Interaction of Klebsiella pneumoniae with tissue macrophages in a mouse infection model and ex-vivo pig organ perfusions: an exploratory investigation.
Interaction of Klebsiella pneumoniae with tissue macrophages in a mouse infection model and ex-vivo pig organ perfusions: an exploratory investigation.
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DOI：
10.1016/s2666-5247(21)00195-6
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发表时间：
2021-12
期刊：
影响因子：
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通讯作者：
Oggioni MR
中科院分区：
文献类型：
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作者：
Wanford JJ;Hames RG;Carreno D;Jasiunaite Z;Chung WY;Arena F;Di Pilato V;Straatman K;West K;Farzand R;Pizza M;Martinez-Pomares L;Andrew PW;Moxon ER;Dennison AR;Rossolini GM;Oggioni MR
K1 和 K2 型胶囊型高毒力肺炎克雷伯菌 (hvKp) 菌株会引起与肝脓肿相关的侵袭性感染，这种感染很难治疗，并且经常与复发性感染相关。其他肺炎克雷伯菌菌株（非 hvKp），包括已获得碳青霉烯类耐药的谱系，没有表现出这种病理学。在这项工作中，我们旨在检验以下假设：巨噬细胞内复制是 hvKp 感染中脓肿形成的关键机制。在这项探索性研究中，为了研究脓肿形成的病理生理学，小鼠静脉内感染了 106 个集落形成单位 (CFU) 的 hvKp 分离株（6 株）或非 hvKp 分离株（7 株）。通过荧光显微镜对感染后 30 分钟、6 小时和 24 小时收集的小鼠冷冻保存器官切片进行定量，对肝脏和脾脏中的细胞内细菌复制和中性粒细胞流入进行定量，目的是提供细菌与肝脏库普弗细胞和脾脏中不同组织巨噬细胞关联的数据。使用猪肝和脾感染的离体模型的微生物学和显微镜分析来确认巨噬细胞内的复制。用肝素化的自体猪血灌注猪器官，并注射6·5 × 107 CFU的hvKp K2序列25型菌株GMR151。感染前和感染后30分钟、1小时、2小时、3小时、4小时和5小时收集的血液和组织活检用于测量细菌计数并通过免疫组织化学分析鉴定细菌的亚细胞定位。我们发现，hvKp 抵抗吞噬细胞介导的清除，并在感染后 6 小时在小鼠肝脏巨噬细胞中复制形成簇，每个库普弗细胞平均有 7·0 个细菌 (SD 6·2)；然而，非 hvKp 被有效清除（平均每个细胞 1·5 个细菌 [SD 1·1]）。 HvKp 感染促进中性粒细胞募集到感染部位，这在肝脏中早在感染后 24 小时就导致脓肿形成的组织病理学迹象。猪器官与人体器官在功能和解剖学上高度相似，实验为 hvKp 在肝巨噬细胞内复制的倾向提供了强有力的证据。这些发现表明肺炎克雷伯菌对肝脏中先天免疫过程的破坏以及对库普弗细胞介导的清除的抵抗力可以解释hvKp菌株引起肝脓肿的倾向。牛津大学和沃尔夫森皇家学会拨款资助的生物安全设施。
Hypervirulent Klebsiella pneumoniae (hvKp) strains of capsule type K1 and K2 cause invasive infections associated with hepatic abscesses, which can be difficult to treat and are frequently associated with relapsing infections. Other K pneumoniae strains (non-hvKp), including lineages that have acquired carbapenem resistance, do not manifest this pathology. In this work we aimed to test the hypothesis that within-macrophage replication is a key mechanism underpinning abscess formation in hvKp infections. In this exploratory investigation, to study the pathophysiology of abscess formation, mice were intravenously infected with 106 colony forming units (CFU) of either hvKp isolates (six strains) or non-hvKp isolates (seven strains). Intracellular bacterial replication and neutrophil influx in liver and spleen was quantified by fluorescence microscopy of sliced cryopreserved organs of mice collected 30 min, 6 h, and 24 h after infection with the aim to provide data of bacterial association to Kupffer cells in the liver and to the different tissue macrophages in the spleen. Microbiological and microscopy analysis of an ex-vivo model of pig liver and spleen infection were used to confirm within-macrophage replication. Pig organs were perfused with heparinised, autologous pig's blood and injected with 6·5 × 107 CFU of hvKp K2 sequence type 25 strain GMR151. Blood and tissue biopsies collected before infection and 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h after infection were used to measure bacterial counts and to identify the subcellular localisation of bacteria by immunohistochemistry analysis. We show that hvKp resisted phagocyte-mediated clearance and replicated in mouse liver macrophages to form clusters 6 h after infection, with a mean of 7·0 bacteria per Kupffer cell (SD 6·2); however, non-hvKp were efficiently cleared (mean 1·5 bacteria per cell [SD 1·1]). HvKp infection promoted neutrophil recruitment to sites of infection, which in the liver resulted in histopathological signs of abscess formation as early as 24 h post-infection. Experiments in pig organs which share a high functional and anatomical resemblance to human organs, provided strong evidence for the propensity of hvKp to replicate within the hepatic macrophages. These findings show subversion of innate immune processes in the liver by K pneumoniae and resistance to Kupffer cell mediated clearance as an explanation for the propensity of hvKp strains to cause hepatic abscesses. University of Oxford and a Royal Society Wolfson grant funded biosafety facility.