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Bioscience Horizons 2008 1(1):28-37; doi:10.1093/biohorizons/hzn008
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© Oxford University Press 2008

Gene expression of Pseudomonas aeruginosa and MRSA within a catheter-associated urinary tract infection biofilm model

Michael John Howard Goldsworthy*

School of Biosciences, Exeter University, Geoffrey Pope Building, Stocker Road, Exeter, EX4 4QD, UK

* Corresponding author: 6 Woodbine Terrace, Exeter, Devon, EX4 4LJ, UK. Tel: +44 01392 269170. Email: m.j.h.goldsworthy{at}ex.ac.uk

Supervisors: Sara Burton and Hilary Lappin-Scott, School of Biosciences, Exeter University, Geoffrey Pope Building, Stocker Road, Exeter, EX4 4QD, UK.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Catheter-associated urinary tract infections (CAUTI) have frequently been studied in monocultures in vitro. However, many CAUTI are due to the presence of a mixed microbial community and not just a single population, especially within patients subjected to long-term catheterization. This can potentially have important clinical implications in regards to treatment strategy and outcome. This study revealed that when Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) (both commonly found in CAUTI) were grown together as a mixed culture within a CAUTI model in comparison to their respective monocultures, accelerated biofilm development was observed. Virulence gene expression analysis within P. aeruginosa and MRSA monoculture and mixed biofilms was performed through use of real-time quantitative PCR. It was revealed that production of P. aeruginosa exotoxin A was increased 1839-fold when P. aeruginosa and MRSA were grown together as a mixed biofilm. Significant expression of {alpha}-haemolysin by MRSA was not observed in either culture. It is proposed that both the biofilm-forming capability and virulence gene expression of microbes within CAUTI mixed biofilms may differ substantially to the respective microbial monocultures. This highlights the importance of developing specific treatment strategies for CAUTI when polymicrobial communities are present.

Key words: Pseudomonas aeruginosa, MRSA, urinary tract infection, Biofilm, exotoxin A, {alpha}-haemolysin


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Catheter-associated urinary tract infections (CAUTI) are recognized as the most prevalent nosocomial infection and present a large clinical and economic burden upon the health system with more than 1 million cases per year recorded in US hospitals and nursing homes.1 Between 15 and 25% of patients admitted to hospital are predicted to have a catheter in place at some time during their treatment.2 Approximately one-third of these patients will have the catheter in place for less than a day and although bacteriuria may be exhibited in these patients, it is predominantly asymptomatic and is unlikely to present a problem clinically.2, 3 In patients with urinary catheters in place for 2–10 days, bacteriuria has been proposed to develop in approximately 26% of patients. Of these patients, around 24% are expected to be symptomatic and approximately 4% are expected to develop bacteraemia.4 For patients with long-term catheterization, bacteriuria is almost inevitable and without treatment may lead to more severe complications such as acute pyelonephritis and bladder cancer.2 CAUTI are directly responsible for approximately 1000 deaths per year in the USA and contribute to an additional 6500 deaths.5 Despite relatively low mortality rates, CAUTI represent a large problem in hospitals due to additional hospital days (approximately 900 000 per year in the US) and treatment costs.2

Clinical observations have established that the microbial populations within CAUTI frequently develop as biofilms, directly attaching to the surface of catheters.6 Biofilms are surface-associated, matrix-enclosed, microbial communities proposed to be a primitive survival mechanism.7 The formation of a biofilm may protect the population within from a diverse array of environmental stresses.8 Clinically, biofilms can present a huge problem due to the antibiotic resistance often conferred by their formation. Further complications due to their formation within infections include phagocytosis inhibition, protection from exposure to antibodies and impairment of lymphocyte function.9

Approximately 12% of hospital-acquired urinary infections can be accounted for by Pseudomonas aeruginosa,10 although significant antibiotic resistance is rare.11 However, studies of CAUTI antibiotic resistance are often performed in vitro, with samples taken directly from the urine. These results may therefore only represent the planktonic microbial population and not consider the phenotypically distinct populations within biofilms.12

P. aeruginosa possesses a hierarchical signalling cascade containing interlinking quorum sensing systems involved in regulating many families of genes, including those involved in biofilm maturation.13, 14 At low cell densities P. aeruginosa constitutively expresses lasI, producing N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL).15 An increase in population density is paralleled by an increase in concentration of 3OC12-HSL. Once a threshold concentration is reached, the acyl-homoserine lactone (AHL) directly interacts with the LuxR-type transcriptional regulator LasR. This complex then binds to specific sequences within promoters known as Lux-boxes, resulting in the activation of genes encoding exoproteases, siderophores, exotoxins and genes involved in biofilm formation.16, 17 Below the Las system in P. aeruginosa QS hierarchy lays the Rhl circuitry. This second quorum sensing system relies on the LasR-3OC12-HSL complex to upregulate the expression of rhlR. Upregulation of this transcriptional regulator allows it to bind to N-butanoylhomoserine lactone (C4-HSL), synthesized by rhlI.18 This results in a multimer that directs biosynthesis of rhamnolipid, among other proteins, many of which are associated with virulence,18 and expression of the stationary-phase sigma factor RpoS;13 Whiteley, Parsek and Greenberg19 suggested, however, that RpoS may not directly regulate RhlI but may provide a role in regulation earlier in the quorum sensing signalling cascade. RpoS has shown to directly affect the expression of approximately 40% of quorum sensing-controlled genes in P. aeruginosa including lasI13, 19 and is therefore a vital component of the molecular machinery involved in controlling biofilm formation and virulence. Numerous virulence genes are downregulated significantly upon deletion of RpoS, suggesting that an intimate relationship exists between RpoS expression and pathogenecity.20 The impact of RpoS upregulation in regards to biofilm growth, however, is seemingly inhibitory, with recent studies showing RpoS mutant strains forming ‘better’ biofilms with increased antibiotic resistance.21

Exotoxin A (toxA) of P. aeruginosa is a chromosomally encoded secreted toxin capable of inhibiting protein synthesis of infected patients through the ADP-ribosylation of cellular elongation factor 2.22, 23 Biochemical models have revealed three distinct domains within the toxin: an N-terminal domain involved in recognizing and binding target cells, a central domain involved in exotoxin secretion and possibly its translocation into eukaryotic vesicles, and a C-terminal domain, proposed to be the catalytic site of the toxin involved in ADP-ribosylation.24 Ultimately, the action of this toxin results in localized tissue damage and bacterial invasion.10 Rumbaugh et al.25 revealed that P. aeruginosa isolates recovered from patients suffering from urinary tract infections produced significant levels of exotoxin A. Equally, upregulation of exotoxin A may be observed within biofilms due to the action of quorum sensing.26 Taken together, this information suggests that exotoxin A may play a significant role as a virulence factor of P. aeruginosa within CAUTI.

MRSA has shown to account for around 30% of hospital-acquired infections27 and approximately 10% of CAUTI.11 Staphylococci have demonstrated significant levels of antibiotic resistance within CAUTI and therefore represent a huge problem—both economically, due to treatment costs and clinically—within such infections.11, 28

Staphylococcal biofilm formation has been reported to have two stages: initial attachment and maturation.29, 30 Attachment requires the action of cell-wall-associated adhesions, notably, the microbial surface components recognizing adhesive matrix molecules.31 Maturation of the biofilm is the result of numerous processes, including microcolony formation, polysaccharide intercellular adhesion (PIA) secretion (a vital extracellular matrix component) and microbial communication through the action of quorum sensing.31, 32

The accessory gene regulator (agr) quorum sensing system is central to both the biofilm forming capability and virulence of MRSA.33, 34 Functionality of the agr relies on the expression of two primary transcripts—RNAII and RNAIII—that originate from the P2 and P3 promoters, respectively, and are expressed at low cell densities.35, 36 The P2 operon harbours the quorum sensing machinery of the regulatory system and encodes four genes, agrA, agrB, agrC and agrD.37 Both the agrB and AgrD gene products are essential in the generation of the signal molecule, an autoinducing peptide (AIP) of 7–9 amino acids in length.38 AgrD is encoded in a propeptide form and must undergo proteolytic digestion by the action of AgrB. AgrB has further been proposed to be involved in thioester bond formation and secretion of the AIP.36 A two-component regulatory system is formed by the gene products of agrA and agrC.31 AgrC, a histidine kinase, represents the transmembrane component of the agr and acts through the binding of extracellular AIP. Subsequently, AgrC functions to modulate the activity of AgrA.39 AgrA acts as a response regulator and upregulates both the P2 and P3 operons significantly during the late-log phase of bacterial growth.31 RNAIII encodes the toxin {delta}-haemolysin and is transcribed at the mid-exponential phase of growth.40 Upregulation of RNAIII results in an increase in expression of numerous virulence genes including TSS toxin-1 and {alpha}-haemolysin.41, 42 Expression of agr is positively associated with virulence; agr mutant studies have shown significant depletion in pathogenicity within S. aureus isolates.43, 44 However, only recently has the role of agr in biofilm formation been elucidated. Interestingly, it appears that agr downregulation (or possibly inactivation) is imperative for biofilm formation in S. aureus infections.37 Loss of agr function within staphylococcal infections has therefore been postulated to result in the conversion of acute symptoms into those of a chronic nature.31

The secreted toxin {alpha}-haemolysin (hla) is the best characterized of the S. aureus cytotoxins and has shown to have dermonecrotic and neurotoxic potential.45 After targeting host cells, {alpha}-haemolysin integrates into the membrane and forms cylindrical heptamers.46 Following the formation of this complex, erythrocyte lysis may occur. Numerous regulators of {alpha}-haemolysin have been described, including the staphylococcal accessory regulator (sarA), sae, sarT and, as mentioned, the agr.42, 47 Expression of sarA has shown to be required for maximum expression of hla.31 Furthermore, sarA has been proposed as an essential component of the biofilm forming machinery of S. aureus48 and has demonstrated a role in the upregulation of the agr.49, 50 The sae system acts in a similar manner to sarA, positively acting upon the expression of hla. However, unlike sarA, it appears to act downstream of the agr.31 In contrast to both sae and sarA, sarT possesses an established role in the inhibition of {alpha}-haemolysin production. SarT expression has shown to be inhibited by the expression of both agr and sarA.42

In conjunction with its contribution towards virulence, {alpha}-haemolysin has shown to play an essential role in biofilm formation. Studies have demonstrated that S. aureus isolates deficient in {alpha}-haemolysin are unable to colonize plastic surfaces under both static and flow conditions.51 From this study, it was proposed that this toxin is directly involved in cell-to-cell interactions during the process of biofilm formation. In relation to CAUTI, Ando et al.30 revealed that MRSA isolates from such infections produce significant levels of {alpha}-haemolysin. Taking this into account, it is likely that {alpha}-haemolysin plays a critical role in the pathogenicity of S. aureus within CAUTI.

Biofilm formation and virulence gene expression within S. aureus and P. aeruginosa monocultures has been well-studied in vitro.14, 18, 37 However, clinical observations have revealed that a high proportion of CAUTI consist of a mixed microbial consortia, most notably within patients subjected to long-term catheterization whose infections frequently become polymicrobial.2, 11 Knowledge of bacterial interactions within monocultures may therefore have only limited applicability towards the understanding and treatment of CAUTI. This study attempts to elucidate the impact that growth of S. aureus and P. aeruginosa within mixed culture has upon biofilm formation when compared with growth within their respective monocultures. Furthermore, quantitation of hla expression by S. aureus and toxA expression by P. aeruginosa within monoculture and mixed culture systems will be performed through quantitative gene analysis.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Strains and culturing conditions
P. aeruginosa NCIMB 10777 and methicillin resistant S. aureus EMRSA16 strains were obtained from cryotubes. Initially, strains were plated onto nutrient agar and were subsequently cultured in an artificial urine medium52 at 37°C in order to simulate the conditions of urinary catheter infections.

Biofilm growth
Biofilms were grown in glass flow cells that were supplied with a constant flow of artificial urine medium52 (Fig. 1). Media was pumped through silastic tubing at a rate of 30 ml/h to the flow cell that was mounted on a heated stage (37°C). Prior to media flow, an overnight inoculum was introduced to the system and incubated for 3 h to allow cell attachment. Once the flow was initialized, biofilm growth was established for 18 h. Micrographs of the biofilm were taken at 15-min intervals. These were used to establish biofilm growth dynamics by analysing the surface area coverage over the 18-h period using Scion Image software. For the mixed culture biofilm inoculum it was necessary to obtain overnight monocultures of each bacterial strain. Growth curves showed P. aeruginosa and MRSA to have similar growth rates when cultured in artificial urine (data not shown) and so equal volumes of the monocultures were used to create the mixed culture inoculum. Thus, 5 ml samples of each monoculture was aseptically pipetted into 100 ml artificial urine prior to seeding the flow cell.


Figure 1
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  		Figure 1. Flow cell equipment set-up.

 
Biofilm extraction
Subsequent to biofilm growth, mRNA fixation was carried out in preparation for quantitative gene expression analysis. The flow cell was flushed with a fixing solution containing 9 ml artificial urine and 1 ml ethanol:phenol (20:1, v/v) and then placed on ice for 1 h. Biofilm samples were then collected using a sterile loop and resuspended in 300 µl of the mRNA fixing solution. The sample was centrifuged at 13 000 rpm at 4°C for 10 min. The supernatant was then removed, leaving the pelleted cells.

mRNA extraction
Extraction of mRNA was performed using a bead beating protocol. Reaction buffer and acid washed beads were added to the pellet prior to mechanical lysis of the cells carried out using Tissue Lyser. Further extraction was performed using the RNeasy mini kit (Qiagen) according to manufacturer's instructions.

Residual genomic DNA was removed via the addition of 1 µl DNase, 1 µl nuclease-free water and 2 µl 10x reaction buffer to 16 µl of the mRNA preparation. This sample was incubated at 37°C for 30 minutes. Addition of 2 µl stopper solution to the sample followed by incubation at 65°C for 10 min was performed to complete the reaction.

cDNA synthesis
Reverse transcription (RT) was performed to create a cDNA copy of the biofilm transcriptome. This was carried out according to the manufacturer's instructions of the Invitrogen RT–PCR kit.

Real-time quantitative polymerase chain reaction
To analyse the relative amounts of target gene expression in a mixed culture biofilm as compared to the bacterial species in biofilms of their respective monocultures, real-time quantitative polymerase chain reaction (RT-qPCR) was performed. This process was undertaken on the Bio-Rad Mini-Opticon using SYBR green fluorescent dye. The reaction mixture consisted of 12.5 µl of iQ SYBR Green supermix, 1 µl forward primer, 1 µl reverse primer, 9.5 µl nuclease-free water and 1 µl of the cDNA template. Control samples were created by using 10.5 µl nuclease-free water and no cDNA template. Both the 16s and target gene primers had been previously designed (Table 1) (R. Goldstone, personal communication).


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  		Table 1. qPCR Primers

 
Reaction conditions for 16s gene amplification were optimized previously for both strains (R. Goldstone, personal communication). An initial stage of heating at 95°C for 5 min was performed, followed by 40 cycles of 95°C for 1 min, 55°C for 1 min and 72°C for 30 s.

Reaction conditions for MRSA hla amplification were obtained in accordance with Renzoni et al.53 An initial stage of heating at 95°C for 5 min was followed by 40 cycles of 95°C for 30 s and 60°C for 30 s.

Reaction conditions for P. aeruginosa toxA amplification consisted of heating at 95°C for 5 min, followed by 40 cycles of 95°C for 1 min, 60°C for 1 min and 72°C for 30 s for every qPCR a plate read-out was taken after each cycle. A melting curve using 1°C increments was also performed following the 40 cycles in order to determine that the correct gene was amplified.

Fluorescent antibody tagging
In order to differentiate between bacterial species within a mixed community biofilm, Texas red labelled polyclonal antibodies raised against P. aerguinosa (Abcam) were directly added to the biofilm. Prior to tagging, dilutions of both the primary and secondary antibodies were performed in order to optimize the process. The biofilm was initially fixed by addition of 2–4% v/v formalin. The flow cell was then cut into three sections. The primary antibody was added to each glass section then left in the dark for 30 min at room temperature. The biofilm was washed with PBS before secondary antibody addition. The sections were again left in the dark for 30 min at room temperature. Fluorescent microscopy was undertaken using a suppression BP 635/40 nm filter and excitation 575/30 nm BP filter.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Biofilm development
P. aeruginosa displayed slow progressive biofilm development when grown as a monoculture, starting with a low level of initial attachment and growing to give area coverage of approximately 30% after 18 h (Figs 2 and 6). This development was contrasted by MRSA biofilm growth. A high level of initial attachment was observed within this system (27% area coverage) and significant detachment events occurred over the course of 18 h, the biofilm diminishing over time (Figs 3 and 6). When grown as a mixed-culture, the biofilm developed rapidly, giving 100% area coverage after approximately 12 h (Figs 4 and 6). Figure 5 displays the different relative abundances of P. aeruginosa observed within fields of the biofilm that showed 100% area coverage. All micrographs reveal only partial area coverage by P. aeruginosa, suggesting that both strains contribute towards the biomass of the biofilm within the CAUTI model.


Figure 2
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  		Figure 2. P. aeruginosa biofilm flow cell micrographs showing initial attachment (A), growth after 9 h (B) and growth after 18 h (C).

 


Figure 3
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  		Figure 3. MRSA biofilm flow cell micrographs showing initial attachment (A), growth after 9 h (B) and growth after 18 h (C).

 


Figure 4
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  		Figure 4. MRSA and P. aeruginosa mixed-culture biofilm flow cell micrographs showing initial attachment (A), growth after 9 h (B) and growth after 18 h (C).

 


Figure 5
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  		Figure 5. Fluorescent micrographs taken from randomly selected fields of the MRSA and P. aeruginosa mixed-culture biofilm after staining with Texas red labelled polyclonal antibodies raised against P. aeruginosa (Abcam).

 


Figure 6
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  		Figure 6. Area coverage displayed by P. aeruginosa (blue), MRSA (red) and MRSA and P. aeruginosa mixed-culture (green) biofilms (calculations were based on appropriate image modifications (not shown) made using Scion Image software).

 
Pseudomonas aeruginosa 16s rDNA and toxA gene expression
P. aeruginosa 16s rDNA displayed similarly high levels of expression in both the monoculture and mixed culture biofilms (Fig. 7A and B), giving mean Ct values of 7.705 and 6.255, respectively. Amplification of toxA differed more greatly between the monocultures and mixed cultures (Fig. 7C and D), with the mixed culture giving a lower Ct value (mean = Ct 24.08) compared to that of the monoculture (mean Ct = 33.475) indicating that its expression is enhanced when grown in the presence of MRSA.


Figure 7
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  		Figure 7. Real-time quantitative analysis of 16s rDNA, toxA and hla genes obtained from P. aeruginosa and MRSA monoculture and mixed culture biofilms. Samples containing cDNA of the target gene (red and green), control samples containing mRNA preparations subjected to reverse transcription in the absence of reverse transcriptase (blue and orange) and a control containing free water (pink) were tested. The horizontal dashed line denotes the threshold line and was set manually to 0.03. This value was used to determine cyclic threshold (Ct)—the cycle number at which gene product first appears.

 
To calculate the precise difference in expression of toxA between the two cultures the following equation was used:


Formula

where E (PCR amplification efficiencies) = 2.

It was revealed that toxA expression was hugely influenced by growth of P. aeruginosa alongside MRSA. Its expression was calculated to be 1839-fold greater in mixed culture than in monoculture. Contamination was evident, to varying degrees, in both 16s rDNA and toxA gene amplification experiments (Fig. 7A–D). However, samples differed significantly enough to controls to suggest that the apparent massive upregulation of toxA observed when P. aeruginosa is grown in competition with MRSA within a CAUTI model is indeed real.

MRSA 16s rDNA and hla gene expression
Expression of MRSA 16s rDNA was substantially lower in monoculture (mean Ct = 22.05) compared to that of P. aeruginosa (Fig. 7A and E). In mixed culture, only one MRSA sample displayed 16s rDNA gene amplification (Fig. 7F), giving a Ct value of 15.64. No significant amplification of hla was revealed in either culture (Fig. 7G and H). Only in monoculture was hla amplification evident (Fig. 7G). However, the Ct value of 37.83 for this sample was not greatly different to any of the control samples and its amplification differed greatly compared to its complementing sample (no gene product produced). The reliability of this result is therefore contestable. Again, due to the sensitivity of qPCR, contamination was observed in the control samples and controls of all MRSA gene amplification experiments (Fig. 7E–H). Due to the inconsistencies observed in both 16s rDNA and hla gene amplification and the very low level of hla expression observed in both cultures, the effect of growth of MRSA in the presence of P. aeruginosa on hla expression within a CAUTI model could not be quantitatively determined.

Melting curves were produced for all gene amplification experiments to confirm that the correct fragments were amplified. All sample melting curves revealed single peaks corresponding to the melting point of the amplified gene (data not shown). Control samples and controls showing gene amplification produced single peaks mirroring those of the samples, suggesting that contamination was most likely derived from genomic DNA of the respective sample (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
It has been demonstrated that cell-to-cell communication through quorum sensing mechanisms may occur not only within species of microbes but also between them.54, 55 In this study, it is demonstrated that the biofilm-forming capabilities of P. aeruginosa and MRSA within a CAUTI model are significantly enhanced when these organisms are grown together as a mixed culture, compared to growth as monocultures. Furthermore, production of exotoxin A by P. aeruginosa is substantially increased when grown in a mixed culture MRSA biofilm.

P. aeruginosa displayed slow progressive biofilm growth in the monoculture CAUTI model. This contrasted to the growth of the MRSA monoculture which showed a high rate of initial attachment followed by a decrease in biofilm area coverage due to large detachment events. The unexpected decline of MRSA biofilm area coverage may be explained by the bacterium's tendency to release large clumps of biofilm; an alternative dispersal strategy to that of P. aeruginosa which sheds only single cells or small clumps.7 The mixed biofilm developed at a rate several times that of either monoculture.

Due to the difficulties of calculating the relative abundances of P. aeruginosa and MRSA within the CAUTI mixed biofilm, analysing genome regulation and determining relative rates of growth was problematic. However, both strains contributed significantly to the mixed biofilm biomass. It is therefore proposed that both P. aeruginosa and MRSA undergo alterations in biofilm-forming gene expression when grown in each others presence. Qazi et al.56 reported that long chain AHLs (most notably 3OC12-HSL) display high-affinity attachment to S. aureus membranes, binding to an uncharacterized receptor in a specific manner. Significant inhibition of both sarA and agr was subsequently observed when S. aureus was exposed to varying concentrations of purified 3OC12-HSL and P. aeruginosa isolates. SarA and the agr have opposite effects on S. aureus biofilm development (Fig. 8). Although the expression of SarA has often been proposed to be vital for staphylococcal biofilm formation,48, 57 reports have suggested that mutation of this gene merely reduces the bacterium's capacity to form a biofilm.58 This is important when considering the impact of agr on biofilm growth. Studies have revealed that S. aureus biofilm detachment events are largely due to the action of agr-induced extracellular factors.29, 34 Cessation of MRSA clumping dispersal through LasI-inhibition of the agr may have significantly contributed to the extensive biofilm growth observed in the mixed biofilm. However, this outcome would only hold true if inhibition of SarA does not inhibit biofilm growth altogether. S. aureus strains containing independent knock-out mutations of sarA and agr may therefore be used in future applications to confirm the contribution that these two genes have towards S. aureus biofilm development in a mixed biofilm CAUTI model.


Figure 8
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  		Figure 8. P. aeruginosa and MRSA gene map showing interactions within and between the two strains. Black arrows indicate activation or upregulation and red lines indicate repression. Dashed lines represent an inconclusive role in regulation.

 
Caizza and O' Toole51 suggested that {alpha}-haemolysin plays an important role in S. aureus biofilm development. Quantitative gene analysis revealed no significant hla expression by S. aureus in either the mixed culture or monoculture biofilms. These results indicate that hla is not essential for biofilm formation in S. aureus. However, as no measurable expression of hla was detected in either culture, it is possible that faults in primer design or in reaction conditions may have given a set of false negative results.

Expression of toxA by P. aeruginosa in the mixed biofilm was calculated to be 1839-fold more than in monoculture. This upregulation may not be directly caused by the presence of MRSA but by the extensive biofilm growth observed as a result of their coexistence. Both LasI and RpoS have demonstrated roles in the regulation of toxA (Fig. 8).20, 59 LasI accumulation has shown to be density-dependent14 and so the increase in exotoxin A expression observed within the CAUTI mixed biofilm may be partly due to the increase in biomass. RpoS provides a significant role in P. aeruginosa gene regulation, but only during stationary phase when sigma factor switching may occur.60 Upregulation of toxA in mixed culture may be due to early onset of stationary phase as a result of the presence of MRSA and switching of P. aeruginosa sigma factor to RpoS.

Iron-chelating siderophores such as pyochelin and pyoverdine (Pvd) have demonstrated important roles in the promotion of growth and the expression of virulence factors within P. aeruginosa.61–63 PvdS, an alternative sigma factor, is critical in the upregulation of exotoxin A and is only expressed when P. aeruginosa is subjected to iron-deplete conditions.64, 65 In mixed culture, P. aeruginosa may function to create iron-replete local biofilm conditions by lysing MRSA cells.66 Therefore, in a mixed biofilm, only low levels of exotoxin A expression should be observed. However, the spatio-temporal dynamics of microbial biofilm development are complex and so microenvironmental conditions may change greatly over short periods of time.67 It is proposed that during logarithmic phase and early stationary phase, extensive P. aeruginosa biofilm growth may result as a direct consequence of the liberation of iron through MRSA lysis. However, following proliferation, iron uptake by P. aeruginosa and MRSA may create pockets of iron-deplete conditions within the biofilm, especially within populations closer to the substratum where nutrients fail to penetrate. This may result in the activation of iron acquisition systems within surrounding P. aeruginosa populations, ultimately resulting in upregulation of toxA.59 The lower level of toxA expression observed within the P. aeruginosa monoculture may therefore be directly related to the low level of biofilm proliferation. Clearly, a complicated relationship exists between biofilm formation and expression of virulence factors. Therefore, understanding the temporal patterning of virulence gene expression would lead to clarification of gene regulatory mechanisms of clinical significance.

This study reveals that the pathogenicity of bacteria within CAUTI may be altered substantially when in the presence of other microbes. This clearly has critical clinical implications and highlights the importance in the correct identification of microbes within CAUTI. These observations may be crucial in the understanding and treatment of CAUTI, especially within patients subjected to long-term catheterization where infections are frequently polymicrobial.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
I gratefully acknowledge funding from the University of Exeter School of Biosciences and Environmental Microbiology and Ecology research group.


   Acknowledgements
 
This work was carried out in the microbiology laboratories of Exeter University under the supervision of Hilary Lappin-Scott and Sara Burton. Further assistance and direction on this project was given by Peter Splatt and Natasha Mohajeri.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Submitted on 28 September 2007; accepted on 21 December 2007


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