Collaborators: Ameet Singh and Scott Weese - Ontario Veterinary College

Surgical site infections (SSIs) are an inherent risk of any surgical procedure and SSIs caused by Staphylococcus pseudintermedius are becoming the most common nosocomial infections in canines at the Ontario Veterinary College. An important underlying pathogenic factor for the development of SSIs is the ability of the bacteria to form a biofilm. Bacterial biofilms are complex communities of bacteria embedded within a self-produced carbohydrate matrix attached to biological or non-biological surfaces that can greatly impact the ability to treat infections. Clarithromycin eliminates biofilms formed by a wide variety of bacteria and has an effective break-point of 8mg/l on methicillin-susceptible S.pseudintermedius strains. In this study, we investigated the in-vitro efficacy of clarithromycin on 20 methicillin-resistant S.pseudintermedius (MRSP) isolates in-order to test eradication therapies against SSI related infections. MRSP isolates were sub-cultured and inoculated into tryptic soy-broth before addition to microtiter plates. Biofilm formation was quantified first through removal of planktonic bacteria followed by staining, then heat fixing, and finally with elution of biofilm-embedded bacteria before completion of an OD570 reading. To characterize the adhesion, MRSP isolates were grown on stainless steel orthopaedic screws exposed to antibiotics at various time points using Scanning Electron Microscopy (SEM). Visual and image processing evaluation of the SEM images revealed the ability of the MRSPs to form biofilm on the surface and between the screw threads. The quantitative assay results (P > 0.5126) suggest that the influence of clarithromycin in the remediation of MRSP biofilms was insignificant after a 24h growth period. The results of our study indicate that the MRSP biofilms exhibits higher resistance to clarithromycin in therapeutic doses.

Bacterial biofilms have garnered much attention in recent years due to their importance in a wide range of both natural and engineered processes such as infection, water treatment, food processing, and oil pipelining. Here, we use a microfluidic device to quantify the effects of fluid shear force on the biofilm morphology of Shewanella oneidensis, a metal reducing bacteria of interest for several bioremediation and energy applications.

The composition of microbial communities within and around plant species is dependent on dynamic physical and chemical signaling events that occur within the local environment and at the root surface.  Visualization and quantification of these events in natural systems is challenging. However, emerging technologies that combine advances in nanostructure fabrication, microfluidics and imaging provide a means of recreating these events within model systems.  These systems mimic aspects of their natural counterparts while providing tractable experimental platforms in which both individual cellular responses and population dynamics can be recorded and analyzed.

Model systems, amenable to imaging, that allow dynamic modulation of local physicochemical cues in a controllable manner have been developed to recreate the interactions between microbes and their hosts.  A nanostructured microfluidic platform has been created in order to examine the chemotactic responses of isolates to specific plant-associated signals.  This platform is created using a combination of electron beam lithography and anisotropic silicon etching techniques.  It can be easily replicated via silicone molding and facilitates the physical tracking of hundreds of microbes within a quasi-two-dimensional space that confines microbes within the focal volume of a conventional phase contrast microscope without significantly impeding natural motility.

The attachment of bacteria to host roots is an initial step in colonization and is influenced by several factors including bacterial transport, surface recognition, and local shear forces. A quantitative understanding of the affinity of proteobacteria for root surfaces and a clear picture of the initial adhesion kinetics associated with attachment is essential to characterize biofilm formation and root colonization. The dual function of imaging and measuring interaction forces makes Atomic Force Microscopy (AFM) a unique tool for studying bacterial adhesion. The adhesion between the cell surface and the AFM tip can be measured from the extension and the retraction of the force curve cycle using functionalized cantilever tips. The mechanisms involved in the bacterial attachment and motility behavior were elucidated by force spectroscopy measurements and microfluidic systems. The developed analytical techniques quantified the adhesion forces which helped to explain the spatial and temporal dynamics of the colonization of Populus by proteobacteria. The role of the extracellular polymeric substance and the pili in facilitating the bacterial adhesion during biofilm formation are being analyzed.

In this study, confocal laser scanning microscopy was used to examine the spatial and temporal dynamics of microbial colonization of Populus roots using microbial isolates expressing GFP. Standard molecular methods were used to introduce GFP into microbial isolates. Atomic force microscopy was also used to characterize the morphology, surface characteristics, and dynamics of biofilm formation of selected microbes isolated from the Populus rhizosphere. Gamma-proteobacteria isolate YR343 was observed to attach to Poplar roots after approximately 5 hours co-culture. The cells were observed to grow and form colonies on the surface of the root. Microbial isolate GM30 was examined during biofilm formation using atomic force microscopy. Expression of pili over the time course of biofilm formation and distinct morphotypes were revealed by the AFM image analysis. These colonization studies provide direct evidence that microbes collected from the rhizosphere directly associate with Populus roots. The dynamics of colonization were observed in real time using GFP-expressing microbes.

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