Bacterial Community Structure of Biofilms on Artificial Surfaces in an Estuary

This study examined bacterial community structure of biofilms on stainless steel and polycarbonate in seawater from the Delaware Bay. Free-living bacteria in the surrounding seawater were compared to the attached bacteria during the first few weeks of biofilm growth. Surfaces exposed to seawater were analyzed by using 16S rDNA libraries, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE). Community structure of the free-living bacterial community was different from that of the attached bacteria according to FISH and DGGE. In particular, alpha-proteobacteria dominated the attached communities. Libraries of 16S rRNA genes revealed that representatives of the Rhodobacterales clade were the most abundant members of biofilm communities. Changes in community structure during biofilm growth were also examined by DGGE analysis. We hypothesized that bacterial communities on dissimilar surfaces would initially differ and become more similar over time. In contrast, the compositions of stainless steel and polycarbonate biofilms were initially the same, but differed after about 1 week of biofilm growth. These data suggest that the relationship between surface properties and biofilm community structure changes as biofilms grow on surfaces such as stainless steel and polycarbonate in estuarine water.     

Bacterial Community Composition of Stream Biofilms in Spatially Variable-Flow Environments

Streams are highly heterogeneous ecosystems, in terms of both geomorphology and hydrodynamics. While flow is recognized to shape the physical architecture of benthic biofilms, we do not yet understand what drives community assembly and biodiversity of benthic biofilms in the heterogeneous flow landscapes of streams. Within a metacommunity ecology framework, we experimented with streambed landscapes constructed from bedforms in large-scale flumes to illuminate the role of spatial flow heterogeneity in biofilm community composition and biodiversity in streams. Our results show that the spatial variation of hydrodynamics explained a remarkable percentage (up to 47%) of the variation in community composition along bedforms. This suggests species sorting as a model of metacommunity dynamics in stream biofilms, though natural biofilm communities will clearly not conform to a single model offered by metacommunity ecology. The spatial variation induced by the hydrodynamics along the bedforms resulted in a gradient of bacterial beta diversity, measured by a range of diversity and similarity indices, that increased with bedform height and hence with spatial flow heterogeneity at the flume level. Our results underscore the necessity to maintain small-scale physical heterogeneity for community composition and biodiversity of biofilms in stream ecosystems.Biofilms (attached and matrix-enclosed microbial communities) are an important form of microbial life in streams and rivers, where they can greatly contribute to ecosystem functions and even large-scale carbon fluxes (1, 3). Streams are inherently heterogeneous and are characterized by a largely unidirectional downstream flow of water that controls the dispersal of suspended microorganisms (21), biofilm community composition (7), architecture (2), and metabolism (13), for instance. However, we do not understand how diverse microorganisms assemble into biofilm communities based on flow heterogeneity and related dispersal in these ecosystems.Dispersal, as the propagation and immigration of biota, can have important consequences for biodiversity and ecosystem functioning in heterogeneous landscapes (18, 25). Landscape topography and turbulent transport affect dispersal, a relationship that is well studied in the dispersal of plant seeds (31) but not in the microbial world. Only recently have microbial ecologists begun to understand the role of dispersal in large-scale biogeographic patterns (29) and metacommunity ecology (24, 44). This growing body of research on microbial dispersal and its consequences for spatial patterns of community assembly and composition rests entirely on free-living bacteria, while no comparable data exist for microbial biofilms. The confirmation of detachment as an intrinsic behavior in many biofilms has led to the appreciation of dispersal as an insurance policy for these microbial communities to seed new habitats during resource limitation or aging of the parental biofilm (4). However, microbial ecology lacks conceptual models to predict postemigration processes, such as cell propagation, immigration, and community assembly during colonization of new surfaces. The perception of biofilms as microbial landscapes and, at the same time, as integrated parts of the landscape they inhabit offers the possibility to test models for habitat selection by dispersal cells (4). In this study, we focused on the assembly of biofilm communities by dispersal cells in spatially variable-flow environments; we did not measure dispersal as the emigration of cells from established biofilms. We adopted metacommunity ecology as a framework that encapsulates environmental heterogeneity and dispersal (18) to illuminate the mechanisms underlying community assembly.If the effects of microbial diversity on ecosystem functions are to be understood, we need to address the proper spatial resolution at which microorganisms assemble into communities and at which their functioning becomes manifest. In streams, this is typically at the level of habitats and microhabitats ranging from meters to centimeters, where characteristic geomorphological features (e.g., bedforms) and induced hydrodynamic fields develop and where spatial variations in biofilm metabolism become apparent (13). The ensemble of these small-scale variations translates into the landscape heterogeneity of the streambed.The aim of this study was to test whether spatial flow heterogeneity generating diverse microhabitats induces spatial species turnover and increases the biodiversity of microbial biofilms. Microbial metacommunity ecology predicts mass effects rather than species sorting to drive community composition in ecosystems with low residence time, such as streams (14, 18, 24). To test this prediction, we constructed six streambed landscapes from bedforms of defined dimensions differing in height; the mean flow (at flume scale) was kept constant, whereas the spatial heterogeneity of flow increased across the gradient of the six landscapes. The inoculum (i.e., the stream water and naturally contained microorganisms) and water chemistry were equal in all flumes. This allowed us to isolate flow heterogeneity as a potential driver of biofilm community composition in a high-energy ecosystem. We used terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA gene sequences from winter and summer communities and related bacterial community composition and microbial biomass to the hydrodynamics in representative microhabitats using causal modeling and forward selection of explanatory variables (9, 23).     

Stratification of Activity and Bacterial Community Structure in Biofilms Grown on Membranes Transferring Oxygen

Previous studies have shown that membrane-aerated biofilm (MAB) reactors can simultaneously remove carbonaceous and nitrogenous pollutants from wastewater in a single reactor. Oxygen is provided to MABs through gas-permeable membranes such that the region nearest the membrane is rich in oxygen but low in organic carbon, whereas the outer region of the biofilm is void of oxygen but rich in organic carbon. In this study, MABs were grown under similar conditions but at two different fluid velocities (2 and 14 cm s⁻¹) across the biofilm. MABs were analyzed for changes in biomass density, respiratory activity, and bacterial community structure as functions of biofilm depth. Biomass density was generally highest near the membrane and declined with distance from the membrane. Respiratory activity exhibited a hump-shaped profile, with the highest activity occurring in the middle of the biofilm. Community analysis by PCR cloning and PCR-denaturing gradient gel electrophoresis of 16S rRNA genes demonstrated substantial stratification of the community structure across the biofilm. Population profiles were also generated by competitive quantitative PCR of gene fragments specific for ammonia-oxidizing bacteria (AOB) (amoA) and denitrifying bacteria (nirK and nirS). At a flow velocity of 14 cm s⁻¹, AOB were found only near the membrane, whereas denitrifying bacteria proliferated in the anoxic outer regions of the biofilm. In contrast, at a flow velocity of 2 cm s⁻¹, AOB were either not detected or detected at a concentration near the detection limit. This study suggests that, under the appropriate conditions, both AOB and denitrifying bacteria can coexist within an MAB.     

Effects of Photosynthesis on Bacterial Phosphatase Production in Biofilms

The fraction of bacteria displaying phosphatase activity within natural photosynthetic biofilms was examined in relation to phosphorus limitation and algal photosynthesis. An artificial substrate that forms a fluorescent precipitate was used in conjunction with the nucleic acid stain DAPI to enumerate extracellular phosphatase expression by biofilm bacteria exposed to different photosynthetic activities and phosphorus supplies. The proportion of bacteria displaying phosphatase activity changed in response to the presence or absence of algal photosynthesis. In general, phosphate-deprived biofilms had positive linear trends in bacterial phosphatase activity (p <0.001), with greater proportions of bacteria displaying phosphatase under photosynthetic inhibition compared to active photosynthesis. Under sufficient phosphate supplies, biofilms had negative linear trends (p <0.05) or were lower in the proportion of bacteria displaying phosphatase activity in the presence of algal photosynthesis, whereas bacterial phosphatase activity was generally maintained when photosynthesis was inhibited. it is suggested that the amount of extracellular organic carbon released within the biofilm matrix during photosynthesis indirectly affected bacterial phosphatase synthesis.     

Biophysical Controls on Community Succession in Stream Biofilms

Biofilm formation is controlled by an array of coupled physical, chemical, and biotic processes. Despite the ecological relevance of microbial biofilms, their community formation and succession remain poorly understood. We investigated the effect of flow velocity, as the major physical force in stream ecosystems, on biofilm community succession (as continuous shifts in community composition) in microcosms under laminar, intermediate, and turbulent flow. Flow clearly shaped the development of biofilm architecture and community composition, as revealed by microscopic investigation, denaturing gradient gel electrophoresis (DGGE) analysis, and sequencing. While biofilm growth patterns were undirected under laminar flow, they were clearly directed into ridges and conspicuous streamers under turbulent flow. A total of 51 biofilm DGGE bands were detected; the average number ranged from 13 to 16. Successional trajectories diverged from an initial community that was common in all flow treatments and increasingly converged as biofilms matured. We suggest that this developmental pattern was primarily driven by algae, which, as “ecosystem engineers,” modulate their microenvironment to create similar architectures and flow conditions in all treatments and thereby reduce the physical effect of flow on biofilms. Our results thus suggest a shift from a predominantly physical control to coupled biophysical controls on bacterial community succession in stream biofilms.     

Homeostatic Interplay between Bacterial Cell-Cell Signaling and Iron in Virulence

Pathogenic bacteria use interconnected multi-layered regulatory networks, such as quorum sensing (QS) networks to sense and respond to environmental cues and external and internal bacterial cell signals, and thereby adapt to and exploit target hosts. Despite the many advances that have been made in understanding QS regulation, little is known regarding how these inputs are integrated and processed in the context of multi-layered QS regulatory networks. Here we report the examination of the Pseudomonas aeruginosa QS 4-hydroxy-2-alkylquinolines (HAQs) MvfR regulatory network and determination of its interaction with the QS acyl-homoserine-lactone (AHL) RhlR network. The aim of this work was to elucidate paradigmatically the complex relationships between multi-layered regulatory QS circuitries, their signaling molecules, and the environmental cues to which they respond. Our findings revealed positive and negative homeostatic regulatory loops that fine-tune the MvfR regulon via a multi-layered dependent homeostatic regulation of the cell-cell signaling molecules PQS and HHQ, and interplay between these molecules and iron. We discovered that the MvfR regulon component PqsE is a key mediator in orchestrating this homeostatic regulation, and in establishing a connection to the QS rhlR system in cooperation with RhlR. Our results show that P. aeruginosa modulates the intensity of its virulence response, at least in part, through this multi-layered interplay. Our findings underscore the importance of the homeostatic interplay that balances competition within and between QS systems via cell-cell signaling molecules and environmental cues in the control of virulence gene expression. Elucidation of the fine-tuning of this complex relationship offers novel insights into the regulation of these systems and may inform strategies designed to limit infections caused by P. aeruginosa and related human pathogens.     

Nanoparticle-Encapsulated Chlorhexidine against Oral Bacterial Biofilms

Background

Chlorhexidine (CHX) is a widely used antimicrobial agent in dentistry. Herein, we report the synthesis of a novel mesoporous silica nanoparticle-encapsulated pure CHX (Nano-CHX), and its mechanical profile and antimicrobial properties against oral biofilms.

Methodology/Principal Findings

The release of CHX from the Nano-CHX was characterized by UV/visible absorption spectroscopy. The antimicrobial properties of Nano-CHX were evaluated in both planktonic and biofilm modes of representative oral pathogenic bacteria. The Nano-CHX demonstrated potent antibacterial effects on planktonic bacteria and mono-species biofilms at the concentrations of 50–200 µg/mL against Streptococcus mutans, Streptococcus sobrinus, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Enterococccus faecalis. Moreover, Nano-CHX effectively suppressed multi-species biofilms such as S. mutans, F. nucleatum, A. actinomycetemcomitans and Porphyromonas gingivalis up to 72 h.

Conclusions/Significance

This pioneering study demonstrates the potent antibacterial effects of the Nano-CHX on oral biofilms, and it may be developed as a novel and promising anti-biofilm agent for clinical use.     

Distribution of Bacterial Growth Activity in Flow-Chamber Biofilms

In microbial communities such as those found in biofilms, individual organisms most often display heterogeneous behavior with respect to their metabolic activity, growth status, gene expression pattern, etc. In that context, a novel reporter system for monitoring of cellular growth activity has been designed. It comprises a transposon cassette carrying fusions between the growth rate-regulated Escherichia coli rrnBP1 promoter and different variant gfp genes. It is shown that the P1 promoter is regulated in the same way in E. coli and Pseudomonas putida, making it useful for monitoring of growth activity in organisms outside the group of enteric bacteria. Construction of fusions to genes encoding unstable Gfp proteins opened up the possibility of the monitoring of rates of rRNA synthesis and, in this way, allowing on-line determination of the distribution of growth activity in a complex community. With the use of these reporter tools, it is demonstrated that individual cells of a toluene-degrading P. putida strain growing in a benzyl alcohol-supplemented biofilm have different levels of growth activity which develop as the biofilm gets older. Cells that eventually grow very slowly or not at all may be stimulated to restart growth if provided with a more easily metabolizable carbon source. Thus, the dynamics of biofilm growth activity has been tracked to the level of individual cells, cell clusters, and microcolonies.     

Cell-Cell Interactions during Vascular Development

Received 1 September 2001/ Accepted in revised form 15 October 2001     

Bacterial Influences on Animal Origins

Animals evolved in seas teeming with bacteria, yet the influences of bacteria on animal origins are poorly understood. Comparisons among modern animals and their closest living relatives, the choanoflagellates, suggest that the first animals used flagellated collar cells to capture bacterial prey. The cell biology of prey capture, such as cell adhesion between predator and prey, involves mechanisms that may have been co-opted to mediate intercellular interactions during the evolution of animal multicellularity. Moreover, a history of bacterivory may have influenced the evolution of animal genomes by driving the evolution of genetic pathways for immunity and facilitating lateral gene transfer. Understanding the interactions between bacteria and the progenitors of animals may help to explain the myriad ways in which bacteria shape the biology of modern animals, including ourselves.The first bacteria evolved more than 3 billion years ago and dominated the biosphere continually thereafter, shaping the environment in which animals would eventually evolve more than 2 billion years later (Narbonne 2005; Knoll 2011). Because animals evolved in seas filled with bacteria and have lived in close association with bacteria throughout their evolutionary history, it is likely that diverse interactions with bacteria (including predation on bacteria, harboring bacterial commensals, and infection with bacterial pathogens) influenced animal origins. Nonetheless, although the potential contributions of global environmental change and genome evolution to animal origins have received a fair amount of attention (Hoffman et al. 1998; Knoll and Carroll 1999; Knoll 2003; King 2004; Canfield et al. 2007; Shen et al. 2008; Srivastava et al. 2008, 2010; Richter and King 2013), relatively little is known about how the interactions of animal progenitors with the abundant bacteria in their environment may have influenced the evolution of animals (McFall-Ngai 1999; Moran 2007; Hughes and Sperandio 2008; McFall-Ngai et al. 2013). We review here the current state of knowledge about ancient bacterial interactions and consider how these associations may have shaped the biology and evolution of the earliest animals.     

Solutions to the Public Goods Dilemma in Bacterial Biofilms

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Shear Rate Moderates Community Diversity in Freshwater Biofilms

The development of freshwater multispecies biofilms at solid-liquid interfaces occurs both in quiescent waters and under conditions of high shear rates. However, the influence of hydrodynamic shear rates on bacterial biofilm diversity is poorly understood. We hypothesized that different shear rates would significantly influence biofilm diversity and alter the relative proportions of coaggregating and autoaggregating community isolates. In order to study this hypothesis, freshwater biofilms were developed at five shear rates (<0.1 to 305 S⁻¹) in a rotating concentric cylinder reactor fed with untreated potable water. Eubacterial diversity was assessed by denaturing gradient gel electrophoresis (DGGE) and culturing on R2A agar. Fifty morphologically distinct biofilm strains and 16 planktonic strains were isolated by culturing and identified by partial 16S rRNA gene sequencing, and their relatedness was determined by the construction of a neighbor-joining phylogenetic tree. Phylogenetic and DGGE analyses showed an inverse relationship between shear rate and bacterial diversity. An in vitro aggregation assay was used to assess the relative proportions of coaggregating and autoaggregating species from each biofilm. The highest proportion of autoaggregating bacteria was present at high shear rates (198 to 305 S⁻¹). The intermediate shear rate (122 S⁻¹) selected for the highest proportion of coaggregating bacteria (47%, or 17 of a possible 36 coaggregation interactions). Under static conditions (<0.1 S⁻¹), 41 (33%) of a possible 125 coaggregation interactions were positive. Few coaggregation (3.3%) or autoaggregation (25%) interactions occurred between the 16 planktonic strains. In conclusion, these data show that shear rates affect biofilm diversity as well as the relative proportions of aggregating bacteria.     

Interactions of Botryococcus braunii Cultures with Bacterial Biofilms

Unicellular microalgae generally grow in the presence of bacteria, particularly when they are farmed massively. This study analyzes the bacteria associated with mass culture of Botryococcus braunii: both the planktonic bacteria in the water column and those forming biofilms adhered to the surface of the microalgal cells (∼10⁷–10⁸ culturable cells per gram microalgae). Furthermore, we identified the culturable bacteria forming a biofilm in the microalgal cells by 16S rDNA sequencing. At least eight different culturable species of bacteria were detected in the biofilm and were evaluated for the presence of quorum-sensing signals in these bacteria. Few studies have considered the implications of this phenomenon as regards the interaction between bacteria and microalgae. Production of C4-AHL and C6-AHL were detected in two species, Pseudomonas sp. and Rhizobium sp., which are present in the bacterial biofilm associated with B. braunii. This type of signal was not detected in the planktonic bacteria isolated from the water. We also noted that the bacterium, Rhizobium sp., acted as a probiotic bacterium and significantly encouraged the growth of B. braunii. A direct application of these beneficial bacteria associated with B. braunii could be, to use them like inoculants for large-scale microalgal cultures. They could optimize biomass production by enhancing growth, particularly in this microalga that has a low growth rate.     

Terrestrial Runoff Controls the Bacterial Community Composition of Biofilms along a Water Quality Gradient in the Great Barrier Reef

16S rRNA gene molecular analysis elucidated the spatiotemporal distribution of bacterial biofilm communities along a water quality gradient. Multivariate statistics indicated that terrestrial runoff, in particular dissolved organic carbon and chlorophyll a concentrations, induced shifts of specific bacterial communities between locations and seasons, suggesting microbial biofilms could be suitable bioindicators for water quality.     

Rate of Bacterial Mortality in Aquatic Environments

A method is proposed which provides a minimum estimate of the rate of bacterial mortality in growing natural populations of planktonic bacteria. This estimate is given by the rate of decrease of radioactivity from the DNA of a [³H]thymidine-labeled natural assemblage of bacteria after all added thymidine has been exhausted from the medium. Results obtained from river water, estuarine water, and seawater show overall bacterial mortality rates in the range 0.010 to 0.030 h⁻¹, in good agreement with the range of growth rates measured in the same environments. Use of selective filtration through Nuclepore filters (pore size, 2 μm) allowed us to determine the contribution of microzooplankton grazing to overall bacterial mortality. Grazing rates estimated by this method ranged from 0 to 0.02 h⁻¹.     

Differential Effects of Distinct Bacterial Biofilms in a Cave Environment

Current microbial surveys using molecular methods provide us with critical information on the major components of natural bacterial communities. However, limited investigation has been performed on the influence of bacterial metabolism on the environment. In this study, we analyzed the pH generated by distinct bacterial communities in a cave environment. Different bacterial biofilms developing on the walls of the cave were visually distinguished by their colorations (e.g., white, yellow, and gray) and mineral depositions, and previous studies have reported on their bacterial diversity and distribution. Using pH microelectrodes, we carried out in situ measurements and were able to detect differences among these bacterial biofilms. White biofilms and carbonate depositions resulted in alkaline pH values. Gray biofilms also increased the pH although these values remained lower than in white biofilms. A combination of gray–white biofilms resulted in alkaline pH values with highest values at the white edge of the colonies. Yellow biofilms generated a slightly acid pH. These results suggest that different bacterial communities can lead to distinct effects on their environment, for instance, precipitation or dissolution of carbonates in caves. These results add information about metabolic response to current knowledge from bacterial diversity surveys, providing information on the interaction between complex bacterial communities and the geological substrate.     

High Bacterial Diversity in Epilithic Biofilms of Oligotrophic Mountain Lakes

Benthic microbial biofilms attached to rocks (epilithic) are major sites of carbon cycling and can dominate ecosystem primary production in oligotrophic lakes. We studied the bacterial community composition of littoral epilithic biofilms in five connected oligotrophic high mountain lakes located at different altitudes by genetic fingerprinting and clone libraries of the 16S rRNA gene. Different intra-lake samples were analyzed, and consistent changes in community structure (chlorophyll a and organic matter contents, and bacterial community composition) were observed along the altitudinal gradient, particularly related with the location of the lake above or below the treeline. Epilithic biofilm genetic fingerprints were both more diverse among lakes than within lakes and significantly different between montane (below the tree line) and alpine lakes (above the tree line). The genetic richness in the epilithic biofilm was much higher than in the plankton of the same lacustrine area studied in previous works, with significantly idiosyncratic phylogenetic composition (specifically distinct from lake plankton or mountain soils). Data suggest the coexistence of aerobic, anaerobic, phototrophic, and chemotrophic microorganisms in the biofilm, Bacteroidetes and Cyanobacteria being the most important bacterial taxa, followed by Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Chlorobi, Planctomycetes, and Verrucomicrobia. The degree of novelty was especially high for epilithic Bacteroidetes, and up to 50?% of the sequences formed monophyletic clusters distantly related to any previously reported sequence. More than 35?% of the total sequences matched at <95?% identity to any previously reported 16S rRNA gene, indicating that alpine epilithic biofilms are unexplored habitats that contain a substantial degree of novelty within a short geographical distance. Further research is needed to determine whether these communities are involved in more biogeochemical pathways than previously thought.     

Structural and Functional Dynamics of Sulfate-Reducing Populations in Bacterial Biofilms

We describe the combined application of microsensors and molecular techniques to investigate the development of sulfate reduction and of sulfate-reducing bacterial populations in an aerobic bacterial biofilm. Microsensor measurements for oxygen showed that anaerobic zones developed in the biofilm within 1 week and that oxygen was depleted in the top 200 to 400 μm during all stages of biofilm development. Sulfate reduction was first detected after 6 weeks of growth, although favorable conditions for growth of sulfate-reducing bacteria (SRB) were present from the first week. In situ hybridization with a 16S rRNA probe for SRB revealed that sulfate reducers were present in high numbers (approximately 10⁸ SRB/ml) in all stages of development, both in the oxic and anoxic zones of the biofilm. Denaturing gradient gel electrophoresis (DGGE) showed that the genetic diversity of the microbial community increased during the development of the biofilm. Hybridization analysis of the DGGE profiles with taxon-specific oligonucleotide probes showed that Desulfobulbus and Desulfovibrio were the main sulfate-reducing bacteria in all biofilm samples as well as in the bulk activated sludge. However, different Desulfobulbus and Desulfovibrio species were found in the 6th and 8th weeks of incubation, respectively, coinciding with the development of sulfate reduction. Our data indicate that not all SRB detected by molecular analysis were sulfidogenically active in the biofilm.