Table_3_Comparative Genomic Analysis of 19 Clinical Isolates of Tigecycline-Resistant Acinetobacter baumannii.DOCX

To assess the genomic profiles of tigecycline (Tgc)-resistant Acinetobacter baumannii, including antibiotic resistance (AR) genes and virulence factors (VF), whole-genome shotgun sequencing was performed on 19 Tgc-resistant (TgcR) A. baumannii strains collected in a tertiary hospital during the early phase of the clinical introduction of Tgc in China from late 2012 to mid-2014. The major sample types containing TgcR strains were sputum and drain fluid. Data from an average of 624 Mbp of sequence was generated on each bacterial genome, with Q30 quality of 90%, and an average coverage of 96.6%. TCDC-AB0715 was used as a reference genome. The genome sequences were annotated for functional elements including AR genes, VFs, genome islands, and inserted sequences before they were comparatively analyzed. The antibiotic susceptibility phenotypes of the strains were examined by a broth microdilution method to determine the minimal inhibitory concentration (MIC) of strains against major clinical antibiotics. The AR genes (ARGs) were annotated using the Comprehensive Antibiotic Resistance Database (CARD). Thirty-three ARGs were shared by all 19 TgcR strains, and 24 ARGs were distributed differently among strains. A total of 391 VFs were found to be diversely distributed in all TgcR strains. Based on ARG number distribution, the 19 TgcR strains were divided into several groups. Highly differentiated genes included gpi, mphG, armA, msrE, adec, catB8, aadA, sul1, blaOXA–435, aph3i, and blaTEM–1, which may represent gene markers for TgcR A. baumannii sub-types. In addition, when compared with Tgc-sensitive (TgcS) strains collected during the same period, TgcR strains featured enrichment of ARGs including aph6id, aph3ib, and teta. Compared with 26 other whole-genome sequences of A. baumannii deposited in GeneBank, TgcR strains in this study commonly lacked the EF-Tu mutation for elfamycin resistance. Previous investigation of three A. baumannii strains isolated from one patient indicated genomic exchange and a homologous recombination event associated with generation of tigecycline resistance. This study further analyzed additional TgcR strains. Phylogenetic analysis revealed a close evolutionary relationship between 19 TgcR strains and to isolates in East and Northeast China. In short, the comprehensive functional and comparative genomic analysis of 19 clinical TgcR A. baumannii strains isolated in the early stage of Tgc usage in China revealed their close phylogenetic relationship yet variable genetic background involving multiple resistance mechanisms. Using a simple ARG or VF gene number diversity method and marker genes, TgcR strain sub-types can be identified. The distinct characteristics of TgcR A. baumannii strains with versatile genomic resistance and regulation patterns raise concern regarding prediction and control of Tgc resistance in the clinic.

为评估替加环素（Tgc）耐药鲍曼不动杆菌的基因组特征，包括抗生素耐药基因（AR）和致病性因子（VF），对2012年末至2014年中期在中国临床引入Tgc的早期阶段，从一家三级医院收集的19株Tgc耐药（TgcR）鲍曼不动杆菌菌株进行了全基因组鸟枪法测序。含有TgcR菌株的主要样本类型包括痰液和引流液。每个细菌基因组生成了平均624兆碱基对的序列数据，Q30质量达到90%，平均覆盖率为96.6%。TCDC-AB0715被用作参考基因组。在基因组序列进行比较分析之前，对功能元件进行了注释，包括AR基因、VF、基因组岛和插入序列。通过肉汤微量稀释法检查菌株的抗生素敏感性表型，以确定菌株对主要临床抗生素的最小抑菌浓度（MIC）。使用综合抗生素耐药数据库（CARD）对AR基因（ARGs）进行了注释。所有19株TgcR菌株共享33个ARGs，而24个ARGs在菌株间分布不同。在所有TgcR菌株中发现了391个VF，分布各异。根据ARG数量分布，将19株TgcR菌株分为几个组。高度分化的基因包括gpi、mphG、armA、msrE、adec、catB8、aadA、sul1、blaOXA-435、aph3i和blaTEM-1，这些可能代表TgcR鲍曼不动杆菌亚型的基因标记。此外，与同期收集的Tgc敏感（TgcS）菌株相比，TgcR菌株富含aph6id、aph3ib和teta等ARGs。与GeneBank中存储的26个其他鲍曼不动杆菌全基因组序列相比，本研究中的TgcR菌株普遍缺乏与elfamycin耐药性相关的EF-Tu突变。先前对一位患者分离的三个鲍曼不动杆菌菌株的研究表明，与替加环素耐药性的产生相关的基因组交换和同源重组事件。本研究进一步分析了额外的TgcR菌株。系统发育分析揭示了19株TgcR菌株与东中和东北地区的分离株之间紧密的进化关系。简而言之，对19株在中国Tgc使用早期阶段分离的临床TgcR鲍曼不动杆菌菌株进行的综合功能和比较基因组分析，揭示了它们紧密的进化关系和涉及多种耐药机制的可变遗传背景。通过简单的ARG或VF基因数量多样性方法和标记基因，可以识别TgcR菌株亚型。具有多变基因组耐药和调控模式的TgcR鲍曼不动杆菌菌株的独特特征，引起了临床预测和控制替加环素耐药性的关注。