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To develop an easy-to-use method for the rapid detection of antibiotic-resistant bacteria. Here, a new immunochromatographic assay specific for aminoglycoside 6′-N-acetyltransferase AAC(6′)-Iae was designed. AAC(6′)-Iae is a significant marker molecule for multidrug-resistant (MDR) Pseudomonas aeruginosa isolates in Japan.Monoclonal antibodies specific for AAC(6′)-Iae were used to construct the assay. The assessment of the assay was performed using 116 P. aeruginosa clinical isolates obtained from hospitals in the Kanto area of Japan where little was known about AAC(6′)-Iae producers. PCR analyses of the aac(6′)-Iae and class 1 integron, antimicrobial susceptibility testing and PFGE analysis were performed to characterize positive strains.The detection limit of the assay was 1.0 × 105 cfu. Of 116 clinical isolates, 60 were positive for AAC(6′)-Iae using the assay. The results of assessment with clinical isolates were fully consistent with those of aac(6′)-Iae PCR analyses, showing no false positives or negatives. All positive strains detected by the assay showed MDR phenotypes that were resistant to several classes of antibiotic. PFGE analysis showed that 59 of 60 positive strains tightly clustered, and these included clonal expansions.The developed assay is an easy-to-use and reliable detection method for AAC(6′)-Iae-producing MDR P. aeruginosa. This approach may be applicable for screening and investigation of antibiotic-resistant bacteria as an alternative to PCR analysis.