Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics.
Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics.
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DOI：
10.1128/aem.00113-17
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发表时间：
2017-05-01
影响因子：
4.4
通讯作者：
Bjarnsholt T
中科院分区：
文献类型：
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作者：
Sønderholm M;Kragh KN;Koren K;Jakobsen TH;Darch SE;Alhede M;Jensen PØ;Whiteley M;Kühl M;Bjarnsholt T
藻酸盐珠代表了用于扩散限制细菌生长的简单且高度可重复的体外模型系统。在这项研究中，藻酸盐珠接种铜绿假单胞菌，并跟踪长达72小时。共聚焦显微镜显示，铜绿假单胞菌形成了与在囊性纤维化肺和慢性伤口中离体观察到的体内聚集体大小相似的致密簇。细菌聚集体主要生长在珠粒周边，并且朝向珠粒中心的尺寸和丰度减小。微传感器测量结果表明，O2浓度迅速下降，并达到缺氧下100 μm的海藻酸钠珠表面。这种梯度在补充有NO3−作为替代电子受体的珠粒中得到缓解，从而允许更深地生长到珠粒中。浮游和藻酸盐包裹的铜绿假单胞菌之间的基因表达谱比较证实，该细菌经历了缺氧和缺氧的生长条件。此外，藻酸盐包封的铜绿假单胞菌表现出较低的呼吸速率比无张力对应物，并显示出对抗生素的高耐受性。铜绿假单胞菌在藻酸盐珠中的接种和生长代表了简单且灵活的体内样生物膜模型系统，其中细菌生长表现出体内生物膜的中心特征。这是通过在具有O2限制生长的二级基质中形成小细胞聚集体观察到的，通过添加NO3−作为替代电子受体和降低呼吸速率以及增强对抗生素处理的耐受性来缓解。重要性铜绿假单胞菌已被深入研究了几十年，由于其参与慢性感染，如囊性纤维化和慢性伤口，在那里它形成生物膜。许多研究致力于表面上的生物膜形成;然而，在慢性感染中，大多数生物膜形成不附着于表面的细胞的小聚集体，而是嵌入宿主材料中。在这项研究中，细菌被封装在小的藻酸盐珠中，并形成类似于在慢性细菌感染中观察到的聚集体。我们的研究结果表明，聚集体暴露于陡峭的氧气梯度，氧气耗尽区，硝酸盐可以作为氧气的替代品，使生长在氧气耗尽区。这一点很重要，因为在低氧条件下缓慢生长可能使细菌对抗生素具有耐受性。该模型提供了一种替代表面生物膜模型，并增加了生物膜不依赖于表面形成的理解。
Alginate beads represent a simple and highly reproducible in vitro model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ∼100 μm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3− as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3− as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment. IMPORTANCE Pseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.