Commensal streptococci serve as a reservoir for β-lactam resistance genes in Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, septicemia, and middle ear infections. The incidence of S. pneumoniae isolates that are not susceptible to penicillin has risen worldwide and may be above 20% in some countries. Beta-lactam antibiotic resistance in pneumococci is associated with significant sequence polymorphism in penicillin-binding proteins (PBPs). Commensal streptococci, especially S. mitis and S. oralis, have been identified as putative donors of mutated gene fragments. However, no studies have compared sequences of the involved pbp genes in large collections of commensal streptococci with those of S. pneumoniae. We therefore investigated the sequence diversity of the transpeptidase region of the three pbp genes, pbp2x, pbp2b, and pbp1a in 107, 96, and 88 susceptible and nonsusceptible strains of commensal streptococci, respectively, at the nucleotide and amino acid levels to determine to what extent homologous recombination between commensal streptococci and S. pneumoniae plays a role in the development of beta-lactam resistance in S. pneumoniae. In contrast to pneumococci, extensive sequence variation in the transpeptidase region of pbp2x, pbp2b, and pbp1a was observed in both susceptible and nonsusceptible strains of commensal streptococci, conceivably reflecting the genetic diversity of the many evolutionary lineages of commensal streptococci combined with the recombination events occurring with intra- and interspecies homologues. Our data support the notion that resistance to beta-lactam antibiotics in pneumococci is due to sequences acquired from commensal Mitis group streptococci, especially S. mitis. However, several amino acid alterations previously linked to beta-lactam resistance in pneumococci appear to represent species signatures of the donor strain rather than being causal of resistance.

FIG 1

(a to c) Comparative analyses of pbp genes in Mitis group streptococci. Left columns show minimum evolution trees generated in MEGA of the transpeptidase region of pbp2x (a), pbp2b (b), and pbp1a (c) genes of susceptible and nonsusceptible strains of commensal streptococci and S. pneumoniae. Bootstrap values of 500 replications are shown. Clusters CX1 to CX8 (pbp2x), CB1 to CB5 (pbp2B), and CA1 to CA9 (pbp1a) identified by the BratNextgen software for detecting recombination are highlighted. In the pbp2x tree, +, −, or (−) after a strain name denotes the presence of, absence of, or a nonfunctional murM gene. The middle columns show recombination segments identified by the BratNextgen software over the length of the sequenced part of the transpeptidase region of the pbp genes. Blocks with the same color are hypothesized to be of the same origin. The numbers at the bottom of the middle columns indicate the region of the genes used for the analysis according to the positions in sequences of S. pneumoniae R6. Right columns show the susceptibility of six beta-lactam antibiotics (benzylpenicillin [pen], piperacillin [pip], oxacillin [oxa], cephalothin [cep], cefuroxime [cef], and cefotaxime [ctx]) indicated as susceptible (green), intermediate (yellow), and resistant (red) according the thresholds given by EUCAST (28) and shown in Table 2.

FIG 1

(a to c) Comparative analyses of pbp genes in Mitis group streptococci. Left columns show minimum evolution trees generated in MEGA of the transpeptidase region of pbp2x (a), pbp2b (b), and pbp1a (c) genes of susceptible and nonsusceptible strains of commensal streptococci and S. pneumoniae. Bootstrap values of 500 replications are shown. Clusters CX1 to CX8 (pbp2x), CB1 to CB5 (pbp2B), and CA1 to CA9 (pbp1a) identified by the BratNextgen software for detecting recombination are highlighted. In the pbp2x tree, +, −, or (−) after a strain name denotes the presence of, absence of, or a nonfunctional murM gene. The middle columns show recombination segments identified by the BratNextgen software over the length of the sequenced part of the transpeptidase region of the pbp genes. Blocks with the same color are hypothesized to be of the same origin. The numbers at the bottom of the middle columns indicate the region of the genes used for the analysis according to the positions in sequences of S. pneumoniae R6. Right columns show the susceptibility of six beta-lactam antibiotics (benzylpenicillin [pen], piperacillin [pip], oxacillin [oxa], cephalothin [cep], cefuroxime [cef], and cefotaxime [ctx]) indicated as susceptible (green), intermediate (yellow), and resistant (red) according the thresholds given by EUCAST (28) and shown in Table 2.

FIG 1

(a to c) Comparative analyses of pbp genes in Mitis group streptococci. Left columns show minimum evolution trees generated in MEGA of the transpeptidase region of pbp2x (a), pbp2b (b), and pbp1a (c) genes of susceptible and nonsusceptible strains of commensal streptococci and S. pneumoniae. Bootstrap values of 500 replications are shown. Clusters CX1 to CX8 (pbp2x), CB1 to CB5 (pbp2B), and CA1 to CA9 (pbp1a) identified by the BratNextgen software for detecting recombination are highlighted. In the pbp2x tree, +, −, or (−) after a strain name denotes the presence of, absence of, or a nonfunctional murM gene. The middle columns show recombination segments identified by the BratNextgen software over the length of the sequenced part of the transpeptidase region of the pbp genes. Blocks with the same color are hypothesized to be of the same origin. The numbers at the bottom of the middle columns indicate the region of the genes used for the analysis according to the positions in sequences of S. pneumoniae R6. Right columns show the susceptibility of six beta-lactam antibiotics (benzylpenicillin [pen], piperacillin [pip], oxacillin [oxa], cephalothin [cep], cefuroxime [cef], and cefotaxime [ctx]) indicated as susceptible (green), intermediate (yellow), and resistant (red) according the thresholds given by EUCAST (28) and shown in Table 2.

FIG 2

(a to c) Codons with amino acid changes in the transpeptidase region of PBP2x (a), PBP2b (b), and PBP1a (c) compared to the S. pneumoniae R6 reference strain. Only amino acids that differ from the reference sequence of the R6 strain are shown. Percentages of isolates in each group that have amino acid changes in the specified position are indicated. Amino acid positions further marked with colors or an asterisk indicate sites previously associated with resistance in S. pneumoniae and commensal streptococci (see reference 8). Orange, laboratory mutants; green, clinical isolates; purple, both laboratory mutants and clinical isolates. Amino acid alterations linked to reduced beta-lactam susceptibility in a study by Chewapreecha et al. (21) are marked with an asterisk. The color scale bar represents the percentage of isolates with altered amino acids compared to S. pneumoniae R6 in each position.