The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a nonvirion form of the coat protein.

Research into naturally occurring antimicrobial substances has yielded effective treatments. One area of interest is peptides and proteins produced by invertebrates as part of their defence system, including the contents of mollusc mucous. Mucous produced by the African giant land snail, Achatina fulica has been reported to contain two proteins with broad spectrum anti-bacterial activity. Mucous from the brown garden snail, Helix aspersa appears to have skin regenerationproperties. This study sought to investigate the antimicrobial properties of H.aspersa mucous.Mucous was collected from H.aspersa snails, diluted in PBS and centrifuged, with the supernatant tested against a wide range of organisms in a disc diffusion antimicrobial assay. This was followed up with comparative experiments involving A.fulica , including bacteriophage assays. Mucous from both species of snail was passed through a series of protein size separation columns in order to determine the approximate size of the antimicrobial substance. Electrophoresis was also carried out on the H.aspersa mucous.Results indicated that H.aspersa mucous had a strong antibacterial effect against several strains of Pseudomonas aeruginosa and a weak effect against Staphylococcus aureus. Mucous from A.fulica also inhibited the growth of S.aureus, but the broad spectrum of activity reported by other workers was not observed. Antimicrobial activity was not caused by bacteriophage. Size separation experiments indicated that the antimicrobial substance(s) in H.aspersa were between 30 and 100 kDa. Electrophoresis revealed two proteins in this region -30-40 kDa and 50-60kDa. These do not correspond with antimicrobial proteins previously reported in A.fulica. This study found one or more novel antimicrobial agents in H.aspersa mucous, with a strong effect against P.aeruginosa . 2

b; Queen Victoria NHS Foundation Trust, East Grinstead, West Sussex, United Kingdom c Proteus mirabilis forms extensive crystalline biofilms on urethral catheters that occlude urine flow and frequently complicate the management of long-term-catheterized patients. Here, using random transposon mutagenesis in conjunction with in vitro models of the catheterized urinary tract, we elucidate the mechanisms underpinning the formation of crystalline biofilms by P. mirabilis. Mutants identified as defective in blockage of urethral catheters had disruptions in genes involved in nitrogen metabolism and efflux systems but were unaffected in general growth, survival in bladder model systems, or the ability to elevate urinary pH. Imaging of biofilms directly on catheter surfaces, along with quantification of levels of encrustation and biomass, confirmed that the mutants were attenuated specifically in the ability to form crystalline biofilms compared with that of the wild type. However, the biofilm-deficient phenotype of these mutants was not due to deficiencies in attachment to catheter biomaterials, and defects in later stages of biofilm development were indicated. For one blocking-deficient mutant, the disrupted gene (encoding a putative multidrug efflux pump) was also found to be associated with susceptibility to fosfomycin, and loss of this system or general inhibition of efflux pumps increased sensitivity to this antibiotic. Furthermore, homologues of this system were found to be widely distributed among other common pathogens of the catheterized urinary tract. Overall, our findings provide fundamental new insight into crystalline biofilm formation by P. mirabilis, including the link between biofilm formation and antibiotic resistance in this organism, and indicate a potential role for efflux pump inhibitors in the treatment or prevention of P. mirabilis crystalline biofilms.

h 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 (ϳ3-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.A frequent complication associated with long-term urethral catheterization is the encrustation and blockage of catheters due to infection with Proteus mirabilis, which can be isolated from around 45% of catheter-associated urinary tract infections (CAUTI) (1, 2). Blockage stems from the ability of P. mirabilis to form dense biofilms on catheter surfaces and the production of a potent urease enzyme which generates ammonia through hydrolysis of urea (1,3,4). Ammonia production elevates urinary pH, causing the precipitation of calcium and magnesium phosphates and the subsequent formation of crystals which become trapped within developing biofilms (1, 5). Once embedded in the biofilm, crystal growth is stabilized and enhanced by the biofilm matrix (6, 7). As this process continues, the biofilm gradually becomes mineralized, leading to development of extensive crystalline biofilm structures which ultimately block catheters (1, 5). If blockage is unnoticed, it can lead to reflux of infected urine to the upper urinary tract and the onset of serious clinical complications, including pyelonephritis, septicemia, and shock (1, 8).Although catheters containing antimicrobial coatings are currently available, their efficacy in preventing infection during even short-term use remains questionable, and all...