title: Multiple mutations and increased RNA expression in tetracycline-resistant Streptococcus pneumoniae as determined by genome-wide DNA and mRNA sequencing.
abstract: OBJECTIVE
  The objective of this study was to characterize chromosomal mutations associated with resistance to tetracycline in Streptococcus pneumoniae.
  
  
  METHODS
  Chronological appearance of mutations in two S. pneumoniae R6 mutants (R6M1TC-5 and R6M2TC-4) selected for resistance to tetracycline was determined by next-generation sequencing. A role for the mutations identified was confirmed by reconstructing resistance to tetracycline in a S. pneumoniae R6 WT background. RNA sequencing was performed on R6M1TC-5 and R6M2TC-4 and the relative expression of genes was reported according to R6. Differentially expressed genes were classified according to their ontology.
  
  
  RESULTS
  WGS of R6M1TC-5 and R6M2TC-4 revealed mutations in the gene rpsJ coding for the ribosomal protein S10 and in the promoter region and coding sequences of the ABC genes patA and patB. These cells were cross-resistant to ciprofloxacin. Resistance reconstruction confirmed a role in resistance for the mutations in rpsJ and patA. Overexpression of the ABC transporter PatA/PatB or mutations in the coding sequence of patA contributed to resistance to tetracycline, ciprofloxacin and ethidium bromide, and was associated with a decreased accumulation of [(3)H]tetracycline. Comparative transcriptome profiling of the resistant mutants further revealed that, in addition to the overexpression of patA and patB, several genes of the thiamine biosynthesis and salvage pathway were increased in the two mutants, but also in clinical isolates resistant to tetracycline. This overexpression most likely contributes to the tetracycline resistance phenotype.
  
  
  CONCLUSIONS
  The combination of genomic and transcriptomic analysis coupled to functional studies has allowed the discovery of novel tetracycline resistance mutations in S. pneumoniae.
authors: Centre de recherche en Infectiologie du Centre de recherche du CHU de Québec and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada.
authors: Andréanne
authors: A
authors: Lupien
authors: Centre de recherche en Infectiologie du Centre de recherche du CHU de Québec and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada.
authors: Hélène
authors: H
authors: Gingras
authors: Centre de recherche en Infectiologie du Centre de recherche du CHU de Québec and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada.
authors: Michel G
authors: MG
authors: Bergeron
authors: Centre de recherche en Infectiologie du Centre de recherche du CHU de Québec and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada.
authors: Philippe
authors: P
authors: Leprohon
authors: Centre de recherche en Infectiologie du Centre de recherche du CHU de Québec and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada marc.ouellette@crchul.ulaval.ca.
authors: Marc
authors: M
authors: Ouellette
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D013296:Streptococcus pneumoniae / Q000187:drug effects* / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology*; D013753:Tetracycline Resistance
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D013752:Tetracycline
keywords: ABC transporters; RNA-seq; S. pneumoniae; genomic; rpsJ; thiamine