Strain-Level Resolution and Pneumococcal Carriage Dynamics by Single Molecule Real Time (SMRT) Sequencing of The Plyncr Marker: A Longitudinal Study In Swiss Infants.

Abstract

Background: Pneumococcal carriage has often been studied from a serotype perspective, however, little is known about strain-specific carriage and inter-strain interactions. Here, we examined strain-level carriage and co-colonization dynamics in a Swiss birth cohort by PacBio single molecule real time (SMRT) sequencing of the plyNCR marker and assessed changes four years after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13).Methods: A total of 872 nasal swab (NS) samples were collected biweekly from 47 healthy infants during the first year of life. Pneumococcal carriage was determined based on the quantitative real-time polymerase chain reaction (qPCR) targeting the lytA gene. The plyNCR marker was amplified from 214 samples having lytA-based carriage for pneumococcal strain resolution. Amplicons were sequenced using SMRT technology and sequences were analyzed with the DADA2 pipeline. In addition, pneumococcal serotypes were determined using conventional, multiplex PCR (cPCR). Results: PCR-based plyNCR amplification demonstrated a 94.2% sensitivity and 100% specificity for S. pneumoniae if compared to lytA qPCR. Overall carriage prevalence was 63.8% and pneumococcal co-colonization (≥2 plyNCR amplicon sequence variants (ASVs)) was detected in 38/213 (17.8%) sequenced samples with the relative proportion of the least abundant strain(s) ranging from 1.1% to 48.8% (median, 17.2%; IQR, 5.8-33.4%). The median age to first acquisition was 147 days and having ≥2 siblings increased the risk of acquisition. Conclusion: The plyNCR amplicon sequencing is species specific and enables pneumococcal strain-resolution. We therefore recommend its application for longitudinal strain-level carriage studies of Streptococcus pneumoniae.