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Identification of a "hot spot" for integration of mobile elements in Enterococcus faecium

Abstract number: O196

Top J., van Schaik W., Bonten M., Willems R.

Objectives: The aim of the current study was to determine the integration site and exact size of the esp containing putative pathogenicity island (PAI) of Enterococcus faecium strain E1162 and to investigate sequence heterogeneity adjoining the PAI integration site in PAI positive and negative isolates.

Methods: The genome sequences of the PAI-negative strain E1071 and the PAI-positive strain E1162 have been determined in our laboratory (Van Schaik et al., in preparation). From the genome sequences, regions that were homologous to the flanking sequence of the esp containing PAI in E1162 were identified in E1071 and in another PAI negative strain, E. faecium DO, which has a publicly available genome sequence. This allowed us to identify the PAI integration site. Subsequently, PAI integration and sequence heterogeneity adjoining the integration site was determined using normal and long-range PCR in 17 PAI-positive and 34 PAI-negative strains using combinations of PAI specific primers and primers specific for the flanking genes.

Results: DNA alignments between E1162 and DO revealed integration in E1162 of a 61-kb large DNA fragment, containing the esp gene, in the 3' end of an open reading frame with high identical to orf1671 of E. faecium DO. This integration resulted in a 54-bp duplication. At the 5' end of the E1162 PAI a 22-kb region with high similarity (up to 100%) to an E. faecalis mobile element was found, while the 3'-end is homologous to orf13 to orf23 of Tn916. Interestingly, in strain E1071 another 8.3-kb element with no homology to the PAI of E1162 was integrated at the same position. PCR demonstrated that in all 17 PAI-positive isolates the PAI was integrated in the DO orf1671 homolog. In 23/33 PAI-negative strains, PCR confirmed no integration at this site, however, in one isolate PCR indicated integration of a fragment of at least 13-kb. In 9 strains no PCR product using primers spanning the integration site was obtained suggesting either integration of DNA elements too large to amplify or polymorphisms at the primer annealing site.

Conclusions: The striking variety of mobile DNA inserted into the DO orf1671 homolog suggests that this site may be a "hot spot" for integration of exogenously acquired genetic elements in E. faecium.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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