Site of Isolation Determines Biofilm Formation and Virulence Phenotypes of Serotype 3 Streptococcus pneumoniae Clinical Isolates

Claudia Trappetti,1 Erika Van der Maten,1 Zarina Amin,1 Adam J. Potter,1 Austen Y. Chen,1 Andrew J. Lawrence,2 Adrienne W. Paton,1 and James C. Paton1

1School of Molecular and Biomedical Science, University of Adelaide, S.A., 5005, Australia; and 2SA Pathology, Women’s and Children’s Hospital, North Adelaide, S.A., 5006..

Running head: Pneumococcal Niche Adaptation

Word counts: Abstract, 197; Main text, 3494.

Footnotes:
Potential Conflicts of Interest: the authors do not have any conflicts of interest.

Financial support: Program Grant 565526 from the National Health and Medical Research Council (NHMRC) of Australia; Garnett Passe and Rodney Williams Memorial Foundation.

Reprints or correspondence: Prof. James C. Paton, School of Molecular and Biomedical Science, University of Adelaide, S.A., 5005, Australia. Phone: 61-8-83135929; Fax: 61-8-83133262; E-mail: james.paton @adelaide.edu.au.
ABSTRACT
Background. Streptococcus pneumonaie is a diverse species causing invasive as well as localized infections that result in massive global morbidity and mortality. Strains vary markedly in pathogenic potential, but the molecular basis is obscured by the diversity and plasticity of the pneumococcal genome. Methods. S. pneumoniae serotype 3 blood (n = 12) or ear (n = 13) isolates were multi-locus sequence typed (MLST) and assessed for biofilm formation and virulence phenotype. Results. Blood and ear isolates exhibited similar MLST type distribution, but differed markedly in phenotype. Blood isolates formed robust biofilms only at pH 7.4, which was enhanced in Fe(III)-supplemented medium. Conversely, ear isolates formed biofilms only at pH 6.8, and Fe(III) was inhibitory. Biofilm formation paralleled luxS expression and genetic competence. In a mouse intranasal challenge model, blood isolates did not stably colonize the nasopharynx, but spread to the blood; none spread to the ear. Ear isolates colonized the nasopharynx at higher levels and also spread to the ear compartment in a significant proportion of animals; none caused bacteremia. Conclusions. Pneumococci of the same serotype and MLST type exhibit distinct phenotypes in accordance with clinical site of isolation, indicative of stable niche-adaptation within a clonal lineage.

Key words: pneumococcus, virulence phenotype, pH-dependent biofilm formation, luxS, niche adaptation, clonal lineage.
INTRODUCTION
Streptococcus pneumoniae (the pneumococcus) is responsible for massive global morbidity and mortality. It is a major cause of pneumonia, meningitis and sepsis, especially in young children and the elderly. S. pneumoniae also causes less serious, but highly prevalent infections such as otitis media (OM) and sinusitis [1-5]. The World Health Organisation estimates that 1.6 million people, of whom 0.7 to 1 million are under the age of 5, die of pneumococcal diseases each year, with the highest incidence in developing countries. Indeed, S. pneumoniae accounts for more deaths worldwide than any other single pathogen [6-7]. In spite of this mortality, S. pneumoniae is part of the commensal nasopharyngal flora of humans. Most colonised individuals are asymptomatic, and carriers are the principal reservoirs for transmission of S. pneumoniae in the community. In a small proportion of carriers, which nevertheless translates into globally significant total case numbers, S. pneumoniae invades from its nasopharyngeal beachhead to cause disease. This may occur, for example, by aspiration into the lungs to cause pneumonia, by direct invasion of the blood, or by ascension of the Eustachian tube to access the middle ear and cause OM [1, 8-9]. However, the mechanisms whereby pneumococci transition from commensal to pathogen are poorly understood. OM is one of the most common paediatric diagnoses [10] and