Characterization of a novel plasmid-mediated cephalosporinase (CMY-9) and its genetic environment in an Escherichia coli clinical isolate

Abstract

An Escherichia coli strain, HKYM68, which showed resistance to broad-spectrum cephalosporins was isolated from a sputum specimen in Japan. The high-level resistance of the strain to ceftazidime, cefpirome, and moxalactam was carried by a self-transferable plasmid. The beta-lactamase gene responsible for the resistance was cloned and sequenced. The deduced amino acid sequence of this gene product, CMY-9, had a single amino acid substitution (E85D), the residue reported to be part of the recognition site for the R1 side chain of beta-lactams, compared with the amino acid sequence of CMY-8 and also had 78% identity with the amino acid sequence of CepH, a chromosomal cephalosporinase of Aeromonas hydrophila. A sul1-type class 1 integron containing an aacA1-orfG gene cassette was identified upstream of bla(CMY-9) and ended with a truncated 3' conserved segment. The following 2.1 kb was almost identical to the common region of integrons In6 and In7 and the integron of pSAL-1, except that orf513 encoding a putative transposase was identified instead of orf341 due to addition of a single nucleotide. bla(CMY-9) was closely located downstream of the end of the common region. These observations are indicative of the exogenous derivation of bla(CMY-9) from some environmental microorganisms such as aeromonads.

FIG. 1.

Dendrogram for plasmid-mediated cephalosporinases and the chromosomal cephalosporinases from which they may have originated. The dendrogram was calculated with the CLUSTAL W program. Branch lengths correspond to the number of amino acid exchanges.

FIG. 2.

Schematic comparison of the genetic environment of bla_CMY-9 with integrons In7, In6, and In60 and the integron of pSAL-1. The arrangements of the common regions of In6 and In7 are based on updated sequences (GenBank accession nos. L06822 and L06418, respectively).

FIG. 3.

Comparison of the upstream nucleotide sequences of six plasmid-mediated cephalosporinase genes reported from East Asia, along with the partial sequence upstream of the 5′ end of bla_CepH. Sequence identity among the cephalosporinase genes is indicated with asterisks. Sequence identity between bla_CMY-10 and bla_CepH is indicated with dots. The −35 and −10 regions of putative promoter sequences of the cephalosporinase genes are shown. Horizontal arrows indicate a direct duplication of 18 bp found upstream of bla_CMY-11. GenBank accession nos. are as follows: CMY-9, AB061794; CMY-8, AF167990; MOX-1, D13304; CMY-1, X92508; CMY-10, AF381615; CMY-11, AF381619; CepH, AJ276030.