Insertion Sequence�CDriven Diversification Creates a Globally Dispersed Emerging Multiresistant Subspecies of E. faecium

Enterococcus faecium, an ubiquous colonizer of humans and animals, has evolved in the last 15 years from an avirulent commensal to the third most frequently isolated nosocomial pathogen among intensive care unit patients in the United States. E. faecium combines multidrug resistance with the potential of horizontal resistance gene transfer to even more pathogenic bacteria. Little is known about the evolution and virulence of E. faecium, and genomic studies are hampered by the absence of a completely annotated genome sequence. To further unravel its evolution, we used a mixed whole-genome microarray and hybridized 97 E. faecium isolates from different backgrounds (hospital outbreaks (n = 18), documented infections (n = 34) and asymptomatic carriage of hospitalized patients (n = 15), and healthy persons (n = 15) and animals (n = 21)). Supported by Bayesian posterior probabilities (PP = 1.0), a specific clade containing all outbreak-associated strains and 63% of clinical isolates was identified. Sequencing of 146 of 437 clade-specific inserts revealed mobile elements (n = 74), including insertion sequence (IS) elements (n = 42), phage genes (n = 6) and plasmid sequences (n = 26), hypothetical (n = 58) and membrane proteins (n = 10), and antibiotic resistance (n = 9) and regulatory genes (n = 11), mainly located on two contigs of the unfinished E. faecium DO genome. Split decomposition analysis, varying guanine cytosine content, and aberrant codon adaptation indices all supported acquisition of these genes through horizontal gene transfer with IS16 as the predicted most prominent insert (98% sensitive, 100% specific). These findings suggest that acquisition of IS elements has facilitated niche adaptation of a distinct E. faecium subpopulation by increasing its genome plasticity. Increased genome plasticity was supported by higher diversity indices (ratio of average genetic similarities of pulsed-field gel electrophoresis and multi locus sequence typing) for clade-specific isolates. Interestingly, the previously described multi locus sequence typing�Cbased clonal complex 17 largely overlapped with this clade. The present data imply that the global emergence of E. faecium, as observed since 1990, represents the evolution of a subspecies with a presumably better adaptation than other E. faecium isolates to the constraints of a hospital environment.

屎肠球菌（Enterococcus faecium）是人和动物体内普遍存在的定植共生菌，在过去15年间已从无致病性的共生菌株演变为美国重症监护病房患者中分离率位列第三的医院感染病原体。屎肠球菌兼具多重耐药性与向致病性更强的细菌水平转移耐药基因的能力。目前学界对屎肠球菌的演化机制与致病机理知之甚少，而完整注释基因组序列的缺失极大阻碍了相关基因组学研究的开展。为进一步阐明该菌的演化历程，本研究采用混合全基因组微阵列技术，对97株不同来源的屎肠球菌分离株进行杂交实验，涵盖医院暴发株（n=18）、确诊感染株（n=34）、住院患者无症状定植株（n=15）、健康人群定植株（n=15）以及动物来源株（n=21）。在贝叶斯后验概率（Bayesian posterior probabilities, PP=1.0）的支持下，研究团队鉴定出一个特异性进化分支，该分支包含所有医院暴发相关菌株以及63%的临床分离株。研究人员对437个分支特异性插入序列中的146个进行测序，结果显示这些序列主要分布于未完成组装的屎肠球菌DO基因组的两个重叠群（contig）上，涵盖移动遗传元件（n=74，其中插入序列（insertion sequence, IS）元件n=42、噬菌体基因n=6、质粒序列n=26）、假定蛋白编码基因（n=58）与膜蛋白编码基因（n=10），以及抗生素耐药基因（n=9）与调控基因（n=11）。分裂分解分析、异常的鸟嘌呤-胞嘧啶（G+C）含量以及偏离正常的密码子适应指数，均支持这些基因通过水平基因转移获得，其中IS16被预测为最关键的插入元件（灵敏度98%，特异性100%）。上述研究结果表明，插入序列元件的获取通过提升基因组可塑性，促进了特定屎肠球菌亚群的生态位适应。分支特异性分离株的多样性指数（脉冲场凝胶电泳（pulsed-field gel electrophoresis）与多位点序列分型（multi locus sequence typing）的平均遗传相似性比值）更高，这进一步验证了基因组可塑性增强的结论。值得注意的是，此前报道的基于多位点序列分型的克隆复合群17（clonal complex 17），与该进化分支高度重合。本研究数据提示，自1990年以来全球范围内出现的屎肠球菌流行，实则代表了一个亚种的演化：该亚种相较于其他屎肠球菌分离株，对医院环境的约束条件具备更优异的适应能力。