Biofilms are a huge problem in medicine and in industry. The dispersal of biofilms has long posed a problem to biologists, chemists, and engineers. Recently, a paper<cite>Phage</cite> 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.
==== Project 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 biofilms, we aim to use a cell free approach, as it was thought that using bacteria to detect biofilms might result in biofilm forming on the catheters, if traces of the detector are not cleaned properly. We will also characterise the detector system within the cell free system and in vesicles and compare them to the detector system in E. coli. <br>
There are three major assumptions that may prove problematic in this proposal:
Detection of biofilms will be done using the lux system. We will simulate the presence of biofilms by AHL producing E. coli. The sensitivity of the lux promoter under different conditions (concentration of AHL and temperature) will be assayed by a fluorescent reporter. The detection system will be implemented and tested in E coli, in vitro and in veso. Hrp system may also be incorporated into the system to amplify the fluorescent output, as it is thought that the vessicles may reduce the synthesis of the fluorescent protein.
# That the saboteur bacteria will be able to penetrate the biofilm
# That the saboteur bacteria will survive in the biofilm
# 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.
====Project plan====
Our plan is to make the basic construct of the detector system and test it in the three chassis mentioned above. The results can then be compared to determine the best chassis for the system. For each chassis, the system will be tested for: <br>
*the response time for different temperatures and AHL concentrations
*the variation in reporter expression for different concentrations of AHL
*the variation in reporter expression for different temperatures
*the lifespan of the system in different temperatures
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.
We will also test atleast two reporters with our system and the final design would have the reporter best suited to our requirements. The reporter we need must:<br>
*have a fast response time (steady state reponse)
*would respond to very low concentrations of AHL
<br> Different reporters and their advantages and disadvantages can be found [[IGEM:IMPERIAL/2007/Projects/Reporters| here]]
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.
<br>The AHL will intially be induced directly into the detector solution for each of the experiment, to verify that the system responds to it. Later the AHL can be introduced into the system by creating a signalling system within an E coli. To fully test the system for its application, the detector needs to be tested on a biofilm. For this purpose a biofilm might be grown in the lab, but this test will only be carried out of time allows it. Figure 1 on the right shows the final infector detector construct. <br>
<br> However, more detailed information concerning the previously investigated Biofilms can be found here:
[IGEM:IMPERIAL/2007/Projects/Biofilm Detector/FutureApps Future extensions of Biofilm Detector]<br>
This is important to consider in future applications of the "Biofilm Detector".
==== MRSA Biofilms ====
To improve the efficiency of the detector, HRP may be incorporated into the design. The HRP system will not only work to amplify the output signal but also increase the repsonse time of the system. This stage can only be completed after the HRP system has been fully characterised.
Findings suggest that strongly biofilm-producing MRSA strains are associated with nosocomial infection, such as surgical site infection and pneumonia. In particular, agr-2 and agr-3 produced strong biofilms, while agr-1 produced weak biofilms<cite>agr</cite>
The agr gene is a factor of the quorum-sensing system in staphylococci that senses the density of bacteria. Agr-positive bacteria are less likely to produce a biofilm (6%) while agr-negative are more likely (78%).<cite>MRSA-agr</cite>
The intracellular adhesion locus (ica) is present in MRSA, and is necessary for the formation of biofilm.<cite>ica ica2</cite> If we can detect its expression, we may be able to detect biofilm presence.<cite>detection</cite>
The problem now is to find a way to detect the presence and expression of the ica locus.<cite>icalocus icadetection</cite> Perhaps the regulation<cite>icareg</cite> might be a good place to start.
===== The MRSA Problem =====
While hygiene procedures offer a proactive approach to containing MRSA, surveillance could enable the UK to make better use of the monies and efforts directed at reducing the MRSA threat. Measurement is a key tool for any operation to be able to gauge the success, efficacy or effectiveness of any tactic aimed at fulfilling a strategy. This is no less true of disease control.<cite>CountryDoctor</cite>
Public health surveillance has four objectives:
# detect and monitor adverse events
# assess risk and protective factors
# evaluate preventive interventions
# provide information that helps implement effective prevention strategies
(From the [http://www.cdc.gov/ncidod/dhqp/nnis.html National Nosocomial Infection Surveillance System])
It has been shown that active surveillance of MRSA decreases the incidence of infection. Active surveillance culture is important for identifying hidden reservoirs of MRSA. Contact isolation can prevent new colonization and infection and lead to a significant reduction of morbidity and healthcare costs.<cite>Surv</cite>
Although current surveillance methods are cost-effective when applied selectively in hospitals where MRSA is endemic, the procedure is lengthy and involved. In addition to the routines of preparation and transportation to the lab, samples take 24-48 hours to culture before analysis can take place.<cite>Surv</cite> (Microbiologic Analysis) Specifically, MRSA presence is tested by the following techniques:[http://www.cdc.gov/ncidod/dbmd/abcs/meth-lab-char.htm]
* Antimicrobial susceptibility testing
* Pulsed-field gel electrophoresis and typing
* Screening for SCC mec type II vs IV
* PCR detection of genes for TSST1, PVL and selected enterotoxins
This procedure is costly in time, money, and allocation of hospital resources (suspected patients must remain in isolation before results come out).
Thus, there is room for improvement in MRSA surveilance strategies. A surveillance method that is faster and does not require use of a laboratory could have a great impact. Considering the costs involved, a simple binary detection mechanism that can reliably confirm the presence of ''Staphylococcus aureus'' - regardless of methicillin resistance status - would allow for faster screening of patients. Since 43.6% ([http://www.rivm.nl/earss/database/ EARSS] 2005) of UK hospital ''S. aureus'' infections are methicillin resistant, only about half of the positively identified patients would prove to be false-positives. Those samples flagged as carriers of ''S. aureus'' can be sent for further analysis.
Some MRSA infection routes:
*
*
*
*
*
* Airborne, via acanthamoeba[http://en.wikipedia.org/wiki/Acanthamoeba]<cite>amoeba</cite>
=====Detection of MRSA=====
As hinted above, a few processes have been studied already as a possible avenue for detecting the presence of Staphilococcus aureus. However, the fact that S. aureus is a gram-positive organism makes things very complicated. agr, ica, and luxS have been looked at. luxS may have potential, but someone with more biochemical knowledge than Dirk must look at it and decide whether it is worth pursuing.
Another possible avenue is to use Bacillus subtilis as the detection system. As it is a gram-positive model organism, it may well be capable of detecting the signalling molecules used by S. aureus. In fact, BS is commonly used to work with apparatus normally found in SA for characterisation of parts. Other features that are catching about BS:
* Can we experiment on MRSA? Or do we use some other strand of SA?
==== On Pseudomonas aeruginosa ====
* Useful text describing the organism: [http://textbookofbacteriology.net/pseudomonas.html]
* We wanted to consider the possibility of being able to use ''Pseudomonas aeruginosa'' as our target. Within ''Pseudomonas aeruginosa'' the three pathways mentioned above involved within this organism.
===== Las and Rhl system =====
*The las and rhl systems within the ''Pseudomonas aeruginosa'' are similar to that seen in the lux system. They both depend on a type of AHL molecule that can move to and accumulate in the extracellular environment. In addition the same principle of a gene to synthesis the AHL and a gene encoding a AHL dependent transcriptional activator.
* These systems were both considered for detection, however as our knowledge increased of these pathways, so did the possible problems:
# The las is only produced at high levels in the early growth stages of biofilm development.
# The rhl system maintains a constant low level of expression throughout biofilm formation.
# Both lad and rhl are mainly expressed in the lowest layers of the biofilm and so detection from the exterior of the biofilm will be a problem.
# Finally ''Pseudomonas aeruginosa'' are found within most environments, e.g. on the skin. This causes a massive problems of false positives.
==== On Escherichia coli ====
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[http://textbookofbacteriology.net/e.coli.html].
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====
====Application of System====
Line 160:
Line 75:
From [http://www.biofilmsonline.com/cgi-bin/biofilmsonline/ed_facts_primer.html Biofilms Online]
From [http://www.biofilmsonline.com/cgi-bin/biofilmsonline/ed_facts_primer.html 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.
'''Obsolete Items'''
*Can generally split them down into three catagories;
#Signaling molecules - This really concerns quorum sensing.
[[IMAGE:Agr.JPG|thumb|right| Diagram of the Agr system ]]
*Two possible targets for quorum sensing are the:
Anthony's comments {{hide|
# Las pathway
This page needs to be re-written.
# Rhl pathway
# 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.
#Specifcity:For starters we are targeting urinary catheter infections so there needs to be more of this and not other areas in which biofilms are problems. Let's make this introduction specific to urinary catheter infections.
===== ''Staphylococcus aureus'' =====
#References:There are no references in this introduction, considering the amount of references found on the references page this is both confusing and annoying as what we have said here has no weight
#Background:Although we have references on it there is no talk here of what has been done to target urinary catheter infecions so far. For example one paper in the reference section talks about using herapin coating as a preventative stratergy and i've found another paper which discusses using silver based catheters as a preventative measure.
*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.
#Mystery People: Let's get some people on board with the project and mention them in the introduction. We've got 3 profs on the refence page but there are not mentioned at all here.
*However, upon further investigation we came across several problems:
}}
#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.
# In addition we failed to find a way to specifically targeted ''Staphylococcus aureus'' biofilm signaling or indeed the adhesion and exopolysacchorides of the biofilm.
<biblio>
#1 pmid=12409047
</biblio>
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.
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 biofilms, we aim to use a cell free approach, as it was thought that using bacteria to detect biofilms might result in biofilm forming on the catheters, if traces of the detector are not cleaned properly. We will also characterise the detector system within the cell free system and in vesicles and compare them to the detector system in E. coli.
Detection of biofilms will be done using the lux system. We will simulate the presence of biofilms by AHL producing E. coli. The sensitivity of the lux promoter under different conditions (concentration of AHL and temperature) will be assayed by a fluorescent reporter. The detection system will be implemented and tested in E coli, in vitro and in veso. Hrp system may also be incorporated into the system to amplify the fluorescent output, as it is thought that the vessicles may reduce the synthesis of the fluorescent protein.
Project plan
Our plan is to make the basic construct of the detector system and test it in the three chassis mentioned above. The results can then be compared to determine the best chassis for the system. For each chassis, the system will be tested for:
Figure 1: Proposed system
the response time for different temperatures and AHL concentrations
the variation in reporter expression for different concentrations of AHL
the variation in reporter expression for different temperatures
the lifespan of the system in different temperatures
We will also test atleast two reporters with our system and the final design would have the reporter best suited to our requirements. The reporter we need must:
have a fast response time (steady state reponse)
would respond to very low concentrations of AHL
Different reporters and their advantages and disadvantages can be found here
The AHL will intially be induced directly into the detector solution for each of the experiment, to verify that the system responds to it. Later the AHL can be introduced into the system by creating a signalling system within an E coli. To fully test the system for its application, the detector needs to be tested on a biofilm. For this purpose a biofilm might be grown in the lab, but this test will only be carried out of time allows it. Figure 1 on the right shows the final infector detector construct.
To improve the efficiency of the detector, HRP may be incorporated into the design. The HRP system will not only work to amplify the output signal but also increase the repsonse time of the system. This stage can only be completed after the HRP system has been fully characterised.
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.
Specifcity:For starters we are targeting urinary catheter infections so there needs to be more of this and not other areas in which biofilms are problems. Let's make this introduction specific to urinary catheter infections.
References:There are no references in this introduction, considering the amount of references found on the references page this is both confusing and annoying as what we have said here has no weight
Background:Although we have references on it there is no talk here of what has been done to target urinary catheter infecions so far. For example one paper in the reference section talks about using herapin coating as a preventative stratergy and i've found another paper which discusses using silver based catheters as a preventative measure.
Mystery People: Let's get some people on board with the project and mention them in the introduction. We've got 3 profs on the refence page but there are not mentioned at all here.
