IGEM:IMPERIAL/2007/Projects/Biofilm Detector/FutureApps

MRSA Biofilms

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[1]

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%).[2]

The intracellular adhesion locus (ica) is present in MRSA, and is necessary for the formation of biofilm.[3, 4] If we can detect its expression, we may be able to detect biofilm presence.[5]

The problem now is to find a way to detect the presence and expression of the ica locus.[6, 7] Perhaps the regulation[8] 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.[9] Public health surveillance has four objectives:

1. detect and monitor adverse events
2. assess risk and protective factors
3. evaluate preventive interventions
4. provide information that helps implement effective prevention strategies

(From the 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.[10]

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.[10] (Microbiologic Analysis) Specifically, MRSA presence is tested by the following techniques:[1]

• 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% (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[2][11]

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:

• it is naturally competent
• it is not considered a human pathogen

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1360299 http://www.journals.uchicago.edu/JID/journal/issues/v190n2/31437/31437.web.pdf http://www.jbc.org/cgi/content/abstract/276/4/2658

Questions Arising

• Can we get hold of the required DNA sequences?
• Can we experiment on MRSA? Or do we use some other strand of SA?

On Pseudomonas aeruginosa

• Useful text describing the organism: [3]

• 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:

1. The las is only produced at high levels in the early growth stages of biofilm development.
2. The rhl system maintains a constant low level of expression throughout biofilm formation.
3. 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.
4. Finally Pseudomonas aeruginosa are found within most environments, e.g. on the skin. This causes a massive problems of false positives.