Kelly Schwartz

Most microbes in nature are thought to exist as surface-associated communities in biofilms. 1 Bacterial biofilms are encased within a matrix and attached to a surface. 2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates) 3 or growing biofilms for visualization (flow cells) 4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor. Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions. 5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing. 6,7,8,9 The rotating disk reactor consists of a teflon disk containing recesses for removable coupons. 10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials. 9,11,12,13 1. The drip flow biofilm reactor (available from Biosurface Technologies or custom designed versions can usually be made by university machine shops, see Figure 1) is assembled and autoclaved. Assembly involves affixing coupons in chambers and securing chamber lids. The chamber, along with biofilm medium (tryptic soy broth 2 grams/L and glucose 2 grams/L), and influent nutrient tubing are sterilized by autoclaving. 2. Inoculation of the drip flow reactor is preformed by placing the reactor on a flat surface, clamping the effluent tubing lines, filling each chamber with 10 mL tryptic soy broth and adding 10 μL of a S. aureus culture grown overnight in tryptic soy broth. The inoculated reactor is then place in a 37°C incubator for 18 hours. 3. After 18 hours of incubation, the effluent tubing is unclamped and the reactor is placed on a wooden block cut to a 10° angle. 4. Aseptically connect the influent nutrient tubing to the bottle containing the continuous flow nutrient broth. Feed the tubing line through the pump and prime the tubes by running the pump at a maximum speed (will vary depending on pump model). 5. Once the influent tubing is primed stop the pump and attach connect needles (22 gauge, 1 inch) to the end of each tube. Wipe the chamber inlet stopper with an ethanol wipe and aseptically insert the needles through the inlet stopper. 6. Turn on the pump and allow the media to slowly drip (flow rate ~125 μL/minute) over the coupons. The media should flow downward along the coupon from the inlet stopper port to the effluent port. Operate the reactor in continuous flow for 2-5 days (depending on the application), occasionally checking the reactor for proper drainage. 7. To harvest the drip flow reactor biofilms, stop the pump and carefully remove the needles from the reactor. The reactor can then be placed on a flat surface and the coupons can be aseptically removed using sterile forceps. If microscopy is desired, the coupons can now be processed accordingly (Figure 2B is a scanning electron micrograph of a S. aureus biofilm grown in a drip biofilm reactor). If quantification of the biofilm biomass or physiology studies are the goal of the study, the biofilm can be removed from the coupon using a cell scraper. While holding the coupons with forceps, gently scrape the biofilm off the coupon into a conical tube containing phosphate buffered saline using a cell scraper. Note: to quantify the colony forming units in the biofilms, it is necessary to homogenize the harvested biofilms with a tissue homogenizer to disaggregate clumps and form a homogenous suspension. Various models of tissue homogenizers are suitable for this application. We utilize a Fisher Scientific Tissuemiser Homogenizer (product # 15-338-420) at full speed for 1 minute to homogenize biofilm samples. Failure to homogenize the biofilm will result in an underestimation of the colony forming units present in the sample. 1. The rotating disk biofilm reactor (available from available from Biosurface Technologies or can be custom made, see Figure 3) is assembled and autoclaved. Assemble the reactor by first place the spinning disk coupons into the slots of the spinning disc and placing it into a 1-liter glass beaker with an overflow port. A number 15-rubber stopper with holes drilled in it to allow media flow and aeration is used as the reactor cap. The reactor, biofilm media (tryptic soy broth 2 g/L and glucose 2 g/L), and inlet tubing are then sterilized by autoclaving.

Staphylococcus aureus is an opportunistic pathogen that colonizes the skin and mucosal surfaces of mammals. Persistent staphylococcal infections often involve surface-associated communities called biofilms. Here we report the discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity. Biochemical and genetic analysis has revealed that these fibers have amyloid-like properties and consist of small peptides called phenol soluble modulins (PSMs). Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress. Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not. This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms.

The aggregation of proteins into amyloid fibers is a common characteristic of many neurodegenerative disorders including Alzheimer's, Parkinson's, and prion diseases. Amyloid formation was originally characterized in these systems and is traditionally viewed as a consequence of protein misfolding and aggregation. An emerging field of study brings functional amyloids, like those produced by bacteria, into the scientific mainstream, and demonstrates a ubiquitous role for amyloids in living systems. This review aims to summarize what is known about the bacterial amyloids and their interactions within various host environments.

Persistent staphylococcal infections often involve surface-associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the coordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA) and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus, this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology.