Molecular characterization of plasmid-mediated oxytetracycline resistance in Aeromonas salmonicida

Abstract

Using broth conjugation, we found that 19 of 29 (66%) oxytetracycline (OT)-resistant isolates of Aeromonas salmonicida transferred the OT resistance phenotype to Escherichia coli. The OT resistance phenotype was encoded by high-molecular-weight R-plasmids that were capable of transferring OT resistance to both environmental and clinical isolates of Aeromonas spp. The molecular basis for antibiotic resistance in OT-resistant isolates of A. salmonicida was determined. The OT resistance determinant from one plasmid (pASOT) of A. salmonicida was cloned and used in Southern blotting and hybridization experiments as a probe. The determinant was identified on a 5.4-kb EcoRI fragment on R-plasmids from the 19 OT-resistant isolates of A. salmonicida. Hybridization with plasmids encoding the five classes (classes A to E) of OT resistance determinants demonstrated that the OT resistance plasmids of the 19 A. salmonicida isolates carried the class A resistance determinant. Analysis of data generated from restriction enzyme digests showed that the OT resistance plasmids were not identical; three profiles were characterized, two of which showed a high degree of homology.

FIG. 1

Plasmid profiles of OT-resistant and -sensitive isolates of A. salmonicida. Lanes 1 and 17, markers (HindIII digest of λ DNA): lanes 2 and 16, blank; lanes 4 to 14, plasmid DNA from A. salmonicida OT-resistant isolates MT1402, MT1431, MT1434, MT1427, MT1401, MT1407, MT1432, MT0918, MT1425, MT1406, and MT1413, respectively; lanes 3 and 15, plasmid DNA from OT-sensitive isolates 256/91 and AS20, respectively. The arrow indicates the position of the large plasmids. Not all plasmids were routinely obtained at sufficiently high yields to be detected by photography (lanes 6 and 10).

FIG. 2

(a) Southern blot analysis of plasmid DNA prepared from E. coli transformants digested with EcoRI and hybridized with probe AST. Strong hybridization occurred with a 5.4-kb fragment in lanes 2 to 11 and 13, which contained plasmid DNA from the following transformants: MT0404x, MT0321x, MT1407x, MT0903x, MT1410x, MT0900x, MT1431x, MT0164x, Banksx, MT1427x, and 718x, respectively. Lane 1 contained plasmid DNA from OT-sensitive A. salmonicida isolate 96. Lane 12 contained markers (HindIII digest of DIG-labeled λ DNA). (b) Southern blot analysis of plasmid DNA prepared from E. coli transformants digested with EcoRI and hybridized with probe TA. Strong hybridization occurred with a 5.4-kb fragment in lanes 2 to 9, which contained plasmid DNA from the following transformants: MT1407x, MT1410x, MT0164x, MT1427x, MT0350x, MT1413x, MT0404x, and 718x, respectively. Lane 1 contained markers (HindIII digest of DIG-labeled λ DNA), and lane 10 contained plasmid DNA from the OT-sensitive isolate A. salmonicida 96.

FIG. 3

Restriction endonuclease fragment patterns generated by EcoRI digestion of plasmid pRAS1 (from E. coli transformant 718x) and R-plasmids from E. coli transformants MT0320x, MT0903x, MT0906x, MT1407x, MT1410x, and MT1432x. Lane 1, markers (HindIII digest of λ DNA); lane 2, 718x; lane 3, MT0320x; lane 4, MT0903x; lane 5, MT0906x; lane 6, MT1407x; lane 7, MT1410x; lane 8, MT1432x; lane 9, markers (1-kb DNA ladder).