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:Eight low-passage-number Streptococcus pneumoniae clinical isolates, each of a different serotype and a different multilocus sequence type, were obtained from pediatric participants in a pneumococcal vaccine trial. Comparative genomic analyses were performed with these strains and two S. pneumoniae reference strains. Individual genomic libraries were constructed for each of the eight clinical isolates, with an average insert size of approximately 1 kb. A total of 73,728 clones were picked for arraying, providing more than four times genomic coverage per strain. A subset of 4,793 clones were sequenced, for which homology searches revealed that 750 (15.6%) of the sequences were unique with respect to the TIGR4 reference genome and 263 (5.5%) clones were unrelated to any available streptococcal sequence. Hypothetical translations of the open reading frames identified within these novel sequences showed homologies to a variety of proteins, including bacterial virulence factors not previously identified in S. pneumoniae. The distribution and expression patterns of 58 of these novel sequences among the eight clinical isolates were analyzed by PCR- and reverse transcriptase PCR-based analyses, respectively. These unique sequences were nonuniformly distributed among the eight isolates, and transcription of these genes in planktonic cultures was detected in 81% (172/212) of their genic occurrences. All 58 novel sequences were transcribed in one or more of the clinical strains, suggesting that they all correspond to functional genes. Sixty-five percent (38/58) of these sequences were found in 50% or less of the clinical strains, indicating a significant degree of genomic plasticity among natural isolates.
Project description:BACKGROUND: Streptococcus pneumoniae serotype 14 is one of the most common pneumococcal serotypes that cause invasive pneumococcal diseases worldwide. Serotype 14 often expresses resistance to a variety of antimicrobial agents, resulting in difficulties in treatment. To gain insight into the evolution of virulence and antimicrobial resistance traits in S. pneumoniae from the genome level, we sequenced the entire genome of a serotype 14 isolate (CGSP14), and carried out comprehensive comparison with other pneumococcal genomes. Multiple serotype 14 clinical isolates were also genotyped by multilocus sequence typing (MLST). RESULTS: Comparative genomic analysis revealed that the CGSP14 acquired a number of new genes by horizontal gene transfer (HGT), most of which were associated with virulence and antimicrobial resistance and clustered in mobile genetic elements. The most remarkable feature is the acquisition of two conjugative transposons and one resistance island encoding eight resistance genes. Results of MLST suggested that the major driving force for the genome evolution is the environmental drug pressure. CONCLUSION: The genome sequence of S. pneumoniae serotype 14 shows a bacterium with rapid adaptations to its lifecycle in human community. These include a versatile genome content, with a wide range of mobile elements, and chromosomal rearrangement; the latter re-balanced the genome after events of HGT.
Project description:Linezolid is a member of a novel class of antibiotics, with resistance already being reported. We used whole-genome sequencing on three independent Streptococcus pneumoniae strains made resistant to linezolid in vitro in a step-by-step fashion. Analysis of the genome assemblies revealed mutations in the 23S rRNA gene in all mutants including, notably, G2576T, a previously recognized resistance mutation. Mutations in an additional 31 genes were also found in at least one of the three sequenced genomes. We concentrated on three new mutations that were found in at least two independent mutants. All three mutations were experimentally confirmed to be involved in antibiotic resistance. Mutations upstream of the ABC transporter genes spr1021 and spr1887 were correlated with increased expression of these genes and neighboring genes of the same operon. Gene inactivation supported a role for these ABC transporters in resistance to linezolid and other antibiotics. The hypothetical protein spr0333 contains an RNA methyltransferase domain, and mutations within that domain were found in all S. pneumoniae linezolid-resistant strains. Primer extension experiments indicated that spr0333 methylates G2445 of the 23S rRNA and mutations in spr0333 abolished this methylation. Reintroduction of a nonmutated version of spr0333 in resistant bacteria reestablished G2445 methylation and led to cells being more sensitive to linezolid and other antibiotics. Interestingly, the spr0333 ortholog was also mutated in a linezolid-resistant clinical Staphylococcus aureus isolate. Whole-genome sequencing and comparative analyses of S. pneumoniae resistant isolates was useful for discovering novel resistance mutations.
Project description:Phenotypic, genotypic, and antimicrobial characteristics of six phenotypically distinct human clinical isolates that most closely resembled the type strain of Streptococcus halichoeri isolated from a seal are presented. Sequencing of the 16S rRNA, rpoB, sodA, and recN genes; comparative whole-genome analysis; conventional biochemical and Rapid ID 32 Strep identification methods; and antimicrobial susceptibility testing were performed on the human isolates, the type strain of S. halichoeri, and type strains of closely related species. The six human clinical isolates were biochemically indistinguishable from each other and showed 100% 16S rRNA, rpoB, sodA, and recN gene sequence similarity. Comparative 16S rRNA gene sequencing analysis revealed 98.6% similarity to S. halichoeri CCUG 48324(T), 97.9% similarity to S. canis ATCC 43496(T), and 97.8% similarity to S. ictaluri ATCC BAA-1300(T). A 3,530-bp fragment of the rpoB gene was 98.8% similar to the S. halichoeri type strain, 84.6% to the S. canis type strain, and 83.8% to the S. ictaluri type strain. The S. halichoeri type strain and the human clinical isolates were susceptible to the antimicrobials tested based on CLSI guidelines for Streptococcus species viridans group with the exception of tetracycline and erythromycin. The human isolates were phenotypically distinct from the type strain isolated from a seal; comparative whole-genome sequence analysis confirmed that the human isolates were S. halichoeri. On the basis of these results, a novel subspecies, Streptococcus halichoeri subsp. hominis, is proposed for the human isolates and Streptococcus halichoeri subsp. halichoeri is proposed for the gray seal isolates. The type strain of the novel subspecies is SS1844(T) = CCUG 67100(T) = LMG 28801(T).
Project description:The major virulence factor of the pneumococcus, and target for conjugate vaccines, is the polysaccharide capsule, which is usually encoded by the highly variable cps locus. Serotype 37 is an unusual pneumococcal type in which the single ?-glucosyltransferase gene responsible for serotype capsule production (tts) is located outside of the capsular operon region. Using a previously described automated whole genome sequence (WGS)-based serotyping bioinformatics tool, PneumoCaT, we identified and investigated seven clinical isolates (three from blood cultures) of non-pneumococcal streptococci containing a highly homologous tts and included them in a study panel of 20 isolates which included a 11 further clinical isolates of S. pneumoniae serotype 37, a reference strain of serotype 37 and the S. pseudopneumoniae type strain BAA 960T. The seven non-pneumococcal isolates generated novel alleles at all pneumococcal MLST loci and gave low percentage similarity (<45%) to S. pneumoniae or S. pseudopneumoniae species by comparison of short sequence patterns in genomic data (k-mer analysis). The S. pseudopneumoniae BAA-960T isolate generated two novel alleles in the MLST and gave a high similarity (>99%) to the reference sequence for BAA-960T. Twelve isolates gave high similarity (>77%) to the Streptococcus pneumoniae 5652-06 serotype 19A reference genome sequence and had previously reported MLST alleles. Each of the seven clinical non-pneumococcal strains and all of the 12 S. pneumoniae possessed a ?-glycosyltransferase gene (tts) with >95% similarity to the pneumococcal tts reference DNA sequence with 20-22 non-synonymous SNPs. All but two strains in which the tts gene was detected gave positive reactions for serotype 37 in slide agglutination tests with serotype 37 typing sera. Phylogenetic analysis using both SNP and MLST data showed distinct clades corresponding to strains identified as pneumococcus or non-pneumococcus by kmer WGS analysis. Extended k-mer database analysis and ribosomal MLST placed the non-pneumococcal isolates within the S. mitis group. Biochemical and bile solubility assays showed differences between the unusual isolates and S. pneumoniae. All isolates had detectable pneumolysin (ply) genes, but only those that identified as pneumococcus contained the genes for autolysin (lytA) or the ABC transporter lipoprotein A (piaA) with >80% coverage and >95% similarity. Here we report the existence of a novel group of strains distinct from S. pneumoniae, but which can express a pneumococcal serotype 37 capsular polysaccharide which can be associated with clinical disease.