Presented at the Neonatal Society 2004 Spring Meeting.

Beveridge C, Saunders N, Herbert M  (introduced by Andrew Wilkinson)

Neonatal Unit, Department of Paediatrics, University of Oxford, John Radcliffe Hospital, Oxford, UK

Purpose: We aimed to develop a sub-microarray to study gene expression in Group B Streptococcus. The long-term goal is to identify key regulators of virulence that can be targeted to develop novel means of preventing GBS disease.

Experimental design: We used the two publicly available GBS genomes (1, 2) (serotypes III and V) to identify the genes encoding all the known regulators and virulence genes of GBS, many other surface-expressed proteins, and genes encoded by 14 putative pathogenicity islands. 384 probes were amplified by PCR and spotted onto glass slides (Genetix Qarray Mini microarrayer). To test the array, we undertook Comparative Genome Hybridisation (CGH) with DNA from a collection of GBS colonising or disease causing strains (n=22). DNA from each strain was directly Cy-dye labelled, hybridised to the array, and scanned (Genepix 4000B). We have also extracted RNA from GBS grown under disease-related conditions using RNAprotect (Qiagen) and the FastPrep (Qbiogene) system.

Results: The gene content of 22 strains of GBS (15 colonising, 3 early-onset sepsis, 4 late-onset sepsis) was assessed by CGH (figure 1). All the GBS strains had all the genes encoding the regulators and virulence genes that we included in the array. The putative pathogenicity islands were only present in a fraction of the strains. There was no relationship between the presence of an island and the disease-causing capacity of that strain. We have obtained good RNA profiles from GBS grown under disease-related conditions (figure 2).

 
Figure1: Representative CGH of two GBS strains. Most spots are
yellow but red or green spots indicate genes present in only one strain.

Figure 2: RNA profiles of GBS.

Conclusions: Our array is suitable for assessing expression of the regulators and virulence genes of GBS. These genes are present in all strains tested, indicating that they are essential to the organism. However, genes found on putative pathogenicity islands were not present in all strains, implying that they do not contain genes that confer virulence on the organism.

References
1. Glaser P. Genome sequence of serotype III Streptococcus agalactiae, a pathogen causing invasive neonatal disease. Mol Microbiol 2002; 45:1499-513.
2. Tettelin H. Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae. Proc Natl Acad Sci U S A, 2002; 99:12391-6.