Table_1_Colistin Resistant A. baumannii: Genomic and Transcriptomic Traits Acquired Under Colistin Therapy.DOCX

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.

尽管基于粘菌素的疗法构成了多重耐药性革兰氏阴性菌感染管理方案的基石，但至少在鲍曼不动杆菌（Ab）中，粘菌素耐药性仍属罕见，特别是作为完全耐药性。本研究对两对临床广泛耐药性（XDR）粘菌素敏感/耐药（COL-S/R）Ab菌株进行了基因组学和转录组学分析，这些菌株在接触粘菌素治疗后发展出了COL耐药性。菌株的分子特征表明，所有菌株均属于PFGE-A、ST-281、OXA-23产生者、Global Clone-II，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/磺胺甲噁唑耐药，对替加环素敏感，这与通过下一代测序（NGS）获得的耐药基因库一致。COL-R与COL-S Ab的比较基因组学分析，映射至鲍曼不动杆菌ATCC 17978和鲍曼不动杆菌ACICU参考基因组，揭示了菌株间紧密的基因亲缘关系，特别是在菌株对分离株之间，以及COL-R菌株中独特的常见非同义单核苷酸多态性（nsSNPs）。此外，发现了pmrB和pmrC nsSNPs。值得注意的是，我们首次从临床COL-R Ab中恢复了先前仅在“体外”突变体中描述的lpxC和lpxD nsSNPs，这些突变体与粘菌素耐药性相关。COL-R与COL-S Ab的比较转录组学分析显示，对粘菌素耐药性启动的响应在单个COL-R菌株与其COL-S亲本之间高度可变，仅存在七个过度表达的共同转录本，即PgaB脂蛋白（用于生物膜基质的产生）、二酰甘油激酶（参与膜来源的寡糖循环中的脂质回收）、膜非核糖体肽合成酶、脂质A磷酸乙醇胺转移酶PmrC和三个假设蛋白。对“COL-R相关基因”的转录分析以及RNA测序数据证实了pmrCAB过度表达导致净细胞电荷增加，以及lpxACD在COL-R中的低表达导致LPS产生减少，这是粘菌素耐药性的主要机制。本研究报道了COL-R Ab的基因组和转录组特征，反映了多种直接和间接潜在适应抗菌压力的相互作用，包括与内在或适应性粘菌素耐药性相关的基因中SNP积累热点位点的出现、表面粘附蛋白和孔蛋白以及参与不同途径（如生物膜产生、氧化应激反应、广泛耐药性和COL耐药性）的过度表达基因。