Centre de recherche en Infectiologie du Centre de recherche du CHUL and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, Canada.
J Antimicrob Chemother. 2013 Aug;68(8):1718-27. doi: 10.1093/jac/dkt113. Epub 2013 Apr 21.
To identify non-penicillin-binding protein (PBP) mutations contributing to resistance to the third-generation cephalosporin cefotaxime in Streptococcus pneumoniae at the genome-wide scale.
The genomes of two in vitro S. pneumoniae cefotaxime-resistant isolates and of two transformants serially transformed with the genomic DNA of cefotaxime-resistant mutants were determined by next-generation sequencing. A role in cefotaxime resistance for the mutations identified was confirmed by reconstructing resistance in a cefotaxime-susceptible background.
Analysis of the genome assemblies revealed mutations in genes coding for the PBPs 2x, 2a and 3, of which pbp2x was the only mutated gene common to all mutants. The transformation of altered PBP alleles into S. pneumoniae R6 confirmed the role of PBP mutations in cefotaxime resistance, but these were not sufficient to fully explain the levels of resistance. Thirty-one additional genes were found to be mutated in at least one of the four sequenced genomes. Non-PBP resistance determinants appeared to be mostly lineage specific. Mutations in spr1333, spr0981, spr1704 and spr1098, encoding a peptidoglycan N-acetylglucosamine deacetylase, a glycosyltransferase, an ABC transporter and a sortase, respectively, were implicated in resistance by transformation experiments and allowed the reconstruction of the full level of resistance observed in the parent resistant strains.
This whole-genome analysis coupled to functional studies has allowed the discovery of both known and novel cefotaxime resistance genes in S. pneumoniae.
在全基因组范围内鉴定导致肺炎链球菌对第三代头孢菌素头孢噻肟耐药的非青霉素结合蛋白（PBP）突变。
通过下一代测序技术，对两株体外头孢噻肟耐药肺炎链球菌分离株和两株连续转化头孢噻肟耐药突变体基因组 DNA 的转化子的基因组进行了测序。通过在头孢噻肟敏感背景下重建耐药性，证实了所鉴定的突变在头孢噻肟耐药中的作用。
对基因组组装的分析显示，编码 PBPs 2x、2a 和 3 的基因发生了突变，其中 pbp2x 是所有突变体共有的唯一突变基因。将改变的 PBP 等位基因转化为肺炎链球菌 R6 证实了 PBP 突变在头孢噻肟耐药中的作用，但这些突变不足以完全解释耐药水平。在四个测序基因组中，至少有一个基因组中发现了 31 个额外的基因突变。非 PBP 耐药决定因素似乎主要是谱系特异性的。spr1333、spr0981、spr1704 和 spr1098 基因突变，分别编码肽聚糖 N-乙酰葡萄糖胺脱乙酰酶、糖基转移酶、ABC 转运蛋白和分选酶，转化实验表明这些突变与耐药有关，并允许重建在亲本耐药菌株中观察到的全部耐药水平。
这项全基因组分析与功能研究相结合，发现了肺炎链球菌中已知和新的头孢噻肟耐药基因。
