Article Figures & Data
Figures
- Fig. 1.
Amino acid alignments of SMB-1 sequence with those of other subclass B3 MBLs. The residues involved in zinc binding are highlighted with a dark background. The signal peptide of SMB-1 is shown with an underline. An asterisk indicates amino acid residues conserved among all subclass B3 MBLs. Proteins (GenBank accession no.) are the following: SMB-1 (AB636283), AMO1 (ACS83721), BJP-1 (NP772870), CAU-1 (CAC87665), CRB11 (ACS83724), FEZ-1 (CAB96921), GOB-1 (ABO21417), L1 (ABO60992), POM-1 (ADC79555), THIN-B (CAC33832), and CAR-1 (Q6D395).
- Fig. 2.
Tree view exhibiting the similarity of SMB-1 with the other subclass B3 MBLs. The tree was constructed using ClustalW, version 1.83 (http://clustalw.ddbj.nig.ac.jp/top-j.html), and was provided by the DNA Data Bank of Japan (DDBJ). Sequences incorporated to draw the tree were the same as those used for Fig. 1. The 0.1 scale represents a genetic unit reflecting 10% of the amino acid substitutions, and it was calculated with the ClustalW program.
- Fig. 3.
Localization of the blaSMB-1 gene on I-CeuI-digested total DNA of S. marcescens strain 10mdr148 separated by PFGE. Lane: M, CHEF DNA size standard marker (Bio-Rad); A, I-CeuI-digested total DNA of S. marcescens strain 10mdr148 stained with ethidium bromide; B, hybridization of I-CeuI-digested total DNA of S. marcescens strain 10mdr148 with probe specific for 16S rRNA gene; and C, hybridization of I-CeuI-digested total DNA of S. marcescens strain 10mdr148 with probe specific for the blaSMB-1 gene.
- Fig. 4.
Schematic representation of the generic environment of the blaSMB-1 gene. The 5′ end of the qacEΔ1 gene in the second 3′-CS was deleted of 143 bp that are present in the first 3′-CS.
Tables
- Table 1.
Primers used in this study
Primer Sequencea Nucleotide positionb ampC-F 5′-ATG ACG AAA GTG AAC CGC-3′ 1-18 ampC-R 5′-CCT GGA CGA TGT GGT AAG-3′ 1103-1120 CTX-M-3F 5′-ACC GTC ACG CTG TTG TTA G-3′ 47-64 CTX-M-3R 5′-CTT TCT GCC TTA GGT TGA G-3′ 806-824 IMP-1F 5′-ACC GCA GCA GAG TCT TTG CC-3′ 49-68 IMP-1R 5′-ACA ACC AGT TTT GCC TTA CC-3′ 616-635 IMP-2F 5′-GTT TTA TGT GTA TGC TTC C-3′ 16-34 IMP-2R 5′-AGC CTG TTC CCA TGT AC-3′ 677-693 VIM-2F 5′-ATG TTC AAA CTT TTG AGT AAG-3′ 1-21 VIM-2R 5′-CTA CTC AAC GAC TGA GCG-3′ 784-801 NDM-1F 5′-TTG CCC AAT ATT ATG CAC CC-3′ 7-26 NDM-1R 5′-ATT GGC ATA AGT CGC AAT CC-3′ 407-426 16S rRNA-10F 5′-GTT TGA TCC TGG CTC A-3′ 11-26 16S rRNA-800R 5′-TAC CAG GGT ATC TAA TCC-3′ 785-802 SMB-F 5′-CAG CAG CCA TTC ACC ATC TA-3′ 79-98 SMB-R 5′-GAA GAC CAC GTC CTT GCA CT-3′ 551-570 SMB-CloF 5′-CCC AAG CTT TCC GCC GAC TTG GCG CAG-3′ SMB-CloR 5′-GGG GTA CCA AGA CCG ATT TAG CCG GC-3′ PET-1 5′-GGA ATT CCA TAT GAA AAT CAT CGC TTC CC-3′ PET-2 5′-CCC AAG CTT TCA GCG TTT CTC GCT GGC C-3′ - Table 2.
Result of susceptibility testing
Antimicrobial agent MIC (μg/ml) against strain: S. marcescens 10mdr148 E. coli KAM32 (pCL-SMB) E. coli KAM32 (pCL1920) Ampicillin >256 >256 2 Piperacillin >256 >256 0.25 Cephalothin >256 256 8 Cephaloridine >256 64 2 Cefazolin >256 256 1 Cefuroxime >256 >256 0.25 Cefotaxime >256 16 ≤0.06 Ceftazidime 256 256 0.25 Cefepime >256 0.5 ≤0.06 Cefoxitin >256 >256 2 Cefmetazole >256 256 0.5 Flomoxef >256 64 ≤0.06 Aztreonam >256 0.13 0.13 Imipenem >32 8 0.25 Meropenem >32 16 0.03 Panipenem >32 32 0.5 Biapenem >32 16 0.25 Amikacin 12 NDa ND Gentamicin 1.5 ND ND Ciprofloxacin 1 ND ND Moxifloxacin 0.5 ND ND Tigecycline 1 ND ND ↵a ND, not determined.
- Table 3.
Kinetic parameters and inhibition profile of SMB-1a
Substrate or chelating agent Km (μM) kcat (s−1) kcat/Km (M−1·s−1) Relative kcat/Kmd IC50 (μM) Ampicillin 102 247 2.4 × 106 100 Piperacillin 380 68 1.8 × 105 7.5 Cephalothin 15 28 1.9 × 106 79 Cefuroxime 22 30 1.4 × 106 58 Cefotaxime 35 31 8.9 × 105 37 Ceftazidime 57 4.4 7.7 × 104 3.2 Cefepime 747 2.7 3.6 × 103 0.15 Cefoxitin 26 39 1.5 × 106 63 Aztreonam NHb NDc ND ND Imipenem 133 518 3.9 × 106 163 Meropenem 144 604 4.2 × 106 175 Dipicolinic acid 2.2 1,10-o-Phenanthroline 156 EDTA 14
