PDF(Pubmed)
Abstract：
It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, extracellular beta-lactamases produced by resistant cells that subsequently degrade penicillin and related antibiotics allow neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show in multiple bacterial species that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface-grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss was still observed. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated.IMPORTANCEPlasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid-containing cell is grown in the presence of antibiotic. Here, we find that growth of plasmid-containing bacteria on a surface and to a lesser extent in liquid culture in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation.
摘要：
青霉素的分离和使用已经过去了近一个世纪，预示着各种不同抗生素的发现。除了临床应用，这些抗生素一直是必不可少的实验室工具，允许选择和维持编码同源抗性基因的实验室质粒。然而,抗生素耐药机制还可以作为公共物品发挥作用。例如,耐药细胞产生的细胞外β-内酰胺酶随后降解青霉素和相关抗生素,使邻近的无质粒易感细菌能够在抗生素治疗中存活.在实验室条件下的实验中，这种合作机制如何影响质粒的选择尚不清楚。这里,我们在多种细菌物种中表明，使用质粒编码的β-内酰胺酶导致表面生长细菌中质粒的显着治愈。此外，对于氨基糖苷磷酸转移酶和四环素反转运蛋白耐药机制，这种治愈也很明显。或者，液体生长中的抗生素选择导致更强大的质粒维持，尽管仍观察到质粒丢失。这种质粒丢失的最终结果是产生含质粒和无质粒细胞的异质群体。导致实验混乱，没有得到广泛的重视。IMPORTANCE质粒常规用于微生物学中作为细胞生物学的读数或操纵细胞功能的工具。这些研究的核心是假设实验中的所有细胞都含有质粒。宿主细胞中的质粒维持通常取决于质粒编码的抗生素抗性标记。这在含质粒的细胞在抗生素存在下生长时提供了选择性优势。这里,我们发现，在存在三个不同的抗生素家族的情况下，含质粒的细菌在表面和较小程度的液体培养物中的生长导致大量无质粒细胞的进化，依赖于含质粒细胞的抗性机制。此过程产生了无质粒和含质粒的细菌的异质种群，这个结果可能会混淆进一步的实验。
