New typing assay based on PCR amplification of IS257-ileS2 junctions for tracking high-level mupirocin resistance dissemination in staphylococci
Abstract number: P1711
Pérez Roth E., Alcoba Florez J., Rivero Pérez B., Méndez Álvarez S.
Objectives: Transfer of ileS2-carrying plasmids plays a critical role in the dispersion of mupirocin resistance. Here we have investigated the presence, location, and orientation of insertion sequence IS257 flanking the ileS2 gene and we have evaluated the utility of PCR amplification of IS257-ileS2 junctions for tracking high-level mupirocin resistance.
Methods: Forty-eight MRSA clinical isolates each carrying one of nine well characterized ileS2-carrying plasmids encoding high-level mupirocin resistance belonging to four structural groups was included. Specific primers were designed and used for the amplification of the upstream and downstream IS257-ileS2 junctions. IS257-ileS2 junctions confirmation was carried out by Southern blotting and hybridization with two probes specific for the ileS2 and an IS257 internal probe. Filter mating and curing experiments, as well as hybridization with specific DNA probes were performed to investigate the antibiotic resistance gene content of the plasmids.
Results: Using the same amplification conditions four single PCRs were performed for each S. aureus clinical isolate harbouring an ileS2-encoding plasmid to detect the existence and orientation of IS257 copies flanking the ileS2 gene. In all nine plasmids the ileS2 gene was found to be flanked by IS257 copies. Considering upstream and downstream regions, plasmids belonging to different groups have distinc IS257-ileS2 organizations. Isolates carrying plasmids of the same group showed an identical organization of the IS257-ileS2 spacers and thus the same amplification patterns. As in the case of pGO400, pUSA03, and pV0308 ileS2-encoding plasmids, S1 and S2 plasmids showed directly repeated copies of IS257 but upstream amplification patterns differed in size. S3 group plasmids have inverted copies of IS257, while in S4 plasmids, both IS257 copies were in opposite orientation to ileS2. Interestingly, IS257-ileS2 amplification patterns correlate with the ileS2-hybiridizaton polymorphs. However, the antibiotic resistance gene content vary for plasmids of the same group.
Conclusion: Our study highlights the value of IS257-ileS2 junctions heterogeneity for distinguishing ileS2-carrying plasmids through PCR amplification. This novel assay offers a rapid, simple, and feasible method for typing ileS2-carrying plasmids, and hopefully it could serve as a useful tool for clinicians and epidemiologists in their efforts to prevent and control high-level mupirocin resistance.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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