Quantitative real-time PCR study of the influence of probiotic culture soluble fraction on the expression of Pseudomonas aeruginosa quorum sensing genes

The opportunistic pathogen Pseudomonas (Ps.) aeruginosa causes severe infections, particularly in immunocompromised individuals and patients with cystic fibrosis (CF). A serious side effect of antibiotic therapy in Ps. aeruginosa infections is the development of resistance to antibiotics. During the infection process Ps. aeruginosa forms biofilms, rendering bacterial cells more resistant to disinfectants, antibiotics and the action of host immune defense effectors. Pseudomonas aeruginosa employs the intercellular communication system, known as quorum sensing (QS) to coordinate the expression of tissue-damaging factors. Since the QS systems controls the production of different virulence factors, it is possible that the inhibition of its regulatory activity to severely compromise the ability of Ps. aeruginosa to cause infections in humans. Many studies have shown that some probiotic strains exhibit inhibitory activity on different virulence properties of pathogenic bacteria (adherence to cellular or inert substrate, soluble virulence factors expression). The aim of the present study was to investigate by real-time RT-qPCR the influence of probiotic culture soluble factors on the QS genes expression in 30 Ps. aeruginosa strains isolated from patients hospitalized in the National Institute for Cardiovascular Infections, Prof. C.C. Iliescu Fundeni Hospital, Bucharest. The results of the real time RT-qPCR have shown that in all Ps. aeruginosa strains grown in the presence of probiotic culture sterile filtrates, the level of QS genes expression was reduced comparatively with those from control cultures. In conclusion, these results proved that the inhibition of virulence factors regulation mechanisms by soluble molecules secreted by probiotics could represent an interesting way pathogenicity and virulence attenuation in Ps. aeruginosa nosocomial strains.     

Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system

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.     

Factors influencing the adhesive properties of Pseudomonas aeruginosa

The aim of this study was to evaluate different factors which may influence surface and adhesive properties of Pseudomonas aeruginosa strains isolated from patients with urinary tract infections. P. aeruginosa strains exhibited moderate surface hydrophobicity as shown by "salting out" with ammonium sulfate and hydrophobic interaction chromatography. The ability to haemagglutinate red blood cells by P. aeruginosa strains was increased when the cells were treated with papain and neuraminidase. The ability of all tested strains to attach to plastic and glass surfaces was independent on incubation temperature. There was no significant difference in the ability of any particular P. aeruginosa strains to adhere to Vero cells in culture. In this study no correlation between hydrophobic properties, intensity of haemagglutination, and ability to attach to Vero cells in culture was observed.     

Quorum sensing: dynamic response of Pseudomonas aeruginosa to external signals

A recent study suggests that the opportunistic pathogen Pseudomonas aeruginosa can actively monitor the host immune system. The P. aeruginosa outer membrane protein OprF was found to bind specifically to the cytokine interferon-gamma (IFN-gamma), and this interaction upregulated production of virulence factors through a cell-cell communication system known as quorum sensing (QS). Taken together with previous findings that P. aeruginosa QS can alter the host immune response (e.g. by activation of IFN-gamma), these data illustrate an exciting new element of bacteria-host interactions in which the P. aeruginosa quorum-sensing system both senses and modulates the host immune state.     

Central role of quorum sensing in regulating the production of pathogenicity factors in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic human pathogen, causing various infections that are often very persistent. P. aeruginosa infections are the major cause of death in cystic fibrosis patients. Infections are difficult to treat since P. aeruginosa is resistant to most antibiotics and its antibiotic susceptibility is decreased when it is present in biofilms. P. aeruginosa produces many exoproducts (including toxins and hydrolytic enzymes) that are involved in virulence. Recent research has elucidated many mechanisms and pathways that regulate the production of these virulence factors. The regulation is extremely complex and many components are influenced by environmental conditions. Quorum sensing is a key regulatory system, which itself is affected by many other regulators. Targeting the regulation of pathogenicity factors provides a novel strategy for combating P. aeruginosa infections. Degradation of acyl homoserine lactones, the signaling molecules of the quorum-sensing system, is a promising therapeutic treatment option.     

A bacterial cell to cell signal in the lungs of cystic fibrosis patients

Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of mortality in cystic fibrosis (CF) patients. This bacterium has numerous genes controlled by cell to cell signaling, which occurs through a complex circuitry of interconnected regulatory systems. One of the signals is the Pseudomonas Quinolone Signal (PQS), which was identified as 2-heptyl-3-hydroxy-4-quinolone. This intercellular signal controls the expression of multiple virulence factors and is required for virulence in an insect model of P. aeruginosa infection. Previous studies have implied that the intercellular signals of P. aeruginosa are important for human disease, and our goal was to determine whether PQS was produced during human infections. In this report, three types of samples from CF patients infected with P. aeruginosa were analyzed for the presence of PQS. Sputum, bronchoalveolar lavage fluid, and mucopurulent fluid from distal airways of end-stage lungs removed at transplant, all contained PQS, indicating that this cell to cell signal is produced in vivo by P. aeruginosa infecting the lungs of CF patients.     

7-fluoroindole as an antivirulence compound against Pseudomonas aeruginosa

The emergence of antibiotic resistance has necessitated new therapeutic approaches for combating persistent bacterial infection. An alternative approach is regulation of bacterial virulence instead of growth suppression, which can readily lead to drug resistance. The virulence of the opportunistic human pathogen Pseudomonas aeruginosa depends on a large number of extracellular factors and biofilm formation. Thirty-one natural and synthetic indole derivatives were screened. 7-fluoroindole (7FI) was identified as a compound that inhibits biofilm formation and blood hemolysis without inhibiting the growth of planktonic P.?aeruginosa cells. Moreover, 7FI markedly reduced the production of quorum-sensing (QS)-regulated virulence factors 2-heptyl-3-hydroxy-4(1H)-quinolone, pyocyanin, rhamnolipid, two siderophores, pyoverdine and pyochelin. 7FI clearly suppressed swarming motility, protease activity and the production of a polymeric matrix in P.?aeruginosa. However, unlike natural indole compounds, synthetic 7FI did not increase antibiotic resistance. Therefore, 7FI is a potential candidate for use in an antivirulence approach against persistent P.?aeruginosa infection.     

Distribution of Pseudomonas aeruginosa in a riverine ecosystem

The distribution of Pseudomonas aeruginosa in navigation pool 8 of the upper Mississippi River was investigated by acetamide broth enrichment of water, sediment, and swab (solid-water interface) samples. Among the 152 P. aeruginosa isolates, serological type 1 was most prevalent (34.2%), and a small number (13.2%) showed carbenicillin resistance. Pigmentation was variable, with only 44.7% elaborating typical blue-green pigment. P. aeruginosa was most commonly isolated from sediment, with solid-water interfaces (aufwuchs samples) also exhibiting high frequencies of isolation. Current velocity, oxygen and nutrient availability, surface tension, desiccation, and negative phototropism were important factors in the riverine distribution of this epibacterium.     

The Action of Ethylenediaminetetra-acetic Acid on Pseudomonas aeruginosa

It has been shown that ethylenediaminetetra-acetic acid (EDTA) has a direct bactericidal action against strains of Pseudomonas aeruginosa and Alcaligenes faecalis . The action against Ps. aeruginosa is considered to take the form of competition for a metal essential to the integrity of the cells: some of the factors influencing this antibacterial action have been discussed. From a study of the release of solutes from cells of Ps. aeruginosa , it was concluded that the action of EDTA takes place at the cell wall of the organism: an effect by EDTA on the isolated walls of sensitive organisms has been demonstrated.     

Distribution of Pseudomonas aeruginosa in a riverine ecosystem.

The distribution of Pseudomonas aeruginosa in navigation pool 8 of the upper Mississippi River was investigated by acetamide broth enrichment of water, sediment, and swab (solid-water interface) samples. Among the 152 P. aeruginosa isolates, serological type 1 was most prevalent (34.2%), and a small number (13.2%) showed carbenicillin resistance. Pigmentation was variable, with only 44.7% elaborating typical blue-green pigment. P. aeruginosa was most commonly isolated from sediment, with solid-water interfaces (aufwuchs samples) also exhibiting high frequencies of isolation. Current velocity, oxygen and nutrient availability, surface tension, desiccation, and negative phototropism were important factors in the riverine distribution of this epibacterium.     

Identification of RegA protein from Pseudomonas aeruginosa using anti-RegA antibody

The product of Pseudomonas aeruginosa regA gene acts as a positive regulator of exotoxin A expression. The protein, RegA, was overproduced in E. coli transformed with an expression vector containing the regA gene. The overproduced RegA accumulated in E. coli in the form of inclusion bodies. The latter were isolated and served as a source of antigen for raising polyclonal antibodies. The antibodies reacted specifically with a P. aeruginosa protein whose molecular weight corresponded to that predicted for RegA from its known DNA sequence, and whose response to modulating factors matched that expected for RegA. The immunodetectable RegA was localized in the membrane fraction of P. aeruginosa strain PA103.     

Biocalorimetric studies of the metabolic activity of Pseudomonas aeruginosa aerobically grown in a glucose-limited complex growth medium

Biocalorimetric experiments were performed to investigate the aerobic growth of Pseudomonas aeruginosa, isolated from tannery saline wastewater. Growth factors (pH, Inoculum size, carbon source, temperature, aeration rate, and agitation rate) were optimized in shaker and calorimeter based on the growth of P. aeruginosa and heat generation rates. A limiting value of 0.2% glucose concentration was found to be optimum for the growth of P. aeruginosa in a complex growth medium, and the heat flux (q(r)) profiles resulting from the metabolic activity of P. aeruginosa further confirmed this observation. The bacterial growth profile was found to correlate well with the metabolic heat generated. Heat-yield values were calculated for both glucose consumption and the growth of P. aeruginosa from the calorimetric results. Metabolic shifts in substrate uptake from glucose to peptone present in growth medium was observed by the variations in heat-flux profile. The calorimetric data presented in this study should be useful in understanding the behavior of the isolated bacterial strain in degrading complex and mixed substrates commonly observed in tannery saline waste stream, and further to extend the results for scale-up studies.     

Elucidating the molecular mechanisms of bacterial virulence using non-mammalian hosts

Several strains of the human opportunistic pathogen Pseudomonas aeruginosa infect plants, nematodes and insects. Our laboratory has developed a multihost pathogenesis system based on the P. aeruginosa clinical isolate PA14, in which non-mammalian hosts are used to screen directly for virulence-attenuated mutants. The majority of PA14 mutants isolated using non-mammalian hosts also displayed reduced virulence in a burned mouse model. Surprisingly, only a few host-specific virulence factors were identified, and many of the P. aeruginosa mutants were attenuated in virulence in all the hosts. These studies illustrate the extensive conservation in the virulence mechanisms used by P. aeruginosa to infect evolutionarily diverged hosts, and validate the multihost method of screening for virulence factors relevant to mammalian pathogenesis. Through the use of genetically tractable hosts, the multihost pathogenesis model also provides tools for elucidating host responses and dissecting the fundamental molecular interactions that underlie bacterial pathogenesis.     

惰性材料表面细菌生物膜构建的研究

目的构建惰性材料塑料输液管内壁细菌生物膜体外模型,观察细菌生物膜的结构,探讨输液管内壁细菌生物膜形成影响因素。方法建立铜绿假单胞菌生物膜和铜绿假单胞菌、肺炎克雷伯菌混合生物膜,分别于培养1、3和7d用扫描电镜动态观察生物膜形成过程。结果混合菌生物膜的生长速度高于铜绿假单胞菌单独成膜。结论输液管是形成细菌生物膜的良好支持材料,混合细菌培养可以加速细菌形成生物膜。     

Expression and purification of Pseudomonas aeruginosa SecA N-terminal domain: stimulation of ATPase activity of the SecAL43P mutant protein

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium which secretes a wide range of hydrolytic enzymes, toxins, and virulence factors into the extracellular medium. Although P. aeruginosa possesses numerous specific systems for the export of proteins across its double-membrane envelopes, the Sec system is still the major and essential mechanism. However, very little is known about its molecular basis. We constructed, cloned, and expressed the N-terminal 236 amino acids of PaSecA domain (PaSecAN236), and SecAL43P mutants of P. aeruginosa in Escherichia coli BL21.19 (secA(ts)). Here, we describe the purification of PaSecAN236 by using osmotic shock as the first step to efficiently release targeted protein from cells, followed by cation-exchange and size exclusion columns to obtain homogeneous PaSecAN236. The purified PaSecA N-terminal domain was functional in stimulating the ATPase activity of mutant SecAL43P protein of P. aeruginosa.     

Interspecies communication between Burkholderia cepacia and Pseudomonas aeruginosa

Burkholderia cepacia and Pseudomonas aeruginosa are opportunistic pathogens that commonly cause pulmonary infections in cystic fibrosis patients and occasionally co-infect patients' lungs. Both organisms possess quorum-sensing systems dependent on N-acyl homoserine lactone (N-acyl-HSL). Cross-feeding assays demonstrated that P. aeruginosa and B. cepacia were able to utilize heterologous N-acyl-HSL signaling molecules. The ability of quorum-sensing genes from one species to complement the respective quorum-sensing mutations in the heterologous species was also examined. These studies suggest that B. cepacia CepR can use N-acyl-HSLs synthesized by RhlI and LasI and that P. aeruginosa LasR and RhlR can use N-acyl-HSLs synthesized by CepI. It is possible that a mixed bacterial population of B. cepacia and P. aeruginosa can coordinately regulate some of their virulence factors and influence the progression of lung disease due to infection with these organisms.     

Characterization of pseudomonads isolated from diseased fleece

A total of 59 Pseudomonas isolates was obtained from 11 samples of diseased fleece taken from live sheep. All but four of the isolates could be assigned to one of nine Pseudomonas species, of which P. aeruginosa, P. alcaligenes, P. mendocina , and P. putida were the most common. P. aeruginosa was found in four of the fleece samples and, when present, appeared to predominate. Although several of the isolates of P. aeruginosa lacked the ability to produce pyocyanin (and some produced neither pyocyanin nor fluorescein), nearly all produced several virulence factors. Of the other pseudomonads, many produced proteinase, esterase, and catalase, several were able to grow at 42 degrees C and reduce nitrate, and some also produced lipase and hemolysin and, like P. aeruginosa, might serve to initiate (or sustain) the dermatitis frequently associated with fleece rot in sheep.