Comparison of Enterococcus faecalis Biofilm Removal Efficiency among Bacteriophage PBEF129, Its Endolysin, and Cefotaxime.

Enterococcus faecalis is a Gram-positive pathogen which colonizes human intestinal surfaces, forming biofilms, and demonstrates a high resistance to many antibiotics. Especially, antibiotics are less effective for eradicating biofilms and better alternatives are needed. In this study, we have isolated and characterized a bacteriophage, PBEF129, infecting E. faecalis. PBEF129 infected a variety of strains of E. faecalis, including those exhibiting antibiotic resistance. Its genome is a linear double-stranded DNA, 144,230 base pairs in length. Its GC content is 35.9%. The closest genomic DNA sequence was found in Enterococcus phage vB_EfaM_Ef2.3, with a sequence identity of 99.06% over 95% query coverage. Furthermore, 75 open reading frames (ORFs) were functionally annotated and five tRNA-encoding genes were found. ORF 6 was annotated as a phage endolysin having an L-acetylmuramoyl-l-alanine amidase activity. We purified the enzyme as a recombinant protein and confirmed its enzymatic activity. The endolysin's host range was observed to be wider than its parent phage PBEF129. When applied to bacterial biofilm on the surface of in vitro cultured human intestinal cells, it demonstrated a removal efficacy of the same degree as cefotaxime, but much lower than its parent bacteriophage.

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.

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.

Enhancement of the direct antimicrobial activity of Lysep3 against Escherichia coli by inserting cationic peptides into its C terminus.

Phage lysins are considered promising antimicrobials against resistant bacterial infections. Some lysins have been reported for the prevention and treatment of Gram-positive bacterial infection. Gram-negative bacterial phage lysins, however, can only destroy the bacterial cell wall from inside because of the obstruction of the bacterial outer membrane that prevents direct hydrolysis of the bacterial wall peptidoglycan from the outside, severely restricting the development of lysins against Gram-negative bacteria. In this study, genetic engineering techniques were used to fuse a 5 cationic amino acid polypeptide (KRKRK), a 10 cationic amino acid polypeptide (KRKRKRKRKR), a 15 cationic amino acid polypeptide (KRKRKRKRKRKRKRK), and a polypeptide including both cationic and hydrophobic amino acids (KRKRKFFVAIIP) to the C-terminus of the Escherichia coli phage lysin Lysep3 to obtain four fusion lysins (5aa, 10aa, 15aa, Mix). The bactericidal effects of those four lysins on E. coli were then compared in vitro. Our results showed that the fusion of hydrophobic and positively charged amino acids, Mix, can kill E. coli effectively; the fusion of positively charged amino acids alone at the C-terminus (5aa, 10aa, 15aa) also showed bactericidal activity against E. coli from the outside, with the bactericidal activity gradually increasing with the positive charge at the C-terminus of the lysin. Collectively, improving the positive charge at the C-terminus of E. coli bacteriophage lysin Lysep3 increases its bactericidal ability from outside E. coli, providing a new practical method for the development of anti-Gram-negative bacterial lysins.

Prophages in enterococcal isolates from renal transplant recipients: renal failure etiologies promote selection of strains.

Infections caused by commensal bacteria may be fatal for the patients under immunosuppressive therapy. This results also from difficulty in identification of high risk strains. Enterococcal infections are increasingly frequent but despite many studies on virulence traits, the difference between commensal and pathogenic strains remains unclear. Prophages are newly described as important elements in competition between strains during colonization, as well as pathogenicity of the strains. Here we evaluate a difference in presence of pp4, pp1, and pp7 prophages and ASA (aggregation substance) gene expression in enterococcal isolates from renal transplant recipients (RTx) with different etiology of the end-stage renal failure. Prophages sequence was screened by PCR in strains of Enterococcus faecalis isolated from urine and feces of 19 RTx hospitalized at Medical University of Gdansk and 18 healthy volunteers. FLOW-FISH method with use of linear locked nucleic acid (LNA) probe was used to assess the ASA gene expression. Additionally, ability of biofilm formation was screened by crystal violet staining method. Presence of prophages was more frequent in fecal isolates from immunocompromised patients than in isolates from healthy volunteers. Additionally, both composition of prophages and ASA gene expression were related to the etiology of renal disease.

CD27 deficiency is associated with combined immunodeficiency and persistent symptomatic EBV viremia.

BACKGROUND: CD27 is a lymphocyte costimulatory molecule that regulates T-cell, natural killer (NK) cell, B-cell, and plasma cell function, survival, and differentiation. On the basis of its function and expression pattern, we considered CD27 a candidate gene in patients with hypogammaglobulinemia. OBJECTIVE: We sought to describe the clinical and immunologic phenotypes of patients with genetic CD27 deficiency. METHODS: A molecular and extended immunologic analysis was performed on 2 patients lacking CD27 expression. RESULTS: We identified 2 brothers with a homozygous mutation in CD27 leading to absence of CD27 expression. Both patients had persistent symptomatic EBV viremia. The index patient was hypogammaglobulinemic, and immunoglobulin replacement therapy was initiated. His brother had aplastic anemia in the course of his EBV infection and died from fulminant gram-positive bacterial sepsis. Immunologically, lack of CD27 expression was associated with impaired T cell-dependent B-cell responses and T-cell dysfunction. CONCLUSION: Our findings identify a role for CD27 in human subjects and suggest that this deficiency can explain particular cases of persistent symptomatic EBV viremia with hypogammaglobulinemia and impaired T cell-dependent antibody generation.

Isolation and characterization of a novel Enterococcus faecalis bacteriophage phiEF24C as a therapeutic candidate.

Vancomycin-resistant Enterococcus faecalis (VRE) has become a significant threat in nosocomial settings. Bacteriophage (phage) therapy is frequently proposed as a potential alternative therapy for infections caused by this bacterium. To search for candidate therapeutic phages against Enterococcus faecalis infections, 30 Enterococcus faecalis phages were isolated from the environment. One of these, virulent phage phiEF24C, which has a broad host range, was selected for analysis. The plaque-forming ability of phiEF24C was virtually unaffected by differences in the clinical host strains. Furthermore, the phage had a shorter latent period and a larger burst size than ordinary tailed phages, indicating that phiEF24C has effective lytic activity against many Enterococcus faecalis strains, including VRE. Morphological and genomic analyses revealed that phiEF24C is a large myovirus (classified as family Myoviridae morphotype A1) with a linear double-stranded DNA genome of c. 143 kbp. Analyses of the N-terminal amino acid sequences of the virion proteins, together with the morphology and the genome size, speculated that phiEF24C is closely related to other myoviruses of Gram-positive bacteria that have been used experimentally or practically for therapy or prophylaxis. Considering these results, phiEF24C may be a potential candidate therapeutic phage against Enterococcus faecalis infections.

Phage therapy: facts and fiction.

Recent examples of the use of bacteriophages in controlling bacterial infections are presented, some of which show therapeutic promise. The therapeutic use of bacteriophages, possibly in combination with antibiotics, may be a valuable approach. However, it is also quite clear that the safe and controlled use of phage therapy will require detailed information on the properties and behavior of specific phage-bacterium systems, both in vitro and especially in vivo. In vivo susceptibility of bacterial pathogens to bacteriophages is still largely poorly understood and future research on more phage-bacterium systems has to be undertaken to define the requirements for successful phage treatments.