Biofilm dispersion is a survival mechanism where bacteria transition to a planktonic lifestyle. Dispersion is not characterized in Enterococcus faecalis (E. faecalis), a human intestinal tract bacterium. In semi-batch culture experiments, we determined steady state biofilms formed after 48 hours when E. faecalis was grown in 10% Tryptic Soy Broth (TSB) at 37oC without shaking. Next, we challenged biofilm cultures with a 10-fold step increase in nutrients after 4 days of growth to avoid interference from self-induced dispersion and found a statistically significant dispersion response. To evaluate the effect of bile acids on nutrient-induced dispersion, we exposed dispersed cells to the primary bile acid cholic acid or the secondary bile acid lithocholic acid. Lithocholic acid retained biofilm growth during nutrient-induced dispersion and thus may cause increased biofilm formation and preservation in the gut. To evaluate E. faecalis antibiotic tolerance, we conducted dose finding assays with the oxazolidinone linezolid and glycopeptide vancomycin. Mid-log cultures were grown for 18 hours in 100% TSB and shaken prior to treatment. E. faecalis displayed antibiotic tolerance when treated with 50 µg/ml of linezolid, resulting in a 0.5 log reduction. Our study has revealed novel findings on the growth and dispersion response of E. faecalis biofilms.
Clostridioides difficile is a virulent pathogen known to be susceptible to vancomycin, although less is known about how microbial interactions might influence the effectiveness of antibiotics. Here we test how the susceptibility of C. difficile to vancomycin is affected when in co-culture with Vancomycin-resistant Enterococcus faecium (VRE). We show that the in vitro co-culture of C. difficile with VRE reduces C. difficile’s susceptibility to vancomycin. In Brain Heart Infusion (BHI) media, susceptibility is reduced compared to Sporulation Media (SM), suggesting different environments affect antibiotic effectiveness. These findings contribute to our understanding of how microbial interactions and the environment affects antibiotic effectiveness.
Pseudomonas aeruginosa (Pa) is an opportunistic pathogen that causes severe infections in immunocompromised individuals, including those with cystic fibrosis (CF). Pa forms antibiotic-resistant biofilms, making infections difficult to treat and often lethal. In the CF lung, elevated Ca2+ concentrations are encountered by Pa, which our group has shown to induce virulence factor production. To understand the molecular mechanisms behind this regulation, we identified the novel inner membrane protein CarP, which plays a crucial role in Ca2+-induced pyocyanin production, oxidative stress tolerance, Ca2+ homeostasis, and host immune interactions. Although CarP’s molecular function remains unknown, we hypothesize it interacts with other proteins. To test this, we utilized a FLAG tag co-immunoprecipitation approach, generating two expression constructs: membrane-bound and periplasmic CarP, each with a 3xFLAG tag fused to the C-terminus. Pull-down assays will be performed with each construct, and periplasmic and cytoplasmic samples will be analyzed by mass spectrometry to identify CarP’s protein-binding partners. This research will provide important insights into CarP’s molecular function and the mechanisms of Ca2+-induced virulence in Pa.
The Hippo signaling pathway controls the regulation of organ growth and development. Dysfunctions of this pathway are related to various types of cancers, making it a critical area of study for cancer research and the development of regenerative medicines. This study investigates the manipulation of gene Minibrain (mnb). Mnb is a kinase that phosphorylates other proteins. Understanding the function of Mnb will ultimately allow us to understand its role within the Hippo pathway. Mnb is thought to affect the function and localization of the kinase Warts (Wts), which determines Yorkie (Yki) activation and consequently the outcomes of the Hippo pathway. (Fig. 3) We hypothesize that high levels of mnb will cause overgrowth and affect Wts localization, and vice versa. We employed gene manipulation techniques using the UAS/Gal 4 system (Fig. 1). Using immunofluorescence and confocal microscopy, we analyzed the effects of different levels of mnb on Yki. Additionally, we assessed the phenotypic consequences of the varying levels of mnb on wing growth. These findings will guide further exploration of the Hippo signaling pathway and its broader implications.
