Background

Metabolic flexibility is a prerequisite for successful transition of Streptococcus pneumoniae (Spn) from colonizing to invasive state. The aim of this study was to investigate the common metabolic features of invasive Spn strains and propose vaccine candidate antigens.

Methods

The dataset includes 1058 PubMLST Spn strains. MLST concatenates were used to build maximum likelihood phylogeny. Sequence clusters (SCs) were identified with RhierBAPS. Genomes (n=512) were analyzed with GenomeComparator. The resulting gene variant matrix was used to run classification algorithm to find associations of gene variants with genotypes and invasiveness. GWAS was performed with Samtools mpileup.

Results

Pneumococcal population was clustered into three groups (A,B1,B2) (Fig.1,2). Isolates from blood were more prevalent in B2 group. Clusterization was based on AccC, CiaH variants and SCs, correspondingly divided by variants of ComX1, TrpE and CshA. StrH variants were associated with both SCs and invasiveness. Mentioned groups have eleven highly conservative membrane proteins with unknown function and possibly high vaccine potential.

Conclusions

Pathways of fatty acid synthesis are involved in virulence regulation. A number of known vaccine targets are confirmed and novel ones (surface and membrane proteins with unknown function) are proposed.

Background

Sustaining herd protection via reduced dose schedules may ameliorate pneumococcal conjugate vaccine costs. However, there is limited understanding of pneumococcal transmission pathways and their role in herd immunity. We aimed to develop and validate phylogenetic methods for detecting the occurrence and direction of pneumococcal transmission.

Methods

Based on the timing of serotype-specific carriage within a household, 10 likely transmission pairs and the corresponding transmission direction were identified from a longitudinal study of nasopharyngeal carriage in the UK and sequenced by whole genome sequencing. Any metadata were blinded, and linkage and the transmission direction inferred from the genomic data alone using Phyloscanner.

Results

Unblinding revealed that transmission pair linkage via genomics was identical to that based on epidemiological criteria. One instance of co-colonization was detected and only the dominant serotype was transmitted. All transmission pairs had moderate to strong phylogenetic signals suggesting transmission direction, however, only 6/10 directions were concordant with the epidemiological metadata.

Conclusions

Phylogenetics did successfully predict transmission pairs in this small sample. To improve the inference of transmission direction we will consider factors including sequencing coverage, degrees of intra-host diversity, and phylogenetic uncertainty, although concordance with epidemiolocal metadata may be limited by imperfect sensitivity of culture-based tests for pneumococcal detection.

Background

In 2010, Brazil introduced PCV10 into the national children’s immunization program. This study describes the genomic population structure of invasive SPN before and after PCV10 introduction.

Methods

As part of Global Pneumococcal Sequencing (GPS) project, 466 (pre-PCV10:n=232, post-PCV10:n=234; <5-year-olds:n=310, ≥5-year-olds:n=156) invasive SPN collected through national laboratory surveillance were whole-genome sequenced.

Results

The study identified 65 GPS clusters (GPSCs): 49 (88%) GPS previously described and 16 (12%) were Brazilian clusters. 36 GPSCs (55%) were non-PCV10 lineages, 11 (17%) PCV10/non-PCV10 and 18 (28%) PCV10. In both periods, the most frequent lineage was GPSC6/CC156/PMEN3/14-9V. Post-vaccine non-PCV10 lineages GPSC16/CC66/9N-15A, GPSC12/CC180/PMEN3/3 and GPSC32/CC218/PMEN24/12F increased; in <5-year-olds, GPSC1/CC320/DLV-PMEN14/19A, GPSC47/CC386/DLV-PMEN20/6C and GPSC51/CC458/3; and ≥5-year-olds GPSC3/CC53/PMEN33/8 were predominant (Figure-1).

SPN penicillin nonsusceptibility was predicted in 40%; 127 PBP combinations were identified (51 predicted MIC≥0.125mg/L); cotrimoxazole (folA+folP alterations), macrolide (mef/ermB/ermB+mef) and tetracycline (tetM/tetO/tetS/M) resistance were predicted in 46%, 13% and 21% SPN, respectively. In <5-year-olds, a penicillin (p=0.0169) and cotrimoxazole (p<0.0001) resistance reduction and an increase in tetracycline (p=0.019) were observed. Post-PCV10, PBP15-12-18(2mg/L) was frequent in lineage GPSC6/CC156/PMEN3/14-9V; among <5-year-olds the PBP13-11-16(4mg/L) in GPSC1/CC320/DLV-PMEN14/19A and PBP2-53-77(0.125mg/L) in GPSC47/ST386/DLV-PMEN20/6C were predominant.

Conclusions

Post-PCV10, important non-PCV10 lineages, GPSC1/CC320/DLV-PMEN14/19A and GPSC47/ST386/DLV-PMEN20/6C associated with multidrug resistance and GPSC12/CC180/PMEN31/3, GPSC3/CC53/PMEN33/8 and GPSC32/CC218/PMEN24/12F were identified in Brazil.

Background

Serotype 1 Streptococcus pneumoniae is a common cause of IPD in India. The present study describes the phylogeny, clonality and antimicrobial susceptibility pattern of the isolates collected in the pre-PCV era in India.

Methods

21 invasive serotype 1 pneumococcal isolates collected across India during 2009-2016, were sequenced on Illumina platform. Phylogenetic tree was built with REALPHY 1.12. Abricate software with VFDB was used to analyse virulence genes and CDC Pneumococcal specific pipeline was used for antimicrobial resistance gene identification.

Results

Population structure analysis of the strains showed that 20 of them belong to sequence cluster GPSC2 and one to GPSC31. MLST resolved the isolates to 8 known STs and four clonal complexes CC217, CC5191, CC5316 and CC303. CC217 (n=16) was the most prevalent clonal complex, followed by CC5191 (n= 3). 90% (n=19) of the isolates harboured the virulence factors, lytA, nanA, hasC, pspA, srtG1, srtG2. Phenotypic and genomic analysis demonstrated sensitivity to penicillin, erythromycin and vancomycin of all 21 isolates; six isolates were resistant to cotrimoxazole and one to tetracycline.

Conclusions

With the introduction of pneumococcal vaccine in the national immunization programme in 2017, this study provides baseline data for future analyses.

Background

After PCV10 introduction into the national children’s immunization program in 2010 in Brazil, Spn19A increased as cause of DPI and NCC. This study describes the genomic population structure of Spn19A before and after PCV10 introduction.

Methods

Spn19A (n=454) collected from <5-year and ≥50-year through national laboratory surveillance (DPI:n=403) and from 1-<2-year NCC (n=51) were MLST characterized (pre-PCV10:n=68, post-PCV10:n=386). Of these, 50 Spn19A (pre-PCV10:n=20, post-PCV10:n=30) were whole-genome sequenced (WGS).

Results

This study identified 8 clonal complexes (CCs), with the prevalent lineages CC1118 (51.5%, 35/68) and MDR-CC320 (60.9%, 235/386) in pre-PCV10 and post-PCV10 periods, respectively. The WGS showed 11 GPS clusters (GPSCs), with GPSC1 (32.0%, 16/50) and GPSC204 (30.0%, 15/50) prevalent and related to MDR-CC320 and CC1118, respectively. GPSC1/CC320 (87.5%, 14/16) lineage was associated to penicillin (MIC≥2.0µg/mL) and ceftriaxone (MIC≥1.0µg/mL) nonsusceptibility, presenting pbp1a+pbp2b+pbp2x mutations, and 100.0% (16/16) of cotrimoxazole resistance (folA+folP alterations). Only in the GPSC1/CC320 lineage was identified the pilus-1 and pilus-2 genes and the transposon Tn2010, which carry macrolide (mefE+msrD+ermB) and tetracycline (tetM) resistance genes.

Conclusions

The study showed the expansion of the lineage GPSC1/CC320-Spn19A-MDR after PCV10 introduction in Brazil. Besides vaccine impact, the presence of MDR and the pilus-1 and pilus-2 genes could collaborate with the expansion.

Background

Pneumococcal Conjugate Vaccine (PCV) use has resulted in decrease of vaccine serotypes (VTs) and emergence of non-vaccine types (NVTs). We applied whole genome sequence (WGS) to predict serotype, sequence type (ST) and antibiotic resistance of NVT invasive pneumococcal isolates collected during the pre-vaccine era from Indian population.

Methods

96 NVT invasive isolates (2009-2017) collected across the country were sequenced on Illumina platform. Bioinformatic pipelines SeroBA and CDC pneumococcal pipeline for AMR calls were used for data analysis.

Results

Serotypes 15B (n=11), 24 (n=9) were dominant NVT types followed by 8 (n=8) and 34,10A,11A,16F (n=5). MLST resolved strains into 67 known STs. ST13727 (n=6) and ST2234 (n=5) were most common. Strains clustered in 45 clonal complexes and 16 singletons. The dominant clonal complex CC230 (n=12) was from serotypes 15B,15C,24,10A and 11A. 78(81%) of isolates were multidrug-resistant. Resistance genes for tetracycline (n=44), cotrimoxazole (n=41), erythromycin (n=34), penicillin (n=13) and chloramphenicol (n=2) were identified.

Conclusions

With the introduction of PCV in 2018 in national immunization program our data provides information for post-vaccination assessments. With higher valency vaccines coming to market by Indian manufacturers, knowledge of PCV13 NVT disease is important to identify serotypes to expand vaccine coverage in India.

Background

The invasive pneumococcal disease remains one of the leading causes of morbidity and mortality worldwide. In this study, we investigated high-resolution population genetic structure of S. pneumoniae isolates in Moscow, using the genomic definition of pneumococcal lineages (Global Pneumococcal Sequence Clusters (GPSCs)), serotypes and antimicrobial resistance patterns.

Methods

Eighty-seven pneumococcal isolates were recovered from cerebrospinal fluid and nasopharyngeal swabs of patients with meningitis and upper respiratory tract infections, ages one to 93 years in Moscow between 2011-2015. The serotypes and multilocus sequence types (MLSTs) were derived from whole-genome sequencing data using ARIBA and MLSTcheck. GPSCs were assigned by popPUNK. Antibiotic susceptibility was predicted based on genotypes.

Results

Sixty-seven sequence types identified in the collection belonged to 39 clonal complexes (CCs) and 10 singletons. Overall, 42 GPSCs were identified. The two prevalent lineages were GPSC1 (CC320) and GPSC7 (CC437). Twenty-two serotypes were found and their associated GPSCs are shown in Figure 1. The major GPSC contributing to multidrug resistance was GPSC1, which expressed 19F/19A serotypes (Figure 2).

Conclusions

The pneumococcal collection showed high diversity in population structure. Ongoing surveillance is needed to monitor the dynamics of the pneumococcal population in Russia following the introduction of PCV13 immunization in 2014.

Background

Bangladesh has been generating pneumococcal data since last 30 years to make an evidence-based data for vaccine introduction. This study is aimed to make a genomic characterization of pneumococcus isolated from pre-vaccine period.

Methods

Whole-genome sequencing data of total 525 pneumococcus isolated from IPD, during 2002 to 2015, were analyzed using previously established methods.

Results

Overall, 57 serotypes were identified, and most predominant serotypes were 2, 1, 14, 23F, 5, 19F, 12A and 45 which accounted for 50% of isolates. Serotype coverage were 47% for PCV10+6A, 50% for PCV13 and 58% for PCV20. The population was genetically diverse with 108 known and 61 new Sequence Types (STs), encompassing in 89 GPSCs. Among them, GPSC96 (serotype 2, n=66, 11.6%), GPSC 2 (serotype 1, n=48, 9%), GPSC 9 (serotype 14, n=32, 6%) were most predominant. Significant increase in resistance has observed for Erythromycin (0%-60%). Resistance is commonly seen in GPSC 10, 43, 101 and 482 mainly among serotype 19F, 23F, 6B and 7B, respectively.

Conclusions

Pneumococcus in Bangladesh is diverse and different in respect of serotype, ST and GPSC. This data will work as the baseline population to monitor vaccine induced changes in molecular epidemiology.

Background

Severe pneumonia is the major cause of childhood mortality worldwide. Streptococcus pneumoniae remains a leading pathogen in severe acute lower respiratory illness (ALRI) in children globally. Increased incidence of severe ALRI in defined genetic groups suggests heritable components to ALRI susceptibility. We aimed to identify novel susceptibility alleles to severe childhood ALRI.

Methods

In a case-control study, we consented and collected saliva samples from children presenting with ALRI (Cases) and their healthy biological parents or siblings (Controls). Whole exome analysis was performed on 6 children and their controls from Papua New Guinea.

Results

A rare aspartate to tyrosine variant allele of COQ6, COQ6^D→Y, was found in a homozygous state in 3 of 6 ALRI cases. The variant was either present in a heterozygous state or absent in controls and enriched above background levels. COQ6 is a required enzyme in ubiquinone biosynthesis. Current work on additional samples seek to validate a genetic association of COQ6^D→Y with ALRI.

Conclusions

A novel single nucleotide variant in a ubiquinone biosynthesis enzyme is highly enriched in pediatric populations very susceptible to severe ALRI. Validating COQ6^D→Y variant as causative to severe ALRI will inform novel interventional trials using nutritional supplements to treat high risk children, thereby improving ALRI outcomes.

Background

Serotype 2 was a major cause of pneumococcal pneumonia about 100 years ago and then disappeared. Recently, serotype 2 re-emerged in many countries, including Bangladesh and associated with meningitis. This study aims to understand genomic and epidemiological characteristics of newly emerged serotype 2 strains.

Methods

Whole-genome sequencing was performed on 146 isolates (invasive= 125, carriage= 8 and other= 5, unknown= 8) collected between 1989 and 2017. Data were analyzed for comparative genomics, antimicrobial resistance and molecular typing.

Results

Isolates were from 16 countries, mostly in Asia (n=93), Africa (n=23) and Oceania (n=26). Bangladesh (n=66) and Papua New Guinea (n=26) contributed 63% of the isolates. Among the known clinical conditions, 80% (91/113) were from meningitis. All isolates belonged to GPSC96 lineage and descended from two predominant sequence types: ST74 found in Asia and Africa, and ST1504 found in Papua New Guinea and Israel. Almost all isolates were sensitive to all antibiotics. No significant genetic differences were detected between invasive and carriage isolates.

Conclusions

Our findings don’t explain why the recent increase in serotype 2 occurred but exclude an outbreak or emergence of an antimicrobial-resistant strain as the cause. These isolates have unusually high propensity to be invasive, mostly causing meningitis.

Background

Streptococcus pneumoniae is a leading cause of meningitis. Intense inflammatory response observed in meningitis is partially attributed to zinc metalloprotease encoded by zmpB in pneumococcal strains. We aimed to study allelic variations of zmpB among isolates obtained from Meningitis patients

Methods

36 cerebrospinal fluid (CSF) isolates collected across the country from 2009-2016 were sequenced on Illumina platform. The CRL in-house bioinformatics pipeline was used to extract gene sequences, alignment and phylogeny analysis. SeroBA was used to determine serotype. Allelic variations of zmpB gene was analyzed by comparing the identity with the virulent strain S. pneumoniae TIGR4.

Results

36 pneumococcal isolates belong to 24 serotypes with 19F(n=5) as dominant type. Non-PCV13 vaccine serotypes constituted 50% of the isolates(n=18). The isolates were assigned to 28 sequence clusters, among them GPSC10(n=4) & GPSC2(n=3) were predominant.32 of 36 isolates showed 21 to 88% of sequence variation, while remaining 4 isolates showed sequence similarity of 98% with the TIGR4. Allelic variations did not affect the protein coding region analysis and conserved domains of ZmpB protein was identified in all isolates.

Conclusions

The findings provide insight on the allelic variations of zmpB, indicating there is a high degree of polymorphism in the sequence of zmpB in pneumococci causing meningitis.

Background

Streptococcus pneumoniae is a human opportunistic pathogen responsible for morbidity and mortality worldwide. Pneumococcal surface protein, Choline-binding protein A (CbpA) plays a key biological role in nasopharyngeal colonization and modulating the immune response to pneumococci. We have analyzed the genetic diversity of cbpA in invasive isolates.

Methods

264 invasive S.pneumoniae isolates collected from 2010-2018, were sequenced on Illumina Platform. The CRL in-house bioinformatics pipeline was used to extract gene sequences, alignment and phylogeny analysis. Allelic variations of CbpA gene was analyzed by comparing the identity with a well-defined virulent strain of S. pneumoniae TIGR4.

Results

Gene cbpA was identified in 261(99%) of the 264 genomes. The sequences were highly polymorphic at both nucleotide and amino acid levels. Similarity of cbpA gene ranged from 65 – 98%, while 80- 99% homology was observed at amino acid level. Amino acid residues with similar physicochemical properties aligned allowing the identification of broadly conserved CbpA domains.

Conclusions

Due to high polymorphism at the cbpA locus, analysis of this loci from different isolates highlights how sequence diversity correlates with structural variation. The conserved epitope regions of the CbpA protein fragments can be exploited to develop more efficacious serotype-independent vaccines.

Background

Determining the host molecular genetic characteristics of pneumococcal colonisation may inform the development of new clinical interventions which could interrupt pneumococcal transmission and establishment of disease. We performed a genome-wide association study to identify the genes associated with pneumococcal carriage.

Methods

DNA collected from healthy Nepalese children were genotyped using Illumina Global Screening Arrays. Array data underwent QC and filtering before undergoing imputation using the HRC R1.1 2016 reference panel. Nasopharyngeal swabs collected from participants were processed for the presence of pneumococci by conventional microbiological techniques. Association analysis was performed using PLINK 1.9.

Results

Following filtering, 1355 carriers (cases) and 766 non-carriers (controls) were analysed. 10 variants within a single region, were associated with pneumococcal carriage (p<10^-8). The variant that had the strongest association with pneumococcal carriage (MAF carriers = 0.07 vs MAF non-carriers = 0.13, OR 0.52, 95% CI 0.42-0.64, p=2.3x10^-8), is within an intergenic region between PPFIA2 and CCDC59.

Conclusions

We identified host genetic variants associated with pneumococcal carriage. Further studies confirming this association and its biological role in pneumococcal carriage are needed.

Background

Pneumococcal infections remain a major cause of infections among children. In this study, a part of the Global Pneumococcal Sequencing (GPS) Project, we described the genomic structure of the pneumococcal population in Casablanca, Morocco, before and after vaccination introduction.

Methods

DNA of 47 invasive isolates from children < 5 years old (32 isolates from pre-vaccine period, 15 isolates from post-vaccine period) were whole genome sequenced. Sequences were assembled, annotated and pangenome characterized. Core genome based phylogenetic trees were constructed. Finally, PopPUNK was used to group our isolates in different GPS Clusters (GPSC).

Results

Pangenomic analysis revealed 1,396, 1,465 and 1,357 core genes for pre-vaccination period, post-vaccination period and the two periods combined, respectively; and 2,915, 2,405, 3,432 accessory genes for the same periods. Phylogenomic analysis showed two major clusters GPSC6 (serotype 14) and GPSC10 (Serotype 19A and non-vaccine serogroup 24). Both clusters are non-susceptible to penicillin. The two serogroup 24 isolates in GPSC10 were found in pre-vaccine (n=1) and post-vaccine periods (n=1), and both were resistant to erythromycin and tetracycline.

Conclusions

The pneumococcal population in Casablanca was highly diverse regardless of serotype and period isolation. Several minor homogeneous and/or heterogeneous clusters were found with two major clusters.

Background

Widespread use of pneumococcal conjugate vaccines (PCVs) perturb the bacterial population structure, the consequences of which include changes in nasopharyngeal competition among pneumococci. Bacteriocins are antimicrobial peptides produced by bacteria, and are believed to inhibit competing bacterial strains. Twenty putative pneumococcal bacteriocins have been identified: this study aims to compare the bacteriocin distribution among pneumococci recovered pre- and post-PCV10 introduction in two countries.

Methods

Carriage and disease pneumococci collected in Iceland (n=1,916) and Kenya (n=3,257) before and after PCV10 introduction were sequenced and genomes were screened for twenty different bacteriocin sequences. Significant changes in the prevalence of genetic lineages, serotypes, and bacteriocins post-PCV10 introduction were assessed.

Results

PCV10 use led to a significant reduction in the prevalence of vaccine serotypes and associated genetic lineages in both countries. Overall, 18 of 20 bacteriocins were detected in 1% to 100% of genomes. The prevalence of six bacteriocins was significantly altered in the Kenyan dataset post-PCV10, and three of these also changed in the Icelandic dataset. These bacteriocins were associated with genetic lineages that also significantly changed in prevalence.

Conclusions

The bacteriocin distribution changed significantly following PCV10 introduction in Iceland and Kenya. The biological relevance of these changes to competition dynamics is currently under investigation.

Background

Streptococcus pneumoniae serotype 19F is one of the invasive serotypes causing IPD in Indonesia where it is commonly found as multi-drug resistant (MDR). However, relationship between its genetic variation and multi-drug resistant is still not well described. Therefore, we performed multilocus sequence typing on multidrug resistant serotype 19F to describe sequence type of multi-drug resistance serotype 19F in Indonesia.

Methods

We used 25 archived isolates previously described as serotype 19F. Antimicrobial susceptibility testing was performed using disk diffusion according to CLSI guidelines. Multi-locus sequence typing was performed by sequencing targeting seven house-keeping genes; aroE, gdh, gki, recP, spi, xpt, and ddl. Sequence type (ST) was determined by allele number combination of seven house-keeping genes.

Results

S. pneumoniae serotype 19F isolates were resistant to tetracycline (89%), Sulfametoxazole/Trimethophrim (72%), macrolide (52%), and chloramphenicol (60%) where 56% of isolates was MDR. We identified 5 sequence types where ST 9192 as dominant sequence type (44%) while others were ST 320 (20%), ST 242 (16%), ST 236 (12%), and ST 271 (4%) belonged to MDR strain.

Conclusions

Serotype 19F has been resistant to common antibiotic used in Indonesia where sequence type 320, 242, 236 and 271 are sequence type for MDR strain of serotype 19F

Background

We investigated the use of whole genome sequencing (WGS) to predict antibiotic resistance of circulating S. pneumoniae (pneumococcus) carriage isolates collected among PCV7 and PCV10-vaccinated toddlers in the Netherlands.

Methods

All Pneumococcal strains (n=737) isolated between 2009-2018 from 24-months old carriers were subjected to WGS. Genome assembly of short reads into contigs was performed using SPAdes, which were subsequently assigned a taxonomic origin with Kraken2. All DNA sequences identified as pneumococcus were used for resistance gene identification with RGI. Predicted resistance genes with ≥50 bitscore and ≥80% sequence identity were further analyzed and compared against phenotypical antibiotic resistance data. Susceptibility testing was performed according to EUCAST guidelines.

Results

Based on WGS data 9.8% (72/737) of pneumococcal isolates were predicted to harbor resistance genes. Genetic markers of antibiotic resistance predicted by WGS were in concordance with resistotypes determined using the EUCAST-recommended method. Lack of susceptibility to penicillins was primarily observed for serotypes 19A (13% of 128) and 23B (19% of 63), respectively.

Conclusions

We have observed good congruence between antibiotic resistance prediction with WGS and standard phenotypical antibiotic sensitivity testing. Antibiotic resistance among pneumococcal carriage isolates in the Netherlands is relatively rare and is characterized by circulation of resistant serotype 19A and 23B strains.

Background

Interruption of vaccine serotype transmission underlies effectiveness of pneumococcal conjugate vaccine (PCV) programmes at population level. Streptococcus pneumoniae (Sp) nasal carriage density is affected by upper respiratory tract viral infections and PCV. The impact of carriage density on transmission is unknown.

In a multi-centre prospective randomised stepped-wedge trial, we have used the Live Attenuated Influenza Vaccine (LAIV) to modulate Sp carriage density in 2-year-olds and assess impact on household transmission.

Methods

410 families were recruited across 10 UK sites. Families were randomised 1:1 to early or late (4 weeks later) index-child LAIV; saliva and nasopharyngeal samples (NPS) were collected 2-weekly over 2 months. Samples are being analysed for Sp by real-time quantitative PCR (lytA), culture amplification and microarray.

Results

SP nasal carriage rate, densities and density range are highest in our index children (Table 1 for baseline (visit 1) and Figure 1 for visits 1-5).

Conclusions

The range of Sp carriage density is very wide with rates and densities in keeping with the literature. The impact of density on Sp transmission will be analysed when full results are available, June 2020.

Background

Recurrent invasive pneumococcal disease (rIPD) presents clinical management and diagnostic challenges as the infection may persist because of treatment failure or acquisition of a new strain of Streptococcus pneumoniae. This study aimed to characterise rIPD and differentiate cases of endogenous relapse over new exogenous infections.

Methods

A total of 50 rIPD episodes diagnosed >31 days apart (105 isolates) were investigated. All isolates were serotyped and subjected to whole genome sequencing.

Results

The same causal serotype was documented in 28 episodes. The majority of genomes associated with these relapses (25/28) had high homology (0-13 single nucleotide polymorphisms (SNPs)) between core genes, compared to rIPD caused by a different serovar (>1000 SNPs). The time between rIPD episodes was a predictor of relapse, with a significantly shorter time period between IPD infections (median time 11 months versus 1.5 years, P=0.0034). However, the acquisition of point mutations or mobile elements enabling resistance only occurred in three relapse cases.

Conclusions

Genomic comparisons of pneumococci associated with rIPD improved differentiation between relapse and reinfection, compared to traditional serotyping and streamlined follow-up investigations. High core genome and resistome homology were detected in relapse cases suggesting that the development of antibiotic resistance did not play a major role in rIPD.

Background

Identifying the molecular characteristics of pneumococci associated with disease may inform development of new clinical interventions. We aimed to perform a bacterial genome-wide association study to identify pneumococcal genes associated with carriage among children with pneumonia.

Methods

DNA from nasopharyngeal pneumococcal isolates obtained from Nepalese children admitted to hospital with pneumonia (cases) and healthy community-based children (controls), underwent whole-genome-sequencing on the Wellcome Sanger Institutes core sequencing pipeline. The association of variants from sequences mapped against the S. pneumoniae ATCC700669 genome, with cluster of orthologous groups using a fixed effects model, was performed using a python based sequence element enrichment analysis.

Results

245 case and 597 control isolates were sequenced. 405461 variants were identified and 31708 tested after filtering. 20 variants from colonising bacteria had a strong association (p<10^-8) with pneumonia. 18/20 of these variants were located within the lacE2 gene. The variant with the strongest association, presence of an A allele at position 1066739, was identified in 240/597 (40%) of controls and 150/245 (61%) of cases (p=10^-10).

Conclusions

In this study in Nepal the pneumococcal gene lacE2 was associated with colonisation in children with pneumonia. Studies examining the role of lacE2 in the pathogenesis of pneumococcal pneumonia are needed.

Background

The 10-valent pneumococcal conjugate vaccine (PCV10) was introduced into the Nepalese infant immunisation schedule in August 2015. We aimed to examine how PCV10 introduction in affected pneumococcal lineages.

Methods

DNA from randomly selected nasopharyngeal pneumococcal isolates of healthy community-based Nepalese children in the Kathmandu valley pre- (2009-2014) and post-PCV10 (2017-2018) introduction, underwent whole-genome-sequencing on the Wellcome Sanger Institutes core sequencing pipeline. Isolates were clustered into lineages based on shared sequence and gene content using Population Partitioning Using Nucleotide K-mers (PopPUNK) software.

Results

313 and 284 pre- and post-PCV10 isolates were sequenced. There was a significant reduction in the proportion of PCV10 serotypes when comparing pre 73/313 (23.3%) with post 37/284 (13%) PCV10 samples (p=0.0014). Overall 122 distinct lineages were identified, 98 pre- and 74 post-PCV10. Simpson's index of diversity for the lineages was 0.992 and 0.987 pre- and post-PCV10 respectively. Within the 3 largest PCV10 serotype lineages there were no examples of non-PCV10 serotype isolates pre-vaccination, whereas all 3 lineages contained non-PCV10 serotypes post-vaccination.

Conclusions

PCV10 serotype prevalence significantly declined following PCV10 introduction. However, strain diversity remained high post-PCV10 and there is evidence suggestive of vaccine escape via capsular-switching among lineages possessing predominantly vaccine-covered capsules prior to PCV10 introduction.

Background

Our understanding on bacterial genetic determinants of pneumococcal disease manifestation is still limited. We aim to confirm the previous findings and identify additional bacterial variants associated with meningitis.

Methods

We sequenced IPD isolates identified through the Active Bacterial Core surveillance (ABCs) in the United States from 2016 to 2017. We evaluated the association between meningitis and a previously reported pneumococcal pbp1b641C allele by using a mixed-effects logistic regression model accounting for population structure (represented by multi-locus sequence type) and potential confounders (pneumococcal serotype, antibiotic resistance, and patient age). We also performed a k-mer based bacterial genome-wide association study (GWAS).

Results

Among all 5560 sequenced IPD isolates, 371 (6.7%) were from meningitis cases. Among the 576 isolates carrying the pbp1b641C allele, 86 (14.9%) were meningitis. After adjusting for covariates, the pbp1b641C allele was significantly associated with meningitis (OR=1.76, 95% CI,1.10-2.81). Pneumococcal genome contents explained 8.2% (95% CI,1.6%-13.2%) of variation in meningitis in the GWAS. Additional pneumococcal mutations that showed significant association were identified in loci including SP_1448 (conserved hypothetical protein, p=1.7×10^-8), SP_2167 (L-fuculokinase, p=3.6×10^-8), and SP_0647 (phosphotransferase, p=1.0×10^-7).

Conclusions

IPD manifestation varied significantly according to pneumococcal genomes. More knowledge on such mutations could help better understand bacterial pathogenesis and clinical outcome.

Background

Worldwide Streptococcus pneumoniae serotype 19F, often multi-drug resistant, has emerged as an important pathogen associated with invasive pneumococcal disease (IPD). The aim of the study was to characterize invasive serotype 19F isolates collected from India in pre-vaccine era.

Methods

Among 480 pneumococcal isolates collected across India from 2010-2018, 38 belonged to serotype 19F (8%). These were sequenced on Illumina Platform. The sequence data was analysed for serotype, clonal complex, pilus islets and MLST using the CDC pipeline

Results

Overall, 11 STs encompassing in 4 GPSCs and 3 clonal complexes (CCs) were identified. The most prevalent strain of serotype 19F was GPSC1 (n=31, CC320), followed by GPSC10 (n=3, CC10879). CC320 was the major clonal complex (n=33) with ST236 (n=7), ST271 (n=7), ST320 (n=7), ST2697 (n=7), ST2854 (n=2) and ST651, ST1396, ST8359 (n=1 each). A majority of GPSC1 isolates (30/31) had pilus 1 & 2 while GPSC10 isolates were negative for both. All GPSC1 isolates and GPSC10 isolates were resistance to at least three antibiotic classes

Conclusions

This analysis identified CC320 as the major lineage among serotype 19F isolates pre-PCV vaccination in India. Overall, serotype 19F isolates were found to be multi-drug resistant with a high percentage of pili genes present.

Background

Several studies have demonstrated limited PCV13 efficacy against carriage of serotype 3 Streptococcus pneumoniae. In Blantyre, no direct vaccine effect was identified on serotype 3 carriage 8 years after PCV13 introduction, and population-based serology showed limited induction of serotype 3-specific antibodies by vaccine or natural exposure. We hypothesised that sequence variability of the CPS locus could at least partly explain these observations.

Methods

CPS loci of 540 serotype 3 isolates (from Africa, Asia, Europe and America) were analysed. ML-phylogeny, antimicrobial resistance (tetM, mefA, ermB, cat gene presence and pbpX allelic profiles) and MLST were calculated. Colony morphology was assessed in microaerophilic and anaerobic conditions.

Results

We identified a serotype 3 CPS locus variant in 82% of Blantyre isolates (2015-2019), characterized by an 8-gene cluster deletion but unchanged colony morphology. This variant appeared independently in at least 3 distinct phylogenetic clusters, including ST700 and ST3214. These strains are characterised by the presence of AMR genes and higher MIC to penicillin. The missing CPS genes have hypothetical protein annotation.

Conclusions

This CPS variant segregated with an AMR genotype, which may have contributed to its emergence. Whether this CPS variation will influence immunogenicity remains to be determined.

Background

Streptococcus pneumoniae is the most common cause of pneumonia in children, including empyema, a severe complication. Over 90 serotypes of pneumococcus exist, but only a subset cause empyema. Virulence determinants of empyema remain largely unknown.

Methods

For viable pneumococcal isolates from invasive pneumococcal disease at Primary Children’s Hospital (Salt Lake City, UT) from 1996-2018, we performed Illumina sequencing, de novo genome assembly (SPADES), annotation (PROKKA), and serotyping via Quelling and in silico (SeroBA). ROARY was used for pan-genome assembly, and SCOARY for microbial GWAS. Maximum likelihood phylogeny was calculated using RAxML.

Results

354 pneumococcal isolates were analyzed from 37 serotypes and a spectrum of phenotypes including pneumonia (n=84), empyema (n=54), CNS infection (n=50), and isolated bacteremia (n=68). 6462 genes comprised the pneumococcal pan-genome, with only 23% present in 99% of isolates. Serotype and empyema phenotype clustered roughly by phylogeny. Specific capsule synthesis and diverse metabolic regulatory genes were statistically correlated with the empyema phenotype.

Conclusions

Genetic clustering of empyema isolates within and across serotypes supports genetic determinants of virulence. Altered metabolic regulation may confer optimal fitness for pleural disease. Further validation and characterization of critical determinants of empyema will inform future efforts at prevention and treatment.

Background

The rarity of serotype 19A among invasive pneumococcal disease (IPD) supported choice of PCV-10 for Bangladesh’s program. However, concern remains about emergence of this serotype, particularly multi-drug resistant (MDR) sequence type (ST) 320, after widespread PCV10 use. Therefore, we tracked the molecular epidemiology of 19A among pneumococci from Bangladeshi children over time.

Methods

We analyzed 64 isolates (54 pre-PCV and 10 post-PCV) spanning 2005-2018; isolates were from IPD (n=23), carriage (n=23) and otitis media (n=18). Isolates were subjected to whole genome sequencing and STs determined. Antimicrobial resistance patterns were determined phenotypically and genotypically.

Results

Overall, a total of 24 STs were identified, including 3 novel STs. Among the identified STs, ST12473 (16.4%), ST2464 (16.4%) and ST12891 (11.5%) were most common. No ST320 strains were detected. Multidrug resistance (erythromycin, co-trimoxazole and tetracycline) was only seen in ST12473. In addition, ST2464 was predominantly non-susceptible to co-trimoxazole and tetracycline.

Conclusions

The surveillance identified two drug resistant STs but not ST320. However, considering the increased rate of serotype 19A seen elsewhere following PCV7 and PCV10 use, we should continue surveillance and molecular investigation of this serotype.

Background

Introduction: Variants at complement genes predispose to infections and/or hyperinflammation (complementopathies). We analyzed the association of variants at complement genes with susceptibility to community-acquired pneumonia (CAP).

Methods

Methods. Common variants at the genes C2, C3, CFH and CFB associated with several complementopathies; C2 Del28bp, C5 p.V802I and CFH p.E936D, predisposing to infectious diseases; Copy number variation (CNV) at C4 (C4A, C4B, C4S and C4L). Taqman SNP genotyping assays and Taqman Copy Number assays. 932 adults with CAP (356 had pneumococcal CAP -P-CAP) and 851 controls.

Results

Results. The rs2230199 (C3_102R) alleles and genotypes associated with CAP (p=0.000005, OR 1.6) and P-CAP (p=0.0001, OR 1.72). The rs547154 at C2 (tagging FB_R32Q) associated with CAP (p=0.002, OR 1.35) and P-CAP (p=0.0002, OR 1.59). No association of CNV at C4 or other variants with P-CAP was observed.

Conclusions

Conclusion. The functional variants C3_102G and FB_32R, which activate the alternative pathway more efficiently, have been previously associated to several complementopathies. It was hypothesized that the low activity C3_102R and FB_32Q variants could associate with bacterial infections. Our data show that the C3_102R and FB_32Q variants do associate with predisposition to pneumococcal CAP in adults.

Background

Despite inclusion in PCV13, Streptococcus pneumoniae serotype 3 (ST3) continues to cause significant morbidity and mortality. In Native American communities in the southwest US in 2015-2017, the ST3 carriage prevalence among children was 2.7% and the incidence of ST3 invasive pneumococcal disease (IPD) among adults was 9.0/100,000. An emerging lineage of ST3 belonging to Clonal Complex (CC) 180, termed clade II, has recently increased.

Methods

We analyzed the genomic epidemiology of 202 ST3 isolates collected from 133 adults and 69 children with carriage (n=71) or IPD (n=131) from 1999–2018. Using phylogenetics based on whole-genome sequencing data, we determined clade membership of each isolate and assessed how the population structure changed over time.

Results

The percent of isolates belonging to clade II increased from 22.3% (n=94) in 1999-2010 to 65.7% (n=108) in 2010-2018 (Figure A). Carriage isolates were comprised equally by three clades (CC180 clade Ia n=24 and II n=23 and non-CC180 n=24); however, IPD isolates were more likely to be clade II (1a n=37, II n=69, non-CC180 n=25; OR=2.32, 95% CI 1.27-4.25) (Figure B).

Conclusions

Overall, we find that ST3 clade II has increased significantly since the introduction of PCV13 and is found more commonly in invasive disease compared to other clades.

Background

Our ongoing 14-year surveillance (2006-2020) in Rochester NY has tracked pneumococcal serotypes causing acute otitis media (AOM) in children age 6-36 months.

Methods

Pneumococci were identified by culture of middle ear fluid using tympanocentesis and nasopharyngeal swabs. Serotypes were determined by Quellung and whole genome sequencing (WGS) (using Illumina NextSeq500 or Pacific Biosciences RSII). Assemblies of 35B strains were aligned using LR216049 as reference. A dot plot was used to map overall sequence similarity between “old 35B” isolated prior to 2015 and “new 35B” isolated after 2015.

Results

Serotype 35B case/carriage ratio of old 35B was 0.13 and 1.17 for new 35B, p=0.0001, suggesting increased virulence. New 35B accounts for almost 25% of all pneumococcal isolates from children with AOM, MLST was the same for old and new 35B strains (type 558). 2149 genes were consistently identified among 39 strains of 35B with WGS. Specifically WGS alignments revealed a 20 kb region encoding 30 proteins that was inverted in the old 35B (2 strains) strains compared to the new 35B (3 strains). Further analysis is ongoing.

Conclusions

35B has emerged as a dominant otopathogen in recent years among children in Rochester NY. Increased virulence appears associated with genomic change.