Preferential feeding by the ciliates Chilodonella and Tetrahymena spp. and effects of these protozoa on bacterial biofilm structure and composition

Protozoa are important components of microbial food webs, but protozoan feeding preferences and their effects in the context of bacterial biofilms are not well understood. The feeding interactions of two contrasting ciliates, the free-swimming filter feeder Tetrahymena sp. and the surface-associated predator Chilodonella sp., were investigated using biofilm-forming bacteria genetically modified to express fluorescent proteins. According to microscopy, both ciliates readily consumed cells from both Pseudomonas costantinii and Serratia plymuthica biofilms. When offered a choice between spatially separated biofilms, each ciliate showed a preference for P. costantinii biofilms. Experiments with bacterial cell extracts indicated that both ciliates used dissolved chemical cues to locate biofilms. Chilodonella sp. evidently used bacterial chemical cues as a basis for preferential feeding decisions, but it was unclear whether Tetrahymena sp. did also. Confocal microscopy of live biofilms revealed that Tetrahymena sp. had a major impact on biofilm morphology, forming holes and channels throughout S. plymuthica biofilms and reducing P. costantinii biofilms to isolated, grazing-resistant microcolonies. Grazing by Chilodonella sp. resulted in the development of less-defined trails through S. plymuthica biofilms and caused P. costantinii biofilms to become homogeneous scatterings of cells. It was not clear whether the observed feeding preferences for spatially separated P. costantinii biofilms over S. plymuthica biofilms resulted in selective targeting of P. costantinii cells in mixed biofilms. Grazing of mixed biofilms resulted in the depletion of both types of bacteria, with Tetrahymena sp. having a larger impact than Chilodonella sp., and effects similar to those seen in grazed single-species biofilms.     

Architecture and spatial organization in a triple-species bacterial biofilm synergistically degrading the phenylurea herbicide linuron

Members of a triple-species 3-(3,4-dichlorophenyl)-1-methoxy-1-methyl urea (linuron)-mineralizing consortium, i.e. the linuron- and 3,4-dichloroaniline-degrading Variovorax sp. WDL1, the 3,4-dichloroaniline-degrading Comamonas testosteroni WDL7 and the N,O-dimethylhydroxylamine-degrading Hyphomicrobium sulfonivorans WDL6, were cultivated as mono- or multi-species biofilms in flow cells irrigated with selective or nonselective media, and examined with confocal laser scanning microscopy. In contrast to mono-species biofilms of Variovorax sp. WDL1, the triple-species consortium biofilm degraded linuron completely through apparent synergistic interactions. The triple-species linuron-fed consortium biofilm displayed a heterogeneous structure with an irregular surface topography that most resembled the topography of linuron-fed mono-species WDL1 biofilms, indicating that WDL1 had a dominating influence on the triple-species biofilm architecture. This architecture was dependent on the carbon source supplied, as the biofilm architecture of WDL1 growing on alternative carbon sources was different from that observed under linuron-fed conditions. Linuron-fed triple-species consortium biofilms consisted of mounds composed of closely associated WDL1, WDL7 and WDL6 cells, while this association was lost when the consortium was grown on a nonselective carbon source. In addition, under linuron-fed conditions, microcolonies displaying associated growth developed rapidly after inoculation. These observations indicate that the spatial organization in the linuron-fed consortium biofilm reflected the metabolic interactions within the consortium.     

Development and dynamics of Pseudomonas sp. biofilms

Pseudomonas sp. strain B13 and Pseudomonas putida OUS82 were genetically tagged with the green fluorescent protein and the Discosoma sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of P. putida OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.     

基于COMSTAT软件对微生物被膜进行定量分析的方法与意义

目的:微生物被膜是一种具有协调性、功能性和高度结构性的膜状复合物,它可以为微生物提供良好的生存环境,免受外界因素的干扰。研究发现,具有产生微生物被膜能力的细菌治病性明显增强,而现今缺乏一种分析微生物被膜的有效手段。本文探讨利用COMSTAT软件对微生物被膜进行定量分析的方法和意义,为对微生物被膜定性定量分析提供支持手段,从而为研究微生物被膜致病性提供方法理论基础。方法:以葡萄球菌为研究模型,利用激光扫描共聚焦显微镜成像技术,结合COMSTAT微生物被膜分析软件对微生物被膜的单位面积生物量、基质覆盖率、平均厚度、粗糙系数等方面进行定量分析,研究了该葡萄球菌的生物被膜生长变化过程,并考察了抗生素对其生物被膜的抑制作用。结果:在葡萄球菌生物被膜生长过程中,生物量、平均厚度以及平均扩散距离等结构指标数值都有明显增加,而粗糙度和表面积与生物量比值呈现降低趋势,表明了微生物被膜由发生向成熟的转化过程。与此同时,经10μg/mL和100μg/mL的卡那霉素处理得到的葡萄球菌微生物被膜生长受到明显抑制,且随着卡那霉素的浓度增加,抑制效果随之增加。结论:本文运用COMSTAT软件的分析方法首次从生物量、平均厚度等结构指标数值的角度描述了葡萄球菌生物被膜,从而有效评价微生物被膜发生、发展、成熟以及崩解的生长过程。该技术在研究微生物被膜形成的理论机制方面存在潜在价值,可以为研究微生物被膜治病性提供理论基础,具有理论指导意义。     

Analysis of size distribution and areal cell density of ammonia-oxidizing bacterial microcolonies in relation to substrate microprofiles in biofilms

A fine-scale in situ spatial organization of ammonia-oxidizing bacteria (AOB) in biofilms was investigated by combining molecular techniques (i.e., fluorescence in situ hybridization (FISH) and 16S rDNA-cloning analysis) and microelectrode measurements. Important parameters of AOB microcolonies such as size distribution and areal cell density of the microcolonies were determined and correlated with substrate microprofiles in the biofilms. In situ hybridization with a nested 16S rRNA-targeted oligonucleotide probe set revealed two different populations of AOB, Nitrosomonas europaea-lineage and Nitrosospira multiformis-lineage, coexisting in an autotrophic nitrifying biofilm. Nitrosospira formed looser microcolonies, with an areal cell density of 0.51 cells microm(-2), which was half of the cell density of Nitrosomonas (1.12 cells microm(-2)). It is speculated that the formation of looser microcolonies facilitates substrate diffusion into the microcolonies, which might be a survival strategy to low O(2) and NH(4) (+) conditions in the biofilm. A long-term experiment (4-week cultivation at different substrate C/N ratios) revealed that the size distribution of AOB microcolonies was strongly affected by better substrate supply due to shorter distance from the surface and the presence of organic carbon. The microcolony size was relatively constant throughout the autotrophic nitrifying biofilm, while the size increased by approximately 80% toward the depth of the biofilm cultured at the substrate C/N = 1. A short-term ( approximately 3 h) organic carbon addition experiment showed that the addition of organic carbon created interspecies competition for O(2) between AOB and heterotrophic bacteria, which dramatically decreased the in situ NH(4) (+)-uptake activity of AOB in the surface of the biofilms. This result might explain the spatial distribution of AOB microcolony size in the biofilms cultured at the substrate C/N = 1. These experimental results suggest O(2) and organic carbon were the main factors controlling the spatial organization and activity of AOB in biofilms. These findings are significantly important to further improve mathematical models used to describe how the slow-growing AOB develop their niches in biofilms and how that configuration affects nitrification performance in the biofilm.     

Formation and growth of heterotrophic aerobic biofilms on small suspended particles in airlift reactors

In this article, the conditions for aerobic biofilm formation on suspended particles, the dynamics of biofilm formation, and the biomass production during the start-up of a Biofilm Airlift Suspension reactor (BAS reactor) have been studied. The dynamics of biofilm formation during start up in the biofilm airlift suspension reactor follows three consecutive stages: bare carrier, microcolonies or patchy biofilms on the carrier, and biofilms completely covering the carrier. The effect of hydraulic retention time and of substrate loading rate on the formation of biofilms were investigated. To obtain in a BAS reactor a high biomass concentration and predominantly continuous biofilms, which completely surround the carrier, the hydraulic retention time must be shorter than the inverse of the maximum growth rate of the suspended bacteria. At longer hydraulic retention times, a low amount of attached biomass can be present on the carrier material as patchy biofilms. During the start-up at short hydraulic retention times the bare carrier concentration decreases, the amount of biomass per biofilm particle remains constant, and biomass increase in the reactor is due to increasing numbers of biofilm particles. The substrate surface loading rate has effect only on the amount of biomass on the biofilm particle. A higher surface load leads to a thicker biofilm.A strong nonlinear increase of the concentration of attached biomass in time was observed. This can be explained by a decreased abrasion of the biofilm particles due to the decreasing concentration of bare carriers. The detachment rate per biofilm area during the start-up is independent of the substrate loading rate, but depends strongly upon the bare carrier concentration.The Pirt-maintenance concept is applicable to BAS reactors. Surplus biomass production is diminished at high biomass concentrations. The average maximal yield of biomass on substrate during the experiments presented in this article was 0.44 +/- 0.08 C-mol/C-mol, the maintenance value 0.019 +/- 0.012 C-mol/(C-mol h). The lowest actual biomass yield measured in this study was 0.15 C-mol/C-mol. (c) 1994 John Wiley & Sons, Inc.     

Enhanced biofilm formation and 3-chlorobenzoate degrading activity by the bacterial consortium of Burkholderia sp. NK8 and Pseudomonas aeruginosa PAO1

Aims:  To characterize biofilm formation of a chlorobenzoates (CBs) degrading bacterium, Burkholderia sp. NK8, with another bacterial species, and the biodegradation activity against CBs in the mixed-species biofilm.
Methods and Results:  Burkholderia sp. NK8 was solely or co-cultured with each of five other representative bacteria in microtitre dishes. Biofilm formation involving the strain NK8 was synergistically promoted by co-culturing with only Pseudomonas aeruginosa PAO1. Epifluorescent microscopy revealed that cells of the bacterial strain NK8 were viable and distributed randomly in the mixed-species biofilms. Enumeration of the attached cells on the surface of wells revealed that cells of the strain NK8 increased approx. 10-fold by the co-culture with the strain PAO1 compared to those by monoculture of the strain NK8, and the degradation activity of 3-chlorobenzoate by the dual-species biofilms was more promoted than that by the strain NK8-monocultured biofilms.
Conclusions:  Enhanced biofilm formation of Burkholderia sp. NK8 by the bacterial consortium occurred, but is determined by the partner bacterial species. The mixed-species biofilms have the advantage to degrade CBs on a solid surface.
Significance and Impact of the Study:  This study provides a significance of bacterial consortia on the biofilm formation and the degradation activity of Burkholderia sp. NK8, which contribute for complete degradation of chlorinated aromatics.     

Waveguide evanescent field scattering microscopy: bacterial biofilms and their sterilization response via UV irradiation

Waveguide Evanescent Field Scattering (WEFS) microscopy is introduced as a new and simple tool for label‐free, high contrast imaging of bacteria and bacteria sensors. Bacterial microcolonies and single bacteria were discriminated both by their bright field images and by their evanescent scattering intensity. By comparing bright field images with WEFS images, the proportion of planktonic: sessile (i.e., “floating”: attached) bacteria were measured. Bacteria were irradiated with UV light, which limited their biofilm forming capability. A quantitative decrease in attachment of individual, sessile bacteria and in attached, microcolony occupied areas was easily determined within the apparent biofilms with increasing UV dose. WEFS microscopy is an ideal tool for providing rapid quantitative data on biofilm formation. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Grazing resistance of Pseudomonas aeruginosa biofilms depends on type of protective mechanism, developmental stage and protozoan feeding mode

In a previous study we identified microcolony formation and inhibitor production as the major protective mechanisms of Pseudomonas aeruginosa biofilms against flagellate grazing. Here we compared the efficacy of these two key protective mechanisms by exposing biofilms of the non-toxic alginate overproducing strain PDO300 and the wild-type toxic strain PAO1 to a range of feeding types commonly found in the succession of protozoans associated with natural biofilms. Alginate-mediated microcolony formation conferred effective protection for strain PDO300 against the suspension feeding flagellate Bodo saltans and, as reported earlier, the surface feeding flagellate Rhynchomonas nasuta, both of which are considered as early biofilm colonizers. However, microcolonies of mature PDO300 biofilms were highly susceptible to late biofilm colonizers, the surface-feeding amoeba Acanthamoeba polyphaga and the planktonic ciliate Tetrahymena sp., resulting in a significant reduction of biofilm biomass. Mature biofilms of strain PAO1 inhibited growth of flagellates and A. polyphaga while the grazing activity of Tetrahymena sp. remained unaffected. Our findings suggest that inhibitor production of mature P. aeruginosa biofilms is effective against a wider range of biofilm-feeding predators while microcolony-mediated protection is only beneficial in the early stages of biofilm formation.     

A stochastic mechanism for biofilm formation by Mycoplasma pulmonis

Bacterial biofilms are communities of bacteria that are enclosed in an extracellular matrix. Within a biofilm the bacteria are protected from antimicrobials, environmental stresses, and immune responses from the host. Biofilms are often believed to have a highly developed organization that is derived from differential regulation of the genes that direct the synthesis of the extracellular matrix and the attachment to surfaces. The mycoplasmas have the smallest of the prokaryotic genomes and apparently lack complex gene-regulatory systems. We examined biofilm formation by Mycoplasma pulmonis and found it to be dependent on the length of the tandem repeat region of the variable surface antigen (Vsa) protein. Mycoplasmas that produced a short Vsa protein with few tandem repeats formed biofilms that attached to polystyrene and glass. Mycoplasmas that produced a long Vsa protein with many tandem repeats formed microcolonies that floated freely in the medium. The biofilms and the microcolonies contained an extracellular matrix which contained Vsa protein, lipid, DNA, and saccharide. As variation in the number of Vsa tandem repeats occurs by slipped-strand mispairing, the ability of the mycoplasmas to form a biofilm switches stochastically.     

Diversity in surface colonization behavior in marine bacteria

Using laminar flow chambers and time-lapse video imaging, colonization of surfaces by four marine bacteria revealed a diverse range of morphological characteristics and cell-cell interactions. The strain SW5 formed a compact, multilayered single- and double-cell biofilm on hydrophobic surfaces but developed long multicellular chains on hydrophilic surfaces. The morphologically similar SW8 showed unusual proximal vertical packing of cells on both substrata.Vibrio sp strain S14 exhibited cyclical colonization-detachment events on both substrata.Pseudomonas sp strain S9 initially displayed reversible and then irreversible adhesion apparently triggered by a cell density phenomenon that led to the development of regular microcolonies on both substrata with individual cells translocating between the colonies. The length of time bacteria were exposed to and their density at a surface influenced behavioral traits, with diverse and distinctive species-specific behavioral events.     

Structural organization and dynamics of exopolysaccharide matrix and microcolonies formation by Streptococcus mutans in biofilms

Aims: To investigate the structural organization and dynamics of exopolysaccharides (EPS) matrix and microcolonies formation by Streptococcus mutans during the biofilm development process. Methods and Results: Biofilms of Strep. mutans were formed on saliva‐coated hydroxyapatite (sHA) discs in the presence of glucose or sucrose (alone or mixed with starch). At specific time points, biofilms were subjected to confocal fluorescence imaging and computational analysis. EPS matrix was steadily formed on sHA surface in the presence of sucrose during the first 8 h followed by a threefold biomass increase between 8 and 30 h of biofilm development. The initial formation and further development of three‐dimensional microcolony structure occurred concomitantly with EPS matrix synthesis. Tridimensional renderings showed EPS closely associated with microcolonies throughout the biofilm development process forming four distinct domains (i) between sHA surface and microcolonies, (ii) within, (iii) covering and (iv) filling the spaces between microcolonies. The combination of starch and sucrose resulted in rapid formation of elevated amounts of EPS matrix and faster assembly of microcolonies by Strep. mutans, which altered their structural organization and susceptibility of the biofilm to acid killing (vs sucrose‐grown biofilms; P < 0·05). Conclusions: Our data indicate that EPS modulate the development, sequence of assembly and spatial distribution of microcolonies by Strep. mutans. Significance and Impact of the Study: Simultaneous visualization and analysis of EPS matrix and microcolonies provide a more precise examination of the structural organization of biofilms than labelling bacteria alone, which could be a useful approach to elucidate the exact mechanisms by which Strep. mutans influences oral biofilm formation and possibly identify novel targets for effective antibiofilm therapies.     

Structural changes in S. epidermidis biofilms after transmission between stainless steel surfaces

Transmission is a main route for bacterial contamination, involving bacterial detachment from a donor and adhesion to receiver surfaces. This work aimed to compare transmission of an extracellular polymeric substance (EPS) producing and a non-EPS producing Staphylococcus epidermidis strain from biofilms on stainless steel. After transmission, donor surfaces remained fully covered with biofilm, indicating transmission through cohesive failure in the biofilm. Counter to the numbers of biofilm bacteria, the donor and receiver biofilm thicknesses did not add up to the pre-transmission donor biofilm thickness, suggesting more compact biofilms after transmission, especially for non-EPS producing staphylococci. Accordingly, staphylococcal density per unit biofilm volume had increased from 0.20 to 0.52 μm^–3 for transmission of the non-EPS producing strain under high contact pressure. The EPS producing strain had similar densities before and after transmission (0.17 μm^–3). This suggests three phases in biofilm transmission: (1) compression, (2) separation and (3) relaxation of biofilm structure to its pre-transmission density in EPS-rich biofilms.     

Interactions between non-phospholipid liposomes containing cetylpyridinium chloride and biofilms of Streptococcus mutans: modulation of the adhesion and of the biodistribution

Cetylpyridinium chloride (CPC) is a surfactant that binds strongly to bacteria and bacterial biofilms. In this study, fluorescence-based techniques were used to determine the penetration and adhesion of CPC when it was introduced in liposomes. In spite of a reduced adhesion as compared to pure CPC micelles, CPC-containing liposomes adhered significantly to the biofilms of Streptococcus mutans. In contrast, no binding was observed for liposomes that were composed of phosphatidylcholine-cholesterol. The influence of the charge of the liposome on its adhesion to biofilms was studied using cholesterol (Chol) and cholesterol sulfate (Schol). In spite of similar binding to the biofilms, positively charged CPC/Chol liposomes were located mainly in the core of the biofilm microcolonies, whereas the negatively charged CPC/Schol liposomes were mainly concentrated at their periphery. This effect may be attributed to the different availability of the CPC head group. In summary, this work demonstrates the high potential for tailoring drug nanovectors by modulating sterol selection in order to selectively target and bind biofilms.     

生物被膜的物理特性及其表征

生物被膜涉及到人类生产生活的方方面面。生物被膜的形成有时是有益的,可用于生物降解、生物催化等;但同时也造成了诸多不利的影响,医疗领域中的感染性疾病、工业生产中的生物污损等均与生物被膜的形成有关。生物被膜形成过程中的物理性质决定着生物被膜的形态结构以及机械稳定性,对它在应对外界环境刺激并得以生存具有重要的意义。本文介绍了生物被膜形成初期和发展过程中的物理性质以及相应的表征手段。其中,细菌的表面粘附由细菌的近界面运动行为及细菌与表面的相互作用决定,并对生物被膜的初期形成起关键的作用。此外,机械性能测试发现成熟的生物被膜可看作具备粘弹性的聚合物。     

Bacteria can form interconnected microcolonies when a self-excreted product reduces their surface motility: evidence from individual-based model simulations

Recent experimental observations of Pseudomonas aeruginosa, a model bacterium in biofilm research, reveal that, under specific growth conditions, bacterial cells form patterns of interconnected microcolonies. In the present work, we use an individual-based model to assess the involvement of bacteria motility and self-produced extracellular substance in the formation of these patterns. In our simulations, the pattern of interconnected microcolonies appears only when bacteria motility is reduced by excreted extracellular macromolecules. Immotile bacteria form isolated microcolonies and constantly motile bacteria form flat biofilms. Based on experimental data and computer simulations, we suggest a mechanism that could be responsible for these interconnected microcolonies.     

Biofilms: strategies for metal corrosion inhibition employing microorganisms

Corrosion causes dramatic economic loss. Currently widely used corrosion control strategies have disadvantages of being expensive, subject to environmental restrictions, and sometimes inefficient. Studies show that microbial corrosion inhibition is actually a common phenomenon. The present review summarizes recent progress in this novel strategy: corrosion control using beneficial bacteria biofilms. The possible mechanisms may involve: (1) removal of corrosive agents (such as oxygen) by bacterial physiological activities (e.g., aerobic respiration), (2) growth inhibition of corrosion-causing bacteria by antimicrobials generated within biofilms [e.g., sulfate-reducing bacteria (SRB) corrosion inhibition by gramicidin S-producing Bacillus brevis biofilm], (3) generation of protective layer by biofilms (e.g., Bacillus licheniformis biofilm produces on aluminum surface a sticky protective layer of γ-polyglutamate). Successful utilization of this novel strategy relies on advances in study at the interface of corrosion engineering and biofilm biology.     

Effects of Disinfection on Legionella spp., Eukarya, and Biofilms in a Hot Water System

Legionella species are frequently detected in hot water systems, attached to the surface as a biofilm. In this work, the dynamics of Legionella spp. and diverse bacteria and eukarya associated together in the biofilm, coming from a pilot scale 1 system simulating a real hot water system, were investigated throughout 6 months after two successive heat shock treatments followed by three successive chemical treatments. Community structure was assessed by a fingerprint technique, single-strand conformation polymorphism (SSCP). In addition, the diversity and dynamics of Legionella and eukarya were investigated by small-subunit (SSU) ribosomal cloning and sequencing. Our results showed that pathogenic Legionella species remained after the heat shock and chemical treatments (Legionella pneumophila and Legionella anisa, respectively). The biofilm was not removed, and the bacterial community structure was transitorily affected by the treatments. Moreover, several amoebae had been detected in the biofilm before treatments (Thecamoebae sp., Vannella sp., and Hartmanella vermiformis) and after the first heat shock treatment, but only H. vermiformis remained. However, another protozoan affiliated with Alveolata, which is known as a host cell for Legionella, dominated the eukaryal species after the second heat shock and chemical treatment tests. Therefore, effective Legionella disinfection may be dependent on the elimination of these important microbial components. We suggest that eradicating Legionella in hot water networks requires better study of bacterial and eukaryal species associated with Legionella in biofilms.     

牙菌斑生物膜的形成与控制

牙菌斑生物膜是附着于牙釉质表面,由复杂的微生物群落构成的一种聚集体。牙菌斑生物膜的形成与生长对口腔健康有着直接或间接的影响,许多研究证实口腔疾病如龋齿和牙周病都与细菌的积累及牙菌斑的形成有关。在牙菌斑生物膜形态建成过程中,牙齿表面最初的定殖菌对生物膜的微生物组成和结构至关重要,这些初级定殖菌决定了后续与之结合形成共生体的微生物种类和数量。不同的微生物组成可能在与生物膜形成相关的口腔病理状况中发挥不同的作用。因此,本文就牙菌斑生物膜的生长及控制进行综述,介绍其微生物的早期定殖和成熟过程、以及通过物理和化学方法对牙菌斑生物膜的控制,以期为了解牙菌斑生物膜的形成机制及相关口腔疾病的预防和治疗提供有价值的参考。     

The role of quorum sensing mediated developmental traits in the resistance of Serratia marcescens biofilms against protozoan grazing

Resistance against protozoan grazers is a crucial factor that is important for the survival of many bacteria in their natural environment. However, the basis of resistance to protozoans and how resistance factors are regulated is poorly understood. In part, resistance may be due to biofilm formation, which is known to protect bacteria from environmental stress conditions. The ubiquitous organism Serratia marcescens uses quorum sensing (QS) control to regulate virulence factor expression and biofilm formation. We hypothesized that the QS system of S. marcescens also regulates mechanisms that protect biofilms against protozoan grazing. To investigate this hypothesis, we compared the interactions of wild-type and QS mutant strains of S. marcescens biofilms with two protozoans having different feeding types under batch and flow conditions. Under batch conditions, S. marcescens forms microcolony biofilms, and filamentous biofilms are formed under flow conditions. The microcolony-type biofilms were protected from grazing by the suspension feeder, flagellate Bodo saltans, but were not protected from the surface feeder, Acanthamoeba polyphaga. In contrast, the filamentous biofilm provided protection against A. polyphaga. The main findings presented in this study suggest that (i) the QS system is not involved in grazing resistance of S. marcescens microcolony-type biofilms; (ii) QS in S. marcescens regulates antiprotozoan factor(s) that do not interfere with the grazing efficiency of the protozoans; and (iii) QS-controlled, biofilm-specific differentiation of filaments and cell chains in biofilms of S. marcescens provides an efficient mechanism against protozoan grazing.