Enterococcus faecalis (EF) is both a common commensal of the human gastrointestinal tract (GI) and a leading cause of hospital acquired infections1. Systemic infections with multi-drug resistant enterococci occur subsequent to GI colonization2. Preventing colonization by multi-drug resistant EF could therefore be a valuable approach to limiting infection. However, little is known about mechanisms EF uses to colonize and compete for stable gastrointestinal niches. Pheromone-responsive, conjugative plasmids encoding bacteriocins are common among enterococcal strains3, and could modulate niche competition among enterococci or between enterococci and the intestinal microbiota. We developed a model of mouse gut colonization with EF without disrupting the microbiota, to evaluate the role of the conjugative plasmid pPD1 expressing bacteriocin 214 on enterococcal colonization. Here we show that EF harboring pPD1 replaces indigenous enterococci and outcompetes EF lacking pPD1. Furthermore, in the intestine, pPD1 is transferred to other EF strains by conjugation, enhancing their survival. Moreover, colonization with an EF strain carrying a conjugation-defective pPD1 mutant resulted in clearance of vancomycin-resistant enterococci, without plasmid transfer. Therefore bacteriocin expression by commensal bacteria can influence niche-competition in the GI tract, and bacteriocins, delivered by commensals that occupy a precise intestinal bacterial niche, may be an effective therapeutic approach to specifically eliminate intestinal colonization by multi-drug resistant bacteria, without profound disruption of the indigenous microbiota.

Enterococcus faecalis is a Gram-positive commensal bacterium of the gastrointestinal tract. E. faecalis is also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite the clinical significance of the enterococci, genetic analysis has been restricted by limitations inherent in the available genetic tools. To facilitate genetic manipulation of E. faecalis, we developed a conjugative delivery system for high-frequency introduction of cloned DNA into target strains of E. faecalis and a host-genotype-independent counterselectable marker for use in markerless genetic exchange. We used these tools to construct a collection of E. faecalis mutant strains carrying defined mutations in several genes, including ccfA, eep, gelE, sprE, and an alternative sigma factor (sigH). Furthermore, we combined these mutations in various permutations to create double mutants, triple mutants, and a quadruple mutant of E. faecalis that enabled tests of epistasis to be conducted on the pheromone biosynthesis pathway. Analysis of cCF10 pheromone production by the mutants revealed that both the ccfA2 and Δeep10 mutations are epistatic to mutations in gelE/sprE. To our knowledge, this represents the first example of epistasis analysis applied to a chromosomally encoded biosynthetic pathway in enterococci. Thus, the advanced tools for genetic manipulation of E. faecalis reported here enable efficient and sophisticated genetic analysis of these important pathogens.

Enterococcus faecalis is a gram-positive opportunistic pathogen known to form biofilms in vitro. In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices. However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown. Recent work has suggested that a specific cell surface protein (Esp) of E. faecalis is critical for biofilm formation by this organism. However, in the same study, esp-deficient strains of E. faecalis were found to be capable of biofilm formation. To test the hypothesis that Esp is dispensable for biofilm formation by E. faecalis, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains. Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of esp, but also in the absence of the entire pathogenicity island that harbors the esp coding sequence. Using scanning electron microscopy to evaluate biofilms of E. faecalis OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels. Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions. Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by E. faecalis. In summary, E. faecalis forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein.

Antibiotic-resistant enterococci are major causes of hospitalacquired infections. The emergence of Enterococcus faecalis as a significant nosocomial pathogen is a consequence of its inherent resistance to certain antibiotics and of its ability to survive and proliferate in the intestinal tract. Genetic determinants of E. faecalis conferring these properties are largely unknown. Here we show that PrkC, a one-component signaling protein containing a eukaryotic-type Ser/Thr kinase domain, modulates inherent antimicrobial resistance and intestinal persistence of E. faecalis. An E. faecalis mutant lacking PrkC grows at a wild-type rate in the absence of antimicrobial stress but exhibits enhanced sensitivity to cell-envelope-active compounds, including antibiotics that target cell-wall biogenesis and bile detergents. Consistent with its bile sensitivity, the mutant was also impaired at persistence in the intestine of mice. Thus, PrkC regulates key physiological processes in E. faecalis associated with its success as a nosocomial pathogen. The predicted domain architecture of PrkC comprises a cytoplasmic kinase domain separated by a transmembrane segment from extracellular domains thought to bind uncross-linked peptidoglycan, suggesting that PrkC is a transmembrane receptor that monitors the integrity of the E. faecalis cell wall and mediates adaptive responses to maintain cell-wall integrity. Given its role in modulating traits of E. faecalis important for its ability to cause nosocomial infections, we suggest that the one-component signaling protein PrkC represents an attractive target for the development of novel therapies to prevent infections by antibioticresistant enterococci.cell-envelope stress ͉ one-component system ͉ signal transduction ͉ cephalosporin resistance ͉ bile resistance

Enterococcus faecalis is a gram-positive commensal bacterium of the gastrointestinal tract and an important opportunistic pathogen. Despite the increasing clinical significance of the enterococci, most of the genetic analysis of these organisms has focused on mobile genetic elements, and existing tools for manipulation and analysis of the core E. faecalis chromosome are limited. We are interested in a comprehensive analysis of the genetic determinants for biofilm formation encoded within the core E. faecalis genome. To identify such determinants, we developed a substantially improved system for transposon mutagenesis in E. faecalis based on a mini-mariner transposable element. Mutagenesis of wild-type E. faecalis with this element yielded predominantly mutants carrying a single copy of the transposable element, and insertions were distributed around the entire chromosome in an apparently random fashion. We constructed a library of E. faecalis transposon insertion mutants and screened this library to identify mutants exhibiting a defect in biofilm formation. Biofilm-defective mutants were found to carry transposon insertions both in genes that were previously known to play a role in biofilm formation and in new genes lacking any known function; for several genes identified in the screen, complementation analysis confirmed a direct role in biofilm formation. These results provide significant new information about the genetics of enterococcal biofilm formation and demonstrate the general utility of our transposon system for functional genomic analysis of E. faecalis.