Potential of phage EF-N13 as an alternative treatment strategy for mastitis infections caused by multidrug-resistant Enterococcus faecalis.

Bovine mastitis is the most common and costly disease affecting dairy cattle throughout the world. Enterococcus faecalis is one of the environmental origin mastitis-causing pathogens. The treatment of bovine mastitis is primarily based on antibiotics. Due to the negative impact of developing antibiotic resistance and adverse effects on soil and water environments, the trend toward use of nonantibiotic treatments is increasing. Phages may represent a promising alternative treatment strategy. However, it is unknown whether phages have therapeutic effects on E. faecalis-induced mastitis. Thus, the objective of this study was to investigate the degree of protection conferred by a phage during murine mastitis caused by multidrug-resistant E. faecalis. Enterococcus faecalis was isolated from the milk of dairy cows with mastitis, and a phage was isolated using the E. faecalis isolates as hosts. The bactericidal ability of the phage against E. faecalis and the ability to prevent biofilm formation were determined in vitro. The therapeutic potential of the phage on murine mastitis was evaluated in vivo. We isolated 14 strains of E. faecalis from the milk of cows with mastitis, all of which exhibited multidrug resistance, and most (10/14) could form strong biofilms. Subsequently, a new phage (EF-N13) was isolated using the multidrug-resistant E. faecalis N13 (isolated from mastitic milk) as the host. The phage EF-N13 belongs to the family Myoviridae, which has short latent periods (5 min) and high bursts (284 pfu/cell). The genome of EF-N13 lacked bacterial virulence-, antibiotic resistance-, and lysogenesis-related genes. Furthermore, bacterial loading in the raw milk medium was significantly reduced by EF-N13 and was unaffected by potential IgG antibodies. In fact, EF-N13 could effectively prevent the formation of biofilm by multidrug-resistant E. faecalis. All of these characteristics suggest that EF-N13 has potential as mastitis therapy. In vivo, 1 × 105 cfu/gland of multidrug-resistant E. faecalis N13 resulted in mastitis development within 24 h. A single dose of phage EF-N13 (1 × 104, 1 × 105, or 1 × 106 pfu/gland) could significantly decrease bacterial counts in the mammary gland at 24 h postinfection. Histopathological observations demonstrated that treatment with phage EF-N13 effectively alleviated mammary gland inflammation and damage. This effect was confirmed by the lower levels of proinflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor-α in the mammary gland treated with phage EF-N13 compared with those treated with phosphate-buffered saline. Overall, the data underscored the potential of phage EF-N13 as an alternative therapy for bovine mastitis caused by multidrug-resistant E. faecalis.

vB-ApyS-JF1, the First Trueperella pyogenes Phage, Shows Potential as an Alternative Treatment Strategy for Trueperella pyogenes Infections.

Trueperella pyogenes (T. pyogenes) is an important opportunistic animal pathogen that causes huge economic losses to the animal husbandry industry. The emergence of bacterial resistance and the unsatisfactory effect of the vaccine have prompted investigators to explore alternative strategies for controlling T. pyogenes infection. Due to the ability of phages to kill multidrug-resistant bacteria, the use of phage therapy to combat multidrug-resistant bacterial infections has attracted attention. In this study, a T. pyogenes phage, vB-ApyS-JF1 (JF1), was isolated from sewage samples, and its whole genome and biological characteristics were elucidated. Moreover, the protective effect of phage JF1 on a mouse bacteremic model caused by T. pyogenes was studied. JF1 harbors a double-stranded DNA genome with a length of 90,130 bp (30.57% G + C). The genome of JF1 lacked bacterial virulence-, antibiotic resistance- and lysogenesis-related genes. Moreover, the genome sequence of JF1 exhibited low coverage (<6%) with all published phages in the NCBI database, and a phylogenetic analysis of the terminase large subunits and capsid indicated that JF1 was evolutionarily distinct from known phages. In addition, JF1 was stable over a wide range of pH values (3 to 11) and temperatures (4 to 50°C) and exhibited strong lytic activity against T. pyogenes in vitro. In murine experiments, a single intraperitoneal administration of JF1 30 min post-inoculation provided 100% protection for mice against T. pyogenes infection. Compared to the phosphate-buffered saline (PBS) treatment group, JF1 significantly (P < 0.01) reduced the bacterial load in the blood and tissues of infected mice. Meanwhile, treatment with phage JF1 relieved the pathological symptoms observed in each tissue. Furthermore, the levels of the inflammatory cytokines tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-6 (IL-6) in the blood of infected mice were significantly (P < 0.01) decreased in the phage-treated group. Taken together, these results indicate that phage JF1 demonstrated great potential as an alternative therapeutic treatment against T. pyogenes infection.

Microsphere-based suspension array for simultaneous recognition and quantification of multiple cancer-associated miRNA via DNAzyme-Mediated signal amplification.

Herein, a novel suspension array of polystyrene (PS) beads for simultaneous recognition and quantification of multiple cancer-associated microRNAs (miRNAs) using flow cytometry has been reported. The suspension array contained three moieties, streptavidin-modified PS beads, biotin-labeled substrate strands (17S) of the 17-8 DNAzyme and two split DNAzyme parts (PA, PB). 17S was labeled with 6-carboxyfluorescein (FAM) and Dabcyl on both sides of the ribonucleic acid. Once the target miRNAs appear, they can bind with the corresponding PA and PB to form an active secondary structure of DNAzyme. The active DNAzyme can cleave 17S and remove Dabcyl from the bead's surface, thus recovering the FAM's fluorescence intensity. Furthermore, the released target miRNA can autonomously move to the neighboring inactive DNAzyme for further cleavage, thus amplifying the fluorescence signal. Therefore, the target miRNAs can be quantified by reading the fluorescence intensity output from flow cytometry. The PS beads-based suspension array for the target miRNA in buffer shows good selectivity and high sensitivity. Via binding with a different pair of PA and PB, this suspension sensor array has successfully typed and quantified cancer-associated miRNAs of miR-21, miR-155, miR-335, and miR-122 in buffer and serum conditions.

A spherical nucleic acid-based two-photon nanoprobe for RNase H activity assay in living cells and tissues.

We report here a two-photon nanoprobe for the detection of RNase H activity in living cells and ex vivo tissues by combining a two-photon dye with a spherical nucleic acid (SNA) featuring a DNA/RNA duplex corona and a gold nanoparticle core.