Pneumococcal genome sequencing tracks a vaccine escape variant formed through a multi-fragment recombination event

Abstract

Streptococcus pneumoniae ('pneumococcus') causes an estimated 14.5 million cases of serious disease and 826,000 deaths annually in children under 5 years of age(1). The highly effective introduction of the PCV7 pneumococcal vaccine in 2000 in the United States(2,3) provided an unprecedented opportunity to investigate the response of an important pathogen to widespread, vaccine-induced selective pressure. Here, we use array-based sequencing of 62 isolates from a US national monitoring program to study five independent instances of vaccine escape recombination(4), showing the simultaneous transfer of multiple and often large (up to at least 44 kb) DNA fragments. We show that one such new strain quickly became established, spreading from east to west across the United States. These observations clarify the roles of recombination and selection in the population genomics of pneumococcus and provide proof of principle of the considerable value of combining genomic and epidemiological information in the surveillance and enhanced understanding of infectious diseases.

Figure 1

Resequencing of pneumococcal vaccine escape recombinants: comparison of recombinant and putative recipient and donor sequences. (A) Vaccine escape recombinants P1-P5 were resequenced using the Affymetrix CustomSeq platform, a microarray-based approach covering a selected 12% of the pneumococcus genome (300 kbp). The genomes are coloured by inferred origin of genomic intervals (blue - recipient; yellow - donor). The capsular locus transferred in a different form in each recombinant is evident as a large yellow block centred on the same location towards the left of each genome. Other yellow blocks indicate extra transferred sequences. The recipient genomes in all cases were highly similar. (B) Whole-genome structure of recombinant P1. Representatives of recombinant P1 and its best-matching prospective parents were sequenced on the Illumina GAIIx platform. Sequence reads were assembled de novo and aligned to the TIGR4 reference sequence and a heuristic algorithm was implemented in which three or more donor-like single nucleotide variants encompassing no more than one recipient-like variant was used to identify donor genomic fragments (blue - recipient; yellow - donor). This approach detected 15 imports of ≥50bp, including 8 that did not overlap previously detected fragments. The scale in (B) is different to that in (A) because the reference sequences differ slightly. In each panel the smallest fragments (triangles) have been magnified to a minimum size of 5kb for visibility.

Figure 2

Spread of P1 vaccine escape recombinant through space and time. Incidence of P1 vaccine escape recombinants (red) and other 19A (blue) as a proportion of all pneumococci in invasive pneumococcal disease among children under 5 years of age. Data are shown for the years 2003-2007; left to right panels for each state in 10 ABCs monitoring states. Data from 2003 in New Mexico are missing (grey box) because surveillance started there in 2004. P1 was first detected in New York and Connecticut from where it has spread westwards.