Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.

Phage infection and sub-lethal antibiotic exposure mediate Enterococcus faecalis type VII secretion system dependent inhibition of bystander bacteria.

Bacteriophages (phages) are being considered as alternative therapeutics for the treatment of multidrug resistant bacterial infections. Considering phages have narrow host-ranges, it is generally accepted that therapeutic phages will have a marginal impact on non-target bacteria. We have discovered that lytic phage infection induces transcription of type VIIb secretion system (T7SS) genes in the pathobiont Enterococcus faecalis. Membrane damage during phage infection induces T7SS gene expression resulting in cell contact dependent antagonism of different Gram positive bystander bacteria. Deletion of essB, a T7SS structural component, abrogates phage-mediated killing of bystanders. A predicted immunity gene confers protection against T7SS mediated inhibition, and disruption of its upstream LXG toxin gene rescues growth of E. faecalis and Staphylococcus aureus bystanders. Phage induction of T7SS gene expression and bystander inhibition requires IreK, a serine/threonine kinase, and OG1RF_11099, a predicted GntR-family transcription factor. Additionally, sub-lethal doses of membrane targeting and DNA damaging antibiotics activated T7SS expression independent of phage infection, triggering T7SS antibacterial activity against bystander bacteria. Our findings highlight how phage infection and antibiotic exposure of a target bacterium can affect non-target bystander bacteria and implies that therapies beyond antibiotics, such as phage therapy, could impose collateral damage to polymicrobial communities.

Structural and functional insights into a novel two-component endolysin encoded by a single gene in Enterococcus faecalis phage.

Using bacteriophage-derived endolysins as an alternative strategy for fighting drug-resistant bacteria has recently been garnering renewed interest. However, their application is still hindered by their narrow spectra of activity. In our previous work, we demonstrated that the endolysin LysIME-EF1 possesses efficient bactericidal activity against multiple strains of Enterococcus faecalis (E. faecalis). Herein, we observed an 8 kDa fragment and hypothesized that it contributes to LysIME-EF1 lytic activity. To examine our hypothesis, we determined the structure of LysIME-EF1 at 1.75 Å resolution. LysIME-EF1 exhibits a unique architecture in which one full-length LysIME-EF1 forms a tetramer with three additional C-terminal cell-wall binding domains (CBDs) that correspond to the abovementioned 8 kDa fragment. Furthermore, we identified an internal ribosomal binding site (RBS) and alternative start codon within LysIME-EF1 gene, which are demonstrated to be responsible for the translation of the truncated CBD. To elucidate the molecular mechanism for the lytic activity of LysIME-EF1, we combined mutagenesis, lytic activity assays and in vivo animal infection experiments. The results confirmed that the additional LysIME-EF1 CBDs are important for LysIME-EF1 architecture and its lytic activity. To our knowledge, this is the first determined structure of multimeric endolysin encoded by a single gene in E. faecalis phages. As such, it may provide valuable insights into designing potent endolysins against the opportunistic pathogen E. faecalis.

Enterococcus phage Nonaheksakonda infecting clinical isolates of Enterococcus faecalis represents a new lineage in the family Siphoviridae.

Enterococcus phage Nonaheksakonda was isolated from wastewater, using a vancomycin-resistant strain of the opportunistic pathogen Enterococcus faecalis (VRE) as a host. Nonaheksakonda is a lytic phage infecting E. faecalis V583 and clinical isolates with at least four different multi-locus sequence types (MLSTs). The genome is a 41.9-kb double-stranded DNA molecule (34.6% GC) with 74 coding sequences. Comparative analysis revealed only one close relative, Enterococcus phage heks. All other phages had low protein similarity and shared less than 54% nucleotide sequence identity with phage Nonaheksakonda. The most similar phages were all classified and unclassified efquatroviruses. We propose that the phages Nonaheksakonda and heks represent a novel genus within the family Siphoviridae, order Caudovirales, for which we propose the name "Nonaheksakondavirus".

Intrinsic resistance of Enterococcus faecalis strains to ΦEf11 phage endolysin is associated with the presence of ΦEf11 prophage.

The use of bacteriophage-encoded murein hydrolases (endolysins) is being actively explored as a means of controlling multidrug-resistant pathogens. Previously, we isolated and characterized one such enzyme, the phage ΦEf11 ORF28 lysin, which demonstrated profound antimicrobial activity against many strains of Enterococcus faecalis. Although the lysin is eminently active against many vancomycin-resistant enterococal (VRE) strains, and displays lower minimum inhibitory concentrations than vancomycin against vancomycin-sensitive strains, there is a subset of E. faecalis strains that is not affected by the lysin. Currently, there is no explanation for the disparate sensitivity to ORF28 lysin among E. faecalis strains. In the present investigation, we show that the intrinsic insensitivity of the insusceptible strains to the lysin is associated with the presence of a ΦEf11 prophage. Of the strains harboring phage ΦEf11 genes (N = 28), 68% were insensitive to the lysin, whereas 91% of the strains (N = 75) lacking detectable ΦEf11 genes demonstrated lysin sensitivity. Furthermore, curing a lysin-resistant, lysogenic E. faecalis strain resulted in a lysin-sensitive derivative, whereas lysogenizing a wild-type non-lysogenic strain converted it from lysin sensitivity to lysin resistance. Our results suggest that lysin resistance comes about through lysogenic conversion of non-lysogenic, lysin-sensitive strains.

Biological characteristics and whole-genome analysis of the Enterococcus faecalis phage PEf771.

Enterococcus faecalis is a common pathogen causing refractory periapical periodontitis and secondary intraradicular infections. In this study, E. faecalis YN771 isolated from a re-treated root canal at a stomatology department was used as the host bacterium and was co-cultured with wastewater from the same department and patient samples to isolate a phage that lyses E. faecalis. We studied the biological and genomic characteristics of this phage. Transmission electron microscopy showed that this phage's head is icosahedral in structure, with a head diameter of around 98.4 nm, and a contractile tail of around 228.5 nm in length and a diameter of 17.3 nm. The phage was identified as a member of the Myoviridae family and named PEf771. It is sensitive to proteinase K but resistant to chloroform and Triton X-100. Its lytic cycle is 45 min, burst size is 78, optimal multiplicity of infection is 0.1, lysis spectrum is narrow, and host strain specificity is strong. Its optimal growth temperature is 37 °C, most suitable pH is 6.0, and is sensitive to ultraviolet radiation. Whole-genome sequencing of PEf771 indicated it has a genome size of 151 052 bp, with a GC content of 36.97%, and encodes 197 proteins plus 26 tRNAs. PEf771 is most closely related to E. faecalis phage EFDG1. Phage PEf771 has strong host specificity and lytic ability, so it is important to further characterize this phage and its interaction with E. faecalis.

Characterization of the Bacteriophage vB_EfaS-271 Infecting Enterococcus faecalis.

A newly isolated bacteriophage infecting Enterococcus faecalis strains has been characterized, including determination of its molecular features. This phage, named vB_EfaS-271, has been classified as a Siphoviridae member, according to electron microscopy characterization of the virions, composed of a 50 nm-diameter head and a long, flexible, noncontractable tail (219 × 12.5 nm). Analysis of the whole dsDNA genome of this phage showed that it consists of 40,197 bp and functional modules containing genes coding for proteins that are involved in DNA replication (including DNA polymerase/primase), morphogenesis, packaging and cell lysis. Mass spectrometry analysis allowed us to identify several phage-encoded proteins. vB_EfaS-271 reveals a relatively narrow host range, as it is able to infect only a few E. faecalis strains. On the other hand, it is a virulent phage (unable to lysogenize host cells), effectively and quickly destroying cultures of sensitive host bacteria, with a latent period as short as 8 min and burst size of approximately 70 phages per cell at 37 °C. This phage was also able to destroy biofilms formed by E. faecalis. These results contribute to our understanding of the biodiversity of bacteriophages, confirming the high variability among these viruses and indicating specific genetic and functional features of vB_EfaS-271.

Identification of Novel Bacteriophages with Therapeutic Potential That Target Enterococcus faecalis.

The Gram-positive opportunistic pathogen Enterococcus faecalis is frequently responsible for nosocomial infections in humans and represents one of the most common bacteria isolated from recalcitrant endodontic (root canal) infections. E. faecalis is intrinsically resistant to several antibiotics routinely used in clinical settings (such as cephalosporins and aminoglycosides) and can acquire resistance to vancomycin (vancomycin-resistant enterococci). The resistance of E. faecalis to several classes of antibiotics and its capacity to form biofilms cause serious therapeutic problems. Here, we report the isolation of several bacteriophages that target E. faecalis strains isolated from the oral cavity of patients suffering root canal infections. All phages isolated were Siphoviridae with similar tail lengths (200 to 250 nm) and icosahedral heads. The genome sequences of three isolated phages were highly conserved with the exception of predicted tail protein genes that diverge in sequence, potentially reflecting the host range. The properties of the phage with the broadest host range (SHEF2) were further characterized. We show that this phage requires interaction with components of the major and variant region enterococcal polysaccharide antigen to engage in lytic infection. Finally, we explored the therapeutic potential of this phage and show that it can eradicate E. faecalis biofilms formed in vitro on a standard polystyrene surface but also on a cross-sectional tooth slice model of endodontic infection. We also show that SHEF2 cleared a lethal infection of zebrafish when applied in the circulation. We therefore propose that the phage described here could be used to treat a broad range of antibiotic-resistant E. faecalis infections.

Bacteriophage Resistance Alters Antibiotic-Mediated Intestinal Expansion of Enterococci.

Enterococcus faecalis is a human intestinal pathobiont with intrinsic and acquired resistance to many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multidrug-resistant enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism against E. faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both conserved and strain-specific genes. We discovered that exposure to phage selective pressure favors mutation in nonconserved epa genes both in culture and in a mouse model of intestinal colonization. Despite gaining phage resistance, epa mutant strains exhibited a loss of resistance to cell wall-targeting antibiotics. Finally, we show that an E. faecalisepa mutant strain is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal dysbiosis, and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing the intestine and sensitized to vancomycin, preventing their overgrowth during antibiotic treatment.

Therapeutic Effects of Intravitreously Administered Bacteriophage in a Mouse Model of Endophthalmitis Caused by Vancomycin-Sensitive or -Resistant Enterococcus faecalis.

Endophthalmitis due to infection with Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Given that the frequency of this condition caused by vancomycin-resistant Enterococcus faecalis has been increasing, the development of novel therapeutics is urgently required. We have demonstrated the therapeutic potential of bacteriophage ΦEF24C-P2 in a mouse model of endophthalmitis caused by vancomycin-sensitive (EF24) or vancomycin-resistant (VRE2) strains of E. faecalis Phage ΦEF24C-P2 induced rapid and pronounced bacterial lysis in turbidity reduction assays with EF24, VRE2, and clinical isolates derived from patients with E. faecalis-related postoperative endophthalmitis. Endophthalmitis was induced in mice by injection of EF24 or VRE2 (1 × 104 cells) into the vitreous. The number of viable bacteria in the eye increased to >1 × 107 CFU, and neutrophil infiltration into the eye was detected as an increase in myeloperoxidase activity at 24 h after infection. A clinical score based on loss of visibility of the fundus as well as the number of viable bacteria and the level of myeloperoxidase activity in the eye were all significantly decreased by intravitreous injection of ΦEF24C-P2 6 h after injection of EF24 or VRE2. Whereas histopathologic analysis revealed massive infiltration of inflammatory cells and retinal detachment in vehicle-treated eyes, the number of these cells was greatly reduced and retinal structural integrity was preserved in phage-treated eyes. Our results thus suggest that intravitreous phage therapy is a potential treatment for endophthalmitis caused by vancomycin-sensitive or -resistant strains of E. faecalis.

Complete genome analysis of the novel Enterococcus faecalis phage vB_EfaS_AL3.

This work describes the characterization and genome annotation of a new lytic Enterococcus faecalis siphovirus, vB_EfaS_AL3 (referred to as AL3), isolated from wastewater samples collected in Liaoning Province, China. The genome of phage AL3 is composed of linear double-stranded DNA that is 40,789 bp in length with a G + C content of 34.84% and 61 putative protein-coding genes. Phylogenetic and comparative genomic analyses indicate that phage AL3 should be considered a novel phage.

Loss-of-Function Mutations in epaR Confer Resistance to ϕNPV1 Infection in Enterococcus faecalis OG1RF.

Enterococcus faecalis is a Gram-positive opportunistic pathogen that inhabits the human gastrointestinal tract. Because of the high frequency of antibiotic resistance among Enterococcus clinical isolates, interest in using phage to treat enterococcal infections and to decolonize high-risk patients for antibiotic-resistant Enterococcus is rising. Bacteria can evolve phage resistance, but there is little published information on these mechanisms in E. faecalis In this report, we identified genetic determinants of E. faecalis resistance to phage NPV1 (ϕNPV1). We found that loss-of-function mutations in epaR confer ϕNPV1 resistance by blocking phage adsorption. We attribute the inability of the phage to adsorb to the modification or loss of an extracellular polymer in strains with inactivated epaR Phage-resistant epaR mutants exhibited increased daptomycin and osmotic stress susceptibilities. Our results demonstrate that in vitro spontaneous resistance to ϕNPV1 comes at a cost in E. faecalis OG1RF.

Characterization and genome analysis of novel phage vB_EfaP_IME195 infecting Enterococcus faecalis.

Enterococcus faecalis is one of the main bacteria in the human and animal intestine but is also classed as an opportunistic pathogen. During normal growth, E. faecalis produces natural antibiotics and is conducive to human health. As ectopic parasites, E. faecalis is capable of causing infective endocarditis, neonatal sepsis, bloodstream infections, bacteremia, and intraabdominal infections. With the incidence of antibiotic resistance reaching crisis point, it is imperative to find alternative treatments for multidrug-resistant infections. Using phage for pathogen control is a promising treatment option to combat bacterial resistance. In this study, a lytic phage, designated vB_EfaP_IME195, was isolated from hospital sewage using a clinical multidrug-resistant Enterococcus faecalis strain as an indicator. The one-step growth curve with the optimal multiplicity of infection of (MOI) 0.01 revealed a latent period of ~ 30 min and a burst size of ~ 120 plaque-forming units (pfu) per cell. Transmission electron microscopy showed that the phage belongs to the family Podoviridae. Phage vB_EfaP_IME195 has a linear, double-stranded DNA genome of 18,607 bp with a G + C content of 33% and 27 coding sequences (GenBank accession no. KT932700). Run-off sequencing experiments showed that the phage has a unique 59-bp inverted repeat sequences at the terminal ends. BLASTn analysis revealed that vB_EfaP_IME195 shares 92% identity (93% genome coverage) with unpublished E. faecalis phage Idefix. This study reported a novel E. faecalis phage with unique genome termini containing inverted repeats. The isolation and characterization of this novel lytic E. faecalis phage provides the basis for the development of new therapeutic agents like phage cocktails for multidrug-resistant E. faecalis infection, and its unique genomic feature would also provide valuable knowledge and insight for further phage genome analysis.

Phage-mediated dispersal of biofilm and distribution of bacterial virulence genes is induced by quorum sensing.

The microbiome and the phage meta-genome within the human gut are influenced by antibiotic treatments. Identifying a novel mechanism, here we demonstrate that bacteria use the universal communication molecule AI-2 to induce virulence genes and transfer them via phage release. High concentrations (i.e. 100 µM) of AI-2 promote dispersal of bacteria from already established biofilms, and is associated with release of phages capable of infecting other bacteria. Enterococcus faecalis V583ΔABC harbours 7 prophages in its genome, and a mutant deficient in one of these prophages (i.e. prophage 5) showed a greatly reduced dispersal of biofilm. Infection of a probiotic E. faecalis strain without lytic prophages with prophage 5 resulted in increased biofilm formation and also in biofilm dispersal upon induction with AI-2. Infection of the probiotic E. faecalis strain with phage-containing supernatants released through AI-2 from E. faecalis V583ΔABC resulted in a strong increase in pathogenicity of this strain. The polylysogenic probiotic strain was also more virulent in a mouse sepsis model and a rat endocarditis model. Both AI-2 and ciprofloxacin lead to phage release, indicating that conditions in the gastrointestinal tract of hospitalized patients treated with antibiotics might lead to distribution of virulence genes to apathogenic enterococci and possibly also to other commensals or even to beneficial probiotic strains.

A genomic virulence reference map of Enterococcus faecalis reveals an important contribution of phage03-like elements in nosocomial genetic lineages to pathogenicity in a Caenorhabditis elegans infection model.

In the present study, the commensal and pathogenic host-microbe interaction of Enterococcus faecalis was explored using a Caenorhabditis elegans model system. The virulence of 28 E. faecalis isolates representing 24 multilocus sequence types (MLSTs), including human commensal and clinical isolates as well as isolates from animals and of insect origin, was investigated using C. elegans strain glp-4 (bn2ts); sek-1 (km4). This revealed that 6 E. faecalis isolates behaved in a commensal manner with no nematocidal effect, while the remaining strains showed a time to 50% lethality ranging from 47 to 120 h. Principal component analysis showed that the difference in nematocidal activity explained 94% of the variance in the data. Assessment of known virulence traits revealed that gelatinase and cytolysin production accounted for 40.8% and 36.5% of the observed pathogenicity, respectively. However, coproduction of gelatinase and cytolysin did not increase virulence additively, accounting for 50.6% of the pathogenicity and therefore indicating a significant (26.7%) saturation effect. We employed a comparative genomic analysis approach using the 28 isolates comprising a collection of 82,356 annotated coding sequences (CDS) to identify 2,325 patterns of presence or absence among the investigated strains. Univariate statistical analysis of variance (ANOVA) established that individual patterns positively correlated (n = 61) with virulence. The patterns were investigated to identify potential new virulence traits, among which we found five patterns consisting of the phage03-like gene clusters. Strains harboring phage03 showed, on average, 17% higher killing of C. elegans (P = 4.4e(-6)). The phage03 gene cluster was also present in gelatinase-and-cytolysin-negative strain E. faecalis JH2-2. Deletion of this phage element from the JH2-2 clinical strain rendered the mutant apathogenic in C. elegans, and a similar mutant of the nosocomial V583 isolate showed significantly attenuated virulence. Bioinformatics investigation indicated that, unlike other E. faecalis virulence traits, phage03-like elements were found at a higher frequency among nosocomial isolates. In conclusion, our report provides a valuable virulence map that explains enhancement in E. faecalis virulence and contributes to a deeper comprehension of the genetic mechanism leading to the transition from commensalism to a pathogenic lifestyle.

A novel termini analysis theory using HTS data alone for the identification of Enterococcus phage EF4-like genome termini.

BACKGROUND: Enterococcus faecalis and Enterococcus faecium are typical enterococcal bacterial pathogens. Antibiotic resistance means that the identification of novel E. faecalis and E. faecium phages against antibiotic-resistant Enterococcus have an important impact on public health. In this study, the E. faecalis phage IME-EF4, E. faecium phage IME-EFm1, and both their hosts were antibiotic resistant. To characterize the genome termini of these two phages, a termini analysis theory was developed to provide a wealth of terminal sequence information directly, using only high-throughput sequencing (HTS) read frequency statistics. RESULTS: The complete genome sequences of phages IME-EF4 and IME-EFm1 were determined, and our termini analysis theory was used to determine the genome termini of these two phages. Results showed 9 bp 3' protruding cohesive ends in both IME-EF4 and IME-EFm1 genomes by analyzing frequencies of HTS reads. For the positive strands of their genomes, the 9 nt 3' protruding cohesive ends are 5'-TCATCACCG-3' (IME-EF4) and 5'-GGGTCAGCG-3' (IME-EFm1). Further experiments confirmed these results. These experiments included mega-primer polymerase chain reaction sequencing, terminal run-off sequencing, and adaptor ligation followed by run-off sequencing. CONCLUSION: Using this termini analysis theory, the termini of two newly isolated antibiotic-resistant Enterococcus phages, IME-EF4 and IME-EFm1, were identified as the byproduct of HTS. Molecular biology experiments confirmed the identification. Because it does not require time-consuming wet lab termini analysis experiments, the termini analysis theory is a fast and easy means of identifying phage DNA genome termini using HTS read frequency statistics alone. It may aid understanding of phage DNA packaging.

Targeting Enterococcus faecalis biofilms with phage therapy.

Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment.

Genomic annotation for the temperate phage EFC-1, isolated from Enterococcus faecalis KBL101.

The temperate phage EFC-1 was newly isolated from a mitomycin-C-induced lysate of Enterococcus faecalis KBL101. EFC-1 has an isometric head and a long tail. The phage belongs to the family Siphoviridae according to its genomic structure and morphology. The phage EFC-1 has 40,286 base pairs of double-stranded DNA and a G+C content of 35.05 %. Bioinformatic analysis of the phage revealed 60 putative open reading frames (ORFs). The genome of the temperate phage EFC-1 was not significantly similar to that of previously reported bacteriophages from E. faecalis.

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria.

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria.

Genome analysis of Enterococcus faecalis bacteriophage IME-EF3 harboring a putative metallo-beta-lactamase gene.

Lytic Enterococcus faecalis bacteriophage IME-EF3 was isolated from hospital sewage, and its genome was sequenced using high-throughput sequencing. Genomic analysis and electron microscopy suggested that IME-EF3 was a member of the family Siphoviridae. The phage has an isometric head and a long non-contractile tail with a 41 kb linear double-stranded DNA genome. The genome encodes 69 putative proteins, with 32 annotated functionally, including proteins related to phage structure, packaging, transcription, replication, and a lysis module. Interestingly, a metallo-beta-lactamase gene responsible for multi-drug resistance was found in the genome of IME-EF3. The possibility of horizontal gene transfer of the metallo-beta-lactamase gene suggests that phage IME-EF3, although lytic, might not be suitable for phage therapy unless one would devise a way to delete the metallo-beta-lactamase gene. Hence, whole genome sequencing should always be a prerequisite for identifying a phage therapy candidate.