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Mechanisms of Resistance

Association of Amino Acid Substitutions in Penicillin-Binding Protein 3 with β-Lactam Resistance in β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae

Kimiko Ubukata, Yumi Shibasaki, Kentarou Yamamoto, Naoko Chiba, Keiko Hasegawa, Yasuo Takeuchi, Keisuke Sunakawa, Matsuhisa Inoue, Masatoshi Konno
Kimiko Ubukata
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morookacho, Kohoku-ku, Yokohama,
Institute of Microbial Chemistry, Kamiohsaki, Shinagawa-ku, and
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Yumi Shibasaki
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morookacho, Kohoku-ku, Yokohama,
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Kentarou Yamamoto
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morookacho, Kohoku-ku, Yokohama,
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Naoko Chiba
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morookacho, Kohoku-ku, Yokohama,
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Keiko Hasegawa
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morookacho, Kohoku-ku, Yokohama,
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Yasuo Takeuchi
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morookacho, Kohoku-ku, Yokohama,
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Keisuke Sunakawa
Department of Infectious Diseases and
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Matsuhisa Inoue
Department of Bacteriology, School of Medicine, Kitasato University, Kitasato, Sagamihara, Japan
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Masatoshi Konno
Teikyo University School of Medicine,Tokyo, and
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DOI: 10.1128/AAC.45.6.1693-1699.2001
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  • Fig. 1.
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    Fig. 1.

    PBP profiles of AMP-susceptible and BLNAR H. influenzae strains (A) and Rd strains transformed with theftsI gene encoding PBP 3 (B). Membrane fractions were labeled with [3H]PEN for 10 min at 30°C and were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography (I). PBPs were named according to the proposal of Parr and Bryan (19), our data on PBP affinity, and morphologic observations. AMP MICs are as follows: for strain MT053 (lane 1) and MT196 (lane 2), 0.125 μg/ml; for strain MT066 (lane 3), 2 μg/ml; for strain ME870 (lane 4), 4 μg/ml; for strain MT040 (lane 5), 2 μg/ml; for strain ME1133 (lane 6), 4 μg/ml; for strain ME587 (lane 7), 1 μg/ml; for strain KK01 (lane 8), 1 μg/ml; for strain ME495 (lane 9), 1 μg/ml. (B) lane 1, recipient Rd strain of H. influenzae; lane 2, transformed strain Rd3A/ME870-11; lane 3, transformed strain Rd3A/ME870-12; lane 4, BLNAR strain ME870.

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    Fig. 2.

    Fluorograms of PBPs of H. influenzae TK422 pretreated with AMP (A), CTX (B), and FRM (C). A 30-μl aliquot of each membrane fraction was incubated with 3 μl of each of the nonradioactive β-lactams diluted to various concentrations for 10 min and then further incubated with 3 μl of [3H]PEN for another 10 min. PBPs were visualized by autoradiography after exposure of the dried gels to X-OMAT film for 20 days at −80°C. The numbers identifying the lanes are concentration (in micrograms per milliliter).

  • Fig. 3.
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    Fig. 3.

    Deduced amino acid sequence of part of PBP 3 from the strains listed in Table 1. Only amino acids that differed from the Rd amino acid sequence are shown. Boxes represent the conserved amino acid motifs of Ser-Thr-Val-Lys (STVK), Ser-Ser-Asn (SSN), and Lys-Thr-Gly (KTG).

  • Fig. 4.
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    Fig. 4.

    Deduced amino acid sequence of part of PBP 4s from the strains listed in Table 1. Only amino acids that differed from the Rd amino acid sequence are shown. Boxes indicate conserved amino acid sequences of Ser-Thr-Gln-Lys (STQK), Ser-Asp-Asn (SDN), and Lys-Thr-Gly (KTG).

Tables

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  • Table 1.

    Serotype, β-lactamase production, PBP affinity, and susceptibility to β-lactam antibiotics of the H. influenzae strains useed in this study

    Assigned groupStrainSourceaSerotypeβ-LactamasePBP affinityMIC (μg/ml)
    PBP 3A PBP 3B PBP 4AMP CEC CTX CRO CPD CDR CDN CPN FRMMEM
    Control MT196 NaNSb−NcNN 0.125 0.5 0.008 0.004 0.0160.25 0.008 0.008 0.25 0.031
    MT053 NaNS − N N N 0.25 1 0.008 0.0040.031 0.125 0.008 0.008 0.25 0.031
    TK422 Na NS − N N N 0.5 20.031 0.008 0.063 0.5 0.016 0.016 0.50.063
    TC01 CSF b +NDdND ND 8 2 0.0160.004 0.063 0.5 0.016 0.016 0.5 0.031
    AK01 CSF b + ND ND ND 8 20.016 0.008 0.063 0.5 0.008 0.016 0.250.031
    I ME2014 Ot NS −DeN N 1 32 0.0630.016 0.25 2 0.031 0.063 2 0.031
    ME1547 Na NS − D N N 1 640.063 0.008 0.5 4 0.031 0.125 1 0.063
    ME692 Na NS − D N N 1 320.063 0.016 0.5 4 0.063 0.125 0.5 0.125
    ME1503 Na NS − D N N 1 640.063 0.016 1 8 0.063 0.125 2 0.125
    H17 Na NS − D N N 1 320.063 0.008 0.25 2 0.031 0.125 1 0.125
    N130 Na NS − D N N 1 320.063 0.016 0.25 2 0.031 0.125 1 0.25
    H2 Na NS − D N N 4 64 0.250.031 1 2 0.063 0.25 1 0.025
    ME1495Na NS + ND ND ND 64 64 0.1250.016 0.5 4 0.063 0.125 1 0.031
    H7Na NS + ND ND ND 64 64 0.1250.031 4 8 0.063 0.5 1 0.25
    IIND01 CSF b − D N N 1 32 0.0630.016 0.25 8 0.031 0.125 2 0.25
    KK01 CSF b − D N N 1 640.063 0.031 0.25 4 0.031 0.125 0.50.125
    ME570 Na NS − D D N 164 0.063 0.016 1 2 0.031 0.125 10.25
    ME587 Na NS − D D N 164 0.063 0.008 0.25 4 0.031 0.063 10.25
    ME495 Na NS − D N N 164 0.125 0.031 1 4 0.063 0.25 20.125
    MT273 Na NS − D N N 232 0.063 0.016 0.5 2 0.031 0.125 80.5
    MT160 Na NS − D N N 232 0.063 0.008 0.5 2 0.031 0.125 20.5
    ME267 Ot NS − D D N 232 0.063 0.016 0.25 1 0.031 0.063 20.25
    ME040 Ot NS − D N D 264 0.063 0.063 1 4 0.063 0.25 20.25
    ME1210 Na NS − D D N 464 0.125 0.031 1 4 0.063 0.25 20.25
    MT313 Na NS − D N N 464 0.125 0.031 1 4 0.063 0.25 20.25
    ME1976 Na NS + ND ND ND 6432 0.063 0.016 0.25 2 0.016 0.125 10.063
    III MT066 Na NS − D DN 2 64 1 0.25 4 8 0.25 1 20.25
    MT070 Na NS − D D N 264 1 0.25 4 8 0.125 1 4 0.125
    ME1133 Na NS − D N D 4 641 0.25 8 16 0.25 2 2 0.25
    ME870Na NS − D D N 4 64 2 0.254 16 0.5 2 4 0.5
    • ↵a Na, nasopharynx; Ot, otorrhea; CSF; cerebrospinal fluid.

    • ↵b NS, not specified.

    • ↵c N, PBP affinity is normal.

    • ↵d ND, not determined.

    • ↵e D, PBP affinity is decreased.

  • Table 2.

    Deduced amino acid substitutions identified in PBP 3s from 25 BLNAR strains and 5 AMP-susceptible strains of H. influenzae

    Assigned groupStrainAmino acid substitution
    Asp-350 Ser-357 Met-377 Ser-385 Leu-389 Arg-517Asn-526
    Control MT196
    MT053
    TK422
    TC01
    AK01
    I ME2014His
    ME1547 Asn ThrHis
    ME692 Asn Asn His
    ME1503 Asn Asn His
    H17Asn His
    N130 Asn AsnHis
    H2 Asn Asn His
    ME1495 Asn His
    H7Asn Asn IIe Thr His
    II ND01Asn Asn Lys
    ME570 AsnLys
    ME587 AsnLys
    KK01 Asn AsnLys
    ME495 Asn AsnLys
    MT273 Asn AsnLys
    MT160 Asn AsnLys
    ME267 Asn AsnLys
    MT040 Asn AsnLys
    ME1210 Asn AsnLys
    MT313 Asn AsnLys
    ME1976 AsnLys
    III MT066 Asn AsnIIe Thr Phe Lys
    MT070 Asn AsnIle Thr Phe Lys
    ME1133 Asn AsnIle Thr Phe Lys
    ME870 Asn AsnIle Thr Phe Lys
  • Table 3.

    Susceptibilities to β-lactam antibiotics of H. influenzae strains transformed with ftsI DNA fragment

    Assigned groupStrainaMIC (μg/ml)
    AMP CEC CTX CDR CPD CDN CPN MEM
    Rd (ATCC51097) 0.25 1 0.0080.25 0.063 0.008 0.016 0.031
    IRd3A/H171 16 0.063 1 0.250.063 0.063 0.125
    II Rd3A/KK01116 0.063 1 0.5 0.031 0.063 0.125
    IIIRd3A/ME8702 16 0.5 2 2 0.1251 0.25
    • ↵a H. influenzae Rd (ATCC 51097) (the recipient) was transformed with PCR-amplifiedftsI gene DNA fragments from group I, II, and III strains as the donors. Transformants were selected on plates containing 5 or 10 μg of CEC per ml. As an example of the transformant nomenclature, Rd3A/ME870 corresponds to Rd strain containing theftsI gene from strain ME870.

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Association of Amino Acid Substitutions in Penicillin-Binding Protein 3 with β-Lactam Resistance in β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae
Kimiko Ubukata, Yumi Shibasaki, Kentarou Yamamoto, Naoko Chiba, Keiko Hasegawa, Yasuo Takeuchi, Keisuke Sunakawa, Matsuhisa Inoue, Masatoshi Konno
Antimicrobial Agents and Chemotherapy Jun 2001, 45 (6) 1693-1699; DOI: 10.1128/AAC.45.6.1693-1699.2001

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Association of Amino Acid Substitutions in Penicillin-Binding Protein 3 with β-Lactam Resistance in β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae
Kimiko Ubukata, Yumi Shibasaki, Kentarou Yamamoto, Naoko Chiba, Keiko Hasegawa, Yasuo Takeuchi, Keisuke Sunakawa, Matsuhisa Inoue, Masatoshi Konno
Antimicrobial Agents and Chemotherapy Jun 2001, 45 (6) 1693-1699; DOI: 10.1128/AAC.45.6.1693-1699.2001
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KEYWORDS

ampicillin
Anti-Bacterial Agents
Bacterial Proteins
Carrier Proteins
Haemophilus influenzae
Hexosyltransferases
Multienzyme Complexes
Muramoylpentapeptide Carboxypeptidase
penicillin resistance
Peptidyl Transferases
beta-lactamases

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