Molecular Characterization of Class 1, 2 and 3 Integrons in spp. Clinical Isolates in Poland - Isolation of a New Plasmid and Identification of a Gene for a Novel Fusion Protein.

Purpose: Gram-negative rods of the genus play an increasing role as etiological agents of healthcare-associated infections (HAI) in humans. These bacteria are characterized by natural and acquired resistance to several groups of antibacterial agents. The aim of the study was to characterize class 1, 2 and 3 integrons in the clinical isolates of spp. in Poland.

Methods: The study comprised 112 clinical strains of , isolated from patients hospitalized in Poland in 2010-2012. Identification of strains was confirmed using MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) system. Detection of class 1, 2 and 3 integrase DNA sequence was performed by multiplex-PCR. Amplicons obtained in the PCR reactions were purified and then sequenced bidirectionally.

Results: Among the analyzed strains, was a predominant species (103/112, 92.0%). All three classes of integrase DNA sequence were detected in the analyzed strains of spp. DNA sequence of class 3 integron, besides integrase gene, revealed three gene cassettes ( , ). BLAST analysis of DNA sequence revealed that class 3 integron was carried on 9448 bp plasmid which was named pPCMI3 - whole sequence of its DNA was submitted to GenBank NCBI (National Center for Biotechnology Information) - NCBI MH569711.

Conclusion: In this study, we identified a new plasmid pPCMI3 harboring class 3 integron. This is the first report of a gene coding for a novel fusion protein, which consists of OXA β-lactamase and acetyltransferase aac(6')-Ib-cr. In the analyzed strains, class 1 and 2 integrons were also detected. Among the strains with class 1 integron, nine contained cassette array 5'CS-aadA2-ORF-dfrA12-3'CS, and two - cassette array 5'CS-aacC1-ORF-ORF-aadA1-3'CS, which were not previously reported in spp.