Acceleration of Enterococcus faecalis Biofilm Formation by Aggregation Substance Expression in an Ex Vivo Model of Cardiac Valve Colonization

Infectious endocarditis involves formation of a microbial biofilm in vivo. Enterococcus faecalis Aggregation Substance (Asc10) protein enhances the severity of experimental endocarditis, where it has been implicated in formation of large vegetations and in microbial persistence during infection. In the current study, we developed an ex vivo porcine heart valve adherence model to study the initial interactions between Asc10+ and Asc10− E. faecalis and valve tissue, and to examine formation of E. faecalis biofilms on a relevant tissue surface. Scanning electron microscopy of the infected valve tissue provided evidence for biofilm formation, including growing masses of bacterial cells and the increasing presence of exopolymeric matrix over time; accumulation of adherent biofilm populations on the cardiac valve surfaces during the first 2–4 h of incubation was over 10-fold higher than was observed on abiotic membranes incubated in the same culture medium. Asc10 expression accelerated biofilm formation via aggregation between E. faecalis cells; the results also suggested that in vivo adherence to host tissue and biofilm development by E. faecalis can proceed by Asc10-dependent or Asc10-independent pathways. Mutations in either of two Asc10 subdomains previously implicated in endocarditis virulence reduced levels of adherent bacterial populations in the ex vivo system. Interference with the molecular interactions involved in adherence and initiation of biofilm development in vivo with specific inhibitory compounds could lead to more effective treatment of infectious endocarditis

AhrC and Eep are biofilm infection-associated virulence factors in enterococcus faecalis

Enterococcus faecalis is part of the human intestinal microbiome and is a prominent cause of health care-associated infections. The pathogenesis of many E. faecalis infections, including endocarditis and catheter-associated urinary tract infection (CAUTI), is related to the ability of clinical isolates to form biofilms. To identify chromosomal genetic determinants responsible for E. faecalis biofilm-mediated infection, we used a rabbit model of endocarditis to test strains with transposon insertions or in-frame deletions in biofilm-associated loci: ahrC, argR, atlA, opuBC, pyrC, recN, and sepF. Only the ahrC mutant was significantly attenuated in endocarditis. We demonstrate that the transcriptional regulator AhrC and the protease Eep, which we showed previously to be an endocarditis virulence factor, are also required for full virulence in murine CAUTI. Therefore, AhrC and Eep can be classified as enterococcal biofilm-associated virulence factors. Loss of ahrC caused defects in early attachment and accumulation of biofilm biomass. Characterization of ahrC transcription revealed that the temporal expression of this locus observed in wild-type cells promotes initiation of early biofilm formation and the establishment of endocarditis. This is the first report of AhrC serving as a virulence factor in any bacterial species

Enterococcus faecalis Endocarditis Severity in Rabbits Is Reduced by IgG Fabs Interfering with Aggregation Substance

Background: Enterococcus faecalis is a significant cause of infective endocarditis, an infection of the heart endothelium leading to vegetation formation (microbes, fibrin, platelets, and host cells attached to underlying endothelial tissue). Our previous research determined that enterococcal aggregation substance (AS) is an important virulence factor in causation of endocarditis, although endocarditis may occur in the absence of AS production. Production of AS by E. faecalis causes the organism to form aggregates through AS binding to enterococcal binding substance. In this study, we assessed the ability of IgGs and IgG Fabs against AS to provide protection against AS + E. faecalis endocarditis. Methodology/Principal Findings: When challenged with AS + E. faecalis, 10 rabbits actively immunized against AS + E. faecalis developed more significant vegetations than 9 animals immunized against AS 2 E. faecalis, and 9/10 succumbed compared to 2/9 (p,0.005), suggesting enhanced aggregation by IgG contributes significantly to disease. IgG antibodies against AS also enhanced enterococcal aggregation as tested in vitro. In contrast, Fab fragments of IgG from rabbits immunized against purified AS, when passively administered to rabbits (6/group) immediately before challenge with AS + E. faecalis, reduced total vegetation (endocarditis lesion) microbial counts (7.9610 6 versus 2.0610 5, p = 0.02) and size (40 mg versus 10, p = 0.05). In vitro, the Fabs prevented enterococcal aggregation. Conclusions/Significance: The data confirm the role of AS in infective endocarditis formation and suggest that use of Fab

A Novel Core Genome-Encoded Superantigen Contributes to Lethality of Community-Associated MRSA Necrotizing Pneumonia

Bacterial superantigens (SAg) stimulate T-cell hyper-activation resulting in immune modulation and severe systemic illnesses such as Staphylococcus aureus toxic shock syndrome. However, all known S. aureus SAgs are encoded by mobile genetic elements and are made by only a proportion of strains. Here, we report the discovery of a novel SAg staphylococcal enterotoxin-like toxin X (SElX) encoded in the core genome of 95% of phylogenetically diverse S. aureus strains from human and animal infections, including the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 clone. SElX has a unique predicted structure characterized by a truncated SAg B-domain, but exhibits the characteristic biological activities of a SAg including Vβ-specific T-cell mitogenicity, pyrogenicity and endotoxin enhancement. In addition, SElX is expressed by clinical isolates in vitro, and during human, bovine, and ovine infections, consistent with a broad role in S. aureus infections of multiple host species. Phylogenetic analysis suggests that the selx gene was acquired horizontally by a progenitor of the S. aureus species, followed by allelic diversification by point mutation and assortative recombination resulting in at least 17 different alleles among the major pathogenic clones. Of note, SElX variants made by human- or ruminant-specific S. aureus clones demonstrated overlapping but distinct Vβ activation profiles for human and bovine lymphocytes, indicating functional diversification of SElX in different host species. Importantly, SElX made by CA-MRSA USA300 contributed to lethality in a rabbit model of necrotizing pneumonia revealing a novel virulence determinant of CA-MRSA disease pathogenesis. Taken together, we report the discovery and characterization of a unique core genome-encoded superantigen, providing new insights into the evolution of pathogenic S. aureus and the molecular basis for severe infections caused by the CA-MRSA USA300 epidemic clone

Storylines: an alternative approach to representing uncertainty in physical aspects of climate change

As climate change research becomes increasingly applied, the need for actionable information is growing rapidly. A key aspect of this requirement is the representation of uncertainties. The conventional approach to representing uncertainty in physical aspects of climate change is probabilistic, based on ensembles of climate model simulations. In the face of deep uncertainties, the known limitations of this approach are becoming increasingly apparent. An alternative is thus emerging which may be called a ‘storyline’ approach. We define a storyline as a physically self-consistent unfolding of past events, or of plausible future events or pathways. No a priori probability of the storyline is assessed; emphasis is placed instead on understanding the driving factors involved, and the plausibility of those factors. We introduce a typology of four reasons for using storylines to represent uncertainty in physical aspects of climate change: (i) improving risk awareness by framing risk in an event-oriented rather than a probabilistic manner, which corresponds more directly to how people perceive and respond to risk; (ii) strengthening decision-making by allowing one to work backward from a particular vulnerability or decision point, combining climate change information with other relevant factors to address compound risk and develop appropriate stress tests; (iii) providing a physical basis for partitioning uncertainty, thereby allowing the use of more credible regional models in a conditioned manner and (iv) exploring the boundaries of plausibility, thereby guarding against false precision and surprise. Storylines also offer a powerful way of linking physical with human aspects of climate change

Active immunization against AS⁺<i>E. faecalis</i> worsens infectious endocarditis after challenge with AS⁺ enterococci.

<p>^a−Determined with use of Fishers Exact Test.</p><p>^b−1+ vegetations were small in size and number (approximately 10 mg); 2+ vegetations were moderate in size and number (approximately 50 mg); and 3+ vegetations were large in size and numbers, often obstructing the aorta (>75 mg).</p

Enterococcal aggregation substance enhances vegetation production in endocarditis in rabbits.

<p>Enterococcal aggregation substance enhances vegetation production in endocarditis in rabbits.</p

Differential ability of Staphylococcus aureus to cause infective endocarditis and lethal sepsis in rabbits

Staphylococcus aureus is a major cause of infective endocarditis (IE) and sepsis. In addition, 50% of IE survivors develop strokes and metastatic abscesses due to the release of emboli from infected valves. Both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains cause IE and sepsis, and they may be categorized by pulsed-field gel electrophoresis, for example clonal types USA200, 300, and 400. We hypothesize that secreted virulence factors contribute to their differential ability to cause IE and/or sepsis. Rabbits are an excellent model for studying IE, which over the course of 4 days are monitored for development of vegetations (the hallmark signs of IE), and sepsis, as S. aureus are administered intravenously. Rabbit cardiac physiology is similar to humans, and rabbits exhibit susceptibility to superantigens and cytolysins produced by these clonal types of S. aureus. We examined the differential ability of community-associated MRSA and MSSA strains (5 USA200 or related strain FRI1169, 3 USA300, and 2 USA400) to cause vegetations versus lethal sepsis in rabbits. USA200 and related strain FRI1169 exhibited intermediate LD50s in sepsis (5x106-5x108) colony-forming units (CFUs), and 4/5 caused significant IE. In contrast, USA300 strains were highly effective in causing lethal sepsis (LD50s of 1 x 106-5 x 107 CFUs) but were minimally capable of causing IE. USA300 variant strain Newman was not highly lethal (LD50 of 2 x 109 CFUs) but was highly effective in causing IE. USA400 strains were both highly lethal (LD50s of 1 x 107-5 x 107 CFUs) and highly effective causes of IE. Additional studies showed that phenol soluble modulins produced by FRI1169 were important for sepsis but did not contribute to IE. Our studies show that these clonal groups of S. aureus have differential abilities to cause IE and lethal sepsis and suggest that secreted virulence factors, including superantigens and cytolysins, account for these differences

Recovery of Sephadex G100 fractions of S. sanguinis 133-79 tryptic digest.

a<p>PRP was preincubated with the indicated fraction at a final concentration of 0.1 mg/ml.</p>b<p>Proteins were incubated with 50 µM of AMP, ADP or ATP for 15 minutes at 37°C at a final concentration of 10 µg/ml.</p