• 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.
    • Comparative Microbial Adherence to Various Implant Dental Restorative Materials

      Alshehri, Malek Rofidi A; Masri, Radi, 1975- (2018)
      The oral cavity contains the most complex microbial community of the body, which has more than 700 bacterial species. These microbial species colonize different habitats in the oral cavity. The biological interaction between dental restorative materials and the encompassing oral microbes is one of the most important factors for their clinical prognosis. Many studies have shown that there are distinctive interactions between the rate of microbial formation and the restoration material itself. Currently, there are several Implant Dental Restorative Materials on the market like poly-methyl methacrylate, feldspathic porcelain, dental zirconia, and dental composite resin. The purpose of this study was to comparatively evaluate initial adherence for C. albicans and S. aureus on five different implant dental restorative materials. Ten samples/group were constructed as 5mm x 5mm x 2.5 mm rectangles and were fabricated as per the manufacturer's instructions for each groups. Five groups were made from different types of materials: Polymethyl methacrylate denture material processed with the compression molding technique (PMMA), computer-aided design and computer-aided manufactured dental acrylic (CAD/CAM PMMA), feldspathic porcelain, dental zirconia, and pink dental composite resin. There was a significant difference between the five groups for C. Albicans (F=891.16, p=.0005). CFUs/ml for the pink dental composite resin were significantly higher than the other four groups. However, The dental zirconia group showed the lowest CFUs/ml for initial adherence between all the groups. For S. aureus, CFUs/ml for the pink dental composite resin was significantly higher than the other four groups. Heat polymerized PMMA and the CAD/CAM PMMA group showed significantly higher CFUs/ml than Feldspathic porcelain and dental zirconia groups. In conclusion, there was a significant difference between the five groups for initial adherence of C. albicans and S. aureus. The dental zirconia group showed the lowest initial adherence and the pink dental composite resin group showed the highest initial adherence for both microbes. Within limitation for this study, understanding what is the best for final dental restorative material from aspect of how much it could harbor microbes is an important factor in the final success of treatment and this study investigated this aspect under control conditions.
    • IL-17/Th17 responses and their influence on oral microbial communities

      Abusleme, Loreto; Moutsopoulos, Niki (2017)
      T helper 17 (Th17) cells are key regulators of immunity and inflammation at mucosal surfaces. This T cell subset has been reported to mediate protective responses, but also immunopathology at the oral cavity. However, the requirements for Th17 cells to arise at this site remained minimally explored. Our initial objective was to examine the role of oral microbial communities in local Th17 induction, as the development of these cells had been shown to be determined by commensal bacteria at other barriers. To this end, we established assays to analyze the murine oral microbiome and found that Th17 accumulation occurred independently from microbial colonization at the oral mucosa, underlining the unique properties of each tissue barrier. Th17 cells are relevant mediators in mucosal defense against extracellular bacteria and fungi. Yet, their role in shaping human microbial communities at the oral barrier was not well-understood. To appreciate how Th17 immunity affects the oral microbiome establishment, we performed studies in a patient cohort with Autosomal-Dominant Hyper IgE Syndrome (AD-HIES) that exhibits impaired Th17 differentiation due to loss-of-function mutations in the transcription factor STAT3. We confirmed that AD-HIES patients display a significant susceptibility to oral fungal infections. Our characterization of their mycobiome during active candidiasis revealed a dominance of Candida albicans, alongside a generalized depletion of health-associated bacteria with the exception of Streptococcus, which were overrepresented. These results demonstrate the critical role of Th17 immunity in the containment of C. albicans as a commensal and suggest a synergistic relationship of C. albicans with streptococci, that may influence oral disease susceptibility. Finally, to understand the effects of excessive Th-17/IL-17 inflammatory responses on oral microbial communities, we evaluated patients with Leukocyte Adhesion Deficiency type-I (LAD-I), that present with severe periodontitis driven by IL-17 mediated immunopathology. Our findings revealed that exaggerated IL-17 responses are linked to dysbiosis of subgingival communities. Additionally, IL-23/IL-17 blockade treatment of an LAD-I patient demonstrated reversal of microbial shifts with immune-modulation, suggesting a causative role for IL-17 responses in LAD-associated microbial dysbiosis. In sum, our studies reveal critical roles for the IL-17/Th17 responses in establishment of the oral bacteriome/mycobiome in health and disease.
    • In Vitro and in Vivo Characterization of Candida albicans and Streptococcus mutans Interactions

      Khoury, Zaid; Jabra-Rizk, Mary Ann; 0000-0001-9596-3560 (2019)
      The oral cavity is a complex environment harboring diverse microbial species that often co-exist within biofilms formed on oral surfaces. Within a biofilm, inter species interactions can be synergistic in that the presence of one organism generates a niche for another enhancing colonization. Among these species are the opportunistic fungal pathogen Candida albicans and the bacterial species Streptococcus mutans, the causative microorganisms strongly linked to the development of oral candidiasis and dental caries, respectively. Recent studies have reported the enhanced prevalence of C. albicans in children with early childhood caries indicating that this fungal-bacterial interaction may have clinical implications. In this study, we aimed to elucidate and characterize this interaction between these diverse species. Specifically, we designed in vitro and in vivo studies to validate the hypothesis that the presence of C. albicans in the oral cavity augments S. mutans colonization, potentially mediating dental caries development. Using various C. albicans mutant strains and a GFP-tagged S. mutans, metabolic viability and fluorescent biofilm assays were performed to assess S. mutans recovery from mixed biofilms and to elucidate the mechanisms of interactions. Additionally, to visualize the architecture of formed biofilms, confocal scanning laser fluorescent and electron scanning microscopy were used. Importantly, a clinically relevant mouse model of oral co-infection was developed to demonstrate C. albicans mediated enhanced S. mutans colonization in a host. The findings demonstrated significantly higher recovery of S. mutans from biofilms with C. albicans in vitro. Images revealed a high bacterial affinity to C. albicans, and secreted fungal cell wall polysaccharides were identified as the key factor mediating biofilm formation, particularly mannans. Importantly, analyses of harvested tissue demonstrated significantly higher S. mutans recovery from teeth of co-infected mice compared to mice infected only with S. mutans. Collectively, the findings strongly indicate that the presence of C. albicans in the oral environment may impact the development of dental caries and should be considered as a factor in evaluating the risk of caries. Results obtained in this thesis will support future studies using animal models of dental caries to further characterize this relationship in a closely related model in our laboratory.
    • Interactions Between Candida albicans and Streptococcus mutans in the Oral Cavity

      Al Saud, Hadeel; Jabra-Rizk, Mary Ann (2017)
      The oral microbial communities are some of the most complex microbial floras in the human body. Occurrence of oral disease results from disturbance of the equilibrium of this ecosystem. Dental caries or tooth decay is the most common oral disease particularly in children characterized by irreversible destruction of the tooth structure. These processes are the result of interactions between the various microbial species embedded in the biofilm formed on tooth surface known as dental plaque. The bacterial species Streptococcus mutans has long been considered the etiologic agent of caries, however our in vitro findings demonstrated that S. mutans avidly adheres to C. albicans forming mixed biofilms on abiotic surfaces. As mucosal biofilms are implicated in a wide variety of infections, it is imperative that we understand the implications of the interactions between colonizing microbial species, as they co-exist on host tissue surfaces. To that end, in this project, we developed a clinically relevant mouse model of oral co-infection to investigate the interactions between C. albicans and S. mutans in a host. In this model, experimental animals were orally infected with C. albicans then they were exposed to S. mutans through drinking water, whereas control animals received S. mutans. Four days post infection with C. albicans, animals were euthanized and tongues and teeth were harvested and assessed for microbial burden as well as tissue histopathology analysis. The combined findings from the study demonstrated that in the presence of C. albicans, a significantly higher numbers of S. mutans were recovered from tongues and teeth of mice compared to mice infected with only S. mutans. Histopathology of tongue sections revealed massive invasion of tissue by the invasive C. albicans hyphae along with accumulation of large amounts of S. mutans. In contrast, in mice with S. mutans alone, minimal amount of S. mutans was seen adhering to the tongues. The combined findings from this study indicate a potential role for C. albicans in mediating cariogenic development via its physical interactions with S. mutans. Therefore, future studies should focus on developing novel therapeutic strategies geared towards prevention of adhesion receptors to block adhesive microbial reactions.
    • Protection of the Oral Mucosa by Salivary Histatin-5 against Candidiasis in a Murine Model of Oral Infection

      Al Shaye, Sara Yousef; Jabra-Rizk, Mary Ann (2011)
      The oral cavity is a primary target for opportunistic infections, particularly oral candidiasis caused by Candida albicans. A commensal fungus commonly colonizing mucosal surfaces, under conditions of immune dysfunction, C. albicans can become a pathogen causing recurrent infections. Yet, the role of host oral innate immunity in the development of candidiasis has not been fully elucidated. Specifically, the host salivary antimicrobial peptide histatin-5 (Hst-5) has been proposed to play a protective role in the oral cavity against C. albicans. However, investigations demonstrating its efficacy on oral tissue are lacking. To this end, in this study, an ex vivo and in vivo murine model of oral infection were developed. Viable C. albicans counts and histopathological analyses demonstrated a significant protective effect for Hst-5 on mouse oral tissue against C. albicans. More importantly, host saliva exerted a comparable anticandidal effect. However, this effect was neutralized upon treatment of saliva with proteases and C. albicans, previously shown to degrade Hst-5, indicating that Hst-5 is likely the salivary component responsible for the observed protection. Combined, the findings from this study demonstrate for the first time the efficacy of salivary Hst-5 in protecting host oral tissue against C. albicans infection, thereby affirming the therapeutic potential of this natural host peptide.
    • Therapeutic Evaluation of a Novel Topical Antimicrobial Formulation against Candida-Associated Denture Stomatitis in an Experimental Rat Model

      Sultan, Ahmed; Jabra-Rizk, Mary Ann; 0000-0001-5286-4562 (2019)
      Candida-associated denture stomatitis (DS), caused by the fungal species Candida albicans, is the most common manifestation of oral candidiasis and is prevalent in up to 70% of denture wearers. DS tends to be a persistent and recurrent oral condition as a consequence of the ability of C. albicans to adhere to denture material and invade associated palatal tissue. There are currently no effective therapeutic strategies targeting DS, and despite antifungal therapy, infection is often re-established after treatment ceases. Therefore, it has become crucial to identify novel therapeutic approaches. Antimicrobial peptides have attracted significant attention as candidates for drug development due to their potent antimicrobial and anti-inflammatory properties, lack of toxicity and lack of development of drug resistance. Specifically, histatin-5 (Hst-5), naturally produced and secreted by host salivary glands, has demonstrated potent antifungal activity, including against strains resistant to traditional antifungals. However, our laboratory has previously demonstrated vulnerability for Hst-5 to proteolysis by C. albicans secreted proteolytic enzymes at specific amino acid residues. Therefore, to generate a resistant derivative of Hst-5, we engineered a variant (K11R-K17R) with substitutions in the amino acid residues at the cleavage sites. The new peptide proved to be more stable, and unlike the native Hst-5, resistant to proteolysis by C. albicans proteases. Importantly, for clinical application, we designed a polymer-based bioadhesive hydrogel as a delivery system for the peptide and developed a therapeutic formulation specifically designed for oral topical application. The potency of the new formulation in inhibiting C. albicans adherence and biofilm formation on denture acrylic material was demonstrated in vitro indicating a potential clinical applicability against DS. To that end, using 3D digital design and printing technology, we engineered and fabricated a universal intraoral device that was successfully used in the animals to develop clinical disease mimicking DS as in humans. Using the novel animal model, we established the clinical utility of the formulation for the prevention of biofilm formation on denture device and DS development. Importantly, in addition to DS, the formulation can also be used for treatment of other forms of candidiasis as well as serve in augmenting host natural immune defenses.
    • 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.