Journal: Scientific Reports

Article Title: Isolation, functional characterization and antibiofilm properties of a lytic Enterococcus phage RG1 against multidrug resistant E. faecium

doi: 10.1038/s41598-025-21701-3

Figure Lengend Snippet: Isolation and characterization of the phage RG1. ( A ) Formation of plaques with a diameter of 2.1 ± 0.3 mm on Enterococcus faecium ATCC 35667 lawn by the phage RG1 using the double agar method. ( B ) Host Range analysis of phage RG1 against (a) E. faecium ATCC 35667 (b) E. faecalis (c) Pseudomonas aeruginosa (d) Klebsiella oxytoca (e) Burkholderia cepacia (f) Deinococcus radiodurans (g) Proteus mirabilis (h) Staphylococcus aureus. ( C ) Transmission Electron Microscopy-based morphological analysis of the phage RG1 showing an icosahedral head with a diameter of 53.96 ± 1.56 nm and a non-contractile tail with a length of 196.0 ± 6.86 nm. ( D ) Effect of different temperatures (for 1 h), ( E ) pH (for 1 h) and ( F ) chloroform concentrations (for 10 min) on the stability and viability of the phage RG1 was measured using 0.1 ml of ~ 10 8 PFU/ml of phage in double agar method. The X-axis denotes various environmental conditions, and the Y-axis indicates titer (Log10 PFU/ml) of the bacteriophage RG1. ND means ‘not detected’. Statistical analysis from three independent experiments was performed using GraphPad Prism software (version 5.01). The p values less than 0.05 and 0.01 were indicated as (*) and (**), respectively.

Article Snippet: The phage RG1 lysis kinetics against E. faecium ATCC 35667 at different MOIs suggested that a phage titer lower than optimal MOI is sufficient to inhibit the bacterial growth in liquid broth.

Techniques: Isolation, Transmission Assay, Electron Microscopy, Software

Journal: Scientific Reports

Article Title: Isolation, functional characterization and antibiofilm properties of a lytic Enterococcus phage RG1 against multidrug resistant E. faecium

doi: 10.1038/s41598-025-21701-3

Figure Lengend Snippet: The multiplicity of infection and one-step growth curve analysis of the phage RG1. ( A ) Estimation of phage titer (Log10 PFU/ml) at different MOIs of the phage RG1 against E. faecium ATCC 35667 by the double agar method. ( B ) Measurement of Phage titer (Log10 PFU/ml) at different time points and MOI of 0.001 to develop the one-step growth curve for the phage RG1, which indicates the latent period and burst size of this bacteriophage with the host E. faecium ATCC 35667. The experiments were performed in triplicate to calculate the Mean ± SD, and plotted.

Article Snippet: The phage RG1 lysis kinetics against E. faecium ATCC 35667 at different MOIs suggested that a phage titer lower than optimal MOI is sufficient to inhibit the bacterial growth in liquid broth.

Techniques: Infection

Journal: Scientific Reports

Article Title: Isolation, functional characterization and antibiofilm properties of a lytic Enterococcus phage RG1 against multidrug resistant E. faecium

doi: 10.1038/s41598-025-21701-3

Figure Lengend Snippet: Mapping of the phage RG1 genomic DNA. The circular whole genome map of the phage RG1 comprises 41,364 base pairs of linear dsDNA visualized with the Proksee tool ( http://stothard.afns.ualberta.ca/cgview_server/ ). Different Open reading frames (ORFs) were categorized into replication and DNA metabolism, Cell lysis, Structural proteins, Assembly proteins and Hypothetical proteins, are illustrated with different colors in the genome map.

Article Snippet: The phage RG1 lysis kinetics against E. faecium ATCC 35667 at different MOIs suggested that a phage titer lower than optimal MOI is sufficient to inhibit the bacterial growth in liquid broth.

Techniques: Lysis

Journal: Scientific Reports

Article Title: Isolation, functional characterization and antibiofilm properties of a lytic Enterococcus phage RG1 against multidrug resistant E. faecium

doi: 10.1038/s41598-025-21701-3

Figure Lengend Snippet: Comparative genomic analysis of the phage RG1 with other Enterococcus phages. ( A ) Heat map obtained from the VIRIDIC tool depicted the average nucleotide identity (ANI) percentage between RG1 and the 15 most humongous bacteriophages. The ANI values were computed based on a matrix of Hadamard values reflecting pairwise alignment coverage and percentage identity, with RG1 highlighted in red. ( B ) The intergenomic comparison through synteny between the phage RG1 and 15 other Enterococcus phages was conducted by using the EasyFig tool. The ORFs for Host cell lysis, DNA replication and metabolism, structural proteins, assembly proteins, additional functions and hypothetical proteins were depicted in red, green, yellow, orange, pink and grey colours, respectively. Genetic similarity profiles between the phage RG1 and other phages are depicted in greyscale, indicating percent homology.

Article Snippet: The phage RG1 lysis kinetics against E. faecium ATCC 35667 at different MOIs suggested that a phage titer lower than optimal MOI is sufficient to inhibit the bacterial growth in liquid broth.

Techniques: Comparison, Lysis

Journal: Scientific Reports

Article Title: Isolation, functional characterization and antibiofilm properties of a lytic Enterococcus phage RG1 against multidrug resistant E. faecium

doi: 10.1038/s41598-025-21701-3

Figure Lengend Snippet: Antibacterial and antibiofilm activity of the phage RG1 against E. faecium ATCC 35667. ( A ) Antibacterial activity of the phage RG1 was monitored by studying growth kinetics of E. faecium without and with bacteriophage at different MOIs (0.01, 0.1, 1, and 10). The optical density (600 nm) was measured at every 20 min up to 4 h in a sterile microtiter plate at 37 ℃. Three technical replicates were used to calculate Mean ± SD, and plotted. ( B ) Growth rate (% per hour) at each MOI was calculated from the data of Fig. A using formula (Nt = N0 * (1 + r)t) where Nt is absorbance at time t, N0 is absorbance at start of the growth, r is growth rate, and t is time passed. ( C ) Bacteriophage was co-incubated (CI) for 24 h with host bacterium as well as post-infected (PI) for 2 h and 4 h on pre-existing 24 h old biofilm at MOIs of 0.1 and 1. Antibiofilm activity of the phage RG1 against E. faecium was investigated using the crystal violet assay as described in the method. Four technical replicates were taken to calculate Mean ± SD, and analysed by t -test using GraphPad Prism (version 5.01). The p values less than 0.05, 0.01 and 0.001 were indicated as (*), (**) and (***), respectively.

Article Snippet: The phage RG1 lysis kinetics against E. faecium ATCC 35667 at different MOIs suggested that a phage titer lower than optimal MOI is sufficient to inhibit the bacterial growth in liquid broth.

Techniques: Activity Assay, Sterility, Incubation, Infection, Crystal Violet Assay

Journal: Scientific Reports

Article Title: Isolation, functional characterization and antibiofilm properties of a lytic Enterococcus phage RG1 against multidrug resistant E. faecium

doi: 10.1038/s41598-025-21701-3

Figure Lengend Snippet: Impact of different sugars on the stability of E. faecium and host-phage interaction. Effect of various concentrations (0.0%, 0.1%, 0.2%, 0.5%, and 1.0%) of different monosaccharide such as Ribose ( A ), Glucose ( B ), Fructose ( C ); disaccharides like Maltose ( D ), Sucrose ( E ), Lactose ( F ), Trehalose ( G ); and sugar alcohols such as Sorbitol ( H ) & Mannitol ( I ) on growth rate (% per hour) of E. faecium clinical strain 15667 was measured in the absence and presence of the phage RG1 at an MOI of 0.1. The growth kinetics was performed in triplicate for 4 h to generate the data for growth rate calculation, and represented as Mean ± SD. Statistical significance was estimated either by one-way ANOVA or t -test using GraphPad Prism (version 5.01). The p values less than 0.05, 0.01 and 0.001 were indicated as (*), (**) and (***), respectively.

Article Snippet: The phage RG1 lysis kinetics against E. faecium ATCC 35667 at different MOIs suggested that a phage titer lower than optimal MOI is sufficient to inhibit the bacterial growth in liquid broth.

Techniques:

Journal: Scientific Reports

Article Title: Isolation, functional characterization and antibiofilm properties of a lytic Enterococcus phage RG1 against multidrug resistant E. faecium

doi: 10.1038/s41598-025-21701-3

Figure Lengend Snippet: Antibacterial and antibiofilm activity of the phage RG1 towards the clinical isolates of E. faecium . ( A ) The growth kinetics of 19 clinical and one ATCC 35667 strain of E. faecium was performed in the absence and presence of the phage RG1 at an MOI of 0.1 in triplicate using 96-well microtiter plate for 280 min at 37 ℃ (Fig. S2). The growth rate (% per hour) was estimated from data of the growth curve as described in the methodology, and plotted for different clinical isolates of E. faecium . ( B ) Biofilm disruption activity of the phage RG1 against ATCC 35667 and 19 clinical isolates of E. faecium was measured using the crystal violet assay by 4 h incubation of bacteriophage (10 7 PFU/ml) on the 24 h old biofilm. Four technical replicates were used for this experiment to calculate Mean ± SD, and plotted. Statistical significance was estimated either by one-way ANOVA or t-test using GraphPad Prism (version 5.01). The p values less than 0.05, 0.01 and 0.001 were indicated as (*), (**) and (***), respectively.

Article Snippet: The phage RG1 lysis kinetics against E. faecium ATCC 35667 at different MOIs suggested that a phage titer lower than optimal MOI is sufficient to inhibit the bacterial growth in liquid broth.

Techniques: Activity Assay, Disruption, Crystal Violet Assay, Incubation

Journal: Scientific Reports

Article Title: Isolation, functional characterization and antibiofilm properties of a lytic Enterococcus phage RG1 against multidrug resistant E. faecium

doi: 10.1038/s41598-025-21701-3

Figure Lengend Snippet: Biocontrol assay using different MOIs of the phage RG1. Approximately 1 × 10 4 CFU/ml culture of E. faecium clinical strain 15166 was infected with phage RG1 at varying MOIs viz. 0.1, 1 and 10, and grown for 0, 4, 8 and 24 h. Viable cells (CFU/ml) were counted at each time point after spread plating and overnight incubation at 37 ℃. Experiment was carried out in triplicate to calculate Mean ± SD, and analysed for statistical significance by t -test using GraphPad Prism (version 5.01). The p values less than 0.05, 0.01 and 0.001 were represented as (*), (**) and (***), respectively.

Article Snippet: The phage RG1 lysis kinetics against E. faecium ATCC 35667 at different MOIs suggested that a phage titer lower than optimal MOI is sufficient to inhibit the bacterial growth in liquid broth.

Techniques: Infection, Incubation