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Sir, The use of antibiotics in aquaculture production has been associated with the isolation of antibiotic-resistant bacteria in fish as well as pollution of the fish farm environment. Previously, 25 tetracycline-resistant bacteria isolated from four freshwater Chilean salmon farms were characterized. Fifteen of the isolates carried one of seven different tetracycline (tet) genes [tet(A), tet(B), tet(E), tet(H), tet(l), tet(34) and tet(35)] and 10 had unknown tet genes. Over the past 12 years, 13 new tet genes have been characterized, which prompted us to re-examine the 10 tetracycline-resistant bacteria that were previously negative for the 22 tet genes. The isolates were originally identified using biochemical assays, but were re-identified using 16S ribosomal DNA sequencing as previously described. Four of the isolates (C8, L7, O193 and Q52) had a different genus identification using 16S than from the original manuscript (.99% identity by 16S with sequencing of amplicons 694–711 bp) (Table 1). L7 identification as Gram-positive Corynebacterium minutissimum by 16S was verified by Gram stain and the VITEK 2 microbial identification system (bioMérieux, Inc., Durham, NC, USA) (Table 1). The other bacteria included three Acinetobacter spp., two Providencia rettgeri, two Pseudomonas spp., one Psychrobacter sp. and one Stenotrophomonas sp. (Table 1). PCR assays for tet(39), tet(W) and tet(X) were done as previously described using primers and conditions described previously. – 10 The tet(39) gene, which codes for an efflux protein, was first isolated from Acinetobacter spp. collected from freshwater trout farms, from sewage and from Gram-positive and Gram-negative bacteria isolated from a polluted river in south-western Nigeria. It is one of the few new tet genes to code for an efflux protein that is found in both Gram-negative and Gram-positive species. The tet(W) gene, which codes for a ribosomal protection protein, has been isolated in both Gram-positive and Gramnegative bacteria, is primarily found in environmental isolates and is so far the fourth most commonly identified tet gene in bacteria. The tet(X) gene codes for an enzyme that breaks down tetracycline and is found primarily in Gram-negative environmental bacteria. Six of the isolates were tet(39) positive and included isolates from each of the four fish farms. At farm CC1, all three tet(39)positive isolates came from salmon fingerlings and represented three different genera. One of two isolates was tet(39) positive from farm CC2 from the tank water and one of three isolates from farm CC3. One isolate from farm CC4 was also tet(39) positive and was isolated from pelletized feed (Table 1). All three of the Acinetobacter spp. isolates (O213, CH90 and Q75), each from a different fish farm, were tet(39) positive. One Pseudomonas sp. isolate (O275), the Psychrobacter sp. isolate (O193) and the C. minutissimum isolate (L7) were also tet(39) positive. Two tet(39) PCR amplicons (694–711 bp) (L7 and O193) were Research letters

Tetracycline resistance gene tet(39) identified in three new genera of bacteria isolated in 1999 from Chilean salmon farms