Diversity of Mosaic pbp2x Families in Penicillin-Resistant Streptococcus pneumoniae from Iran and Romania

Abstract

Penicillin-resistant Streptococcus pneumoniae strains are found at high rates in Romania and Iran. The mosaic structure of PBP2x was investigated in 9 strains from Iran and in 15 strains from Romania to understand their evolutionary history. Mutations potentially important for β-lactam resistance were identified by comparison of the PBP2x sequences with the sequence of the related PBP2x of reference penicillin-sensitive S. mitis strains. Two main PBP2x mosaic gene families were recognized. Eight Iranian strains expressed PBP2x variants in group 1, which had a mosaic block highly related to PBP2x of the Spain^23F-1 clone, which is widespread among international penicillin-resistant S. pneumoniae clones. A second unique PBP2x group was observed in Romanian strains; furthermore, three PBP2x single mosaic variants were found. Sequence blocks of penicillin-sensitive strain S. mitis 658 were common among PBP2x variants from strains from both countries. Each PBP2x group contained specific signature mutations within the transpeptidase domain, documenting the existence of distinct mutational pathways for the development of penicillin resistance.

Keywords: Streptococcus pneumoniae; mosaic genes; penicillin resistance; penicillin-binding proteins.

FIG 1

Mosaic structures of the PBP2x variants. Mosaic PBP2x structures were deduced by comparison with the reference PBP2x sequences of S. pneumoniae R6 (white sequence blocks) and S. mitis strains M3 (red sequence blocks) and 658 (green sequence blocks). Highly similar sequences (<5% difference) are shown in the same color; reference sequences are indicated by the color code at the bottom. The active-site motifs of PBP2x within the transpeptidase domain (aa 266 to 616) are shown on top; the gray-shaded areas indicate the central transpeptidase domain. Mutations at sites 338, 394, and 550, close to active-site motifs, are marked by black arrowheads. The strains are indicated on the right. Green dots, strains from Iran; red dots, strains from Romania. Highly similar variants are indicated by brackets on the right; black arrows on the left indicate single PBP2x variants. (A) PBP2x containing sequences similar to those of pbp2x of the Spain^23F-1 clone (group 1) and the single PBP2x variant RO116; (B) group 2 pbp2x and the single PBP2x variants RO67 and IR158, including related sequences of S. mitis strains B6 and 578.

FIG 2

Alignment of PBP2x sequences. Only sites of the transpeptidase domain that differ from PBP2x of S. pneumoniae R6 are shown. The vertical numbers in the first three rows indicate the amino acid positions. The asterisks above the amino acid position mark relevant mutations (see the text for details). The PBP2x variants of group 1 and group 2 are boxed. The color code for the mutations is as follows: light blue, mutations close to active-site motifs (aa 338, 394, and 550); yellow and green, signature mutations of group 1 and group 2 PBP2x, respectively; gray, mutations that occur only in particular variants. The reference sequences of S. mitis (Sm) strains M3 and 658 are shaded in pink and green, respectively. Black arrows on the right mark distinct PBP2x variants; brackets on the left indicate highly similar sequences. Green dots, strains from Iran; red dots, strains from Romania. ¹, PBP2x of the Spain^23F-1 clone is identical to PBP2x of the Spain^9V-3 clone and S. pneumoniae CGSP14 (6); ², a PBP2x variant identical to that in RO31 is present in RO58, RO61, and RO106. SA^19A-13, South Africa^19A-13 clone.

FIG 3

Positions of the signature mutations in group 1 and group 2 PBP2x variants. The structure of the transpeptidase domain of the acylated form of PBP2x of S. pneumoniae R6 with cefuroxime is shown (PDB accession number 1QMF) (20). The positions of the mutations are indicated. Light pink, site 338 close to active-site serine 337; white, active-site residues (S337, S395, and K547); red, position 371, which is common to both protein groups; yellow, the cefuroxime molecule in the active-site cavity.