Klebsiella pneumoniae is of growing public health concern due to the emergence of strains that are multidrug resistant, virulent, or both. Taxonomically, the K. pneumoniae complex ("Kp") includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. Here, we analyzed 1,001 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR (zur-khe intergenic region) assay, was developed and used to detect Kp in 96 environmental samples. The results were compared to a culture-based method using Simmons citrate agar with 1% inositol medium coupled to matrix-assisted laser desorption ionization–time of flight mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed. The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 x 10^–1 CFU g^–1 after enrichment for 24 h in lysogeny broth supplemented with ampicillin, and it was 1.5 x 10³ to 1.5 x 10⁴ CFU g^–1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method. Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 multilocus sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific, and sensitive novel method to detect the presence of Kp in complex matrices and indicates that Kp isolates from environmental samples differ from clinical isolates.

IMPORTANCE The Klebsiella pneumoniae species complex Kp includes human and animal pathogens, some of which are emerging as hypervirulent and/or antibiotic-resistant strains. These pathogens are diverse and classified into seven phylogroups, which may differ in their reservoirs and epidemiology. Proper management of this public health hazard requires a better understanding of Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in complex matrices such as food or the environment has been difficult due to a lack of accurate and sensitive methods. Here, we describe a novel method based on real-time PCR which enables detection of all Kp phylogroups with high sensitivity and specificity. We used this method to detect Kp isolates from environmental samples, and we show based on genomic sequencing that they differ in antimicrobial resistance and virulence gene content from human clinical Kp isolates. The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, food, animal and human sources.

由于出现了多药耐药，强毒或两者兼而有之的菌株，肺炎克雷伯菌引起了越来越多的公共卫生关注。分类学中，肺炎克雷伯氏菌复合物（“的Kp”）包括七个遗传谱系，具有的Kp1（肺炎克雷伯菌 狭义）是医学上突出。Kp可以存在于土壤和植被等环境资源中，可以作为动物和人类感染的储存库。然而，目前缺乏在复杂基质中检测Kp的筛选方法限制了对Kp生态学的研究。在这里，我们分析了1,001个基因组序列，发现现有的分子检测靶标缺乏对Kp的特异性。一种新颖的实时PCR方法，ZKIR（zur-khe开发了一种用于检测96个环境样品中Kp的方法。将该结果与使用柠檬酸西蒙斯琼脂和1％肌醇培养基结合基质辅助激光解吸电离-飞行时间质谱鉴定的基于培养的方法进行了比较。进行了环境Kp的全基因组测序。ZKIR分析对于48个测试的Kp参考菌株为阳性，而88个非Kp菌株为阴性。加有氨苄青霉素的溶菌培养液中富集24 h后，加标土壤微观样品中Kp的检出限为1.5 x 10 ^–1 CFU g ^–1，范围为1.5 x 10 ³至1.5 x 10 ⁴ CFU g在土壤DNA提取后直接^–1。ZKIR分析比培养方法灵敏。在43％的环境样品中检测到Kp。分离株的基因组分析显示，优势群为Kp1（65％）和Kp3（32％），遗传多样性高（23种多基因座序列类型），抗生素耐药性或毒力基因几乎不存在，并且频率高（50） ％）的3型O抗原。这项研究表明ZKIR分析是检测复杂基质中Kp的准确，特异性和灵敏的新方法，并且表明来自环境样品的Kp分离株与临床分离株不同。

重要性的肺炎克雷伯菌物种复合体Kp包括人类和动物病原体，其中一些正在以高毒力和/或抗生素抗性菌株出现。这些病原体是多种多样的，分为七个系统群，它们的贮藏和流行病学可能不同。正确管理这种公共卫生危害，需要对Kp生态和向人类的传播途径有更好的了解。迄今为止，由于缺乏准确而灵敏的方法，很难在食物或环境等复杂基质中检测这些微生物。在这里，我们描述了一种基于实时PCR的新颖方法，该方法能够以高灵敏度和特异性检测所有Kp系统族。我们使用这种方法来检测环境样品中的Kp分离物，并且我们根据基因组测序结果显示，它们与人类临床Kp分离株的抗药性和毒力基因含量不同。ZKIR PCR测定法将能够快速筛选多个样品中是否存在Kp，从而有助于追踪这些致病菌株在环境，食物，动物和人类来源中的扩散方式。