Genomic Epidemiology of Penicillin-Nonsusceptible Pneumococci with Nonvaccine Serotypes Causing Invasive Disease in the United States

Abstract

Conjugate vaccination against seven pneumococcal serotypes (PCV7) reduced disease prevalence due to antibiotic-resistant strains throughout the 2000s. However, diseases caused by resistant nonvaccine type (NVT) strains increased. Some of these emerging strains were derived from vaccine types (VT) that had changed their capsule by recombination. The introduction of a vaccine targeting 13 serotypes (PCV13) in 2010 has led to concern that this scenario will repeat itself. We generated high-quality draft genomes from 265 isolates of NVT pneumococci not susceptible to penicillin (PNSP) in 2009 and compared them with the genomes of 581 isolates from 2012 to 2013 collected by the Active Bacterial Core surveillance (ABCs) of the Centers for Disease Control and Prevention (CDC). Of the seven sequence clusters (SCs) identified, three SCs fell into a single lineage associated with serogroup 23, which had an origin in 1908 as dated by coalescent analysis and included isolates with a divergent 23B capsule locus. Three other SCs represented relatively deep-branching lineages associated with serotypes 35B, 15A, and 15BC. In all cases, the resistant clones originated prior to 2010, indicating that PNSP are at present dominated by descendants of NVT clones present before vaccination. With one exception (15BC/ST3280), these SCs were related to clones identified by the Pneumococcal Molecular Epidemiology Network (PMEN). We conclude that postvaccine diversity in NVT PNSP between 2009 and 2013 was driven mainly by the persistence of preexisting strains rather than through de novo adaptation, with few cases of serotype switching. Future surveillance is essential for documenting the long-term dynamics and resistance of NVT PNSP.

Keywords: genomic epidemiology; nonvaccine serotype; penicillin; vaccine.

FIG 1

Distribution of the 846 isolates across sampling sites, year, ST, and serotype. (A) Map of the United States showing the 10 sampling sites. The proportions of isolates for each SC are calculated based on sampling site, with colors corresponding to the colors on the map (B), year of collection (C), ST (D), and serotype (E). The number of isolates and the r/m ratio per SC are also indicated.

FIG 2

Core genome phylogeny and distribution of genes coding for resistance against other antibiotic classes. (A) The maximum-likelihood tree was generated using the concatenated alignment of 719 core genes, using S. pneumoniae ATCC 700669 as an outgroup to root the tree. The inner ring delineates the seven SCs identified using hierBAPS. The heights of the bars in the outer ring correspond to MIC values for penicillin, with bars scaled with respect to the highest value of 8 μg/ml. (B) The maximum-likelihood tree is identical to the phylogeny in panel A. The branches are colored according to the hierBAPS membership. Outer rings show the presence (colored) or absence (gray) of the resistance gene. Shown are the distributions of genes conferring resistance to aminoglycosides (aphIII and sat4A), macrolides-lincosamides-streptogramin (ermB/C, msrD, and mefA), phenicols (cat), and tetracyclines (tetM). ermC was detected in only a single isolate and was therefore included in the ermB ring.

FIG 3

Serotype switching in PMEN clones in SC7. (Left) Maximum-likelihood phylogeny of SC7 on the basis of point mutations, with polymorphic sites due to recombination excluded. Colored branches indicate isolates that have an ST identical to that of recognized PMEN clones. The serotypes of these isolates are indicated on the right of the tree. (Middle) Sampling sites and years of collection for each of the isolates represented by the colored branches on the tree. The colors on the table correspond to the colored branches on the tree. (Right) Nomenclature of PMEN clones that have STs identical to those found in the NVT PNSP population. The names indicate the country or region and serotype of the first clone that was first identified.

FIG 4

Bayesian phylogeny and population dynamics of SC1. Bayesian maximum clade credibility phylogeny of SC1 based on nonrecombinogenic regions of the core genome. Divergence date (median estimate with 95% highest posterior density dates in brackets) is indicated in blue on the tree. Sampling sites, year of isolation, and serotypes of each isolate are shown on the right of the tree. Inset: a Bayesian skyline plot showing changes in effective population size N_e over time (median is in black and 95% confidence intervals are in blue). Results for the other SCs are shown in Fig. S5 in the supplemental material.

FIG 5

Geographical structure of NVT PNSP. Pairwise genetic distances, which delineate separation on the basis of point mutations alone, were calculated between isolates within the same SC from phylogenetic trees in Fig. 3 and Fig. S7 in the supplemental material. The proportions of all pairwise comparisons of isolates originating from the same location were calculated and plotted against the genetic distances. The resulting values are plotted as black data points, with the blue lines representing the curves with approximately exponential decays. The red data points represent the outcomes of 100 permutations in which the same statistics were calculated when the locations of the isolates were randomized.