Department of Chemical and Materials Engineering, San José State University, San José, California, USA.
Department of Biology, The University of Texas at San Antonio, San Antonio, Texas, USA.
mSphere. 2021 Apr 28;6(2):e00186-21. doi: 10.1128/mSphere.00186-21.
Many microbes in their natural habitats are found in biofilm ecosystems attached to surfaces and not as free-floating (planktonic) organisms. Furthermore, it is estimated that nearly 80% of human infections are associated with biofilms. Biofilms are traditionally defined as three-dimensional, structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. While this view of biofilm largely arises from studies under static or flow conditions, observations have indicated that this view of biofilms is essentially true only for foreign-body infections on catheters or implants where biofilms are attached to the biomaterial. In mucosal infections such as chronic wounds or cystic fibrosis or joint infections, biofilms can be found unattached to a surface and as three-dimensional aggregates. In this work, we describe a high-throughput model of aggregate biofilms of methicillin-resistant (MRSA) using 96-well plate hanging-drop technology. We show that MRSA forms surface-independent biofilms, distinct from surface-attached biofilms, that are rich in exopolymeric proteins, polysaccharides, and extracellular DNA (eDNA), express biofilm-related genes, and exhibit heightened antibiotic resistance. We also show that the surface-independent biofilms of clinical isolates of MRSA from cystic fibrosis and central catheter-related infections demonstrate morphological differences. Overall, our results show that biofilms can form by spontaneous aggregation without attachment to a surface, and this new system can model surface-independent biofilms that may more closely mimic the corresponding physiological niche during infection. The canonical model of biofilm formation begins with the attachment and growth of microbial cells on a surface. While these models reasonably mimic biofilms formed on foreign bodies such as catheters and implants, this is not the case for biofilms formed in cystic fibrosis and chronic wound infections, which appear to present as aggregates not attached to a surface. The hanging-drop model of biofilms of methicillin-resistant (MRSA), the major causative organism of skin and soft tissue infections, shows that these biofilms display morphological and antibiotic response patterns that are distinct from those of their surface-attached counterparts, and biofilm growth is consistent with their location. The simplicity and throughput of this model enable adoption to investigate other single or polymicrobial biofilms in a physiologically relevant setting.
许多微生物在其自然栖息地中存在于生物膜生态系统中，附着在表面上，而不是作为自由漂浮（浮游）生物。此外，据估计，近 80%的人类感染与生物膜有关。生物膜传统上被定义为三维结构的微生物群落，附着在表面上，并被外多聚物基质包裹。虽然这种生物膜的观点主要来自于静态或流动条件下的研究，但观察结果表明，这种生物膜的观点基本上只适用于导管或植入物上的异物感染，生物膜附着在生物材料上。在黏膜感染如慢性伤口或囊性纤维化或关节感染中，生物膜可以在没有附着在表面的情况下发现，并作为三维聚集物存在。在这项工作中，我们使用 96 孔板悬滴技术描述了耐甲氧西林金黄色葡萄球菌（MRSA）聚集生物膜的高通量模型。我们表明，MRSA 形成了与表面无关的生物膜，与附着在表面上的生物膜不同，富含外多聚物蛋白、多糖和细胞外 DNA（eDNA），表达与生物膜相关的基因，并表现出更高的抗生素耐药性。我们还表明，来自囊性纤维化和中央导管相关感染的 MRSA 临床分离株的非附着表面生物膜表现出形态学差异。总的来说，我们的结果表明，生物膜可以通过自发聚集而无需附着在表面上形成，并且这个新系统可以模拟可能更接近感染期间相应生理龛的非附着表面生物膜。生物膜形成的经典模型始于微生物细胞在表面上的附着和生长。虽然这些模型合理地模拟了导管和植入物等异物上形成的生物膜，但对于囊性纤维化和慢性伤口感染中形成的生物膜并非如此，这些生物膜似乎作为不附着在表面的聚集物出现。耐甲氧西林金黄色葡萄球菌（MRSA）生物膜的悬滴模型，是皮肤和软组织感染的主要病原体，表明这些生物膜显示出与附着在表面上的生物膜不同的形态和抗生素反应模式，并且生物膜的生长与其位置一致。该模型的简单性和高通量使其能够采用该模型在生理相关环境中研究其他单一或多微生物生物膜。
