Biofilms : Introduction

Biofilms are ‘surface-associated communities of microorganisms embedded within a self-produced extracellular matrix’ - that is to say, under stress conditions, bacteria will begin to secrete extracellular polymeric substances (EPS) such as DNA, polysaccharides and proteins1, which form a matrix containing the cells: a biofilm. As a result, bacteria in biofilms become much more resistant to stresses, such as pH and temperature fluctuations, the presence of antimicrobials, starvation, desiccation and shear stress2.

Biofilm Formation Diagram
Figure 1: The initial attachment of a biofilm, its maturation and its dispersal (Adapted from Ref.3)

Biofilms : The Problem

Biofilms are notoriously associated with many problems, from medical issues, such as catheter-associated urinary tract infections (CAUTIs), to industrial issues, such as Microbially Induced Corrosion (MIC) resulting in structural failure.

Mindmap of Biofilm Associated Problems
Figure 2: The problems associated with biofilms

A problem that stood out was the issue of zoonotic diseases, in which pathogenic biofilms such as Salmonella and Campylobacter form on the walls and floors in farms. This leads to constant exposure of farm animals to the bacteria and thus an increase in the spread of disease, with chickens recognised as the primary vector. To prevent animal disease, farmers often use prophylactics such as antibiotics; however, this leads to the development of Anti-Microbial Resistance (AMR), which is recognised by the World Health Organisation (WHO) as one of the top 10 threats to global health, due to the association of AMR with over 5 million deaths annually4. Over 60% of the global use of antibiotics is on farms5; however, as much as 30 to 90% of the used antibiotics are excreted6 and potentially released to the environment. This contributes to constant exposure to antimicrobials in the environment, leading to further AMR development. Despite all of this, disease treatment still costs farmers around the globe at least US$ 24 billion7 annually, this is expected to increase by 65% if left unchecked8.

Stat 1 - 1 in 10 Suffer From Foodbourne Diseases Each Year, According to WHO Stat 2 - $1.56 Billion Spent on Campylobacteriosis, an Emerging & Underreported Zoonosis Stat 3 - 10 Million Annual Deaths By 2050 Due to AMR

Current Solutions

As described earlier, biofilms are more resilient to most stresses, including many attempts to clean. The use of disinfectants and antimicrobials, as well as mechanical methods of cleaning and more novel methods such as the use of DNases that degrade the biofilm matrix all struggle to completely remove the biofilm, and in leaving a few cells can lead to biofilm regrowth. Also, as most biofilms are only detected visually, this means they're sometimes only cleaned once the biofilms have reached a substantial size.


There are also other problems specific to each case, such as the ineffectiveness of cleaning solutions in metal pipes as they would simply be washed away, and the requirement for non-toxicity in medical and agricultural cases. One current cleaning method of metal pipes is the use of ‘pigs’9, cylindrical machines that are able to move through pipes and mechanically break off any large biofilms that have formed; but as described previously, these are unable to be completely removed and are thus required to be cleaned frequently, during which the pipes are usually shut down.


In the case of farms, many countries such as the UK have begun to recognise to problem of AMR development from the use of prophylactic antibiotics; however, this leaves farmers without many effective solutions, and thus rely upon cleaning their farms excessively, which is labour-intensive and also sometimes involves the use of biocides which present their own problems to the environment.

Our Solution

Our solution to the general idea of harmful biofilms is based on the general idea of engineering a positive biofilm, a non-harmful and beneficial biofilm, to take up the available space and prevent the formation of secondary, harmful biofilms. For this, we decided to use Bacillus subtilis, a gram-positive and non-pathogenic biofilm-forming bacteria, due to its inherent ability to form protective biofilms, such as those on plant roots10. It is also generally regarded as safe, being BSL-1, and is also already used as a probiotic for both humans and animals11.


With our chassis decided, we now need to optimise its properties in order to sustain a resilient and effective protective layer. The main aspect to improve was the biofilm-forming capability of our bacteria, which we were able to do by overexpressing the components of the biofilms matrix, assisted by our Dry Lab model of the relevant genetic circuit used to predict the genes that had the largest impact. Another thing to improve was the resilience of our biofilm - B. subtilis has a tendency to sporulate, which involves the dispersal and detachment of the biofilm, hence we decided to place the gene that initiates sporulation under an inducible promoter. This prevents the biofilm from naturally sporulating, and thus increasing its resilience.


This helped us create a general protective biofilm that could be employed against multiple problematic biofilms; however, we also wanted to create a specialised protective biofilm for the case of pathogenic biofilms in farms. Pathogenic biofilms are a problem as they often are exposed, such as on the walls in animal pens in farms. Whilst this presents ease of access to clean them, a problem is knowing when to clean. Most farms will either disinfect on a regular basis, which can be excessive and generally wasteful, or clean once they can see biofilms growing, except by this point the biofilm has grown to significant sizes and been present and exposed for a long time.


As such, we decided to introduce a biosensor into our protective biofilm. Enteric pathogens usually release a signalling molecule called autoinducer-3 (AI-3) for quorum signalling. Many pathogenic bacteria express a protein named QseC, which is a receptor for AI-3, and QseB, a response regulator that forms a two-component system with QseC12. By having B. subtilis express both proteins and having a GFP gene downstream of a QseB-activated promoter, the bacteria can act as a biosensor for AI-3. As an extension to this, in Dry Lab, we wanted to use molecular dynamics to see if this system from E. coli would work with the B. subtilis RNA polymerase.

B. Max : The Final Product

To conclude, we present B. max - an engineered B. subtilis strain capable of forming protective biofilms that can coat surfaces and protect against secondary colonisation of harmful bacteria. We see this being able to be applied to combat different problematic biofilms, and have specialised ours to be able to detect pathogenic biofilms with a biosensor for AI-3 (more details on our Engineering page).


On our Discussion page, we talk through the potential next steps we might have taken given more time, as well as potential routes for the future teams if they wish to build upon our idea.

References