Project description:Serotype 1 Streptococcus pneumoniae is a leading cause of invasive pneumococcal disease (IPD) worldwide, with the highest burden in developing countries. We report the whole-genome sequencing analysis of 448 serotype 1 isolates from 27 countries worldwide (including 11 in Africa). The global serotype 1 population shows a strong phylogeographic structure at the continental level, and within Africa there is further region-specific structure. Our results demonstrate that region-specific diversification within Africa has been driven by limited cross-region transfer events, genetic recombination and antimicrobial selective pressure. Clonal replacement of the dominant serotype 1 clones circulating within regions is uncommon; however, here we report on the accessory gene content that has contributed to a rare clonal replacement event of ST3081 with ST618 as the dominant cause of IPD in the Gambia.
Project description:Streptococcus pneumoniae strains comprise >90 serotypes. Here we describe establishment of a MassTag PCR assay designed to serotype S. pneumoniae and demonstrate its utility in tests using 31 paired lung aspirate and nasopharyngeal aspirate samples from children with pneumonia in the Gambia. Serotypes 1, 5, and 14 in were implicated in 90% of lung infections. With 5 exceptions, serotypes found in lung aspirates were also found in nasopharyngeal aspirates.
Project description:Streptococcus pneumoniae is a commensal human pathogen and the causative agent of various invasive and noninvasive diseases. Carriage of the pneumococcus in the nasopharynx is thought to be mediated by biofilm formation, an environment where isogenic populations frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. We employed metabolic characterization and whole-genome sequencing of biofilm-derived S. pneumoniae serotype 22F pneumococcal SCVs to investigate diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles, and increased biofilm formation compared to the ancestral strain. Whole-genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51 to 264 bp, one insertion resulting in a coding frameshift, and seven nonsense single-nucleotide substitutions that result in a truncated gene product. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae and therefore may have important implications for colonization, carriage, and persistence of the organism. Furthermore, recurrent mutation of the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development.
Project description:Streptococcus pneumoniae serotype 5 is among the most common serotypes causing invasive pneumococcal disease (IPD) in The Gambia. We anticipate that introduction of the 13-valent pneumococcal conjugate vaccine (PCV-13) into routine vaccination in The Gambia will reduce serotype 5 IPD. However, the emergence of new clones that have altered their genetic repertoire through capsular switching or genetic recombination after vaccination with PCV-13 poses a threat to this public health effort. In order to monitor for potential genetic changes post-PCV-13 vaccination, we established the baseline population structure, epidemiology, and antibiotic resistance patterns of serotype 5 before the introduction of PCV-13.Fifty-five invasive S. pneumoniae serotype 5 isolates were recovered from January 2009 to August 2011 in a population-based study in the Upper River Region of The Gambia. Serotyping was done by latex agglutination and confirmed by serotype-specific Polymerase Chain Reaction (PCR). Genotyping was undertaken using Multilocus Sequence Typing (MLST). Antimicrobial sensitivity was done using disc diffusion. Contingency table analyses were conducted using Pearson's Chi(2) and Fisher's exact test. Clustering was performed using Bionumerics version 6.5.MLST resolved S. pneumoniae serotype 5 isolates into 3 sequence types (ST), namely ST 289(6/55), ST 3339(19/55) and ST 3404(30/55). ST 289 was identified as the major clonal complex. ST 3339, the prevalent genotype in 2009 [84.6% (11/13)], was replaced by ST 3404 [70.4% (19/27)] in 2010 as the dominant ST. Interestingly, ST 3404 showed lower resistance to tetracycline and oxacillin (P < 0.001), an empirical surrogate to penicillin in The Gambia.There has been an emergence of ST 3404 in The Gambia prior to the introduction of PCV-13. Our findings provide important background data for future assessment of the impact of PCV-13 into routine immunization in developing countries, such as The Gambia.
Project description:BACKGROUND:The 13-valent pneumococcal vaccine (PCV13) was introduced in Cambodia in January 2015. There are limited data concerning the common serotypes causing invasive pneumococcal disease (IPD). Knowledge of the circulating pneumococcal serotypes is important to monitor epidemiological changes before and after vaccine implementation. METHODS:All episodes of IPD defined by the isolation of Streptococcus pneumoniae from blood, cerebrospinal fluid or other sterile site in Cambodian children admitted to the Angkor Hospital for Children in Siem Reap, Northwestern Cambodia, between 1st January 2007 and 1st July 2012 were retrospectively studied. Streptococcus pneumoniae isolates that could be retrieved underwent phenotypic typing and whole genome sequencing. RESULTS:There were 90 Cambodian children hospitalized with IPD with a median (IQR) age of 2.3 years (0.9-6.2). The case fatality was 15.6% (95% CI 8-23). Of 50 Streptococcus pneumoniae isolates available for further testing, 46% were penicillin non-susceptible and 8% were ceftriaxone non-susceptible, 78% were cotrimoxazole resistant, 30% were erythromycin resistant and 30% chloramphenicol resistant. There were no significant changes in resistance levels over the five-year period. The most common serotypes were 1 (11/50; 22%), 23F (8/50; 16%), 14 (6/50; 12%), 5 (5/50; 10%) and 19A (3/50; 6%). Coverage by PCV7, PCV10 and PCV13 was 44%, 76% and 92% respectively. We identified novel multilocus sequence types and resistotypes using whole genome sequencing. CONCLUSIONS:This study suggests IPD is an important disease in Cambodian children and can have a significant mortality. PCV13 coverage of the serotypes determined in studied strains was high and consistent with another recent study. The phenotypic resistance patterns observed were similar to other regional studies. The use of whole genome sequencing in the present study provides additional typing and resistance information together with the description of novel sequence types and resistotypes.
Project description:Small noncoding RNAs (sRNAs) play important roles in gene regulation in both prokaryotes and eukaryotes. Thus far, no sRNA has been assigned a definitive role in virulence in the major human pathogen Streptococcus pneumoniae. Based on the potential coding capacity of intergenic regions, we hypothesized that the pneumococcus produces many sRNAs and that they would play an important role in pathogenesis. We describe the application of whole-genome transcriptional sequencing to systematically identify the sRNAs of Streptococcus pneumoniae. Using this approach, we have identified 89 putative sRNAs, 56 of which are newly identified. Furthermore, using targeted genetic approaches and Tn-seq transposon screening, we demonstrate that many of the identified sRNAs have important global and niche-specific roles in virulence. These data constitute the most comprehensive analysis of pneumococcal sRNAs and provide the first evidence of the extensive roles of sRNAs in pneumococcal pathogenesis.
Project description:BACKGROUND:Whole genome sequencing has emerged as a useful tool for identification and molecular characterization of pathogens. MinION (Oxford Nanopore) is a real-time third generation sequencer whose portability, affordability and speed in data production make of it an attractive device for whole genome sequencing. The objective of this study is to evaluate MinION sequencer for pathogen identification and molecular characterization of Streptococcus pneumoniae isolated at a children's Hospital. Whole genome sequencing of 32?Streptococcus pneumoniae invasive isolates, previously characterized by standard methods (Quellung reaction, Multiplex PCR and Sanger-MLST), were performed. DNA was extracted using ZymoBIOMICS DNA Microprep kit. Quantification and purity of DNA was assessed by Qubit and Nanodrop, respectively. Library preparation was performed using the Rapid Barcoding Kit. Real-time workflow EPI2ME platform "What's it in my pot" was used for species identification. Fast5 sequences were converted into FASTQ by Albacore software. Reads were assembled using CANU software. PathogenWatch, genomic epidemiology and pubmlst online tools were used for capsular typing and/or whole genome-MLST profile. RESULTS:Rapid identification of Streptococcus pneumoniae was achieved by "What's in my pot". Capsular typing was correctly assigned with PathogenWatch in all 32 isolates at serogroup level and 24 at serotype level. Whole genome-MLST results obtained by genomic epidemiology and pubmlst were consistent with double locus variant clonal complex obtained by Sanger-MLST in 31 isolates. CONCLUSION:MinION sequencer provides a rapid, cost-effective and promising pathway for performing WGS by a pocked-sized device for epidemiological purposes but improving its sequencing accuracy will make it more appealing to be used in clinical microbiology laboratories.
Project description:Correct identifications of isolates and strains of the Mitis-Group of the genus Streptococcus are particularly difficult, due to high genetic similarity, resulting from horizontal gene transfer and homologous recombination, and unreliable phenotypic and genotypic biomarkers for differentiating the species. Streptococcus pneumoniae and Streptococcus pseudopneumoniae are the most closely related species of the clade. In this study, publicly-available genome sequences for Streptococcus pneumoniae and S. pseudopneumoniae were analyzed, using a pangenomic approach, to find candidates for species-unique gene markers; ten species-unique genes for S. pneumoniae and nine for S. pseudopneumoniae were identified. These species-unique gene marker candidates were verified by PCR assays for identifying S. pneumoniae and S. pseudopneumoniae strains isolated from clinical samples. All determined species-level unique gene markers for S. pneumoniae were detected in all S. pneumoniae clinical isolates, whereas fewer of the unique S. pseudopneumoniae gene markers were present in more than 95% of the clinical isolates. In parallel, taxonomic identifications of the clinical isolates were confirmed, using conventional optochin sensitivity testing, targeted PCR-detection for the "Xisco" gene, as well as genomic ANIb similarity analyses for the genome sequences of selected strains. Using mass spectrometry-proteomics, species-specific peptide matches were observed for four of the S. pneumoniae gene markers and for three of the S. pseudopneumoniae gene markers. Application of multiple species-level unique biomarkers of S. pneumoniae and S. pseudopneumoniae, is proposed as a protocol for the routine clinical laboratory for improved, reliable differentiation, and identification of these pathogenic and commensal species.
Project description:The bacterium Streptococcus pneumoniae is one of the leading causes of fatal infections affecting humans. Intriguingly, phylogenetic analysis shows that the species constitutes one evolutionary lineage in a cluster of the otherwise commensal Streptococcus mitis strains, with which humans live in harmony. In a comparative analysis of 35 genomes, including phylogenetic analyses of all predicted genes, we have shown that the pathogenic pneumococcus has evolved into a master of genomic flexibility while lineages that evolved into the nonpathogenic S. mitis secured harmonious coexistence with their host by stabilizing an approximately 15%-reduced genome devoid of many virulence genes. Our data further provide evidence that interspecies gene transfer between S. pneumoniae and S. mitis occurs in a unidirectional manner, i.e., from S. mitis to S. pneumoniae. Import of genes from S. mitis and other mitis, anginosus, and salivarius group streptococci ensured allelic replacements and antigenic diversification and has been driving the evolution of the remarkable structural diversity of capsular polysaccharides of S. pneumoniae. Our study explains how the unique structural diversity of the pneumococcal capsule emerged and conceivably will continue to increase and reveals a striking example of the fragile border between the commensal and pathogenic lifestyles. While genomic plasticity enabling quick adaptation to environmental stress is a necessity for the pathogenic streptococci, the commensal lifestyle benefits from stability. Importance: One of the leading causes of fatal infections affecting humans, Streptococcus pneumoniae, and the commensal Streptococcus mitis are closely related obligate symbionts associated with hominids. Faced with a shortage of accessible hosts, the two opposing lifestyles evolved in parallel. We have shown that the nonpathogenic S. mitis secured harmonious coexistence with its host by stabilizing a reduced genome devoid of many virulence genes. Meanwhile, the pathogenic pneumococcus evolved into a master of genomic flexibility and imports genes from S. mitis and other related streptococci. This process ensured antigenic diversification and has been driving the evolution of the remarkable structural diversity of capsular polysaccharides of S. pneumoniae, which conceivably will continue to increase and present a challenge to disease prevention.
Project description:The aim of our study was to investigate phenotypic and genotypic features of streptococci misidentified (misID) as Streptococcus pneumoniae, obtained over 20 years from hospital patients in Poland. Sixty-three isolates demonstrating microbiological features typical for pneumococci (optochin susceptibility and/or bile solubility) were investigated by phenotypic tests, lytA and 16S rRNA gene polymorphism and whole-genome sequencing (WGS). All isolates had a 6-bp deletion in the lytA 3' terminus, characteristic for Mitis streptococc and all but two isolates lacked the pneumococcal signature cytosine at nucleotide position 203 in the 16S rRNA genes. The counterparts of psaA and ply were present in 100% and 81.0% of isolates, respectively; the spn9802 and spn9828 loci were characteristic for 49.2% and 38.1% of isolates, respectively. Phylogenetic trees and networks, based on the multilocus sequence analysis (MLSA) scheme, ribosomal multilocus sequence typing (rMLST) scheme and core-genome analysis, clearly separated investigated isolates from S. pneumoniae and demonstrated the polyclonal character of misID streptococci, associated with the Streptococcus pseudopneumoniae and Streptococcus mitis groups. While the S. pseudopneumoniae clade was relatively well defined in all three analyses, only the core-genome analysis revealed the presence of another cluster comprising a fraction of misID streptococci and a strain proposed elsewhere as a representative of a novel species in the Mitis group. Our findings point to complex phylogenetic and taxonomic relationships among S. mitis-like bacteria and support the notion that this group may in fact consist of several distinct species.