Isolation, characterization and therapeutic efficacy of lytic bacteriophage ZK22 against Salmonella Typhimurium in mice

Abstract

Background: Salmonella enterica serovar Typhimurium is one of the most common serovars of Salmonella associated with clinical cases. It not only leads to diarrhea and mortality raised in livestock and poultry farming, but also poses a risk to food safety.

Results: In this study, a lytic bacteriophage named ZK22 was isolated and identified from sewage. It exhibited favorable capability against 20 strains of S. Typhimurium. The genome of ZK22 consisted of a double-stranded DNA with a total length of 47,066 base pairs and a GC content of 45.71%. A total of 78 coding sequences were predicted, with no virulence genes or drug resistance genes predicted. Based on blastn similarity analysis, ZK22 belongs to the genus Skatevirus of the class Caudoviricetes, as a long-tailed Siphovirus. Biological characteristics of ZK22 indicated that the optimal multiplicity of infection (MOI) of bacteriophage ZK22 to S. Typhimurium was 10^{- 2} (PFU/cell), with an incubation period of around 10 min and the burst size of 393 PFU/cell. The physicochemical resistance results of ZK22 demonstrated that it maintained stability under temperatures ranging from 4 °C to 70 °C and under pH conditions ranging from 3 to 12. After inoculating S. Typhimurium at 37 °C, co-culture mixed with the optimal multiplicity of infection exhibited the lowest OD600 within 12 h, demonstrating the exceptional antibacterial effect. Inoculation of phage ZK22 in mice infected with S. Typhimurium was performed to assess its therapeutic efficacy in vivo. The results showed that phage ZK22 at a dose of 10⁸ PFU/mL increased the survival rates of infected mice, effectively suppressed the dissemination and colonization of the host bacteria in the mice, and alleviated the inflammatory response caused by the infection.

Conclusions: In summary, bacteriophage ZK22 presents favorable application prospects as a potential biological agent for the control of Salmonella infections in livestock and poultry farming.

Keywords: Salmonella Typhimurium; Antibacterial effect; Biological agent; Lytic bacteriophage; Therapeutic efficacy.

Fig. 1

Morphology of phage ZK22. (A) Plaques of ZK22 on double-layer agar plates of S. Typhimurium SS10. (B) Transmission electron micrograph of ZK22. (magnification 40,000×)

Fig. 2

CG view of phage ZK22 genome annotation. All known functional proteins identified through blastp alignment have been labeled in the figure, with the hypothetical proteins are omitted from labeling

Fig. 3

Evolutionary relationship of phage ZK22. PhyloSuitev1.2.3 was used to construct the phylogenetic relationships of the top 50 bacteriophages with the highest similarity from the database. Phage with a red-font label and asterisk in the front represent ZK22

Fig. 4

MOI and one-step growth curve of phage ZK22. (A) Multiplicity of infection of phage ZK22. (B) One-step growth curve of phage ZK22

Fig. 5

Temperature and pH stability assay of phage ZK22. (A) Temperature stability. (B) pH stability

Fig. 6

In vitro bacteriostatic efficacy of phage ZK22. ZK22 was mixed with the LB cultures of the host bacterium SS10 at MOIs of 10¹, 10⁰, 10^− 1, 10^− 2, and 10^− 3. The optical density at 600 nm was measured every 0.5 h for a continuous period of 12 h

Fig. 7

In vivo therapeutic efficacy of phage ZK22. (A) Survival rates of mice treated with different titers of phage ZK22. n = 5. (B) Bacterial load in mice treated with 10⁹ PFU/mL of phage ZK22. n = 4. (C) Relative expression of hepatic inflammatory factors in mice. *P < 0.05