Comparative genomic analysis of multidrug-resistant Streptococcus pneumoniae isolates

Abstract

Introduction: Multidrug resistance in Streptococcus pneumoniae has emerged as a serious problem to public health. A further understanding of the genetic diversity in antibiotic-resistant S. pneumoniae isolates is needed.

Methods: We conducted whole-genome resequencing for 25 pneumococcal strains isolated from children with different antimicrobial resistance profiles. Comparative analysis focus on detection of single-nucleotide polymorphisms (SNPs) and insertions and deletions (indels) was conducted. Moreover, phylogenetic analysis was applied to investigate the genetic relationship among these strains.

Results: The genome size of the isolates was ~2.1 Mbp, covering >90% of the total estimated size of the reference genome. The overall G+C% content was ~39.5%, and there were 2,200-2,400 open reading frames. All isolates with different drug resistance profiles harbored many indels (range 131-171) and SNPs (range 16,103-28,128). Genetic diversity analysis showed that the variation of different genes were associated with specific antibiotic resistance. Known antibiotic resistance genes (pbps, murMN, ciaH, rplD, sulA, and dpr) were identified, and new genes (regR, argH, trkH, and PTS-EII) closely related with antibiotic resistance were found, although these genes were primarily annotated with functions in virulence as well as carbohydrate and amino acid transport and metabolism. Phylogenetic analysis unambiguously indicated that isolates with different antibiotic resistance profiles harbored similar genetic backgrounds. One isolate, 14-LC.ER1025, showed a much weaker phylogenetic relationship with the other isolates, possibly caused by genomic variation.

Conclusion: In this study, although pneumococcal isolates had similar genetic backgrounds, strains were diverse at the genomic level. These strains exhibited distinct variations in their indel and SNP compositions associated with drug resistance.

Keywords: SNPs; Streptococcus pneumoniae; antimicrobial resistance; insertions/deletions; phylogenetic analysis; whole-genome sequencing.

Figure 1

Phylogenetic relationships of S. pneumoniae isolates based on single-nucleotide polymorphisms from whole DNA sequences. Notes: A tree representing the isolates. The branch lengths show the evolutionary distances among the isolates. The bootstrap values of the nodes represent the reliability of a branch being formed by all isolates in this branch, and values >70% are considered to be reliable. Resistant profiles: 14LC.ER1023-25, PEN-CXM-CRO-MAC–SXT; 14LC. ER1026-28, CXM-CRO-MAC-SXT; 14LC.ER1029-31, CXM-CRO-MAC; 14LC.ER1032-34, CXM-MAC-SXT; 14LC.ER1035-37, CXM-MAC; 14LC.ER1038-40, MAC-SXT; 14LC.ER1041-43, MAC; and 14LC.ER1044-47, NONE. Abbreviations: PEN, penicillin; CXM, cefuroxime; CRO, ceftriaxone; MAC, macrolides (erythromycin and azithromycin) and clindamycin; SXT, sulfamethoxazole–trimethoprim.

Figure 2

Principle component analysis of the whole genomes of 25 pneumococcal isolates. Notes: MA, PEN-CXM-CRO-MAC–SXT; MB, CXM-CRO-MAC-SXT; MC, CXM-CRO-MAC; MD, CXM-MAC-SXT; ME, CXM-MAC; MF, MAC-SXT; MG, MAC; and CK, NONE. Abbreviations: PEN, penicillin; CXM, cefuroxime; CRO, ceftriaxone; MAC, macrolides (erythromycin and azithromycin) and clindamycin; SXT, sulfamethoxazole–trimethoprim.

Figure 3

Indel and SNP variation in the complete genome of S. pneumoniae R6 compared with 25 S. pneumoniae clinical isolates. Notes: The distribution of nonsynonymous mutations (SNPs and indels) in the genome sequences of 25 isolates compared with the S. pneumoniae R6 reference strain is shown. Protein-coding genes on the sense and anti-sense strands are shown in rings II and III (from inside to outside). The arrow directions indicate the translational directions. The distribution of nonsynonymous mutations in the genomes of 25 isolates (from isolate 14LC-ER1023 to 14LC-ER1047) are shown from ring IV to ring XXVII. Green bars represent nonsynonymous SNPs; red bars represent nonsynonymous indels. Abbreviation: SNP, single-nucleotide polymorphisms.

Figure 4

(A) SNPs changes in PBPs associated with penicillin resistance in S. pneumoniae. (B) SNPs changes in PBPs associated with cephalosporins resistance in S. pneumoniae. (C) SNP changes associated with sulfamethoxazole–trimethoprim resistance in S. pneumonia. Abbreviations: SNPs, single-nucleotide polymorphisms; PBPs, penicillin-binding proteins; PEN, penicillin; CXM, cefuroxime; CRO, ceftriaxone; SXT, sulfamethoxazole–trimethoprim.