Genetic analysis of the cap locus of Haemophilus influenzae serotypeE

Abstract number: P1324

Giufrè M., Cardines R., Accogli M., Mastrantonio P., Cerquetti M.

Objectives: Although immunisation of infants with conjugate vaccines against H. influenzae serotype b (Hib) has dramatically decreased the incidence of invasive Hib disease in developed countries, the potential for emergence of non-vaccine preventable strains has been suggested. Invasive disease caused by H. influenzae serotype e (Hie) strains has been recently observed in Italy, suggesting the importance of further molecular investigations on Hie genetic locus for the capsule biosynthetic genes (cap locus). The sequence of the entire Hie cap locus has not been previously described. In the present study, the Hie cap locus was characterised.

Methods: The location of the Hie cap locus within the chromosome was investigated in 11 invasive Hie isolates by PCR amplification and sequencing of the DNA regions flanking the ends of the locus. The cap locus from the invasive strain Hie 274 was sequenced. To encompass the entire cap locus, overlapping amplicons, ranging from 1500 bp to 9 kb, were generated by PCR. Each amplicon was then subcloned into pCR4-TOPO and the insert from each plasmid was sequenced. In all isolates, the copy number of the Hie cap locus was determined by Southern blot analysis.

Results: In each isolate, the chromosomal location of the Hie cap locus was identical to that previously described for H. influenzae serotype f strain 7000222 and associated with the same flanking genes (sodC at the 5prime; end and HI1637 at the 3' end). No sequences reminiscent of the IS1016 element were found to flank the locus. The Hie cap locus included 14 ORFs organised in three distinct functional regions (I, II and III). Both regions I and III showed high sequence identity to the previously described corresponding regions from Hib and Hif. Eight new ORFs (named ecs1 to ecs8) were identified in the Hie capsule-specific region II. All isolates were found to contain only one copy of the Hie cap locus.

Conclusion: Chromosomal location of the Hie cap locus is conserved among our isolates. Although Hie strains belong to the major phylogenetic division I (majority of serotypes a, b strains and all serotypes c, d, and e strains), the Hie cap locus appeared to be located in the same chromosomal site as that of serotype f strains. The Hie cap locus is not flanked by direct repeats of IS1016 and, accordingly, it is not amplified. Genes within the locus are organised in three distinct regions as other group II capsule biosynthetic clusters.

Session Details

Date: 16/05/2009
Time: 00:00-00:00
Session name: 19th European Congress of Clinical Microbiology and Infectious Diseases
Location: Helsinki, Finland, 16 - 19 May 2009
Presentation type:
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