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Journal of Clinical Microbiology, March 2007, p. 979-989, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01772-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Outbreaks of Multidrug-Resistant Pseudomonas aeruginosa in Community Hospitals in Japan{triangledown}

Jun-Ichiro Sekiguchi,1 Tsukasa Asagi,2 Tohru Miyoshi-Akiyama,1 Atsushi Kasai,2 Yukie Mizuguchi,1 Minako Araake,1 Tomoko Fujino,1 Hideko Kikuchi,2 Satoru Sasaki,2 Hajime Watari,3 Tadashi Kojima,3 Hiroshi Miki,2 Keiji Kanemitsu,4 Hiroyuki Kunishima,4 Yoshihiro Kikuchi,2 Mitsuo Kaku,4 Hiroshi Yoshikura,5 Tadatoshi Kuratsuji,1,6 and Teruo Kirikae1*

Department of Infectious Diseases, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japan,1 National Hospital Organization, Sendai Medical Center, Miyagino 2-8-8, Miyagino, Sendai 938-8520, Japan,2 Eiken Chemical Co., Ltd., 1-33-8 Hongo, Bunkyo, Tokyo 113-8408, Japan,3 Department of Infection Control and Laboratory Diagnostics, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aoba, Sendai, Miyagi 980-8574, Japan,4 Ministry of Health, Labor, and Welfare, Kasumigaseki 1-2-2, Chiyoda, Tokyo 100-8916, Japan,5 National Research Institute for Child Health and Development, Okura 2-101, Setagaya, Tokyo 157-8535, Japan6

Received 28 August 2006/ Returned for modification 23 October 2006/ Accepted 6 November 2006


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ABSTRACT
 
We previously reported an outbreak in a neurosurgery ward of catheter-associated urinary tract infection with multidrug-resistant (MDR) Pseudomonas aeruginosa strain IMCJ2.S1, carrying the 6'-N-aminoglycoside acetyltransferase gene [aac(6')-Iae]. For further epidemiologic studies, 214 clinical isolates of MDR P. aeruginosa showing resistance to imipenem (MIC ≥ 16 µg/ml), amikacin (MIC ≥ 64 µg/ml), and ciprofloxacin (MIC ≥ 4 µg/ml) were collected from 13 hospitals in the same prefecture in Japan. We also collected 70 clinical isolates of P. aeruginosa that were sensitive to one or more of these antibiotics and compared their characteristics with those of the MDR P. aeruginosa isolates. Of the 214 MDR P. aeruginosa isolates, 212 (99%) were serotype O11. We developed a loop-mediated isothermal amplification (LAMP) assay and a slide agglutination test for detection of the aac(6')-Iae gene and the AAC(6')-Iae protein, respectively. Of the 212 MDR P. aeruginosa isolates, 212 (100%) and 207 (98%) were positive in the LAMP assay and in the agglutination test, respectively. Mutations of gyrA and parC genes resulting in amino acid substitutions were detected in 213 of the 214 MDR P. aeruginosa isolates (99%). Of the 214 MDR P. aeruginosa isolates, 212 showed pulsed-field gel electrophoresis patterns with ≥70% similarity to that of IMCJ2.S1 and 83 showed a pattern identical to that of IMCJ2.S1, indicating that clonal expansion of MDR P. aeruginosa occurred in community hospitals in this area. The methods developed in this study to detect aac(6')-Iae were rapid and effective in diagnosing infections caused by various MDR P. aeruginosa clones.


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INTRODUCTION
 
Pseudomonas aeruginosa causes nosocomial infections as a result of its ubiquitous nature, ability to survive in moist environments, and resistance to many antibiotics and antiseptics. A serious problem is the emergence of multidrug-resistant (MDR) P. aeruginosa strains resistant to ß-lactams, aminoglycosides, and quinolones (34, 39, 46). Although intrinsically sensitive to ß-lactams (e.g., ceftazidime [CAZ] and imipenem [IPM]), aminoglycosides (e.g., amikacin [AMK] and tobramycin), and fluoroquinolones (e.g., ciprofloxacin [CIP] and ofloxacin [OFX]), P. aeruginosa resistant to these antibiotics has emerged and is widespread (34, 39, 46).

We previously reported a nosocomial outbreak of catheter-associated urinary tract infection involving new MDR P. aeruginosa strain IMCJ2.S1, which occurred in a neurosurgery ward of a hospital located in the Tohoku area of Japan (46). This strain showed broad-spectrum resistance to aminoglycosides, ß-lactams, fluoroquinolones, tetracyclines, sulfonamide, and chlorhexidine. We found that IMCJ2.S1 harbored a novel class 1 integron, In113, containing an array of three gene cassettes of the metallo-ß-lactamase (MBL) blaIMP-1 gene, aminoglycoside 6'-acetyltransferase aac(6')-Iae gene, and aminoglycoside 3'-adenylyltransferase aadA1 gene (46). This strain possessed mutations of the gyrA (83Thr->Ile) and parC (87Ser->Leu) genes involving amino acid substitutions, resulting in high-level resistance to fluoroquinolones.

In the geographic area where the MDR P. aeruginosa outbreak occurred (46), hospitals and a commercial clinical laboratory were surveyed for similar organisms. Because 99% of the MDR P. aeruginosa isolates analyzed were found to harbor the aac(6')-Iae gene, we developed a loop-mediated isothermal amplification (LAMP) assay (31) and a slide agglutination assay to detect the aac(6')-Iae gene and AAC(6')-Iae protein, respectively. These methods were evaluated for their usefulness in detecting new MDR P. aeruginosa strains.


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MATERIALS AND METHODS
 
Bacterial strains. Criteria for multidrug resistance of P. aeruginosa were in accordance with the Law Concerning the Prevention of Infections and Medical Care for Patients with Infections of the Japanese Ministry of Health, Labor, and Welfare; the criteria are resistance to imipenem (MIC ≥ 16 µg/ml), amikacin (MIC ≥ 64 µg/ml), and ciprofloxacin (MIC ≥ 4 µg/ml). The criterion for amikacin resistance (MIC ≥ 64 µg/ml) was different from that of a guideline of the Clinical and Laboratory Standards Institute (MIC ≥ 32 µg/ml) (4). Two hundred eighty-four clinical isolates of P. aeruginosa were obtained from 284 inpatients in 13 hospitals in Japan during the period October 2003 to September 2004; 214 isolates were MDR, and 70 were non-MDR. Information regarding the origins of the specimens was available for 99 of the 214 MDR isolates: 72 (73%) were from urine specimens, 18 (18%) were from respiratory tract specimens, 5 (5%) were from feces, 2 (2%) were from catheter tips, and 2 (2%) were from wounds. Of the 72 isolates from urine, 55 were from patients with urinary catheters. All P. aeruginosa isolates were originally identified by the submitting laboratories. Isolates that did not have typical characteristics (pigment and colony morphology) for P. aeruginosa were analyzed biochemically with an API 20NE kit (API-bioMerieux, La Balme les Grottes, France) to confirm identity as P. aeruginosa. P. aeruginosa M207 possessing blaIMP-1, P. aeruginosa NCB326 possessing blaIMP-2, and Acinetobacter baumannii NCB0211-439 possessing blaVIM-2 were provided by Y. Arakawa (National Institute of Infectious Diseases, Tokyo, Japan). Escherichia coli strain TOP10 (Invitrogen Corp., Carlsbad, CA) was used as the host for recombinant plasmids.

Serotyping. The O serotypes of the isolates were determined with a slide agglutination test kit containing three polyvalent antisera and 14 monovalent antisera (Denka Seiken Co., Tokyo, Japan). The kit was not in conformity with the International Antigenic Typing Scheme (IATS) (26) and was not applicable to some O types in the IATS. Therefore, we applied the standard classification of O types from A to N proposed by the Serotyping Committee for the Japan Pseudomonas aeruginosa Society (12).

Antimicrobial susceptibility. We obtained AMK and IPM from Banyu Pharmaceutical Co. (Tokyo, Japan), arbekacin [1-N-(S)-4-amino-2-hydroxybutyl dibekacin; ABK] from Meiji Seika Kaisha, Ltd. (Tokyo, Japan), aztreonam (AZL) from Eizai (Tokyo, Japan), CAZ from GlaxoSmithKline K. K. (Tokyo, Japan), CIP and OFX from Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan), doripenem (DRPM) from Shionogi & Co., Ltd. (Osaka, Japan), gentamicin (GEN) and streptomycin (STR) from Nacalai Tesque, Inc. (Kyoto, Japan), meropenem (MEM) from Sumitomo Pharmaceutical Co., Ltd. (Osaka, Japan), piperacillin (PIP) and piperacillin-tazobactam (TZP) from Tomiyama Pure Chemical Industries, Ltd. (Tokyo, Japan), and polymyxin B (PL-B) from Sigma-Aldrich (St. Louis, MO). Arbekacin is an aminoglycoside antibiotic and has been used for the treatment of methicillin-resistant Staphylococcus aureus infections in Japan (51). Values for MICs at which 50% of isolates were inhibited (MIC50) and MIC90 were determined by the microdilution method according to the Clinical Laboratory Standards Institute (CLSI, formally NCCLS; standard M7-A6) (4) except for ABK, PL-B, and STR, for which breakpoints (≥4 µg/ml) were obtained from the published data (16, 30, 46).

Screening for MBL-producing P. aeruginosa. P. aeruginosa isolates were screened for the presence of MBL by a double-disk synergy test with disks containing sodium mercaptoacetic acid, according to the method of Arakawa et al. (2).

Immunologic detection of AAC(6')-Iae. To detect AAC(6')-Iae produced by P. aeruginosa, we developed a new method with AAC(6')-Iae antibody-conjugated beads. Recombinant AAC(6')-Iae was purified as reported previously (46) and used for immunization of Japanese white rabbits. Antibody against AAC(6')-Iae was affinity purified from rabbit antisera with an N-hydroxysuccinimide-Sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ) conjugated to recombinant AAC(6')-Iae. Purified antibody was coupled to Polybead carboxylated microspheres (2.022 µm in diameter; Polysciences, Inc., Warrington, PA) according to the manufacturer's instructions. Antibody-conjugated beads were suspended at 2.5% (vol/vol) in 0.1 M phosphate buffer (pH 7.4) containing 0.1% sodium azide. Agglutination tests were performed with P. aeruginosa isolates grown on N-acetyl-L-cysteine agar medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). Bacterial cells suspended in distilled water were mixed with the antibody-conjugated beads. To confirm the specificity of the agglutination test, P. aeruginosa isolates were analyzed by conventional Western blotting with AAC(6')-Iae antibody.

PCR of class 1 integrons. Class 1 integrons responsible for multidrug resistance in P. aeruginosa (21, 34, 46) were detected and characterized by PCR as described previously (24). Primer pairs designed to amplify the gene cassette of In113 (46) and three primer pairs specific for blaIMP-1, blaIMP-2, and blaVIM-2 (47) were used. Positive controls were P. aeruginosa IMCJ2.S1 for class 1 integron In113, P. aeruginosa M207 for blaIMP-1, P. aeruginosa NCB326 for blaIMP-2, and A. baumannii NCB0211-439 for blaVIM-2. PCR was performed with a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA). Genomic DNA was extracted as described by Sambrook et al. (44). When unexpected sizes of PCR products were obtained, the PCR products were cloned into cloning vector pCRII (Invitrogen Corp.) for DNA sequencing.

LAMP assay of aac(6')-Iae. The LAMP assay amplifies DNA with high specificity under isothermal conditions (31). To identify P. aeruginosa isolates possessing aac(6')-Iae, we designed four primers (FIP, 5'-CAA TAC AAA TGT TTT CGG CGC TAC GTC ACT CCA AAA GGC TAC-3'; BIP, 5'-TAA ACG ATG AAT TGT GTG GTT GGG TTG GAT GTA GTT CCC AAG TT-3'; F3, 5'-TCA CAC ATA AAT TTC GAT TCT TG-3'; and B3, 5'-ACC AAA TCC CTT ATT TTG ATG TT-3') for the LAMP assay. To extract DNA from P. aeruginosa isolates, a colony on N-acetyl-L-cysteine agar medium was suspended in 100 µl distilled water and boiled for 5 min. The bacterial suspension was then centrifuged at 12,000 x g for 2 min, and DNA in the supernatant was used for the LAMP assay. The LAMP reaction was performed with a Loopamp DNA amplification kit (Eiken Chemical Co., Ltd., Tokyo, Japan). The LAMP reaction mixture (12.5 µl), supplemented with 1.6 µM FIP and BIP primers, 0.2 µM F3 and B3 primers, 2x reaction mixture (6.25 µl), 4 U Bst DNA polymerase, 8 µg monomeric cyanine (YO-PRO-1), and 1.0 µl DNA sample, was incubated at 63°C for 45 min in a real-time thermal cycling system (Roter-Gene 2000; Corbett Research, Mortlake, New South Wales, Australia). Amplified DNA was monitored at 510 nm during the incubation. Alternatively, 25 µl of the reaction mixture was incubated at 63°C for 45 min on a block incubator (Advanced Science and Technology Enterprise Corp., Tokyo, Japan). After incubation, 10 µl of 1/100-diluted SYBR Green I nucleic acid gel stain (BioWhittaker Molecular Applications, Rockland, ME) was added to the reaction mixture. A change in color from orange to green indicated positive amplification.

PCR of QRDRs. The gyrA, gyrB, parC, and parE quinolone resistance-determining regions (QRDRs) were amplified by PCR with primers from and according to the methods described previously (1, 11, 20, 28). PCR products were sequenced with the same primers.

DNA sequencing. DNA sequences determined by the dideoxy chain termination method with an ABI PRISM 3100 sequencer (Applied Biosystems), and deduced protein sequences were subjected to homology searches in the DNA Data Bank of Japan (DDBJ), GenBank, and EMBL databases with FASTA and BLAST.

Pulsed-field gel electrophoresis (PFGE). Chromosomal DNA was prepared by the procedure of Grundmann et al. (10) and digested overnight with 10 U SpeI (Takara Bio, Inc., Shiga, Japan). The DNA fragments were separated on 1.0% agarose gels in 0.5x Tris-borate-EDTA buffer with a CHEF Mapper system (Bio-Rad Laboratories, Hercules, CA) at 6 V/cm for 20 h. The obtained fingerprinting patterns, normalized to the molecular weight markers, were analyzed by the unweighted-pair-group method with Molecular Analyst Fingerprinting Plus software, version 1.6 (Bio-Rad Laboratories, Inc.), to obtain average linkage-based dendrograms.

Statistical analysis. Results of a PCR assay, a LAMP assay, and an agglutination test were analyzed by chi-square test. A P value of <0.01 was considered statistically significant.


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RESULTS
 
Distribution of MDR P. aeruginosa among hospitals. Nineteen hospitals and one clinical laboratory center from a single prefecture (population size, 2,360,000) participated in this study. MDR P. aeruginosa was isolated from 13 hospitals (Fig. 1). A total of 214 MDR P. aeruginosa isolates were obtained; 73 (34%), 38 (18%), and 22 (10%) were obtained from hospitals NA, CB, and CA, respectively, indicating that the spread of MDR P. aeruginosa was relatively limited. Seventy non-MDR P. aeruginosa isolates from the same hospitals were used for comparative analysis.


Figure 1
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  		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.

Serotyping. Ten serotypes were identified (Table 1): 222 were O11, 14 were O1, 10 were O10, 8 were B, 7 were M, 5 were O4, 4 were O3, 4 were O6, and 1 each was O9 and C. Six additional isolates showed agglutination with polyvalent antiserum but not with any of the monovalent antisera, i.e., they were nontypeable. A total of 212 of the 214 MDR P. aeruginosa isolates (99%) were serotype O11, whereas 70 of the non-MDR isolates were of a variety of serotypes, including O1, O3, O4, O6, O9, O10, O11, B, C, and M. These results indicated that serotype O11 was predominant for MDR P. aeruginosa in this prefecture.


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  		TABLE 1. Phenotypic and genotypic characterization of 284 clinical isolates of P. aeruginosa

Antimicrobial susceptibility tests. Most of the MDR P. aeruginosa isolates were resistant to all antimicrobials tested, except for GEN and PL-B (Tables 1 and 2). Rates of drug resistance were as follows: AMK, 100%; ABK, 91.6%; AZL, 99.5%; CAZ, 100%; CIP, 100%; DRPM, 99.1%; GEN, 57.5%; IPM, 100%; MEM, 100%; OFX, 100%; PIP, 100%; PL-B, 28%; STR, 100%; TZP, 100%. Rates of drug resistance among the non-MDR isolates were less than 63%, except that for STR, which was 98.6%. MIC50 and MIC90 values for MDR isolates were high, except those for ABK, GEN, and PL-B, and MIC50 and MIC90 values for non-MDR isolates were low, except those for AMK.


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  		TABLE 2. MIC50 and MIC90 values and percent antimicrobial resistance for 284 samples of P. aeruginosa

MBL production. MBL confers bacterial resistance to all ß-lactams except AZL (53). Of the 284 isolates, 213 (75%) produced MBL and all except one were MDR isolates.

AAC(6')-Iae production. AAC(6')-Iae was first identified in MDR P. aeruginosa strain IMCJ2.S1 (46). We developed a slide agglutination test with AAC(6')-Iae antibody-conjugated beads. P. aeruginosa IMCJ2.S1 showed a positive result within 30 s (Fig. 2, lane 2), whereas AAC(6')-Iae-negative P. aeruginosa strain ATCC 27853 did not (Fig. 2, lane 4). Two hundred seventeen isolates were positive for the production of AAC(6')-Iae in this test (Table 3). The results of the slide agglutination test were in complete agreement with Western blotting data obtained with AAC(6')-Iae antibody (data not shown).


Figure 2
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  		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.


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  		TABLE 3. Comparison of PCR, LAMP, and agglutination test results for the detection of MDR P. aeruginosa isolates belonging to genotype cluster Aa

Detection of class 1 integrons. PCR assay with primers 5'-cs and 3'-cs (24), which are specific for the 5' conserved segments (CS) (49) and the 3' CS (49) of class 1 integrons, respectively, showed that 230 of the 284 isolates were positive. Amplified band sizes ranged from 0.8 kb to 2.5 kb (data not shown). All of these 230 isolates yielded a single band. Of these isolates, 212 yielded a 2.5-kb band, which is the same as that of the class 1 integron In113 (46). Sixteen isolates yielded a 0.8-kb band, and the remaining two yielded a 1.8-kb band and a 1.7-kb band. For the 212 isolates showing a 2.5-kb band, the presence of In113 was confirmed by PCR with specific primers, as described previously. MBL genes blaIMP-2 and blaVIM-2 are frequently found in Japan and are often associated with integrons (47). Therefore, we screened the 284 MDR P. aeruginosa isolates for blaIMP-2 and blaVIM-2 by PCR. None of the 284 isolates were positive for blaIMP-2 or blaVIM-2.

The regions between the 5' CS and 3' CS of amplicons of unexpected sizes were sequenced, and the gene cassettes were identified (Table 1). Of 16 isolates showing an 0.8-kb band, three possessed a single gene cassette containing aac(6')-31, encoding 6'-N-aminoglycoside acetyltransferase type IV (R. E. Mendes, unpublished data; DDBJ/EMBL/GenBank accession no. AJ640197) (Table 1). This gene cassette was 639 nucleotides (nt) and contained a 65-nt 59-base-element (be) site, for site-specific cointegration events (35). Nine isolates possessed an aac(6')-31-like1 cassette identical to aac(6')-31, with the exception of a C-to-T substitution at nt 269 in the coding region. Four isolates possessed an aac(6')-31-like2 cassette identical to aac(6')-31, with the exception of a C-to-A substitution at nt 269. One isolate showing a 1.8-kb band possessed an array of three gene cassettes (Table 1). Of them, the first cassette was an aac(6')-31-like3 cassette similar to aac(6')-31 except for T-to-C and A-to-T substitutions at nt 57 and 266, respectively. The second cassette was 855 nt and contained the aminoglycoside adenylyltransferase gene aadA6 (29) and a 60-nt 59-be site. The third cassette was 320 nt and contained open reading frame orfD, of unknown function (29). The aadA6 and orfD cassettes were identical to those of In51 reported previously (29). One isolate showing a 1.7-kb band possessed two gene cassettes of blaIMP-1 (33) and aadA1 (25) (Table 1).

Resistance to fluoroquinolones. Amino acid alterations to GyrA, GyrB, ParC, and ParE QRDRs of the 284 isolates are listed in Table 4. Amino acid replacement in the QRDR of GyrA (83Thr->Ile or 87Asp->Asn, Gly, or Tyr) was detected in 254 of the 284 isolates (89.4%). Of these 254 isolates, 8 possessed a mutation of GyrA alone. The remaining isolates possessed additional substitutions in GyrA, GyrB, ParC, and ParE. The 83Thr->Ile substitution in GyrA was the predominant replacement (251 of 284 isolates, 88.4%), in agreement with previous data on fluoroquinolone-resistant P. aeruginosa isolates (1, 22, 28). A double mutation of GyrA, 83Thr->Ile and 87Asp->Asn or Gly, was detected in nine isolates.


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  		TABLE 4. Amino acid changes in gyrA, gyrB, parC, and parE genes in 284 clinical isolates of P. aeruginosa

Amino acid replacement in the QRDR of ParC (87Ser->Leu or 91Glu->Lys) was detected in 244 of the 284 isolates (85.9%). All of these 244 isolates possessed additional mutations. The 87Ser->Leu substitution was the predominant replacement (242 of 284 isolates, 85.2%) and has been implicated in fluoroquinolone resistance of P. aeruginosa (1, 22, 28). A double mutation of ParC, 87Ser->Leu and 91Glu->Lys, was detected in three isolates. We found an 83Pro->Leu, 85Gly->Asp, and 88Ala->Pro alterations in one isolate each (Table 4).

Amino acid replacement in the QRDR of GyrB (468Glu->Asp) was detected in 70 of the 284 isolates (24.6%). No double mutations in GyrB were detected. Lee et al. (22) recently reported that 468Glu->Asp was a predominant alteration of GyrB, and isolates with this alteration, in addition to GyrA (83Thr->Ile) and ParC (87Ser->Leu) substitutions, showed a high level of resistance to CIP (MIC > 64 µg/ml). Our results were in accordance with their findings. We also found a 458Ala->Thr alteration in four isolates and a 496Ile->Val alteration in one isolate. These alterations are probably not associated with CIP resistance in P. aeruginosa because they were found in CIP-susceptible isolates.

Amino acid replacement in the QRDR of ParE (425Ala->Val or 459Glu->Asp or both) was detected in 30 of the 284 isolates (10.6%). All isolates possessed multiple mutations of ParE. Lee et al. (22) speculated that the 459Glu->Asp mutation of ParE is associated with moderate or high-level fluoroquinolone resistance in P. aeruginosa. The 425Ala->Val mutation has been reported in fluoroquinolone-resistant isolates of P. aeruginosa (1). Other mutations leading to amino acid changes were found at codons 419 (Asp->Asn, 1 isolate), 427 (Gln->Leu, 1 isolate), and 457 (Ser->Alg, 1 isolate). The fluoroquinolone resistance associated with these mutations remains to be determined.

Analysis of the aac(6')-Iae gene by the LAMP method. To detect aac(6')-Iae, we developed a gene-specific LAMP assay. The index strain IMCJ2.S1 was used to standardize the method. Visual inspection showed that the LAMP assay successfully amplified the target sequence of the aac(6')-Iae gene of P. aeruginosa IMCJ2.S1 (Fig. 3A). Real-time kinetics of the LAMP reaction showed that the amplification signal could be detected on average by 18 min; fluorescence increased in the positive samples, following a sigmoid curve (Fig. 3B). Agarose gel electrophoresis of the LAMP products (Fig. 3C) showed a ladder-like pattern on the gel due to the formation of a mixture of stem-loop DNAs of various stem lengths, which are characteristic of LAMP products.


Figure 3
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  		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.

A total of 284 isolates, including 214 MDR P. aeruginosa isolates, were tested by the LAMP assay (Table 3). A total of 212 isolates were positive by the LAMP assay (Table 3). The results of the LAMP assay were in complete concordance with the PCR data, indicating that the PCR can be replaced by the LAMP method for detection of aac(6')-Iae-carrying P. aeruginosa. These results, together with ones of the agglutination test (Table 3), indicate that multidrug resistance was strongly associated with the presence of aac(6')-Iae and AAC(6')-Iae production in the P. aeruginosa isolates (P < 0.0001).

Genotyping by PFGE. The 284 isolates, including 214 MDR isolates, were typed by PFGE. One hundred thirty-three different PFGE types, designated from A1 to AL, were distinguished (Table 1). Fourteen types, A1, A2, A12, A14, A18, A21, A25, A27, A37, A41, A42, A43, A44, and A60, were identified in more than 2 isolates (Fig. 1), and type A1, which represented 83 of the isolates (29%), was the most prevalent and widely disseminated (Fig. 1), suggesting prefecture-wide clonal dissemination. Types A1, A12, A14, A21, A27, A37, and A38 were identified at two or more hospitals. Cluster analysis of the PFGE restriction patterns showed three large clusters, A, B, and C, sharing ≥70% similarity (Fig. 4). Of the 214 MDR isolates, 211 belonged to cluster A, comprising types A1 to A67, indicating that multidrug resistance was associated with one genotype, cluster A (Fig. 4 and Table 3). Fifteen isolates belonged to cluster B comprising types B1 to B14, and 10 isolates belonged to cluster C, comprising types C1 to C8. The PFGE patterns of the 35 non-MDR isolates varied greatly.


Figure 4
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  		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.


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DISCUSSION
 
A clonal expansion of P. aeruginosa resistant to three antibiotics, carbapenems, amikacin, and fluoroquinolones, has been reported (4, 14, 36, 37, 46). However, previous surveillance studies in Japan have not shown clonal expansion involving multiple hospitals (19, 52). The present study showed clonal expansion of MDR P. aeruginosa in hospitals in the Tohoku area of Japan. To our knowledge, this is the first description of a large-scale, community-wide outbreak of nosocomial infection caused by a single P. aeruginosa clone with high-level resistance to a large number of antibiotics. The routes of transmission of the MDR P. aeruginosa clone remain unclear. P. aeruginosa that can be recovered from the hospital environment could be a possible source of nosocomial infection (6, 42, 54). Patient-to-patient transmission has been documented among patients with cystic fibrosis (5, 42, 54). Catheter-associated urinary tract infections appeared widespread among the hospitals in our study; the majority of the isolates (approximately 70%) were obtained from urine specimens, and approximately 80% of these were from patients with urinary catheters.

Most MDR isolates tested (205 of 214; Table 1) showed a serotype of O11. This was not surprising because these isolates belonged to a single cluster, as revealed by PFGE analysis (Fig. 4). P. aeruginosa is categorized into 31 chemotypes, including 20 IATS serotypes and subtypes (48). Thus far, however, particular serotypes, such as serotypes O12 and O11, appear to have been preferentially associated with P. aeruginosa outbreaks (9, 23, 38, 41). A clone of P. aeruginosa belonging to serogroup O12, which was resistant to both carbenicillin and gentamicin, was predominant in outbreaks involving six hospitals in Athens in 1987 (23). Later, O12 isolates resistant to these two drugs were reported in European countries (9, 38, 41). P. aeruginosa O12 resistant to ciprofloxacin and ceftazidime and/or fosfomycin was implicated in hospital outbreaks in France during the period 1993 to 1994 (3). P. aeruginosa serotype O11 caused hospital outbreaks in the 1980s in the United States (8) and in 1994 and 1995 in Greece (50). P. aeruginosa O11 was implicated in folliculitis caused by the use of whirlpools and hot tubs in the 1970s and 1980s in the United States and Canada (40). More recently, hospital outbreaks caused by MDR P. aeruginosa serotype O11 occurred in Belgium (5) and in Japan (46). Different strains of serotype O11 were involved in the above-mentioned outbreaks because their PFGE profiles were quite different. In addition, the Japanese strains produced IMP-1 carbapenemase (46), but the Belgian strains did not (5). It is not known why P. aeruginosa strains belonging to particular serotypes of O12 and O11 were involved in these outbreaks.

We analyzed several features including serotype, antimicrobial susceptibility, MBL production, prevalence of aac(6')-Iae, structure of class 1 integrons, resistance to fluoroquinolones, and genotype based on PFGE analysis for MDR P. aeruginosa isolates. Results indicated that aac(6')-Iae is a good candidate marker for MDR P. aeruginosa infection. To detect the aac(6')-Iae gene and its product, we developed a LAMP-based detection assay and an agglutination assay. LAMP is a nucleic acid amplification method which relies on autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment (31). The amplification products are stem-loop DNA structures with several inverted repeats of the target and cauliflower-like structures with multiple loops. LAMP assays are simple and short and do not require expensive equipment. LAMP assays have been applied to the analysis of various infectious agents such as hepatitis B virus (7), Mycobacterium tuberculosis (15), severe acute respiratory syndrome coronavirus (13), E. coli O157:H7 (27), Clostridium difficile (18), Bordetella pertussis (17), Salmonella enterica (32), Mycoplasma pneumoniae (43), and Streptococcus pneumoniae (45). The LAMP assay developed in this study was as sensitive and specific as PCR. Though less sensitive and specific than the LAMP assay, the agglutination assay for AAC(6')-Iae is sufficiently accurate to detect MDR P. aeruginosa (98% of MDR P. aeruginosa isolates were positive). The agglutination assay is simpler and cheaper than the LAMP assay and is also useful in detecting MDR P. aeruginosa in the clinical setting.

MDR P. aeruginosa may have spread across Japan as a result of the increasing use of carbapenems such as IPM, aminoglycosides such as AMK, and fluoroquinolones such as CIP. Nationwide surveillance for MDR P. aeruginosa is under way. At the hospital level, monitoring for environmental sources of bacteria, cleaning of contaminated surfaces of treatment rooms and bathrooms, review of infection control measures in the treatment of urine, and avoidance of unnecessary measurements of urine are considered effective in preventing P. aeruginosa nosocomial infections. Although the mode of transmission between hospitals is unknown, the movement of infected patients from one hospital to another is a possibility. Thirty-one patients infected with MDR P. aeruginosa had been transferred from other hospitals to the hospitals participating in the present study.


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ACKNOWLEDGMENTS
 
We thank M. Nakano (Jichi Medical School, Japan) for comments on the manuscript and Y. Arakawa and N. Shibata (National Institute of Infectious Diseases, Japan) for providing P. aeruginosa M207 possessing blaIMP-1, P. aeruginosa NCB326 possessing blaIMP-2, and Acinetobacter baumannii NCB0211-439 possessing blaVIM-2. We also thank H. Oikawa, T. Suzuki, I. Kurokawa, T. Sato, N. Onodera, K. Hiratsuka, M. Chiba, M. Utagawa, H. Mikami, M. Tachiya, K. Sasaki, S. Nakanowatari, M. Sasaki, Y. Abe, M. Koseki, N. Yaguchi, T. Nakamura, Y. Sato, and Y. Suzuki for providing clinical isolates of P. aeruginosa. Doripemen was kindly provided by Shionogi & Co., Ltd., Osaka, Japan.

This study was supported by Health Sciences Research grants from the Ministry of Health, Labor, and Welfare of Japan (H16-JRYO- IPPAN-011 and H18-SHINKO-11).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious Diseases, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japan. Phone: (81) 3 3202 7181, ext. 2838. Fax: (81) 3 3202 7364. E-mail: tkirikae{at}ri.imcj.go.jp. Back

{triangledown} Published ahead of print on 22 November 2006. Back


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Journal of Clinical Microbiology, March 2007, p. 979-989, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01772-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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