Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions
Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions
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DOI：
10.1128/aem.72.4.2885-2895.2006
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发表时间：
2006-04-01
影响因子：
4.4
通讯作者：
Hilbi, H
中科院分区：
文献类型：
--
作者：
Mampel, J;Spirig, T;Hilbi, H
嗜肺军团菌在水生栖息地存活了很长时间，在那里细菌与生物膜结合并在原生动物捕食者体内复制。虽然嗜肺乳杆菌是原生动物细胞内生长的范例，但在没有原生动物的情况下，细菌是否在生物膜内形成或复制尚不清楚。在这项研究中，我们分析了嗜肺乳杆菌在支持无菌复制的富培养基中的表面粘附和生物膜形成。用共聚焦显微镜和结晶紫染色分析了嗜肺乳杆菌毒株JR32与临床分离株和环境分离株的生物膜形成情况。菌株JR32在玻璃表面和直立聚苯乙烯孔上形成生物膜，以及在“反向”微量滴度板的引脚上形成生物膜，表明生物膜的形成不仅仅是由于细菌的沉淀。缺乏备选sigma因子或28的嗜肺乳杆菌fliA突变体的生物膜形成减少，这表明细菌因子是必需的。生物量的积累与光密度在600 nm处的增加一致，并在细菌达到固定生长阶段时停止。如果每天更换两次培养基，嗜肺乳杆菌在逆系统中既不生长也不形成生物膜。然而，加入棘阿米巴后，细菌增殖并粘附在表面。无根（表面附着）和浮游（自由游动）嗜肺乳杆菌表达p -半乳糖苷酶活性的程度相似，因此，观察到的表面附着细菌增殖不足不是由于蛋白质合成或代谢活性受损。绿色荧光蛋白（GFP）和dsred标记的嗜肺乳杆菌共培养导致细胞在基质上和聚集体上随机分布，没有观察到大规模的克隆生长细菌斑块。我们的研究结果表明，嗜肺乳杆菌在富培养基中形成生物膜是由于浮游细菌的生长，而不是由于无根细菌的生长。与这一结论一致，gfp标记的嗜肺乳杆菌最初粘附在连续流动的腔室系统中，但随着时间的推移分离；脱落与流速相关，没有生物量的积累。在这些条件下，嗜肺乳杆菌在由短恩培多杆菌或微杆菌形成的生物膜中持续存在，但在由肺炎克雷伯菌或其他环境细菌形成的生物膜中不存在，这表明细菌之间的特定相互作用调节了粘附性。
Legionella pneumophila persists for a long time in aquatic habitats, where the bacteria associate with biofilms and replicate within protozoan predators. While L. pneumophila serves as a paradigm for intracellular growth within protozoa, it is less clear whether the bacteria form or replicate within biofilms in the absence of protozoa. In this study, we analyzed surface adherence of and biofilm formation by L. pneumophila in a rich medium that supported axenic replication. Biofilm formation by the virulent L. pneumophila strain JR32 and by clinical and environmental isolates was analyzed by confocal microscopy and crystal violet staining. Strain JR32 formed biofilms on glass surfaces and upright polystyrene wells, as well as on pins of "inverse" microtiter plates, indicating that biofilm formation was not simply due to sedimentation of the bacteria. Biofilm formation by an L. pneumophila fliA mutant lacking the alternative sigma factor or 28 was reduced, which demonstrated that bacterial factors are required. Accumulation of biomass coincided with an increase in the optical density at 600 nm and ceased when the bacteria reached the stationary growth phase. L. pneumophila neither grew nor formed biofilms in the inverse system if the medium was exchanged twice a day. However, after addition of Acanthamoeba castellanii, the bacteria proliferated and adhered to surfaces. Sessile (surface-attached) and planktonic (free-swimming) L. pneumophila expressed P-galactosidase activity to similar extents, and therefore, the observed lack of proliferation of surface-attached bacteria was not due to impaired protein synthesis or metabolic activity. Cocultivation of green fluorescent protein (GFP)- and DsRed-labeled L. pneumophila led to randomly interspersed cells on the substratum and in aggregates, and no sizeable patches of clonally growing bacteria were observed. Our findings indicate that biofilm formation by L. pneumophila in a rich medium is due to growth of planktonic bacteria rather than to growth of sessile bacteria. In agreement with this conclusion, GFP-labeled L. pneumophila initially adhered in a continuous-flow chamber system but detached over time; the detachment correlated with the flow rate, and there was no accumulation of biomass. Under these conditions, L. pneumophila persisted in biofilms formed by Empedobacter breve or Microbacterium sp. but not in biofilms formed by Klebsiella pneumoniae or other environmental bacteria, suggesting that specific interactions between the bacteria modulate adherence.