Characterization of Pneumococcal Colonization Dynamics and Antimicrobial Resistance Using Shotgun Metagenomic Sequencing in Intensively Sampled South African Infants

Abstract

Background: There remains a significant proportion of deaths due to pneumococcal pneumonia in infants from low- and middle-income countries despite the marginal global declines recorded in the past decade. Monitoring changes in pneumococcal carriage is key to understanding vaccination-induced shifts in the ecology of carriage, patterns of antimicrobial resistance, and impact on health. We longitudinally investigated pneumococcal carriage dynamics in PCV-13 vaccinated infants by collecting nasopharyngeal (NP) samples at 2-weekly intervals from birth through the first year of life from 137 infants. As a proof of concept, 196 NP samples were retrieved from a subset of 23 infants to explore strain-level pneumococcal colonization patterns and associated antimicrobial-resistance determinants. These were selected on the basis of changes in serotype and antibiogram over time. NP samples underwent short-term enrichment for streptococci prior to total nucleic acid extraction and whole metagenome shotgun sequencing (WMGS). Reads were assembled and aligned to pneumococcal reference genomes for the extraction of pneumococcal and non-pneumococcal bacterial reads. Pneumococcal contigs were aligned to the Antibiotic Resistance Gene-ANNOTation database of acquired AMR genes. In silico pneumococcal capsular and multilocus sequence typing were performed. Results: Of the 196 samples sequenced, 174 had corresponding positive cultures for pneumococci, of which, 152 were assigned an in silico serotype. Metagenomic sequencing detected a single pneumococcal serotype in 85% (129/152), and co-colonization in 15% (23/152) of the samples. Twenty-two different pneumococcal serotypes were identified, with 15B/15C and 16F being the most common non-PCV13 serotypes, while 23F and 19A were the most common PCV13 serotypes. Twenty-six different sequence types (STs), including four novel STs were identified in silico. Mutations in the folA and folP genes, associated with cotrimoxazole resistance, were detected in 89% (87/98) of cotrimoxazole-non-susceptible pneumococci, as well as in the pbp1a and pbp2x genes, in penicillin non-susceptible ST7052^15B/15C isolates. Conclusions: Metagenomic sequencing of NP samples is a valuable culture-independent technique for a detailed evaluation of the pneumococcal component and resistome of the NP microbiome. This method allowed for the detection of novel STs, as well as co-colonization, with a predominance of non-PCV13 serotypes in this cohort. Forty-eight resistance genes, as well as mutations associated with resistance were detected, but the correlation with phenotypic non-susceptibility was lower than expected.

Keywords: Streptococcus pneumoniae; multi locus sequence typing; nasopharyngeal; pneumococcal conjugate vaccine; resistance determinants; serotypes; shotgun metagenomic sequencing.

Figure 1

This figure indicates the number of samples selected for metagenomic sequencing and serotype assignments for the 196 nasopharyngeal (NP) samples selected from 23 infants (shown in rows 1 to 23). None of the infants were colonized until 4 weeks of age. Small blue circles represent the NP samples that were collected but not included for shotgun metagenomic sequencing. Large circles (colors represent serotype group) represent the collected NP samples selected for shotgun metagenomic sequencing. Split circles represent samples with co-colonization with multiple serotypes. in silico serotypes are displayed for samples selected for shotgun metagenomic sequencing. (-) NP sample not collected. (nt) Non-typeable.

Figure 2

A maximum likelihood whole genome phylogenetic tree of pneumococcal isolates recovered from 23 infants. Bootstrap values are shown on each branch and the scale bar represents the number of SNPs. Circles with the same color represent longitudinal samples from the same infant. Numbers in brackets indicate the age in weeks at each time-point. The sequence type (ST) detected in each sample is shown as a number, followed by the associated serotype. Serotypes indicated in red were among co-colonizing strains and had genomes with lower coverage than the other co-detected strain. Five out of 15 samples that had low reads mapping to pneumococcal genomes are indicated in blue text. NT, Non-typeable. Completed pneumococcal genomes available on the NCBI database were included and the R6 genome was used as a reference. The samples clustered according to ST but not serotype. Persistent colonization with the same genotype was common.

Figure 3

Shotgun sequence-derived molecular sequence type and resistome (from direct sequencing of NP swabs), and phenotypic antimicrobial susceptibility results (from pneumococcal isolates) in 159 out of 196 NP samples obtained from 23 infants (assigned 1 to 23, top row). (A) in silico sequence types (STs) and associated serotypes detected in longitudinal NP samples from each infant. (B) pbp gene mutations and associated penicillin phenotypic susceptibility profiles; light blue color indicates wild-type pbp genes; the dark blue color indicates detected mutations within or close to the conserved motifs for the pbp genes. (C) Macrolide-resistance genes and associated erythromycin phenotypic susceptibility profiles; the dark blue color indicates detected resistance genes. (D) folA and folP gene mutations and associated cotrimoxazole phenotypic susceptibility profiles; light blue color indicates wild-type folA and folP genes; dark blue color indicates detected mutations, in folA and folP, that reduce the affinity of trimethoprim or sulfamethoxazole. (E) Detected tetracycline resistance gene; dark blue color indicates detected resistance gene. Gray color in (B–E) indicates that no genes were detected.