Draft Genome Sequences of Five Multilocus Sequence Types of Nonencapsulated Streptococcus pneumoniae.

Nonencapsulated Streptococcus pneumoniae can colonize the human nasopharynx and cause conjunctivitis and otitis media. Different deletions in the capsular polysaccharide biosynthesis locus and different multilocus sequence types have been described for nonencapsulated strains. Draft genome sequences were generated to provide insight into the genomic diversity of these strains.

GENOME ANNOUNCEMENT

Colonization of the human nasopharynx by Streptococcus pneumoniae is a prerequisite for pneumococcal pneumonia, meningitis, and otitis media (1, 2). More than 50% of children under five years old in the United States have been colonized by S. pneumoniae (3, 4). The incidence of invasive pneumococcal disease (IPD) has significantly decreased since the introduction of conjugate vaccines based on seven, and more recently, 13 capsular polysaccharides (5, 6). However, the incidences of carriage and IPD have increased for some capsular serotypes not covered by the conjugate vaccines (7, 8), and the incidence of carriage has increased for nonencapsulated and nontypeable pneumococci (9, 10).

Some nonencapsulated strains possess nonsense mutations in the capsular polysaccharide biosynthesis locus, cps (11). However, Park et al. (12) identified two structural variations in nonencapsulated S. pneumoniae called null capsule clades (NCCs), which are caused by deletions of cps. In one of these variations, the cps genes are replaced by the gene for pneumococcal surface protein K, which is involved in murine nasal colonization (12, 13). Using multilocus sequence typing, nonencapsulated strains are found to belong to different sequence types (STs) and different clonal complexes, suggesting that they are not all closely related (12).

Five nonencapsulated strains of S. pneumoniae, representing different cps deletions and STs, were selected for whole-genome sequencing: S. pneumoniae MNZ11b, a subline of MNZ11 (12), and S. pneumoniae MNZ37 (NCC1; ST8966 and ST1106); S. pneumoniae MNZ14 and MNZ85 (NCC2a; ST448 and ST2315); and S. pneumoniae MNZ41 (NCC2b; ST6153). Genomic DNA was isolated with a DNeasy kit (Qiagen), and double-stranded DNA (dsDNA) was quantified with a Qubit fluorometric assay (Invitrogen). Multiplexed paired-end libraries (2 × 150 bp) were prepared using a Nextera XT DNA sample preparation kit (Illumina). Sequencing was done with an Illumina MiSeq. CLC Genomics Workbench v6.0 software was used for quality trimming of the reads and de novo assembly.

An average of 7.86 million paired-end reads per strain was collected. The number of scaffolds, scaffold N50, and total sequence length, respectively, for each strain were as follows: 54, 73.44 kb, and 2.02 Mb for MNZ11b; 72, 61.10 kb, and 2.13 Mb for MNZ14; 66, 50.17 kb, and 1.95 Mb for MNZ37; 97, 38.75 kb, and 2.13 Mb for MNZ41; and 97, 41.84 kb, and 2.15 Mb for MNZ85. Each scaffold from each strain had an average coverage of >146×. Open reading frames were identified and annotated with the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (14). The number of putative protein-coding genes and tRNAs, respectively, detected for each strain were as follows: 2,090 and 29 for MNZ11b, 2,167 and 33 for MNZ14, 1,997 and 31 for MNZ37, 2,185 and 27 for MNZ41, and 2,190 and 15 for MNZ85. The 5S, 16S, and 23S rRNA genes for each strain were assembled into a single contig, so consensus sequences for these genes were reported.

These genome sequences represent a diverse sample of nonencapsulated pneumococci. Previously reported deletions of cps (12) are confirmed by these sequences. This additional sequence information may aid future vaccine designs that may become necessary as nonencapsulated and other pneumococcal populations respond to the ecological effects of the current conjugate vaccines.

Nucleotide sequence accession numbers.

The GenBank accession no. for version 1 of these sequences are ASJF00000000, ASJO00000000, ASJP00000000, ASJQ00000000, and ASJW00000000 for strains MNZ85, MNZ14, MNZ37, MNZ41, and MNZ11b, respectively.