Isolation, characterization and liposome-loaded encapsulation of a novel virulent Salmonella phage vB-SeS-01

Abstract

Introduction: Salmonella is a common foodborne pathogenic bacterium, displaying facultative intracellular parasitic behavior, which can help the escape against antibiotics treatment. Bacteriophages have the potential to control both intracellular and facultative intracellular bacteria and can be developed as antibiotic alternatives.

Methods: This study isolated and characterized vB-SeS-01, a novel Guernseyvirinae phage preying on Salmonella enterica, whose genome is closely related to those of phages SHWT1 and vB-SenS-EnJE1. Furthermore, nine phage-carrying liposome formulations were developed by film hydration method and via liposome extruder.

Results and discussion: Phage vB-SeS-01 displays strong lysis ability against 9 out of 24 tested S. enterica strains (including the pathogenic "Sendai" and "Enteritidis" serovars), high replicability with a burst size of 111 ± 15 PFU/ cell and a titre up to 2.1 × 10¹¹ PFU/mL, and broad pH (4.0 ~ 13.0) and temperature (4 ~ 80°C) stabilities. Among the nine vB-SeS-01 liposome-carrying formulations, the one encapsulated with PC:Chol:T80:SA = 9:1:2:0.5 without sonication displayed the optimal features. This formulation carried up to 10¹¹ PFU/mL, with an encapsulation rate of 80%, an average size of 172.8 nm, and a polydispersity index (PDI) of 0.087. It remained stable at 4°C and 23°C for at least 21 days and at 37°C for 7 days. Both vB-SeS-01 and vB-SeS-01-loaded liposomes displayed intracellular antimicrobial effects and could reduce the transcription level of some tested intracellular inflammatory factors caused by the infected S. enterica sv. Sendai 16,226 and Enteritidis 50041CMCC.

Keywords: Guernseyvirinae; Salmonella enterica; intracellular antibacterial effect; liposome; phage.

Figure 1

Morphological characteristics of vB-SeS-01. (A) Phage morphology under TEM. Scale bar: 50 nm. (B) Plaque forming ability. The phage suspensions were serially diluted (10⁸ to 10³ PFU/ml) and 10 μL of aliquot were spotted on different strains. *The strain Salmonella enterica sv. Sendai 16,226 was used as the propagation host of vB-SeS-01.

Figure 2

Characterization of vB-SeS-01. (A) One-step growth curve. Phage vB-SeS-01 was added to Salmonella enterica sv. Sendai 16,226 at a MOI of 0.001. (B) pH stability. The phage suspensions were resuspended with reaction buffers at different pH (from 2.0 to 13.0) and kept for 1 h at 4°C. (C) Thermal stability. The phage suspensions were pre-treated at different temperatures (4, 20, 30, 37, 50, 60, 70, 80, and 90°C) for 1 h. (D) Effect of chloroform on phage. 5% chloroform was added into the phage suspensions. Phage titers were determined after treatment. Data are means ± standard error of means (SEMs) of three parallel samples for each trial.

Figure 3

Phylogenetic analysis and genome comparison. (A) Phylogenetic tree based on the amino acid sequences of the Terminal Large Subunits (TLS). The dendrogram was constructed using the MEGA X program via the neighbor-joining method, with Poisson model, and tested by a bootstrap of 1,000. (B) Genome comparison between vB-SeS-01, vB_SenS_EnJE1 and SHWT1 using Easyfig v2.2.5. Green: structural proteins; red: host lysis; yellow: transcriptional regulation; blue: DNA replication and regulation; purple: other function; grey: unknown function. The grey scale refers to the identities of the amino acid sequences between the compared CDSs.

Figure 4

TEM of empty-loaded liposomes (A) and phage-loaded liposomes (B). Black arrows indicate empty liposome drops without phage-loaded which displayed as smooth spheres with a diameter of 85.26 and 195.06 nm, respectively. White arrow indicates a phage-loaded liposome which is rough and irregularly, and red arrows indicate phage virions located on its surface.

Figure 5

Particle size distribution of vB-SeS-01-liposomes at different temperatures. (A) 4°C; (B) 23°C; (C) 37°C. The different colors refer to the incubation times (in days) as indicated on the top right side. The intensity of the particle size distribution was measured using Zetasizer Nano ZSE (Malvern Instruments, UK), which measure the particle sizes of numerous liposome particles in three sets of 20 cycles and then yield the intensity values based on the distribution (Supplementary Table S5).

Figure 6

Stability of free vB-SeS-01 and vB-SeS-01-liposomes at different conditions. (A) At 4°C; (B) 23°C; (C) 37°C; (D) at simulated intestinal fluid. Data are means ± SEMs of three parallel samples for each trial.

Figure 7

Efficacy of free vB-SeS-01 and vB-SeS-01-liposomes against Salmonella enterica sv. Sendai 16,226 (A) and Enteritidis CMCC50041 (B) in HeLa cells. (A) At 1 h. (B) At 3 h. Data are means ± SEMs of three parallel samples for each trial (*p < 0.05).

Figure 8

Effects of free vB-SeS-01 and vB-SeS-01-liposomes on relative transcriptional level of cytokine factors infected by Salmonella enterica sv. Sendai 16,226 (A) and Enteritidis CMCC50041 (B). The free phages were added at a MOI = 10, and the phage-loaded liposomes were added at a concentration of 100 μg/mL with an equal phage titer of 1 × 10⁹ PFU/mL. The treated time was 1 h. Data are means ± SEMs of three parallel samples for each trial (*p < 0.05, **p < 0.01).