Genetic-biochemical analysis and distribution of the Ambler class A beta-lactamase CME-2, responsible for extended-spectrum cephalosporin resistance in Chryseobacterium (Flavobacterium) meningosepticum

Abstract

In vitro synergy between extended-spectrum cephalosporins and either clavulanic acid or cefoxitin was found for Chryseobacterium meningosepticum isolates during a double-disk assay on an agar plate. An extended-spectrum beta-lactamase (ESBL) gene from a C. meningosepticum clinical isolate was cloned and expressed in Escherichia coli DH10B. Its protein conferred resistance to most beta-lactams including extended-spectrum cephalosporins but not to cephamycins or to imipenem. Its activity was strongly inhibited by clavulanic acid, sulbactam, and tazobactam, as well as by cephamycins and imipenem. Sequence analysis of the cloned DNA fragment revealed an open reading frame (ORF) of 891 bp with a G+C content of 33.9%, which lies close to the expected range of G+C contents of members of the Chryseobacterium genus. The ORF encoded a precursor protein of 297 amino acids, giving a mature protein with a molecular mass of 31 kDa and a pI value of 9.2 in E. coli. This gene was very likely chromosomally located. Amino acid sequence comparison showed that this beta-lactamase, named CME-2 (C. meningosepticum ESBL), is a novel ESBL of the Ambler class A group (Bush functional group 2be), being weakly related to other class A beta-lactamases. It shares only 39 and 35% identities with the ESBLs VEB-1 from E. coli MG-1 and CBL-A from Bacteroides uniformis, respectively. The distribution of bla(CME-2) among unrelated C. meningosepticum species isolates showed that bla(CME-2)-like genes were found in the C. meningosepticum strains studied but were absent from strains of other C. meningosepticum-related species. Each C. meningosepticum strain produced at least two beta-lactamases, with one of them being a noninducible serine ESBL with variable pIs ranging from 7.0 to 8.5.

FIG. 1

Schematic representation of recombinant plasmid pBS1, which encodes the CME-2 β-lactamase from C. meningosepticum PINT. The thin line represents the cloned insert from C. meningosepticum PINT containing the CME-2 β-lactamase gene, with the arrow indicating its translational orientation (thick line), and the dotted lines indicate the vector pBK-CMV. The three internal probes (probes S1, S2, and S3) used for hybridization experiments are also represented by double-headed arrows.

FIG. 2

Nucleotide sequence of the 1,525-bp fragment of pBS1 containing the 891-bp coding region for bla_CME-2. The deduced amino acid sequence is designated in single-letter code below the nucleotide sequence. The seven conserved boxes described by Joris et al. (14) and the conserved SDN loop are boxed. The start and stop codons of the gene are in boldface type and are underlined.

FIG. 3

Amino acid sequence alignment of CME-2 compared with those of selected class A β-lactamases. The numbering is according to Ambler (1). Roman numerals designate the boxes described by Joris et al. (14): box I, positions ABL 45 to 50; box II, positions ABL 70 to 73; box III, position ABL 105; box IV, position ABL 111; box V, position ABL 166; box VI, position ABL 210; box VII, positions ABL 234 to 236. The β-lactamases included in the alignment are TEM-3 from K. pneumoniae CFF104, SHV-2 from K. ozaenae, MEN-1 from E. coli MEN, TOHO-1 from E. coli TUH12191, NMC-A from E. cloacae NOR-1, L-2 from S. maltophilia 1275IID, CEP-A from B. fragilis CS30, CFX-A from B. vulgatus CLA341, VEB-1 from E. coli MG-1, CBL-A from B. uniformis WAL-7088, and PER-1 from P. aeruginosa RNL-1. Dashes indicate gaps within the alignment.

FIG. 4

Dendrograms obtained for 12 Ambler class A ESBLs by the parsimony method (44). Branch lengths are drawn to scale and are proportional to the number of amino acid changes. The percentages at the branching points (underlined) refer to the number of times that a particular node was found in 100 bootstrap replications (the asterisks indicate uncertainty for nodes with bootstrap values of less than 50%). The distance along the vertical axis has no significance. Abbreviations for β-lactamases are given in the legend to Fig. 3. Percent amino acid identities to CME-2 are indicated in parentheses.

FIG. 5

Autoradiogram after Southern hybridizations of C. meningosepticum genomic DNAs. The DNAs were restricted with XmnI, and the probe consisted of the 372-bp DraI-XmnI fragment (S1) of pBS1 (A), the 306-bp XmnI-DraI fragment (S2) of pBS1 (B), or the 667-bp DraI fragment (S3) of pBS1 (C) (see Fig. 1 for the locations of S1, S2, and S3). Lanes: 1, C. meningosepticum PINT; 2, C. meningosepticum CIP 6058; 3, C. meningosepticum AMA; 4, C. meningosepticum GEO; 5, C. meningosepticum CIP 7830; 6, C. meningosepticum CIP 6059; 7, C. meningosepticum CIP 7905; 8, C. meningosepticum AB 1572; 9, C. meningosepticum H01J100; 10, E. coli DH10B (negative control). The sizes of the DNA fragments may be deduced from the scale (1.5 to 4 kb) shown on the left sides of the gels.