Long Persistence of a Streptococcus pneumoniae 23F Clone in a Cystic Fibrosis Patient

Abstract

Streptococcus pneumoniae isolates of serotype 23F with intermediate penicillin resistance were recovered on seven occasions over a period of 37 months from a cystic fibrosis patient in Berlin. All isolates expressed the same multilocus sequence type (ST), ST10523. The genome sequences of the first and last isolates, D122 and D141, revealed the absence of two phage-related gene clusters compared to the genome of another ST10523 strain, D219, isolated earlier at a different place in Germany. Genomes of all three strains carried the same novel mosaic penicillin-binding protein (PBP) genes, pbp2x, pbp2b, and pbp1a; these genes were distinct from those of other penicillin-resistant S. pneumoniae strains except for pbp1a of a Romanian S. pneumoniae isolate. All PBPs contained mutations that have been associated with the penicillin resistance phenotype. Most interestingly, a mosaic block identical to an internal pbp2x sequence of ST10523 was present in pbp2x of Streptococcus mitis strain B93-4, which was isolated from the same patient. This suggests interspecies gene transfer from S. pneumoniae to S. mitis within the host. Nearly all genes expressing surface proteins, which represent major virulence factors of S. pneumoniae and are typical for this species, were present in the genome of ST10523. One exception was the hyaluronidase gene hlyA, which contained a 12-nucleotide deletion within the promoter region and an internal stop codon. The lack of a functional hyaluronidase might contribute to the ability to persist in the host for an unusually long period of time. IMPORTANCEStreptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can result in severe diseases due to a unique repertoire of pathogenicity factors that are rare in closely related commensal streptococci. We investigated a penicillin-resistant S. pneumoniae clone of serotype 23F isolated from a cystic fibrosis patient on multiple occasions over an unusually long period of over 3 years that was present without causing disease. Genome comparisons revealed an apparent nonfunctional pneumococcus-specific gene encoding a hyaluronidase, supporting the view that this enzyme adds to the virulence potential of the bacterium. The 23F clone harbored unique mosaic genes encoding penicillin resistance determinants, the product of horizontal gene transfer involving the commensal S. mitis as donor species. Sequences identical to one such mosaic gene were identified in an S. mitis strain from the same patient, suggesting that in this case S. pneumoniae played the role of donor.

Keywords: 23F clone; Streptococcus pneumoniae; cystic fibrosis; hyaluronidase; penicillin-binding proteins; persistence.

FIG 1

Schematic representation of the mosaic genes for PBP2x, PBP1a, and PBP2b from S. pneumoniae and S. mitis strains. (A) Mosaic sequence blocks in PBP2x. PBP2x genes of ST10523 (S. pneumoniae strains D122, D139, D141, and D219) are identical; pbp2x of S. pneumoniae Spain^23F-1 is included for comparison. The pbp2x gene of S. pneumoniae HMC3243 from Romania, which is partially identical to pbp2x of ST10523, was also included. The colors indicate closely related sequence portions with >95% identity to pbp2x reference sequences from penicillin-sensitive S. mitis (Sm.) strains M3, 658, NCTC10712, and SV01 and S. oralis (So) ATCC 35307 (76), as indicated by the color coded boxes. White indicates sequences related to the penicillin-sensitive strain S. pneumoniae R6, and gray sequences are highly divergent from R6 sequences and other pbp2x reference sequences. The domain structure of PBP2x with the three homology boxes is shown above; the central penicillin-binding domain is indicated by the black bar. Black arrows represent mutations A₃₃₈ and E₅₅₂ in pbp2x. The regions that are identical between pbp2x of ST10523, S. mitis B93-4, and S. pneumoniae HMC3243 are indicated by the shaded area. (B) Deduced amino acid sequences of the transpeptidase domain of PBP2x; PBP2x of S. pneumoniae R6 was used as a reference. The positions of the amino acids are indicated vertically in the top three rows. Only positions with altered residues are shown; residues identical to those in strain R6 are indicated by dots. The mutations A₃₃₈ and E₅₅₂ are highlighted by black arrowheads; white arrowheads indicate amino acids which are not present in PBP2x of the penicillin-sensitive reference strains (see text for details).

FIG 2

The HlyA gene of S. pneumoniae R6 and ST10523. The HlyA gene of S. pneumoniae R6, including upstream sequences (57 nt) to codon 72, is indicated. The −35 region, the −10 region, and the start codon ATG are underlined. The deduced amino acid sequence is indicated below. In the hlyA region of D219 (ST10523), only nucleotides and deduced amino acids that differ from the reference R6 sequence are shown. The 12-nt deletion upstream of the ATG start codon and the 4-nt deletion in the D219 sequence are indicated by open arrows. Vertical numbers in the first three rows refer to codons; numbers 1, 2, and 3 in the fourth row indicate the first, second, and third positions in the respective codon.