Epidemiology

Outbreaks of Multidrug-Resistant Pseudomonas aeruginosa in Community Hospitals in Japan

Jun-Ichiro Sekiguchi, Tsukasa Asagi, Tohru Miyoshi-Akiyama, Atsushi Kasai, Yukie Mizuguchi, Minako Araake, Tomoko Fujino, Hideko Kikuchi, Satoru Sasaki, Hajime Watari, Tadashi Kojima, Hiroshi Miki, Keiji Kanemitsu, Hiroyuki Kunishima, Yoshihiro Kikuchi, Mitsuo Kaku, Hiroshi Yoshikura, Tadatoshi Kuratsuji, Teruo Kirikae
DOI: 10.1128/JCM.01772-06

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Distribution of 214 isolates of MDR P. aeruginosa among 13 hospitals in Japan. Double capital letters indicate the locations of the hospitals that participated in this MDR P. aeruginosa survey.

  • FIG. 2.
    FIG. 2.

    Slide agglutination test with AAC(6′)-Iae antibody-conjugated beads. Lane 1, AAC(6′)-Iae positive control; lane 2, P. aeruginosa IMCJ2.S1 positive control; lane 3, 50 mM HEPES buffer negative control as solvent of AAC(6′)-Iae; lane 4, P. aeruginosa ATCC 27853 negative control.

  • FIG. 3.
    FIG. 3.

    LAMP assay to detect MDR P. aeruginosa isolates possessing the aac(6′)-Iae gene encoding the aminoglycoside acetyltransferase AAC(6′)-Iae. P. aeruginosa IMCJ2.S1 and ATCC 27853 were used as positive and negative controls, respectively. (A) Visual inspection analysis of LAMP products. Lane 1, P. aeruginosa IMCJ2.S1; lane 2, P. aeruginosa ATCC 27853. (B) Real-time amplification monitoring of aac(6′)-Iae-specific LAMP. The amplification signal was detected at an average of 18 min, as indicated by the continuous increase in fluorescence. Increased fluorescence was not observed in the negative control. (C) Acrylamide gel electrophoresis of LAMP product. Lane 1, LAMP product of the 204-bp target sequence of the aac(6′)-Iae gene of P. aeruginosa IMCJ2.S1; lane 2, P. aeruginosa ATCC 27853 negative control; lane M, 1-kbp ladder.

  • FIG. 4.
    FIG. 4.

    Cluster analysis based on the PFGE patterns of 284 clinical isolates of P. aeruginosa from the 13 hospitals in the present study. Clustering was carried out with Molecular Analyst FingerprintingPlus software, version 1.6, as described in Materials and Methods.

Tables

  • TABLE 1.

    Phenotypic and genotypic characterization of 284 clinical isolates of P. aeruginosa

    No. of isolatesSusceptibility to:SerotypeGene cassette(s) of the class 1 integronPFGE type(s)
    β-LactamsAminoglycosidesFQsa
    PIPTZPCAZIPMMEMAMKGENCIPOFX
    MDR-P. aeruginosab
        120RRRRRRRRRO11blaIMP-1, aac(6′)-Iae, aadA1A1, A2, A4, A5, A7, A8, A9, A10, A12, A14, A15, A16, A18, A20, A21, A24, A25, A27, A28, A30, A31, A32, A33, A38, A41, A42, A43, A44, A45, A46, A48, A51, A54, A56, A62, A64, E2
        85RRRRRRSRRO11blaIMP-1, aac(6′)-Iae, aadA1A1, A2, A6, A11, A12, A13, A17, A18, A19, A21, A22, A23, A25, A26, A27, A34, A35, A36, A37, A39, A40, A41, A44, A47, A52, A53, A55, A58, A60, A61, A63, A65, A66, A67
        1RRRRRRRRRO1blaIMP-1, aac(6′)-Iae, aadA1A1
        2RRRRRRSRRO1blaIMP-1, aac(6′)-Iae, aadA1A38, A50
        1RRRRRRRRRMblaIMP-1, aac(6′)-Iae, aadA1A57
        3RRRRRRSRRMblaIMP-1, aac(6′)-Iae, aadA1A3, A29, A37
        1RRRRRRRRRO10aac(6′)-31-like1B13
        1RSRRRRSRRO1V
    Non-MDR-P. aeruginosa
        1RSSSRRSRRO11A49
        1SSSSSSSRRO11A59
        1RRSRRSRRRO1aac(6′)-31-like2B1
        1SSRRRSRRRO1aac(6′)-31-like2B1
        1SSSRRSSRRO1aac(6′)-31-like2B1
        1RSSRRSRRRO1aac(6′)-31-like2B2
        1SSSRRSRRRO1aac(6′)-31B6
        1SSSRRSRRRO1aac(6′)-31-like1B8
        1RSSRRSSRRO1aac(6′)-31-like1B7
        1SSSSSSSRRO6aac(6′)-31-like1B3
        1SSSSSSSRRO10aac(6′)-31-like1B4
        1SSSRRSRRRO10aac(6′)-31-like1B5
        1SSSRRSSRRO10aac(6′)-31-like1B9
        1SSSRSSRRRO10aac(6′)-31B12
        1RSSRSSSRRO10aac(6′)-31-like1B14
        1SSSSSSSRRNTcaac(6′)-31B10
        1RSSRRSSRRMaac(6′)-31-like1B11
        2RRRRRSSRRNTC1
        1RRRRRSSRRO3C2
        2RRRRRSSRRO3C4
        1SSSRRSSRRO1C3
        1SSRRRSSRRO1C7
        1RRRRRSSRRBC5
        1SSSSSSSSSBC6
        1RRRRRSSRRO11C8
        1SSSSSSSSRO4D1
        1SSSSSSSRRO4D2
        1SSSSSSSRSO11D3
        1SSSRRSSRRO11E1
        1RSSRRSSRRMF1
        1SSSRSSSRRO4F2
        1RSSRRSSSRO11G1
        1RSSSRSSRRO11G2
        1RSRRRSSRRO11H1
        1RRRSSSSSSBH2
        2SSSRRSSSSO10I
        1SSSSSSSSSO4J1
        1SSSSSSSSSO3J2
        1SSSSSSSSSNTK1
        1SSSSSSSSSO6K2
        1RRRSSSSSRO9L1
        1SSSSSSSRRBL2
        1RSSSSSRRRO11aac(6′)-31-like3, aadA6, orfDM
        1RRRRRSRRRBblaIMP-1, aadA1N
        1RSSSSSSSSO1O
        1RSRRRSSSSO6P
        1SSSSSSSSSCQ
        1RRSRRSSSRO10R
        1SSSSSSSSSO4S
        1SSSSSSSSSO11T
        1SSSSSSSSSO11U
        1SSSSSSSSSO11W
        1SSSSSSSSSO11Z
        1SSSSSSSSSO11AA
        1SSSSSSSSSO11AJ
        1SSSSSSSSSMX
        1SSRSSSSSSO1Y
        1SSSSSSSSSO10AB
        1RSRSSSSSRBAC
        1SSSSSSSSSO6AD
        1RRRSSRSSSO11AE
        1SSSSSRRSSO11AF
        1RRSSSSSSSNTAG
        1RSSSSSSSSBAH
        1SSSRSSSSRO1AI
        1SSSSSSSSSBAK
        1SSSSSSSSSNTAL

    • ↵a FQs, fluoroquinolones.

    • ↵b Numbers of MDR isolates showing a respective PFGE type are shown in Fig. 1.

    • ↵c NT, nontypeable.

  • TABLE 2.

    MIC50 and MIC90 values and percent antimicrobial resistance for 284 samples of P. aeruginosa

    Antimicrobial agentBreakpoint for resistance (μg/ml)MDR isolatesa (n = 214)Non-MDR isolates (n = 70)
    % ResistantRange (μg/ml)MIC50 (μg/ml)MIC90 (μg/ml)% ResistantRange (μg/ml)MIC50 (μg/ml)MIC90 (μg/ml)
    PIP≥128100128->512>512>51241.41->51264512
    TZP≥128/4100128->512512>51221.40.5-25632128
    CAZ≥3210032->512>512>51225.71->512864
    IPM≥1610032->51225651247.10.25->512832
    DRPM≥1699.12->512>512>51234.3<0.125->512832
    MEM≥1610032->512512>51244.3<0.125->512432
    AZT≥3299.516->51212812852.90.5-1283264
    ABK≥491.62-164824.3<0.125-1618
    AMK≥3210032-2561282562.90.25-256216
    GEN≥1657.50.25->32161612.9<0.125->128116
    STR≥4100512->512>512>51298.62->51232128
    CIP≥410016->12864>12851.4<0.125->128464
    OFX≥810032->128>128>12862.9<0.125->12816>128
    PL-B≥428.02-82422.91-824

    • ↵a Isolates defined as resistant to three antibiotics, imipenem (MIC ≥ 16 μg/ml), amikacin (MIC ≥ 32 μg/ml), and ciprofloxacin (MIC ≥ 4 μg/ml).

  • TABLE 3.

    Comparison of PCR, LAMP, and agglutination test results for the detection of MDR P. aeruginosa isolates belonging to genotype cluster Aa

    IsolatesNo. of isolates with indicated result by:
    PCRLAMPAgglutination test with AAC(6′)-Iae antibody-conjugated beads
    PositiveNegativeTotalPositiveNegativeTotalPositiveNegativeTotal
    MDR P. aeruginosa
        Cluster A212021221202122075212
        Other022022022
    Non-MDR P. aeruginosa
        Cluster A022022022
        Other0686806868105868
    Total212722842127228421765284

    • ↵a In all tests and combinations, the multidrug resistance of the isolates was positively associated with the positive results of aac(6′) tests based on chi-square tests (P < 0.0001).

  • TABLE 4.

    Amino acid changes in gyrA, gyrB, parC, and parE genes in 284 clinical isolates of P. aeruginosa

    No. of strains (n = 284)MIC (μg/ml) of:Replacement in QRDRsj
    GyrA at position:ParC at position:GyrB at position:ParE at position:
    CIPOFX83Thr (ACC)87Asp (GAC)87Ser (TCG)91Glu (GAG)Other468Glu (GAG)Other425Ala (GCG)459Glu (GAG)Other
    MDR P. aeruginosa
        1>128>128Ile (ATC)—aLeu (TTG)—83Pro→LeubAsp (GAT)——Asp (GAT)—
        25128->128>128Ile (ATC)—Leu (TTG)——Asp (GAT)——Asp (GAT)—
        1128>128Ile (ATC)—Leu (TTG)——Asp (GAT)———427Gln→Leug
        3732-128128->128Ile (ATC)—Leu (TTG)——Asp (GAT)————
        1>128>128Ile (ATC)Asn (AAC)Leu (TTG)Lys (AAG)——————
        11632Ile (ATC)—Leu (TTG)—85Gly→Aspc—————
        14716->12832->128Ile (ATC)—Leu (TTG)———————
        13264Ile (ATC)————————457Ser→Algh
    Non-MDR P. aeruginosa
        564->128>128Ile (ATC)—Leu (TTG)——Asp (GAT)————
        432-12864->128Ile (ATC)Asn (AAC)Leu (TTG)———————
        1128>128Ile (ATC)Asn (AAC)Leu (TTG)Lys (AAG)——————
        1>128>128Ile (ATC)Asn (AAC)—Lys (AAG)——————
        164>128Ile (ATC)Asn (AAC)Leu (TTG)—————Asp (GAT)—
        164128Ile (ATC)Gly (GGC)Leu (TTG)—88Ala→Prod—————
        1332-6464->128Ile (ATC)—Leu (TTG)———————
        216-3232-128Ile (ATC)—Leu (TTG)————Val (GTG)——
        116128Ile (ATC)—Leu (TTG)———458Ala→Thre———
        116128Ile (ATC)——Lys (AAG)——————
        116128Ile (ATC)——————Val (GTG)——
        18128Ile (ATC)—————458Ala→Thr——419Asp→Asni
        1216——Leu (TTG)———458Ala→Thr———
        6<0.25-0.51-8——————458Ala→Thr———
        2<0.250.25——————496Ile→Valf———
        1464—————Asp (GAT)————
        50.5-48-16Ile (ATC)—————————
        21-22-8—Tyr (TAC)————————
        1<0.250.25——————————
        20<0.25-16<0.25-64—Asn (AAC)————————

    • ↵a —, no amino acid change.

    • ↵b 83Pro→Leu, Pro at position 83 of ParC changed to Leu (CCG→CTG).

    • ↵c 85Gly→Asp, Gly at position 85 of parC changed to Asp (GGC→GAC).

    • ↵d 88Ala→Pro, Ala at position 88 of ParC changed to Pro (GCC→CCC).

    • ↵e 458Ala→Thr, Ala at position 453 of GyrB changed to Thr (GCG→ACG).

    • ↵f 496Ile→Val, Ile at position 496 of GyrB changed to Val (ATG→GTC).

    • ↵g 427Gln→Leu, Gln at position 427 of ParE changed to Leu (CAG→CTG).

    • ↵h 457Ser→Arg, Ser at position 457 of ParE changed to Arg (AGC→AGG).

    • ↵i 419Asp→Asn, Asp at position 419 of ParE changed to Asn (GAC→AAC).

    • ↵j Mutated nucleotides are underlined.

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