• Clinical and Therapeutic Implications of Biofilm-associated Fungal-Bacterial Interactions: Candida albicans and Staphylococcus aureus

      Kong, Eric Fei; Jabra-Rizk, Mary Ann (2017)
      Biofilm-associated polymicrobial infections, particularly those involving fungi and bacteria, are responsible for significant morbidity and mortality and tend to be challenging to treat. Candida albicans and Staphylococcus aureus are considered leading microbial pathogens primarily due to their ability to form biofilms on indwelling medical devices. However, the impact of mixed species biofilm growth on therapy remains largely understudied. Here, we demonstrate that in mixed biofilms, C. albicans confers S. aureus significantly enhanced tolerance to antimicrobials mediated by impairment of drug diffusion through the biofilm matrix composed of C. albicans secreted fungal cell wall polysaccharides. Further, we demonstrated a key role for the C. albicans secreted quorum sensing molecule farnesol in modulating S. aureus response to therapy via modulation of drug efflux pumps. Importantly, farnesol was also found to inhibit the production of the carotenoid pigment staphyloxanthin, an important virulence factor in S. aureus involved in protection against oxidative stress. The significance of this inhibition was established through demonstration of the enhanced susceptibility of the depigmented S. aureus cells to oxidants and macrophage phagocytic killing. Theoretical computational binding models indicated that the mechanism for pigment inhibition may be due to farnesol competitively inhibiting the binding of farnesyl diphosphate to CrtM, an essential enzyme in the biosynthesis of staphyloxanthin. To begin to explore the implications of this fungal-bacterial interactions in a host, we utilized a clinically relevant subcutaneous catheter mouse model where central venous catheter segments infected in vitro with S. aureus and C. albicans individually or in combination, are implanted subcutaneously in mice. Vancomycin treatment of mice with infected catheters demonstrated that where therapy significantly reduced S. aureus recovery from catheters in mice infected with S. aureus, in co-infected animals, vancomycin had no impact on S. aureus recovery, underscoring the therapeutic implications of mixed biofilm-associated infections. The combined findings from this work provide lacking mechanistic insights into interspecies interactions in biofilm with great clinical relevance. The understanding of the interspecies dynamics of signaling central to the persistence and antimicrobial resistance of polymicrobial infections will greatly aid in overcoming limitations of current therapies and in designing novel therapeutic strategies to target these complex infections. Therefore, future research should focus on designing animal model systems to study the role of secreted quorum sensing molecules in mediating these interactions in vivo, and the impact of these interactions on pathogenesis.
    • Confirmation of vaccine candidate expression in multiple Staphylococcus aureus strains

      Muench, Anna Rachael; Shirtliff, Mark (2012)
      Staphylococcus aureus has emerged as an important pathogen due to its ability to form persistent infections through the biofilm mode of growth. S. aureus is responsible for thousands infections per year, which include a vast array of diseases and is rapidly developing the ability to become antibiotic resistant resulting in failure of our current methods of treatment. This antibiotic resistance is increased during biofilm formation when the bacteria form a polysaccharide matrix, which prevents infection clearance through antimicrobial means or by the host immune response. This project analyzed 14 Staphylococcus aureus strains and one Staphylococcus epidermidis strain obtained from NARSA (Network on Antimicrobial Resistance in Staphylococcus aureus) detecting the genomic presence and expression of three previously identified immunogenic proteins - glucosaminidase, SA0486 (a hypothetical lipoprotein), and SA0688 (an ABC transporter lipoprotein) - which have been incorporated into a vaccine described by Brady et al 2011. The strains were analyzed for the presence of the three specific genes through PCR and gel electrophoresis and for the expression of these proteins under biofilm growth conditions through western blot analysis. All strains contain the genetic code for these antigens and express at least 2 of the 3 antigens under biofilm growth conditions. Two strains were further analyzed for expression of these antigens under planktonic growth at 2.5hrs, 5hrs, and 24hrs comparing them to biofilm growth utilizing western blot analysis. These strains showed varying expression of the antigens under planktonic conditions with SA0688 expressed under all growth conditions in both strains, glucosaminidase with a gradual increase in expression as growth time increase in both strains, and SA0486 showing different expression patterns in the two strains but with the highest level at 5hrs and 24hrs. To truly understand the expression patterns under planktonic growth, further analysis will be required. The data generated provided the evidence of the universal nature of the vaccine previously developed in the Shirtliff laboratory described in Brady et al 2011.
    • Development of a New Generation of Dental Rechargeable Nanocomposites with Anti-caries Properties

      Al Dulaijan, Yousif Ali; Xu, Huakun H. (2018)
      Dental composites are popular for tooth cavity restorations due to their aesthetics, conservative approach, and direct-filling abilities. However, composite restorations have limited lifetime due to several limitations, including secondary caries, fracture, minimal abrasion and wear resistance and higher marginal leakage. Indeed, secondary caries is the primary reason for composite restoration failure. Besides, several studies have shown that conventional dental composites accumulate more biofilms/plaque when compared to other restorative materials. Therefore, this dissertation aims to develop a new generation of dental composites with antibacterial effects, protein-repellent activities, and remineralization properties. Recently, a rechargeable composite was developed, but this composite has no antibacterial or protein-repellent activities. In this dissertation projects, the nanoparticles of amorphous calcium and phosphate (NACP) as remineralizing agent, dimethylaminohexadecyl methacrylate (DMAHDM) as an antibacterial monomer, and 2- metha-cryloyloxyethyl phosphorylcholine (MPC) as a protein-repellent agent were incorporated into the rechargeable composite for the first time. Mechanical properties of the new nanocomposites were evaluated. The characterization of protein adsorption was measured. A human saliva microcosm biofilm model was used to determine biofilm metabolic activity, lactic acid, and colony-forming units (CFU). Calcium (Ca) and Phosphate (P) initial ion release, recharge and re-release were investigated. All rechargeable nanocomposites have good mechanical properties that were compared to those of a commercial composite. The rechargeable nanocomposites containing MPC showed the ability to reduce protein adsorption, as well as the biofilm metabolic activity, lactic acid, and CFU. The rechargeable nanocomposites containing DMAHDM showed strong antibacterial properties through the great inhibition of biofilm metabolic activity and lactic acid, and CFU. The incorporation of bioactive agents did not compromise the Ca and P initial ion release and rechargeability. The release was maintained at the same level with increasing number of recharge cycles, indicating long-term ion release. Therefore, this new generation of rechargeable nanocomposites with long-term Ca and P ion release, antibacterial and protein-repellent activities will provide the needed therapeutic effects to remineralize and strengthen the tooth structures, prolong the restoration longevity, and inhibit secondary caries.
    • Implications of underperforming light energy delivery for posterior composite: insights into delivered radiant exposure, degradation characteristics, and biofilm formation.

      Maktabi, Haifa; Melo, Mary Anne (2018)
      Poor curing of composites due to incorrect position of a curing unit may trigger a negative pathway of events related to the bacterial growth-stimulating effect of released methacrylate monomers. An increased bacterial adhesion and biofilm formation as a result of incomplete polymerization may contribute to premature failure of posterior composite via secondary caries formation. The objectives of each chapter are: 1) to review the literature concerning the light curing process and implications for failures on posterior composites (chapter 1), 2) to investigate in vitro how the variations on light curing tip placement and irradiance exposure can impact the degree of conversion of conventional 2mm-increment composite and S. mutans biofilm formation (chapter 2). In summary, this set of studies support the importance of proper light curing for an acceptable clinical performance of posterior composites.
    • Nanotechnology-Based Dental Materials for Root Caries Management: Design Concepts and Advanced Strategies to Modulate Dysbiotic Patient-derived Oral Biofilms

      Balhaddad, Abdulrahman Abubaker; Melo, Mary Anne; Xu, Huakun H.; 0000-0001-6678-7940 (2021)
      The distinctive challenges associated with root caries demand innovative interventions to preserve the tooth structure and surrounding soft tissues. This dissertation is composed of a set of manuscripts aiming to advance the anti-biofilm approaches to prevent root caries from two perspectives: (i) invasive approach via novel bioactive resin composites, and (ii) non-invasive approach via magnetic field-guided antimicrobial photodynamic therapy (MF-aPDT). The first chapter provided a general introduction concerning the clinical burden of root caries, current treatment modalities, and their limitations. In the second chapter, I provided an overview of contact-killing monomers and bioactive fillers in restorative dentistry. Then, in chapter three, we developed bioactive resin composite formulations containing dimethylaminohexadecyl methacrylate (DMAHDM) antibacterial monomer and 20% nano-sized amorphous calcium phosphate (NACP) and subjected them to a series of mechanical/physical tests and antibacterial assays. We found that the DMAHDM-NACP resin composites were associated with a potent antibacterial action against cariogenic and periodontal biofilms, as 2 to 6-log reduction was observed. Other virulence factors, as lactic acid production, and polysaccharide production, were also reduced. The mechanical properties, physical characteristics, surface features, and polymerization behavior were comparable to the commercial control at baseline testing and after one year of aging. We concluded that the designed bioactive formulations might present a pathway to preven recurrent caries and the onset of periodontal diseases around dental restorations. In chapter four, we reviewed the most recent updates related to the implementation of nanotechnology to enhance antimicrobial photodynamic therapy (aPDT). Then, in chapter five, we investigated the impact of encapsulating superparamagnetic iron oxide nanoparticles (SPIONs) and toluidine blue ortho (TBO) inside a microemulsion, named MagTBO, to enhance the TBO’s penetration and antibacterial action against S. mutans and saliva-derived biofilms. Besides, the ability of magnetic field (MF) navigation to serve as a biofilm penetration strategy was also investigated. The MagTBO microemulsions were synthesized successfully and demonstrated excellent biocompatibility and thermodynamic stabilities. Furthermore, the MagTBO microemulsions demonstrated more remarkable and significant antibacterial action than conventional aPDT, especially when the MF is applied. Thus, this approach can be an adjunctive technique to control dental caries and other oral diseases.
    • Polymicrobial Colonization of Athletic Mouth Guards Following Mechanical Chew Simulation.

      Richards, Samuel Ian; Masri, Radi, 1975- (2015)
      Mouth guard material is used for athletic mouth guards, temporary dental splints, whitening trays, medicinal depositories and catheter tubing. This study evaluated biofilm formation on mouth guard materials after simulated use to ascertain the propensity of mouth guards to harbor bacteria. Three different mouth guards were evaluated (n=18/group): two boil-and-bite guards, Shock Doctor® (SD) Nano 3D and The Wrightguard™ (TWG) and one custom 100% EVA Buffalo guard (EVA). In vitro wear of the fabricated guards were simulated via a chewing simulator for 120,000 cycles per guard. The most visibly worn 8x10mm section of each guard was determined and cut to standardize specimen size. All guard specimens were incubated with equal cell densities of both S. aureus (SA) and Candida albicans (CA) strains and colony-forming units (cells/ml) were measured as a quantification of biofilm growth. A one-way ANOVA with Tukey's-HSD test was used to analyze biofilm retention (CFU/ml) on non-simulated and simulated EVA, SD, and TWG guards. Neither EVA (2.89±1.09 - 3.28±0.74; p=.486) nor TWG (10.22±3.31 - 11.17±5.46; p=.725) groups exhibited a significant increase in CFUs after 6 months of simulated use. In contrast, SD guards (3.33±1.55 - 9.72±5.37; p=.019) exhibited a significant increase in mean CFUs of CA when non-simulated guards were compared to simulated guards. Like with CA, neither EVA (3.11±1.90 - 3.50±1.63; p=.712) nor TWG (9.83±4.09 - 12.56±5.58; p=.358) groups exhibited significant increases in adherent SA following 6 months of simulated use. However, Shock Doctor (5.11±2.20 - 9.67±4.77; p=.06) guards had a significant increase in adherent SA CFUs when non-simulated guards were compared to simulated guards. With chewing simulation parameters remaining constant for all guard types, significantly greater adherence could indicate a greater change in surface topography/roughness from simulated use. While EVA guards exhibited the lowest measured adherence of CA and SA, and TWG exhibited the highest CA and SA levels of adherence, neither of their respective material compositions undergo surface changes as readily as SD guards.The increased propensity of both fungal and bacterial species to adhere to mouth guards after continuous use indicates a potential concern for regular mouth guard users.
    • Toluidine Blue Ortho @ Magnetic Photosensitizing Nanoplatform: Antibiofilm Effect against Thick Constant-depth-film-fermenter Biofilms

      Balhaddad, Abdulrahman A.; Mokeem, Lamia S.; Weir1, Michael D.; Xu, Hockin H. K.; Melo, Mary Anne S. (2022-04-27)
    • Vancomycin tolerance and host responses in Staphylococcus aureus-Candida albicans dual-species biofilm infections

      Allison, Devon Lea; Shirtliff, Mark (2017)
      The polymorphic fungus Candida albicans and gram-positive bacterium Staphylococcus aureus are biofilm-forming organisms commonly found in immunocompromised patients, with each organism ranked by the CDC in the top causes of mortality for US hospitals. Murine models have demonstrated that C. albicans and S. aureus can form a polymicrobial biofilm on epithelial tissue and facilitate systemic infection of both organisms from the oral cavity. While many studies have examined host responses to each organism in mono-species infections, few have sought to determine the impact of dual-species interaction, despite most infections being polymicrobial in nature. In addition to collaborative pathogenesis, previous research has shown S. aureus gains tolerance to the glycopeptide antibiotic vancomycin when co-cultured with C. albicans. However, the mechanism behind this phenomenon has not been well elucidated. We hypothesized that interaction of C. albicans and S. aureus induces specific changes within both organisms that allow for increased antimicrobial tolerance, survival and virulence in dual-species biofilms. To examine this interaction further, we conducted two global transcriptomics studies (in vitro and in vivo) and one lipidomic study to analyze changes in genetic and lipid profiles in pathogen and host. In vitro transcriptomics revealed that genes in C. albicans remained mostly unchanged but numerous genes in S. aureus were differentially expressed. These results paralleled our in vivo transcriptomic analysis in our murine model of oral co-infection. From these results, we tested multiple S. aureus mutants to determine their vancomycin tolerance and found that mutation of a single stress response gene, clpP (proteolytic subunit), abolished acquisition of staphylococcal antimicrobial resistance. This induction of clpP-dependent antimicrobial tolerance was induced by farnesol produced by C. albicans at physiologically relevant concentrations. Finally, we used our murine co-infection model determine if cells of the innate immune system may be involved in systemic S. aureus infection. Organ culture and flow cytometry analysis revealed intracellular S. aureus in labeled macrophages and neutrophils within lymph nodes of only dual-species infected mice. These findings demonstrate the importance of the dual-species biofilm phenotype, its impact on antibiotic treatment, and its modulation of the host immune response to promote infection.