Genomic analyses of DNA transformation and penicillin resistance in Streptococcus pneumoniae clinical isolates

Abstract

Alterations in penicillin-binding proteins, the target enzymes for β-lactam antibiotics, are recognized as primary penicillin resistance mechanisms in Streptococcus pneumoniae. Few studies have analyzed penicillin resistance at the genome scale, however, and we report the sequencing of S. pneumoniae R6 transformants generated while reconstructing the penicillin resistance phenotypes from three penicillin-resistant clinical isolates by serial genome transformation. The genome sequences of the three last-level transformants T2-18209, T5-1983, and T3-55938 revealed that 16.2 kb, 82.7 kb, and 137.2 kb of their genomes had been replaced with 5, 20, and 37 recombinant sequence segments derived from their respective parental clinical isolates, documenting the extent of DNA transformation between strains. A role in penicillin resistance was confirmed for some of the mutations identified in the transformants. Several multiple recombination events were also found to have happened at single loci coding for penicillin-binding proteins (PBPs) that increase resistance. Sequencing of the transformants with MICs for penicillin similar to those of the parent clinical strains confirmed the importance of mosaic PBP2x, -2b, and -1a as a driving force in penicillin resistance. A role in resistance for mosaic PBP2a was also observed for two of the resistant clinical isolates.

FIG 1

Scaled representation of the S. pneumoniae R6 genome showing recombinant sequence segments acquired from CCRI-18209 (A), CCRI-1983 (B), and 55938 (C). The background genome is shown as a gray circle. The inner circle indicates chromosomal positions in a clockwise fashion. Recombinant sequence segments are shown as colored bands according to the level of transformation at which they were acquired, as follows: white bands with gray labels, T1 transformants; black bands and nonboxed black labels, T2 transformants; green bands and labels, T3 transformants; blue bands and labels, T4 transformants; red bands and labels, T5 transformants; black-boxed labels, recombinant sequence segments acquired as two partial segments during two distinct transformation steps. Asterisks indicate spr0304 (pbp2x), spr0329 (pbp1a), spr1517 (pbp2b), spr1823 (pbp2a), and spr1239 alpha-amylase.

FIG 2

Multiple recombination events occurred at pbp2x and pbp2b loci during selection for increased penicillin resistance in the transformant series derived from S. pneumoniae CCRI-18209 (A), CCRI-1983 (B), and 55938 (C). Recombinant sequence segments are shown as colored bands according to the level of transformation at which they were acquired by S. pneumoniae R6. White bands were acquired by T1 transformants, black bands were acquired by T2 transformants, and red bands were acquired by T5 transformants. Gray bands and dashed lines represent sequences from the recipient S. pneumoniae R6 genome. Genes located within recombinant sequence segments are represented by gray arrows. Numbering on top of bars indicates S. pneumoniae R6 chromosome coordinates in increments of 1 kb. Numbering below bars indicates nucleotide positions of penicillin-binding protein genes and the positions within the genes corresponding to the junctions between distinct recombinant sequence segments.

FIG 3

Distribution of the lengths of the 62 recombinant sequence segments transferred to the last-level transformants T2-18209, T3-55938, and T5-1983 from their respective parent clinical isolates. The lengths of multigene recombinant sequence segments and of single (or partial) gene units are indicated on the y axis.