Weber Robert E, Fuchs Stephan, Layer Franziska, Sommer Anna, Bender Jennifer K, Thürmer Andrea, Werner Guido, Strommenger Birgit
Department of Infectious Diseases, Robert Koch-Institute, Wernigerode, Germany.
Methodology and Research Infrastructure, Genome Sequencing, Robert Koch-Institute, Berlin, Germany.
Front Microbiol. 2021 Feb 15;12:639660. doi: 10.3389/fmicb.2021.639660. eCollection 2021.
As next generation sequencing (NGS) technologies have experienced a rapid development over the last decade, the investigation of the bacterial genetic architecture reveals a high potential to dissect causal loci of antibiotic resistance phenotypes. Although genome-wide association studies (GWAS) have been successfully applied for investigating the basis of resistance traits, complex resistance phenotypes have been omitted so far. For this especially refers to antibiotics of last resort like daptomycin and ceftaroline. Therefore, we aimed to perform GWAS for the identification of genetic variants associated with DAP and CPT resistance in clinical isolates.
MATERIALS/METHODS: To conduct microbial GWAS, we selected cases and controls according to their clonal background, date of isolation, and geographical origin. Association testing was performed with PLINK and SEER analysis. By using analysis, we also searched for rare genetic variants in candidate loci that have previously been described to be involved in the development of corresponding resistance phenotypes.
GWAS revealed MprF P314L and L826F to be significantly associated with DAP resistance. These mutations were found to be homogenously distributed among clonal lineages suggesting convergent evolution. Additionally, rare and yet undescribed single nucleotide polymorphisms could be identified within and putative candidate genes. Finally, we could show that each DAP resistant isolate exhibited at least one amino acid substitution within the open reading frame of . Due to the presence of strong population stratification, no genetic variants could be associated with CPT resistance. However, the investigation of the staphylococcal cassette chromosome (SCC) revealed various SNPs to be putatively linked with CPT resistance. Additionally, some CPT resistant isolates revealed no mutations, supporting the hypothesis that further and still unknown resistance determinants are crucial for the development of CPT resistance in .
We hereby confirmed the potential of GWAS to identify genetic variants that are associated with antibiotic resistance traits in However, precautions need to be taken to prevent the detection of spurious associations. In addition, the implementation of different approaches is still essential to detect multiple forms of variations and mutations that occur with a low frequency.
在过去十年中，随着下一代测序（NGS）技术的迅速发展，对细菌遗传结构的研究显示出剖析抗生素耐药表型因果位点的巨大潜力。尽管全基因组关联研究（GWAS）已成功应用于研究耐药性状的基础，但复杂的耐药表型至今仍被忽视。这尤其适用于达托霉素和头孢洛林等最后手段的抗生素。因此，我们旨在进行GWAS，以鉴定临床分离株中与达托霉素（DAP）和头孢洛林（CPT）耐药相关的基因变异。
材料/方法：为了进行微生物GWAS，我们根据克隆背景、分离日期和地理来源选择病例和对照。使用PLINK和SEER分析进行关联测试。通过分析，我们还在先前描述的与相应耐药表型发展相关的候选基因座中搜索罕见的基因变异。
GWAS显示MprF P314L和L826F与DAP耐药显著相关。这些突变在克隆谱系中均匀分布，表明趋同进化。此外，在候选基因和假定的候选基因中可以鉴定出罕见且尚未描述的单核苷酸多态性。最后，我们可以证明每个DAP耐药分离株在的开放阅读框内至少表现出一个氨基酸替换。由于存在强烈的群体分层，没有基因变异与CPT耐药相关。然而，对葡萄球菌盒式染色体（SCC）的研究揭示了各种SNP可能与CPT耐药相关。此外，一些CPT耐药分离株未显示突变，支持了进一步的未知耐药决定因素对CPT耐药发展至关重要的假设。
我们在此证实了GWAS在鉴定与抗生素耐药性状相关的基因变异方面的潜力。然而，需要采取预防措施以防止检测到虚假关联。此外，实施不同方法对于检测低频发生的多种形式的变异和突变仍然至关重要。
