A novel type of AmpC beta-lactamase, ACC-1, produced by a Klebsiella pneumoniae strain causing nosocomial pneumonia

Abstract

A Klebsiella pneumoniae strain resistant to oxyimino cephalosporins was cultured from respiratory secretions of a patient suffering from nosocomial pneumonia in Kiel, Germany, in 1997. The isolate harbors a bla resistance gene located on a transmissible plasmid. An Escherichia coli transconjugant produces a beta-lactamase with an isoelectric point of 7.7 and a resistance phenotype characteristic of an AmpC (class 1) beta-lactamase except for low MICs of cephamycins. The bla gene was cloned and sequenced. It encodes a protein of 386 amino acids with the active site serine of the S-X-X-K motif at position 64, as is characteristic for class C beta-lactamases. Multiple alignment of the deduced amino acid sequence with 21 other AmpC beta-lactamases demonstrates only very distant homology, reaching at maximum 52.3% identity for the chromosomal AmpC beta-lactamase of Serratia marcescens SR50. The beta-lactamase of K. pneumoniae KUS represents a new type of AmpC-class enzyme, for which we propose the designation ACC-1 (Ambler class C-1).

FIG. 1

Double-disk test to check inducibility of ACC-1 synthesis. (a) Inducibility test with K. pneumoniae KUS. (b) Inducibility test with Enterobacter cloacae WG7250. Disks: 1, cefoxitin; 2, ceftazidime; 3, cefotaxime; 4, cefotetan; 5, aztreonam.

FIG. 2

Isoelectric focusing of β-lactamase ACC-1. The ACC-1 producing wild-type, transconjugant, and transformant strains revealed a band at a pI lower than 7.8 (SHV-4) but slightly above 7.6 (SHV-2), at about 7.7 (a). This band was able to inactivate ceftazidime, as shown by a bioassay (b). Lanes for panel a: A, K. pneumoniae KUS producing ACC-1; B, E. coli C600 R⁺ producing ACC-1; C, E. coli DH5α T⁺ producing ACC-1; D, E. coli R⁺ producing SHV-2. Lanes for panel b: A, E. coli R⁺ producing SHV-4; B, E. coli R⁺ producing SHV-2; C, K. pneumoniae KUS producing ACC-1; D, E. coli C600 R⁺ producing ACC-1; E, E. coli DH5α T⁺ producing ACC-1.

FIG. 3

Nucleotide sequence of the bla_ACC-1 gene (pMVP-8). The deduced amino acid sequence of ACC-1 is shown in the line below the nucleotide triplets. Amino acids of the signal peptide are written in small letters. The β-lactamase active site S-L-S-K, the conserved triad K-T-G, and the class C-typical motif Y-X-N are underlined. The putative −10 and −35 promoter regions upstream of the start codon are underlined. The asterisk indicates the stop codon.

FIG. 4

Multiple alignment of amino acid sequences of the chromosomal AmpC β-lactamase of S. marcescens (32), CMY-2 (6), FOX-2 (8), and ACC-1 (this study).

FIG. 5

Dendrogram for 22 AmpC (group 1) β-lactamases (calculated by Clustal V using the neighbor-joining method of Saitou and Nei [34]). According to the identity of their amino acid sequences with CMY-2, the group 1 β-lactamases might be subclassified into 1a to 1i.