Serotype-Switch Variant of Multidrug-Resistant Streptococcus pneumoniae Sequence Type 271

We discovered 3 invasive, multidrug-resistant Streptococcus pneumoniae isolates of vaccine-refractory capsular serotype 3 that recently arose within the successful sequence type 271 complex through a serotype switch recombination event. Mapping genomic recombination sites within the serotype 3/sequence type 271 progeny revealed a 55.9-kb donated fragment that encompassed cps3, pbp1a, and additional virulence factors.

serotype switch with serotype 19F, emerged as the most common cause of IPD in the United States after introduction of PCV7 (3). After introduction of PCV13, IPD caused by serogroup 19 CC271 greatly decreased (2,4).
To reveal genomic regions within the 3 serotype 3/ST271 progeny resulting from recombination, we fi rst identifi ed likely recipient and donor strains involved in the serotype switch event (Appendix 2, https://wwwnc.cdc.gov/EID/article/27/6/20-3629-App2.pdf). Phylogenetic analysis using publicly available genome sequences revealed single contig ST271 genomes that shared highest relatedness with 3/ST271. Strain A026 (19F/ST271) recovered in China during 2006-2008 was the most highly related putative genetic recipient ( Figure,   The most closely related ST271 single contig reference is A026 (also indicated with blue arrow). Branch colors: yellow, additional 19F/ ST271 Active Bacterial Core surveillance (ABCs) isolates and single contig references; red, single-locus variant single contig references or ABCs isolates; orange, double-locus variant single contig reference or ABCs isolates; blue, ST320 ABCs isolates. Zero-, single-, and double-locus variant, single contig references were identified by using the PubMLST database (https://pubmlst.org). Scale bar corresponds to 1,062 single nucleotide polymorphisms. B) Phylogenetic alignment of the 3 recombinant serotype 3/ST271 isolates and closest known genomic matches of the ST271 recipient lineage and a schematic of recombinant genome fragments, represented by rectangular blocks, that were predicted by Gubbins (10). Block locations and sizes are relative to the aligned genomes; red blocks represent sites in common between >2 isolates (1 site, <150 bp in length, was not counted among the 17 total shown), blue blocks sites unique to a given isolate, and gray blocks the serotype-switch fragment that replaced the corresponding cps19F region within the recipient 19F/ST271 strain. The cps3 locus was not identified using Gubbins because of its complete divergence from cps19F and instead was identified using ProgressiveMauve (12) within the encompassed 55.9 kb fragment (panel C). Gray block contains cps3, pbp1a (PBP1A-17), trigger factor, and choline binding protein G genes. C) Schematic illustrating ancestral recombination event between the 3/ST700 donor (B20605) and 19F/ST271 recipient (A026) to yield the 3/ST271 progeny (20155315-S-ABC). Deduced crossover points, including coordinates in the progeny, are shown. luxS and spsB genes are shown as blue/red hybrids. The minimum genes of the cps3 operon required for polysaccharide capsuale biosynthesire shown in gray (ugd, wchE, gtaB). Genes with arrows in black differ between B20605 and 73D36881 and are absent in the 3/ST271 isolates. ST, sequence type.
susceptible to antimicrobial drugs (4), the likely serotype 3/ST700 donor strain was predicted to have intermediate penicillin resistance attributable to the mosaic pbp1a gene (PBP1A-17), which was co-transferred with the donor cps3 locus to the 19F/ST271 recipient (Appendix 1). Serotype 3/ST700 isolates are documented in several countries in Africa but not in the United States (https://pubmlst.org/spneumoniae).
When we compared the genes encoded in the serotype-switch region of the recipient with the corresponding region from the progeny and donor, we found they encoded near-identical virulence factors as identified by using the Virulence Factors Database (http://www.mgc.ac.cn/VFs/main.htm). The tig and cbpG genes have roles in pathogenesis and are cell wall surface-localized, highly conserved among S. pneumoniae, and immunogenic (13)(14)(15). Capsular polysaccharides provide the basis of approved S. pneumoniae vaccines. The limited efficacy of the serotype 3 component of PCV13 is probably attributable to high-level expression (thickness) of the serotype 3 capsule and its shedding by the bacteria (6,7). At the nucleotide level, tig 20155315-S-ABC and tig A026 share 99.1% identity (11 SNPs apart); cbpG 20155315-S-ABC and cbpG A026 share 51.0% identity, and cps3 and cps19F operons are highly divergent (5). Moreover, the pbp1a gene within the 55.9-kb genomic fragment is identical between the 3 3/ ST271 isolates and donor 3/ST700 strains and exhibits only 81.7% identity with the putative recipient 19F/ST271 strains. Recombination introduced a distinct virulence factor (replacement of the serotype 19F capsule with a structurally and serologically unique serotype 3 capsule) and introduced diversity in the surface protein virulence factors expressed in the serotype 19F/ST271 lineage.
To understand the effects of recombination, a comparison of fitness and virulence of progeny and parental strains in mouse models might be valuable. Our study highlights the existence of the 3/ ST271 strain, because currently no vaccine is available and few antimicrobial drugs are predicted to optimally target this strain should it gain traction. Asymptomatic nasopharyngeal carriage serves as the major reservoir of highly common noninvasive infections and precedes invasive infections. Three nearly isogenic invasive isolates of this clone appearing over an expanse of 3 years in 2 different states is a concerning sign of a successful foothold within the carriage reservoir. All 3 cases were caused by bacteremic pneumonia in middle-aged patients. Of particular interest is the isolation of this strain 16 months apart from blood specimens from the same person (isolates 20155315 and 20170822), which is a rare occurrence and suggests a failure to eradicate the organism and long-term carriage or potentially the reacquisition of the strain from persons in the community. The recovery of still another invasive isolate of this same clone in 2018 further suggests a level of fitness required for long-term survival in the carriage reservoir and potential to cause IPD. Further, after the review process of this manuscript, ABCs has reported recovery of 2 additional closely related 3/ST271 case isolates during 2019 from Minnesota (B. Beall, unpub. data, 2020). This new strain complex has unique features potentially guiding its success, including high antimicrobial resistance, a capsule ineffectively targeted by PCV13, and undefined parameters inherent to the predominant invasive lineage of the post-PCV7 era. These observations emphasize the clear need for a pneumococcal vaccine that effectively targets serotype 3.

About the Author
Dr. Scherer is an assistant professor in the Division of Infectious Diseases, Department of Medicine, School of Medicine, Emory University, Atlanta, Georgia, USA. Her research interests include understanding what immune responses generate durable immunity against infectious diseases, with a focus on antibody and B cell responses to viral and bacterial pathogens and their vaccines.