Influence of Bacterial Outer Membrane Vesicles on Struvite Crystal Growth
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Urinary tract infections (UTIs) are a very serious health concern, affecting millions of people each year. They lead to almost 10 million doctor’s visits and hundreds of thousands of hospital admissions every year in the United States (Mittal 2009). One of the common causatives of UTIs is Pseudomonas aeruginosa, a gram negative, aerobic, rod-shaped bacterium (Balcht 1994). As a potential way to deliver virulence factors, P. aeruginosa secrete outer membrane vesicles (OMV), which are formed through direct interaction of Pseudomonas quinolone signal (PQS) with the lipopolysaccharide component of the outer membrane (Schertzer 2012, Mashburn-Warren et. al. 2008). Strains used in this research have mutations in these genes in the pathway that result in the inhibition of the formation of OMV. Commonly seen in bacterial urinary tract infections is the presence of struvite crystals. These crystals precipitate in alkaline urine, and eventually form larger stones. Alkaline urine is caused by the hydrolysis of urea to ammonia via the enzyme urease (Le Corre et. al. 2005). The purpose of this research is to study if the presence of outer membrane vesicles produced in P. aeruginosa induce struvite crystal formation. Various strains of P. aeruginosa, with and without OMV, were grown in BHI (Brain Heart Infusion agar) and suspended in artificial urine. Since P. aeruginosa lacks urease, the urease action was mimicked by titrating artificial urine with ammonium hydroxide, inducing struvite crystal formation. The presence and shape (crystal habit) of struvite crystals was confirmed via phase contrast microscopy. Transmission electron microscopy (TEM) was used to confirm the presence of OMV. When crystals were measured using imaging flow cytometry, there was no significant difference in crystal numbers in intact and fragmented P. aeruginosa cells regardless of the presence or absence of OMV; although the number of crystals formed was elevated in controls lacking bacteria. This in vitro data suggests that P. aeruginosa, regardless of OMV production, does not enhance struvite formation in artificial urine.