Article Figures & Data
Figures
- 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. coliDH5α T+ producing ACC-1; D, E. coliR+ producing SHV-2. Lanes for panel b: A, E. coli R+ producing SHV-4; B, E. coliR+ 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 theblaACC-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.
Tables
- Table 1.
Bacterial strains used in this study
Strain Plasmid Characteristics pI(s) of β-lactamases K. pneumoniaeKUS pMVP-8 Clinical isolate from sputum, Kiel, Germany 5.4, 7.7 E. coli C600 Rifampin resistant None E. coliMV1190 lac− None E. coli C600 pMVP-8 Transconjugant of KUS 5.4, 7.7 E. coli MV1190 pBC Chloramphenicol-resistant cloning vector None E. coliMV1190 Transformant of KUS containing a 2.3-kb fragment cloned into pBC 7.7 - Table 2.
Antibiotic susceptibilities of the wild-type K. pneumoniae KUS, the transconjugant E. coli C600 R+, the transformant E. coli MV1190 T+, and the recipient E. coliC600 R−
Antibiotic MIC (μg/ml) for: Wild-type K. pneumoniae KUS Transconjugant E. coliC600 R+ Recipient E. coli C600 R− Transformant E. coli MV1190 T+ Host E. coli MV1190 T− Ceftazidime 64 16 0.13 32 0.06 Plus clavulanate 32 16 0.13 16 0.06 Plus sulbactam 32 16 0.13 16 0.06 Plus tazobactam 32 16 0.13 32 0.06 Plus BRL 42715 1 0.5 0.13 1 0.06 Plus Ro 47-8284 1 0.5 0.13 0.5 0.06 Cefotaxime 16 4 0.06 8 0.03 Cefotetan 4 1 0.13 2 0.06 Plus clavulanate 4 1 0.06 2 0.06 Plus sulbactam 4 1 0.06 2 0.06 Plus tazobactam 4 1 0.06 2 0.06 Plus BRL 42715 1 0.25 0.06 0.5 0.06 Plus Ro 47-8284 0.5 0.25 0.06 0.5 0.06 Cefoxitin 4 2 2 4 2 Moxalactam 1 0.25 0.06 1 0.13 Flomoxef 2 0.5 0.06 1 0.13 Cefpirome 4 0.25 0.03 1 0.03 Cefepime 0.25 0.13 0.016 0.25 0.016 Aztreonam 2 0.5 0.06 1 0.06 Piperacillin 512 256 0.5 32 0.5 Plus tazobactam 64 16 0.5 32 0.5 Temocillin 2 4 4 4 4 Imipenem 0.13 0.25 0.25 0.13 0.25 Meropenem 0.03 0.03 0.03 0.03 0.03 - Table 3.
Kinetic data for ACC-1
Substrate Vmax (nmol of substrate/ min/μg of protein) RelativeVmax Km(μM) Vmax/Km Cephaloridine 30.7 ± 3.0 100 122 ± 16 0.25 Nitrocefin 63.7 ± 2.2 208 28 ± 3.7 2.3 Piperacillin 0.17 ± 0.05 0.55 1.4 ± 0.6 0.12 Ceftazidime <0.025 ≤0.1 17a <0.01 Cefotaxime <0.01 <0.02 NDb ND Cefotetan <0.001 <0.01 ND ND Cefoxitin <0.002 <0.01 ND ND - Table 4.
Identity of amino acid sequences of ACC-1 to other class C β-lactamases
β-Lactamase (reference)a % Identity with: ACC-1 S. marcescens AmpC FOX-2 CMY-1 P. aeruginosa AmpC C. freundii AmpC E. coli AmpC Enterobacter cloacae AmpC M. morganii AmpC ACC-1 100 52.3 46.4 43.9 43.7 39.5 38.8 37.9 37.2 S. marcescens AmpC (31) 100 45.6 44.5 44.9 38.3 38.9 41.5 39.8 FOX-2 (8) 100 74.9 53.7 41.8 42.0 43.9 46.3 CMY-1 (7) 100 54.4 41.2 41.1 43.1 43.5 P. aeruginosa AmpC (26) 100 41.9 42.7 44.1 54.4 C. freundii AmpC (25) 100 76.7 73.2 57.2 E. coli AmpC (24) 100 69.8 57.9 Enterobacter cloacae AmpC (17) 100 55.1 M. morganii AmpC (1) 100 ↵a One representative of each group of the seven AmpC types (Fig. 5) was selected for comparison.
