Article Figures & Data
Figures
- Fig. 1.
Agarose gel electrophoresis of plasmid DNAs fromE. cloacae 8009. Lane A, HindIII-digested bacteriophage λ DNA; lane B, E. cloacae 8009. L, large plasmid; M, medium-size plasmid; S, small plasmid.
- Fig. 2.
Construction of recombinant plasmids.
- Fig. 3.
Alignment of deduced amino acid sequence of SFO-1 (AmpA) with the sequences of homologous enzymes. The amino acid numbering for the class A β-lactamase is used (1). The S-X(T)-X(S)-K motif with the active-site serine is shaded. Asterisks indicate the conserved amino acid residues among these class A sequences.
- Fig. 4.
Alignment of deduced amino acid sequence of AmpR of SFO-1 with homologous AmpR sequences. Asterisks indicate the conserved amino acid residues.
Tables
- Table 1.
Strains used in this study
Host Plasmid Phenotype Restriction enzyme insert (length [kb]) Vector Markera Nickname E. cloacae8009 pFCX300L AmpA AmpR Cryptic CTX E. cloacae 8009 pFCX300M AmpA AmpR Cryptic CTX E. cloacae8009 pFCX300S Cryptic E. coli DH10B E. coliDH10B pFCX310 AmpA Sau3AI (2.3) pHSG396 CP, CTX TF2-2 E. coliDH10B pFCX320 AmpA Sau3AI (2.3) pHSG396 CP, CTX TF2-3 E. coli DH10B pFCX300L AmpA AmpR CTX TF-L E. coli DH10B pFCX300M AmpA AmpR CTX TF-M E. coli DH10B pFCX301 AmpA AmpR EcoRI (10) pHSG396 CP, CTX TF-L7 E. coliDH10B pFCX302 AmpA AmpR EcoRI (10) pHSG396 CP, CTX TF-M4 E. cloacae 199S AmpC− E. cloacae 199S pFCX310 AmpA Sau3AI (2.3) pHSG396 CP, CTX E. cloacae199S pFCX320 AmpA Sau3AI (2.3) pHSG396 CP, CTX E. cloacae 199 AmpC (inducible) E. cloacae199C AmpC (constitutive) ↵a CTX, cefotaxime; CP, chloramphenicol; Rif, rifampin.
- Table 2.
Susceptibilities of E. cloacae 8009 and various transformants to β-lactams
Strain Presence of ampR MIC (μg/ml) Cefuroxime Cefotaxime Ceftizoxime Cefoperazone Ceftazidime Cefoselis Cefoxitin Moxalactam Aztreonam Imipenem E. cloacae8009 + >100 100 6.25 >100 3.13 12.5 >100 0.78 12.5 0.78 E. coliDH10B − 6.25 0.05 ≦0.025 0.1 0.2 ≦0.025 3.13 0.1 0.1 0.2 E. coliDH10B/pFCX310 − >100 50 1.56 50 3.13 3.13 3.13 0.2 25 0.39 E. coliDH10B/pFCX320 − >100 50 1.56 100 3.13 6.25 3.13 0.2 25 0.2 E. coliDH10B/pFCX300L + 50 0.78 0.05 0.78 0.2 0.1 3.13 0.1 0.78 0.2 E. coliDH10B/pFCX300M + >100 6.25 0.2 6.25 1.56 0.78 6.25 0.2 6.25 0.39 E. coliDH10B/pFCX301 + >100 25 0.78 25 3.13 3.13 6.25 0.39 25 0.2 E. coliDH10B/pFCX302 + >100 25 1.56 50 3.13 3.13 6.25 0.39 25 0.2 E. cloacae199Sa 3.13 0.05 ≦0.025 0.2 0.1 ≦0.025 0.78 0.05 ≦0.025 0.05 E. cloacae199S/pFCX310 >100 25 0.39 50 1.56 1.56 0.78 0.05 6.25 0.05 E. cloacae199S/pFCX320 >100 50 1.56 >100 3.13 6.25 1.56 0.1 12.5 0.05 E. cloacae199b 6.25 0.2 0.1 0.39 0.2 0.05 >100 0.1 0.1 0.39 E. cloacae199Cc >100 50 50 25 50 1.56 >100 3.13 25 0.2 E. coliDH5α − 3.13 ≦0.025 ≦0.025 ≦0.025 0.05 ≦0.025 3.13 0.1 ≦0.025 0.2 E. coliDH5α/FEC-1 − >100 >100 6.25 >100 12.5 100 3.13 0.39 50 0.2 E. coliKU3290d + >100 25 50 50 100 1.56 >100 12.5 25 0.2 - Table 3.
Susceptibilities of E. cloacae 8009 and various transformants to other antibiotics
Strain MIC (μg/ml) Gentamicin Amikacin Kanamycin Streptomycin Chloramphenicol Ofloxacin STa E. cloacae8009 0.39 1.56 1.56 3.13 3.13 0.05 0.39 E. coliDH10B 0.2 0.78 1.56 >100 3.13 <0.025 0.39 E. coliDH10B/pFCX310 0.1 0.2 0.78 >100 >100 <0.025 0.39 E. coliDH10B/pFCX320 0.2 0.78 1.56 >100 >100 <0.025 0.39 E. coliDH10B/pFCX300L 0.2 1.56 1.56 >100 3.13 <0.025 0.39 E. coliDH10B/pFCX300M 0.2 0.78 1.56 >100 3.13 <0.025 0.39 E. coliDH10B/pFCX301 0.39 0.78 1.56 >100 >100 <0.025 0.39 E. coliDH10B/pFCX302 0.2 0.78 1.56 >100 >100 <0.025 0.39 E. cloacae199Sb 0.2 0.78 0.78 1.56 12.5 0.05 1.56 E. cloacae199S/pFCX310 0.2 0.39 0.78 1.56 >100 0.05 1.56 E. cloacae199S/pFCX320 0.2 0.39 0.78 1.56 >100 0.05 1.56 - Table 4.
Kinetic parameters of SFO-1 and FEC-1 for β-lactam antibiotics
Substrate SFO-1 FEC-1 Km (μM) Relative Vmaxa Relative Vmax/ Kma Km(μM) Relative Vmaxa Relative Vmax/ Kma Cephaloridine 110 100 100 152 100 100 Cephalothin 60 234 428 134 198 225 Cefamandole 171 241 154 122 125 156 Cefotiam 27 50 203 38 43 170 Cefoperazone 6.6 15 246 2.8 2.6 139 Cefpiramide 26 71 297 33 36 167 Cefuroxime 13 23 195 27 32 179 Ceftizoxime 217 12 5.8 821 12 2.3 Cefotaxime 2.2 16 790 61 23 59 Cefmenoxime 21 20 108 84 61 110 Ceftriaxone 19 15 84 27 14 80 Ceftazidime 785 0.47 0.065 393 0.13 0.05 Ampicillin 40 19 51 30 17 89 ↵a Values relative to that for hydrolysis of cephaloridine, which was set equal to 100.
- Table 5.
Susceptibility of SFO-1 to β-lactamase inhibitors
Enzyme Bush type IC50(μg/ml)a Clavulanic acid Imipenem SFO-1 2e 0.1 0.35 FEC-1 2e 0.0022 0.13 Chromosomal β-lactamase of P. vulgaris 2e 0.6 0.62 TEM-1 2b 1.0 14 Chromosomal β-lactamase of E. cloacae 1 12 0.24 Chromosomal β-lactamase ofE. coli 1 7.8 1.4 ↵a IC50, concentration that inhibits 50% of activity after 10 min of incubation at 37°C; nitrocefin was used as the substrate.
- Table 6.
β-Lactamase inducibility in E. cloacae 8009 and various transformants
Expt. no. and strain Phenotype Inducera β-Lactamase activityb Inducibility and inducibility ratioc μM/min · mg of protein Ratiod Expt 1 E. cloacae 8009 AmpA AmpR − 340 1.0 Inducible + 2,400 7.1 7.1 E. coliDH10B − <1 <0.003 Constitutive + <1 <0.003 1.0e E. coli DH10B/pFCX300L (TF-L) AmpA AmpR − 22 0.064 Inducible + 210 0.64 9.6 E. coliDH10B/pFCX300M (TF-M) AmpA AmpR − 119 0.35 Inducible + 1,720 5.1 14.5 E. coliDH10B/pFCX301 (TF-L7) AmpA AmpR − 700 2.1 Inducible + 6,500 19 9.2 E. coliDH10B/pFCX302 (TF-M4) AmpA AmpR − 620 1.9 Inducible + 7,400 22 11.9 Expt 2 E. cloacae 8009 AmpA AmpR − 260 1.0 Inducible + 2,500 9.6 9.3 E. coliDH10B/pFCX310 (TF2-2) AmpA − 280 1.1 Constitutive + 280 1.1 1.0 E. coliDH10B/pFCX320 (TF2-3) AmpA − 350 1.3 Constitutive + 350 1.3 1.0 ↵a The inducer was imipenem-cilastatin at 0.1 μg/ml.
↵b Cefotaxime (100 μM) was used as a substrate.
↵c Induced activity/uninduced activity for each set.
↵d Activity of extract/uninduced activity ofE. cloacae 8009.
↵e Nitrocefin was used as the substrate for calculation of the inducibility ratio for this strain.
