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Pseudomonas aeruginosa is an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors. P. aeruginosa infections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains. In this study, we analyzed whether the amoeba Dictyostelium discoideum can be used as a simple model system to analyze the virulence of P. aeruginosa strains. The virulent wild-type strain PAO1 was shown to inhibit growth of D. discoideum. Isogenic mutants deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while rhl quorum-sensing mutants permitted growth of Dictyostelium cells. Therefore, in this model system, factors controlled by the rhl quorum-sensing system were found to play a central role. Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis of D. discoideum cells. By using this simple model system, we predicted that certain antibiotic-resistant mutants of P. aeruginosa should show reduced virulence. This result was confirmed in a rat model of acute pneumonia. Thus, D. discoideum could be used as a simple nonmammalian host system to assess pathogenicity of P. aeruginosa.The bacterium Pseudomonas aeruginosa is an important causative agent of nosocomial infections, including severe pneumonia (10) and bacteremia. This opportunistic pathogen also colonizes the lungs of cystic fibrosis patients and leads to progressive lung damage, respiratory failure, and eventually death (3, 12). The seriousness of P. aeruginosa infections is further exacerbated by the rapid selection of antibiotic-resistant strains following antibiotic treatment (14).Studies in mammalian hosts have shown that quorum sensing is important for the virulence of P. aeruginosa (28,37,42). Secreted components essential for Pseudomonas virulence, such as proteases, rhamnolipids, pyocyanin, and exotoxin A, are under the control of two quorum-sensing systems, las and rhl ( Fig. 1) (31, 43). When the bacterial cell density reaches a certain threshold, the accumulation in the medium of signaling autoinducer molecules (3-oxo-C12-homoserine lactone [HSL] and C4-HSL) induces the las and rhl pathways, respectively, leading to transcription of virulence genes. Both systems involve a transcriptional regulator (LasR and RhlR, respectively) and an autoinducer synthase (LasI and RhlI, respectively). The las quorum-sensing system can also induce the transcription of rhlR and consequently activate, to some degree, the rhl quorum-sensing system (21).Strategies to develop innovative treatments against Pseudomonas infections rely on the elucidation of virulence and antibiotic resistance mechanisms. These studies involve the characterization of mutant strains and analysis of their virulence.However, the assessment of bacterial pathogenicity in mammalian hosts is time-consuming and expensive. Therefore, alternative yet equally relevant host systems would be extremely useful. Pseudomonas is remarkable in its ability to infect a number of alt...

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Rare disease patients experience particular obstacles in accessing high quality healthcare. These obstacles include but are not limited to: (i) lack of scientific knowledge of their disease, (ii) lack of access to correct diagnosis, (iii) delays in diagnosis, (iv) lack of appropriate multidisciplinary healthcare, (v) lack of quality information and support at the time of diagnosis, (vi) undue social consequences, (vii) inequities and difficulties in access to treatment, rehabilitation and care, (viii) dissatisfaction with and loss of confidence in medical and social services, (ix) denied treatment by health professionals and (x) lack of availability of orphan drugs. Three surveys and their subsequent analysis, conducted by the European Organisation for Rare Diseases (EURORDIS), a non-governmental patient driven alliance of European patient organisations, demonstrate several of these obstacles by describing the experience of rare disease patients across 18 rare diseases and over 24 European countries as well as highlighting inequalities that exist between them.

The virulence of Pseudomonas aeruginosa is partly controlled by the las quorum-sensing system. A rat model of acute pneumonia was used to investigate the pathophysiological impact of this system by comparing the virulence of the wild-type virulent laboratory strain PAO1 with that of its lasR-deleted mutant PAOR. In comparison with PAO1, PAOR was avirulent after an instillation of 106 cfu (mortality rates, 72 versus 0%, respectively; p < 0.0001). A ten-fold higher inoculum slightly increased the mortality rate induced by PAOR (25%), which remained lower than that induced by PAO1 (75%, p = 0.0001). In addition, with both inocula lung and bronchoalveolar lavage bacterial counts were significantly lower in rats infected with PAOR than with PAO1 (p

The disposition of monodesethylamodiaquine was studied in four healthy subjects after a single oral dose of 10 mg/kg amodiaquine base. Amodiaquine was not found in any sample, but the major metabolite monodesethylamodiaquine was detected and was assumed to be the sole derivative that contributed significantly to antimalarial activity in the blood. The best fit for the decay of the metabolite was obtained with a three-compartment model. The half-lives of the first two phases were 3.2 to 11.4 h for t1/2 alpha 1 and 22.7 to 50.3 h for t1/2 alpha 2 in plasma. The half-life of the terminal phase (t1/2 beta) was between 9 and 18.2 days. The concentration in whole blood was 4- to 6-times higher than in plasma. Three schedules (alternate days, weekly, daily) of the conventional prophylactic dose of 10 mg/kg per week were compared in six other healthy subjects. There were significant differences in the plasma monodesethylamodiaquine levels between the three schedules.

The ability of trovafloxacin and ciprofloxacin to select efflux mutants in vivo was studied in a model of acute Pseudomonas aeruginosa pneumonia in rats. Twelve hours after intratracheal inoculation of 10 6 CFU of P. aeruginosa strain PAO1 enmeshed in agar beads, two groups of 12 rats were treated by three intraperitoneal injections of each antibiotic given every 5 h. Dosing regimens were chosen to obtain a comparable area under the concentration-time curve from 0 to infinity/MIC ratio of 27.9 min for trovafloxacin (75 mg/kg of body weight) and of 32.6 min for ciprofloxacin (12.5 mg/kg). Twelve rats were left untreated and served as controls. Rats were sacrificed 12 h after the last injection (34 h after infection) for lung bacteriological studies. Selection of resistant bacteria was determined by plating lung homogenates on Trypticase soy agar plates containing antibiotic. In untreated animals, the frequency of resistant colonies was 10-fold higher than in agar beads. Compared to controls, both treatment regimens resulted in a 2-log reduction of lung bacterial load. The frequency of resistant colonies was 10-fold less with trovafloxacin than with ciprofloxacin at twice the MIC (7.4 ؋ 10 ؊5 versus 8.4 ؋ 10 ؊4 , respectively) (P < 0.05) and at four times the MIC (6.2 ؋ 10 ؊4 versus 5.0 ؋ 10 ؊5 , respectively) (P < 0.05). A multidrug resistance phenotype typical of efflux mutants was observed in all 41 randomly tested colonies obtained from treated and untreated rats. In agreement with in vitro results, trovafloxacin and ciprofloxacin preferentially selected MexCD-OprJ and MexEF-OprN overproducers, respectively. These results demonstrate the differential ability of trovafloxacin and ciprofloxacin to select efflux mutants in vivo and highlight the rapid emergence of those mutants, even without treatment.