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Mechanisms of Resistance

Detection of gyrA Mutations among 335Pseudomonas aeruginosa Strains Isolated in Japan and Their Susceptibilities to Fluoroquinolones

Takashi Takenouchi, Eiko Sakagawa, Mie Sugawara
Takashi Takenouchi
Biological Research Laboratories and
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Eiko Sakagawa
Biological Research Laboratories and
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Mie Sugawara
Biomedical Research Laboratories, Sankyo Co., Ltd., 2-58 Hiromachi 1-chome, Shinagawa-ku, Tokyo 140-8710, Japan
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DOI: 10.1128/AAC.43.2.406
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    Fig. 1.

    Detection of gyrA mutations by PCR-RFLP analysis (a) and PCR-SSCP analysis of SacII site-positive (b) and -negative (c) DNA. Lanes M, molecular size standard (HaeIII-digested φX174); lane +, SacII site-positive DNA fragment; lane −, SacII site-negative DNA fragment; lanes 1, wild type; lane 2, mutant containing alteration of GCC→GCT at codon 67 (silent); lane 3, mutant containing alteration of CGT→CGA at codon 68 (silent); lane 4, mutant containing alteration of CCG→CCA at codon 79 (silent); lane 5, mutant containing alteration of CGC→CGT at codon 91 (silent); lane 6, mutant containing alteration of Asp-87→Asn; lane 7, mutant containing alterations of CCG→CCA at codon 79 (silent) plus Asp-87→Gly; lane 8, mutant containing alterations of Ala-67→Ser plus Asp-87→Gly; lane 9, mutant containing alteration of Gln-106→Leu; lane 10, mutant containing alteration of Thr-83→Ile; lane 11, mutant containing alterations of CGT→CGA at codon 68 (silent) plus Thr-83→Ile; lane 12, mutant containing alterations of Thr-83→Ile plus CGC→CGT at codon 91 (silent); lane 13, mutant containing alterations of Thr-83→Ile plus Asp-87→Gly; lane 14, mutant containing alterations of Thr-83→Ile plus Asp-87→Asn; lane 15, mutant containing alterations of Thr-83→Ile plus Asp-87→His; lane 16, mutant containing alteration of GCG→GCA at codon 84 (silent); lane 17, mutant containing alteration of Ala-84→Pro; lane 18, mutant containing alterations of GCG→GCA at codon 84 (silent) plus Asp-87→Tyr. Lanes 6 through 13, 17, and 18, quinolone-resistant clinical strains; lanes 14 and 15, mutated plasmids derived from pGL2B5; lanes 16 and 18, generous gifts from T. Schollaardt, namely, P4394 and Y4492, respectively.

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  • Table 1.

    Types of point mutation(s) and respective incidence among the 335 clinical isolates

    SacII siteType of mutationAmino acid changeNucleotide changeIncidence (no. [%] of strains)
    PositiveWild typeNoneNone243 (72.5)
    Silent mutationsAla-67→Ala (silent)GCC→GCT2 (0.6)
    Arg-68→Arg (silent)CGT→CGA9 (2.7)
    Pro-79→Pro (silent)CCG→CCA6 (1.8)
    Arg-91→Arg (silent)CGC→CGT5 (1.5)
    Single missense mutations with or without silent mutationAsp-87→AsnGAC→AAC2 (0.6)
    Pro-79, Asp-87→Pro (silent), GlyCCG, GAC→CCA, GGC1 (0.3)
    Gln-106→LeuCAG→CTG1 (0.3)
    Double missense mutationsAla-67, Asp-87→Ser, GlyGCC, GAC→TCC, GGC1 (0.3)
    NegativeSilent mutationAla-84→Ala (silent)GCG→GCA0a
    Single missense mutations with or without silent mutationThr-83→IleACC→ATC60 (17.9)
    Arg-68, Thr-83→Arg (silent), IleCGT, ACC→CGA, ATC2 (0.6)
    Thr-83, Arg-91→Ile, Arg (silent)ACC, CGC→ATC, CGT1 (0.3)
    Ala-84→ProGCG→CCG1 (0.3)
    Ala-84, Asp-87→Ala (silent), TyrGCG, GAC→GCA, TAC0b
    Double missense mutationsThr-83, Asp-87→Ile, GlyACC, GAC→ATC, GGC1 (0.3)
    Thr-83, Asp-87→Ile, AsnACC, GAC→ATC, AAC0c
    Thr-83, Asp-87→Ile, HisACC, GAC→ATC, CAC0c
    Total335 (100)
    • ↵a Found in strain P4394 (13).

    • ↵b Found in strain Y4492 (13).

    • ↵c Constructed by site-directed mutagenesis by introduction into plasmid pGL2B5.

  • Table 2.

    Susceptibilities of 336 clinical isolates of P. aeruginosa to 10 fluoroquinolones

    DrugMIC (μg/ml) for strains with the following GyrA alterationsa (type of change):
    PAO1Wild type (n = 265)bThr-83→Ile (PU→NH) (n = 63)Ala-84→Pro (NH→NH) (n = 1)Asp-87→Gly (−1c→PU) (n = 1)Asp-87→Asn (−1→PU) (n = 2)Asp-87→Tyrd (−1→PU) (n = 1)Gln-106→Leu (PU→NH) (n = 1)Ala-67, Asp-87→Ser, Gly (NH, −1→PU, PU) (n = 1)Thr-83, Asp-87→Ile, Gly (PU, −1→NH, PU) (n = 1)
    Range50%90%Range50%90%
    Norfloxacin0.390.20–1000.783.131.56–4001002002512.51.56, 12.512.52512.5200
    Ofloxacin0.780.39–1001.566.253.13–800502002512.53.13, 2512.52512.5200
    Enoxacin0.780.20–2001.566.253.13–>400100>40025253.13, 25502525200
    Ciprofloxacin0.100.025–500.200.780.39–40025506.253.130.78, 3.136.253.133.1350
    Tosufloxacin0.200.05–>250.391.560.10–>25>25>256.256.250.78, 1.563.131.566.25>25
    Lomefloxacin0.780.39–2001.566.250.78–80020040050256.25, 50255025400
    Fleroxacin0.780.20–2001.566.253.13–>1,6001004002512.53.13, 25252512.5400
    Sparfloxacin0.780.20–2001.563.131.56–>1,60010020012.512.51.56, 12.512.5256.25200
    Gatifloxacin0.780.20–501.563.131.56–2005010012.56.251.56, 6.256.2512.56.2550
    CS-9400.390.10–500.783.131.56–800501006.253.130.78, 12.53.1312.56.25100
    • ↵a Silent mutation is neglected.

    • ↵b n indicates number of strains.

    • ↵c −1, negatively charged.

    • ↵d Strain Y4492.

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Detection of gyrA Mutations among 335Pseudomonas aeruginosa Strains Isolated in Japan and Their Susceptibilities to Fluoroquinolones
Takashi Takenouchi, Eiko Sakagawa, Mie Sugawara
Antimicrobial Agents and Chemotherapy Feb 1999, 43 (2) 406-409; DOI: 10.1128/AAC.43.2.406

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Detection of gyrA Mutations among 335Pseudomonas aeruginosa Strains Isolated in Japan and Their Susceptibilities to Fluoroquinolones
Takashi Takenouchi, Eiko Sakagawa, Mie Sugawara
Antimicrobial Agents and Chemotherapy Feb 1999, 43 (2) 406-409; DOI: 10.1128/AAC.43.2.406
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KEYWORDS

anti-infective agents
DNA Topoisomerases, Type II
Pseudomonas aeruginosa

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