Darren R. Korber

Confocal Laser Microscopy and Digital Image Analysis in Microbial Ecology

Microbial ecologists have extensively explored the potential applications of light microscopy for more than five decades (Henrici and Johnson, 1935; Perfil’ev and Gabe, 1969; Casida, 1969, 1972, 1975, 1976; Staley, 1971; Caldwell and Hirsch, 1973; Caldwell et al., 1973, 1975; Caldwell and Tiedje, 1975a,b; Labeda et al., 1976; Hirsch, 1977, 1980; Geesey et al., 1978; Marshall, 1986). Now traditional microscopy has given way to “microvisualization” (Friedhoff, 1991) greatly accelerating research. Microorganisms are no longer merely photographed; instead, they are digitally “imaged” using fluorescent molecular probes, confocal laser microscopy, and computer image analysis. The chemical and biological relationships between a microorganism and its microenvironment are seen directly, nondestructively, in situ, and in “real time” (Lawrence and Caldwell, 1990). Consequently, it is no longer necessary to disrupt microbial communities when studying the molecular or behavioral aspects of their ecology.

Behavior ofPseudomonas fluorescens within the hydrodynamic boundary layers of surface microenvironments

Phase, darkfield, and computer-enhanced microscopy were used to observe the surface microenvironment of flow cells during bacterial colonization. Microbial behavior was consistent with the assumptions used previously to derive surface colonization kinetics and to calculate surface growth and attachment rates from cell number and distribution. Surface microcolonies consisted of closely packed cells. Each colony contained 2n cells, where n is the number of cell divisions following attachment. Initially, cells were freely motile while attached, performing circular looping movements within the plane of the solid-liquid interface. Subsequently, cells attached apically, maintained a fixed position on the surface, and rotated. This type of attachment was reversible and did not necessarily lead to the formation of microcolonies. Cells became irreversibly attached by progressing from apical to longitudinal attachment. Longitudinally attached cells increased in length, then divided, separated, moved apart laterally, and slid next to one another. This resulted in tight cell packing and permitted simultaneous growth and adherence. After approximately 4 generations, individual cells emigrated from developing microcolonies to recolonize the surface at new locations. Surface colonization byPseudomonas fluorescens can thus be subdivided into the following sequential colonization phases: motile attachment phase, reversible attachment phase, irreversible attachment phase, growth phase, and recolonization phase.

Next-Generation Sequencing of Microbial Communities in the Athabasca River and Its Tributaries in Relation to Oil Sands Mining Activities

ABSTRACT The Athabasca oil sands deposit is the largest reservoir of crude bitumen in the world. Recently, the soaring demand for oil and the availability of modern bitumen extraction technology have heightened exploitation of this reservoir and the potential unintended consequences of pollution in the Athabasca River. The main objective of the present study was to evaluate the potential impacts of oil sands mining on neighboring aquatic microbial community structure. Microbial communities were sampled from sediments in the Athabasca River and its tributaries as well as in oil sands tailings ponds. Bacterial and archaeal 16S rRNA genes were amplified and sequenced using next-generation sequencing technology (454 and Ion Torrent). Sediments were also analyzed for a variety of chemical and physical characteristics. Microbial communities in the fine tailings of the tailings ponds were strikingly distinct from those in the Athabasca River and tributary sediments. Microbial communities in sediments taken close to tailings ponds were more similar to those in the fine tailings of the tailings ponds than to the ones from sediments further away. Additionally, bacterial diversity was significantly lower in tailings pond sediments. Several taxonomic groups of Bacteria and Archaea showed significant correlations with the concentrations of different contaminants, highlighting their potential as bioindicators. We also extensively validated Ion Torrent sequencing in the context of environmental studies by comparing Ion Torrent and 454 data sets and by analyzing control samples.

Effect of laminar flow velocity on the kinetics of surface recolonization by Mot+ and Mot−Pseudomonas fluorescens

Computer-enhanced microscopy (CEM) was used to monitor bacteria colonizing the inner surfaces of a 1×3 mm glass flow cell. Image analysis provided a rapid and reliable means of measuring microcolony count, microcolony area, and cell motility. The kinetics of motile and nonmotilePseudomonas fluorescens surface colonization were compared at flow velocities above (120μm sec−1) and below (8μm sec−1) the strains maximum motility rate (85μm sec−1). A direct attachment assay confirmed that flagellated cells undergo initial attachment more rapidly than nonflagellated cells at high and low flow. During continuous-flow slide culture, neither the rate of growth nor the timing of recolonization (cell redistribution within surface microenvironments) were influenced by flow rate or motility. However, the amount of reattachment of recolonizing cells was both flow and motility dependent. At 8μm sec−1 flow, motility increased reattachment sixfold, whereas at 120μm sec−1 flow, motility increased reattachment fourfold. The spatial distribution of recolonizing cells was also influenced by motility. Motile cells dispersed over surfaces more uniformly (mean distance to the nearest neighbor was 47.0μm) than nonmotile cells (mean distance was 14.2μm) allowing uniform biofilm development through more effective redistribution of cells over the surface during recolonization. In addition, motile cell backgrowth (where cells colonize against laminar flow) occurred four times more rapidly than nonmotile cell backgrowth at low flow (where rate of motility exceeded flow), and twice as rapidly at high flow (where flow exceeded the rate of motility). The observed backgrowth of Mot+ cells against high flow could only have occurred as the result of motile attachment behavior. These results confirm the importance of motility as a behavioral mechanism in colonization and provides an explanation for enhanced colonization by motile cells in environments lacking concentration gradients necessary for chemotactic behavior.

Function of EPS

The production of extracellular polymeric substances (EPS) involves a significant investment of carbon and energy by microorganisms. Considering the tendency in nature to conserve rather than to waste, this expenditure of energy (in some cases more than 70% — see Harder and Dijkhuizen 1983) is likely to hold benefits to the producers of EPS, as well as those organisms associated with them. Bacteria are very efficient in converting nutrients into EPS; it has been calculated (Underwood et al. 1995) that a single Azotobacter cell can produce enough EPS to coat more than 500 particles with a 0.4 µm diameter per day. The size of a single cell is typically 1–2 µm by 0.5 µm, and often much smaller, and therefore this number is impressive. The importance of EPS has long been recognized and a variety of functions have been attributed to EPS as far as the benefits they provide to cells, either living as single organisms, in binary associations, or in heterogeneous communities. However, Christensen and Characklis (1990) implied that there is a lack of knowledge of the properties of EPS in biofilms, as well as their role in biofilm ecology. The aim of this chapter is to provide an overview of some of the progress that has been made in recent years to elucidate the functional role of EPS.

Analysis of spatial variability within mot+ and mot− pseudomonas fluorescens biofilms using representative elements

The spatial variability for most measurable parameters contained within biofilms is very large. Therefore a procedure for determining statistically representative regions of analysis is desirable. Scanning confocal laser microscopy, a computer‐controlled xy stage, and fluorescence exclusion staining were used to obtain a series of optical thin sections of biofilms formed by motile (mot+) and nonmotile (mot−) Pseudomonas fluorescens on the surfaces of glass flow cells. Based on a representative elementary area (REA) analysis procedure, the images were used to construct montages large enough to encompass the range of variation in biofilm cell area. The minimum area of analysis required to be representative varied with depth in the biofilm and between the strains used. Biofilms consisting of mot− P. fluorescens were variable. Thus, large area (REA ≥ × 105‐μm−) were required for statistically valid comparisons of cell distribution. REAs for the mot+ biofilm reflected a more uniform distribution of cells at al...

Do Bacterial Communities Transcend Darwinism

Until the development of fluorescent molecular probes and confocal laser microscopy, there were few alternatives to isolating microorganisms from their communities prior to laboratory study. Isolation was necessary to obtain a sufficient amount of homogeneous cell material for chemical analyses, yet it constrained most laboratory work to the molecular, cellular, or organismal level. However, fluorescent probes and other molecular techniques now allow the analysis of individual microorganisms without isolation (Olsen et al., 1986; Pace et al., 1986; Caldwell et al., 1992a). This affords the opportunity to perform community-level laboratory experiments that are not possible with plants and animals due to their large size. However, inconsistencies between evolutionary ecology (Mayr, 1993; Krassilov, 1994; Kauffman, 1993, 1995), ecosystem ecology (Maynard-Smith, 1991; Loehle and Pechman, 1988; Schulze and Mooney, 1993), microbial ecology (Margulis, 1990; Caldwell, 1993; Caldwell and Costerton, 1996), germ theory (Caldwell, 1995; Caldwell et al., 1997a), and information theory (Rasmussen, 1988, 1991; Rasmussen et al., 1990; Yockey, 1990, 1995; Kelly, 1994) make it difficult to formulate testable hypotheses that are relevant in understanding ecology at the community level. Consideration of communities as units of proliferation (and hence as units of evolution) requires a more generalized theory of life, amenable to the formulation of community-level hypotheses and tests.

Planktonic or biofilm growth affects survival, hydrophobicity and protein expression patterns of a pathogenic Campylobacter jejuni strain.

The effect of planktonic or biofilm modes of growth on survival, hydrophobicity and cellular protein expression patterns of a pathogenic Campylobacter jejuni strain were determined. This was achieved by growing the strain in brain heart infusion broth (with 1% yeast extract), or attached to glass beads in the same medium, at 37 degrees C for 48 h under microaerophilic conditions. Cells from the broth or the bead surfaces were stored at different temperatures (4, 10, 25 and 37 degrees C) for 28 days in phosphate buffered saline (PBS) and monitored at appropriate time intervals for culturable numbers and hydrophobicity by standard methods. In addition, cells were inoculated onto the surface of two processed meat products (a bologna and a summer sausage) vacuum packaged and stored at 4 degrees C for 28 days. Numbers of culturable cells were monitored at appropriate time intervals by standard methods. Cells from the broth or the bead surfaces were also examined for protein expression using two-dimensional protein electrophoresis. Results indicated that numbers of culturable cells in phosphate buffered saline decreased from approximately 6 log colony forming units (cfu) g(-1) to undetectable levels within 14-day storage in a temperature dependent manner. Hydrophobicity of broth grown cells decreased from 15% to 0% adherence to xylene over the same time in a temperature independent manner. Cells grown in a biofilm mode initially displayed a <0.3% adherence to xylene which was maintained during storage. Furthermore, cells grown in the biofilm mode decreased in number more rapidly on storage in buffer than their counterparts grown in broth. Numbers of culturable cells on meat decreased from approximately 5 log cfu g(-1) to undetectable levels within 14-day storage in a product dependent manner, with the most rapid decrease observed for the more acidic summer sausage. Cells grown in a biofilm mode decreased in number more rapidly on storage than broth grown cells. The protein expression patterns differed between planktonic and biofilm cells with seven unique and 12 up-regulated protein spots expressed in a growth mode specific manner. A number of the differentially expressed spots were tentatively identified, by comparison to existing literature, as surface- and stress-associated proteins. Despite the elicitation of some putative stress proteins, this study importantly indicates that biofilm cells of C. jejuni are less resistant to stress than their planktonic counterparts and may lack a sophisticated adaptive stress-resistance response. These findings have implication in determining the risks of infection associated with C. jejuni contamination on food.

Adaptive Resistance and Differential Protein Expression of Salmonella enterica Serovar Enteritidis Biofilms Exposed to Benzalkonium Chloride

ABSTRACT The development of adaptive resistance of Salmonella enterica serovar Enteritidis ATCC 4931 biofilms following exposure to benzalkonium chloride (BC) either continuously (1 μg ml−1) or intermittently (10 μg ml−1 for 10 min daily) was examined. Biofilms adapted to BC over a 144-h period could survive a normally lethal BC challenge (500 μg ml−1 for 10 min) and then regrow, as determined by increases in biofilm thickness, total biomass, and the ratio of the viable biomass to the nonviable biomass. Exposure of untreated control biofilms to the lethal BC challenge resulted in biofilm erosion and cell death. Proteins found to be up-regulated following BC adaptation were those involved in energy metabolism (TpiA and Eno), amino acid and protein biosynthesis (WrbA, TrxA, RplL, Tsf, Tuf, DsbA, and RpoZ), nutrient binding (FruB), adaptation (CspA), detoxification (Tpx, SodB, and a probable peroxidase), and degradation of 1,2-propanediol (PduJ and PduA). A putative universal stress protein (YnaF) was also found to be up-regulated. Proteins involved in proteolysis (DegQ), cell envelope formation (RfbH), adaptation (UspA), heat shock response (DnaK), and broad regulatory functions (Hns) were found to be down-regulated following adaptation. An overall increase in cellular protein biosynthesis was deduced from the significant up-regulation of ribosomal subunit proteins, translation elongation factors, and amino acid biosynthesis protein and down-regulation of serine endoprotease. The cold shock response, stress response, and detoxification are suggested to play roles in the adaptive resistance of Salmonella serovar Enteritidis biofilms to BC.

Behavioral Strategies of Surface-Colonizing Bacteria

Bacterial survival and reproductive success in many systems requires colonization of a surface and/or integration into a biofilm community. Success in a community context requires morphological, physiological, and genetic attributes that have only recently been explored. Previously, many aspects of microbial behavior at interfaces have been explained in terms of physicochemical interactions. Indeed, Van Loosdrecht et al. (1989) concluded that virtually no studies have shown a bacterial response to a surface. However, many studies past and present have shown specific responses to the surface environment, including chemoadherence, morphogenesis, gene induction, and variable rates of polymer production (McCarter et al., 1988, 1992; Vandevivere and Kirchman, 1993). The induction of many genetic pathways has been shown to be surface-specific phenomena. Ample evidence has also been provided for behavioral strategies that only function during surface colonization and growth (Kjelleberg et al., 1982; Lawrence et al., 1987, 1991, 1992; Lawrence and Caldwell, 1987; Power and Marshall, 1988; Lawrence and Korber, 1993). These strategies represent essential elements for multicellular community growth and are expressed as specific adaptations, life cycle alternation between attached and planktonic growth, as well as formation and maintenance of microcolonies, aggregates, and consortia.