Insight into the genetic characteristics of pneumococcal isolates

Streptococcus pneumoniae is a human pathogen and a major contributor to morbidity and mortality worldwide. It has been estimated that it is responsible for between 1-2 million deaths annually. The normal niche for the pneumococci is the nasopharynx where the bacteria can residue for months without causing any symptoms. Different studies have shown that up to 70% of children attending day care centers may be carriers. S. pneumoniae can be divided into different serotypes based on differences in the capsular polysaccharide. Also the bacteria can be further divided into genetically related clones using methods such as pulsed field gel electrophoresis (PFGE) and multi locus sequence typing (MLST). When using MLST parts of seven housekeeping genes are sequenced, and isolates belong to the same sequence type (ST) if they have identical sequences. Isolates of different serotypes have different ability to cause invasive disease, Invasive Disease Potential (IPD).

The overall aim of this work was to genetically characterize clinical pneumococcal isolates and to correlate their genetics to ability to spread, fitness and invasiveness. Clinical isolates with known origin were characterized using MLST and microarray. In addition growth rates, as a measurement for fitness, were determined for some isolates. Also mutants with deletion in specific genes or region of genes were created and compared to wild type TIGR4 in an intranasal murine model of infection.

We established that isolates of the same or related ST have similar genetic content. We also observed a trend that isolates belonging to serotypes associated with a high IDP have fewer genetic differences than isolates associated with lower IDP. We found the accessory genome of S. pneumoniae to be about 34% of the combined genomes of R6 and TIGR4, which were used as reference isolates. We determined that a huge part of the accessory genome is located to smaller gene clusters around the genome, termed accessory regions (ARs). In addition to previously recognized regions we identified 5 new ARs giving a total of 41. We also found that by determining the presence or absence of a set of 25 accessory genes it was possible to determine the genetic relationship among the isolates. Furthermore, we found that virulence associated genes (found in signature tagged mutagenesis screens) may be absent in invasive human isolates. We tried to correlate the presence of different ARs with the IDP of the isolates. Two regions, AR6 and AR32, were found to be present in a higher proportion among isolates having a high IDP than those associated with a low IDP. However none of these regions were essential for mouse virulence in a serotype 4 background.

We believe that the capacity to cause invasive disease is due to a combination of different regions, including the capsule, and that there is a redundancy amongst them. We did not find any gene or set of genes that correlated to the fitness of the bacteria. We did however observe that fitness in vitro is highly correlated to virulence in vivo. This was true for clinical isolates as well as constructed mutants. We also determined that the internationally successful clone of ST156 (Spain9V-3) constitute around 50% of all PNSP (penicillin non-susceptible pneumococci) isolates in Sweden. The clone is seen with several different capsular types, where types 9V and 14 are most common. We found that presence of pili is a common trait for isolates of ST156 and that, pili constitutes an advantage in a competition model of infection. Furthermore, at least 70% of PNSP isolates in Sweden were found to harbor the pilus islet. In conclusion, there is considerable redundancy in gene functions associated with virulence and fitness of pneumococci, but specific ARs (or combinations of ARs), such as the pili may constitute one explanation for the success in spread by certain clones.

List of scientific papers

I. Dagerhamn J, Blomberg C, Browall S, Sjöström K, Morfeldt E, Henriques-Normark B (2008). Determination of accessory gene patterns predicts the same relatedness among strains of Streptococcus pneumoniae as sequencing of housekeeping genes does and represents a novel approach in molecular epidemiology. J Clin Microbiol. 46(3): 863-8. Epub 2007 Dec 26
https://pubmed.ncbi.nlm.nih.gov/18160453

II. Blomberg C, Dagerhamn J, Dahlberg S, Browall S, Fernebro J, Morfeldt E, Normark S, Henriques Normark B. (2008). Pattern of accessory regions and invasive disease potential in Streptococcus pneumoniae. [Submitted]

III. Sjöström K, Blomberg C, Fernebro J, Dagerhamn J, Morfeldt E, Barocchi MA, Browall S, Moschioni M, Andersson M, Henriques F, Albiger B, Rappuoli R, Normark S, Henriques-Normark B (2007). Clonal success of piliated penicillin nonsusceptible pneumococci. Proc Natl Acad Sci USA. 104(31): 12907-12. Epub 2007 Jul 20
https://pubmed.ncbi.nlm.nih.gov/17644611

IV. Fernebro J, Blomberg C, Morfeldt E, Wolf-Watz H, Normark S, Normark BH (2008). The influence of in vitro fitness defects on pneumococcal ability to colonize and to cause invasive disease. BMC Microbiol. 8: 65
https://pubmed.ncbi.nlm.nih.gov/18423011