• 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.
    • Evaluation of a Pentavalent Vaccine Against Staphylococcus aureus in a Murine Model of Chronic Peritoneal Abscess

      Sanjari, Salar; Shirtliff, Mark (2014)
      The majority (65%) of human bacterial infections involve biofilms, a complex heterogeneous community of microorganisms. Staphylococcus aureus causes a broad range of local and systemic infectious diseases including chronic biofilm-associated infections. The high morbidity, mortality and the ever-increasing associated healthcare costs have persuaded scientists to search for a silver bullet. Furthermore, emergence of antibiotic resistance and associated financial justifications have convinced many researchers and pharmaceutical companies that a vaccination approach can be a far more effective preventive approach against staphylococcal infections. As such, many researchers have focused their efforts to develop a vaccine against the free-floating, planktonic, S. aureus. In a new approach, we evaluated the efficacy of a pentavalent vaccine with four biofilm and one planktonic specific recombinant antigen against S. aureus in a murine model of chronic peritoneal abscess. Intraperitoneal challenge with 3x108 CFUs of S. aureus (MRSA M2) in BALB/c mice resulted in 17% (n=2) mortality over 21 days in the vaccinated group and 92% (n=11) in the control group (Fig. 1) (p< 0.001). 80% (n=8) of the survived animals in the vaccinated group cleared the infection. Obtained results confirm the effectiveness of our pentavalent vaccine that targets both planktonic and biofilm-associated proteins in this specific model.
    • Th17-associated immune responses are required for resolution of Staphylococcus aureus nasal carriage

      Archer, Nate; Shirtliff, Mark (2013)
      The anterior nares of humans are the major reservoir for Staphylococcus aureus colonization. Approximately 20% of the healthy human population is persistently and 80% intermittently colonized with S. aureus in the nasal cavity. Previous studies have shown a strong causal connection between S. aureus carriage and increased risk of nosocomial infection, as well as increased carriage due to immune dysfunction. However, the immune responses that permit persistence or mediate clearance are undefined. We developed a carriage model in C57BL/6J mice and showed that clearance begins 14 days post-inoculation. In contrast, SCID mice that have a deficient adaptive immune response are unable to eliminate S. aureus even after 28 days post-inoculation. Furthermore, decolonization was found to be T-cell mediated, but B-cell independent by evaluating carriage clearance in TCR-beta/delta KO and IgH-mu KO mice, respectively. Up-regulation of IL-1beta, KC, IL-17A and IL-17F occurred following inoculation with intra-nasal S. aureus. IL-17A production was crucial for clearance since IL-17A-deficient mice were unable to eliminate S. aureus carriage. In addition, inoculation of IL-17A/F KO mice displayed a significant inability to control S. aureus growth in the nares. Subsequently, cell differential counts were evaluated from nasal lavage fluid obtained from wild type and IL-17A-deficient colonized mice. These counts displayed IL-17A-dependent neutrophil migration. Antibody-mediated depletion of neutrophils in colonized mice caused reduced clearance compared to isotype treated controls. Th17-associated responses are reported to induce antimicrobial peptide production at epithelial surfaces. Therefore, we utilized RT-PCR to determine nasal tissue expression following inoculation with S. aureus of three antimicrobial peptides with anti-staphylococcal activity, mouse CRAMP, beta-defensin-3 (mBD-3), and beta-defensin-14 (mBD-14). We elucidated an IL-17A-dependent up-regulation of antimicrobial peptides post-S. aureus inoculation, and enhanced nasal tissue expression of mouse beta-defensin-3 upon IL-17A stimulation. Additionally, we discovered that ex-vivo nasal tissue supernatants have anti-staphylococcal activity that is solute-dependent and heat-sensitive. Our data suggest that the Th17-associated immune response is required for nasal decolonization. This response is T-cell dependent, mediated via IL-17F and IL-17A production, neutrophil influx, and potentially antimicrobial peptide induction. Th17-associated immune responses may be targeted for strategies to mitigate distal infections originating from persistent S. aureus carriage in humans.
    • 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.