Chromosomally encoded blaCMY-2 located on a novel SXT/R391-related integrating conjugative element in a Proteus mirabilis clinical isolate

Abstract

Integrating conjugative elements (ICEs) are mobile genetic elements that can transfer from the chromosome of a host to the chromosome of a new host through the process of excision, conjugation, and integration. Although SXT/R391-related ICEs, originally demonstrated in Vibrio cholerae O139 isolates, have become prevalent among V. cholerae isolates in Asia, the prevalence of the ICEs among gram-negative bacteria other than Vibrio spp. remains unknown. In addition, SXT/R391-related ICEs carrying genes conferring resistance to extended-spectrum cephalosporins have never been described. Here we carried out a genetic analysis of a cefoxitin-resistant Proteus mirabilis clinical isolate, TUM4660, which revealed the presence of a novel SXT/R391-related ICE, ICEPmiJpn1. ICEPmiJpn1 had a core genetic structure showing high similarity to that of R391 and carried xis and int genes completely identical to those of R391, while an IS10-mediated composite transposon carrying bla(CMY-2) was integrated into the ICE. A nucleotide sequence identical to the 3' part of ISEcp1 was located upstream of the bla(CMY-2) gene, and other genes observed around bla(CMY-2) in earlier studies were also present. Furthermore, the nucleotide sequences of hot spot 2 and hot spot 4 in ICEPmiJpn1 showed high similarity to that of hot spot 2 in SXT(MO10) and with a part of the nucleotide sequence found in P. mirabilis ATCC 29906, respectively. ICEPmiJpn1 was successfully transferred to Escherichia coli, Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Citrobacter koseri in conjugation experiments. These observations suggest that ICEs may contribute to the dissemination of antimicrobial resistance genes among clinically relevant Enterobacteriaceae, which warrants careful observation of the prevalence of ICEs, including SXT/R391-related ICEs.

FIG. 1.

Localization of bla_CMY-2 and int in P. mirabilis TUM4660 and its transconjugant. (A) Whole genomic DNAs of P. mirabilis TUM4660 (lane 1), E. coli ML4909 (lane 2), and E. coli TUM4670 (lane 3) were digested with I-CeuI, and the restricted fragments were subjected to pulsed-field gel electrophoresis. DNA fragments were transferred to a nylon membrane and hybridized with probes specific to the 23S rRNA gene (B), bla_CMY-2 (C), and int (D).

FIG. 2.

Schematic representation of genes conserved between ICEPmiJpn1 and R391 and genes specific to ICEPmiJpn1 and R391. The gray arrows represent genes conserved between ICEPmiJpn1 and R391. The black and white arrows represent genes specific to ICEPmiJpn1 and R391 (not to scale), respectively. Thin black arrows represent primers used for PCR amplification of variable regions. Primers: 1, PMLE1; 2, intFor1; 3, intRev1; 4, orf14-Rev2; 5, orf14-For1; 6, CMY-Rev; 7, CMY-For; 8, orf14-Rev1; 9, orf14-For2; 10, traI-Rev; 11, HS1F; 12, HS1R; 13, HotS2F; 14, HotS2R; 15, HotS4F; 16, HotS4R; 17, HotS3F2; 18, TraF2R.