Cloning and nucleotide sequence analysis of the ampicillin resistance gene on a conjugative R plasmid from the fish pathogen Photobacterium damselae subsp. piscicida

Abstract

Transferable resistance to various drugs was investigated in strains of Photobacterium damselae subsp. piscicida from Japan. Drug resistance was transferred via two plasmids of 100 and 50 kilobases (kb) or three plasmids of 100, 50, and 40 kb. Resistance to ampicillin was transferred on the 50-kb plasmid. An ampicillin resistance gene in the 50-kb plasmid pPDP8517 from strain PP8517 was cloned from a 1.8-kb Hinc II fragment of the plasmid. The nucleotide sequence of the coding and flanking region of the ampicillin resistance gene was determined to be 1,736 base pairs. The nucleotide sequence identified an open reading frame (ORF) encoding 282 amino acid residues with a calculated molecular mass of 31,292 daltons. The ampicillin resistance gene's ORF was found to be a β-lactamase bearing low levels of amino acid identities (<63%) relative to other β-lactamases. This ORF was very high in amino acid identities with class A β-lactamases compared with those of class B, C, and D β-lactamases. Four important structural features were conserved in all class A β-lactamases present in the deduced amino acid sequence of the ampicillin resistance gene. These results suggest that the P. damselae subsp. piscicida ampicillin resistance gene is a class A β-lactamase encoded on an R plasmid that is distinctly different from other known β-lactamases. Moreover, Southern blot analysis suggested that P. damselae strains with the ampicillin resistance gene are widely distributed in marine fish farms in the Kyushu area in Japan.