Salmonella enteritidis: AmpC plasmid-mediated inducible beta-lactamase (DHA-1) with an ampR gene from Morganella morganii

Abstract

DHA-1, a plasmid-mediated cephalosporinase from a single clinical Salmonella enteritidis isolate, conferred resistance to oxyimino-cephalosporins (cefotaxime and ceftazidime) and cephamycins (cefoxitin and moxalactam), and this resistance was transferable to Escherichia coli HB101. An antagonism was observed between cefoxitin and aztreonam by the diffusion method. Transformation of the transconjugant E. coli strain with plasmid pNH5 carrying the ampD gene (whose product decreases the level of expression of ampC) resulted in an eightfold decrease in the MIC of cefoxitin. A clone with the same AmpC susceptibility pattern with antagonism was obtained, clone E. coli JM101(pSAL2-ind), and its nucleotide sequence was determined. It contained an open reading frame with 98. 7% DNA sequence identity with the ampC gene of Morganella morganii. DNA sequence analysis also identified a gene upstream of ampC whose sequence was 97% identical to the partial sequence of the ampR gene (435 bp) from M. morganii. The gene encoded a protein with an amino-terminal DNA-binding domain typical of transcriptional activators of the LysR family. Moreover, the intercistronic region between the ampC and ampR genes was 98% identical to the corresponding region from M. morganii DNA. AmpR was shown to be functional by enzyme induction and a gel mobility-shift assay. An ampG gene was also detected in a Southern blot of DNA from the S. enteritidis isolate. These findings suggest that this inducible plasmid-mediated AmpC type beta-lactamase, DHA-1, probably originated from M. morganii.

FIG. 1

Double-disk synergy test with the transformant E. coli JM101(pSAL2-ind). (A) Synergy test with 10 μg of clavulanate (disk 6); (B) synergy test with 20 μg of RO48-1220 (disk 7). Disks: 1, cefotaxime; 2, ceftazidime; 3, moxalactam; 4, cefotetan; 5, aztreonam.

FIG. 2

Nucleotide sequences of the ampC and ampR genes and of the intercistronic region from pSAL2-ind and the deduced amino acid sequence of DHA-1. Putative −35 and −10 regions of the promoters of the two genes are boxed.

FIG. 3

Schematic representation of multiple-sequence alignments of 25 class C β-lactamases calculated with Clustal W. Amino acids sequences were directly obtained from the GenBank, EMBL, or SWISS-PROT database.

FIG. 4

Gel mobility-shift assay of the S. enteritidis ampR and ampC intercistronic region with cell protein extracts containing the AmpR regulator protein. The intercistronic sequence, an end-labeled 173-bp PCR product, was retarded with various concentrations of protein extracts from the transformant pSAL2-ind. Lane A, 2.6 μg; lane B, 5.2 μg; lane C, 7.8 μg; lane D, 10.4 μg; lane E, 13 μg; lane F, 0 μg. The position of the retarded complexes containing the intercistronic region is indicated by the arrow.

FIG. 5

Southern blot analysis of DNA from S. enteritidis KF92 probed with ampG from E. coli HB101. Lane A, clinical isolate of K. oxytoca; lane B, clinical isolate of K. pneumoniae; lane C, clinical isolate of S. enteritidis; lane D, S. enteritidis KF92; lane E, E. coli HB101.