目的：肺炎克雷伯菌是临床引起血流感染的重要致病菌，尤其是高毒力碳青霉烯 耐药肺炎克雷伯菌（hv-CRKP），本课题的主要目的是揭示 hv-CRKP 的分子流 行病学和 hv-CRKP 的进化规律及关键影响因子。黏菌素作为临床治疗 CRKP 的 最后一线用药之一，对其耐药率的监测具有重要临床意义。本课题旨在报道中国 革兰阴性菌黏菌素耐药率及商品化检测系统性能评价结果，同时对肺炎克雷伯菌 黏菌素异质性耐药率及形成机制进行深入研究。 方法：本研究纳入 239 株中国多中心血流感染来源的肺炎克雷伯菌的全基因组 测序数据和 Genbank 数据库中 1014 株肺炎克雷伯菌的完整基因组进行 hvCRKP 的分子流行病学研究，通过单核苷酸多态性鉴定、系统发育、平均核苷酸 一致性和质粒结构分析进行 hv-CRKP 进化路径的预测及关键影响因子的挖掘； 通过接合、敲除、克隆、转录组检测、抗氧化应激能力检测、巨噬细胞吞噬、肺 癌上皮细胞毒性检测及粘附侵袭实验、动物实验和生长曲线测定实验进行了 hvCRKP 关键因子 iroBCDN 的功能验证实验。以肉汤微量稀释法为金标准，通过 对采集自临床病例及家畜的 257 株革兰阴性菌株进行黏菌素 MIC 检测，评价 VITEK 2 COMPACT、Phoenix M50、Bio-kont 抗菌药物敏感性检测系统性能。 使用固定浓度法和 PAP 法进行肺炎克雷伯菌黏菌素异质性耐药的筛查，通过全 基因组测序、转录组测序、RT-qPCR、外排泵抑制剂进行异质性耐药机制的研 究。 结果：中国多中心临床血流感染来源的肺炎克雷伯菌的全基因组二代测序结果显 示 ST11-K64 是我国目前最主要的 hv-CRKP 克隆株，主要由 ST11-K64 CRKP 从 hvKP 获得 pLVPK-like 毒力质粒进化而来。研究结果发现 ST11-K64 hv-CRKP 毒力质粒中缺失了 iroBCDN 基因簇，而 hvKP 毒力质粒中存在 iroBCDN 基因 簇，具有 iroBCDN 缺失的截短型 IncHI1B/IncFIB 型毒力质粒的 hv-CRKP 菌株 更具有临床流行优势。 通过接合实验、细胞实验及动物感染模型证实了 iroBCDN 的缺失并未降低 hv-CRKP 菌株的毒力，并进一步提高了质粒接合转移效率，促使非接合性毒力 质粒从 hvKP 转移到 ST11-K64 CRKP，形成 hv-CRKP。转录组分析结果显示 iroBCDN 缺失的菌株氧化应激相关基因表达上调，并通过实验验证发现其具有 高抗氧化能力和巨噬细胞内存活能力。 目前临床常用微生物药物敏感性检测系统中，国产 Bio-kont 系统和 Phoenix M50 系统对黏菌素的检测性能优于 Vitek 2 系统，但都不符合 CLSI 标准。对于mcr-1 阳性大肠杆菌的黏菌素药敏检测，Bio-kont 和 Phoenix M50 表现出优异 的性能，没有分类错误，而 Vitek 2 表现出较高的 VME。 本课题发现我国院内血流感染病原肺炎克雷伯菌以 ST11 型菌株为主，而社 区获得性血流感染以 ST23 型菌株为主。生存分析发现 ST11 型、携带碳青霉烯 酶基因的菌株为高风险型，入住 ICU 与来自腹部或呼吸道的原发感染与高死亡 风险存在相关性。目前全国各地区的肺炎克雷伯菌株对头孢他啶-阿维巴坦、黏 菌素、多黏菌素与替加环素敏感性较高，对头孢曲松、复方新诺明、头孢吡肟和 左氧氟沙星的敏感性较差。 中国目前多黏菌素 B 和黏菌素耐药率均为 3.09%，敏感菌株中，对黏菌素 的异质性耐药率为 6.18%。外排泵抑制剂 CCCP 对黏菌素异质性耐药表型具有 一定的抑制作用。本研究未发现基因水平的黏菌素异质性耐药发生机制，但在转 录水平上，发现黏菌素耐药相关基因 phoPQ 的表达上调被确认为主要的耐药机 制。转录组分析进一步揭示，双组份系统通路的最终环节——操纵子 arnBCADTEF 的表达上调在所有菌株的耐药亚群中均显著存在。此外，核糖体 相关通路基因的富集现象表明，核糖体在异质性耐药表型中表现出活跃的调控作 用。通过小鼠腹腔感染模型，本研究发现在免疫正常的小鼠体内，异质性耐药表 型并未导致显著的死亡率增加或治疗失败。 结论：本课题创新性发现 iroBCDN 基因簇缺失的毒力质粒驱动 CRKP 通过接合 获得毒力基因为中国 hv-CRKP 的重要进化路径，并整合多组学和体内外进化研 究，解析该质粒通过增强细菌抗氧化能力、增强巨噬细胞内存活能力促进 hvCRKP 形成。另一方面发现中国肺炎克雷伯菌黏菌素异质性耐药率较为低，主要 由双组份系统 phoPQ 转录水平上调引起。对黏菌素异质性耐药的机制深入研究 有利于临床对异质性耐药菌株进行有效诊断、治疗和预防。

Objectives: Klebsiella pneumoniae is an important pathogen causing bloodstream infection in clinic. In recent years, hypervirulent carbapenem resistant Klebsiella pneumoniae (hv-CRKP) has seriously threatened clinical and public health. The main purpose of this project is to reveal the molecular epidemiology of hv-CRKP, especially the characteristics of resistance plasmids and virulence plasmids, and further reveal the evolutionary features and key influencing factors of hv-CRKP. As one of the last drugs used in clinical treatment of CRKP, monitoring of its resistance rate is of great clinical significance. This project also aims to report the colistin resistance rate of Gram-negative bacteria in China and the performance evaluation of commercial antimicrobial susceptible testing systems. At the same time, the colistin heteroresistance rate and formation mechanism of Klebsiella pneumoniae are studied in depth, and the colistin heteroresistance detection process based on growth-metabolism combined with flow mode Raman activated cell sorter is further established to assist in studying the internal mechanism of colistin heteroresistance. Methods: This study included the whole genome sequencing data of 239 Klebsiella pneumoniae from multi-center bloodstream infections in China and the complete genomes of 1014 Klebsiella pneumoniae from Genbank for molecular epidemiological research on hv-CRKP. The evolutionary pathway of hv-CRKP was predicted and key influencing factors were found through single nucleotide polymorphism identification, phylogeny, average nucleotide consistency and plasmid structure analysis. The functional validation experiment of the key factor iroBCDN of hv-CRKP was carried out through conjugation, deletion, cloning, transcriptome detection, antioxidant stress capacity detection, macrophage phagocytosis, lung cancer epithelial cell toxicity detection and adhesion and invasion experiments, animal experiments and growth curve determination experiments. Using the broth microdilution method as the gold standard, the performance of VITEK 2 COMPACT, PhoenixTM M50 and Bio-kont antimicrobial susceptible detection systems in colistin MIC determination was evaluated by with 257 Gram-negative strains collected from clinical cases and livestock. The fixed concentration method and population analysis profiling (PAP) method were used to screen for heteroresistance to colistin in Klebsiella pneumoniae. The mechanism of heteroresistance was studied by whole genome sequencing, transcriptome sequencing, RT-qPCR, and efflux pump inhibitors. A new procedure for detecting colistin heteroresistance was established by combining growth curve monitoring, Raman spectroscopy detection, flow cytometry sorting and other technologies. Results: The whole genome sequencing results of Klebsiella pneumoniae from clinical bloodstream infection in multiple centers in China showed ST11-K64 is currently the most important hv-CRKP clone in China, and it is mainly evolved from ST11-K64 CRKP acquiring the pLVPK-like virulence plasmid from hvKP. It is worth noting that the iroBCDN gene cluster is missing in the virulence plasmid from the ST11-K64 hv-CRKP, while the it is present in the virulence plasmid of hvKP. It was further found that compared with the hv-CRKP strain with complete iroBCDN and IncHI1B/IncFIB replicons, the hv-CRKP strain with a truncated IncHI1B/IncFIB virulence plasmid with iroBCDN deletion has a greater clinical epidemic advantage. Bacterial tests, cell interaction experiments and animal infection models confirmed that the loss of iroBCDN did not reduce the virulence of the hv-CRKP strain, and further improved the efficiency of plasmid conjugation, prompting the non-conjugative virulence plasmid to transfer from hvKP to ST11-KL64 CRKP to form hv-CRKP. Transcriptome analysis results showed that the expression of oxidative stress-related genes in the strain with iroBCDN deletion was upregulated, and it also showed higher antioxidant capacity and survival ability in macrophages in the experiment. Among the currently commonly used clinical antimicrobial susceptibility detection systems, the Bio-kont system and Phoenix M50 system have better detection performance for colistin than the Vitek 2 system, but neither meets the CLSI standard. For the colistin susceptibility test of mcr-1-positive Escherichia coli, Bio-kont and Phoenix M50 showed excellent performance without classification errors, while Vitek 2 showed a higher VME. In this study, it was found that the pathogenic Klebsiella pneumoniae in hospital-acquired bloodstream infection was mainly ST11 strain, while the community-acquired bloodstream infection was mainly ST23 strain. Survival analysis found that ST11 strains carrying carbapenase genes were high-risk, and ICU admission and primary infection from the abdomen or respiratory tract are associated with a higher risk of death. Klebsiella pneumoniae strains from different regions are all highly susceptible to ceftazidime-avibactam, colistin, polymyxin B and tigacycline, but are less susceptible to ceftriaxone, cotrimoxazole, cefepime and levofloxacin. In this project, the current resistance rates of polymyxin B and colistin in China are both 3.09%, and the hetero resistance rate to colistin among susceptible strains is 6.18%. The efflux pump inhibitor CCCP has a certain inhibitory effect on the heteroresistance phenotype of colistin. This study did not find the mechanism of colistin heteroresistance at the genomic level, but at the transcriptional level, the upregulation of the expression of the colistin resistance-related gene phoPQ was found to be the main resistance mechanism. Transcriptome analysis further revealed that the upregulation of the expression of the operon arnBCADTEF, the final component of the two component system pathway, was significantly present in the resistant subclones of all strains. In addition, the enrichment of ribosome-related pathway genes indicated that the ribosome showed an active regulatory role in the heteroresistance phenotype. Through the mouse intraperitoneal infection model, this study found that the heteroresistance phenotype did not lead to a significant increase in mortality or treatment failure in mice with normal immunity. This project innovatively proposed a growth-metabolism-based colistin heteroresistance detection procedure combined with a flow Raman-activated cell sorter, which monitors whether there are highly resistant subclones in on clone through growth and heavy water (D2O) metabolism, and can estimate the proportion of resistant subgroups. Compared with the PAP method, it has higher sensitivity (10-6), faster detection speed (within 48h), more minute biological structure information (protein, phospholipid and nucleic acid) and more biological metabolism information (deuterium metabolism). It successfully observed three subgroups with different phenotypes (S, X, R) in the population with heteroresistance, and analyzed the homology of S and X subgroups through protein, lipid and nucleic acid metabolism information, while R subgroup exists independently. It is proposed that S and X have conversion when bacteria adapt to the colistin pressure environment. Conclusion: This project innovatively discovered that the pLVPK virulence plasmid with the iroBCDN gene cluster deleted drives CRKP to obtain virulence genes through conjugation, which is an important evolutionary path for hv CRKP in China. It also integrates multi-omics and in vivo and in vitro evolution studies to analyze how the iroBCDN deletion plasmid promotes the formation of hv-CRKP by enhancing the antioxidant capacity of bacteria and enhancing the survival ability in macrophages. On the other hand, it was found that the heteroresistance rate of colistin in Chinese Klebsiella pneumoniae was relatively low, which was mainly caused by the upregulation of the two component system PhoPQ at the transcriptional level. In addition, the growth metabolism-based colistin heteroresistance detection process combined with flow Raman-activated cell sorter proposed in this study can monitor the heteroresistance of colistin in clinical Klebsiella pneumoniae more quickly and sensitively, which is conducive to the effective diagnosis, treatment and prevention of heteroresistant strains in clinical practice