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 Manenzhe et al. Pneumococcal Colonization Dynamics in a Birth Cohort 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.

Strain-level resolution and pneumococcal carriage dynamics by single-molecule real-time (SMRT) sequencing of the plyNCR marker: a longitudinal study in Swiss infants

“…To our knowledge, this is the first carriage study that provides adequate resolution of "unique" nasopharyngeal pneumococcal strains in a longitudinal infant sampling without a prior culture step. Only two other studies have attempted to characterize S. pneumoniae at the strain level using infant longitudinal samples [3,4]. However, both studies employed a metagenomic approach, which requires (i) a general enrichment process for Streptococcus sp., (ii) a high coverage that is mostly outbalanced by host DNA, (iii) a short-read sequencing library requiring assembly, and (iv) a more computationally intensive (and mostly database-dependent) approach [31], all of which may be cost-limiting for large-scale studies.…”

“…Previous studies have attempted to ascertain pneumococcal profiles from nasopharyngeal samples via shotgun metagenomics [3,4]; however, resolution of "unique" pneumococcal strains remained limited, as shotgun metagenomics requires a very high read coverage to detect minor variants, which is even more challenging to achieve with samples that have a high quantity of host DNA. Alternative approaches are therefore needed for a thorough exploration of S. pneumoniae strains and their dynamics during carriage.…”

Expansion of pneumococcal serotype 23F and 14 lineages with genotypic changes in capsule polysaccharide locus and virulence gene profiles post introduction of pneumococcal conjugate vaccine in Blantyre, Malawi

“…The predominant serotype 23F lineage before PCV13 introduction in Malawi was GPSC20 ST802, a lineage found on multiple continents (52,53). However, post-PCV13 introduction, there was a decrease in ST802, and an expansion of GPSC14 ST2059, which, to date, has only been reported from neighbouring South Africa and Mozambique (52,54). Intriguingly, data from South Africa suggests that 23F ST2059 has a more invasive phenotype (19).…”

Expansion of pneumococcal serotype 23F and 14 lineages with genotypic changes in capsule polysaccharide locus and virulence gene profiles post introduction of pneumococcal conjugate vaccine in Blantyre, Malawi

“…The predominant serotype 23F lineage before PCV13 introduction in Malawi was GPSC20 ST802, a lineage found on multiple continents [ 60 61 ]. However, post-PCV13 introduction, there was a decrease in ST802, and an expansion of GPSC14 ST2059, which, to date, has only been reported from neighbouring South Africa and Mozambique [ 60 62 ]. Intriguingly, data from South Africa suggests that 23F ST2059 has a more invasive phenotype [ 27 ].…”