CathExe Project Description

Harnessing quorum sensing for biofilm prevention in catheters.

DESCRIPTION

Background

What are CAUTIs?

Urinary catheters are a common medical device implemented for a variety of reasons, including urinary retention, incontinence, or surgery. In the National Health Service (NHS) 1 in 5 hospital patients are catheterised, with this increasing to 70% in critical care units [1]. Catheter usage is the predominant risk factor for 70-80% of urinary tract infections (UTIs) - these are referred to as catheter-associated UTIs (CAUTIs) [2]. UTIs are characterised as an infection of the urinary tract system; composed of the urethra, bladder, ureters, and kidneys [3]. Patients with long-term indwelling catheters, which have been in place for 30 days or greater, have close to a 100% chance of developing bacteriuria [4] and a significantly increased risk of developing a CAUTI [5]. This supports the findings that CAUTIs, and linked catheter-associated blood stream infections (CABSIs), are two leading causes of healthcare-associated infection (HCAI) in the NHS England [6]. Currently, to combat this, extensive antimicrobial and antibiotic use is being employed; however, the formation of biofilms renders these treatments inefficient and contributes to their resistance [7].


Due to the high rates of CAUTIs in the healthcare system there have been attempts made to reduce infection rate. With a focus on infections caused by Gram-negative bacteria, 55% of which are caused by Escherichia coli (E. coli) [8]. In the HM Government response to the 2016 Review on Antimicrobial Resistance they pledged to half the number of nosocomial GNBSIs before 2020 [6]. However, minimal progress has been made to reach this goal, which was pushed back to 2023/24 - and a clear strategy to achieve this reduction has not been identified [9].


Therefore, despite the consistent publication rate in CAUTI research, there is a clear separation between the research and the application of such measures in practice. Following on from this, our goal is to target the prevention of CAUTIs in the healthcare system in hopes of reducing HCAIs by up to 1/3 - the proportion attributed catheter usage [10].


An Introduction to Biofilms

Biofilms are an aggregate of one or more microorganisms fixed within a complex matrix [11]. They aid bacterial adherence to both biotic and abiotic surfaces, and are a supportive environment for persister cells [12]. Persister cells are not metabolically active, meaning they are tolerant to antimicrobials which results in chronic infections [11]. The formation of a biofilm can make bacterial cells anywhere from 10 to 1000 times more resistant to antibiotics compared to planktonic cells [13]. In clinical settings this is particularly dangerous as hospitalised patients are often immunocompromised, so are highly vulnerable to infection [14].

Diagram explaining biofilm formation in S. epidermidis
Figure 1: A diagram demonstrating the four stages of biofilm formation

Biofilm formation has 4 distinct stages, depicted in Figure 1. Each of these stages has characteristic features [11]:

  1. Reversible attachment:
    This first step is triggered by interactions between a surface and the planktonic microbial cells. Adherence of the microorganisms is aided by extracellular polymeric substances including exopolysaccharides, various surface proteins, biofilm-associated protein, and autolysin.
  2. Irreversible Attachment:
    As the microorganisms proliferate and the biofilm grows, attachment becomes irreversible. The level of cell-to-cell adhesion increases, and extracellular polymeric substances (EPSs) start to be produced. The EPS matrix is made up of polysaccharides, nucleic acids, and proteins and has various roles including - promoting microbial attachment, enhancing virulence, and acting as a protective barrier from threats.
  3. Maturation:
    At this stage the biofilm becomes a more structured community, with microorganisms being well-protected from external threats such as host defences and antibiotics as the EPS is established. This is accompanied by the release of autoregulators which influences gene expression, increasing virulence.
  4. Cell detachment:
    In the final stage planktonic cells are released from the surface of the biofilm. These dispersed cells are no longer protected, so are sensitive to antimicrobials.

This process occurs on indwelling medical devices, including urethral catheters. These biofilms contain uropathogens and can be found on the catheter and in the surrounding uroepithelial region - see Figure 2.

Diagram explaining biofilm formation in S. epidermidis
Figure 2:Depiction of Foley catheter inside of urethra with a biofilm formed, extending to the bladder.

The Main Offenders


The formation of biofilms is a key step pathology of CAUTIs. There are a broad range of microorganisms associated with the biofilm microenvironment, with a few major players [5]- see Figure 3. Most species depicted are bacterial, although Candida albicans (a fungal species) also contributes to a significant proportion of infections. However, across the literature there is some discrepancy in the most prevalent species, with Staphylococci sp. appearing in a multitude of studies [15]. For example, the biofilm-positive Staphylococcus epidermidis has emerged as a major pathogen associated with nosocomial infections of indwelling medical devices [5].

Diagram explaining biofilm formation in S. epidermidis
Figure 3: Pie chart displaying the most common bacteria that form biofilms in catheters

Our Solution

Due to the high diversity of microbial species associated with biofilm formation, we identified the need for a broad-spectrum anti-biofilm therapy.

Our approach centres on the modification of Lactiplantibacillus plantarum to effectively target the formation of biofilms by uropathogens. L. plantarum is a commensal Gram-positive bacterial species commonly found in urogenital samples of patients with no infection [5]. It is also a desirable probiotic, found in many fermented foods, with multiple beneficial medical properties [16]. For example, it's presence can tackle pathogenic bacteria due to molecules produced, and competition for space and resources. This is due to the production of acids such as lactate and acetate, which moderate the environmental pH and inhibit growth of harmful bacteria [17]. In addition, the cell wall component of L. plantarum-GMNL6, lipoteichoic acid (LTA) inhibits biofilm formation of S. aureus [18].

Therefore, L. plantarum is the ideal therapeutic agent - as it can survive in the urogenital tract whilst providing benefits before any further modifications have been made. By genetically engineering L. plantarum to synthesise specific molecules that inhibit biofilm development, we can precisely customise its application for use on catheters - see Figure 4. Constitutive expression of biofilm-inhibiting molecules by the L. plantarum would prevent initial attachment of biofilm-positive bacterial species onto the catheter.


diagram of our idea
Figure 4:

Overview of the Biofilm Inhibiting Molecules:

Our modified L. plantarum will contain genes encoding various molecules which will holistically prevent uropathogenic bacterial biofilms. These include:

Gram-positive:
  • Quorum Quencher 7 (QQ-7) - Decreases bacterial biofilm formation in an ica-dependent manner via oxidoreductase activity and prevents the yeast to hyphal switch in C. albicans resulting in biofilm inhibition [19].
  • Quorum Quencher 5 (QQ-5) - Prevents the yeast to hyphal switch in C. ablicans, inhibiting biofilm formation and shows some of the markers for bacterial biofilm inhibition found in QQ-7 [19].
  • LuxS - An enzyme tightly linked to activity of the ica operon and exopolysaccharide synthesis [20].

Gram-negative:
  • AHL lactonase - Breaks down N-acyl homoserine lactones (AHLs) of Gram-negative bacteria into N-acyl-homoserine [21].


Getting the L. plantarum onto catheters

We initially planned to adhere our modified L. plantarum to the catheter surface via upregulation of CapA production, however this did not work and can be read about as one of our engineering cycles.
After this setback we looked to our modelling (see 2D and 3D Diffusion, as well as AHL Degradation), for details. This led us to realising a spray coating is the most effective way to apply our molecular cocktail to the catheter.


Target Systems

The ica operon - target of QQ5, QQ7 and LuxS

What is the ica operon?

The ica operon is composed of 4 genes located at the icaADBC locus, as shown in Figure 5. These 4 genes encode enzymes responsible for the synthesis of the exopolysaccharide poly-β(1-6)-N-acetylglucosamine (PNAG) - also known as polysaccharide intercellular adhesin (PIA)[26]. Across bacterial species exopolysaccharides play a critical role in biofilm formation, with ica orthologs present in multiple biofilm-forming species [27].

Diagram explaining biofilm formation in S. epidermidis
Figure 5:

Control of the ica operon

Control of the ica operon has been tightly linked to LuxS activity. LuxS is an autoinducer-2 (AI-2) synthase, with AI-2 molecules acting as the signalling molecule in AI-2 mediated quorum sensing (QS). QS is a cell communication mechanism and there are various systems in bacteria that utilise different signalling molecules [28]. LuxS is also a vital enzyme in the activated methyl cycle, meaning changes in these pathways could also alter various metabolic pathways [29].

LuxS mutants display various altered phenotypes, linked to the response of ica - which can be positive or negative depending on the species or strain of bacteria [29]. Hence, careful consideration is required when choosing how to target the AI-2 control system.


Positive regulation

In species with positive regulation, less AI-2 correlates with reduced biofilm formation; as AI-2 increases expression from the ica promoter meaning PIA production is reduced. A reduction in AI-2 has been associated with increased icaR expression in these species [30]. Although, depending on the species various other mechanisms behind this positive control have been suggested [29].

Species exhibiting positive control include:

  • Staphylococcus epidermidis strain RP62A [30].
  • Escherichia coli
  • Streptococcus pneumonia
  • Salmonella serovar
  • Klebsiella pneumoniae [25]

Negative Regulation

In species with negative regulation of the ica operon, LuxS mutants have increased biofilm formation. Therefore, production of more AI-2 would reduce PIA production by inducing expression of icaR - see Figure 6.

Species exhibiting negative control include:

  • Staphylococcus aureus [29]
  • Staphylococcus epidermidis 1457 [20]
Diagram explaining biofilm formation in S. epidermidis
Figure 6:
ica proteins [27]: icaA: N-acetylglucosaminyl-transferase
  • Catalyses the polymerisation of N-acetylglucosamine (GlcNAc in Figure 6).
icaB: IcaA co-factor
  • Required for optimal efficiency of IcaA [26].
IcaB - Metal-dependent de-N-acetylase
  • Deacetylates the poly-N-acetylglucosamine molecule to form the final PIA molecule.
IcaC - O-succinyl-transferase
  • Is responsible for the addition of a succinyl group.
  • Aids elongation of shorter N-acetylglucosamines.
  • Is associated with the export of the N-acetylglucosamines.
IcaR - The repressor of the ica operon.
  • Binds to the ica promoter, preventing RNA polymerase from binding and transcription from taking place.

How do we aim to target the ica operon?

We are mainly focusing on targeting species with AI-2 positive control of ica as these species are the prominent biofilm-forming strains - see Figure 3. To target these pathways QQ-5 and QQ-7 should be expressed by the L. plantarum. Of these two molecules, QQ-7 was shown to induce the most significant increase in icaR expression. It is suggested, based on results from S. epidermidis RP62A, that QQ-7 has oxidoreductase activity that degrades AI-2. QQ-5 has a similar mode of action, but was found to be more effective against C. albicans than the bacterial species [19].

Despite most species having positive regulation, we still aim to test the effects of LuxS expression. Particularly as Staphylococcus aureus remains a problematic strain implicated in indwelling medical device infections [26].


AHL-mediated QS - target of AHL lactonase

QS is a form of bacterial communication as mentioned above - allowing bacteria to detect and respond to different kinds of stimuli. QS ensures bacteria can develop into a high cell density from a low cell density [31]. However, current research has characterised AHL-based QS to a high degree, see Figure 7, especially when compared to current knowledge of LuxS/AI-2-based QS. By degrading AHLs we could prevent biofilm formation in many of the problematic Gram-negative species including E. coli and P. aeruginosa [32].

Diagram explaining biofilm formation in S. epidermidis Figure 7:

Acyl homoserine lactone (AHL) signal molecules are a wide-spread class of autoinducers in Gram negative bacteria. AHLs are synthesised by the Luxl enzyme and are then able to passively diffuse across the bacterial membrane in both directions. While AHLs diffuse into the cell they are recognised by the LuxR receptor. Following binding to the receptor, this dimerised complex acts as a transcription factor on the Lux box. This activates expression of virulence-associated genes downstream, as well as the Luxl/LuxR AHL system. These virulence genes have a wide variety of functions in the cell, including regulation of biofilm formation.

Diagram and explanation adapted from [32,33].

We are aiming to express AHL lactonase in the L. plantarum. AHL lactonase works outside the cell by degrading AHL into N-acyl homoserine, therefore there would be no AHL to diffuse back into the cell to activate the LuxR receptor [32]. Therefore, preventing the formation of a mature biofilm.



How we chose the project

Timeline of how we chose the project (1) biofilms - we researched biofilms and decided they were a problem we wanted to tackle, although we were unsure how. (2) Magnetosomes - we began developing an idea to deliver drugs into biofilms using a magnetosome based drug delivery system. (3) Biofilms in industry? - We started researching biofilm issues in industry and began accumulating ideas to tackle industry biofilm formation. (4) S. epidermidis - Through searching the iGEM whitelist, we found s.epidermidis, a biofilm former on hospital catheters, and decided to focus our research towards preventing S. epidermidis biofilm formation (5) Quorum sensing - We began to consider targeting quorum sensing to prevent biofilm formation. We spoke to an expert to focus our process. (6) E. coli - We discussed an approach using E. coli as a vessel for RIP delivery within our biofilm treatment. (7) Pitch of ideas to academics - We presented several concept ideas, including our magnetosome and E. coli biofilm prevention projects, to a room of academics from the University of Exeter. They advised us to pursue the bacterial RIP delivery system but that we should reconsider using E.coli in catheters (8) L. plantarum - we finalised our concept, opting to use L. plantarum as our RIP delivery chassis, as opposed to E. coli (9) AI-2 LuxS system - after searching through a considerable amount of literature we decided to move our project away from the highly controversial RIP/RAP mechansism. Instead we chose to increase AI-2 production, using the upregulation of the LuxS gene. (10) AHL mediated quorum sensing - We finalised our new idea by specialising our research to focus on AHL mediated quorum quenchers and enzymes that target this process.


During the process of planning our project we also looked at previous iGEM projects that considered similar areas of research. We were inspired by the iGEM Pasteur Paris 2018 team, who engineered E. coli to prevent S. aureus biofilm formation on implants, and the HKUST 2010 team who proposed using Lactobacillus to sense and reduce S. aureus virulence.



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