Outbreaks of multidrug-resistant Pseudomonas aeruginosa in community hospitals in Japan

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 >or= 16 microg/ml), amikacin (MIC >or= 64 microg/ml), and ciprofloxacin (MIC >or= 4 microg/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 >or=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.

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.

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.

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.

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.