Nucleotide Sequence of the Chromosomal ampC Gene of Enterobacter aerogenes

Abstract

The AmpC ␤-lactamase gene and a small portion of the regulatory ampR sequence of Enterobacter aerogenes 97B were cloned and sequenced. The ␤-lactamase had an isoelectric point of 8 and conferred cephalosporin and cephamycin resistance on the host. The sequence of the cloned gene is most closely related to those of the ampC genes of E. cloacae and C. freundii.Several members of the family Enterobacteriaceae are naturally resistant to cephalosporins due to the production of an inducible, chromosomally encoded cephalosporinase (7). In the uninduced state, transcription of the structural ␤-lactamase gene, ampC, is repressed by the product of the linked ampR gene (6,12,16). Chromosomal ampC-ampR systems have been described for Citrobacter freundii (11, 13), Enterobacter cloacae (4, 6), Morganella morganii (2, 16), and Yersinia enterocolitica (21). The ampC of Escherichia coli (8) lacks the regulatory gene, possibly as the result of a deletion (6).Recently discovered plasmid-borne AmpC cephalosporinases such as MIR-1, ACT-1, and LAT-1 appear to be genetic descendants of these chromosomally encoded AmpC enzymes and have been implicated in the spread of the cephalosporinresistant phenotype to other genera such as Klebsiella (3). In this paper, we describe a hitherto unknown chromosomal ampC from E. aerogenes that was cloned accidentally while searching for a potential plasmid-mediated cephalosporinase.Enterobacter aerogenes isolates 97A and 97B, identified with the Vitek Identification System (bioMérieux Vitek, Inc., Hazelwood, Mo.), were obtained from the urine of a 30-year-old female patient receiving antibiotic therapy (ampicillin, cefazolin, and gentamicin) for recurring urinary tract infections. The specimen containing 97B was collected 17 h after collection of the one containing 97A. Standard disk diffusion tests (14) and Etests (AB Biodisk North America, Inc., Piscataway, N.J.) were used to determine antibiograms and MICs, respectively. Isolate 97B showed greater resistance than 97A to cefuroxime, cefotetan, cefazolin, ceftriaxone, ceftazidime, and ampicillin (AMP)-sulbactam (Table 1). Both isolates exhibited cefoxitin (FOX) resistance, with MICs of Ͼ256 g/ml.In a "shotgun" attempt to clone a possible plasmid-mediated cephamycinase, libraries of plasmid fragments from 97A and 97B were created by standard procedures (9, 19). Plasmid DNAs were combined with the vector pUC19, digested with PstI or KpnI, and ligated. E. coli DH5␣ (Gibco-BRL, Gaithersburg, Md.) transformants were selected on agar containing 16 g of FOX per ml or 50 g of AMP per ml. This experiment produced only one recombinant, pACM200, from the 97B KpnI-fragment library. The construct contained a 10-kb insert (Fig. 1).Deletion subclones of pACM200 were selected on AMP agar and were subsequently tested for growth on FOX agar. FOX resistance was localized to the 2-kb SacI-SalI fragment in pACM204 (Fig. 1). Further subcloning of pACM204 by cutting the BamHI or NruI sites (pACM205, pACM206, and pACM207) eliminated FOX resistance (Table 1).Probe 206 was m...

The complete nucleotide sequence and environmental distribution of the cryptic, conjugative, broad-host-range plasmid pIPO2 isolated from bacteria of the wheat rhizosphere The GenBank accession number for the pIPO2T sequence reported in this paper is AJ297913.

“…The only other topoisomerase found in transfer systems, IncP-1 TraE, is a topoisomerase of type III (Li et al, 1997). ORF17 shares similarity with the conjugative primase PriL of the IncM plasmid pACM1 (Preston et al, 2000). However, the region of similarity matches only part of the PriL molecule, which most likely does not represent the primase moiety but instead represents something else (located towards the C terminus) with as yet unknown function.…”

Dissemination of SHV-12 and Characterization of New AmpC-Type Beta-Lactamase Genes among Clinical Isolates of Enterobacter Species in Korea

“…It demonstrated that six AmpC beta-lactamases are most closely related to AmpC of E. cloacae MHN1 (98.1 to 100% identity of amino acid sequence), followed by MIR-1 (9) (86.6 to 87.1% identity), ACT-1 (3) (87.4 to 87.7% identity), and AmpC of E. aerogenes (20) (74.8 to 75.1% identity). At this time, the number of chromosomal class C betalactamases that have been described is 36 (1).…”

Clonal distribution of multidrug-resistant Enterobacter cloacae

“…Sequence analysis identified an ampC gene sharing 100% nucleotide identity with that of E. aerogenes EA1509E (Preston et al, 2000), and no more than 73.4% nucleotide identity with the ampC genes identified from E. cloacae. As suggested in Fig.…”