Norma UNE-EN 1550:1998+A1:2008

Differences between the test isolate genomes and the TIGR4 genome are illustrated in Appendix 8. Of the R6 genes represented on the microarray which do not feature in the TIGR4 genome it was possible to identify from the hybridization patterns for the 10 test isolates all six regions of diversity described by Bruckner et al (Bruckner et al., 2004). This is illustrated in Appendix 9. For instance, the entire region spr0102 to spr0119 was present in strains ATCC700904 and 07-2839 but appeared almost entirely lacking hybridization for strain 05-1271. The other test isolates demonstrated many different combinations of hybridization or absent hybridization for these genes.

For the region spr0311 to spr0323, only genes from the smaller cluster spr0320 to spr0323 were found to hybridize. Again this could be the entire section (ATCC51916 and 05-1271) or only smaller sections (07-2839, ATCC700904 and BAA-659) or there could be a total lack of hybridization for the entire region (BAA-340, BAA-660, 05-2565, 05-1821 and OXC141).

Similar variable hybridization was seen at the regions of diversity spr0955 to spr0971, spr1403 to spr1404 and spr1618 to spr1621. Only isolate 07-2839 showed hybridization of any genes within the region of diversity spr1184 to spr1198.

In isolate ATCC700904 there appears to be a small region of diversity at spr1549 to spr1550 with hybridization occurring at these probes for this isolate alone. Both these genes code for hypothetical proteins although spr1550 has sequence similarity to a MutR protein. Spr1549 is 393bp in size and spr1550 is 852bp in size. These were noted as highly variable genes coding for a putative regulatory protein by Bruckner et al (Bruckner et al., 2004).

For these regions of diversity in the R6 genome, it would be of interest to perform CGH of DNA from the test strains against R6 DNA as competitive hybridization should then be seen for those genes highlighted in red above in Appendix 9.

Comparative hybridization of TIGR4 DNA against test isolate DNA has elucidated 4 regions of diversity which do not feature in the list of regions of diversity based on the TIGR4 genome identified by Silva et al (Silva et al., 2006) which are shown in Chapter 1 Table 1-2. The first is a 6.3kb region from SP0303 to SP0311 which mainly relates to components of a cellobiose phosphotransferase system. This region features as cluster 2 identified by Bruckner et al (Bruckner et al., 2004). A second region of 5.7kb from SP0726 to SP0730 was identified in isolate ATCC700904 which showed no hybridization, despite hybridizing well for all other strains (Figure 4-2) This was unexpected as the isolate ATCC700904 features in the work of Silva et al where it was named PMEN13 and this region was not identified despite using the same isolate on the same version of microarray with similar hybridization methodologies (Silva et al., 2006). This can be explained as the data analysis methods chosen (such as the normalization methods) are different between the two studies. Several genes within this region (SP0726, SP0728 and SP0729) have been associated with virulence in a serotype 4 mouse bacteraemic pneumonia model (Hava and Camilli, 2002).

Figure 4-2 Demonstration of a new region of diversity (SP0726 – SP0731) in the TIGR4 genome using Genespring GX 7.3.1.

Each coloured bar represents a consecutive gene in the TIGR4 genome. Shades of yellow and orange indicate that hybridization occurred with DNA from both TIGR4 and the test isolate while shades of blue indicate no hybridization occurred with DNA from the test strain but unopposed hybridization by TIGR4 DNA had occurred.

A third shorter region of 5.1kb relates to a putative Type II restriction endonuclease which was identified as cluster 9 by Bruckner et al (Bruckner et al., 2004). It was also identified as present in serotype 6A isolates from cases of invasive pneumococcal disease but not serotypes 6B or 14 by Obert et al (Obert et al., 2006) and was not identified by Silva et al which again is likely to be due to the different data analysis methods utilised as Silva et al had also studied isolate BAA-659 which was named PMEN23 in their analysis (Silva et al., 2006).

The fourth region of diversity identified by this study which was not identified by Silva et al is a 2.9kb region found between SP2180 and SP2183 which was identified as not hybridizing in isolate 07-2839 from Bolivia. This is illustrated in Figure 4-3 below.

Figure 4-3 Demonstration of a new region of diversity (SP2180 – SP2183) in the TIGR4 genome using Genespring GX 7.3.1.

Each coloured bar represents a consecutive gene in the TIGR4 genome. Shades of yellow and orange indicate that hybridization occurred with DNA from both TIGR4 and the test isolate while shades of blue indicate no hybridization occurred with DNA from the test strain but unopposed hybridization by TIGR4 DNA had occurred.

The gene SP2179 falling immediately before this region was identified by Bruckner et al as a highly variable gene related to insertion sequence IS1380. The genes SP2180 and SP2181 have been previously identified as pseudogenes, particularly as SP2180 is interrupted by the IS1380 Spn1 element. However, gene expression studies to be described later in Chapter 10 using serotype 3 and serotype 1 pneumococci show SP2180, SP2182 and SP2183 to be expressed in both TIGR4 and the test isolates while SP2181 was not expressed in any of these expression experiments. SP2181 was expressed though by the strain South Africa 2507 but only in response to the addition of subtherapeutic clarithromycin (Chapter 11). Evidence of their expression under certain conditions does suggest that these genes may be functional.

Bruckner et al identified some regions which were not present in some of their battery of international test strains but were evident in TIGR4 and R6 (Bruckner et al., 2004). SP0505 to SP0508 is such a region. However, this region appears to be present in all the 10

strains tested in this chapter. Bruckner et al also identify the region SP1309 - SP1337 as a, “hot spot,” for recombination (Bruckner et al., 2004). These results also concur with this observation as this is a region of substantial diversity between strains as illustrated below in Figure 4-4. This region is within a slightly larger region of diversity extending from SP1309 - SP1347 which is part of the region of diversity 17 described by Silva et al and clusters 9 and 10 described by Bruckner et al (Bruckner et al., 2004).

Figure 4-4 Demonstration of a, “hot spot,” for recombination events (SP1309 - SP1337) in the TIGR4 genome using Genespring GX 7.3.1.

Each coloured bar represents a consecutive gene in the TIGR4 genome. Shades of yellow and orange indicate that hybridization occurred with DNA from both TIGR4 and the test isolate while shades of blue indicate no hybridization occurred with DNA from the test strain but unopposed hybridization by TIGR4 DNA had occurred.