A number of human infections are characterized by the presence of more than one bacterial species and are defined as polymicrobial diseases. Methods for the analysis of the complex biological interactions in mixed infections with a large number of microorganisms are limited and do not effectively determine the contribution of each bacterial species to the pathogenesis of the polymicrobial community. We have developed a novel Drosophila melanogaster infection model to study microbe–microbe interactions and polymicrobe–host interactions. Using this infection model, we examined the interaction of 40 oropharyngeal isolates with Pseudomonas aeruginosa. We observe three classes of microorganisms, one of which acts synergistically with the principal pathogen, while being avirulent or even beneficial on its own. This synergy involves microbe–microbe interactions that result in the modulation of P. aeruginosa virulence factor gene expression within infected Drosophila. The host innate immune response to these natural-route polymicrobial infections is complex and characterized by additive, suppressive, and synergistic transcriptional activation of antimicrobial peptide genes. The polymicrobial infection model was used to differentiate the bacterial flora in cystic fibrosis (CF) sputum, revealing that a large proportion of the organisms in CF airways has the ability to influence the outcome of an infection when in combination with the principal CF pathogen P. aeruginosa.
Individuals with cystic fibrosis (CF) are commonly colonized with Pseudomonas aeruginosa. The chronic infections caused by P. aeruginosa are punctuated by acute exacerbations of the lung disease, which lead to significant morbidity and mortality. As regulators of virulence determinants, P. aeruginosa quorum-sensing systems may be active in the chronic lung infections associated with CF. We have examined the levels of autoinducer molecules and transcript accumulation from the bacterial populations found in the lungs of patients with CF. We detected biologically active levels of N-(3-oxododecanoyl)-L-homoserine (3-oxo-C12-HSL) and N-butyryl-L-homoserine lactone (C4-HSL) in sputum from CF patients. Interestingly, it appears that C4-HSL is less frequently detected than 3-oxo-C12-HSL in the lungs of patients with CF. We also examined the transcription of the autoinducer synthase gene lasI and showed that it is frequently expressed in the lungs of patients with CF. We observed a significant correlation between the expression of lasI and four target genes of the Las quorum-sensing system. Taken together, our results indicate that quorum-sensing systems are active and may control virulence factor expression in the lungs of patients with CF.Quorum-sensing signaling systems allow bacteria to regulate gene expression in a population-dependent manner. Quorumsensing regulatory mechanisms are widespread; they have been described in numerous gram-positive (18) as well as gramnegative (9, 42) bacteria. In acyl-homoserine lactone-based systems, growing bacteria produce small signaling molecules (autoinducers) that accumulate in the surrounding environment. At a specific cell density, the concentration of autoinducer becomes sufficient to interact with the autoinducer-dependent transcriptional activator protein and alter gene expression.In Pseudomonas aeruginosa, quorum-sensing systems have been extensively studied. Two acyl-homoserine lactone-based systems, the las (10) and rhl (25) systems, have been described. These operate in a hierarchical fashion along with a recently described quinolone signaling system (33) to regulate as much as 4% of the genome (45). LasI and RhlI synthesize the autoinducers N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) (29) and N-butyryl-L-homoserine lactone (C4-HSL) (30), respectively. LasR and RhlR bind DNA to modify transcription of target genes only after a threshold level of their respective autoinducers is reached.Quorum-sensing systems are believed to be central to the pathogenesis of P. aeruginosa (reviewed by Rumbaugh et al. [34]). One such infection in which quorum sensing may play an important role is the P. aeruginosa lung infections associated with cystic fibrosis (CF). These infections may be the perfect environment for the expression of the quorum-sensing systems, as the lungs are a spatially limited environment and P. aeruginosa can grow to high densities (10 7 to 10 8 /ml) (40) in sputum. These conditions should be sufficient to induce expression of P. aeruginosa quorum-sens...
Pseudomonas aeruginosa biofilms are intrinsically resistant to antimicrobial chemotherapies. At present, very little is known about the physiological changes that occur during the transition from the planktonic to biofilm mode of growth. The resistance of P. aeruginosa biofilms to numerous antimicrobial agents that are substrates subject to active efflux from planktonic cells suggests that efflux pumps may substantially contribute to the innate resistance of biofilms. In this study, we investigated the expression of genes associated with two multidrug resistance (MDR) efflux pumps, MexAB-OprM and MexCD-OprJ, throughout the course of biofilm development. Using fusions to gfp, we were able to analyze spatial and temporal expression of mexA and mexC in the developing biofilm. Remarkably, expression of mexAB-oprM and mexCD-oprJ was not upregulated but rather decreased over time in the developing biofilm. Northern blot analysis confirmed that these pumps were not hyperexpressed in the biofilm. Furthermore, spatial differences in mexAB-oprM and mexCD-oprJ expression were observed, with maximal activity occurring at the biofilm substratum. Using a series of MDR mutants, we assessed the contribution of the MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY efflux pumps to P. aeruginosa biofilm resistance. These analyses led to the surprising discovery that the four characterized efflux pumps do not play a role in the antibiotic-resistant phenotype of P. aeruginosa biofilms.
We have investigated a potential role for GacA, the response regulator of the GacA/GacS two‐component regulatory system, in Pseudomonas aeruginosa biofilm formation. When gacA was disrupted in strain PA14, a 10‐fold reduction in biofilm formation capacity resulted relative to wild‐type PA14. However, no significant difference was observed in the planktonic growth rate of PA14 gacA−. Providing gacA in trans on the multicopy vector pUCP‐gacA abrogated the biofilm formation defect. Scanning electron microscopy of biofilms formed by PA14 gacA− revealed diffuse clusters of cells that failed to aggregate into microcolonies, implying a deficit in biofilm development or surface translocation. Motility assays revealed no decrease in PA14 gacA− twitching or swimming abilities, indicating that the defect in biofilm formation is independent of flagellar‐mediated attachment and solid surface translocation by pili. Autoinducer and alginate bioassays were performed similarly, and no difference in production levels was observed, indicating that this is not merely an upstream effect on either quorum sensing or alginate production. Antibiotic susceptibility profiling demonstrated that PA14 gacA− biofilms have moderately decreased resistance to a range of antibiotics relative to PA14 wild type. This study establishes GacA as a new and independent regulatory element in P. aeruginosa biofilm formation.
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