A global genomic perspective on the multidrug-resistant Streptococcus pneumoniae 15A-CC63 sub-lineage following pneumococcal conjugate vaccine introduction

The introduction of pneumococcal conjugate vaccines (PCV7, PCV10, PCV13) around the world has proved successful in preventing invasive pneumococcal disease. However, immunization against Streptococcus pneumoniae has led to serotype replacement by non-vaccine serotypes, including serotype 15A. Clonal complex 63 (CC63) is associated with many serotypes and has been reported in association with 15A after introduction of PCVs. A total of 865 CC63 isolates were included in this study, from the USA (n=391) and a global collection (n=474) from 1998–2019 and 1995–2018, respectively. We analysed the genomic sequences to identify serotypes and penicillin-binding protein (PBP) genes 1A, 2B and 2X, and other resistance determinants, to predict minimum inhibitory concentrations (MICs) against penicillin, erythromycin, clindamycin, co-trimoxazole and tetracycline. We conducted phylogenetic and spatiotemporal analyses to understand the evolutionary history of the 15A-CC63 sub-lineage. Overall, most (89.5 %, n=247) pre-PCV isolates in the CC63 cluster belonged to serotype 14, with 15A representing 6.5 % of isolates. Conversely, serotype 14 isolates represented 28.2 % of post-PCV CC63 isolates (n=618), whilst serotype 15A isolates represented 65.4 %. Dating of the CC63 lineage determined the most recent common ancestor emerged in the 1980s, suggesting the 15A-CC63 sub-lineage emerged from its closest serotype 14 ancestor prior to the development of pneumococcal vaccines. This sub-lineage was predominant in the USA, Israel and China. Multidrug resistance (to three or more drug classes) was widespread among isolates in this sub-lineage. We show that the CC63 lineage is globally distributed and most of the isolates are penicillin non-susceptible, and thus should be monitored.


InTRoDuCTIon
The introduction of pneumococcal conjugate vaccines (PCV7, PCV10, PCV13) in countries around the world has proved successful in the prevention of invasive pneumococcal disease. However, immunization against Streptococcus pneumoniae has led to serotype replacement by non-vaccine serotypes (NVTs), including serotype 15A, and a concomitant increase in antimicrobial resistance associated with some of these serotypes [1][2][3][4][5][6]. In some cases, serotype replacement has occurred through capsular switching, but most often it is due to the expansion of previously recognized clones of NVTs. The expansion of the multidrug-resistant 15A/ sequence type (ST)63 (Sweden 15A -ST63, PMEN25) and related clones (clonal complex 63; CC63) after the introduction of PCVs has been reported in several countries, both among carriage and invasive pneumococcal isolates: Canada [7], Germany [8], Japan [9], Norway [10], the USA [11][12][13] and Taiwan [14].
The Global Pneumococcal Sequencing (GPS) project is a whole-genome sequencing survey of the global pneumococcal population in the context of PCV use. The Centers for Disease Control and Prevention (CDC) Active Bacterial Core Surveillance (ABCs) programme is an active laboratory and population-based surveillance system in the USA for invasive bacterial pathogens of public-health importance, including S. pneumoniae. In this study, we examined the global distribution of the 15A-CC63 sublineage in the context of these two large isolate collections.
Recombination was detected and removed using Gubbins (Genealogies Unbiased By recomBinations In Nucleotide Sequences) [27]. The resulting tree and recombination predictions, along with corresponding metadata, were visualized using Phandango

Impact Statement
Increases in non-pneumococcal conjugate vaccine (non-PCV) serotypes due to expansion of pre-existing clones and emergence of capsule-switch strains have been reported in some countries following the introduction of PCV. In this study, we describe the global distribution of non-vaccine serotype 15A before and after the introduction of PCV. Our findings contribute to the general understanding of pneumococcal molecular epidemiology in the PCV era, as well as the international effort to characterize replacement serotypes. Table 1. Country of origin, vaccine period, age and source information for all isolates *Some isolates are missing age information. †No age data. ‡PCV10 and PCV13 are available in Thailand, but not included in the National Immunization Programme.   [28]. Branch length was then converted to time since most recent common ancestor (MRCA) using BactDating, with a mixed gamma, relaxed clock model [29]. The effective sample size was over 200 for the inferred parameters α, μ and σ.
The chi-square test was used for all statistical comparisons. A comparison of overall serotype distributions among the study isolates from before and after the introduction of one of the PCVs (PCV7, PCV10 or PCV13), revealed a decline in the proportion of serotype 14 and concurrent increase in the proportion of serotype 15A (Fig. 2) All isolates from the Indian subcontinent (n=111) belonged to the 14-CC63 sub-lineage. Since PCVs were introduced in Nepal and Bangladesh in 2015, and in India in 2017, the majority (86.5 %) of our samples from this region predated vaccines. Serotype 14-CC63 was also the predominant sub-lineage in Sub-Saharan Africa, where serotype 14 was expressed by all pre-PCV CC63 isolates (n=89). , mainly because CC63 isolates and 15A isolates of all genotypes were rare in the USA pre-PCV7. Only 8 serotype 15A isolates were collected in 1999, out of 4547 total ABCs samples (surveillance population of 18 550 681) and only 2 of them were CC63. Invasive pneumococcal disease plummeted, but serotype 15A incidence increased modestly, after PCV7 was introduced, then levelled off before PCV13 was introduced, and has remained stable since then [11] (unpublished ABCs data). In 2009, 109 serotype 15A isolates (99 of them CC63, 90.8 %) were found among 4176 ABCs isolates (surveillance population of 29 206 528). Fig. 3 shows a phylogenetic tree of all the study isolates. The serotype 14 clades are very homogeneous, except for two branches of non-typeable carriage isolates from The Gambia. The first branch is composed of two isolates (ST12149 and ST63) with total absence of cps genes and identical pbp2x-dexB-aliA-pbp1a sequences (Supplementary Data S1); most closely related to an ST3310 isolate. The second branch is composed of 14 isolates (ST4040 and ST10967) without cps genes (Supplementary Data S2), and with a dexB-aliA region that most likely emerged from recombination with the previously described NT lineage ST344/GPSC81 (98.8 % identity) [33].
Of interest, the serotype 8-ST63 combination was previously determined to be a recombination of a highly invasive 8-ST53 clone and the multidrug-resistant Sweden 15A -ST63 clone, and widespread in Spain as of 2014 [34]; a single representative of this lineage, from the ABCs collection, was included in our study [16]. The serotype 19A-ST63 combination, which has previously been reported in Portugal [35], Australia [36] and Italy [37], was identified in 10 isolates from Thailand (n=2), Israel (n=1) and the USA (n=7); 6/7 USA isolates were isolated post-PCV13. Additionally, four isolates from Thailand belonged to the 15C-ST8346 lineage, which has been previously reported in Japan [38]. Fig. 4(a, b, c) shows the results of the recombination analysis, including a maximum-likelihood phylogeny reconstructed from the non-recombinant regions of the alignment (Fig. 4a). A total of 129 395 SNPs were identified. Several recombination hotspots were evident in the genome, but the most notable was in pbp2X, which reflected the diversity of PBP types that were identified (Fig. 4c).

Recombination and phylogenetic dating
Root-to-tip analysis showed a significant temporal signal, with R 2 =0.30 and P<0.001, allowing for coalescent analysis (Fig. 5a). A reconstruction of the dated phylogeny (Fig. 5b)
A total of 82 PBP types was identified among isolates in this sub-lineage (166 in total among all isolates in the study), mostly driven by diversity in pbp2X, with 184 (40.5 %) of the isolates represented by the 24-27-28 combination (Table S2). Only nine of these PBP types were associated with multiple STs and serotypes, the rest were associated with a single ST and serotype. Except for three combinations (24-27-28, 24-27-8 and 34-89-147), each PBP type was identified in isolates from a single country.
Full resistance (MIC=2 µg μl −1 ) was predicted for nine PBP types (present in 39 isolates) identified in this sub-lineage. These included the 4-7-7 and 4-74-7 combinations, which were likely acquired by recombination with a 35B-ST558 strain, as described by Metcalf et al. [16]. Of these, seven PBP types were associated with serotype 15A, one with 19A and one with 19F. By comparison, 13 of the PBP types in the 14-CC63 sub-lineage were predicted to confer full resistance to penicillin (present in 30 isolates); 10 of these 13 combinations remained in the post-PCV era.
In addition, 99.1 % of the isolates in the 15A-CC63 sub-lineage carried the ermB and tetM genes, and were thus predicted to be resistant to erythromycin, clindamycin and tetracycline. The ermB gene was carried in a Tn917 cassette [43], inserted into a Tn916-like element carrying tetM [44]; this element was highly conserved within this clade. A similar element has been previously identified in S. pneumoniae [45].
Nonsusceptibility to cotrimoxazole, due to changes within folA and/or folP, was less prevalent, with 60.2 % of the 15A-CC63 sub-lineage isolates predicted as susceptible, 33.0 % as intermediate and only 6.8 % as resistant. Less common resistance genotypes included ten isolates carrying the aph(3')-III gene, which confers resistance to aminoglycosides [46], and one isolate with changes in the rpoB gene, which confers resistance to rifampin [20]. An additional eight isolates had changes in parC, which confers reduced susceptibility to fluoroquinolones, most often reflected by clinically relevant MICs for ciprofloxacin.
Limitations to this study include: varied sampling strategies and timings of vaccine introduction among different countries, lack of post-vaccine sampling in some of the countries included in the GPS collection, and low overall proportion of CC63 isolates among GPS isolates (2.0 %) and ABCs isolates (2.6 %) for the years tested.
We conclude that because the CC63lineage is globally distributed and most of these isolates are PNS, it merits continued close monitoring. The 15A-CC63 sub-lineage in particular, since it is composed almost exclusively (95.8 %) of non-vaccine type multidrug-resistant isolates, has a high invasive potential [19], and is not included in either of the most recently licensed PCVs (PCV15 and PCV20).