New vectors and optimal conditions for allelic exchange in hypervirulent Klebsiella pneumoniae

IF 1.7 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of microbiological methods Pub Date : 2024-11-19 DOI:10.1016/j.mimet.2024.107070

Mia E. Van Allen , Dakshayini G. Chandrashekarappa , X. Renee Bina, James E. Bina

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引用次数: 0

Abstract

The emergence of antibiotic-resistant Klebsiella pneumoniae is a significant global health threat that has led to increased morbidity and mortality. This resistance also hinders basic research, as many strains are no longer susceptible to antibiotics commonly used in microbial genetics. Addressing this requires the development of new genetic tools with alternative selective markers. In this report, we introduce new allelic exchange vectors for use in drug-resistant strains. These vectors feature a conditional R6K origin of replication, an origin of transfer, SacB counter-selection, and alternative selectable markers. We validated the vectors by generating unmarked deletions in the K. pneumoniae KPPR1S bla (β-lactamase) and lacZ (β-galactosidase) genes. During this process, we defined optimized conditions for SacB-mediated allelic exchange in KPPR1S, significantly enhancing the efficiency of mutant generation. Furthermore, we demonstrated that lacZ is dispensable for virulence and that the lacZ mutant can serve as a surrogate for wild-type strains in competition assays using the Galleria mellonella infection model. Our findings provide new tools for the efficient genetic manipulation of K. pneumoniae and other drug-resistant bacteria.

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高病毒性肺炎克雷伯氏菌等位基因交换的新载体和最佳条件。

耐抗生素肺炎克雷伯氏菌的出现对全球健康构成了严重威胁，导致发病率和死亡率上升。这种耐药性也阻碍了基础研究，因为许多菌株不再对微生物遗传学中常用的抗生素敏感。要解决这个问题，需要开发新的遗传工具，并使用替代性选择标记。在本报告中，我们介绍了用于耐药菌株的新型等位基因交换载体。这些载体具有条件 R6K 复制源、转移源、SacB 反选择和替代性选择标记。我们通过在肺炎双球菌 KPPR1S bla（β-内酰胺酶）和 lacZ（β-半乳糖苷酶）基因中产生无标记缺失来验证这些载体。在此过程中，我们确定了 SacB 介导的 KPPR1S 等位基因交换的优化条件，大大提高了突变体的生成效率。此外，我们还证明了 lacZ 对于毒力是不可或缺的，而且在使用 Galleria mellonella 感染模型进行的竞争试验中，lacZ 突变体可作为野生型菌株的替代品。我们的发现为高效遗传操作肺炎克氏菌和其他耐药细菌提供了新工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of microbiological methods 生物-生化研究方法

CiteScore

4.30

自引率

4.50%

发文量

151

审稿时长

29 days

期刊介绍： The Journal of Microbiological Methods publishes scholarly and original articles, notes and review articles. These articles must include novel and/or state-of-the-art methods, or significant improvements to existing methods. Novel and innovative applications of current methods that are validated and useful will also be published. JMM strives for scholarship, innovation and excellence. This demands scientific rigour, the best available methods and technologies, correctly replicated experiments/tests, the inclusion of proper controls, calibrations, and the correct statistical analysis. The presentation of the data must support the interpretation of the method/approach. All aspects of microbiology are covered, except virology. These include agricultural microbiology, applied and environmental microbiology, bioassays, bioinformatics, biotechnology, biochemical microbiology, clinical microbiology, diagnostics, food monitoring and quality control microbiology, microbial genetics and genomics, geomicrobiology, microbiome methods regardless of habitat, high through-put sequencing methods and analysis, microbial pathogenesis and host responses, metabolomics, metagenomics, metaproteomics, microbial ecology and diversity, microbial physiology, microbial ultra-structure, microscopic and imaging methods, molecular microbiology, mycology, novel mathematical microbiology and modelling, parasitology, plant-microbe interactions, protein markers/profiles, proteomics, pyrosequencing, public health microbiology, radioisotopes applied to microbiology, robotics applied to microbiological methods,rumen microbiology, microbiological methods for space missions and extreme environments, sampling methods and samplers, soil and sediment microbiology, transcriptomics, veterinary microbiology, sero-diagnostics and typing/identification.