The majority of bacterial infections involve the formation of biofilms. Biofilm formation is nutrient and growth dependent. Determination of the effects of nutrients on exopolysaccharide production and bacterial growth is labor and time intensive. We tested whether the Bioscreen C (Growth Curves, Inc.) would have utility as a high-throughput tool in the measurement of fundamental phenotype expression, as it relates to growth conditions. Within 48 -72 hr, reproduceble, statistically significant data on the affects of growth conditions on generation time, capsule production and biofilm formation (maximally for 25 different conditions per 24 hr run cycle; n = 4) were obtained. Although all S. aureus strains produced similar amounts of capsule, sarA -and agr -strains grew significantly slower than parent strain (1.6 fold slower) and produced significantly (p < 0.05) less biofilm (~2 fold). E. coli growth rate, biofilm and capsule production in simulated nephropathic urine medium was similar for urine with insulin (20 µU). Addition of insulin to urine medium with proline increased generation time, capsule and biofilm production. Findings from this study show that the Bioscreen C is a rapid, reproducible, and easily manipulated system to concurrently measure bacterial growth, biofilm formation, and capsule production. In addition, there is the potential for further applications of this system by expanding the types of detector dyes used.
During infection, Staphylococcus aureus is exposed to exogenous menaquinone which is essential for the human blood clotting cascade. The effect of exogenous menaquinone on S. aureus phenotypic expression is not known. To test whether menaquinone affects expression of virulence-associated phenotypes, methicillin-sensitive (MSSA) and-resistant (MRSA) S. aureus strains (n = 8) were grown in the presence of menaquinone (0.001-12 µg/ml). Capsule production, biofilm formation (plastic and fibronectin-coated microtiter plates) and carotenoid levels were determined spectrophotometrically after growth in Mueller Hinton broth (MH; 24-hr, 37˚C). All experiments were, at minimum, done in triplicate and repeated twice. Menaquinone at physiologic levels (0.01 µg/ml MH) significantly increased (p < 0.05) biofilm formation on plastic in a manner that was bacterial population size dependent. In addition, menaquinone (0.05-4 µg/ml) significantly increased (p < 0.05) biofilm formation on fibronectin-coated surfaces for four MSSA strains and one MRSA strain by two to six-fold as compared to medium controls. However, menaquinone had no effect on capsule production or cell-associated carotenoid levels. Menaquinone's effect on biofilm formation on fibronectin-coated surfaces appears to be regulated by sarA. These findings are the first to demonstrate that a vitamin at concentrations reported in humans affects S. aureus virulence-associated phenotypes.
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