Genomic characterisation of multidrug-resistant septicaemia-causing Klebsiella pneumoniae to inform patient care and infection control

抗菌素耐药性（AMR）是全球卫生危机。肺炎克雷伯菌是一种革兰氏阴性菌，对碳青霉烯类（治疗难治性感染的最后一线药物的一部分）具有耐药性，被列入世界卫生组织的严重耐药病原体名单。它们导致许多医疗保健相关感染，并与死亡率和发病率增加有关。这在一定程度上是由于它们能够从最初的感染部位迅速变成全身感染，但也因为它们可以在质粒等可移动的遗传元件上共享耐药基因，导致它们迅速将抗菌素耐药性传播给其他革兰氏阴性物种。这可能导致抗菌素治疗失败和患者预后不良，特别是在迅速给予正确抗生素至关重要的情况下，如败血症。这些质粒还可以携带有助于环境生存、调节毒力因子和赋予多重耐药性的基因。因此，质粒的获取可以改变细菌的表型，改变疾病的严重程度和患者的预后。因此，了解质粒进化的机制对于预测毒力的变化和质粒传播的风险是很重要的。在这个项目中，我们将采用长读测序来分析肺炎克雷伯菌临床分离株的基因组，它会导致患者败血症。我们的目标是了解为什么有些菌株会导致致命感染，而另一些则不会。生物信息学分析将用于鉴定染色体和质粒携带的抗菌素耐药性基因、毒力因子和可能导致突变的易出错聚合酶。这将辅以表型评估，如吞噬细胞内存活和抗菌药物敏感性，以了解患者结果的差异。将调查存在消毒剂、干燥条件和表面粘附的环境存活情况，以评估环境存活和作为污染物的传播。我们还将在抗生素的亚抑制浓度下建立长期培养，以评估易出错聚合酶在碳青霉烯酶进化中的作用，这些酶使β -内酰胺类抗生素失活。这将得到频繁测序和表型表征的支持，以确定基因突变和表型表达的变化。我们还将在肠道细菌中进行种间和种内质粒转移以及与无质粒菌株表型相关的变化。这些调查将使人们了解全基因组测序如何影响患者的治疗结果，管理病原体和质粒的传播，并为卫生保健机构的感染控制实践和耐药病原体监测提供信息。这应有助于制定新的战略，以对抗质粒和耐药病原体的传播。

Antimicrobial resistance (AMR) is global health crisis. Klebsiella pneumoniae, a Gram-negative bacteria, that are resistant to carbapenems (part of the last-line drugs for difficult-to-treat infections) are on the World Health Organization's list of critically resistant pathogens. They contribute to many healthcare-associated infections and are associated with increased mortality and morbidity. This is partly due to their ability to rapidly become systemic from the initial infection site, but also because they can share resistance genes on mobile genetic elements, such as plasmids, leading them to rapidly spread AMR to other Gram-negative species. This can lead to failed antimicrobial therapy and negative patient outcomes, especially in cases where rapid administration of the correct antibiotics is crucial, such as sepsis. These plasmids can also carry genes that aid in environmental survival, regulate virulence factors and confer multidrug resistance. Plasmid acquisition can, therefore, change the bacterial phenotype and alter disease severity and patient outcomes. Thus, it is important to understand the mechanisms of plasmid evolution to be able to predict changes in virulence and risk of plasmid spread. In this project, we will employ long-read sequencing to analyse the genomes of clinical isolates of K. pneumoniae where it caused sepsis in patients. We aim to understand why some strains caused fatal infections while others did not. Bioinformatic analyses will be employed to identify both chromosomal and plasmid-borne AMR genes, virulence factors and error-prone polymerases that may lead to mutations. This will be complemented by phenotypic assessments such as survival within phagocytic cells and antimicrobial susceptibility to understand the differences in patient outcomes. Environmental survival in the presence of disinfectants, desiccated conditions and adherence to surfaces will be investigated to assess environmental survival and spread as fomites. We will also set up long-term cultures in the presence of sub-inhibitory concentrations of antibiotics to evaluate the role of error-prone polymerases in carbapenemase evolution, enzymes that inactivate Beta-lactam antibiotics. This will be supported by frequent sequencing and phenotypic characterisation to identify genetic mutations and changes in phenotypic expression. We will also conduct inter- and intra-species plasmid transfer and associated changes in phenotypes to plasmid-free strains in gut bacteria. These investigations will result in an understanding how whole-genome sequencing can influence patient treatment outcomes, manage pathogen and plasmid spread and inform infection control practices and surveillance of resistant pathogens in healthcare settings. This should aid in the development of new strategies to combat the spread of plasmids and resistant pathogens.