Phenotypic and genetic diversification in a multi-species bacterial biofilm community

细菌在自然界中的生存能力大大提高了，因为它们能够在被称为生物膜的表面相关群落中生长。细菌生物膜包括密集的、基质包裹的细胞菌落，它们表现出复杂的化学相互作用和显著的结构形态。在生物膜系统中研究的细菌行为和功能的许多重要特性与在自由活细胞中观察到的根本不同。近年来，我们的研究表明，生物膜内的细菌可以在生物膜内驻留的细菌和从生物膜分离和分散的细胞中迅速获得广泛的表型和基因型多样性。例如，来自生物膜的细菌细胞通常表现出菌落形态、运动性、细胞表面结构特征和生长速度的变化。这种现象可以增强生物膜细胞在不同环境胁迫下的生存和传播能力。这种变异在生物体的几代传代中通常是稳定的，这意味着永久性和可遗传的遗传变化，并对一系列现象产生影响，包括细菌群落的适应性进化、细菌群落的代谢能力以及生物膜细胞建立慢性和耐抗生素感染的能力。因此，对于任何生物膜系统，了解生物膜群体内发生的遗传变异的程度和性质变得至关重要。此外，迄今为止，基因组变异只在单物种生物膜模型中进行了研究；多物种相互作用在基因组多样化中的作用在很大程度上尚未得到研究。该项目的目的是了解在多物种生物膜模型中，生物膜发育过程中细菌之间的相互作用如何影响细菌多样化的过程。我们将使用肺炎链球菌、流感嗜血杆菌和脑膜炎奈瑟菌——这些细菌的生物膜相关多样化尤为重要，因为针对这些生物的结合疫苗的引入为新的、非疫苗变异菌株重新占据这些致病生物以前的生态位提供了潜力。众所周知，与疫苗所覆盖的细菌相关的细菌物种可以通过物种内和物种间重组交换DNA而携带毒力基因。如果发生足够的遗传变异和重组，目前疫苗未覆盖的这些物种的变异菌株可能进化并适应重新占据生态位。我们将使用肺炎链球菌、流感嗜血杆菌和脑膜炎奈瑟菌的分离株，这些分离株以前已经通过荚膜分型、多位点序列分型（MLST）和抗生素敏感性得到了很好的表征。我们将首先开发一个模型系统来研究这些菌株的生物膜形成能力。分离株将使用连续培养流式细胞系统进行监测，该系统允许单种和多种生物膜的生长和微观观察，并通过该系统可以观察基于微菌落的生物膜生长。生物膜结构的生长和发育将在共聚焦激光扫描显微镜下进行监测，生物膜内的细菌种类将通过荧光蛋白标记或荧光原位杂交（FISH）技术进行标记和可视化。在生物膜发育过程中，在不同的时间间隔从流式细胞流出物中收集生物膜来源的细菌将被培养，并监测菌落形态的任何表型变化。为了能够比较生物膜衍生细菌和浮游细菌，还将使用浮游生物培养进行平行研究。将对代表性定植细菌的生物膜衍生和浮游培养进行荚膜血清分型、MLST和抗生素敏感性试验。

The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities known as biofilms. Bacterial biofilms comprise dense, matrix-encased colonies of cells which exhibit complex chemical interactions and significant structural morphology. Many important properties of bacterial behaviour and function studied in biofilm systems are fundamentally different from those observed in free-living cells. Recently our research has demonstrated that bacteria within biofilms can rapidly acquire extensive phenotypic and genotypic diversity both among resident biofilm bacteria and among cells that detach and disperse from biofilms. For example, bacterial cells derived from biofilms commonly exhibit variation in colony morphology, motility, cell-surface structural characteristics and growth rates. This phenomenon can enhance the ability of biofilm cells to persist and spread under diverse environmental stresses. The variation is often stable over several passages of the organism, implying permanent and heritable genetic change, and has implications for a range of phenomena including the adaptive evolution of bacterial communities, the metabolic capability of bacterial consortia, and the ability of biofilm cells to establish chronic and antibiotic-resistant infections. Thus, for any biofilm system, it becomes critical to understand the extent and nature of genetic variation that occurs within the biofilm population. Moreover, genomic variation has to date only been studied in monospecies biofilm models; the role of multispecies interactions on genomic diversification is largely unstudied. The aim of this project is to understand how interactions between bacteria during biofilm development can influence processes of bacterial diversification within a multi-species biofilm model. We will use the bacteria Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis - Biofilm-associated diversification of these bacteria is particularly important because the introduction of conjugate vaccines against these organisms has provided the potential for new, non-vaccine variant strains to re-occupy the previous niches of these pathogenic organisms. It is already known that bacterial species related to those covered by vaccines can harbour virulence genes due to the swapping of DNA by intra and inter-species recombination. If sufficient genetic variation and recombination occurs, variant strains of these species which are not covered by the present vaccines could evolve and adapt to re-occupy the niche. We will use isolates of S. pneumoniae, H. influenzae and N. meningitidis which have been previously well-characterised by capsular typing, multi-locus sequence typing (MLST) and antibiotic susceptibility. We will first develop a model system with which to study the biofilm forming ability of these bacterial strains. Isolates will be monitored using a continuous-culture flow cell system which allows for the growth and microscopic observation of single and multi-species biofilms and by which microcolony-based biofilm growth can be observed. Growth and development of biofilm architecture will be monitored under confocal laser scanning microscopy and bacterial species within biofilms will be tagged and visualised by using fluorescent protein markers or fluorescent in-situ hybridization (FISH) techniques. Biofilm-derived bacteria collected from the flow cell effluent at different time intervals during biofilm development will be cultured and any phenotypic changes in colony morphology monitored. To enable comparison between biofilm-derived and planktonic bacteria, parallel investigations will also be performed using planktonic cultures. Capsular serotyping, MLST and antibiotic susceptibility testing will be performed on biofilm-derived and planktonically-grown cultures of representative colonizing bacteria.