Infection responsive coatings to reduce biofilm formation and encrustation of urinary catheters.

AIMS: The care of patients undergoing long-term urethral catheterization is frequently complicated by Proteus mirabilis infection. This organism forms dense, crystalline biofilms, which block catheters leading to serious clinical conditions. However, there are currently no truly effective approaches to control this problem. Here, we describe the development of a novel theranostic catheter coating, to simultaneously provide early warning of blockage, and actively delay crystalline biofilm formation. METHODS AND RESULTS: The coating comprises of a pH sensitive upper polymer layer (poly(methyl methacrylate-co-methacrylic acid); Eudragit S 100®) and a hydrogel base layer of poly(vinyl alcohol), which is loaded with therapeutic agents (acetohydroxamic acid or ciprofloxacin hydrochloride) and a fluorescent dye, 5(6)-carboxyfluorescein (CF). The elevation of urinary pH due to P. mirabilis urease activity results in the dissolution of the upper layer and release of cargo agents contained in the base layer. Experiments using in vitro models, which were representative of P. mirabilis catheter-associated urinary tract infections, demonstrated that these coatings significantly delay time taken for catheters to block. Coatings containing both CF dye and ciprofloxacin HCl were able to provide an average of ca. 79 h advanced warning of blockage and extend catheter lifespan ca. 3.40-fold. CONCLUSIONS: This study has demonstrated the potential for theranostic, infection-responsive coatings to form a promising approach to combat catheter encrustation and actively delay blockage.

Antibiotic Therapy and the Gut Microbiome: Investigating the Effect of Delivery Route on Gut Pathogens.

The contribution of the gut microbiome to human health has long been established, with normal gut microbiota conferring protection against invasive pathogens. Antibiotics can disrupt the microbial balance of the gut, resulting in disease and the development of antimicrobial resistance. The effect of antibiotic administration route on gut dysbiosis remains under-studied to date, with conflicting evidence on the differential effects of oral and parenteral delivery. We have profiled the rat gut microbiome following treatment with commonly prescribed antibiotics (amoxicillin and levofloxacin), via either oral or intravenous administration. Fecal pellets were collected over a 13-day period and bacterial populations were analyzed by 16S rRNA gene sequencing. Significant dysbiosis was observed in all treatment groups, regardless of administration route. More profound dysbiotic effects were observed following amoxicillin treatment than those with levofloxacin, with population richness and diversity significantly reduced, regardless of delivery route. The effect on specific taxonomic groups was assessed, revealing significant disruption following treatment with both antibiotics. Enrichment of a number of groups containing known gut pathogens was observed, in particular, with amoxicillin, such as the family Enterobacteriaceae. Depletion of other commensal groups was also observed. The degree of dysbiosis was significantly reduced toward the end of the sampling period, as bacterial populations began to return to pretreatment composition. Richness and diversity levels appeared to return to pretreatment levels more quickly in intravenous groups, suggesting convenient parenteral delivery systems may have a role to play in reducing longer term gut dysbiosis in the treatment of infection.

Genomic and Ecogenomic Characterization of Proteus mirabilis Bacteriophages.

Proteus mirabilis often complicates the care of catheterized patients through the formation of crystalline biofilms which block urine flow. Bacteriophage therapy has been highlighted as a promising approach to control this problem, but relatively few phages infecting P. mirabilis have been characterized. Here we characterize five phages capable of infecting P. mirabilis, including those shown to reduce biofilm formation, and provide insights regarding the wider ecological and evolutionary relationships of these phages. Transmission electron microscopy (TEM) imaging of phages vB_PmiP_RS1pmA, vB_PmiP_RS1pmB, vB_PmiP_RS3pmA, and vB_PmiP_RS8pmA showed that all share morphologies characteristic of the Podoviridae family. The genome sequences of vB_PmiP_RS1pmA, vB_PmiP_RS1pmB, and vB_PmiP_RS3pmA showed these are species of the same phage differing only by point mutations, and are closely related to vB_PmiP_RS8pmA. Podophages characterized in this study were also found to share similarity in genome architecture and composition to other previously described P. mirabilis podophages (PM16 and PM75). In contrast, vB_PimP_RS51pmB showed morphology characteristic of the Myoviridae family, with no notable similarity to other phage genomes examined. Ecogenomic profiling of all phages revealed no association with human urinary tract viromes, but sequences similar to vB_PimP_RS51pmB were found within human gut, and human oral microbiomes. Investigation of wider host-phage evolutionary relationships through tetranucleotide profiling of phage genomes and bacterial chromosomes, indicated vB_PimP_RS51pmB has a relatively recent association with Morganella morganii and other non-Proteus members of the Morganellaceae family. Subsequent host range assays confirmed vB_PimP_RS51pmB can infect M. morganii.

An In Vitro Bladder Model for Studying Catheter-Associated Urinary Tract Infection and Associated Analysis of Biofilms.

Urethral catheters are among the most widely used medical devices, applied to manage a wide range of conditions in hospital, community, and care home settings. In long-term catheterized individuals, infection with Proteus mirabilis frequently complicates the care of patients owing to formation of extensive crystalline biofilms. Here we describe the use of an in vitro bladder model of the catheterized urinary tract and associated analyses to study P. mirabilis crystalline biofilm formation. The model originally described by Stickler et al. (1999, 310:494-501, Methods Enzymol) replicates a complete sterile closed drainage system as used in clinical practice, and permits formation of biofilms directly on catheters under conditions representative of those encountered in vivo. Models may be used to replicate either established infection or early stage colonization, and we describe a range of associated methods for quantification and visualization of biofilms formed on catheters. These methods are also easily adapted to study catheter-associated biofilm formation by other urinary tract pathogens.

The potential of nanoflow liquid chromatography-nano electrospray ionisation-mass spectrometry for global profiling the faecal metabolome.

Faeces are comprised of a wide array of metabolites arising from the circulatory system as well as the human microbiome. A global metabolite analysis (metabolomics) of faecal extracts offers the potential to uncover new compounds which may be indicative of the onset of bowel diseases such as colorectal cancer (CRC). To date, faecal metabolomics is still in its infancy and the compounds of low abundance present in faecal extracts poorly characterised. In this study, extracts of faeces from healthy subjects were profiled using a sensitive nanoflow-nanospray LC-MS platform which resulted in highly repeatable peak retention times (<2% CV) and intensities (<15% CV). Analysis of the extracts revealed wide coverage of the faecal metabolome including detection of low abundant signalling compounds such as sex steroids and eicosanoids, alongside highly abundant pharmaceuticals and tetrapyrrole metabolites. A small pilot study investigating differences in metabolomics profiles of faecal samples obtained from 7 CRC, 25 adenomatous polyp and 26 healthy groups revealed that secondary bile acids, conjugated androgens, eicosanoids, phospholipids and an unidentified haem metabolite were potential classes of metabolites that discriminated between the CRC and control sample groups. However, much larger follow up studies are needed to confirm which components of the faecal metabolome are associated with actual CRC disease rather than dietary influences. This study reveals the potential of nanospray-nanoflow LC-MS profiling of faecal samples from large scale cohort studies for uncovering the role of the faecal metabolome in colorectal disease formation.

Emerging medical and engineering strategies for the prevention of long-term indwelling catheter blockage.

Urinary catheters have been used on an intermittent or indwelling basis for centuries, in order to relieve urinary retention and incontinence. Nevertheless, the use of urinary catheters in the clinical setting is fraught with complication, the most common of which is the development of nosocomial urinary tract infections, known as catheter-associated urinary tract infections. Infections of this nature are not only significant owing to their high incidence rate and subsequent economic burden but also to the severe medical consecutions that result. A range of techniques have been employed in recent years, utilising various technologies in attempts to counteract the perilous medical cascade following catheter blockage. This review will focus on the current advancement (within the last 10 years) in prevention of encrustation and blockage of long-term indwelling catheters both from engineering and medical perspectives, with particular emphasis on the importance of stimuli-responsive systems.

Development of a High-Throughput ex-Vivo Burn Wound Model Using Porcine Skin, and Its Application to Evaluate New Approaches to Control Wound Infection.

Biofilm formation in wounds is considered a major barrier to successful treatment, and has been associated with the transition of wounds to a chronic non-healing state. Here, we present a novel laboratory model of wound biofilm formation using ex-vivo porcine skin and a custom burn wound array device. The model supports high-throughput studies of biofilm formation and is compatible with a range of established methods for monitoring bacterial growth, biofilm formation, and gene expression. We demonstrate the use of this model by evaluating the potential for bacteriophage to control biofilm formation by Staphylococcus aureus, and for population density dependant expression of S. aureus virulence factors (regulated by the Accessory Gene Regulator, agr) to signal clinically relevant wound infection. Enumeration of colony forming units and metabolic activity using the XTT assay, confirmed growth of bacteria in wounds and showed a significant reduction in viable cells after phage treatment. Confocal laser scanning microscopy confirmed the growth of biofilms in wounds, and showed phage treatment could significantly reduce the formation of these communities. Evaluation of agr activity by qRT-PCR showed an increase in activity during growth in wound models for most strains. Activation of a prototype infection-responsive dressing designed to provide a visual signal of wound infection, was related to increased agr activity. In all assays, excellent reproducibility was observed between replicates using this model.

Resolution of habitat-associated ecogenomic signatures in bacteriophage genomes and application to microbial source tracking.

Just as the expansion in genome sequencing has revealed and permitted the exploitation of phylogenetic signals embedded in bacterial genomes, the application of metagenomics has begun to provide similar insights at the ecosystem level for microbial communities. However, little is known regarding this aspect of bacteriophage associated with microbial ecosystems, and if phage encode discernible habitat-associated signals diagnostic of underlying microbiomes. Here we demonstrate that individual phage can encode clear habitat-related 'ecogenomic signatures', based on relative representation of phage-encoded gene homologues in metagenomic data sets. Furthermore, we show the ecogenomic signature encoded by the gut-associated ɸB124-14 can be used to segregate metagenomes according to environmental origin, and distinguish 'contaminated' environmental metagenomes (subject to simulated in silico human faecal pollution) from uncontaminated data sets. This indicates phage-encoded ecological signals likely possess sufficient discriminatory power for use in biotechnological applications, such as development of microbial source tracking tools for monitoring water quality.

Fluoxetine and thioridazine inhibit efflux and attenuate crystalline biofilm formation by Proteus mirabilis.

Proteus mirabilis forms extensive crystalline biofilms on indwelling urethral catheters that block urine flow and lead to serious clinical complications. The Bcr/CflA efflux system has previously been identified as important for development of P. mirabilis crystalline biofilms, highlighting the potential for efflux pump inhibitors (EPIs) to control catheter blockage. Here we evaluate the potential for drugs already used in human medicine (fluoxetine and thioridazine) to act as EPIs in P. mirabilis, and control crystalline biofilm formation. Both fluoxetine and thioridazine inhibited efflux in P. mirabilis, and molecular modelling predicted both drugs interact strongly with the biofilm-associated Bcr/CflA efflux system. Both EPIs were also found to significantly reduce the rate of P. mirabilis crystalline biofilm formation on catheters, and increase the time taken for catheters to block. Swimming and swarming motilies in P. mirabilis were also significantly reduced by both EPIs. The impact of these drugs on catheter biofilm formation by other uropathogens (Escherichia coli, Pseudomonas aeruginosa) was also explored, and thioridazine was shown to also inhibit biofilm formation in these species. Therefore, repurposing of existing drugs with EPI activity could be a promising approach to control catheter blockage, or biofilm formation on other medical devices.

Prevention of encrustation and blockage of urinary catheters by Proteus mirabilis via pH-triggered release of bacteriophage.

The crystalline biofilms of Proteus mirabilis can seriously complicate the care of patients undergoing long-term indwelling urinary catheterisation. Expression of bacterial urease causes a significant increase in urinary pH, leading to the supersaturation and precipitation of struvite and apatite crystals. These crystals become lodged within the biofilm, resulting in the blockage of urine flow through the catheter. Here, we describe an infection-responsive surface coating for urinary catheters, which releases a therapeutic dose of bacteriophage in response to elevated urinary pH, in order to delay catheter blockage. The coating employs a dual-layered system comprising of a lower hydrogel 'reservoir' layer impregnated with bacteriophage, capped by a 'trigger' layer of the pH-responsive polymer poly(methyl methacrylate-co-methacrylic acid) (EUDRAGIT®S 100). Evaluation of prototype coatings using a clinically reflective in vitro bladder model system showed that catheter blockage time was doubled (13 h to 26 h (P < 0.05)) under conditions of established infection (108 CFU ml-1) in response to a 'burst-release' of bacteriophage (108 PFU ml-1). Coatings were stable both in the absence of infection, and in the presence of urease-negative bacteria. Quantitative and visual analysis of crystalline biofilm reduction show that bacteriophage constitute a promising strategy for the prevention of catheter blockage, a clinical problem for which there is currently no effective control method.

An in-situ infection detection sensor coating for urinary catheters.

We describe a novel infection-responsive coating for urinary catheters that provides a clear visual early warning of Proteus mirabilis infection and subsequent blockage. The crystalline biofilms of P. mirabilis can cause serious complications for patients undergoing long-term bladder catheterisation. Healthy urine is around pH 6, bacterial urease increases urine pH leading to the precipitation of calcium and magnesium deposits from the urine, resulting in dense crystalline biofilms on the catheter surface that blocks urine flow. The coating is a dual layered system in which the lower poly(vinyl alcohol) layer contains the self-quenching dye carboxyfluorescein. This is capped by an upper layer of the pH responsive polymer poly(methyl methacrylate-co-methacrylic acid) (Eudragit S100®). Elevation of urinary pH (>pH 7) dissolves the Eudragit layer, releasing the dye to provide a clear visual warning of impending blockage. Evaluation of prototype coatings using a clinically relevant in vitro bladder model system demonstrated that coatings provide up to 12h advanced warning of blockage, and are stable both in the absence of infection, and in the presence of species that do not cause catheter blockage. At the present time, there are no effective methods to control these infections or provide warning of impending catheter blockage.

Escherichia coli Nissle 1917 enhances bioavailability of serotonin in gut tissues through modulation of synthesis and clearance.

Accumulating evidence shows indigenous gut microbes can interact with the human host through modulation of serotonin (5-HT) signaling. Here we investigate the impact of the probiotic Escherichia coli Nissle 1917 (EcN) on 5-HT signalling in gut tissues. Ex-vivo mouse ileal tissue sections were treated with either EcN or the human gut commensal MG1655, and effects on levels of 5-HT, precursors, and metabolites, were evaluated using amperometry and high performance liquid chromatography with electrochemical detection (HPLC-EC). Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT. These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures. In contrast, when supernatant recovered from untreated ileal tissue was pre-incubated with EcN, the derivative cell-free supernatant was able to elevate 5-HT overflow when used to treat fresh ileal tissue. Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA. The former pointed to modulation of tryptophan hydroxylase-1 to enhance 5-HT synthesis, while the latter indicates an impact on clearance into enterocytes through SERT. Taken together, these findings show EcN is able to enhance 5-HT bioavailability in ileal tissues through interaction with compounds secreted from host tissues.

Bacteriophage Can Prevent Encrustation and Blockage of Urinary Catheters by Proteus mirabilis.

Proteus mirabilis forms dense crystalline biofilms on catheter surfaces that occlude urine flow, leading to serious clinical complications in long-term catheterized patients, but there are presently no truly effective approaches to control catheter blockage by this organism. This study evaluated the potential for bacteriophage therapy to control P. mirabilis infection and prevent catheter blockage. Representative in vitro models of the catheterized urinary tract, simulating a complete closed drainage system as used in clinical practice, were employed to evaluate the performance of phage therapy in preventing blockage. Models mimicking either an established infection or early colonization of the catheterized urinary tract were treated with a single dose of a 3-phage cocktail, and the impact on time taken for catheters to block, as well as levels of crystalline biofilm formation, was measured. In models of established infection, phage treatment significantly increased time taken for catheters to block (∼ 3-fold) compared to untreated controls. However, in models simulating early-stage infection, phage treatment eradicated P. mirabilis and prevented blockage entirely. Analysis of catheters from models of established infection 10 h after phage application demonstrated that phage significantly reduced crystalline biofilm formation but did not significantly reduce the level of planktonic cells in the residual bladder urine. Taken together, these results show that bacteriophage constitute a promising strategy for the prevention of catheter blockage but that methods to deliver phage in sufficient numbers and within a key therapeutic window (early infection) will also be important to the successful application of phage to this problem.

Disruption of Escherichia coli Nissle 1917 K5 capsule biosynthesis, through loss of distinct kfi genes, modulates interaction with intestinal epithelial cells and impact on cell health.

Escherichia coli Nissle 1917 (EcN) is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants), prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB), or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC), were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health.

Selection of DNA aptamers against uropathogenic Escherichia coli NSM59 by quantitative PCR controlled Cell-SELEX.

In order to better control nosocomial infections, and facilitate the most prudent and effective use of antibiotics, improved strategies for the rapid detection and identification of problematic bacterial pathogens are required. DNA aptamers have much potential in the development of diagnostic assays and biosensors to address this important healthcare need, but further development of aptamers targeting common pathogens, and the strategies used to obtain specific aptamers are required. Here we demonstrate the application of a quantitative PCR (qPCR) controlled Cell-SELEX process, coupled with downstream secondary-conformation-based aptamer profiling. We used this approach to identify and select DNA aptamers targeted against uropathogenic Escherichia coli, for which specific aptamers are currently lacking, despite the prevalence of these infections. The use of qPCR to monitor the Cell-SELEX process permitted a minimal number of SELEX cycles to be employed, as well as the cycle-by-cycle optimisation of standard PCR amplification of recovered aptamer pools at each round. Identification of useful aptamer candidates was also facilitated by profiling of secondary conformations and selection based on putative aptamer secondary structure. One aptamer selected this way (designated EcA5-27), displaying a guanine-quadruplex sequence motif, was shown to have high affinity and specificity for target cells, and the potential to discriminate between distinct strains of E. coli, highlighting the possibility for development of aptamers selectively recognising pathogenic strains. Overall, the identified aptamers hold much potential for the development of rapid diagnostic assays for nosocomial urinary tract infections caused by E. coli.