摘要
       已知来自患有囊性纤维化（CF）的患者的铜绿假单胞菌临床分离株与通过菌落形态，药物敏感性模式和基因组超集可变性而与非CF宿主相关的铜绿假单胞菌不同。长期以来，来自CF患者的铜绿假单胞菌分离株的抗菌药物敏感性总体降低，但其长期以来一直忽视其MIC的异质性。我们使用2013年分离的两组不同的临床菌株（n = 224例来自56例CF患者，n = 130例来自68例非CF患者），与非CF菌株相比，我们证明了在CF铜绿假单胞菌分离株中具有明显的Etest MIC异质性。绿脓杆菌分离株。在对来自9名具有异质性MIC的9名患者的19CF铜绿假单胞菌分离物的全基因组测序的基础上，核心基因组系统发育树证实了患者内的CF铜绿假单胞菌克隆谱系以及相当大的编码序列可变性。尽管外排和孔蛋白基因及其调节子中存在许多异质性突变，但没有染色体外DNA元件或先前表征的抗生素抗性突变可解释铜绿假单胞菌coisolates之间抗微生物MIC的广泛分歧。独特的OprD序列在大多数分离的铜绿假单胞菌分离株中保守，表明对分离菌常见的选择性压力的假单胞菌响应。基因组序列数据还表明，CF假单胞菌高度可变性不完全归因于mutL，mutS和uvr的突变。我们得出这样的结论：数百个适应性突变的净效应，都是克隆相关的分离物对和未共享的，这说明它们的高度不均一的MIC差异。我们推测，这种异质性是在长时间“锁定”在宿主焦点气道环境条件下的假单胞菌样生活方式的指示。重要性囊性纤维化患者可忍受各种微生物的“慢性局灶感染”。一种微生物，铜绿假单胞菌（Pseudomonas aeruginosa），适应宿主并产生对各种抗微生物剂的抗性。有趣的是，随着感染的进展，铜绿假单胞菌的多个同基因菌株出现并且在这些患者的气道内共存。尽管有共同的亲本来源，但铜绿假单胞菌的多种菌株对积极使用的抗微生物剂产生极大不同的易感性模式 - 我们将这种现象定义为“非均相MIC”。通过对显示不均一MIC的铜绿假单胞菌分离株进行测序，我们在药物转运蛋白，DNA错配修复机制和许多其他结构或细胞功能中观察到广泛的同基因基因损伤。加上不均一的中等收入国家，这些遗传病变表现出对宿主选择的共生反应，并提出多细胞合胞体细菌生活方式的进化。目前的实验室标准解释标准没有解决体外异构生长和易感性的出现以及治疗影响。

       Clinical isolates of Pseudomonas aeruginosa from patients with cystic fibrosis (CF) are known to differ from those associated with non-CF hosts by colony morphology, drug susceptibility patterns, and genomic hypermutability. Pseudomonas aeruginosaisolates from CF patients have long been recognized for their overall reduced rate of antimicrobial susceptibility, but their intraclonal MIC heterogeneity has long been overlooked. Using two distinct cohorts of clinical strains (n = 224 from 56 CF patients, n = 130 from 68 non-CF patients) isolated in 2013, we demonstrated profound Etest MIC heterogeneity in CFP. aeruginosa isolates in comparison to non-CF P. aeruginosa isolates. On the basis of whole-genome sequencing of 19 CFP. aeruginosa isolates from 9 patients with heterogeneous MICs, the core genome phylogenetic tree confirmed the within-patient CF P. aeruginosa clonal lineage along with considerable coding sequence variability. No extrachromosomal DNA elements or previously characterized antibiotic resistance mutations could account for the wide divergence in antimicrobial MICs betweenP. aeruginosa coisolates, though many heterogeneous mutations in efflux and porin genes and their regulators were present. A unique OprD sequence was conserved among the majority of isolates of CF P. aeruginosa analyzed, suggesting a pseudomonal response to selective pressure that is common to the isolates. Genomic sequence data also suggested that CF pseudomonal hypermutability was not entirely due to mutations in mutL, mutS, and uvr. We conclude that the net effect of hundreds of adaptive mutations, both shared between clonally related isolate pairs and unshared, accounts for their highly heterogeneous MIC variances. We hypothesize that this heterogeneity is indicative of the pseudomonal syntrophic-like lifestyle under conditions of being "locked" inside a host focal airway environment for prolonged periods. IMPORTANCE Patients with cystic fibrosis endure "chronic focal infections" with a variety of microorganisms. One microorganism, Pseudomonas aeruginosa, adapts to the host and develops resistance to a wide range of antimicrobials. Interestingly, as the infection progresses, multiple isogenic strains of P. aeruginosa emerge and coexist within the airways of these patients. Despite a common parental origin, the multiple strains of P. aeruginosa develop vastly different susceptibility patterns to actively used antimicrobial agents-a phenomenon we define as "heterogeneous MICs." By sequencing pairs of P. aeruginosa isolates displaying heterogeneous MICs, we observed widespread isogenic gene lesions in drug transporters, DNA mismatch repair machinery, and many other structural or cellular functions. Coupled with the heterogeneous MICs, these genetic lesions demonstrated a symbiotic response to host selection and suggested evolution of a multicellular syntrophic bacterial lifestyle. Current laboratory standard interpretive criteria do not address the emergence of heterogeneous growth and susceptibilities in vitro with treatment implications.

https://www.ncbi.nlm.nih.gov/pubmed/29564400