An MIC test of 12 chemotherapeutic agents performed on 175 strains of Pasteurella piscicida collected from cultured yellowtail (Seriola quinqueradiata) in different areas of Japan from 1989 to 1991 revealed 152 strains (87%) with resistance to combinations of ampicillin (AP), chloramphenicol (CP), kanamycin (KM), nalidixic acid (NA), sulfamonomethoxine (SA), tetracycline (TC), and/or trimethoprim (TMP). The remaining 23 strains were sensitive to all the drugs tested: AP, cefazolin, CP, florfenicol (FF), furazolidone, KM, NA, novobiocin, SA, streptomycin, TC, and TMP. FF showed the most effective antibacterial activity against P. piscicida with MICs ranging from 0.004 to 0.6 μg/ml. One hundred and forty‐nine of the 152 resistant strains carried transferable R plasmids encoding one of the Cp Km Sa Tc, Km Sa Tc, Km Sa, and Sa resistance. The most common resistance marker of transferable R plasmids identified in P. piscicida was Km Sa Tc. R plasmids encoding three different resistant markers were very similar on the basis of their digestion patterns with restriction endonucleases. There was homology among the DNAs of nine transferable R plasmids selected. Our findings suggest that multiple drug resistant strains of P. piscicida carrying transferable R plasmids with the same DNA structure are common in yellowtail farms and that the R plasmid has been retained within the P. piscicida population without change in their DNA structure according to geography and year.

Tetracycline (pp-tet), and kanamycin (pp-kan) resistance genes were cloned from a transferable R plasmid of fish pathogen Pasteurella piscicida, and complete nucleotide sequences were determined. The pp-tet was a class D Tet determinant constructed with the tetA resistance gene of 1,182 by encoding a protein with a deduced molecular mass of 41 kDa and the tetR repressor gene of 654 by encoding a product of 24 kDa. The pp-tet was highly homologous to the tet(D) of plasmid RA1 isolated from Aeromonas hydrophila with two nucleotide differences in the tetR, and of plasmid pIP173 from Salmonella ordonez with two nucleotide differences in the tetA. The pp-kan contained 813 by encoding a 31 kDa protein of 271 amino acids, and was classified into type aph-Ic. It was identical to the aphA 7 in the IAB operon of pBWH77, in which was originally found an isolate of Klebsiella pneumoniae, in its nucleotide sequences and hybrid promoter construction. The genes were connected by an insertion sequence IS 26 of 820 bp, and were flanked by repeated copies in direct orientation at the 3' flanking region of the pp-tetA and in inverted orientation at the 3' flanking region of the pp-kan. The genetic elements are organized like a complex transposon by close linkage of the 1S26 and the pp-tet and -kan.

One hundred and fourteen strains of Vibrio anguillarum collected from cultured ayu Plecoglossus altivelis between 1989 and 1991 were studied for their sensitivities to 10 chemotherapeutants. Among the drugs tested florfenicol (FF) was the most effective against all strains. Only one strain was found to be sensitive to all the drugs (ampicillin, chloramphenicol (CP), FF, furazolidone, kanamycin (KM), nalidixic acid, streptomycin (SM), sulfamonomethoxine (SA), tetracycline (TC), and trimethoprim (TMP).The remaining strains showed resistance to various combinations of drugs.Transferable R plasmids were detected from 21 strains encoded with resistance to 7 to 9 drugs.All detected R plasmids encoded with resistance to CP, KM, SA, SM, TC, and TMP. Identical endonuclease digestion patterns were observed in R plasmids detected from various areas, and strong homology among these R plasmid DNAs were also observed.The DNA structure of the R plasmids detected from 1989 to 1991 was not similar to that of previously detected R plasmids from V. anguillarum.The results indicate that multiple drug resistant strains of V. anguillarum were common in ayu farms between 1989 and 1991.

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The chloramphenicol resistance gene (pp-cat) was cloned from a transferable R plasmid of Pasteurella piscicida, pSP9351, and the sequence of the gene was determined. Subcloning and deletion analysis localized the resistance gene, pp-cat, to within a 2.3 kb Hinc11-BamH1 fragment. The fragment as a probe hybridized with the type I chloramphenicol acetyltransferase (CAT) gene and did not hybridize to CAT types II, III, and CAT-VA. The fragment hybridized to transferable R plasmids encoded with resistance to chloramphenicol, which were detected from P. piscicida isolated in different years. Nucleotide sequences of the coding and flanking regions of pp-cat (2031 bp) identified an open reading frame coding type I CAT of a molecular mass of about 25,000 Da. Comparison analysis of the sequences outside the cat open reading frame showed also that pp-cat has homology, in part, with the gene that coding for the endonuclease EcoRII and those that flank the cat gene derived from the Acinetobacter baumannii chromosome.