IGEM:IMPERIAL/2007/Projects/Biofilm Detector

Biofilm Detection: Introduction

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Summary

Biofilms are a huge problem in medicine and in industry. The dispersal of biofilms has long posed a problem to biologists, chemists, and engineers. Our project is based on the detection and possibly destruction of bacterial biofilms that are formed on catheters. To detect the biofilm, we aim to use a cell free approach, which makes the detection of AHL from the biofilm possible, unlike when using E. coli. Part of our project is also to characterise the detector within the cell free system and vessicles and compare them to the detector system in E. coli.

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(confirm) The process of biofilm investigation carried us through a host of different-natured biofilms, including those produced by MRSA and Pseudomonas Aeruginosa. The scope of implementing a bio-film detector, and possibly disperser ("saboteur"), proved too ambitious to undertake over a limited project period of 10 weeks. It was determined, therefore, that the well-studied E-Coli biofilm would be investigated further, and that the Detector would in fact be applied to this biofilm platform. More detailed information concerning the previously investigated Biofilms can be found here: Future Extensions of Biofilm Detector.This is important to consider in future applications of the "Biofilm Detector".

Recently, a paper[1] was published describing the use of an engineered bacteriophage that would also produce DspB, an enzyme that breaks down one of the major structural components of biofilm. However, it was pointed out that bacteriophages target very specific cells, which may not be present in the biofilm under consideration. Furthermore, bacteria often evolve defences against phages. Thus, although the phage technique has proven that the principle of infecting a biofilm is a good strategy to disperse it, it is far from becoming usable outside the tightly controlled lab environments.

This project proposes an alternative method of biofilm dispersal. In short, a bacteria will be modified to express DspB as a response to three input signals - quorum sensing, biofilm presence, and anaerobic conditions. These bacteria can then be sprayed over a biofilm, which they infiltrate, and disperse.

There are three major assumptions that may prove problematic in this proposal:

1. That the saboteur bacteria will be able to penetrate the biofilm
2. That the saboteur bacteria will survive in the biofilm
3. That the quorum sensing mechanism will work

A list of references can be found at the bottom, that may be able to help addressing these concerns.

The Hrp system may be used with its three inputs: biofilm detection, quorum sensing, and anaerobic conditions sensing (inverted in V input). By inverting the anaerobic conditions sensing, a basal level (the 10% leakage of the V-inhibition) of DspB will be produced. As soon as anaerobic conditions are reached, V-expression is inhibited (the inversion) which kicks the Hrp system into full swing.

On Escherichia coli Bio-Films

E. coli is ubiquitous in human intestines, and is normally benign. However, a few strains can cause infections, of three types: urinary tract infection (UTI), neonatal meningitis, and intestinal diseases (gastroenteritis). Of these, urinary tract infection is the most common nosocomial infection, and is caused by E. coli in abou 80% of all cases. Further, the UTI infective strains are well documented[1].

Once again, creating a system that can detect the presence of the particular infectious strains would serve two useful purposes: one, it could help in the surveillance and prevention of e.coli infections. Two, and perhaps more importantly, it could serve as a proof of concept that synthetic biology can be used to produce cheap, reliable, and fast sensors for particular pathogens. Also, E.coli is usually used as an indicator for human fecal contamination of water.

One question is that, if e.coli is used as the chassis for the detection system, how would one avoid having the system activate itself? A method for separating the target e.coli from the system e.coli must be devised.

Other nosocomial infections

Apparently, according to WikiPedia, urinary tract infections account for 40% of nosocomial infections (note, reference not found). Also, the most common cause of urinary tract infections is e.coli (again, note the lacking references - they are mostly about acupuncture, and not directly related to statistics on infections).

Defining the Project

After having several people spend many hour researching the possibility of detecting S. aureus biofilms, it looks like this will be difficult to achieve within the time restrictions of the iGEM project. However, a proof of concept may be achievable instead: we might be able to demonstrate that we can build a system that detects the presence of minimal e.coli biofilms, and then reports back quickly and strongly. The pathway would involve detection, amplification, and reporting. Proving that the concept works with e.coli might then inspire further work into a similar solution for MRSA. At this point, we still have several options going forward: try it with Bacillus Subtilis, change target to Pseudomonae, or change target to e.coli.

Application of System

Motivation

Improved life expectancy of the global population plays a major role in the rising demand for all medical catheters. Over a 100 different types of catheters are available on the market at the moment; involved amongst others in infusion, cardiovascular, renal, haemodynamic monitoring and neurological contexts.

Catheters, specifically central venous catheters (CVCs), are responsible for more device-related infections than any other "internal" medical device. As an illustration, catheter-related blood stream infections (CRBSIs) are both common and costly. Approximately 3 million central lines are placed in the United States each year; resulting in 150,000 cases of CRBSIs annually. In addition, 90% of all CRBSIs occur in clinical situations in which a temporary central venous catheter was used. The cost of treating CRBSIs annually in the United States ranges from $300 million to $2 billion a year.

Detection methods (EM and TEM) indicate their presence both on their outside surface and inner lumen. Colonization and consequent biofilm formation may occur within 3 days. On a shorter scale, within 10 days, biofilm is more extensive; longer-term (up to 30 days) catheters exhibit greater biofilm formation internally. Current detection methods involve the so-called roll-plate technique: (details?). This method, however, is unable to detect the presence of organisms on the inner luminal surface, and its sensitivity is insufficient: not more than 1000 CFU per tip.

The proposed system would involve detection of biofilm formation on the outer surface of the catheter. Its presence would be displayed by way of fluorescence. However, the system could potentially be extended to incorporate an eradication mechanism, exploiting the degradatory protein, DspB.

Biofilms and your health...

• Biofilms are responsible for diseases such as otitis media, the most common acute ear infection in children in the U.S. Other diseases in which biofilms play a role include bacterial endocarditis (infection of the inner surface of the heart and its valves), cystic fibrosis (a chronic disorder resulting in increased susceptibility to serious lung infections), and Legionnaire's disease (an acute respiratory infection resulting from the aspiration of clumps of Legionnella biofilms detached from air and water heating/cooling and distribution systems).
• Biofilms may be responsible for a wide variety of nosocomial (hospital-acquired) infections. Sources of biofilm-related infections can include the surfaces of catheters, medical implants, wound dressings, or other types of medical devices.
• Biofilms are highly resistant to antibiotics. Consequently, very high and/or long-term doses are often required to eradicate biofilm-related infections.
• Biofilms happily colonize many household surfaces, including toilets, sinks, countertops, and cutting boards in the kitchen and bath. Poor disinfection practices and ineffective cleaning products may increase the incidence of illnesses associated with pathogenic organisms associated with normal household activity.

Biofilms and industry...

• Biofilms are responsible for billions of dollars in lost industrial productivity and both product and capital equipment damage each year. For example, biofilms are notorious for causing pipe plugging, corrosion and water contamination.
• Biofilm contamination and fouling occurs in nearly every industrial water-based process, including water treatment and distribution, pulp and paper manufacturing, and the operation of cooling towers.

From Biofilms Online

Biofilm formation on catheters

• AHL is producded by the clinical biofilms
• useful link for method probably!: http://aem.asm.org/cgi/reprint/64/9/3486

Detection Targets

• After looking through the literature tried to identify possible targets for detection of biofilms.
• Can generally split them down into three catagories;

1. Signaling molecules - This really concerns quorum sensing.
2. Exopolysacchorides
3. Attachment molecules
4. more?

Quorum Sensing

Diagram of the Agr system

• Two possible targets for quorum sensing are the:

1. Las pathway
2. Rhl pathway
3. Lux pathway

At the outset of this investigation into biofilms we looked at specific target biofilms that we could target. We tried to consider biofilms that have importance within medicine and in particular, are a problem in terms of disease and detection.

Staphylococcus aureus

• We have identified a pathway that is unique to Staphylococcus aureus this system is called the Arg system. We investigated this and the potential target of this pathway. A mechanism of this pathway is shown to the right.
• However, upon further investigation we came across several problems:

1. This type of quorum sensing is not with soluble 'signal inducers' but small peptides. The small peptides are cannot freely diffuse in and out of cells and require specialized membrane equipment in order for the cells to uptake the peptides. This is a problem because Staphylococcus aureus is a gram positive bacterium and the model organism that we would ideally use is E.coli which is a gram negative bacterium. The difference in membrane structures posses a problem and in previous studies, attempts to apply the gram postive equipment in E.coli has failed.
2. In addition we failed to find a way to specifically targeted Staphylococcus aureus biofilm signaling or indeed the adhesion and exopolysacchorides of the biofilm.

2. Cámara M, Williams P, and Hardman A. Controlling infection by tuning in and turning down the volume of bacterial small-talk. Lancet Infect Dis. 2002 Nov;2(11):667-76. DOI:10.1016/s1473-3099(02)00447-4 |

A biofilm is a organizations of microorganisms on various biotic and abiotic surfaces. They are naturally occurring and are key for many organisms survival, in addition to being key for many organisms infectious ability. There are a variety of fields concerned with biofilms, notably medical and industrial. Medical interests in biofilms are in controlling the growth harmful biofilms forming. In addition, there has been interest for industrial applications, for example, biofilms can grow and block pipes.

In-Veso Biofilm Detector presentation (PPT)