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An electrified catheter to resist encrustation by Proteus mirabilis biofilm.

PURPOSE: We investigated the effect of iontophoresis produced by passing an electric current through silver electrodes attached to catheters on catheter encrustation by crystalline Proteus mirabilis biofilm. MATERIALS AND METHODS: Four glass bladder models were catheterized with 16Fr silicone catheters, of which 3 had 0.25 mm silver wires running through and beside the lumen. Two wired catheters had the silver wires connected to a 9 V direct current source supplying a steady current of 150 microA via a self-regulating circuit. Artificial urine, which had been inoculated with a clinical strain of P. mirabilis isolated from an encrusted catheter, was instilled into the bladder model at 0.5 ml per minute. The models were operated until the test catheters became blocked. Mean blockage time was statistically analyzed by ANOVA. Bacterial colony count, silver ions and pH were assessed every 24 hours. RESULTS: The experiment was repeated 3 times. Time to blockage, colony count, pH and scanning electron microscopy was used to assess encrustation in electrified and control catheters. Time to blockage in electrified vs control catheters was 156 vs 22 hours. The difference in blockage times was statistically significant (p <0.002). The viable bacterial cell count in urine with test catheters vs that in controls was 1.12 x 10(4) vs 2.73 x 10(9) cfu/ml. The pH increased to 9 in control models, whereas it remained less than 6.5 in test models for about 100 hours. CONCLUSIONS: Electrified catheters released ions in urine that have the oligodynamic property of inhibiting bacterial growth. The application of electric current to catheters fitted with silver electrodes significantly decreased the rate at which these devices became encrusted by P. mirabilis. This principle could be used to prevent encrustation on long-term catheters.

Biofilms↗

Overgrowth management in Klippel-Trenaunay-Weber and Proteus syndromes.

Twenty-eight patients with limb overgrowth and the diagnosis of Klippel-Trenaunay-Weber or Proteus syndromes were evaluated retrospectively. These disorders are part of the phakomatosis spectrum of syndromes. The orthopedic problems consisted of asymmetric limb overgrowth, localized gigantism, angular deformities, scoliosis, vascular malformations, and skin anomalies. Systemic abnormalities are common and deserve full evaluation before treatment. Surgical treatment consisted of epiphysiodesis, osteotomies, debulking procedures, and amputation. Mixed results were obtained with surgery, and conservative or supportive treatment should be the primary mode of orthopedic care.

Adolescent↗

Proteus mirabilis phage 5006M: a physical characterization.

This report deals with physical characterization of the generalized transducing Proteus mirabilis phage 5006M. The morphology of the phage is presented, the buoyant density was determined (1.491 g/ml) and the G + C content of the phage DNA was found to be 44%. The phage genome has a length of 14.8 micrometers and mol. wt. of 30.7 x 10(6). Denaturation mapping revealed non-random circular permutation of the phage DNA. The genome exhibits 3.6% terminal redundancy as shown by homoduplex analysis. The existence of concatemeric precursors of phage 5006M DNA is inferred and the results are interpreted in terms of a sequential headful packaging mechanism.

Bacteriophages↗

Proteus mirabilis phages 5006M, 5006M HFT k and 5006M HFT ak: physical comparison of genome characteristics.

The genomes of proteus mirabilis phages 5006M, kanamycin resistance transducing variant 5006M HFT k and kanamycin-ampicillin resistance transducing variant 5006M HFT ak have been compared. Homo- and heteroduplex and partial denaturation mapping analyses were performed. The results confirm a sequential headful packaging mechanism, facilitate mapping of the ampicillin resistance marker, demonstrate a hairpin loop structure in both variants, reveal a common insertion site for 8 X 10(6) mol. wt. non-phage DNA in both variants and implicate a role for the non-inducible cryptic host strain prophage 5006M in the generatin cycle of the variant phages.

Ampicillin↗

Inhibition of virulence factor expression and swarming differentiation in Proteus mirabilis by p-nitrophenylglycerol.

Proteus mirabilis is a common cause of upper urinary tract infections that can involve invasion of host urothelial cells. The ability to invade urothelial cells is coupled closely to swarming, a form of multicellular behaviour in which vegetative bacteria differentiate into hyperflagellate, filamentous swarming cells capable of co-ordinated and rapid population migration. Co-ordinate expression of virulence factors including urease, protease, haemolysin and flagellin during swarm-cell differentiation in P. mirabilis has been reported. To investigate the effects of p-nitrophenylglycerol (PNPG), a potent anti-swarming agent, on the various swarming-associated traits of P. mirabilis and to elucidate the relationships among them, P. mirabilis growth rate, swarming/swimming activity, cell invasion ability and the ability to express various virulence factors were monitored in the presence or absence of PNPG. It was found that PNPG could inhibit the growth rate, swarming differentiation and swarming/swimming activities of P. mirabilis. The expression of virulence factors such as protease, urease, haemolysin and flagellin in P. mirabilis was also inhibited by PNPG. The ability of P. mirabilis to invade human urothelial cells was reduced dramatically in the presence of PNPG. These results suggest that PNPG has the potential to be developed as an agent active against the effects of P. mirabilis infection.

Humans↗

Characterisation of p-nitrophenylglycerol-resistant Proteus mirabilis super-swarming mutants.

p-Nitrophenylglycerol (PNPG) inhibits the co-ordinately regulated activities of swarming behaviour and virulence factor expression in Proteus mirabilis. The inhibitory action of PNPG was investigated by the isolation of Tn5 insertion mutants that could swarm, albeit with much reduced ability, in the presence of PNPG. The mutants exhibited a super-swarming phenotype in the absence of PNPG; i.e., they migrated further in a given time than did the wild-type cells. Cloning and sequence analysis of the mutants indicated that Tn5 was inserted into the rsbA gene, which may encode a membrane sensor histidine kinase of the bacterial two-component signalling system. In the absence of PNPG, the mutants exhibited several swarming-related phenotypes that were different from those of the wild type; they initiated swarming earlier and had a less conspicuous consolidation phase, they differentiated earlier and maintained a differentiated state for longer, they started to express virulence factors earlier and maintained high expression levels of these factors for longer, and they had higher cell invasion ability than the wild type. These mutant phenotypes could be complemented by a plasmid-borne copy of rsbA. Together, these data suggest that RsbA may act as a repressor of swarming and virulence factor expression. In the presence of PNPG, these rsbA-mutated mutants could still swarm, differentiate and express virulence factors, whereas the wild type could not, suggesting that PNPG may target RsbA or RsbA-regulated pathways to exert its inhibitory effect. Together, these data reveal a novel mechanism through which bacteria may negatively regulate swarming differentiation and virulence factor expression and identify a potential target of PNPG action.

Bacterial Proteins↗

The Helicobacter pylori flbA flagellar biosynthesis and regulatory gene is required for motility and virulence and modulates urease of H. pylori and Proteus mirabilis.

Helicobacter pylori and Proteus mirabilis ureases are nickel-requiring metallo-enzymes that hydrolyse urea to NH3 and CO2. In both H. pylori and in an Escherichia coli model of H. pylori urease activity, a high affinity nickel transporter, NixA, is required for optimal urease activity, whereas the urea-dependent UreR positive transcriptional activator governs optimal urease expression in P. mirabilis. The H. pylori flbA gene is a flagellar biosynthesis and regulatory gene that modulates urease activity in the E. coli model of H. pylori urease activity. All flbA mutants of eight strains of H. pylori were non-motile and five had a strain-dependent alteration in urease activity. The flbA gene decreased urease activity 15-fold when expressed in E. coli containing the H. pylori urease locus and the nixA gene; this was reversed by disruption of flbA. The flbA gene decreased nixA transcription. flbA also decreased urease activity three-fold in E. coli containing the P. mirabilis urease locus in a urea- and UreR-dependent fashion. Here the flbA gene repressed the P. mirabilis urease promoter. Thus, FlbA decreased urease activity of both H. pylori and P. mirabilis, but through distinct mechanisms. H. pylori wild-type strain SS1 colonised gerbils at a mean of 5.4 x 10(6) cfu/g of antrum and caused chronic gastritis and lesions in the antrum. In contrast, the flbA mutant did not colonise five of six gerbils and caused no lesions, indicating that motility mediated by flbA was required for colonisation. Because FlbA regulates flagellar biosynthesis and secretion, as well as forming a structural component of the flagellar secretion apparatus, two seemingly unrelated virulence attributes, motility and urease, may be coupled in H. pylori and P. mirabilis and possibly also in other motile, ureolytic bacteria.

Animals↗

Proteus mirabilis converting phage 5006Mpa has an oversized genome.

Proteus mirabilis phage 5006Mpa is a converting variant for ampicillin resistance of phage 5006M. We show here that the ampicillin resistance marker of transposon Tn1 is located on a 9.8% insertion with respect to the wild-type phage genome. This renders the 5006Mpa genome 5% oversized, albeit without loss of wild-type genetic material from the phage population.

Ampicillin↗

A physical analysis of the prophage of Proteus mirabilis PM5006.

Proteus mirabilis phage 5006M was investigated as a prophage by DNA hybridization. A physical map of the prophage was established and the site of integration was localized on the phage genome. Older reports that the phage is cryptic in P. mirabilis could not be verified.

Bacteriophages↗

Extension of a chromosome linkage group of Proteus mirabilis.

Mating procedures for detection of mobilization of the Proteus mirabilis chromosome were re-investigated. The chromosome was mobilized by plasmid D, the previously used hybrid between plasmids P-lac and R1drd19. About a 40-fold increase in recombinant recovery correlated with the absence of swarming during mating and a lower temperature of incubation. The modification introduced was that conjugation was allowed to proceed on a non-selective supplemented minimal medium at 30 degrees C before washing and plating on selective media. Final incubation was also at 30 degrees C. This technique enabled eight additional chromosomal markers to be mapped. Polarized transfer of the chromosome was shown by gradient of transmission experiments using a previously described marker as reference, by linkage analysis with reference to proximal and distal markers and (less successfully) by interrupted mating on solid medium. Markers of plasmid D transferred at high frequency to all recombinants. The plasmid was stable in recombinants and could transfer itself and chromosomal markers of the new hosts in further matings. Resulting recombination of markers occurred at usual frequencies. The marker order, his-1, ser-2, ura-2, pyrB1, trp-3, cysA1, ade-2, ilv-2, cysG1, gly-1, cysC1, argA2, metF2, nalA1, thr-1, leuB2, did not resemble the order of these markers in Escherichia coli.

Chromosome Mapping↗

Mobilization of the Proteus mirabilis chromosome by R plasmid R772.

The P-1 incompatibility group plasmid R772 can mobilize the chromosome of Proteus mirabilis strain PM5006. The decreasing gradient of recombinant recovery frequencies found for markers which were increasingly distal to 0 min with plasmid D donors was not found with R772. Instead, it produced recombinants for all markers at frequencies of about 5 X 10(-5) per donor. This is about 10-fold lower than the plasmid transfer frequency. Recombinants were stable and recombination was only detected over short segments of the chromosome which corresponded to about 10 min on the D plasmid map of the chromosome. All recombinants had inherited R772 and expressed all properties of the plasmid. Attempts to isolate variant plasmids with increased frequencies of recombinant formation were unsuccessful.

Chromosomes, Bacterial↗

Genetic circularity of the Proteus mirabilis linkage map.

The T incompatibility group plasmid R394 can mobilize the chromosome of Proteus mirabilis strain PM5006. It transferred relatively large segments, corresponding to at least 20 min on the D plasmid chromosomal map of the organism. The frequency of recombination for a large number of selected markers was nearly constant at 5 X 10(-6) per donor cell and it is concluded that mobilization takes place from a number of chromosomal sites. All recombinants were R+ and displayed all properties of the plasmid. By analysing crosses for co-inheritance frequencies of unselected markers, a number of chromosomal loci were assembled in linear array. Linkage between markers at the ends of this linkage group was established to markers at the respective termini of the existing D plasmid linkage group. This established a composite circular linkage map of genes of the P. mirabilis strain PM5006 chromosome.

Chromosome Mapping↗

Patterns of mobilization of the Proteus mirabilis chromosome by R plasmids.

R plasmids R40a, Rip69, R447b, R769 belonging to incompatibility groups A-C, M, N, V, respectively, were investigated for chromosomal mobilizing ability in Proteus mirabilis. Plasmids R40a, Rip69 and R447b mediated polarized transfer of markers in a clockwise direction from origins near tyr-1, metF and ser-2, respectively, on the linkage map. The recovery frequency per donor cell of proximal markers approached 1 x 10(-4) for these three plasmids and the efficiency of chromosomal transfer was higher than that of the previously studied plasmid D. The plasmid-guided chromosomal trajectories overlap and it was possible to complement results obtained with plasmid D to assemble a time-of-entry chromosomal map and directly establish the circularity of the linkage group. The map comprises a length of 93 min in terms of transfer time. Plasmid R769 had a different pattern of chromosome transfer. This plasmid produced recombinants for all markers at frequencies of about 4 x 10(-6) per donor. It effected multiple and more or less simultaneous entry of markers and produced recombination over lengths of chromosome rarely corresponding to more than 10 min on the linkage map.

Chromosome Mapping↗

Mobilization of the Proteus morganii chromosome by R plasmids.

R plasmids R702, R711b, R1, D, Rip69, R447b, R471 and R394, belonging to different incompatibility groups, mobilized the Proteus morganii 2815 chromosome. Matings employing plasmids R711b or R702 as sex factors with doubly auxotrophic recipients produced recombinants characterized by the obligatory inheritance of ser-1+, irrespective of the selected marker.

Chromosomes, Bacterial↗

Changes in metabolic activity of Proteus mirabilis during swarming.

Proteus mirabilis in the long, swarming form had altered metabolic activity compared with bacteria in non-swarming phases on solid media. During swarming the rates of incorporation of precursors into DNA, RNA and protein, as measured in broth cultures immediately after harvesting from swarm plates, were lowered. The rates of uptake of these precursors into the bacteria were also lowered, and at the same time the rate of oxygen uptake was reduced to less than 20% of the normal rate, although intracellular ATP concentrations remained constant. The return of macromolecular synthesis and oxygen uptake to preswarming rates corresponded to the end of the active swarm period. The results indicate that in the multiflagellate swarmers of P. mirabilis metabolic activity was lowered to a level necessary to maintain flagella activity but not bacterial growth.

Adenosine Triphosphate↗

Relationship between cyclopropane synthetase and the formation of cyclopropane fatty acids by Proteus vulgaris grown under various respiratory conditions.

In medium saturated with oxygen, the cyclopropane synthetase (unsaturated-phospholipid methyltransferase; EC 2.1.1.16) of Proteus vulgaris was generally synthesized after the mid-exponential phase of growth. The enzyme could also be induced by rapidly limiting the oxygen supply, or by initiating respiration on nitrate or thiosulphate following an initial period of growth in a highly aerobic environment. In each of these 'step-down' situations the specific activity of cyclopropane synthetase rose to a maximum prior to the stationary phase of growth and subsequently decreased. The cyclopropane fatty acids, methylene hexadecanoic acid and methylene octadecanoic acid accumulated throughout exponential growth following the induction of the enzyme. During a 12 h period in the stationary phase there was little synthesis of either of the fatty acids, despite detectable cyclopropane synthetase activity in the cells, indicating that essentially all the fatty acid synthesis was complete prior to entering the stationary phase. When nitrate was used as a respiratory electron acceptors a twofold increase in octadecenoic acid was observed, giving rise to an increase in methylene octadecanoic acid. This increase in octadecanoic acid was not apparent in mutants unable to respire on nitrate.

Cyclopropanes↗

In vitro-constructed RP4-prime plasmids mediate orientated mobilization of the Proteus morganii chromosome.

RP4-prime plasmids containing inserts of Proteus morganii strain 2815 chromosomal DNA (0.5-13 megadaltons in size) were constructed in vitro. When introduced into the latter organism, the hybrid plasmids promoted transfer of chromosomal markers in an orientated manner, with recombinant frequencies from 10(-4) to 10(-6) per donor. Transconjugants expressed the tetracycline-resistant phenotype of the plasmids. Plasmid-guided trajectories overlapped and a circular map of chromosome markers was constructed. Correspondence between size of inserted chromosomal DNA in hybrid plasmids and numbers of markers transferred suggested that homology played a role in chromosome mobilization by these plasmids.

Chromosome Mapping↗

The effect of temperature on the synthesis and assembly of proticine 3 particles by Proteus mirabilis.

Proteus mirabilis CW977 produced high yields of the bacteriocin proticine 3 upon mitomycin C induction of cultures growing at 30 degrees C. The proticine was purified and found to have a relative density of 1.299 and to be composed of 10 proteins assembled into structures resembling contractile phage tails. When induction was performed at 41 degrees C neither proticine particles nor proticine activity was detected, although the growth rate of cells and degree of lysis were indistinguishable from that at 30 degrees C. Failure in proticine production was due to a 41 degrees C sensitive stage occurring between 60 and 90 min after the addition of mitomycin C. During this period at 30 degrees C, two proteins of mol. wt 58 000 and 41 000 were formed. These proteins were associated with events leading to the formation of proticine particles with biological activity. When the production of both proteins was prevented either by chloramphenicol or as a result of mutation or through sampling before they were formed, no proticine particles were found nor proticine activity detected. The synthesis of both proteins was also inhibited at 41 degrees C. Co-electrophoresis of the labelled proteins with unlabelled purified proticine confirmed that the protein of mol. wt 58 000 was a proticine structural protein. The protein of mol. wt 41 000 was not a structural component of proticine and its role, if any, in proticine 3 production is possibly that of an assembly protein.

Bacterial Proteins↗