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Mechanisms of Resistance

Characterization of SFO-1, a Plasmid-Mediated Inducible Class A β-Lactamase from Enterobacter cloacae

Yoshimi Matsumoto, Matsuhisa Inoue
Yoshimi Matsumoto
Department of Microbiology, Kitasato University School of Medicine, Sagamihara, and
Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan
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Matsuhisa Inoue
Department of Microbiology, Kitasato University School of Medicine, Sagamihara, and
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DOI: 10.1128/AAC.43.2.307
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  • Fig. 1.
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    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.
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    Fig. 2.

    Construction of recombinant plasmids.

  • Fig. 3.
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    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.
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    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

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  • Table 1.

    Strains used in this study

    HostPlasmidPhenotypeRestriction enzyme insert (length [kb])VectorMarkeraNickname
    E. cloacae8009pFCX300LAmpA AmpRCrypticCTX
    E. cloacae 8009pFCX300MAmpA AmpRCrypticCTX
    E. cloacae8009pFCX300SCryptic
    E. coli DH10B
    E. coliDH10BpFCX310AmpASau3AI (2.3)pHSG396CP, CTXTF2-2
    E. coliDH10BpFCX320AmpASau3AI (2.3)pHSG396CP, CTXTF2-3
    E. coli DH10BpFCX300LAmpA AmpRCTXTF-L
    E. coli DH10BpFCX300MAmpA AmpRCTXTF-M
    E. coli DH10BpFCX301AmpA AmpREcoRI (10)pHSG396CP, CTXTF-L7
    E. coliDH10BpFCX302AmpA AmpREcoRI (10)pHSG396CP, CTXTF-M4
    E. cloacae 199SAmpC−
    E. cloacae 199SpFCX310AmpASau3AI (2.3)pHSG396CP, CTX
    E. cloacae199SpFCX320AmpASau3AI (2.3)pHSG396CP, CTX
    E. cloacae 199AmpC (inducible)
    E. cloacae199CAmpC (constitutive)
    • ↵a CTX, cefotaxime; CP, chloramphenicol; Rif, rifampin.

  • Table 2.

    Susceptibilities of E. cloacae 8009 and various transformants to β-lactams

    StrainPresence of ampRMIC (μg/ml)
    CefuroximeCefotaximeCeftizoximeCefoperazoneCeftazidimeCefoselisCefoxitinMoxalactamAztreonamImipenem
    E. cloacae8009+>1001006.25>1003.1312.5>1000.7812.50.78
    E. coliDH10B−6.250.05≦0.0250.10.2≦0.0253.130.10.10.2
    E. coliDH10B/pFCX310−>100501.56503.133.133.130.2250.39
    E. coliDH10B/pFCX320−>100501.561003.136.253.130.2250.2
    E. coliDH10B/pFCX300L+500.780.050.780.20.13.130.10.780.2
    E. coliDH10B/pFCX300M+>1006.250.26.251.560.786.250.26.250.39
    E. coliDH10B/pFCX301+>100250.78253.133.136.250.39250.2
    E. coliDH10B/pFCX302+>100251.56503.133.136.250.39250.2
    E. cloacae199Sa3.130.05≦0.0250.20.1≦0.0250.780.05≦0.0250.05
    E. cloacae199S/pFCX310>100250.39501.561.560.780.056.250.05
    E. cloacae199S/pFCX320>100501.56>1003.136.251.560.112.50.05
    E. cloacae199b6.250.20.10.390.20.05>1000.10.10.39
    E. cloacae199Cc>100505025501.56>1003.13250.2
    E. coliDH5α−3.13≦0.025≦0.025≦0.0250.05≦0.0253.130.1≦0.0250.2
    E. coliDH5α/FEC-1−>100>1006.25>10012.51003.130.39500.2
    E. coliKU3290d+>1002550501001.56>10012.5250.2
    • ↵a β-Lactamase nonproducer.

    • ↵b Inducible β-lactamase producer (original strain).

    • ↵c Constitutive β-lactamase producer.

    • ↵d Transformant producing cloned P. vulgaris β-lactamase.

  • Table 3.

    Susceptibilities of E. cloacae 8009 and various transformants to other antibiotics

    StrainMIC (μg/ml)
    GentamicinAmikacinKanamycinStreptomycinChloramphenicolOfloxacinSTa
    E. cloacae80090.391.561.563.133.130.050.39
    E. coliDH10B0.20.781.56>1003.13<0.0250.39
    E. coliDH10B/pFCX3100.10.20.78>100>100<0.0250.39
    E. coliDH10B/pFCX3200.20.781.56>100>100<0.0250.39
    E. coliDH10B/pFCX300L0.21.561.56>1003.13<0.0250.39
    E. coliDH10B/pFCX300M0.20.781.56>1003.13<0.0250.39
    E. coliDH10B/pFCX3010.390.781.56>100>100<0.0250.39
    E. coliDH10B/pFCX3020.20.781.56>100>100<0.0250.39
    E. cloacae199Sb0.20.780.781.5612.50.051.56
    E. cloacae199S/pFCX3100.20.390.781.56>1000.051.56
    E. cloacae199S/pFCX3200.20.390.781.56>1000.051.56
    • ↵a ST, sulfamethoxazole-trimethoprim.

    • ↵b β-Lactamase nonproducer.

  • Table 4.

    Kinetic parameters of SFO-1 and FEC-1 for β-lactam antibiotics

    SubstrateSFO-1FEC-1
    Km (μM)Relative VmaxaRelative Vmax/ KmaKm(μM)Relative VmaxaRelative Vmax/ Kma
    Cephaloridine110100100152100100
    Cephalothin60234428134198225
    Cefamandole171241154122125156
    Cefotiam27502033843170
    Cefoperazone6.6152462.82.6139
    Cefpiramide26712973336167
    Cefuroxime13231952732179
    Ceftizoxime217125.8821122.3
    Cefotaxime2.216790612359
    Cefmenoxime21201088461110
    Ceftriaxone191584271480
    Ceftazidime7850.470.0653930.130.05
    Ampicillin401951301789
    • ↵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

    EnzymeBush typeIC50(μg/ml)a
    Clavulanic acidImipenem
    SFO-12e0.10.35
    FEC-12e0.00220.13
    Chromosomal β-lactamase of P. vulgaris2e0.60.62
    TEM-12b1.014
    Chromosomal β-lactamase of E. cloacae1120.24
    Chromosomal β-lactamase ofE. coli17.81.4
    • ↵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 strainPhenotypeInduceraβ-Lactamase activitybInducibility and inducibility ratioc
    μM/min · mg of proteinRatiod
    Expt 1
     E. cloacae 8009AmpA AmpR−3401.0Inducible
    +2,4007.17.1
     E. coliDH10B−<1<0.003Constitutive
    +<1<0.0031.0e
     E. coli DH10B/pFCX300L (TF-L)AmpA AmpR−220.064Inducible
    +2100.649.6
     E. coliDH10B/pFCX300M (TF-M)AmpA AmpR−1190.35Inducible
    +1,7205.114.5
     E. coliDH10B/pFCX301 (TF-L7)AmpA AmpR−7002.1Inducible
    +6,500199.2
     E. coliDH10B/pFCX302 (TF-M4)AmpA AmpR−6201.9Inducible
    +7,4002211.9
    Expt 2
     E. cloacae 8009AmpA AmpR−2601.0Inducible
    +2,5009.69.3
     E. coliDH10B/pFCX310 (TF2-2)AmpA−2801.1Constitutive
    +2801.11.0
     E. coliDH10B/pFCX320 (TF2-3)AmpA−3501.3Constitutive
    +3501.31.0
    • ↵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.

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Characterization of SFO-1, a Plasmid-Mediated Inducible Class A β-Lactamase from Enterobacter cloacae
Yoshimi Matsumoto, Matsuhisa Inoue
Antimicrobial Agents and Chemotherapy Feb 1999, 43 (2) 307-313; DOI: 10.1128/AAC.43.2.307

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Characterization of SFO-1, a Plasmid-Mediated Inducible Class A β-Lactamase from Enterobacter cloacae
Yoshimi Matsumoto, Matsuhisa Inoue
Antimicrobial Agents and Chemotherapy Feb 1999, 43 (2) 307-313; DOI: 10.1128/AAC.43.2.307
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KEYWORDS

Enterobacter cloacae
beta-lactamases

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