Article Figures & Data
Figures
- Fig. 1.
Amino acid differences between the β-lactamases of the OXA-10 group. Amino acid numbering is according to Huovinen et al. (13); the conserved motifs typical for class D enzymes are boxed. The amino acids shown in boldface contribute to the substrate profile, with asparagine in position 157 leading to extended-spectrum variants. The amino acids of OXA-10 that are not numbered are G20, S27, S50, D55, V89, T107, Y174, A197, E229, T230, S245, and E259; −, absence of an amino acid.
- Fig. 2.
Structure of the integron encoding the β-lactamases OXA-19 and OXA-13-1. Nucleotide sequences are identical in both clusters, except for three differences in the −35 sequences of the promoter and in the aac(6′)-Ib and theoxa genes. Amino acid numbering is from Tran Van Nhieu and Collatz (36) and Huovinen et al. (13), respectively. Arrows represent the following open reading frames:intI1, for integrase;aac(6′)-Ib, for the aminoglycoside acetyltransferases genesaac(6′)-Ib10 on pAZ310 (20) andaac(6′)-Ib9 on pAZ316;oxa, for the OXA variants indicated on the left; andqacEΔ1, for the typically truncated quaternary ammonium resistance-conferring gene associated with the 3′ conserved segment of integrons. The intI1 and qacEΔ1 genes have been only partially sequenced here (indicated in boldface). Data for pAZ309 are from reference 20, and data for pAZ310 and pAZ316 are from the present study. Boldface, differences with respect to the OXA-13-encoding plasmid pAZ309; dashed line, theoxa13-1 region that has been sequenced.
- Fig. 3.
Isoelectric focusing of extended-spectrum OXA-10-related β-lactamases. Enzymes SHV-1 (pIapp, 7.6), SHV-5 (pIapp, 8.2), OXA-13 (pIapp, 8 [18]), and OXA-10 (pIapp, 6.1) were used as pI standards. The two novel β-lactamases, OXA-13-1 and OXA-19, had estimated pIapps of 7.8 and 7.6, respectively. β-Lactamase activity was revealed by overlaying the gel with nitrocefin.
Tables
- Table 1.
Strains and plasmids
Strain or plasmid Genotype or description Resistance phenotypea Reference or source Strain PAe191 Clinical strain ofP. aeruginosa Ti Ca Ak Gm Su Hg This work DH5α E. coli; F′/endA1 hsdR17 (rk−mk+)supE44 thi-1 recA1 gyrA (Nalr) relA1 D(lacIZYA-argF)U169 deoR[f80 dlacD(lacZ)M15] Nal New England Biolabs PAO38Rif Rifampin-resistant derivative of P. aeruginosa PAO38 Ri (MIC, >500 μg/ml) 20 Plasmid pHSS6 2.3-kb plasmid vector Km 32 pK18 2.7-kb plasmid vector Km 25 pNJR3-2 28-kb E. coli-P. aeruginosa shuttle vector Tc 28 pAZ305 23-kbEcoRI fragment obtained from PAe191 total DNA, cloned into pHSS6; oxa19 Km Ti Ca Gmb This work pAZ316 4.0-kb HindIII fragment from pAZ305 cloned into HindIII-digested pNJR3-2;oxa19 Tc Ti Ca Gmb This work pAZ327 1,097-bp NarI-HindIII fragment from pAZ316 cloned intoAccI-HindIII-digested pK18,oxa19 Km Ti Ca This work pAZ309 pNJR3-2 derivative encoding oxa13 Tc Ti Ak 20 pAZ310 In vitro-selected derivative of pAZ309 selected on ceftazidime; oxa13-1 Tc Ti Ca Ak This work pAZ326 1,097-bpNarI-HindIII fragment from pAZ310 cloned intoAccI-HindIII-digested pK18;oxa13-1 Km Ti Ca This work - Table 2.
MICs of different β-lactams for PAe191 and OXA13-1, OXA-19- and OXA-13-producing transformants
Antibiotic + inhibitora MIC (μg/ml) for the following strains (plasmid/enzyme): PAe191 PAO38 (pAZ310/OXA13-1) PAO38 (pAZ316/OXA-19) PAO38 (pAZ309/OXA-13)b PAO38 (pNJR-32) DH5α (pAZ326/OXA13-1) DH5α (pAZ327/OXA19) DH5α (pK18) Ampicillin >512 >512 >512 —c >512 8 64 2 Ticarcillin 512 64 256 256 16 8 64 1 + CLA 512 64 256 256 16 8 32 1 Piperacilin 128 32 64 32 2 2 8 0.5 + TAZ 128 16 64 32 2 0.5 1 0.5 Cefotaxime 128 32 32 16 16 — — — Ceftazidime 512 256 256 2 2 8 16 0.12 + CLA 256 128 128 2 16 2 2 0.06 + IMI 32 16 32 16 16 — — — Aztreonam 64 8 16 8 2 — — — Cefsulodin 64 64 64 32 2 — — — Imipenem 1 1 1 1 1 — — — Amikacin 32 16 8 16 4 — — — Gentamicin 256 4 64 — 4 — — — - Table 3.
Specific activities of OXA-13-1 and OXA-19 fromP. aeruginosa and E. coli producers
Substrate (50 mM) Sp act (mU/mg) PAO38 DH5α pAZ316/ OXA-19 pAZ310/ OXA-13-1 pAZ327/ OXA-19 pAZ326/ OXA-13-1 Ampicillin 999 28 826 30 Ceftazidime 0.9 1.0 1.5 1.8
