Kaplan Jeffrey B, Florjanczyk Aleksandr P, Ochiai Maria, Jones Caleb D, Horswill Alexander R
Department of Biology, American University, Washington DC 20016, USA.
Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
bioRxiv. 2023 Nov 5:2023.11.05.565664. doi: 10.1101/2023.11.05.565664.
Biofilm formation is an important virulence factor for methicillin-resistant (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA. MRSA cells also secrete micrococcal nuclease (Nuc1) which degrades double-stranded DNA. In this study we used a mutant strain to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. Biofilm was quantitated in microplates using a crystal violet binding assay. Extracellular DNA (eDNA) was isolated from colony biofilms and analyzed by agarose gel electrophoresis. In some experiments, broth or agar was supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods, transferring the rods to fresh broth, and enumerating CFUs that detached from the rods. Biofilm sloughing was investigated by culturing biofilms in glass tubes perfused with broth and measuring the sizes of the detached cell aggregates. We found that a mutant strain produced significantly more biofilm and more eDNA than a wild-type strain in both the absence and presence of sub-MIC amoxicillin, mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or a wild-type gene on a plasmid. In the sloughing assay, mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.
生物膜形成是耐甲氧西林金黄色葡萄球菌（MRSA）的一个重要毒力因子。MRSA生物膜的细胞外基质含有大量双链DNA。MRSA细胞还分泌可降解双链DNA的微球菌核酸酶（Nuc1）。在本研究中，我们使用一个突变菌株来研究Nuc1在MRSA生物膜形成和分散中的作用。使用结晶紫结合试验在微孔板中对生物膜进行定量。从菌落生物膜中分离细胞外DNA（eDNA），并通过琼脂糖凝胶电泳进行分析。在一些实验中，在肉汤或琼脂中添加亚抑菌浓度的阿莫西林以诱导生物膜形成。通过将生物膜接种在棒上进行培养，将棒转移到新鲜肉汤中，并对从棒上脱落的菌落形成单位（CFU）进行计数，来定量生物膜侵蚀。通过在灌注肉汤的玻璃管中培养生物膜并测量脱落细胞聚集体的大小，来研究生物膜脱落。我们发现，无论有无亚抑菌浓度的阿莫西林，一个突变菌株产生的生物膜和eDNA都比野生型菌株显著更多，在棒上生长的突变生物膜脱落的细胞明显少于野生型生物膜。通过外源性脱氧核糖核酸酶或质粒上的野生型基因可恢复脱落。在脱落试验中，突变生物膜释放的细胞聚集体明显大于野生型生物膜释放的细胞聚集体。我们的结果表明，Nuc1调节生物膜形成、生物膜脱落以及脱落细胞聚集体的大小。这些过程可能在生物膜相关感染期间MRSA生物膜的传播和后续存活中发挥作用。
