Advances in Biofilm Research: How the Experts are Overcoming Obstacles

Drug Resistance & Development of Antibiofilm Treatments

A major reason that microorganisms, such as bacteria and fungi, form biofilms is to protect themselves from external threats. This often means that complete penetration of biofilms by antibiofilm treatments is difficult, particularly in areas such as the lungs or inside of cells. To overcome the protective effects of biofilms, researchers at institutions associated with the NBIC are examining new methods of antibiofilm drug delivery. One of the major drug delivery methods currently under investigation is the use of nanoparticles. As the name suggests, nanoparticles are very small in size, typically ranging from 1-100 nm. Although certain types of nanoparticles exist in nature, investigators at NBIC institutions have crafted synthetic nanoparticles with antibiofilm drugs inside of them. The advantage of this method is that biofilm-harboring cells take up the nanoparticles and then the antibiofilm drug is released, delivering treatment directly to microbes within the biofilm. This method prevents beneficial microorganisms from being exposed to potentially harmful treatment. The investigators at NBIC institutions hope this targeted approach can reduce the amount of drug required to effectively treat intracellular biofilms and preserve a healthy microbiome.
Mechanisms of biofilm drug resistance

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Figure 1. The protective effects of biofilms. This figure highlights how microbes form biofilms to help protect themselves from external threats, such as immune cells and antimicrobial treatments.
The fight against biofilm drug resistance is incredibly important, as explained to us by the microbiologists. The researchers highlighted that antibiotic treatments are often unable to fully penetrate all the layers of bacterial biofilms. This can lead to surviving bacteria developing the ability to limit the uptake of a drug, modify a drug target so that the drug becomes less effective, inactivate the drug, and/or elicit drug efflux1. Patients prescribed antibiotics often do not complete the entire treatment regime, leading to the proliferation of bacterial strains that are less sensitive or unresponsive to standard antibiotic treatments. These strains must then be treated with stronger medications. The investigators stressed that if this cycle persists, it is probable that some strains of bacteria will become completely resistant to all current treatments. As the old saying goes, “What doesn’t kill you gets stronger and tries again.” Therefore, these groups are invested in studying the mechanisms of biofilm drug resistance in the hopes of leveraging them into more effective prevention and treatment strategies.
Mechanisms of antibiotic resistance

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Figure 2. Mechanisms of antibiotic resistance. Known mechanisms of how bacteria can reduce or eliminate their responsiveness to antibiotic treatments.

Challenges in Biofilm Investigation

Traditionally, to grow microbial strains of interest, microbiologists follow the basic steps outlined below:

Microorganisms are grown in large culture flasks
Colonies are placed onto a sheet of plastic to allow biofilms to form
Biofilms are moved to a 96-well plate
Assays are performed in static wells of the 96-well plate
Biofilms are transferred to a glass slide and imaged

This workflow requires a significant time investment, causes changes in biofilm morphology due to compression by the coverslip, has an imaging throughput of a single biofilm, and perhaps most frustratingly, often yields results that do not transfer to in vivo models.

Most biofilms, particularly those that form within the body, grow under some form of liquid flow. Therefore, shear flow chambers represent an improvement over the traditional workflow by enabling the analysis of biofilms in biological conditions. This can create major benefits, such as improved nutrition for the entire biofilm2 and enhanced biofilm attachment3. Therefore, to overcome the obstacle of in vitro to in vivo translation, some microbiology labs have developed their own shear flow chambers to conduct biofilm assays.

Creating a DIY setup, such as a parallel plate flow chamber, typically involves purchasing a peristaltic pump, creating a specialized flow chamber, attaching a liquid reservoir, creating a pressure sensor, attaching resistance valves, attaching tubing, and running the setup through a dampener. Although these components can be purchased separately, assembly, calibration, cleaning, and maintenance are difficult and time-consuming. In addition, control of the flow rate is difficult and imaging under flow is extremely challenging. Furthermore, the throughput of DIY shear flow systems is often limited to 1-6 samples. This can be a dealbreaker for many labs, as a recent survey conducted by Cell Microsystems found that 60% of respondents required between 11 and 49 samples for their projects to be successful.
DIY shear flow system

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Figure 3. Simplified “DIY” flow cell setup. A typical setup includes flow chamber(s), reservoirs, tubing, 3-way stopcocks, male and female luer adapters, a pressure controller, and a microscope with a camera for imaging.

Despite the benefits of assaying biofilms under shear flow, shear flow devices have not been widely adopted by microbiology labs. In a recent CMS survey of microbiologists, it was found that 100% of respondents were aware that shear flow provides benefits to biofilm investigations; however, only 40% currently use some form of shear flow in their investigations. This is likely due to the difficulty of creating and maintaining a “do-it-yourself” shear flow chamber.

A Shear Flow Solution

To enhance the biological relevance of their biofilm investigations, while also achieving the throughput they need, investigators at many of the institutions associated with the NBIC have adopted BioFlux shear flow systems. The BioFlux systems enable the culture, assay, and imaging of biofilms under shear flow conditions.

BioFlux presents several improvements over DIY flow chambers, including being a complete system with a simple setup that requires no calibration and is easy to clean because the liquid flow is controlled by pneumatic air pressure, not liquid flow through tubes. The shear flow rate is also easily calculated and controlled with a click in the BioFlux software. Additionally, BioFlux eliminates “fluid bursts” that occur at the startup of peristaltic pumps, which can lead to undesired biofilm detachment. Perhaps the most beneficial feature of BioFlux are the microfluidic plates. BioFlux plates are Society for Biomolecular Screening (SBS) standard-sized plates with microfluidic channels embedded into the bottom of the plates, with coverslip glass viewing windows. This means that investigators can leverage up to 24 sequential experiment throughput using most inverted microscopes that are compatible with standard-sized microplates. BioFlux has enabled high-resolution imaging and video creation of non-deformed biofilms under shear flow by investigators at these NBIC affiliated institutions.
General BioFlux workflow

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Figure 4. Biofilm workflow using BioFlux. A general workflow showing how biofilms can be grown and assayed using a BioFlux system. Biofilms can be fixed and imaged or imaged live while under shear flow.

The microbiologists that we interviewed stressed how the experimental throughput combined with the standardization and reproducibility of BioFlux has greatly improved the workflow of their labs. Using this microfluidic shear flow technology, these investigators are able to test a similar number of biofilms as a static workflow, and much more than a DIY shear flow system, while leveraging the benefits of a biologically relevant environment. Together, these benefits have and will continue to empower microbiology labs to accelerate biofilm and antimicrobial discoveries.

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