Tetracycline-resistant (Tet r ) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet r gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tet r genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tet r strains transferred Tet r genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tet r strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.Many different kinds of antibiotics have been used as therapeutic agents in aquaculture in Japan. Intensive work was done until the 1980s to develop guidelines for antibiotic usage in fish farms. The guidelines regulated doses and required a period of drug-free rearing before sale of fish and succeeded in keeping the residual antibiotics in cultured fish to nondetectable levels. However, Samuelsen et al. (35) found that antibiotic-resistant bacteria persisted in fish farm sediments for at least 18 months after chemotherapy. Since the products of aquaculture are consumed by humans and since many antibiotic resistance determinants are encoded by transferable plasmids, cultured fish may serve as a vehicle for transmission of antibiotic resistance to bacteria that are commensal or pathogenic to humans (34).Tetracyclines are among the therapeutic agents most commonly used in human and veterinary treatment. Oxytetracycline is permitted to be mixed with feed for fish, and food sanitation law in Japan permits certain residual levels in fish. Because of the widespread use of tetracycline, resistance to it has been disseminated to many species of marine bacteria (4,6,18,42,46).More than 30 different kinds of tetracycline resistance determinants have been published. Resistance genes have been mainly categorized into two major groups, those responsible for proton-dependent efflux of tetracycline (24) and those conferring ribosomal protection by cytoplasmic proteins (9). Dissemination of the proton-dependent tetracycline efflux protein in aquaculture environments has been reported (5,12,13,14,22,34,37). Previous work has identified the relevant genes by using DNA hybridization or PCR methods (4,12,13,14,15,16,27,28,34,37), but the nucleotide sequences of these determinants remain unkn...

Three variants of the composite transposon Tn10 were extracted from transferable plasmids of fish farm bacteria. These variants were identical in insertions with IS10, but differed in another class I transposon insertion and a region of homologous recombination downstream of tetB.Key words: Plasmid, Tn10, fish farm bacteria Tn10 is a composite transposon consisting of IS10 and the tetB operon carrying a tetracycline resistance gene. Its complete nucleotide sequence is already well known (2) and its distribution has been reported for pathogenic bacteria, and for some bacteria with structural variations (11, 16). The structure of Tn10 on the transferable plasmid from Pantoea agglomerans is heterogeneous (16), and a vestige of Tn10 has been found in an integron on a plasmid from Salmonella enterica (11).The distribution of Tn10 has also been reported for marine bacteria (9), but the variation in Tn10 structure has not yet been well known. Therefore, we examined the structural variation of Tn10 on transferable tetB-carrying plasmids which have been found in fish farm bacteria (4).We isolated four fish farm bacteria harboring transferable tetB-carrying plasmids listed in Table 1 (4). Transconjugant strains were obtained by conjugation between the fish farm bacteria and E. coli HB-101 (Table 1) (4). All strains grew on Luria-Bertani (LB) medium with or without 20 µg mL −1 of tetracycline (TC) at 37°C. The plasmids were extracted from the transconjugants by the Kado and Liu method (8), and used as template DNA for PCR amplification of Tn10 regions with the primers listed in Table 2 and Fig. 1.Although most of the primer sets produced DNA fragments of predicted size in all PCR-amplifications, all of the products of primer set 2 were approximately 1,300 bp larger than the predicted size. Primer set 3 produced no PCR products for TC68 or TC69, and no product was obtained using primer set 7 or 8 for TC21 (Fig. 1). These non-amplifiable regions were cloned as shown in Table 1.Sequencing of the PCR product of primer set 2 indicated the presence of an insertion sequence within the jemA region (Fig. 1). Nucleotide sequences of the insertion and insertion point of all strains were identical. The insertion sequences were identified as an IS10 variant since they showed 87.4% similarity to the sequence of IS10 (2). The tnpA (transposase gene) sequence of the IS10 variant showed 91.1% similarity to the IS10 sequence present in the left and the right terminus of Tn10 of Shigella flexneri (Table 3). The repeat of ACCAAAGCC, flanking the inserted IS10 variant, was different from the typical target sequence of Tn10, NGCTNAGCN, by two nucleotides. ACCAAAGCC did not seem to be a new hot spot for IS10 variants, since the IS10 variant did not contain nucleotide substitutions at codons 134 and 249 that were reported to result in a relaxation of target specificity (1).In addition to the insertion of the identical IS10 variant in the jemA region, all terminal IS10s of the Tn10 variants were identical, except for IS10-right of TC21. The IS10-r...

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