Article Figures & Data
Figures
- Fig. 1.
Nucleotide and protein sequences of the coding region of OXA-17 β-lactamase in comparison to those of OXA-10, -11, -14, and -16 enzymes. The nucleotide sequence shown in full corresponds to that determined in this study and was allocated the GenBank accession no. AF060206. Nucleotide differences in otherbla OXA-10 family genes are marked above thebla OXA-17 sequence, and the deduced amino acid changes are indicated below. The signal peptide extends from amino acid residues 1 to 20, and the proposed cleavage site is shown by a vertical line. The underlined nucleotide sequences represent the primers ABD1, ABD2, ABD3, and ABD4 used for PCR amplification or sequencing. The sequences corresponding to the amplification primers have not been independently determined for OXA-17 and are shown in italics. The three conserved elements previously described in class D β-lactamase are in boldface (14, 17).
Tables
- Table 1.
MICs of β-lactams for P. aeruginosa isolates 871 and 873
Strain MIC (μg/ml)a Pip Pip-Clav Pip-Taz Cb Cb-Clav Cb-Taz Caz Caz-Clav Caz-Taz Ctx Ctr Csl Cpm Cpr Cpz Mox Azt Mem Imp 871 32 32 32 1,024 256 512 128 16 64 64 128 128 32 64 128 32 128 0.5 2 873 64 32 64 >512 512 >512 128 16 32 128 128 64 16 32 128 64 64 0.25 4 PU21(pMLH57)/PER-1b 8 4 8 512 128 256 256 4 128 64 128 128 32 64 32 16 128 1 2 PU21 2 4 4 64 64 64 4 2 2 16 16 2 2 8 8 8 4 1 2 ↵a Azt, aztreonam; Cb, carbenicillin; Clav, clavulanate at 4 μg/ml; Cpm, cefepime; Cpr, Cefpirome; Cpz, cefoperazone; Csl, cefsulodin; Ctx, cefotaxime; Caz, ceftazidime; Ctr, ceftriaxone; Imp, imipenem; Mem, meropenem; Mox, moxalactam (latamoxef); Pip, piperacillin; Taz, tazobactam at 4 μg/ml.
↵b P. aeruginosa PU21 with pMLH57 plasmid coding PER-1 β-lactamase.
- Table 2.
Characteristics of E. coli XL1-blue MRF′ transformants selected with ampicillin (10 μg/ml) from the DNA libraries of isolates 871 and 873 in the pBC SK+ vector
Library No. of colonies Plasmid size (kb)a β-Lactam-ase pI Hybridi-zation Plasmid Isolate 873 HindIII 10 10 7.9 bla OXA-2 p13A 2 9 6.1 bla OXA-10 p13F BamHI 4 11 7.9 bla OXA-2 p13D PstI 2 10 6.1 bla OXA-10 p13E Isolate 871 HindIII 17 10 7.9 bla OXA-2 p15A 6 9 6.1 bla OXA-10 p15D BamHI 4 18 6.1 bla OXA-10 p15C PstI 0 NAb NA NA NA - Table 3.
MICs for representative E. coli XL1-blue transformants with ampicillin resistance cloned from isolates 871 and 873
Plasmid and β-lactamase transformanta MIC (μg/ml)b Amp Pip Pip-Clav Pip-Taz Cb Cb-Clav Cb-Taz Caz Caz-Clav Caz-Taz Ctx Ctr Csl Cpim Cpz Mox Azt Imp p15C/OXA-17 from isolate 871 512 8 1 2 512 64 128 0.5 0.25 0.5 0.5 1 64 0.5 16 1 1 0.06 p13E/OXA-17 from isolate 873 512 8 1 4 512 64 256 1 0.5 0.5 0.5 1 64 0.5 16 2 1 0.12 p15A/OXA-2 from isolate 871 256 16 0.12 0.25 512 8 4 1 0.25 0.25 0.12 0.06 32 0.12 8 0.25 0.06 0.06 p13A/OXA-2 from isolate 873 128 2 0.5 0.25 128 8 4 1 0.12 0.5 0.06 0.6 16 0.12 2 0.12 0.12 0.06 XL1-blue(p10A)/OXA-10 512 16 1 8 1,024 128 128 0.25 0.12 0.25 0.12 0.5 32 0.25 8 0.25 1 0.06 XL1-blue (no plasmid) 4 0.25 0.12 0.12 8 4 8 0.25 0.12 0.25 0.06 0.06 16 0.12 0.12 0.12 0.12 0.06 ↵a Into E. coli XL1-blue.
b Amp, ampicillin; Azt, aztreonam; Cb, carbenicillin; Clav, clavulanate at 4 μg/ml; Cpim, cefepime; Cpz, cefoperazone; Csl, cefsulodin; Ctx, cefotaxime; Caz, ceftazidime; Ctr, ceftriaxone; Imp, imipenem; Mox, moxalactam (latamoxef); Pip, piperacillin and Taz, tazobactam at 4 μg/ml.
- Table 4.
Kinetic parameters for OXA-17 β-lactamase compared with OXA-10
Substrate OXA-17 (linear kinetics) OXA-10a Km (μM) kcat(s−1) kcat/Km b Vss Vo Km (s−1) kcat(s−1) kcat/Km b Km (μM) kcat(s−1) kcat/Km b Penicillin G 34 ± 0.6c 5 ± 0.1 147 63 ± 6 89 ± 10 1,412 −d − − Ampicillin 245 ± 37 26 ± 0.2 106 235 ± 30 587 ± 30 2,500 77 ± 9 531 ± 12 6,896 Carbenicillin 296 ± 31 2 ± 0.4 21 195 ± 13 31 ± 1 159 370 ± 44 126 ± 5 340 Oxacillin 153 ± 13 120 ± 2.0 784 222 ± 16 608 ± 10 2739 96 ± 8 660 ± 10 6,870 Cloxacillin 573 ± 79 20 ± 1.0 35 2,640 ± 323 520 ± 36 196 1,110 ± 100 2,682 ± 150 2,416 Cephaloridine 2,940 ± 182 23 ± 1.0 8 2,340 ± 300 79 ± 8 33 395 ± 83 39 ± 3 98 Cephalothin 286 ± 60 5 ± 0.1 17 38 ± 2 6 ± 0.1 158 − − − Cefotaxime 2,240 ± 427 22 ± 3.0 10 346 ± 19 9 ± 0.2 26 − − − Ceftriaxone 544 ± 52 1 ± 0.05 2 55 ± 2 3 ± 0.3 54 − − − Ceftazidime NDe ND ND ND ND − − − − ↵a OXA-10 β-lactamase was shown previously (8) to give biphasic kinetics, so two sets of kinetics parameters could be determined for the initial (Vo ) and steady-state (Vss ) phases of hydrolysis.
↵b (k cat/Km ) × 1,000 in micromolar−1 second−1.
↵c Values are means ± standard error.
↵d −, results not available.
↵e ND, hydrolysis not detected.
