Table_5_Colistin Resistant A. baumannii: Genomic and Transcriptomic Traits Acquired Under Colistin Therapy.XLSX

Even though colistin-based treatment represents the antimicrobial-regimen backbone for the management of multidrug-resistant Gram-negative infections, colistin resistance is still rare, at least as a full resistance, in Acinetobacter baumannii (Ab). We investigated the genomics and transcriptomics of two clinical Extensively Drug Resistance (XDR) colistin-susceptible/resistant (COL-S/R) Ab strain-pairs in which COL-resistance was developed after exposure to colistin therapy. The molecular characterization of the strains showed that all strains belonged to PFGE-A, ST-281, OXA-23 producers, Global Clone-II, and were resistant to imipenem, meropenem, ampicillin/sulbactam, ciprofloxacin, gentamicin, amikacin, trimethoprim/sulfamethoxazole, and susceptible to tigecycline, in agreement with NGS-acquired resistome. COL-R vs. COL-S Ab comparative genomics, mapping on Ab ATCC 17978 and Ab ACICU Reference Genomes, revealed a closely related genomic phylogeny, especially between strain-pair isolates, and distinctive common genomic non-synonymous SNPs (nsSNPs) in COL-R Ab strains. Furthermore, pmrB and pmrC nsSNPs were found. Notably we recovered, for the first time, lpxC and lpxD nsSNPs previously described only in “in-vitro” mutants and associated with colistin resistance in a clinical COL-R Ab. COL-R vs. COL-S Ab comparative transcriptomics evidenced a strain-dependent response to the colistin resistance onset highly variable among the single COL-R strains vs. their COL-S parents and merely seven common over-expressed transcripts, i.e. the PgaB lipoprotein for biofilm-matrix production, the diacylglycerol kinase for the lipid recycling in the membrane-derived oligosaccharide cycle, a membrane non-ribosomal peptide synthetase, the Lipid A phosphoethanol aminotransferase PmrC, and three hypothetical proteins. The transcript analysis of the “COL-R related genes” and the RNA-seq data confirmed pmrCAB over-expression responsible for a greater positive net cell-charge, and lpxACD under-expression in COL-R causing a decreased LPS production, as main mechanisms of colistin resistance. Our study reports the COL-R Ab genomic and transcriptomic signatures reflecting the interplay between several direct and indirect potential adaptations to antimicrobial pressure, including the occurrence of SNP accumulation hotspot loci in genes related to intrinsic or adaptive colistin resistance, surface adhesion proteins and porins, and over-expressed genes involved in different pathways, i.e. biofilm production, oxidative stress response, extensive drug and COL resistance.

尽管基于多粘菌素（colistin）的治疗方案是多重耐药革兰氏阴性菌感染管理的核心抗菌治疗方案，但在鲍曼不动杆菌（Acinetobacter baumannii，Ab）中，完全性多粘菌素耐药仍较为罕见。本研究对两株临床来源的广泛耐药（Extensively Drug Resistance，XDR）多粘菌素敏感/耐药（COL-S/R）鲍曼不动杆菌菌株对开展基因组学与转录组学分析，这些菌株的多粘菌素耐药性均是在暴露于多粘菌素治疗后诱导产生的。菌株分子特征分析显示，所有受试菌株均隶属于PFGE-A型、序列型281（ST-281）、产OXA-23酶型以及全球克隆II型（Global Clone-II）；其耐药谱为对亚胺培南、美罗培南、氨苄西林/舒巴坦、环丙沙星、庆大霉素、阿米卡星、甲氧苄啶/磺胺甲噁唑均耐药，而对替加环素敏感，这与下一代测序（Next-Generation Sequencing，NGS）获得的耐药组（resistome）分析结果一致。以鲍曼不动杆菌标准菌株ATCC 17978与ACICU的参考基因组为比对模板开展多粘菌素耐药株（COL-R）与敏感株（COL-S）的比较基因组学分析，结果显示菌株间基因组系统发育关系密切，尤其是同一菌株对的分离株之间；且多粘菌素耐药鲍曼不动杆菌菌株存在共有的特征性非同义单核苷酸多态性（non-synonymous SNPs，nsSNPs）。此外，研究还检出了pmrB与pmrC基因的nsSNPs。值得注意的是，本研究首次在临床多粘菌素耐药鲍曼不动杆菌菌株中检出lpxC与lpxD基因的nsSNPs——此前这类突变仅在体外诱变菌株中被报道，且与多粘菌素耐药性相关。多粘菌素耐药株与敏感株的比较转录组学分析显示，多粘菌素耐药性的产生呈现菌株特异性应答模式：不同耐药株与其亲本敏感株之间的转录组差异程度差异显著，且仅存在7种共有的上调转录本，分别为参与生物被膜基质合成的PgaB脂蛋白、参与膜来源寡糖循环中脂质回收的二酰甘油激酶、一种膜结合非核糖体肽合成酶、脂质A磷酸乙醇胺转移酶PmrC，以及3种假设蛋白。对多粘菌素耐药相关基因的转录分析及RNA测序数据证实，pmrCAB操纵子的上调表达可导致细胞净正电荷增加，而多粘菌素耐药株中lpxACD基因的下调表达则会减少脂多糖（Lipopolysaccharide，LPS）的合成——上述二者为多粘菌素耐药的主要分子机制。本研究揭示了多粘菌素耐药鲍曼不动杆菌的基因组与转录组特征，这些特征反映了菌株在抗菌压力下多种直接与间接潜在适应性机制之间的相互作用，包括：与固有或适应性多粘菌素耐药、表面黏附蛋白及孔蛋白相关基因中存在单核苷酸多态性富集热点位点，以及参与不同通路的上调基因——如生物被膜生成、氧化应激应答、广泛耐药及多粘菌素耐药相关通路的基因。