE in various phage types (6). Since YE isolated from the environment remained nontypable by these bacteriophages, a complementary scheme of phage typing was done using bacteriophages isolated from sewage water (5). It is unknown whether bacteriophages isolated from sewage water originated from YE strains or from other bacteria. The YE strains are ubiquitous in the environment (4). During a systematic survey in Granada, attempts were made to isolate YE from domestic animals and from surface water. In addition, studies were conducted to isolate bacteriophages active on YE and to study their host ranges among selected YE strains compared to reference bacteriophages used for phage typing (5). MATERIALS AND MEETHODS Isolation of YE. Stool samples collected in slaughterhouses from 188 pigs, 97 cows, 15 goats, and 14 sheep were suspended into phosphate-buffered saline, stored for 3 weeks at 40C before inoculation into salmonella-shigella medium (Difco), and incubated at 280C for 36 h (8). Identification was performed by the method of Bejot et al. (2). Surface water samples from five different areas around Granada (four rivers and one pond) were studied by the technique described by Harvey et al. (3). Recovery of bacteriophages from surface water samples. Water samples used for isolation of YE were also used for bacteriophage isolation: 10 ml was filtered through a 0.22-,um membrane filter (Millipore Corp.) and 1 ml of each filtrate was mixed with an overnight culture of YE strain 8133 (described in Table 1 ) in peptone water. After incubation at 250C, the lysed cultures were filtered through a 0.22-nm membrane filter (Millipore Corp.), and the peptone waterbacteriophage suspensions were stored at 40C. Research of lysogeny among YE strains isolated in Granada. Attempts to demonstrate lysogeny were performed by the techniques described by Adams (1), either by direct filtration (0.22-pm pore size) of overnight peptone water cultures of YE strains or after treatment by H202. Filtrates were tested and then propagated on YE strain 3827 (chemotype 4, serogroup 03, phage type IXa) which was not lysogenic for any of the YE tested strains. Host range of bacteriophages. (i) Reference phageq _om lysogenic YE. Bacteriophages I, II, V, X, and XII were randomly selected from the 12 phages that had been isolated from lysogenic YE strains and used for phage typing of YE (5). Each bacteriophage was propagated since its isolation on the same YE strain 3827 described above. (ii) Reference phages of the set of complementary phage typing. Bacteriophages 3, 4, 7, 9, 10, and 16 were randomly selected from the 16 phages used for complementary phage typing of YE (5). Each bacteriophage was propagated since its isolation on the same YE strain: phage 3 on YE 4194 (chemotype 2, serogroup 0:5.27, phage type Xz); phage 4 on YE 517 (Table 1); phages 7 and 9 on YE 505 (Table 1); phage 10 on YE 2461 (Table 1); and phage 16 on YE 2462 (chemotype 2, serogroup 09, phage type X3). (iii) New waterborne bacteriophages. All newly isolated bacteriophages were purified by three successive isolations on soft agar and propagated once on YE 8133 (Table 1) by the agar-layer technique described by Adams (1). Test bacterial strains. All bacteriophages were 35 on May 8, 2020 by guest http://aem.asm.org/ Downloaded from