Bacteriolytic Potential of Enterococcus Phage iF6 Isolated from "Sextaphag®" Therapeutic Phage Cocktail and Properties of Its Endolysins, Gp82 and Gp84

Abstract

The number of infections caused by antibiotic-resistant strains of bacteria is growing by the year. The pathogenic bacterial species Enterococcus faecalis and Enterococcus faecium are among the high priority candidate targets for the development of new therapeutic antibacterial agents. One of the most promising antibacterial agents are bacteriophages. According to the WHO, two phage-based therapeutic cocktails and two medical drugs based on phage endolysins are currently undergoing clinical trials. In this paper, we describe the virulent bacteriophage iF6 and the properties of two of its endolysins. The chromosome of the iF6 phage is 156,592 bp long and contains two direct terminal repeats, each 2108 bp long. Phylogenetically, iF6 belongs to the Schiekvirus genus, whose representatives are described as phages with a high therapeutic potential. The phage demonstrated a high adsorption rate; about 90% of iF6 virions attached to the host cells within one minute after the phage was added. Two iF6 endolysins were able to lyse enterococci cultures in both logarithmic and stationary growth phases. Especially promising is the HU-Gp84 endolysin; it was active against 77% of enterococci strains tested and remained active even after 1 h incubation at 60 °C. Thus, iF6-like enterococci phages appear to be a promising platform for the selection and development of new candidates for phage therapy.

Keywords: Enterococcus faecalis; Enterococcus faecium; Kochikohdavirus; Shiekvirus; antibiotic resistance; bacteriophage; endolysins; iF6; phage therapy.

Figure 1

(a) Morphology of Enterococcus phage iF6 plaques on a lawn of the E. faecium FS86 strain. (b) Transmission electron microscopy of Enterococcus phage iF6 virions with uncontracted (left virion) and contracted (right virion) tails. Virions are negatively stained with 1% (w/v) uranyl acetate. Scale bar is 50 nm.

Figure 2

Thermal and pH stability of Enterococcus phage iF6. (a) Phage titers after 1 h incubation at temperatures ranging from 5 to 95 °C. (b) Phage titers after 1 h incubation at pH values ranging from 2.2 to 10. The graphs were created in GraphPad Prism 8.4.3 with a 95% confidence interval. Five independent trials were performed for the experiments.

Figure 3

The growth curves of FS86 upon iF6 infection at different MOIs with additional Ca²⁺ or/and Mg²⁺. Growth kinetics of E. faecium FS86 upon infection at various MOI values in (a) the exponential and (b) the stationary growth phases. An uninfected culture was used as a control. Calcium and magnesium effects on the killing activity of iF6 in (c) the exponential and (d) the stationary phases of growth. The graphs were created with GraphPad Prism 8.4.3. Error bars represent the standard deviation of the means from three independent trials.

Figure 4

Phage infection parameters. (a) Adsorption and (b) one-step growth curve of Enterococcus phage iF6. The graphs were created with GraphPad Prism 8.4.3. Error bars represent the standard deviation for average values from three independent trials. The dotted line marks the interpolation according to (a) the exponential model and (b) the sigmoid model.

Figure 5

The Enterococcus phage iF6 chromosome map. Functionally assigned ORFs are highlighted according to their general functions (see the color scheme at the bottom). Abbreviations: DTRs—direct terminal repeats; RNR—ribonucleotide reductase; NTP pyrophosphorylase—nucleoside triphosphate pyrophosphorylase; NMN transporter—nicotinamide mononucleotide transporter; dNK—deoxyribonucleoside kinase; TerL—terminase, large subunit; NAMLAA—N-acetylmuramoyl-L-alanine amidase; MCP—major capsid protein; HCP—head completion protein; TSP—tail sheath protein; TTP—tail tube protein; VSR endonuclease—very short patch repair endonuclease; TAC—tail assembly chaperone; TMP—tape measure protein; NT—nucleotidyltransferase.

Figure 6

Genome-BLAST Distance Phylogeny (GBDP) phylogram based on the amino-acid sequences of the whole phage proteomes and inferred using formula D6. The nodes are colored according to the legend representing GBDP pseudo-bootstrap support values from 100 replications. The branch lengths are scaled in terms of the GBDP distance (formula D6). The Enterococcus phage iF6 is highlighted in light purple.

Figure 7

The pairwise whole-genome tBLASTx comparison of iF6 and the most closely related phages. The iF6 ORFs are colored according to their general function (see the color scheme at the bottom). Gray areas between the genome maps indicate the level of identity in the range of 40% to 100% (see the gradient scheme on the right).

Figure 8

Determination of packaging mechanism and genome termini. (a) Restriction analysis of iF6 DNA. M—molecular weight markers; red and light blue arrows indicate fragments containing the right and left ends of the iF6 chromosome, respectively. The original full-length gel is presented in Supplementary Information, Figure S5. (b) Sequencing chromatograms of the terminal regions of the PCR product sequences obtained with RAGE for the left and right ends. (c) Schematic representation of the iF6 phage chromosome.

Figure 9

Effect of HU-Gp82 and HU-Gp84 endolysins on the growth kinetics of E. faecium FS86. Enzymes were added to the cell suspension in the early exponential (a) and stationary (b) phases of growth. An uninfected culture of E. faecium FS86 was used as a control. The black arrows indicate the addition of endolysins. The graphs were created using GraphPad Prism 8.4.3. Error bars represent the standard deviation of the means from three independent trials.

Figure 10

The bacteriolytic activity of (a) HU-Gp82 and (b) HU-Gp84 endolysins at different pH values at 35 °C. The residual activity of (c) HU-Gp82 and (d) HU-Gp84 endolysins after 1 h incubation at different temperatures. The graphs were created with GraphPad Prism 8.4.3 with a 95% confidence interval. Five independent trials were performed in the experiments.