Robert J. Martinuzzi

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the Calgary Biofilm Device

Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD — an apparatus that was designed for highthroughput susceptibility testing — allows for structure-function analysis of biofilms under multivariate growth and exposure conditions.

Impact of Silver-Containing Wound Dressings on Bacterial Biofilm Viability and Susceptibility to Antibiotics during Prolonged Treatment

ABSTRACT The long-term antimicrobial efficacy of silver dressings against bacterial biofilms was investigated in a 7-day treatment in vitro model where the protein-rich medium was refreshed daily in order to mimic the conditions found in a wound bed. The use of plate-to-plate transfer assays demonstrated measurable differences in the effectivenesses of several silver dressings on the viability of biofilm bacteria and their susceptibility to antibiotics. Whereas after the first day of treatment, all dressings used resulted in a significant reduction in the number of viable cells in the biofilms and disruption of the biofilm colonies, during prolonged treatment, the efficacy of dressings with hydrophilic base materials diminished with daily transfers, and bacterial populations recovered. For dressings with hydrophobic base materials, the level of efficacy correlated with the silver species loaded. Biofilm bacteria, which survived the initial silver treatment, were susceptible to tobramycin, ciprofloxacin, and trimethoprim-sulfamethoxazole, in contrast to untreated biofilms, which were highly tolerant to the same antibiotics. This acquired susceptibility was unaffected by the longevity of pretreatment with the silver dressings but depended on the dressing used. The antimicrobial efficacy of the dressings correlated with the type of the dressing base material and silver species loaded.

Alternating half-loop shedding in the turbulent wake of a finite surface-mounted square cylinder with a thin boundary layer

The configuration and energetics of the large-scale vortex structure are presented for quasi-periodic shedding in the turbulent wake of a finite (h/d = 4) square-cross-section surface-mounted cylinder protruding from a thin boundary layer (δ/h = 0.18). The three-dimensional large-scale structure is educed from phase averaged x-y and x-z planar data measured with particle image velocimetry (PIV). Simultaneous measurements of the surface pressure difference on either side of the obstacle were used to phase-align the PIV planar measurements. The topology of the educed structures resembles alternating half-loops interconnecting close to the base plate. The time averaging of this unsteady structure gives rise to mean streamwise vortices akin to those presented in the literature for similar geometries. This topological analysis offers a contrasting interpretation of the mean streamwise vorticity, which has, otherwise, been presumed to originate from structures generated at the leading edge of the free-end. The ...

Turbulent Flow Around Two Interfering Surface-Mounted Cubic Obstacles in Tandem Arrangement

The flow around two in-line surface-mounted cubes in a thin laminar boundary layer was experimentally investigated as a function of obstacle spacing for a Reynolds number of 22,000 based on approach velocity and cube height. Mean velocity measurements with Laser Doppler Velocimetry and surface flow patterns, obtained with an oil film technique, show that three distinct mean flow field structures exist based on obstacle spacing. Frequency spectra of velocity and surface pressure fluctuations reveal that these structures are related to three regimes of wake flow periodicity. For small spacings, the shear layer separating from the first cube reattaches on the sides of the second obstacle and wake periodicity can only be detected in the wake of the downstream cube. For a critical spacing range, the fluctuations in the gap and wake lock-in. For larger spacings, a second horseshoe vortex appears at the windward base of the second cube. Observations using dye-injection and smoke-wire techniques are consistent with these results

Poroelastic evaluation of fluid movement through the lacunocanalicular system

A poroelastic lacunocanalicular model was developed for the quantification of physiologically relevant parameters related to bone fluid flow. The canalicular and lacunar microstructures were explicitly represented by a dual-continuum poroelastic model. Effective material properties were calculated using the theory of composite materials. Porosity and permeability values were determined using capillaric and spherical-shell models for the canalicular and lacunar microstructures, respectively. Pore fluid pressure and fluid shear stress were calculated in response to simulated mechanical loading applied over a range of frequencies. Species transport was simulated with convective and diffusive flow, and osteocyte consumption of nutrients was incorporated. With the calculated parameter values, realistic pore fluid pressure and fluid shear stress responses were predicted and shown to be consistent with previous experimental and theoretical studies. Stress-induced fluid flow was highlighted as a potent means of species transport, and the importance of high-magnitude low-frequency loading on osteocyte nutrition was demonstrated. This new model can serve as the foundation for future hierarchical modeling efforts that may provide insight into the underlying mechanisms of mechanotransduction and functional adaptation of bone.

Relation between the activity of anaerobic microbial populations in oil sands tailings ponds and the sedimentation of tailings

Oil sands tailings ponds contain a variety of anaerobic microbes, including methanogens, sulfate- and nitrate-reducing bacteria. Methanogenic activity in samples from a tailings pond and its input streams was higher with trimethylamine (TMA) than with acetate. Methanogens closely affiliated to Methanomethylovorans hollandica were found in the TMA enrichments. Tailings sedimentation increased with methanogenic activity, irrespective whether TMA or acetate was used to stimulate methanogenesis. Increased sedimentation of autoclaved tailings was observed with added pure cultures under methanogenic, as well as under nitrate-reducing conditions, but not under sulfate-reducing conditions. Scanning electron microscopy and energy-dispersive X-ray spectroscopy indicated the presence of microbes and of extracellular polymeric substances in tailings particle aggregates, especially under methanogenic and nitrate-reducing conditions. Hence different classes of microorganisms growing in tailings ponds contribute to increased tailings aggregation and sedimentation. Because addition of nitrate is known to lower methane production by methanogenic consortia, these observations offer the potential to combine lower methane emissions with improved microbially-induced tailings sedimentation.

Metal Ions May Suppress or Enhance Cellular Differentiation in Candida albicans and Candida tropicalis Biofilms

ABSTRACT Candida albicans and Candida tropicalis are polymorphic fungi that develop antimicrobial-resistant biofilm communities that are characterized by multiple cell morphotypes. This study investigated cell type interconversion and drug and metal resistance as well as community organization in biofilms of these microorganisms that were exposed to metal ions. To study this, Candida biofilms were grown either in microtiter plates containing gradient arrays of metal ions or in the Calgary Biofilm Device for high-throughput susceptibility testing. Biofilm formation and antifungal resistance were evaluated by viable cell counts, tetrazolium salt reduction, light microscopy, and confocal laser scanning microscopy in conjunction with three-dimensional visualization. We discovered that subinhibitory concentrations of certain metal ions (CrO42−, Co2+, Cu2+, Ag+, Zn2+, Cd2+, Hg2+, Pb2+, AsO2−, and SeO32−) caused changes in biofilm structure by blocking or eliciting the transition between yeast and hyphal cell types. Four distinct biofilm community structure types were discerned from these data, which were designated “domed,” “layer cake,” “flat,” and “mycelial.” This study suggests that Candida biofilm populations may respond to metal ions to form cell-cell and solid-surface-attached assemblages with distinct patterns of cellular differentiation.

Growth and separation of a start-up vortex from a two-dimensional shear layer

The evolution of an isolated line vortex generated by a starting two-dimensional jet is studied experimentally using time-resolved particle image velocimetry. The vortex growth in this current configuration is not linked to any externally imposed length scales or interactions with other vortical structures or walls that could potentially influence vortex growth. A model for the early-stage vortex growth, based on the transport of circulation from the shear layer into the vortex, is proposed and found to agree well with experimental data. The model provides a scaling scheme for vortex growth using shear-layer characteristic velocity and shear-layer thickness. The vortex growth is limited through a gradual separation of the vortex from the feeding shear layer, arising from decreased shear-layer curvature. This phenomenon is linked to a competition between the shear-layer tendency to remain in the streamwise direction and the induced velocity from the vortex on the shear layer. Finally, a dimensionless numbe...

Staphylococcus aureus biofilm formation and tolerance to antibiotics in response to oscillatory shear stresses of physiological levels

Bacterial infections in the blood system are usually associated with blood flow oscillation generated by some cardiovascular pathologies and insertion of indwelling devices. The influence of hydrodynamically induced shear stress fluctuations on the Staphylococcus aureus biofilm morphology and tolerance to antibiotics was investigated. Fluctuating shear stresses of physiologically relevant levels were generated in wells of a six-well microdish agitated by an orbital shaker. Numerical simulations were performed to determine the spatial distribution and local fluctuation levels of the shear stress field on the well bottom. It is found that the local biofilm deposition and morphology correlate strongly with shear stress fluctuations and maximum magnitude levels. Tolerance to killing by antibiotics correlates with morphotype and is generally higher in high shear regions.

The influence of flow cell geometry related shear stresses on the distribution, structure and susceptibility of Pseudomonas aeruginosa 01 biofilms

The effects of non-uniform hydrodynamic conditions resulting from flow cell geometry (square and rectangular cross-section) on Pseudomonas aeruginosa 01 (PAO1) biofilm formation, location, and structure were investigated for nominally similar flow conditions using a combination of confocal scanning laser microscope (CSLM) and computational fluid dynamics (CFD). The thickness and surface coverage of PAO1 biofilms were observed to vary depending on the location in the flow cell and thus also the local wall shear stress. The biofilm structure in a 5:1 (width to height) aspect ratio rectangular flow cell was observed to consist mainly of a layer of bacterial cells with thicker biofilm formation observed in the flow cell corners. For square cross-section (1:1 aspect ratio) flow cells, generally thicker and more uniform surface coverage biofilms were observed. Mushroom shaped structures with hollow centers and wall breaks, indicative of ‘seeding’ dispersal structures, were found exclusively in the square cross-section tubes. Exposure of PAO1 biofilms grown in the flow cells to gentamicin revealed a difference in susceptibility. Biofilms grown in the rectangular flow cell overall exhibited a greater susceptibility to gentamicin compared to those grown in square flow cells. However, even within a given flow cell, differences in susceptibility were observed depending on location. This study demonstrates that the spanwise shear stress distribution within the flow cells has an important impact on the location of colonization and structure of the resultant biofilm. These differences in biofilm structure have a significant impact on the susceptibility of the biofilms grown within flow channels. The impact of flow modification due to flow cell geometry should be considered when designing flow cells for laboratory investigation of bacterial biofilms.