High-resolution genomics identifies pneumococcal diversity and persistence of vaccine types in children with community-acquired pneumonia in the UK and Ireland

Abstract

Background: Streptococcus pneumoniae is a global cause of community-acquired pneumonia (CAP) and invasive disease in children. The CAP-IT trial (grant No. 13/88/11; https://www.capitstudy.org.uk/ ) collected nasopharyngeal swabs from children discharged from hospitals with clinically diagnosed CAP, and found no differences in pneumococci susceptibility between higher and lower antibiotic doses and shorter and longer durations of oral amoxicillin treatment. Here, we studied in-depth the genomic epidemiology of pneumococcal (vaccine) serotypes and their antibiotic resistance profiles.

Methods: Three-hundred and ninety pneumococci cultured from 1132 nasopharyngeal swabs from 718 children were whole-genome sequenced (Illumina) and tested for susceptibility to penicillin and amoxicillin. Genome heterogeneity analysis was performed using long-read sequenced isolates (PacBio, n = 10) and publicly available sequences.

Results: Among 390 unique pneumococcal isolates, serotypes 15B/C, 11 A, 15 A and 23B1 were most prevalent (n = 145, 37.2%). PCV13 serotypes 3, 19A, and 19F were also identified (n = 25, 6.4%). STs associated with 19A and 19F demonstrated high genome variability, in contrast to serotype 3 (n = 13, 3.3%) that remained highly stable over a 20-year period. Non-susceptibility to penicillin (n = 61, 15.6%) and amoxicillin (n = 10, 2.6%) was low among the pneumococci analysed here and was independent of treatment dosage and duration. However, all 23B1 isolates (n = 27, 6.9%) were penicillin non-susceptible. This serotype was also identified in ST177, which is historically associated with the PCV13 serotype 19F and penicillin susceptibility, indicating a potential capsule-switch event.

Conclusions: Our data suggest that amoxicillin use does not drive pneumococcal serotype prevalence among children in the UK, and prompts consideration of PCVs with additional serotype coverage that are likely to further decrease CAP in this target population. Genotype 23B1 represents the convergence of a non-vaccine genotype with penicillin non-susceptibility and might provide a persistence strategy for ST types historically associated with vaccine serotypes. This highlights the need for continued genomic surveillance.

Keywords: Streptococcus pneumoniae; Beta-lactams; Emerging serotype; Non-vaccine types; Population genomics; Vaccine escape; Vaccine types.

Fig. 1

Flow chart depicting isolate collection and filtering process

Fig. 2

Phylogenetic tree generated from the 390 study isolates’ assemblies. Inner squares represent the presence of amino acid changes in (from inner to outer) penicillin-binding protein (PBP) 1a, PBP2b and PBP2x protein sequences described in the Comprehensive Antibiotic Resistance Database (CARD) to provide non-susceptibility to penicillin. Filled squares represent the presence of all described mutations for PBP1a and PBP2b, and > 4 of the 7 mutations described for PBP2x in CARD. Empty squares depict < 4 PBP2x mutations. Penicillin and amoxicillin susceptibilities are colour-coded (green for susceptible, orange for non-susceptible and red for resistant). Acquired resistance genes are depicted as coloured squares when present in the isolate, potentially conferring resistance to macrolides (brown, blue and purple squares), tetracycline (grey square) and aminoglycosides (light blue square). Outer circles represent serotype of the isolate and Global Pneumococcal Sequence Clusters (only GPSCs presenting > 3 isolates are shown). Finally, pink blocks indicate isolates belonging to the same ST and presenting with different serotypes. Serotype 15B/C was present across 8 STs and is highlighted in blue

Fig. 3

Local popPUNK clustering. Node filling and node border represent penicillin and amoxicillin susceptibility, respectively. Node labels refer to Global Pneumococcal Sequence Clusters

Fig. 4

Flow chart depicting isolate distribution within patients across samples, and table showing in detail the serotypes and penicillin non-susceptibility observed in patients where two different serotypes were found at D1 and D29. Green denotes penicillin susceptibility and orange penicillin non-susceptibility

Fig. 5

Phylogenetic tree generated from penicillin-binding protein 2b amino acid sequences. Coloured squares represent the presence of mutations previously described to confer an increase in MIC. Number of total amino acid modifications, penicillin and amoxicillin susceptibility, serotype, and number of isolates are also depicted. Variants highlighted in blue present a low number of overall amino acid modification while containing the three key modifications described in the CARD database to reduce susceptibility to beta-lactams

Fig. 6

Phylogenetic trees generated from whole genome alignments of serotype 3 (A), serotype 19A (B) and serotype 19F (C) isolates derived from this study (orange shading) and older isolates from the same serotypes. References used were CAPIT119_D1-1, CAPIT226_D1-1 and CAPIT214_D1-1 (marked in red shading), respectively. In red, are shown the large genomic changes predicted to have occurred earlier in evolution, thus being present in a cluster of isolates. In blue, genomic changes predicted only in the branch leaves, that is, in only one isolate. The position of the cps locus in the core genome is indicated with a black rectangle. For serotype 3, the clade to which the isolates belong is also depicted. All serotype 3 isolates belonged to sequence type 180

Fig. 7

Phylogenetic trees generated from whole genome alignments of sequence type (ST) 162 (A), ST177 (B) and ST199 (C) isolates derived from this study (orange shading) and older isolates from the same serotypes. References used were CAPIT086_D1-1, CAPIT292_D1-1 and CAPIT226_D1-1 (marked in red shading), respectively. In red are shown the large genomic changes predicted to have occurred earlier in evolution, thus being present in a cluster of isolates. In blue, genomic changes predicted only in the branch leaves, that is, in only one isolate. The position of the cps locus in the core genome is indicated with a black rectangle