Invasive Serotype 35B Pneumococci Including an Expanding Serotype Switch Lineage, United States, 2015–2016

We used whole-genome sequencing to characterize 199 nonvaccine serotype 35B pneumococcal strains that caused invasive pneumococcal disease (IPD) in the United States during 2015–2016 and related these findings to previous serotype 35B IPD data obtained by Active Bacterial Core surveillance. Penicillin-nonsusceptible 35B IPD increased during post–pneumococcal 7-valent conjugate vaccine years (2001–2009) and increased further after implementation of pneumococcal 13-valent conjugate vaccine in 2010. This increase was caused primarily by the 35B/sequence type (ST) 558 lineage. 35B/ST558 and vaccine serotype 9V/ST156 lineages were implicated as cps35B donor and recipient, respectively, for a single capsular switch event that generated emergent 35B/ST156 progeny in 6 states during 2015–2016. Three additional capsular switch 35B variants were identified, 2 of which also involved 35B/ST558 as cps35B donor. Spread of 35B/ST156 is of concern in view of past global predominance of pathogenic ST156 vaccine serotype strains. Protection against serotype 35B should be considered in next-generation pneumococcal vaccines.

A lthough the dramatic protective effect of the pneumococcal 7-valent conjugate vaccine (PCV7) against invasive pneumococcal disease (IPD) persisted a full decade after its introduction in the United States in 2000, the emergence of 19A and other non-PCV7 serotypes reduced the overall benefit (1,2). Before PCV7 implementation, we observed only 2 different 35B lineages within Active Bacterial Core surveillance (ABCs) (3), a populationbased, multistate program that assesses the effect of invasive bacterial infections and is part of the Emerging Infections Program network of the Centers for Disease Control and Prevention (CDC; Atlanta, GA, USA) (http://www. cdc.gov/abcs/index.html). Both lineages were relatively rare causes of IPD but were geographically widespread in the United States before and after PCV7 introduction (3). One 35B lineage was antimicrobial-susceptible and multilocus sequence type (MLST) 452 (35B/ST452), and the second strain was penicillin-nonsusceptible 35B/ST558. During 1995-2001, the penicillin-nonsusceptible 35B/ ST558 lineage, which had resistant MICs of 0.25-2.0 µg/ mL, accounted for 69% of serotype 35B ABCs isolates (3). During 1999During -2007 in the United States, the proportion of penicillin-nonsusceptible IPD isolates within serotype 35B increased to 84%; 35B/ST558 accounted for this increase (4). Consistent with this observation was a 9-fold increase in carriage of 35B/ST558 in young children in the Atlanta, Georgia, area (5).
After introduction of the 13-valent conjugate vaccine (PCV13) in 2010, 35B became the most common serotype in ABCs, associated with MICs >2 µg/mL for penicillin and amoxicillin in pediatric isolates (6) and in the adult population (B. Beall, unpub. data). Consistent with its status as a major cause of IPD in the post-PCV13 era, the 35B/ST558 lineage is currently commonly found in disease and asymptomatic pneumococcal carriage in many countries (7)(8)(9)(10)(11).
We provide a whole-genome sequence (WGS) pipeline−based resolution and description of current 35B lineages within current ABCs surveillance (6,12), including an invasive 35B variant of the historically successful ST156 lineage. Recently, we identified 2 different 35B isolates recovered during 2009 and 2012 that each appeared to have arisen through a unique capsular switch event involving the same 2 parental strains. This observation was made on the basis of the penicillin-binding protein (PBP) gene types flanking the 35B biosynthetic locus (cps35B) in each of the variants (6).
Only the 35B/ST156 variant detected during 2012 has emerged and has been detected within 6 states. ST156 has a remarkable history of conjugate vaccine evasion. Formerly the primary genotype of PCV7 serotype 9V in the United States during the preconjugate vaccine era (13), 9V greatly decreased after PCV7 implementation (1) and was partially replaced by resistant 19A/ST156 (14). To verify that these isolates originated from a single recombinational serotype switch event involving 35B/ST558 and 9V/ST156 parental strains, we analyzed regions flanking the cps35B locus during 2015-2016, 35B/ST156 progeny and the original strain detected during 2012.

Isolates
The surveillance population of ABCs is ≈32 million persons in 10 states (http://www.cdc.gov/pneumococcal/ surveillance.html). Serotype 35B IPD isolates described include 132 recovered during 2015 and 67 recovered during ABCs in 2016 (Table 1, https://wwwnc.cdc.gov/EID/ article/23/6/17-0071-T1.htm). The listing of 35B isolates from 2016 is incomplete because we typically receive all ABCs isolates recovered during a given year by the following summer. Relevant ST156 lineage isolates of other serotypes recovered during this and previous periods are shown in Table 2. Total numbers of ABCs 35B isolates recovered during 1999-2015 and categorized by patient age, penicillin MIC, and IPD incidence are shown in Table 3.

Phylogeny
Paired-end fastq files were trimmed with Cutadapt version 1.8.1 (17), and draft genome assemblies were constructed by using VelvetOptimiser version 2.2.5 with an optimal kmer value calculated by using VelvetK (18). Core genome single-nucleotide polymorphism (SNP) identification and alignment were performed by using kSNP3.0 (19). A maximum-likelihood phylogenetic tree was generated from the core SNP alignment by using RaxML version 7.3.0 (20). RaxML was run with an ASC_GTRGAMMA DNA substitution model and used the Lewis method for ascertainment bias correction. Node support was assessed by using 500 bootstrap replicates.

Conventional MIC Testing and Serotyping
Serotype 35B isolates recovered during 2015 were subjected to conventional broth dilution testing for determination of antimicrobial MICs. A selection of these isolates were also subjected to conventional serotyping by using CDC typing antisera as described (6).
Analysis of the regions flanking the cps35B locus for all 35B/ST156 lineage isolates obtained during 2015-2016 showed identical recombinational sites at bases 6,453 (left coordinate of progeny reference) ( Figure 3) and 10,836 (right coordinate), which is clearly indicative of a single event within a 35B/ST156 ancestral strain of the 19 progeny shown ( Figure 3). Thus, a double-crossover event replaced the recipient strain cps9V locus and flanking PBP markers (2x-18 and 1a-15) with the cps35B locus and its flanking PBP markers (2x-7 and 1a-4). On the basis of available strain data, the original progeny strain is predicted to have been an ST156 strain with the PBP type 4:12:7; a total of 14 of the 19 35B/ST156 lineage strains still shared these characteristics (Table 1). Five isolates are SLVs or differ in PBP2b type. We detected the small (491 bp) segment (bases 11349-11839 of progeny) ( Figure 3) of clearly recipient lineage origin within the left side of the major recombinational fragment. During a single-double crossover event that facilitates a serotype switch, additional independent doublecrossover events appear to occur concurrently (27). However, these events probably do not occur simultaneously. It appears that the actual serotype switch event involved a shorter donor fragment bordering upon the right side of this small recipient lineage segment (base 11839), followed by a second double-crossover event that bordered upon the left side of the recipient lineage fragment (base 11349).

Postserotype Switch Event Diversification of 35B/ST156 Progeny
Five of the 19 35B/ST156 lineage progeny showed indications of genetic diversification that occurred after the capsular switch event. Four of these isolates have 1 of 3 SLV MLSTs of ST156 (ST9910, ST11584, and ST12921) ( Table 1). Although 18 of the 19 strains were PBP type 4:12:7, the SLV ST12921 variant had PBP type 4:11:7. For this particular strain, it is probable that recombination with a highly penicillin-resistant ST320 strain, prevalent during the post-PCV7 era and having PBP type 13:11:16 (6), simultaneously replaced the pbp2b locus and flanking ddl sequence to change the PBP type to 4:11:7 and the MLST type to ST12921 through transfer of ddl with the resistanceconferring selectable pbp2b allele (28). We also observed an increased MIC for amoxicillin for this PBP type 4:11:7 strain compared with MICs for PBP type 4:12:7 strains (Table 1).
Two isolates (20152877 and 20161763) underwent a postswitch intra-cps35B gene deletion event within the wciG gene (Figure 3), which is predicted to encode an acetyltransferase (26). Although these 2 isolates were serotyped as 35B by using CDC typing antisera, they differed in reactivity with serologic factor 35a compared with the other 17 isolates of this lineage (Table 4). Typing antisera factors 29b and 35c are the CDC Quellung reagents definitive for serotype 35B. The original protocol (29,30) that CDC first followed for serogroup 35 resolution also used factor 35a along with factors 29b and 35c for identification of serotype 35B. We found that the 2 wciG deletion strains did not react with factor 35a, but the other 17 serotype 35B strains reacted strongly with factor 35a. These preliminary data is suggestive of a new serotype within serogroup 35 because this specific factor reactivity pattern has not been observed for serogroup 35 (31). 926 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 6, June 2017 Figure 2. Phylogenetic analysis of potential recipient and serotype switch pneumococcal progeny strains within the ST56 lineage based upon a total alignment of 10,409 core single-nucleotide polymorphisms, United States, 2015-2016. All 20 serotype 35B progeny shown were recovered through Active Bacterial Core surveillance, and all but 2 indicated strains were obtained during 2015-2016. Isolate features are depicted for the 2 major nodes, with exceptions indicated by asterisks within the tree. Bootstrap values are indicated at key nodes. The serotype 9V isolate that was used for the reference recipient sequences described in Figure 3 is indicated as the third isolate from bottom. All isolates above the 9V recipient reference within this major cluster, except where indicated, are also 9V/ST156. Three additional serotype switch ST156 strain types detected by Active Bacterial Core surveillance during 2015-2016 are indicated by asterisks (single isolates of serotypes 31 and 13 and 4 serotype 19A isolates). Scale bar indicates nucleotide substitutions per site.
Further indication of chromosome-wide postswitch diversification of this single clade was shown in the leftflanking region of the cps35B locus. Two progeny strains showed diversification within the first 2-3.7 kb when compared with the other 17 progeny. The 6-kb region immediately to the left of base 1 in all of the progeny strains had <99.2% sequence identity with the most similar potential ST156 parental recipient strains that we analyzed. However, beyond this segment, progeny had sequence identity with the parental strain for >8 kb.

35B/ST156 Variant Lineages Arising through Separate Serotype Switch Events
The 9V/ST156 clade also appears likely to have served as the recipient for an independent serotype switch from the same 2 parental strains (  (6), and we have not obtained additional 35B isolates with these distinguishing features. A third serotype switch event involving a CC156 recipient strain is intuitive from the pipeline data, which indicate that the 35B SLV of ST162 is featured by the β-lactam-susceptible PBP type 0:0:0 (Table 1). ST162 has long been associated with penicillin-susceptible 9V strains (13) and more recently with PI-1 positive and penicillin-susceptible 23B, 15B, and 15C strains (6).

Nonserotype 35B Variants of ST156
Single ST156 isolates of serotypes 31 and 13 were obtained during 2015 ( Figure 2) and showed high relatedness to different 9V subdivisions. Again, the likely recipient background for the presumed capsular switch does not appear likely to involve the 19A/ST156 lineage (Figure 2), which was well-represented during the 2000s after PCV7 implementation (6,14,32).

Discussion
An increase of penicillin-nonsusceptible serotype 35B IPD and carriage caused by 35B/ST558 has been apparent in the United States since the introduction of PCV7 in 2000, and it has shown a major increase after PCV13 implementation (4)(5)(6)(7)(8). This finding is of concern because even strains that are rarely detected in IPD sometimes rapidly emerge. For example, the 19A/ST320 strain was not detected during extensive characterization of pre-PCV7 ABCs isolates (13) Before 2015, we detected only 1 isolate of the 35B/ ST156 lineage that was recovered during 2012 (6). Thus, we feel justified in describing it as newly emergent isolate. A smaller study was recently published (during peer review of this article) that described 78 invasive and 48 noninvasive serotype 35B isolates obtained during 1994-2014 from 8 hospitals in 8 states (33). Our data, which included a population-based sampling of 199 35B isolates obtained during 2015-2016, clearly shows the current national predominance of 35B/ST558 and does not support the observation that 35B/ST156 is the major contributor to post-PCV13 antimicrobial-resistant 35B. Both studies noted the initial appearance and emergence of 35B/ST156 in the post-PCV13 period.
This recent identification of the antimicrobial-resistant 35B/ST156 lineage and its subsequent detection within 6 ABCs sites is a cause for concern. The ST156 lineage has shown a remarkable propensity to persist through undergoing serotype-switch events (12,23,25,32). The penicillinresistant 9V/ST156 lineage was the predominant serotype 9V cause of IPD in the United States during the pre-PCV7 era (13,32). Soon after introduction of PCV7, serotype 9V IPD became rare (1,2), and 19A became the predominant representative of the ST156 lineage within ABCs (14,32). After introduction of PCV13, 35B has become the predominant serotype of the ST156 lineage within the United States (B. Beall, unpub. data).
A distinct antimicrobial-susceptible serotype 35B SLV of ST156 (35B/ST162) is included among 35B ABCs of 35B during 2015-2016 by the β-lactam-susceptible PBP type 0:0:0. Thus, our data indicates that >3 independent serotype switches involving the nonvaccine type cps35B locus and the broad ST156 clonal complex serving as recipient strains have previously occurred. In this study, we demonstrated that all penicillin-nonsusceptible 35B/ST156 lineage isolates obtained during current ABCs (2015-2016) arose through a single ancestral recombination event. This event was facilitated through detailed analyses of crossover points and comparisons of corresponding regions of all progeny isolates with likely parental 35B/ST558 and 9V/ ST156 strains. The genetic plasticity of the ST156 lineage is also highlighted in this study by detection of postserotype switch changes affecting β-lactam resistance (PBP1a type) and capsular serotype (wciZ deletion), which is potentially reflective of recent antimicrobial drug pressure and immunologic selection pressure.
An additional 35B variant within a vaccine serotype lineage is shown with ST1092 that is typically associated with serogroup 6 strains ( Figure 1). Because these putative 35B switch variants were not detected during extensive strain surveillance before and shortly after conjugate 928 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 6, June 2017  Figure 3. ‡Refers to 2 indicated wciG deletion isolates in right column under progeny lineage diagram in Figure 3.
vaccine implementation (3,4,13), it is plausible that these serotype switches occurred after implementation of conjugate vaccine.  Table).
Although conjugate vaccines have a history of providing effective and durable protection against IPD (1,2), the continued emergence and expansion of serotype 35B into different clonal complexes supports continued development of wider spectrum pneumococcal vaccines. Serotype 19A IPD, although relatively uncommon in the pre-PCV7 era, rapidly became the predominant invasive serotype in the post-PCV7 period (14,32,34). Serotype 35B strains have several of the same features that were found among serotype 19A strains before implementation of PCV7 in 2000. These features that could predispose for serotype 35B to continue its increasing trend as a cause of IPD include its lack of inclusion within conjugate vaccine, high carriage rates within children, antimicrobial resistance, clonal expansion, and serotype switching. An experimental 15-valent conjugate vaccine in development includes serotypes 22F and 33F (35), which have increased as causes of IPD in the postconjugate vaccine era. Serotypes 15A, 15B, and 23A are expressed by moderately antimicrobial-resistant clones and are not uncommon causes of IPD (4,32). Although less resistant to β-lactam antimicrobial drugs than 35B/ST558 and 35B/ST156, these strains also present a challenge to address through more encompassing pneumococcal vaccines.