Identification of fitness determinants in Enterococcus faecalis by differential proteomics

Enterococcus (E.) faecalis is found as commensal in healthy humans, in a variety of fermented foods. It can serve as probiotic but also as pathogen causing endocarditis, bacteremia and urinary tract infections. We have employed a proteomic study with E. faecalis strain OG1RF under different growth conditions and in contact to mouse intestinal cells to identify novel latent and adaptive fitness determinants. These relate to changes in catabolic pathways (BudA), protein biosynthesis (AsnS), cellular surface biosynthesis (RmlA) and regulatory mechanisms (OmpR). This knowledge can be used to derive novel evidence-based targets, which can be used to further elucidate gene expression changes enhancing pathogenicity or fitness in a commensal strain and possibly delineate this species into groups of higher and lower risk for applications in a food or a medical context versus improved treatment strategies of the so far hard to cure diseases.     

Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF

Background

Enterococcus faecalis has emerged as a major hospital pathogen. To explore its diversity, we sequenced E. faecalis strain OG1RF, which is commonly used for molecular manipulation and virulence studies.

Results

The 2,739,625 base pair chromosome of OG1RF was found to contain approximately 232 kilobases unique to this strain compared to V583, the only publicly available sequenced strain. Almost no mobile genetic elements were found in OG1RF. The 64 areas of divergence were classified into three categories. First, OG1RF carries 39 unique regions, including 2 CRISPR loci and a new WxL locus. Second, we found nine replacements where a sequence specific to V583 was substituted by a sequence specific to OG1RF. For example, the iol operon of OG1RF replaces a possible prophage and the vanB transposon in V583. Finally, we found 16 regions that were present in V583 but missing from OG1RF, including the proposed pathogenicity island, several probable prophages, and the cpsCDEFGHIJK capsular polysaccharide operon. OG1RF was more rapidly but less frequently lethal than V583 in the mouse peritonitis model and considerably outcompeted V583 in a murine model of urinary tract infections.

Conclusion

E. faecalis OG1RF carries a number of unique loci compared to V583, but the almost complete lack of mobile genetic elements demonstrates that this is not a defining feature of the species. Additionally, OG1RF's effects in experimental models suggest that mediators of virulence may be diverse between different E. faecalis strains and that virulence is not dependent on the presence of mobile genetic elements.     

Evaluation of the Enterococcus faecalis Biofilm-Associated Virulence Factors AhrC and Eep in Rat Foreign Body Osteomyelitis and In Vitro Biofilm-Associated Antimicrobial Resistance

Enterococcus faecalis can cause healthcare-associated biofilm infections, including those of orthopedic devices. Treatment of enterococcal prosthetic joint infection is difficult, in part, due to biofilm-associated antimicrobial resistance. We previously showed that the E. faecalis OG1RF genes ahrC and eep are in vitro biofilm determinants and virulence factors in animal models of endocarditis and catheter-associated urinary tract infection. In this study, we evaluated the role of these genes in a rat acute foreign body osteomyelitis model and in in vitro biofilm-associated antimicrobial resistance. Osteomyelitis was established for one week following the implantation of stainless steel orthopedic wires inoculated with E. faecalis strains OG1RF, ΩahrC, and ∆eep into the proximal tibiae of rats. The median bacterial loads recovered from bones and wires did not differ significantly between the strains at multiple inoculum concentrations. We hypothesize that factors present at the infection site that affect biofilm formation, such as the presence or absence of shear force, may account for the differences in attenuation in the various animal models we have used to study the ΩahrC and ∆eep strains. No differences among the three strains were observed in the planktonic and biofilm antimicrobial susceptibilities to ampicillin, vancomycin, daptomycin, linezolid, and tetracycline. These findings suggest that neither ahrC nor eep directly contribute to E. faecalis biofilm-associated antimicrobial resistance. Notably, the experimental evidence that the biofilm attachment mutant ΩahrC displays biofilm-associated antimicrobial resistance suggests that surface colonization alone is sufficient for E. faecalis cells to acquire the biofilm antimicrobial resistance phenotype.     

Complete genome sequencing and comparative analysis of the linezolid-resistant Enterococcus faecalis strain DENG1

Genome level analysis of bacterial strains provides information on genetic composition and resistance mechanisms to clinically relevant antibiotics. To date, whole genome characterization of linezolid-resistant Enterococcus faecalis isolated in the clinic is lacking. In this study, we report the entire genome sequence, genomic characteristics and virulence factors of a pathogenic E. faecalis strain, DENG1. Our results showed considerable differences in genomic characteristics and virulence factors compared with other E. faecalis strains (V583 and OG1RF). The genome of this LZD-resistant E. faecalis strain can be used as a reference to study the mechanism of LZD resistance and the phylogenetic relationship of E. faecalis strains worldwide.     

In Vivo Assessment of Growth and Virulence Gene Expression during Commensal and Pathogenic Lifestyles of luxABCDE-Tagged Enterococcus faecalis Strains in Murine Gastrointestinal and Intravenous Infection Models

Cytolysin and gelatinase are prominent pathogenicity determinants associated with highly virulent Enterococcus faecalis strains. In an effort to explore the expression profiles of these virulence traits in vivo, we have employed E. faecalis variants expressing the luxABCDE cassette under the control of either the P_16S, cytolysin, or gelatinase promoter for infections of Galleria mellonella caterpillars and mice. Systemic infection of G. mellonella with bioluminescence-tagged E. faecalis MMH594 revealed temporal regulation of both gelatinase and cytolysin promoters and demonstrated that these traits were induced in response to the host environment. Gavage of mice pretreated perorally with antibiotics resulted in efficient colonization of the murine gastrointestinal tract (GIT) in a strain-dependent manner, where the commensal baby isolate EF62 was more persistent than the nosocomial isolate MMH594. A highly significant correlation (R² > 0.94) was found between bioluminescence and the CFU counts in mouse fecal samples. Both strains showed similar preferences for growth and persistence in the ileum, cecum, and colon. Cytolysin expression was uniform in these compartments of the intestinal lumen. In spite of high numbers (10⁹ CFU/g of intestinal matter) in the ileum, cecum, and colon, no evidence of translocation or systemic infection could be observed. In the murine intravenous infection model, cytolysin expression was readily detected in the liver, kidneys, and bladder. At 72 h postinfection, the highest bacterial loads were found in the liver, kidneys, and spleen, with organ-specific expression levels of cytolysin ∼400- and ∼900-fold higher in the spleen and heart, respectively, than in the liver and kidneys. Taken together, this system based on the bioluminescence imaging technology is established as a new, powerful method to monitor the differential regulation of E. faecalis virulence determinants and to study the spatiotemporal course of infection in living animals in real time.     

Conjugal Transfer of Plasmid DNA fromEscherichia colito Enterococci: A Method to Make Insertion Mutations

Shuttle vector pAT18 was transferred by conjugation fromEscherichia coliS17-1 toEnterococcus faecalisOG1RF andEnterococcus faeciumSE34. Transfer was mediated by the transfer functions of plasmid RK2 inE. coliS17-1 and the origin of conjugal transfer (oriT) located on pAT18. TheoriTsequence was then inserted into two plasmids to generate vectors pTEX5235 and pTEX5236. These two vectors cannot replicate in gram-positive bacteria and can be used to make insertion mutants in gram-positive bacteria. An internal sequence from an autolysin gene ofE. faecalisOG1RF was cloned into pTEX5235 and transferred by conjugation fromE. coliS17-1 toE. faecalisOG1RF. The plasmid was found to integrate into the chromosome of OG1RF by a single crossover event, resulting in a disrupted autolysin gene. A cosmid carrying the pyrimidine gene cluster fromE. faecalis,with a transposon insertion inpyrC,was also transferred fromE. coliS17-1 toE. faecalisOG1RF. After selection for the transposon, it was found to have recombined into the recipient chromosome by a double crossover between the cosmid and the chromosome of OG1RF. This resulted in apyrCknockout mutant showing an auxotrophic phenotype.     

Importance of the Collagen Adhesin Ace in Pathogenesis and Protection against Enterococcus faecalis Experimental Endocarditis

Ace is an adhesin to collagen from Enterococcus faecalis expressed conditionally after growth in serum or in the presence of collagen. Here, we generated an ace deletion mutant and showed that it was significantly attenuated versus wild-type OG1RF in a mixed infection rat endocarditis model (P<0.0001), while no differences were observed in a peritonitis model. Complemented OG1RFΔace (pAT392::ace) enhanced early (4 h) heart valve colonization versus OG1RFΔace (pAT392) (P = 0.0418), suggesting that Ace expression is important for early attachment. By flow cytometry using specific anti-recombinant Ace (rAce) immunoglobulins (Igs), we showed in vivo expression of Ace by OG1RF cells obtained directly from infected vegetations, consistent with our previous finding of anti-Ace antibodies in E. faecalis endocarditis patient sera. Finally, rats actively immunized against rAce were less susceptible to infection by OG1RF than non-immunized (P = 0.0004) or sham-immunized (P = 0.0475) by CFU counts. Similarly, animals given specific anti-rAce Igs were less likely to develop E. faecalis endocarditis (P = 0.0001) and showed fewer CFU in vegetations (P = 0.0146). In conclusion, we have shown for the first time that Ace is involved in pathogenesis of, and is useful for protection against, E. faecalis experimental endocarditis.     

Comparative Genomics and Transduction Potential of Enterococcus faecalis Temperate Bacteriophages

To determine the relative importance of temperate bacteriophage in the horizontal gene transfer of fitness and virulence determinants of Enterococcus faecalis, a panel of 47 bacteremia isolates were treated with the inducing agents mitomycin C, norfloxacin, and UV radiation. Thirty-four phages were purified from culture supernatants and discriminated using pulsed-field gel electrophoresis (PFGE) and restriction mapping. From these analyses the genomes of eight representative phages were pyrosequenced, revealing four distinct groups of phages. Three groups of phages, ΦFL1 to 3, were found to be sequence related, with ΦFL1A to C and ΦFL2A and B sharing the greatest identity (87 to 88%), while ΦFL3A and B share 37 to 41% identity with ΦFL1 and 2. ΦFL4A shares 3 to 12% identity with the phages ΦFL1 to 3. The ΦFL3A and B phages possess a high DNA sequence identity with the morphogenesis and lysis modules of Lactococcus lactis subsp. cremoris prophages. Homologs of the Streptococcus mitis platelet binding phage tail proteins, PblA and PblB, are encoded on each sequenced E. faecalis phage. Few other phage genes encoding potential virulence functions were identified, and there was little evidence of carriage of lysogenic conversion genes distal to endolysin, as has been observed with genomes of many temperate phages from the opportunist pathogens Staphylococcus aureus and Streptococcus pyogenes. E. faecalis JH2-2 lysogens were generated using the eight phages, and these were examined for their relative fitness in Galleria mellonella. Several lysogens exhibited different effects upon survival of G. mellonella compared to their isogenic parent. The eight phages were tested for their ability to package host DNA, and three were shown to be very effective for generalized transduction of naive host cells of the laboratory strains OG1RF and JH2-2.Enterococcus faecalis is a member of the natural flora of humans and colonizes the gastrointestinal and vaginal tracts and the oral cavity. In recent years it has emerged as an important opportunistic nosocomial pathogen and is a causative agent of bacteremia, infective endocarditis, and surgical wound and urinary tract infections. The accumulation of acquired antibiotic resistance determinants, in addition to its intrinsic resistance and tenacity, has given rise to the evolution of clinical isolates of E. faecalis that are therapeutically problematic (19). Greater notoriety was afforded to this species following the observed transfer of the conjugative transposon Tn1546 to Staphylococcus aureus, imparting vancomycin resistance (11). Subsequent analysis has revealed that multiple independent E. faecalis-dependent vanA transfers had occurred in the United States prior to 2007 (50). This places enterococci in an important and dynamic position within the health care system, warranting their increased study.The specific determinants that are proposed to contribute to the virulence of E. faecalis are not universally present, and expression of the cognate genes is variable (21, 37). For example, in a recent study of 106 clonally diverse strains of E. faecalis the metallopeptidase gelatinase (GelE) was shown to be expressed in less than 60% of 106 genotypically positive isolates, whereas expression of cytolysin was less frequently observed (expression in ∼25% of isolates, with 30% being genotypically positive) (33). A proposed pathogenicity island identified with E. faecalis V583 (49) is composed of a variable gene set encoding the virulence determinants enterococcal surface protein, cytolysin, and aggregation substance. This highly variable 150-kb mobile element contains many components of unknown function that are hypothesized to facilitate survival and/or transmission in the health care setting (34, 40, 49).Two sequenced and annotated genomes of E. faecalis have been completed and published to date. These are the blood isolate and first-observed vancomycin-resistant strain V583 (40) and the oral isolate OG1RF, used as a common laboratory strain (8). A major difference between these genomes is the presence in V583 of seven regions containing phage-associated sequences. In contrast, OG1RF contains only one phage remnant, which was proposed by McBride et al. (33) to form part of the core genome, a theory supported by the presence in OG1RF of this phage remnant region together with two CRISPR loci. CRISPR sequences provide sequence-specific resistance to bacteriophages via the assembly of phage DNA sequences interspersed as spacers between repeats, in concert with associated cas genes, which collectively operate as an RNA-based gene silencing mechanism (5, 6, 28, 30, 36, 42). This elegant heritable mechanism is proposed to limit horizontal gene transfer of bacteriophage, transposable elements, and conjugative plasmids (9, 10, 32).Within the firmicute division of Gram-positive bacteria, temperate bacteriophages are key vectors for the horizontal transfer of virulence genes. In Staphylococcus aureus, bacteriophages encode and mobilize an impressive array of immune evasion genes (54, 55) and Panton-Valentine leukocidin (43). Several bacteriophage-encoded virulence determinants also contribute to pathogenesis in group A Streptococcus (2, 3, 4).The role of bacteriophages in the virulence of E. faecalis is not clear. Encoded within seven phage-related sequences of strain V583, there are multiple reported homologs of the Streptococcus mitis platelet-binding proteins PblA and PblB (7) and a ferrochelatase (40). In contrast, the absence of mobile genetic elements (MGEs) in strain OG1RF led Bourgogne et al. (8) to speculate that they did not engender virulence in E. faecalis.In this study we determined the morphology and complete genome sequences of eight induced bacteriophages purified from clinical isolates of E. faecalis. We sought to determine the potential carriage of genes that might contribute to the virulence or fitness of this organism and characterize the capacity of these phages to participate in transduction.     

LiaR‐independent pathways to daptomycin resistance in Enterococcus faecalis reveal a multilayer defense against cell envelope antibiotics

The lipopeptide antibiotic daptomycin (DAP) is a key drug against serious enterococcal infections, but the emergence of resistance in the clinical setting is a major concern. The LiaFSR system plays a prominent role in the development of DAP resistance (DAP‐R) in enterococci, and blocking this stress response system has been proposed as a novel therapeutic strategy. In this work, we identify LiaR‐independent pathways in Enterococcus faecalis that regulate cell membrane adaptation in response to antibiotics. We adapted E. faecalis OG1RF (a laboratory strain) and S613TM (a clinical strain) lacking liaR to increasing concentrations of DAP, leading to the development of DAP‐R and elevated MICs to bacitracin and ceftriaxone. Whole genome sequencing identified changes in the YxdJK two‐component regulatory system and a putative fatty acid kinase (dak) in both DAP‐R strains. Deletion of the gene encoding the YxdJ response regulator in both the DAP‐R mutant and wild‐type OG1RF decreased MICs to DAP, even when a functional LiaFSR system was present. Mutations in dak were associated with slower growth, decreased membrane fluidity and alterations of cell morphology. These findings suggest that overlapping stress response pathways can provide protection against antimicrobial peptides in E. faecalis at a significant cost in bacterial fitness.     

Impact of actin on adhesion and translocation of Enterococcus faecalis

This study focuses on the impact of actin on adhesion and translocation of Enterococcus (E.) faecalis OG1RF, E. faecalis Symbioflor^®, and E. faecalis V583. Insight into the role of actin aggregation in the mediation of bacterial adhesion and translocation was provided by a two-chamber translocation assay, which employed Ptk6 cells. Determination of translocation rates, cytochalasin D treatment, and laser scanning confocal microscopic observation revealed actin as a predominant brace for enterococci to pass through the epithelial cell layer. As the three enterococci had moderate adhesion ability to actin, actin-binding proteins were isolated and characterized by LC–MS/MS. The isolated proteins were identified as pyruvate formate lyase, enolase, glyceraldehyde-3-phosphate dehydrogenase, and GroEL. All these proteins belong to two major groups of moonlighting proteins, i.e., proteins, which display additional functions other than their described major biochemical catalysis. Both groups of moonlight proteins were determined to be associated with epithelial cell binding.     

Library Screen Identifies Enterococcus faecalis CcpA,the Catabolite Control Protein A,as an Effector of Ace,a Collagen Adhesion Protein Linked to Virulence

The Enterococcus faecalis cell wall-anchored protein Ace is an important virulence factor involved in cell adhesion and infection. Expression of Ace on the cell surface is affected by many factors, including stage of growth, culture temperature, and environmental components, such as serum, urine, and collagen. However, the mechanisms that regulate or modulate Ace display are not well understood. With interest in identifying genes associated with Ace expression, we utilized a whole-cell enzyme-linked immunosorbent assay (ELISA)-based screening method to identify mutants from a transposon insertion mutant library which exhibited distinct Ace surface expression profiles. We identified a ccpA insertion mutant which showed significantly decreased levels of Ace surface expression at early growth phase versus those of wild-type OG1RF. Confirmation of the observation was achieved through flow cytometry and complementation analysis. Compared to the wild type, the E. faecalis ccpA mutant had an impaired ability to adhere to collagen when grown to early exponential phase, consistent with the lack of Ace expression in the early growth phase. As a key component of carbon catabolite regulation, CcpA has been previously reported to play a critical role in regulating expression of proteins involved in E. faecalis carbohydrate uptake and utilization. Our discovery is the first to associate CcpA with the production of a major E. faecalis virulence factor, providing new insights into the regulation of E. faecalis pathogenesis.     

Molecular Screening of Enterococcus Virulence Determinants and Potential for Genetic Exchange between Food and Medical Isolates

Enterococci are used as starter and probiotic cultures in foods, and they occur as natural food contaminants. The genus Enterococcus is of increased significance as a cause of nosocomial infections, and this trend is exacerbated by the development of antibiotic resistance. In this study, we investigated the incidence of known virulence determinants in starter, food, and medical strains of Enterococcus faecalis, E. faecium, and E. durans. PCR and gene probe strategies were used to screen enterococcal isolates from both food and medical sources. Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E. faecium strains. Medical E. faecalis strains had more virulence determinants than did food strains, which, in turn, had more than did starter strains. All of the E. faecalis strains tested possessed multiple determinants (between 6 and 11). E. faecium strains were generally free of virulence determinants, with notable exceptions. Significantly, esp and gelE determinants were identified in E. faecium medical strains. These virulence determinants have not previously been identified in E. faecium strains and may result from regional differences or the evolution of pathogenic E. faecium. Phenotypic testing revealed the existence of apparently silent gelE and cyl genes. In E. faecalis, the trend in these silent genes mirrors that of the expressed determinants. The potential for starter strains to acquire virulence determinants by natural conjugation mechanisms was investigated. Transconjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. In addition, multiple pheromone-encoding genes were identified in both food and starter strains, indicating their potential to acquire other sex pheromone plasmids. These results suggest that the use of Enterococcus spp. in foods requires careful safety evaluation.     

The bacterial flora of the midgut of two Danish populations of healthy fifth instar larvae of the turnip moth, Scotia segetum

Examination of the midgut bacteria of two Danish populations of healthy fifth instar turnip moth larvae, Scotia (=Agrotis) segetum, living on potatoes and celery gave the following results. The total number of living microorganisms in the midgut varied between 1.0 × 10⁴ and 4.0 × 10⁵. Larvae from celery in N. W. Zeeland always contained Streptococcus faecalis and six members of Enterobacteriaceae, viz., Citrobacter freundii, Klebsiella pneumoniae, Hafnia alvei, Proteus mirabilis, P. vulgaris, and Erwinia amylovora. In larvae from potatoes in E. Jutland, the species consistently present were Streptococcus faecalis and four species of Enterobacteriaceae, viz., Escherichia coli, Erwinia amylovora, E. carotovora var. atroseptica, and one other, probably a member of the E. carotovora group. Streptococcus faecalis is supposed to occur as a mutualist in the alimentary tract, suppressing Gram-positive bacteria.     

Transfer of the Pheromone-Inducible Plasmid pCF10 among Enterococcus faecalis Microorganisms Colonizing the Intestine of Mini-Pigs

A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of Enterococcus faecalis in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of E. faecalis OG1 was investigated in the model. The plasmid encodes resistance to tetracycline. Numbers of recipient, donor, and transconjugant bacteria were monitored by selective plating of fecal samples, and transconjugants were subsequently verified by PCR. After being ingested by the mini-pigs, the recipient strain persisted in the intestine at levels between 10⁶ and 10⁷ CFU per g of feces throughout the experiment. The donor strain, which carried different resistance markers but was otherwise chromosomally isogenic to the recipient strain, was given to the pigs 3 weeks after the recipient strain. The donor cells were initially present in high numbers (10⁶ CFU per g) in feces, but they did not persist in the intestine at detectable levels. Immediately after introduction of the donor bacteria, transconjugant cells appeared and persisted in fecal samples at levels between 10³ and 10⁴ CFU per g until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested E. faecalis cells to other E. faecalis organisms already present in the intestinal environment and that the plasmid subsequently persisted in the intestine.     

Horizontal Transfer of the Tetracycline Resistance Gene <Emphasis Type="Italic">tetM</Emphasis> Mediated by pCF10 Among <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> in the House Fly (<Emphasis Type="Italic">Musca domestica</Emphasis> L.) Alimentary Canal

The house fly (Musca domestica L.) alimentary canal was evaluated for the potential of horizontal transfer of tetM on plasmid pCF10 among Enterococcus faecalis. Two sets of experiments were conducted: (1) house flies without surface sterilization and (2) surface-sterilized flies. Both sets of flies were exposed to E. faecalis OG1RF:pCF10 as donor for 12 h and then E. faecalis OG1SSp as recipient for 1 h. Another group of flies received the recipient first for 12 h followed by exposure to the donor strain for 1 h. House flies were screened daily to determine the donor, recipient, and transconjugant bacterial load for up to 5 days. In addition, the sponge-like mouth parts used for food uptake (labellum) of surface-sterilized house flies were removed and analyzed for donors, recipients, and transconjugants, separately. In both groups of flies (n = 90 flies/group), transfer occurred within 24 h after exposure with a transconjugant/donor rate from 8.6 × 10⁻⁵ to 4.5 × 10¹. Transconjugants were also isolated from the house fly labellum. Our data suggest that the house fly digestive tract provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among enterococci. Our results emphasize the importance of this insect as a potential vector of antibiotic-resistant bacterial strains.     

Bacteriocinogenic properties and in vitro probiotic potential of enterococci from Tunisian dairy products

The aim of this study was to isolate new bacteriocinogenic strains with putative probiotic potential from various Tunisian fermented milks. A total of 44 Gram-positive catalase-negative isolates were colony-purified and screened for antimicrobial activity. Of inhibitory isolates, four were identified as Enterococcus durans and one as Enterococcus faecalis using 16S rRNA gene sequence. The five strains were sensitive to penicillin G, all aminoglycosides tested, to the vancomycin, tetracycline, and chloramphenicol, and E. durans 42G and E. faecalis 61B were resistant to erythromycin. The antimicrobial substances were sensitive to proteolytic enzymes and had good biochemical stability. E. durans 61A showed a good resistance to gastric and small intestinal secretions, but were more sensitive to the duodenal conditions. Considering the safety and the stability under simulated gastrointestinal tract, it appears that the bacteriocinogenic strain E. durans 61A is a good candidate for its application as novel probiotic strain in the food industry.     

Global Metabolic Response of Enterococcus faecalis to Oxygen

Oxygen and oxidative stress have become relevant components in clarifying the mechanism that weakens bacterial cells in parallel to the mode of action of bactericidal antibiotics. Given the importance of oxidative stress in the overall defense mechanism of bacteria and their apparent role in the antimicrobial mode of action, it is important to understand how bacteria respond to this stress at a metabolic level. The aim of this study was to determine the impact of oxygen on the metabolism of the facultative anaerobe Enterococcus faecalis using continuous culture, metabolomics, and ¹³C enrichment of metabolic intermediates. When E. faecalis was rapidly transitioned from anaerobic to aerobic growth, cellular metabolism was directed toward intracellular glutathione production and glycolysis was upregulated 2-fold, which increased the supply of critical metabolite precursors (e.g., glycine and glutamate) for sulfur metabolism and glutathione biosynthesis as well as reducing power for cellular respiration in the presence of hemin. The ultimate metabolic response of E. faecalis to an aerobic environment was the upregulation of fatty acid metabolism and benzoate degradation, which was linked to important changes in the bacterial membrane composition as evidenced by changes in membrane fatty acid composition and the reduction of membrane-associated demethylmenaquinone. These key metabolic pathways associated with the response of E. faecalis to oxygen may represent potential new targets to increase the susceptibility of this bacterium to bactericidal drugs.     

Development of a genomic site for gene integration and expression in Enterococcus faecalis

Enterococcus faecalis, a gram-positive opportunistic pathogen, has become one of the leading causes of nosocomial infections. Normally a resident of the gastrointestinal tract, extensive use of antibiotics has resulted in the rise of E. faecalis strains that are resistant to multiple antibiotics. This, compounded with the ability to easily exchange antibiotic determinants with other bacteria, has made certain E. faecalis infections difficult to treat medically. The genetic toolbox for the study of E. faecalis has expanded greatly in recent years, but has lacked methodology to stably introduce a gene in single copy in a non-disruptive manner for complementation or expression of non-native genes. In this study, we identified a specific site in the genome of E. faecalis OG1RF that can serve as an expression site for a gene of interest. This site is well conserved in most of the sequenced E. faecalis genomes. A vector has also been developed to integrate genes into this site by allelic exchange. Using this system, we complemented an in-frame deletion in eutV, demonstrating that the mutation does not cause polar effects. We also generated an E. faecalis OG1RF strain that stably expresses the green fluorescent protein and is comparable to the parent strain in terms of in vitro growth and pathogenicity in C. elegans and mice. Another major advantage of this new methodology is the ability to express integrated genes without the need for maintaining antibiotic selection, making this an ideal tool for functional studies of genes in infection models and co-culture systems.