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

BMC Microbiology volume 25, Article number: 39 (2025) Cite this article

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.

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Introduction

Salmonella, belonging to the Gram-negative short rod bacteria, is one of the most common zoonotic pathogens. It can cause acute gastroenteritis, septicemia, and other systemic diseases in humans and animals, posing a serious threat to public health and safety [1]. Based on the variations in antigen types it carried, Salmonella can be classified into over 2600 serotypes, with the vast majority being Salmonella enterica [2]. In recent years, large-scale livestock and poultry farming has flourished in many countries. Salmonellosis is highly prone to horizontal transmission through contaminated water sources, feed, and feces, or vertical transmission through breeding [3]. Among different serotypes, Salmonella enterica serovar Typhimurium is one of the most common serotypes in clinical cases as a pan-susceptible serovar. It poses significant hazards to livestock and poultry growth, causing substantial losses to the farming industry, and it has also emerged as a food safety concern, jeopardizing humans health [4]. Meanwhile, amidst the widespread use of antibiotics, Salmonella strains are continuously evolving resistance, highlighting an urgent need within the farming industry for the development of effective alternatives to antibiotics [5].

Bacteriophages are bacterial viruses characterized by their simple structure, consisting only of nucleic acid and proteins, and they require host bacteria for replication. Research on bacteriophages and their bactericidal effects began as early as the beginning of the last century. However, studies on them gradually ceased with the advent of antibiotics. It was only until now, due to antibiotic overuse leading to the emergence of superbugs, that bacteriophage therapy has once again taken the forefront in the development of effective alternatives to antibiotics [6]. Compared to antibiotics, the advantages of bacteriophage therapy lie firstly in its widespread presence in nature, abundant quantity, and ease of isolation. Secondly, bacteriophages exhibit strong specificity towards host bacteria, typically infecting only one or a few strains, without negatively impacting other microbial populations within the same environment [7]. Thirdly, unlike chemical substances, a bacteriophage generates a large number of progenies after completing its own life cycle and repeats this process continuously. This feature provides a higher efficiency compared to antibiotics, which cannot self-amplify [8]. Fourth, once the host bacteria within the organism have been eliminated, the bacteriophages will also be metabolized and excreted from the organism, avoiding any residual presence within [9]. Finally, phages can impose evolutionary constraints on host bacteria during their interaction, requiring the host to cope with phage selection pressure by sacrificing growth rate, reducing virulence, and increasing sensitivity to antibiotics. This evolutionary constraint undoubtedly serves as an indirect yet effective way to help us combat bacterial infections [10]. Since the early 21st century, there has been an exponential growth in research on phage therapy due to the increasing demand for efficient alternatives to antibiotics [11]. Numerous studies have demonstrated that bacteriophages, as readily available and highly effective bacterial viruses, hold great potential in the prevention and treatment of common bacterial diseases [12].

This study utilized Salmonella enterica serovar Typhimurium as the host bacteria and isolated the lytic bacteriophage ZK22 from sewage. The biological characteristics of ZK22 were investigated, and its antibacterial effect was evaluated both in vitro and in vivo, aiming to provide novel strategies for the control of Salmonella infections.

Materials and methods

Bacterial strains and growth condition

This study involved a total of 47 bacterial strains, with S. Typhimurium strain SS10 isolated, identified, and preserved in our laboratory, specifically for bacteriophage isolation and biological characteristics identification. The strains P42, TX12, 06–8, 09–74, and 07–85 were kindly provided by Dr. Jianmin Zhang from the College of Veterinary Medicine, South China Agricultural University. The standard strains used in this study were purchased from American Type Culture Collection (ATCC) and Huankai Biotechnology Co., Ltd. The remaining strains were isolated, identified, and preserved by our laboratory. (Table 1). All strains were cultured on Luria Bertani agar (LB, Huankai Biotechnology Co., Ltd, Guangdong, China) plates at 37 °C for 24 h. Individual colonies were added to 10 mL LB broth (Huankai Biotechnology Co., Ltd, Guangdong, China) and incubated at 37 °C with shaking at 200 rpm for 24 h to create liquid cultures. Strains were preserved in 30% glycerol at -80 °C for long-term storage.

Table 1 Host range of phage ZK22
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Isolation and purification of bacteriophage

The following method was slightly modified based on the approach proposed by Kazibwe et al. [13]. A 10 mL sewage sample collected from a fresh market in southern China was initially filtered through gauze, followed by centrifugation at 12,000 rpm for 15 min to collect the supernatant, which was then filtered through a 0.22 μm syringe filter (Labgic Technology Co., Ltd, Beijing, China) and stored at 4 °C. For the subsequent steps, 5 mL of the filtrate, 5 mL of LB broth, and 100 µL of logarithmic-phase S. Typhimurium SS10 pure culture were mixed and incubated at 37 °C with shaking at 200 rpm for 12 h. After centrifugation of the culture at 12,000 rpm for 15 min, the supernatant was collected and filtered through a 0.22 μm syringe filter and stored at 4 °C. For plaque isolation, 200 µL of the filtered supernatant, 200 µL of logarithmic-phase host bacteria culture, and 5 mL of LB agar at around 50 °C were mixed and poured onto pre-solidified LB agar plates to create double-layered plates. After solidification of the top layer, the plates were inverted and incubated at 37 °C for 4–6 h to observe the formation of bacteriophage plaques.

A large and clear phage plaque was picked up and transferred into 1 mL of sterile saline magnesium buffer (SM buffer, Yuanye Bio-Technology Co., Ltd, Shanghai, China). The solution was dispersed at 37 °C and 200 rpm for 30 min, then filtered through a 0.22 μm syringe filter. After gradient dilution of the filtrate in sterile SM buffer, 200 µL of the diluted solution, 200 µL of S. Typhimurium SS10 in the logarithmic growth phase, and 5 mL of LB agar at around 50 °C were mixed. Then the mixture was poured onto a pre-solidified LB agar to create a double-layer plate, incubating inverted in 37 °C for 4–6 h. Then, large and clear phage plaque was selected for further purification. The purification process was repeated five times, ensuring plaque uniformity in shape and size to obtain a purified phage. The titer was determined using the same method above, with each dilution repeated three times.

Transmission electron microscope (TEM)

20 µL of purified bacteriophage solution (1 × 108 PFU/mL) was pipetted onto a copper grid and allowed to naturally adsorb for 5–10 min. Excess liquid was removed using filter paper, and the grid was left to air dry briefly. Subsequently, 20 µL of 2% phosphotungstic acid solution (NA) was applied onto the copper grid, and it was left to stand for 3–5 min. Excess liquid was again removed with filter paper. The grid was air-dried under an incandescent lamp and observed and photographed under a HT7700 transmission electron microscope (Hitachi, Japan) [14]. Fifteen individual micrographs were analyzed using ImageView software to estimate the average lengths of the bacteriophage head and tail.

Genome extraction and sequencing

The phage sample (1 × 108 PFU/mL) was passed through a 0.22 μm syringe filter. The filtrate was treated with 200 U Benzo DNase (NOVOPROTEIN SCIENTIFIC INC) and 0.1 mg/mL RNase A (Sangon Biotech) followed by heat inactivation of DNases at 65 ℃ for 10 min. Phage genome DNA was extracted by Qiagen MinElute Virus Spin Kit following the manufacture’s instruction. DNA concentration was quantified on a Qubit 3.0 Fluorometer (ThermoFisher Scientific) using Equalbit 1x dsDNA HS Assay Kit (Vazyme Biotech Co.,Ltd).

The extracted genomic DNA was sent to Chengdu Phagetimes Biotech Co. Ltd. (Sichuan, China) for genome sequencing. Shotgun libraries were constructed using VAHTS® Universal Plus DNA Library Prep Kit for Illumina (Vazyme Biotech Co., Ltd). The libraries were quality-checked by an Agilent 4200 Bioanalyzer and sequenced on the Illumina Nova Seq 6000 platform with 2 × 150 bp pair-end reads. To ensure the reliability of subsequent data analysis results, fastp was utilized to filter and control the quality of the raw sequencing data, trimming adapters, removing low-quality reads, and reads with high proportions of N, resulting in clean reads. (The sequencing data quality control information of ZK22 was provided in the supplementary material.) The clean reads were de novo assembled using the metaSPAdes software [15]. The assembled genome was then annotated for coding genes and tRNA using Prokka [16]. Subsequently, protein sequences were compared to the NR database using blastp to obtain information on sequences with high similarity in the NR database. Gene sequences were also compared to the Virulence Factor Database (VFDB, http://www.mgc.ac.cn/VFs/main.htm) using blast to predict virulence genes [17], while the presence of resistance genes on the genome was predicted using Resfinder to assess antibiotic resistance [18].

Using blastn on the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi), the top 50 phage genomes with high similarity to the genome sequence of phage ZK22 were selected. The phylogenetic tree was constructed using PhyloSuitev1.2.3 software to analyze the evolutionary relationships among these phages and the phage ZK22 [19].

Determination of host range

The host range and relative efficiency of plating (EOP) calculation of bacteriophage ZK22 was determined using the spot titration method [20]. After culturing 47 strains of bacteria to logarithmic phase, 200 µL of bacterial culture was mixed with 5 mL of LB agar at approximately 50 °C and poured onto solid LB medium under sterile conditions. After the solidification of agar, the purified bacteriophage solution was diluted in tenfold gradients to an estimated concentration of 102 PFU/mL. Each culture plate was consecutively spotted with six gradients, with each gradient being 10 µL of the dilution. After air-drying, the plates were inverted and incubated at 37 °C for 6 h to count bacteriophage plaques and calculate the relative EOP using the isolation host SS10 as reference. The lytic activity was defined as “high activity” for EOP value > 0.5, “moderate activity” for EOP value ≥ 0.1 and ≤ 0.5, “low activity” for EOP value < 0.1 [21]. The experiment was repeated three times.

Determination of optimal multiplicity of infection (MOI)

The logarithmic-phase host bacterial cultures were mixed with purified bacteriophage solutions at different MOIs: MOI = 103 to MOI = 10− 4. The mixture was then oscillated at 37 °C and 200 rpm for 6 h. After filtration through a 0.22 μm syringe filter, the filtrate was gradient-diluted with sterile SM buffer to prepare double-layer plates. Following incubation, the number of plaques on plates was counted with counts ranging between 30 and 300, and the average was calculated to determine the titer. The highest titer of plaque-forming units represents the optimal multiplicity of infection. The experiment was repeated three times.

One-step growth curve assay

The purified bacteriophage solution was mixed with the logarithmic-phase host bacterial culture according to the optimal multiplicity of infection [22]. After incubating at 37 °C for 10 min to allow adsorption, the mixture was centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatant was discarded to remove the unabsorbed phages, and the deposit was resuspended in 10 mL of preheated LB broth at 37 °C. The suspension was then incubated with shaking at 37 °C and 200 rpm for 120 min. Starting from 0 min, 500 µL of the mixed culture was sampled every 10 min and filtered through a 0.22 μm syringe filter. The sterile SM buffer was used to prepare a double-layer agar plate by gradient dilution for bacteriophage titer determination. The experiment was repeated three times.

The burst size (PFU/cell) = Phage titer at the late stage of lysis / Initial bacterial cells infected.

Temperature and pH stability assay

The purified bacteriophage suspension was diluted to 1.5 × 1010 PFU/mL using sterile SM buffer and aliquoted into 200 µL portions in sterile centrifuge tubes. These tubes were then placed in refrigerator at 4 °C and thermostat water bath at 25 °C, 37 °C, 50 °C, 60 °C, 70 °C, 80 °C. Samples were taken at 0 min, 20 min, 40 min, and 60 min and serially diluted with sterile SM buffer to prepare double-layer agar plates for bacteriophage titer determination. The experiment was repeated three times.

The pH of sterile SM buffer was adjusted to a range of 2–13 using HCl and NaOH solutions. Then, 100 µL of purified bacteriophage suspension at a concentration of 1 × 1011 PFU/mL was mixed with 900 µL of sterile SM buffer at different pH levels to achieve an initial titer of 1 × 1010 PFU/mL. After incubating at 37 °C for 1 h, the mixture was serially diluted and plated on double-layer agar plates for bacteriophage titer determination. The experiment was repeated three times.

In vitro bacteriostatic efficacy

The purified bacteriophage solution was mixed with 100 µL of logarithmic-phase host bacterial culture at a concentration of 108 CFU/mL in a 96-well plate according to MOI = 101, MOI = 100, MOI = 10− 1, and MOI = 10− 2 [23]. Simultaneously, 100 µL of logarithmic-phase host bacterial culture at a concentration of 108 CFU/mL was mixed with 100 µL of LB broth as a positive control. Furthermore, 200 µL of LB broth and 200 µL of sterile water were added as negative control and blank control, respectively. Each group had three replicates. The 96-well plate was incubated in a microplate reader at 37 °C with shaking at 200 rpm for 12 h. The OD600 was measured every 0.5 h.

In vivo therapeutic efficacy

SPF female BALB/c mice aged 6–8 weeks (n = 25, Zhuhai BesTest Bio-Tech Co,. Ltd, China) were divided into 5 groups, with a weight difference of less than 2 g within each group. In animal experiments, all mice were provided with adequate food, water, and a suitable living environment. At the end of the experiment, cervical dislocation was performed for euthanasia, and the animals did not experience any suffering throughout the process [24]. All procedures were guided by the Guidelines for euthanasia of laboratory animals (GB/T 39760 − 2021) and the ARRIVE guidelines.

Survival rates

All mice were deprived of food and water for 12 h prior to the experiment. Negative control group received an intraperitoneal injection of 0.1 mL of bacteriophage ZK22 at a concentration of 1010 PFU/mL, followed by a 0.1 mL sterile PBS injection after 1 h. Positive control group received an intraperitoneal injection of 0.1 mL of host bacteria SS10 at a concentration of 106 CFU/mL, followed by a 0.1 mL sterile PBS injection after 1 h. The remaining treatment groups were injected intraperitoneally with 0.1 mL of host bacteria SS10 at a concentration of 106 CFU/mL, followed by subsequent injections of 0.1 mL of bacteriophage ZK22 at concentrations of 107 PFU/mL, 108 PFU/mL, and 109 PFU/mL after 1 h, respectively. Water and food were immediately restored after the injection, and the mice were observed for 96 h to record disease occurrence and survival rates.

Bacterial loads

Blood, heart, liver, and spleen samples were collected from the negative control group, positive control group, and the treatment group with the highest survival rate to measure the bacterial load. The procedure was as follows: 0.1 g of tissue and 3 sterile steel beads were placed into a sterile 2 mL tube, followed by the addition of 0.9 mL of sterile PBS buffer. Samples were homogenized at 60 Hz for 300 s using a high-throughput tissue homogenizer. The homogenates were then serially diluted with sterile PBS solution, and 100 µL of each dilution was spread onto LB solid culture medium. The plates were inverted and incubated overnight at 37 °C in a constant temperature incubator. Colony counting was conducted the following day.

Relative expression of hepatic inflammatory factors

Liver samples were collected from the negative control group, positive control group, and the treatment group with the highest survival rate to measure the relative expression of hepatic inflammatory factors. Total RNA was extracted from the liver using Trizol reagent (Invitrogen, Carlsbad, CA). The RNA concentration and quality were measured using a microspectrophotometer Q3000 (Quawell Technology, Inc., Sunnyvale, CA). RT-PCR reagent kit (Takara Shuzo Co., Ltd., Kyoto, Japan) was used to generate cDNA. The RT-qPCR conditions were as follows: 95 °C for 5 min; 40 cycles of 95 °C for 10 s and 60 °C for 30 s; and a final stage of 95 °C for 5 s, 65 °C for 5 s, and 95 °C for 5 s. Subsequently, RT-qPCR was performed using the SYBR Green QuantiTect RT-qPCR Kit (Roche, South San Francisco, CA), and each sample was analyzed in triplicate. All primers used in this study are listed in Table 2.

Table 2 Primers for RT-qPCR amplification
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Statistical analysis

The data was analyzed using one-way analysis of variance (ANOVA) with IBM SPSS Statistics V25.0 software (SPSS Inc., Chicago, IL). Differences between groups were determined using Duncan’s multiple range test. Data was presented as mean ± standard deviation (SD), and differences among treatments were considered statistically significant when P < 0.05, and highly significant when P < 0.01. Data was presented using GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA).

Results

Isolation and morphology of phage ZK22

The bacteriophage ZK22 was isolated from sewage using S. Typhimurium SS10 as the host bacterium. Purified ZK22 bacteriophage formed uniform, circular, translucent plaques with halo zones on double-layer agar plates, measuring approximately 1 mm in diameter (Fig. 1A). Transmission electron microscopy revealed that bacteriophage ZK22 had a polyhedral symmetric head and a long non-contractile tail, with a head diameter of 62.86 ± 1.25 nm and a tail length of 164.54 ± 2.62 nm (Fig. 1B).

Fig. 1
figure 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×)

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Genomic analysis of phage ZK22

The whole-genome sequencing results revealed that the genome of bacteriophage ZK22 consisted of a double-stranded DNA with a total length of 47,066 base pairs, and a GC content of 45.71%. The complete genome sequence of the bacteriophage ZK22 was deposited in GenBank database, with the accession number of PQ126513. The complete genome of bacteriophage ZK22 was predicted to contain a total of 78 coding sequences (CDs), comprising 35 CDs encoding hypothetical proteins and 43 CDs showing significant homology to genes encoding known functional proteins, including replication-related proteins (DNA primase, DNA helicase, etc.), structural proteins (tail fibers protein, minor tail protein, major tail protein, etc.), packaging proteins (portal protein, terminase large subunit, etc.), and lytic enzymes (holin, endolysin, etc.). No tRNA-encoding genes were predicted. Additionally, no virulence genes or antibiotic resistance genes were identified (Fig. 2).

In the top 50 phage sequences with high similarity to the phage ZK22 genome identified through blastn, the sequence of ZK22 clustered with OQ267695 Salmonella phage Pu29 and MZ489634 Salmonella phage D10, which both are members of the Skatevirus genus of the class Caudoviricetes. Based on these results and the viral classification provided by the International Committee on Taxonomy of Viruses (ICTV), it was concluded that ZK22 belongs to the class Caudoviricetes, within the genus Skatevirus (Fig. 3).

Fig. 2
figure 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

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Fig. 3
figure 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

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Host range of phage ZK22

The spot titration results indicated that bacteriophage ZK22 exhibited lytic activity only against 20 strains of S. Typhimurium, while showing no lytic activity against S. Pullorum, S. Kentucky, S. Agona, S. Indiana, S. Corvallis, 12 strains of Escherichia coli and 9 strains of Lactobacillus. Meanwhile, the EOP values referenced from SS10 indicated that, among the 20 strains of S. Typhimurium, ZK22 exhibited moderate to high lytic activity against strains SS1-8 and SS12. To sum up, bacteriophage ZK22 is a highly specific phage targeting S. Typhimurium (Table 1).

Optimal MOI and one-step growth curve of phage ZK22

The optimal multiplicity of infection of bacteriophage ZK22 was determined to be 10− 2, resulting in the highest progeny phage titer of 1.29 × 1015 PFU/mL (Fig. 4A).

The one-step growth curve indicated that there was almost no increase in the titer from the start of adsorption until 10 min, suggesting that this period corresponded to the latent phase of the bacteriophage ZK22, during which replication and assembly had not yet been completed. Subsequently, from 10 to 80 min, the titer increased sharply, indicating that the bacteriophage ZK22 had been released from the host cells through lysis, marking this period as the burst period. Based on this, the burst size was calculated to be 393 PFU/cell (Fig. 4B).

Fig. 4
figure 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

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Temperature and pH stability assay of phage ZK22

After incubation for 60 min at 4 °C, 25 °C, and 37 °C, the potency of bacteriophage ZK22 remained relatively unchanged. After incubation at 50 °C for 60 min, the potency decreased from 1.5 × 1010 PFU/mL to 5.3 × 107 PFU/mL, resulting in an average loss of 2.45 log units. At 60 °C for 60 min, the potency decreased to 1.5 × 107 PFU/mL, with an average loss of 2.99 log units. At 70 °C for 60 min, it decreased to 1.1 × 107 PFU/mL, with an average loss of 3.15 log units. These results indicate that the loss of potency after heat treatment was relatively minimal. Complete inactivation of bacteriophage ZK22 was observed after treatment at 80 °C, indicating its excellent thermal stability (Fig. 5A).

Bacteriophage ZK22 exhibited high activity under pH conditions ranging from 3 to 12. Within the pH range of 3 to 5, the potency decreased from 1 × 1010 PFU/mL to approximately 107 PFU/mL, with a maximum loss of 2.82 log units. Within the pH range of 6 to 12, the potency decreased to around 108 PFU/mL, with a maximum loss of 1.84 log units. Notably, at pH 8, the potency decreased by only 0.88 log units and remained as high as 3 × 109 PFU/mL. This suggests that bacteriophage ZK22 possesses excellent acid-base stability (Fig. 5B).

Fig. 5
figure 5

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

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In vitro bacteriostatic efficacy of phage ZK22

The results indicated that in the positive control group, the OD600 of S. Typhimurium SS10 began to increase rapidly at 0 h and continued to grow, with an average OD600 of 1.1326 at the end of the experiment, indicating rapid proliferation. In contrast, the OD600 of co-cultures mixed with bacteriophage ZK22 at MOI = 101, MOI = 100, MOI = 10− 1, and MOI = 10− 2 were consistently lower than the positive control, demonstrating an inhibitory effect of bacteriophage ZK22 on the growth of S. Typhimurium SS10. Specifically, co-cultures with the optimal multiplicity of infection, MOI = 10− 2, showed a slight increase in OD600 only after 3.5 h followed by a decrease at 10.5 h, with an average OD600 of 0.4772 at the end of the experiment, which was the lowest among all the experimental groups, indicating the optimal inhibitory effect when bacteriophage ZK22 was co-cultured with S. Typhimurium SS10 at the optimal multiplicity of infection. In summary, bacteriophage ZK22 exhibited a significant inhibitory effect on the proliferation of S. Typhimurium SS10, which showed a dose-dependent efficacy (Fig. 6).

Fig. 6
figure 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

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In vivo therapeutic efficacy of phage ZK22

During the experimental period, all mice in the negative control group survived, exhibited good growth, and showed no significant clinical symptoms, indicating the relative safety profile of bacteriophage ZK22 when applied in vivo. The treatment group gradually displayed clinical symptoms after bacterial challenge, including disheveled fur, skin cyanosis, purulent discharge from the eye corners, and passing of yellow watery stools. All mice in the positive control group died within 72 h, as did those in the 107 PFU/mL bacteriophage treatment group. However, the survival rates for the 108 PFU/mL and 109 PFU/mL bacteriophage treatment groups were 40% and 60%, respectively, suggesting that bacteriophage ZK22 demonstrates therapeutic efficacy at a dose of 108 PFU/mL, with the survival rates increasing with dosage escalation (Fig. 7A).

Mice treated with 109 PFU/mL of bacteriophage ZK22 showed reduced bacterial loads in the blood, heart, liver, and spleen compared to the untreated positive control group. Specifically, bacterial loads in the blood, heart, and liver were significantly reduced by 1.71, 2.63, and 2.17 log units, respectively (P < 0.05) (Fig. 7B). This demonstrated that bacteriophage ZK22 administration in vivo effectively alleviated bacteremia and reduced bacterial colonization in the heart and liver caused by host bacteria SS10 in mice. Moreover, compared to the positive control group, mice treated with 109 PFU/mL of bacteriophage ZK22 exhibited significant decreases in inflammatory cytokines IL-2, IL-6, and TNF-α in the liver, similar to levels observed in the untreated negative control mice, indicating a pronounced effect of bacteriophage ZK22 in mitigating inflammation induced by host bacterial infection (P < 0.05) (Fig. 7C).

Fig. 7
figure 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

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Discussions

Since the 1940s, the discovery of antibiotics has made significant contributions to human health and livestock farming. However, the overuse and misuse of antibiotics have increasingly become significant risk factors for food safety and public health, primarily due to the emergence of antibiotic resistance [25]. Data suggests that by 2050, around 10 million people worldwide could die due to superbugs [26]. As early as the summit of the Group of 20 in 2019, it was made clear that urgent action was needed to address the global health threat posed by antibiotic resistance [27]. Among them, Salmonella, especially S. Typhimurium, is a significant concern in livestock and poultry farming due to its widespread distribution and health risk [28]. The increasing antimicrobial resistance of Salmonella strains, including S. Typhimurium, has raised concerns, complicating treatment options and threatening both animal and human health. Given these alarming trends, responding to the global challenge of antimicrobial resistance has become a critical priority.

Bacteriophage therapy has emerged as a novel strategy to address the issue of bacterial resistance. As bacterial viruses, bacteriophages possess the ability to lyse pathogenic bacteria, along with advantages such as ubiquity, host specificity, and safety. In comparison to antibiotics and chemically synthesized antimicrobials, bacteriophage therapy offers significant advantages, such as low side effects, high distribution levels, cost-effectiveness and efficacy superior to traditional treatments [29]. In summary, bacteriophages, as a novel biological agent, hold great promise for the prevention and treatment of common bacterial infections in livestock and poultry farming.

In this study, a lytic bacteriophage strain named ZK22 was isolated from sewage sample obtained from a fresh food market in Guangdong Province, China. Bacteriophage ZK22 forms clear, circular plaques with a halo on double-layer agar plates. Host range analysis revealed that phage ZK22 has a specific lytic activity against S. Typhimurium. This specificity renders it more effective in clinical treatments once the serotype is identified. The optimal multiplicity of infection represents the initial ratio of phage to host that yields the highest lytic efficiency. A lower optimal MOI indicates that a lower initial phage potency is required to lyse the maximum number of host bacteria, or to achieve the highest potency of progeny bacteriophages. This reduction in required initial phage potency lowers the future cost of phage commercialization. The optimal MOI of phage ZK22 is 10− 2, while for Pu20, a lytic phage isolated by Zhang et al., it is 10− 1 [30], indicating a lower efficiency compared to ZK22. Conversely, Ge et al. isolated the virulent phage S55 from poultry feces [31], which shares the same optimal infection multiplicity as ZK22, suggesting that fewer phages are needed to lyse a large number of host bacteria. One-step growth curve of phages is advantageous for revealing their lytic efficiency, with shorter latent periods and higher burst sizes indicating stronger lytic efficiency. The burst size of phage ZK22 surpasses many reported phages, such as phage fmb-p1 reported by Wang et al. with a latent period of approximately 20 min and a burst size of 77 ± 4 PFU/cell [32]; phage CKT1 reported by Cui et al. with a latent period of about 10 min and a burst size of 147 PFU/cell [33]; and phage vB_SalP_TR2 reported by Shang et al. with a latent period of around 15 min and a burst size of 211 PFU/cell [34]. This indicates that ZK22 has a short adsorption time, strong lytic ability, and higher lytic efficiency. The physicochemical resistance of bacteriophages is closely linked to their practical applications in production. Bacteriophage ZK22 maintains its efficacy nearly unchanged when exposed to temperatures of 4 °C, 25 °C, and 37 °C, indicating minimal temperature dependence for refrigerated storage, room temperature application, and in vivo use. Furthermore, ZK22 retains high activity even below 70 °C, suggesting its effectiveness in high-temperature environments. Additionally, ZK22 demonstrates sustained potency within a pH range of 3 to 12, indicating good tolerance to conventional chemical solvents. Bacteriophage ZK22 exhibits similar physicochemical resistance to bacteriophage SHWT1, isolated by Tao et al. [35]. Compared to the broad-spectrum bacteriophage L13 and SG3 isolated by Hong et al. [36], ZK22 demonstrates better temperature and pH stability.

The interaction between bacteriophages and bacteria exhibits a certain degree of concentration dependence, which is correlated with the respective growth stages of the host bacteria and bacteriophages [37]. Therefore, observing the continuous effects of bacteriophages on host bacteria at different concentrations is crucial. The in vitro bacteriostatic curve of bacteriophage ZK22 indicates that treatment groups containing bacteriophages exhibit varying degrees of bacteriostatic effects. The treatment group with the optimal multiplicity of infection mixture demonstrates the best bacteriostatic effect at the end of the experiment, and this effect shows a clear dose-dependent relationship. This is similar to the findings of Atterbury et al. with bacteriophage Φ10, where the OD value decreases with increasing MOI when bacteriophage Φ10 is co-cultured with its host S. Typhimurium 4/74, indicating a similar dose-dependent bacteriostatic effect to that of bacteriophage ZK22 [38].

One of the advantages of bacteriophage therapy is its ability to selectively target and kill specific pathogenic bacteria without disrupting the normal microbial communities colonizing the organism, thereby avoiding many side effects. Numerous studies have reported the safety and positive effects of bacteriophages in vivo applications [39, 40]. The study by Easwaran et al. demonstrated the beneficial effects of bacteriophage ΦEcSw in treating intraperitoneal infections in mice, resulting in a significant improvement in mice survival rates following treatment [41]. Bacteriophage ZK22, when administered via intraperitoneal injection to mice, did not cause any deaths or significant clinical symptoms, indicating the safety in vivo application. Treatment with bacteriophage ZK22 after 1 h of infection increased the survival rates of mice, consistent with the findings of Easwaran et al. After 96 h post-infection, the host bacterium was widely distributed in various organs of mice and initiated an initial inflammatory response. Treatment with phage ZK22 significantly reduced bacterial loads in blood and different organs of mice, and notably decreased the levels of IL-2, IL-6, and TNF-α in the liver. This indicates that phage ZK22 can effectively reduce the dissemination and colonization of the host bacterium in infected mice, alleviating the inflammatory response caused by the infection. Similarly, phage vB_EcoM-UFV13 isolated by Vinícius et al. showed comparable effects in a mouse model of E. coli-induced mastitis, significantly reducing local tissue bacterial loads and the relative expression level of IL-6 when MOI = 10, demonstrating significant therapeutic efficacy [42]. Taken together, the improved survival rates of infected mice, reduced bacterial loads, and lower relative expression levels of inflammatory factors suggest that phage ZK22 exhibits promising efficacy in treating acute S. Typhimurium infection in vivo.

Conclusions

A lytic bacteriophage ZK22 was isolated from fresh market sewage in southern China, exhibiting strong specificity towards Salmonella enterica serovar Typhimurium, with no virulence or resistance genes predicted. It demonstrated high lytic efficiency and maintained stable activity over a temperature range of 4 °C to 70 °C and a pH range of 3 to 12. It showed excellent bacteriostatic effects when cultured with its host bacteria under optimal multiplicity of infection at 37 °C in vitro. Furthermore, it enhanced the survival rates of mice infected with S. Typhimurium and significantly inhibited the proliferation and colonization of the pathogen within the mice, mitigating the inflammatory response induced by the infection. Moreover, the future research is needed to uncover the specific recognition and lysis mechanism of bacteriophage ZK22 towards S. Typhimurium.

Data availability

Sequence data that support the findings of this study have been deposited in the GenBank repository, with the accession number of PQ126513.

Abbreviations

LB:

Luria Bertani

SM:

Saline magnesium

TEM:

Transmission electron microscope

MOI:

Multiplicity of infection

CDs:

Coding sequences

VFDB:

Virulence factor database

PFU:

Plaque-forming units

CFU:

Colony-forming units

EOP:

Efficiency of plating

PBS:

Phosphate-buffered saline

ICTV:

International Committee on Taxonomy of Viruses

RT-qPCR:

Real-time quantitative reverse transcription polymerase chain reaction

ATCC:

American Type Culture Collection

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Acknowledgements

The authors would like to thank members of their laboratory for helpful and constructive advice.

Funding

Key Realm R&D Program of Guangdong Province (2020B0202080002). The National Natural Science Foundation of China (32172815).

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Author notes

  1. Yihong Sun and Qing Qu contributed equally to this work.

Authors and Affiliations

  1. Zhongkai University of Agriculture and Engineering, Guangzhou, 510225, Guangdong, China

    Yihong Sun, Qing Qu, Yanhua Huang, Shuo Zhou & Wei Wang

  2. College of Animal Science and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou, 510225, Guangdong, China

    Yihong Sun, Qing Qu, Yanhua Huang, Shuo Zhou, Hua Xiang & Wei Wang

  3. Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, Guangdong, China

    Yihong Sun, Qing Qu, Yanhua Huang, Shuo Zhou & Wei Wang

  4. Veterinary Medicine Institute, Veterinary Public Health Public Laboratory of Guangdong, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China

    Hua Xiang

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Contributions

Y.S. conceived and designed the work, performed the experiments, analyzed the data, prepared figures and wrote the main manuscript text. Q.Q. assisted with the experiment. W.W., S.Z. and H.X. supervised the work, reviewed and edited the manuscript. All authors read and approved the final manuscript.

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Correspondence to Shuo Zhou, Hua Xiang or Wei Wang.

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The animal study protocol was approved by approval of the Animal Management and Ethics Committee of the Zhongkai University of Agricultural Engineering (ethical approval number: ZHKUMO-2023-043).

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Sun, Y., Qu, Q., Huang, Y. et al. Isolation, characterization and therapeutic efficacy of lytic bacteriophage ZK22 against Salmonella Typhimurium in mice. BMC Microbiol 25, 39 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03772-4

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