Infection by the bacterial opportunist Pseudomonas aeruginosa frequently assumes the form of a biofilm, requiring motility for biofilm formation and dispersal and an ability to grow in nutrient-and oxygen-limited environments. Anaerobic growth by P. aeruginosa is accomplished through the denitrification enzyme pathway that catalyzes the sequential reduction of nitrate to nitrogen gas. Mutants mutated in the two-component nitrate sensor-response regulator and in membrane nitrate reductase displayed altered motility and biofilm formation compared to wild-type P. aeruginosa PAO1. Analysis of additional nitrate dissimilation mutants demonstrated a second level of regulation in P. aeruginosa motility that is independent of nitrate sensor-response regulator function and is associated with nitric oxide production. Because motility and biofilm formation are important for P. aeruginosa pathogenicity, we examined the virulence of selected regulatory and structural gene mutants in the surrogate model host Caenorhabditis elegans. Interestingly, the membrane nitrate reductase mutant was avirulent in C. elegans, while nitrate sensor-response regulator mutants were fully virulent. The data demonstrate that nitrate sensing, response regulation, and metabolism are linked directly to factors important in P. aeruginosa pathogenesis.Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium capable of causing infection in the immunocompromised host. The types of infection caused by P. aeruginosa include otitis media (19), infection of burn wounds (37), and lung infection in cystic fibrosis (CF) patients (27). In many instances, infection by P. aeruginosa assumes the form of a biofilm, which is highly resistant to antibiotics and to attack by immune effector cells (20).P. aeruginosa growth in biofilms is characterized by its ability to grow in nutrient-and oxygen-limited environments. Anaerobic growth by P. aeruginosa is accomplished through a denitrification enzyme pathway that catalyzes a four-step sequential reduction of nitrate to nitrogen gas, with nitrite, nitric oxide, and nitrous oxide, respectively, as intermediates. Two different nitrate reductase complexes mediate nitrate reduction to nitrite in P. aeruginosa (4, 68), a plasma membrane-bound nitrate reductase complex encoded by the narK1K2GHJI operon and a periplasmic nitrate reductase encoded by napEFDABC. Reduction of either nitrate or nitrite substrates provides energy for P. aeruginosa anaerobic growth, with nitrate reduction to nitrite via nitrate reductase contributing more significantly to proton motive force and hence energy production (4, 68). A well-described environment for P. aeruginosa growth under anoxic conditions is as a biofilm within the mucus of the CF lung (66). Nitrate and nitrite levels in CF mucus, generated in part by the host inflammatory response to infection, are sufficient to support anaerobic metabolism of P. aeruginosa (27).Biofilm formation and organism dispersal leading to spread of infection by P. aeruginosa are dependent on motility. ...

Pseudomonas aeruginosa is a gram-negative, opportunistic pathogen and a significant cause of acute and chronic infections in patients with compromised host defenses. Evidence suggests that within infections P. aeruginosa encounters oxygen limitation and exists in microbial aggregates known as biofilms. However, there is little information that describes genes involved in anaerobic growth of P. aeruginosa and their association with virulence of this pathogen. To identify genes required for anaerobic growth, random transposon (Tn) mutagenesis was used to screen for mutants that demonstrated the inability to grow anaerobically using nitrate as a terminal electron acceptor. Of approximately 35,000 mutants screened, 57 mutants were found to exhibit no growth anaerobically using nitrate. Identification of the genes disrupted by the Tn revealed 24 distinct loci required for anaerobic growth on nitrate, including several genes not previously associated with anaerobic growth of P. aeruginosa. Several of these mutants were capable of growing anaerobically using nitrite and/or arginine, while five mutants were unable to grow anaerobically under any of the conditions tested. Three mutants were markedly attenuated in virulence in the lettuce model of P. aeruginosa infection. These studies have identified novel genes important for anaerobic growth and demonstrate that anaerobic metabolism influences virulence of P. aeruginosa.

Objective. To determine the feasibility and effectiveness of adding a hand hygiene exercise in selfscreening for Methicillin-Resistant Staphylococcus Aureus (MRSA) nasal colonization to a health care delivery course for first-year pharmacy (P1) students. Design. About one month after students were trained in hand hygiene technique and indications, faculty members demonstrated how to self-screen for MRSA nasal colonization. Students were then asked to screen themselves during the required class time. Aggregated class results were shared and compared to prevalence estimates for the general population and health care providers. Assessment. The 71 students present in class on the day of the self-screening exercise chose to participate. A survey comparing presecreening and postscreening responses indicated incremental improvements in student knowledge and awareness of health care associated infections and motivation to perform hand hygiene. On the written exam, student performance demonstrated improved knowledge compared to previous class years. Conclusion. Self-screening for MRSA nasal colonization in a health care delivery course for P1 students increased students' motivation to perform hand hygiene techniques and follow indications promulgated by the World Health Organization.

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