Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.
Appl Environ Microbiol. 2024 Sep 18;90(9):e0047424. doi: 10.1128/aem.00474-24. Epub 2024 Aug 20.
The continued emergence of antibiotic resistance among bacterial pathogens remains a significant challenge. Indeed, the enhanced antibiotic resistance profiles of contemporary pathogens often restrict the number of suitable molecular tools that are available. We have constructed a series of plasmids that confer resistance to two infrequently used antibiotics with variants of each plasmid backbone incorporating several regulatory control systems. The regulatory systems include both commonly used systems based on the and arabinose-controlled promoters found in , as well as less frequently used systems that respond to tetracycline/anhydrotetracycline and toluic acid. As a test case, we demonstrate the utility of these plasmids for regulated and tunable gene expression in a multidrug-resistant (MDR) isolate of , strain AB5075-UW. The plasmids include derivatives of a freely replicating, broad-host-range plasmid allowing for inducible gene expression as well as a set of vectors for introducing genetic material at the highly conserved Tn7-attachment site. We also modified a set of CRISPR-interference plasmids for use in MDR organisms, thus allowing researchers to more readily interrogate essential genes in currently circulating clinical isolates. These tools will enhance molecular genetic analyses of bacterial pathogens in situations where existing plasmids cannot be used due to their antibiotic resistance determinants or lack of suitable regulatory control systems.
Clinical isolates of bacterial pathogens often harbor resistance to multiple antibiotics, with being a prime example. The drug-resistance phenotypes associated with these pathogens represent a significant hurdle to researchers who wish to study modern isolates due to the limited availability of plasmid tools. Here, we present a series of freely replicating and Tn7-insertion vectors that rely on selectable markers to less frequently encountered antibiotics, apramycin, and hygromycin. We demonstrate the utility of these plasmid tools through a variety of experiments looking at a multidrug-resistant strain of , strain AB5075. Strain AB5075 is an established model strain for present-day , due in part to its genetic tractability and because it is a representative isolate of the globally disseminated multidrug-resistant clade of , global clone 1. In addition to the drug-selection markers facilitating use in strains resistant to more commonly used antibiotics, the vectors allow for controllable expression driven by several regulatory systems, including isopropyl β-D-1-thiogalactopyranoside (IPTG), arabinose, anhydrotetracycline, and toluic acid.
细菌病原体的抗生素耐药性持续出现仍然是一个重大挑战。事实上，现代病原体对抗生素耐药性的增强往往限制了可用的合适分子工具的数量。我们构建了一系列质粒，赋予其对两种不常用抗生素的抗性，每个质粒骨架的变体都包含几种调控控制系统。这些调控系统包括基于 和 在 中发现的阿拉伯糖控制启动子的常用系统，以及对四环素/脱水四环素和甲苯酸有反应的不太常用的系统。作为一个测试案例，我们展示了这些质粒在多药耐药（MDR）分离株 AB5075-UW 中的调控和可调基因表达中的用途。这些质粒包括一种自由复制、广谱质粒的衍生物，允许诱导基因表达，以及一组用于在高度保守的 Tn7-附着位点引入遗传物质的载体。我们还修改了一组用于 MDR 生物的 CRISPR 干扰质粒，从而使研究人员更容易在当前流行的临床分离株中检测必需基因。在由于抗生素耐药决定因素或缺乏合适的调控控制系统而无法使用现有质粒的情况下，这些工具将增强对细菌病原体的分子遗传学分析。
细菌病原体的临床分离株通常对多种抗生素具有耐药性， 是一个主要例子。这些病原体的药物耐药表型代表了一个重大障碍，因为研究人员希望研究现代分离株，由于质粒工具的可用性有限。在这里，我们提出了一系列依赖于选择标记的自由复制和 Tn7 插入载体，用于不太常见的抗生素，即壮观霉素和 Hygromycin。我们通过各种实验展示了这些质粒工具的实用性，这些实验涉及到一种多药耐药株 AB5075。AB5075 株是 目前的一种成熟模型株，部分原因是其遗传可操作性，以及它是全球传播的多药耐药克隆群的代表分离株，全球克隆群 1。除了药物选择标记有助于在更常用抗生素耐药的菌株中使用外，这些载体还允许通过几种调控系统进行可控表达，包括异丙基-β-D-1-硫代半乳糖吡喃糖苷（IPTG）、阿拉伯糖、脱水四环素和甲苯酸。
