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Item Coupon position does not affect Pseudomonas aeruginosa and Staphylococcus aureus biofilm densities in the CDC biofilm reactor(Elsevier BV, 2024-08) Buckner, Elizabeth; Buckingham-Meyer, Kelli; Miller, Lindsey A.; Parker, Albert E.; Jones, Christopher J.; Goeres, Darla M.The CDC Biofilm Reactor method is the standard biofilm growth protocol for the validation of US Environmental Protection Agency biofilm label claims. However, no studies have determined the effect of coupon orientation within the reactor on biofilm growth. If positional effects have a statistically significant impact on biofilm density, they should be accounted for in the experimental design. Here, we isolate and quantify biofilms from each possible coupon surface in the reactor to quantitatively determine the positional effects in the CDC Biofilm Reactor. The results showed no statistically significant differences in viable cell density across different orientations and vertical positions in the reactor. Pseudomonas aeruginosa log densities were statistically equivalent among all coupon heights and orientations. While the Staphylococcus aureus cell growth showed no statistically significant differences, the densities were not statistically equivalent among all coupon heights and orientations due to the variability in the data. Structural differences were observed between biofilms on the high-shear baffle side of the reactor compared to the lower shear glass side of the reactor. Further studies are required to determine whether biofilm susceptibility to antimicrobials differs based on structural differences attributed to orientation.Item Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle(Elsevier BV, 2024-06) Barbosa, Ana; Azevedo, Nuno F.; Goeres, Darla M.; Cerqueira, LauraThere has been considerable discussion regarding the environmental life cycle of Legionella pneumophila and its virulence potential in natural and man-made water systems. On the other hand, the bacterium's morphogenetic mechanisms within host cells (amoeba and macrophages) have been well documented and are linked to its ability to transition from a non-virulent, replicative state to an infectious, transmissive state. Although the morphogenetic mechanisms associated with the formation and detachment of the L. pneumophila biofilm have also been described, the capacity of the bacteria to multiply extracellularly is not generally accepted. However, several studies have shown genetic pathways within the biofilm that resemble intracellular mechanisms. Understanding the functionality of L. pneumophila cells within a biofilm is fundamental for assessing the ecology and evaluating how the biofilm architecture influences L. pneumophila survival and persistence in water systems. This manuscript provides an overview of the biphasic cycle of L. pneumophila and its implications in associated intracellular mechanisms in amoeba. It also examines the molecular pathways and gene regulation involved in L. pneumophila biofilm formation and dissemination. A holistic analysis of the transcriptional activities in L. pneumophila biofilms is provided, combining the information of intracellular mechanisms in a comprehensive outline. Furthermore, this review discusses the techniques that can be used to study the morphogenetic states of the bacteria within biofilms, at the single cell and population levels.