5D Health Protection Group Ltd

Centre of Excellence in Biofilm Science and Technologies

Microorganisms such as bacteria, fungi and yeast can exist as free floating or planktonic entities or they can attach to a biotic or abiotic surface (or each other) to form a biofilm. Within a biofilm a community of microorganisms become encased in an extracellular matrix, called EPS (extracellular polymeric substance), composed of polysaccharides, proteins, lipids, extracellular enzymes, metal ions and extracellular DNA (eDNA). In nature, over 90% of microorganisms exist within a biofilm.

5D have over 28 years experience of researching, managing and engineering biofilms, developing biofilm models and developing technologies to prevent and control. This experience has been gained through working in academia, industry and public health bodies. Please see our publications pages for further information on biofilmology.

Biofilms

When microorganisms attach onto a surface or each other, they develop into a biofilm. The microorganisms that grow within a biofilm are significantly more tolerant to antimicrobial agents and the host’s immune system when compared to their planktonic counterparts. It is important that if technologies are being developed to kill or inhibit microorganisms they also have an effect on these microorganisms when grown within a biofilm. 5D have validated a large number of standard and customised biofilm testing models and methods that have been used to mimic specific industrial sectors. There are a large variety of different biofilm models available to investigate the formation of biofilms and to also study the efficacy of active agents on biofilms. Therefore the in vitro models employed must closely reproduce the in situ conditions as best as possible. The main biofilm models we routinely employ and offer are shown below. However, we do customise and develop many different biofilm models when a customer requires something more bespoke.

The Minimum Biofilm Eradication Concentration (MBEC) assay

The Minimum Biofilm Eradication Concentration (MBEC) assay is a static high throughput screening model producing a large number of reproducible biofilms in a microtitre plate system. The model consists of a plastic lid with 96 pegs with a base which contains 96 individual wells to put in nutrients to grow biofilm which can then be replaced with antimicrobials to evaluate their efficacy on biofilms that have been grown on the pegs. The MBEC assay involves growing biofilms on the individual pegs under batch conditions often combined with the use of constant agitation which can help to stabilise the biofilm. The model is specifically utilised to evaluate the efficacy of liquid based actives and antimicrobials at reducing and controlling microbial adhesion and biofilms that have been grown for either a short period of time (24 hrs - considered an immature biofilm) or a long period of time (more than 24 hrs - considered to be a more mature biofilm).

Centers For Disease Control (CDC) Biofilm Reactor

The CDC Biofilm Reactor model develops reproducible biofilms on 24 individual coupon surfaces under batch or continuous nutrient flow (see figure below). The model consists of eight polypropylene coupon holder rods. The rods can hold 3 coupons each. The rods with attached coupons are suspended into a 1 litre glass vessel with a side arm discharge port. A growth media is added to the glass vessel which is then mixed via a magnetic stir bar to generate shear. Following inoculation of microbes into the media and incubation of the reactor for set periods of time, reproducible biofilms will grow on the coupons.

Following removal of the coupons (with attached biofilm) from the vessel, the coupons are then added into antimicrobial based solutions to evaluate the efficacy on biofilms. Biofilms can be grown in this model from 24 hours through to a number of days depending on a customers need.

Drip Flow Biofilm Reactor

The Drip Flow Biofilm Reactor consists of six parallel test channels which are able to hold a standard glass microscope slide. However, it can be modified to hold a length of catheter. Media is dripped onto the slide to encourage the growth of biofilms. The model is used to reproduce biofilms on glass coupons under defined conditions suitable for testing the efficacy of antimicrobials and materials impregnated with antimicrobials. The model can be adapted to allow for the growth of biofilm on skin and wound dressings. The biofilms represent generalised situations where biofilm exists at the air/liquid interface with a continuous flow of nutrients under low fluid shear. The Drip Flow Reactor can be used for growing and characterising both monoculture and multi-species biofilms. Based on the growth requirements of the organism under investigation, the method can be optimised by changing many of the operational parameters. The reactor model can also be used for micro and biosensor monitoring, dental and material evaluations aswell as for investigating the performance of medical devices.

5D Biofilm Models

Minimum Biofilm Eradication Concentration (MBEC)

In this model the biofilm is grown according to ASTM E2799 – 17 (Standard Test Method for Testing Disinfectant Efficacy against Pseudomonas aeruginosa Biofilm using the MBEC Assay). At present this model has only been validated by ASTM for Pseudomonas aeruginosa. 5D have however advanced and validated this model so it can be customised for different microbes dependent on a customer’s specific needs. It has been modified in particular to evaluate the efficacy of disinfectants, antiseptics, antibiotics and other actives for use with monoculture and mixed culture microorganisms.

5D use the model for many applications. These include studying the effects of bone and dental biocides, evaluating antibiotics, biocides, disinfectants, heavy metal ion efficacy.

Centre for Disease Control (CDC) Biofilm Reactor

In this model the biofilm is grown according to the standard ASTM E2562 - 17 (standard test method for quantification of Pseudomonas aeruginosa biofilm grown with high shear and continuous flow using CDC biofilm reactor).

5D have adapted this model to grow an array of different biofilms using both single and multispecies aerobic and anaerobic biofilms.

Centre for Disease Control (CDC) Biofilm Reactor (Single Tube Assay)

In this model the biofilm is grown and antimicrobials are evaluate for efficacy according to the standard ASTM E2871 - 19 (Standard Test Method for Determining Disinfectant Efficacy Against Biofilm Grown in the CDC Biofilm Reactor Using the single Tube Assay).

Drip Flow Biofilm Reactor

In this model the biofilm is grown according to the standard ASTM E2647 - 13 (standard test method for quantification of Pseudomonas aeruginosa biofilm grown with using drip flow biofilm reactor with low shear and continuous flow). The model has been validated by 5D for use with monoculture and mixed culture biofilms. We have also validated it to be used with monoculture and mixed culture biofilms that are grown from 24 hours or 168 hours.

Drip Flow Biofilm Reactor (adapted for Wound Dressings and Medical Devices)

5D have adapted the Drip Flow Biofilm Reactor for evaluating the antibiofilm performance of wound dressings, with and without antimicrobial agents, and medical device performance with and without antimicrobial and antibiofilm technologies. This model can be used with and without skin. The model has been validated by 5D for use with monoculture and mixed culture biofilms.

Rotator Disk Biofilm Reactor

The model consists of both a rotating disk reactor and concentric cylinder reactor that has been designed to study biofilms and efficacy of antimicrobials under shear stress. The reactor is comprised of a circular disk that is able to hold removable coupons. Within this model reproducible biofilms can be obtained under continuous flow. The biofilm is grown according to ASTM E2196 - 17 (standard test method for quantification of Pseudomonas aeruginosa biofilm grown with medium shear and continuous flow using rotating disk reactor).

Flow Cell Biofilm Model

5D utilise microscopy flow cells to monitor and evaluate the performance of antimicrobials and actives in real time using confocal and fluorescent microscopic analysis.

The flow cells enable the creation of uniform and constant environments for studying in vitro biofilms.

Further Offered 5D Customised Models

5D Filter Disc Mixed Culture Biofilm Model

Within the filer disc model biofilms are grown on a semi-permeable filter, often polycarbonate and then placed often onto an appropriate solid medium such as agar. The filter system allows for the transfer of the biofilm from one media source to another and allows the development of a biofilm at the air-surface interface. Within this model the biofilm grows quite large in a relatively short period of time. Within this model we can evaluate the susceptibility of biofilms to antimicrobials by quantifying colony forming units. Furthermore, we can also study the penetration of antimicrobials through a biofilm. This model can be used to study monoculture swell as mixed culture biofilms. We have employed this model for evaluating the performance of medical devices, in particular wound dressings.

5D Tube Biofilm Reactor

Tube biofilm models are used to study the development of biofilms under flow. Within these models we study the development of biofilms within the lumen of tubes. These models enable us to quantitatively evaluate the performance of antimicrobials and fluid dynamics.

Unlike other biofilm models the tube biofilm models are often not amenable for microscopic analysis.

Robbins Device (modified)

This model is composed of a tube into which pegs are inserted. The pegs are removable from the vessel but form a component of the tube wall. Biofilms form on the removable pegs when the tube is seeded with a batch or continuous fluid. Such systems are often employed to study biofilms in water, wastewater and industrial water systems and have been employed to study biofilms on medical lines.

5D Chamberslide Biofilm Model

This biofilm model consists of removable polystyrene chambers which are attached to a glass slide. We utilise the slide surface to grow biofilms. These chamber biofilms can be grown in 1, 2, 4, or 8-well formats.

5D have validated this model so it can be used to study the formation on monoculture and mixed culture biofilms. We have been able to grow consistent and reproducible biofilm for up to 72 hours. We are able to monitor the development and destruction of biofilms in real time. Under confocal laser microscopy we are able to generate 2D time lapse confocal videos, 3D images, and 2D tile images to visualise the whole chamber where a biofilm is being grown. We have employed this model routinely to evaluate the performance and efficacy of antimicrobials, active solutions and antibiofilm technologies in real-time. For time-kill studies this model is very reproducible.

We have also been able to evaluate both the development and destruction of the extracellular matrix of the biofilm.

5D Advanced Wound Eschar Biofilm Model

5D have developed models that can determine how well wound and skin care products breakdown the components of both eschar and slough. The model combines collagen, fibrin and elastin in a reproducible and validated matrix.

As biofilms are found in slough and eschar within a wound, this model helps to determine whether an antimicrobial based wound dressing has the ability to breakdown biofilms, particularly when there is a high level of biological content. Within this model we are able to grow monoculture and mixed culture biofilms.

5D Dry Biofilm Model

Dry biofilms develop on surfaces, particular within the healthcare setting. Therefore it is likely that dry biofilms are responsible for the spreading of hospital acquired infections. 5D have developed dry biofilm models to evaluate the performance of antimicrobials.

5D Biofilm Formation Potential and Coaggregation Model

Coaggregation is often considered to be the first stage of biofilm formation particularly in dentistry and water. We have validated this model to determine whether active agents have the ability to cause a reduction and/or prevent bacteria from coaggregating and adhering.

5D Biofilm Proliferation and Dissemination Model

5D developed this model to determine the rate of microbial dissemination from materials and devices. As the biofilm is growing on the biomaterial we can determine the doubling time and specific growth rates of biofilms.

By evaluating the dissemination rates from surfaces this helps to determine if a material or device supports biofilms and the rate at which microbes detach and then contaminate other surfaces.

5D Urinary Catheter Biofilm Model

Catheter-associated urinary tract infections (CAUTIs) are one of the most common hospital acquired infections. Urinary tract infections due to catheters are also a significant risk factor for Gram negative bloodstream infections (GNBSI).

Catheters can be characterised as either indwelling (ID) or intermittent catheters (IC). The challenges in the use of IDs are biofilm formation and encrustation. We offer a number of urinary tract models; however, there are very limited models for testing ICs - there are currently no standardised urinary tract model to test the efficacies of ICs.

We can also evaluate bladder washout devices. Examples of models we employ include the microtitre plate assay, the time to kill attached bacteria assay, serial plate transfer test, an in vitro bladder model, an in vitro urinary tract model, an in vitro catheter-associated urinary tract infection (CAUTI) model and the meatus model.

5D Intravascular Catheter Biofilm Model

5D have a number of in-house central venous catheter (CVC) models that are used to evaluate the efficacy of antimicrobial impregnated catheters and also lock-flush and flush antimicrobial solutions.

5D Extracellualr Polymeric Substance Breakdown Model

Extracellular polymeric substances (EPS) is considered 'the House of the Biofilm Cells'. EPS is composed of polysaccharides, proteins, enzymes, genetic material, metal ions and lipids. In this 5D EPS test we utilise a number of different techniques and biofilm models to quantify the levels of EPS and investigate what effects active and antimicrobial agents have on the EPS structure and composition.

It is important that antibiofilm technologies, as well as reducing the biofilm community of microorganisms, also reduce the levels of EPS. EPS is very immunogenic and helps to enhance biofilm reformation.

5D Fungating Nail Biofilm Model

Onychomycosis is a fungal infection of the nail. It is a very difficult infection to treat. This is because the fungi that causes the nail infection i.e Trichophyton rubrum has the ability to grow as biofilms. The fungi grows under the nail and it becomes shielded by the nail plate. As it grows under the nail it forms a mass of hypha. 5D have developed a fungal nail model that can be used to evaluate the efficacy of antibiofilm and antimicrobial agents on fungal biofilms.

5D Skin Biofilm Model

We have developed and utilise a number of skin models to grow biofilms. These models are composed of either individual cell lines of fibroblasts and keratinocytes or ones that have an epidermis and dermis. We have managed to stablise biofilms in these models to evaluate wound dressing efficacy on biofilms. In addition we can use these models to investigate cytotoxic effects, cell migration and investigate the effects of agents on inflammatory markers.

5D Biofilm Prevention Model

Preventing both the formation and reformation of biofilms are fundamental for preventing biofilm related infections. We have developed a number of biofilm prevention models that can be used to investigate if surface coatings and related innovations are able to prevent microbial adhesion.

5D Depth of Penetration and Biofilm Permeation Models

It important to determine if an antimicrobial has the ability to penetrate through barriers in oder to get to the area where a biofilm is located, for example in chronic wounds there is often slough.

As biofilms in a wound are often found within and under slough, it is important to investigate if the antibiofilm technology can pass through a barrier to reach its site of action. We have developed numerous models to investigate this.

Standard Test Method for Determining Antimicrobial Activity and Biofilm Resistance Properties of Tube, Yarn or Fibre Specimens

ASTM E3151-18 is a test method which is a quantitative assay that is used to evaluate the antimicrobial efficacy of samples that are tubular or small segments of yarn or fibres that have been treated with antimicrobials. It is also a quantitative assessment of a sample that is able to resist microbial colonisation and therefore biofilm formation.

Other More Customised 5D Biofilm Models

5D also offer many other customised biofilm models specific to different customers in different industries. For example we have designed biofilm models in reference to otitis media and cystic fibrosis. For further information about some of the most routinely used biofilm models please contact us.