Azithromycin to prevent Pseudomonas aeruginosa ventilator-associated pneumonia by inhibition of quorum sensing: a randomized controlled trial.

PURPOSE: Anti-virulence strategies have not been evaluated for the prevention of bacterial infections. Prolonged colonization of intubated patients with Pseudomonas aeruginosa isolates producing high-levels of the quorum sensing (QS)-regulated virulence factor rhamnolipids has been associated with ventilator-associated pneumonia (VAP). In this pathogen, azithromycin reduces QS-regulated virulence. We aimed to assess whether azithromycin could prevent VAP in patients colonized by rhamnolipids producing isolates. METHODS: In a randomized, double-blind, multicenter trial, intubated colonized patients received either 300 mg/day azithromycin or placebo. Primary endpoint was the occurrence of P. aeruginosa VAP. We further identified those patients persistently colonized by isolates producing high-levels of rhamnolipids and therefore at the highest risk to develop VAP linked to this QS-dependent virulence factor. RESULTS: Ninety-two patients were enrolled; 43 azithromycin-treated and 42 placebo patients were eligible for the per-protocol analysis. In the per-protocol population, the occurrence of P. aeruginosa VAP was reduced in the azithromycin group but without reaching statistical significance (4.7 vs. 14.3 % VAP, p = 0.156). QS-dependent virulence of colonizing isolates was similarly low in both study groups, and only five patients in each arm were persistently colonized by high-level rhamnolipids producing isolates. In this high-risk subgroup, the incidence of VAP was reduced fivefold in azithromycin versus placebo patients (1/5 vs. 5/5 VAP, p = 0.048). CONCLUSIONS: There was a trend towards reduced incidence of VAP in colonized azithromycin-treated patients. In addition, azithromycin significantly prevented VAP in those patients at high risk of rhamnolipid-dependent VAP, suggesting that virulence inhibition is a promising anti-microbial strategy.

Rifampin-bonded vascular grafts and postoperative infections.

Postoperative wound and graft infections remain a major challenge for vascular surgeons. The bonding of antimicrobial substances on the graft material has been considered for many years, but the demonstration of safety and efficacy of these techniques is far from evident. Among the different proposed options, bonding of rifampin to the grafts has been the most evaluated technique, both experimentally and clinically. The objective of this review was to present and analyze the available data on rifampin-bonding and the possible evolutions of this technique to improve the resistance of vascular prostheses.

Effect of clarithromycin in patients with sepsis and ventilator-associated pneumonia.

BACKGROUND: Because clarithromycin provided beneficiary nonantibiotic effects in experimental studies, its efficacy was tested in patients with sepsis and ventilator-associated pneumonia (VAP). METHODS: Two hundred patients with sepsis and VAP were enrolled in a double-blind, randomized, multicenter trial from June 2004 until November 2005. Clarithromycin (1 g) was administered intravenously once daily for 3 consecutive days in 100 patients; another 100 patients were treated with placebo. Main outcomes were resolution of VAP, duration of mechanical ventilation, and sepsis-related mortality within 28 days. RESULTS: The groups were well matched with regard to demographic characteristics, disease severity, pathogens, and adequacy of the administered antimicrobials. Analysis comprising 141 patients who survived revealed that the median time for resolution of VAP was 15.5 days and 10.0 days among placebo- and clarithromycin-treated patients, respectively (P = .011); median times for weaning from mechanical ventilation were 22.5 days and 16.0 days, respectively (p = .049). Analysis comprising all enrolled patients showed a more rapid decrease of the clinical pulmonary infection score and a delay for advent of multiple organ dysfunction in clarithromycin-treated patients, compared with those of placebo-treated patients (p = .047). Among the 45 patients who died of sepsis, time to death was significantly prolonged in clarithromycin-treated compared with placebo-treated patients (p = .004). Serious adverse events were observed in 0% and 3% of placebo- and clarithromycin-treated patients, respectively (P = .25). CONCLUSIONS: Clarithromycin accelerated the resolution of VAP and weaning from mechanical ventilation in surviving patients and delayed death in those who died of sepsis. The mortality rate at day 28 was not altered. Results are encouraging and render new perspectives on the management of sepsis and VAP.

A global survey of antibiotic leftovers in the outpatient setting.

We performed a global survey of outpatients who had taken self-administered antibiotics within the last 12 months in order to identify factors that lead to possession of leftover antibiotics in the community. The study included 4,514 subjects aged 18-99 years. Of 4,192 respondents not currently taking antibiotics, 53.7% admitted having leftover antibiotics, of whom 77.0% saved them, 4.6% gave them away and 18.4% threw them away. Living in a country where antibiotics are dispensed in fixed packs rather than exact numbers of pills as well as believing that leftover antibiotics can be saved and used again were the strongest predictors for possession of leftovers. There was also a marked detrimental effect of lack of information from the doctor and/or pharmacist. This investigation suggests that dispensing of antibiotics in exact numbers of doses should be recommended in addition to the development of relevant information campaigns addressing patients' false beliefs about leftovers and the provision of basic information about the importance of completing antibiotic treatment.

Non-compliance with antibiotic therapy for acute community infections: a global survey.

A global patient survey of non-compliance with antibiotic therapy for acute community infections included 4514 adult respondents (aged 18-99 years) in 11 countries. Admitted non-compliance (ANC) was reported in 912/4088 (22.3%) of cases but varied widely between countries. Multivariate analysis identified five independent variables associated with ANC: country, daily dosage regimen, age, attitudes to doctors and attitudes to antibiotics. ANC ranged from 44.0% in China to 9.9% in The Netherlands, and from 14.9% in those prescribed once-daily regimens to 27.0% for three or more daily doses. There was a negative correlation between ANC and age. Analysis of the attitudes to doctors confirmed previous findings that involving the patient in the management of their infection can improve overall compliance. The study identified seven key attitudes to antibiotic use with the potential to improve compliance. However, there was a poor understanding in 10 of the 11 countries of how non-compliance can increase the potential for resistance development. Segmentation and cluster analysis identified four psychographic profiles influencing compliant behaviour, which varied across countries. The global picture of antibiotic non-compliance and psychographic profiling should help identify areas for targeted, country-specific patient educational programmes as well as those areas where physicians can improve their interaction with their patients.

Regulatory effects of macrolides on bacterial virulence: potential role as quorum-sensing inhibitors.

Pseudomonas aeruginosa is an opportunistic pathogen, and this organism is a major cause of pulmonary damage and mortality in patients with cystic fibrosis (CF), diffuse panbronchiolitis (DPB) and other forms of bronchiectasis. A break-through in the treatment of DPB and associated chronic P. aeruginosa pulmonary infection was realized when a patient with DPB improved dramatically after treatment with erythromycin for years. Now, long-term macrolide therapy has become a first line of treatment in DPB patients, and the immunomodulatory properties have now been extended to other clinical settings, including CF. An important factor in the pathogenesis of chronic P. aeruginosa infection is a bacterial cell-to-cell signaling mechanism, referred to as "quorum sensing", which enables bacteria to coordinately turn on and off specific virulence genes through the production of autoinducer molecules. Interference or blocking of quorum-sensing systems has been considered an attractive therapeutic strategy. Clinical and basic science data suggests the potential of macrolides as relevant inhibitors of the Pseudomonas quorum-sensing system. In fact, certain macrolides strongly suppressed quorum-sensing associated genes and autoinducer production, in addition to inhibition of a variety of virulence factors. In this review, clinical efficacy of macrolides on DPB and CF patients will be briefly summarized. Additionally, the mechanisms of action of macrolides will be discussed from the standpoint of sub-MIC macrolide effects on P. aeruginosa, particularly the ability of this antibiotic to suppress quorum-sensing systems, which may be crucial in the pathogenesis of chronic P. aeruginosa infection.

[How bacteria resist antibiotics: a primary form of collective intelligence?]. / Comment les bactéries résistent aux antibiotiques: une premiere forme d'intelligence collective?

Bacteria produce inhibitory enzymes (beta-lactamases, etc), block antibiotic attachment to target molecules (MRSA, etc.), extrude antibiotics from the cell by active efflux systems (multidrug resistant pseudomonas, etc) or limit antibiotic penetration through the outer membrane in Gram negatives. Genetically, resistance occurs after mutation or horizontal transfers (transformation, transduction, conjugation) of mobile genetic elements (integrons, transposons, phages, plasmids), associated with a risk of epidemic spread. Recent data stress the importance pheromones for facilitating inter-bacterial genetic exchanges. Activated by quinolones and penicillins the SOS response augments the mutation rate (by about 10,000 fold) and liberates mobile genetic elements offering more opportunities to select resistance. These highly pertinent non-darwinian systems raise the hypothesis of a primary form of intelligence developed already 3.8 billions years ago.

[The physician, patient and antibiotics]. / Le médecin, le malade et les antibiotiques.

More than 3.000 randomized patients, who received an antibiotic course for a mild respiratory infection in the last 2 months have been interviewed in 4 European countries about their perceptions of antibiotic therapy and the doctor's skills. Six attitudinal dimensions related to the doctor identified 4 patients type: Involved (30 %), Deferents (23%), Ignored (13%) and Critical (17%). Involved and Deferent patients knew better the rules of good antibiotic use (p<0,01), were more compliant (p<0,01), and received more accurater information from the doctor (p<0,01). Ignored patients keep left over antibiotics for uncontrolled further use most often (p<0,01). A large majority of patients, whatever the category, believed that a flu should be treated with an antibiotic. Germany includes more involved patients, the highest rate of confidence in physician's skills, who was the most informative, but they also had less people knowing the uselessness of antibiotics in flu. Spaniards had more propensity to expect antibiotics from their doctor, showed the lesser level of confidence in their physician's skill, and were the most prone to claim for the benign character of their infection. Critical patients were mostly recruited in France and Italy which also includes the highest rate of ignored patients. French patients were by far the less likely to receive accurate information from their physician. In conclusion, an actual educational deficit has been found in the patients regarding antibiotic use. The physician is in the best position for correcting the deficit. By implicating more the patients in the medical decision, he or she will deflate the ignored category, the most likely to misuse antibiotics, and hence to produce antibiotic resistance.

The Pseudomonas aeruginosa autoinducer N-3-oxododecanoyl homoserine lactone accelerates apoptosis in macrophages and neutrophils.

Quorum-sensing systems are critical regulators of the expression of virulence factors of various organisms, including Pseudomonas aeruginosa. Las and Rhl are two major quorum-sensing components, and they are regulated by their corresponding autoinducers, N-3-oxododecanoyl homoserine lactone (3-oxo-C(12)-HSL) and N-butyryl-L-homoserine lactone (C(4)-HSL). Recent progress has demonstrated the potential of quorum-sensing molecules, especially 3-oxo-C(12)-HSL, for modulation of the host immune system. Here we show the specific ability of 3-oxo-C(12)-HSL to induce apoptosis in certain types of cells. When bone marrow-derived macrophages were incubated with synthetic 3-oxo-C(12)-HSL, but when they were incubated not C(4)-HSL, significant loss of viability was observed in a concentration (12 to 50 micro M)- and incubation time (1 to 24 h)-dependent manner. The cytotoxic activity of 3-oxo-C(12)-HSL was also observed in neutrophils and monocytic cell lines U-937 and P388D1 but not in epithelial cell lines CCL-185 and HEp-2. Cells treated with 3-oxo-C(12)-HSL revealed morphological alterations indicative of apoptosis. Acceleration of apoptosis in 3-oxo-C(12)-HSL-treated cells was confirmed by multiple criteria (caspases 3 and 8, histone-associated DNA fragments, phosphatidylserine expression). Structure-activity correlation experiments demonstrated that the fine structure of 3-oxo-C(12)-HSL, the HSL backbone, and side chain length are required for maximal activity. These data suggest that Pseudomonas 3-oxo-C(12)-HSL specifically promotes induction of apoptosis, which may be associated with 3-oxo-C(12)-HSL-induced cytotoxicity in macrophages and neutrophils. Our data suggest that the quorum-sensing molecule 3-oxo-C(12)-HSL has critical roles in the pathogenesis of P. aeruginosa infection, not only in the induction of bacterial virulence factors but also in the modulation of host responses.

Amino acid residues essential for function of the MexF efflux pump protein of Pseudomonas aeruginosa.

At least four broad-spectrum efflux pumps (Mex) are involved in elevated intrinsic antibiotic resistance as well as in acquired multidrug resistance in Pseudomonas aeruginosa. Substrate specificity of the Mex pumps has been shown to be determined by the cytoplasmic membrane component (MexB, MexD, MexF, and MexY) of the tripartite efflux pump system. Alignment of their amino acid sequences with those of the homologous AcrB and AcrD pump proteins of Escherichia coli showed conservation of five charged amino acid residues located in or next to transmembrane segments (TMS). These residues were mutated in the MexF gene by site-directed mutagenesis and replaced by residues of opposite or neutral charge. MexF proteins containing combined D410A and A411G substitutions located in TMS4 were completely inactive. Similarly, the substitutions E417K (next to TMS4) and K951E (TMS10) also caused loss of activity towards all tested antibiotics. The substitution E349K in TMS2 resulted in a MexF mutant protein which was unable to transport trimethoprim and quinolones but retained partial activity for the transport of chloramphenicol. All mutated MexF proteins were expressed at comparable levels when tested by Western blot analysis. It is concluded that charged residues located in or close to TMS are essential for proper function of MexF.

Detection of Pseudomonas aeruginosa cell-to-cell signals in lung tissue of cystic fibrosis patients.

Chronic Pseudomonas aeruginosa infections lead to progressive lung tissue destruction in cystic fibrosis (CF) patients. Two bacterial cell-to-cell signals, 3-oxo-C(12)-HSL and C(4)-HSL are required for the production of several extracellular virulence factors. 3-oxo-C(12)-HSL is also required for the development of a differentiated biofilm, induces IL-8 production by epithelial cells and possesses immunomodulatory activities. These two signalling molecules are therefore believed to play a role in the pathogenesis of P. aeruginosa infections, but have never been isolated from infected human tissues. We extracted and quantified the two P. aeruginosa cell-to-cell signals from lung tissues of two CF patients infected by P. aeruginosa. 3-oxo-C(12)-HSL and C(4)-HSL were detected in the lung tissues in fmol/gram, respectively pmol/gram concentrations; the ratio C(4)-HSL/3-oxo-C(12)-HSL exceeded 100 in all tissue samples. Random Amplified Polymorphism DNA genotyping revealed that one genotype was present per lung. In vitro the P. aeruginosa isolates from the two lungs produced 3-oxo-C(12)-HSL, whereas some isolates did not produce detectable C(4)-HSL. Our results suggest that both P. aeruginosa cell-to-cell signals were produced in the lung tissue of these two cystic fibrosis patients.

Salmonella typhimurium thyA mutants fail to grow intracellularly in vitro and are attenuated in mice.

Salmonella typhimurium ATCC14028 readily multiplies in professional phagocytes in vitro and is highly virulent in mice. Mutants lacking thymidylate synthase activity (thyA) were isolated and shown to be strictly dependent on thymidine monophosphate precursors in the growth medium. The thyA mutants were found to be virtually incapable of intracellular growth and survival in vitro, both in macrophage-like cell line P338D(1) and in the human epithelial cell line Hep-2, and their virulence was impaired in BALB/c mice. Intraperitoneal immunization of mice with two doses of live S. typhimurium thyA provided protection against a challenge with 10(3) times the 50% lethal dose of the virulent parent strain.