Pathogenesis of Streptococcus pneumoniae.

Abstract

Streptococcus pneumoniae is the principal bacterial cause of otitis media (OM). While OM does not have the high rates of mortality associated with invasive pneumococcal diseases, such as meningitis, it has an extremely high rate of morbidity, with most children suffering at least one episode of OM during their early childhood years. Despite being a major public health burden worldwide, certain communities are more impacted than others. In Australia, remote Aboriginal communities have the highest rates of OM in the world with absolutely no improvement over the last few decades. Therefore, understanding the pathogenesis of pneumococcal OM is of vital importance. At the start of this project, serotype 3 middle ear and nasopharyngeal isolates were obtained from both remote Aboriginal communities and the general Australian population from the Menzies School of Health Research (MSHR) in Darwin and the Women’s and Children’s Hospital (WCH) in Adelaide, respectively. This serotype is an important middle ear pathogen, including in remote Aboriginal communities. In addition, serogroup 11 nasopharyngeal and sinusitis isolates were received from the same sources. Serogroup 11 is rarely reported in OM in remote Australian Aboriginal communities and is generally not as prevalent as serotype 3 in other communities. Furthermore, the serogroup 11 isolates tested were avirulent in mouse invasive disease models, while the serotype 3 isolates tested were virulent. A serotype 11A nasopharyngeal isolate from a remote Aboriginal community was capsule switched to serotype 3. However, in a pneumonia/sepsis mouse model, it was less fit in the middle ears compared to the middle ear isolate from which the serotype 3 capsule locus was derived. This indicated the serotype 3 isolates of this study possess genetic factors apart from serotype that influence OM. Multilocus sequence typing identified two STs in the serotype 3 middle ear isolates (ST180 and ST458). Generally, ST180 is the dominant ST within serotype 3 worldwide, but interestingly, all the isolates typed from MSHR belonged to ST458. The genomes of ST180 and ST458 isolates were analysed using a variety of molecular biological techniques, including a comparison with serogroup 11 isolates by DNA microarray analysis. However, no gene common to one serotype/group but not the other was identified and therefore, the capsule loci remained the only distinguishing feature between serotype 3 and serogroup 11. In order to determine if there were any genes present in the genomes of ST180 and ST458 isolates which were not represented on the microarray slide, PCR-based subtractive hybridisation was employed, and a putative cellobiose phosphotransferase system (PTS) was identified. This PTS is part of a 10 kb island, which includes a sulfatase and ROK family protein. However, there is a large deletion in ST458, which includes the sulfatase and part of the putative cellobiose PTS operon. A large number of strains from a variety of serotype/groups, which included serogroup 11, were found to carry the island through PCR analysis or bioinformatic searches, with most possessing the full island. In ST180, mutagenesis of the island and subsequent virulence studies revealed that the island confers a competitive advantage in a variety of niches, including the ear. Unfortunately, mutagenesis in ST458 was not possible despite numerous attempts. Finally, the availability of next generation sequencing and a fully sequenced genome of a serotype 3 ST180 strain (OXC141), allowed the investigation for genes which had been missed by PCR-based subtractive hybridisation due to limitations with this technique. The genome of an ST458 middle ear isolate (MSHR17) was analysed, along with an unrelated serotype 3 strain (WU2) as a comparison. Both OXC141 and MSHR17 had regions not represented on the microarray slide, but these regions had not been detected by PCR-based subtractive hybridisation due to their absence in the other ST. Therefore, the PTS island remained the only region common to both STs. Nonetheless, the repertoire of regions possessed by ST180 may explain the dominance of this ST in serotype 3 worldwide, while the repertoire possessed by ST458 may mean it is better adapted to the microenvironmental niches encountered in remote Aboriginal communities.