McAndrew Grainne, Barbier Elodie, Rodrigues Carla, Piveteau Pascal, Brisse Sylvain, Reddington Kate
Microbiology, School of Biological and Chemical Sciences, University of Galway, Galway, Ireland.
UMR Agroécologie, INRAE, AgroSup Dijon, Université de Bourgogne, Dijon, France.
Microbiology (Reading). 2025 Jul;171(7). doi: 10.1099/mic.0.001587.
The species complex (KpSC) comprises five closely related bacterial species, namely , , , and . The KpSC is ubiquitous in the environment and is also an important human pathogen, particularly associated with healthcare-associated infections. The accurate detection and differentiation of the KpSC is challenging owing to the close phenotypic and genotypic identity (93-95% average nucleotide identity) shared between these members. Current diagnostic assays either fail to detect and identify all KpSC members or misidentify some KpSC members as . It is currently estimated that ~20% of human infections are caused by members of the KpSC other than . This leads to underreporting of some KpSC members in both clinical and environmental settings, which impacts our understanding of the importance of each species. Furthermore, it limits our understanding of the global and local epidemiological impact of some members of the KpSC. In this study, a rapid multiplex real-time PCR assay (KpSC-ID) was designed and developed to detect all KpSC members while simultaneously identifying the predominant human pathogens , and . Assay performance was verified using a panel of over 1,000 publicly available genome sequences and experimentally validated using a panel of genomic DNA extracted from 54 . The assay displayed excellent specificity against over 1,000 genome sequences tested . During validation, the pan-KpSC assay detected each (29/29) KpSC species and strains tested. For the species-specific assays, 100% specificity was demonstrated in the , and assays, respectively. Sensitivity of 10 genomic equivalents was demonstrated for each assay. Ultimately, the diagnostic assay developed in this study can improve our understanding of the significance of KpSC members, which is important when investigating their routes of transmission and epidemiology.
肺炎克雷伯菌复合种（KpSC）由五个密切相关的细菌物种组成，即肺炎克雷伯菌、产酸克雷伯菌、解鸟氨酸克雷伯菌、植生克雷伯菌和土生克雷伯菌。KpSC在环境中普遍存在，也是一种重要的人类病原体，尤其与医疗保健相关感染有关。由于这些成员之间具有相似的表型和基因型特征（平均核苷酸同一性为93 - 95%），准确检测和区分KpSC具有挑战性。目前的诊断检测方法要么无法检测和识别所有KpSC成员，要么将一些KpSC成员误鉴定为肺炎克雷伯菌。目前估计，约20%的人类感染是由除肺炎克雷伯菌之外的KpSC成员引起的。这导致在临床和环境环境中，一些KpSC成员的报告不足，从而影响我们对每个物种重要性的理解。此外，这也限制了我们对KpSC某些成员的全球和局部流行病学影响的理解。在本研究中，设计并开发了一种快速多重实时PCR检测方法（KpSC-ID），用于检测所有KpSC成员，同时识别主要的人类病原体肺炎克雷伯菌、产酸克雷伯菌和解鸟氨酸克雷伯菌。使用一组超过1000个公开可用的基因组序列验证了检测方法的性能，并使用从54株菌株中提取的基因组DNA进行了实验验证。该检测方法对测试的1000多个基因组序列显示出优异的特异性。在验证过程中，泛KpSC检测方法检测到了所有测试的（29/29）KpSC物种和菌株。对于物种特异性检测，在肺炎克雷伯菌、产酸克雷伯菌和解鸟氨酸克雷伯菌检测中分别显示出100%的特异性。每种检测方法的灵敏度为10个基因组当量。最终，本研究中开发的诊断检测方法可以提高我们对KpSC成员重要性的理解，这在调查它们的传播途径和流行病学方面很重要。
