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Article

Salmonella Phage vB_SpuM_X5: A Novel Approach to Reducing Salmonella Biofilms with Implications for Food Safety

by
Xinxin Jin
1,2,†,
Xiuxiu Sun
1,†,
Qin Lu
2,
Zui Wang
1,2,
Zhenggang Zhang
2,3,
Xiaochun Ling
2,
Yunpeng Xu
2,3,
Ruiqin Liang
2,3,
Junjie Yang
1,2,
Li Li
1,2,
Tengfei Zhang
2,3,
Qingping Luo
2,3,4,* and
Guofu Cheng
1,*
1
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
2
Key Laboratory of Prevention and Control Agents for Animal Bacteriosis (Ministry of Agriculture and Rural Affairs), Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan 430064, China
3
Hubei Provincial Key Laboratory of Animal Pathogenic Microbiology, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan 430064, China
4
Hubei Hongshan Laboratory, Wuhan 430070, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microorganisms 2024, 12(12), 2400; https://doi.org/10.3390/microorganisms12122400
Submission received: 21 October 2024 / Revised: 15 November 2024 / Accepted: 20 November 2024 / Published: 22 November 2024
(This article belongs to the Special Issue Phage–Bacteria Interplay: Phage Biology and Phage Therapy)
Browse Figures
Versions Notes

Abstract

:
Salmonella, a prevalent foodborne pathogen, poses a significant social and economic strain on both food safety and public health. The application of phages in the control of foodborne pathogens represents an emerging research area. In this study, Salmonella pullorum phage vB_SpuM_X5 (phage X5) was isolated from chicken farm sewage samples. The results revealed that phage X5 is a novel Myoviridae phage. Phage X5 has adequate temperature tolerance (28 °C–60 °C), pH stability (4–12), and a broad host range of Salmonella bacteria (87.50% of tested strains). The addition of phage X5 (MOI of 100 and 1000) to milk inoculated with Salmonella reduced the number of Salmonella by 0.72 to 0.93 log10 CFU/mL and 0.66 to 1.06 log10 CFU/mL at 4 °C and 25 °C, respectively. The addition of phage X5 (MOI of 100 and 1000) to chicken breast inoculated with Salmonella reduced bacterial numbers by 1.13 to 2.42 log10 CFU/mL and 0.81 to 1.25 log10 CFU/mL at 4 °C and 25 °C, respectively. Phage X5 has bactericidal activity against Salmonella and can be used as a potential biological bacteriostatic agent to remove mature biofilms of Salmonella or for the prevention and control of Salmonella.

1. Introduction

Salmonella is a common foodborne pathogen that poses a risk to public safety [1]. It is a zoonotic pathogen that causes foodborne outbreaks and epidemics of varying degrees worldwide [2,3]. Salmonella is responsible for 80% to 90% of food-borne bacterial poisonings in China [4]. In Columbia and Puerto Rico, a total of 1135 individuals became ill due to consuming poultry contaminated with Salmonella in the 2021. Among them, 2 people died and 273 were hospitalized [5]. Salmonella pullorum is highly infectious in chicks under three weeks of age, with morbidity and mortality rates reaching up to 100%. This pathogen significantly impacts the performance and intestinal health of poultry, leading to severe economic losses for the poultry industry [6,7]. Currently, the best strategy to reduce pullorosis is to eliminate S. pullorum in chicken farms [8]. Chickens infected with Salmonella represent the main source of Salmonella infection in humans [9]. Salmonella usually contaminates a variety of foods such as milk, eggs, and meat.
Phages or bacteriophages are viruses that invade bacteria. Phages, which are avirulent, specific, and sensitive to drug-resistant bacteria [10,11], have been seldomly studied as a bacteriostatic agent to prevent pathogens from contaminating foods and to ensure food safety [12,13]. The phage mixture provides reference for the research and application of phages against Salmonella infection in chicken [14]. Bacteria in biofilms are not affected by antibiotics. As a result, they are difficult to treat. Few studies have demonstrated the effectiveness of phages and their derivatives in inhibiting S. typhimurium biofilms [15]. However, there is limited information on the application of phages to remove mature biofilms of S. pullorum and reduce S. pullorum contamination in chicken breasts in China. To the best of our knowledge, this is the first study that evaluates the effect of phages on the biofilm of S. pullorum, providing some reference for the control of the bacteria.
In this study, Salmonella phage X5 was isolated from sewage samples obtained from a chicken farm. The morphological and biological characteristics of the phage were determined, and the effect of the phage on the biofilm formation of Salmonella was evaluated. In addition, the bacteriostatic activity of the phage in milk and chicken breast meat was studied, which provided a theoretical basis for the development of bacteriostatic agents in foods.

2. Materials and Methods

2.1. Bacterial Strains and Media

S. pullorum 519 (CVCC 519) was purchased from the China Veterinary Culture Collection Center (CVCC, Beijing, China). The study used seven different strains of tested bacteria, including 80 Salmonella strains containing 3 different serotypes and 4 non-Salmonella strains (Table 1). These strains were stored at the Laboratory of Animal Pathology, College of Veterinary Medicine, Huazhong Agricultural University. Luria–Bertani (LB) broth and LB nutrient agar medium (Qingdao Haibo Biotechnology Company, Qingdao, China) were used for bacterial culture.

2.2. Isolation, Identification and Purification of Phage X5

Chicken feces and sewage samples were collected from a large-scale chicken farm in Hubei Province. Phage isolation was performed as previously described [16]. Briefly, all samples were centrifuged at 11,000× g for 15 min to remove large particulate matter and were then passed through a 0.22-μm filter (Millipore, Burlington, MA, USA) to remove bacterial cells and small impurities. Phage enrichment was achieved by mixing 0.1 mL of S. pullorum 519 solution with 1 mL of filtrate and 10 mL of LB broth. The mixture was cultured overnight in a 37 °C incubator, centrifuged at 11,000× g for 15 min, and passed through a 0.22-μm filter. The traditional double-layer agar (DLA) technique was used to verify the presence of phages [17]. DLA plates were incubated overnight at 37 °C. Positive samples were those with transparent plaques. Clear plaques were selected and purified five times to obtain purified phages. The phages were stored at 4 °C prior to use and stored at −80 °C with 30% glycerol for long-term storage.

2.3. Determination of Host Range of Phage X5

The host range of phage X5 was determined using the dropping method [18]. Except for Clostridium perfringens and Lactobacillus acidophilus, the transparent lawns of the tested strains were formulated with LB nutrient agar. The lawns of C. perfringens were prepared using TSC medium supplemented with 0.05% D-cycloserine, and those of L. acidophilus were prepared using MRS medium with 1.5% agar based on 42 °C. A phage suspension of 5 μL (108 PFU/mL) was dropped on the lawns of the strain. The plates were incubated overnight at 37 °C to form transparent lytic plaques. Plaque formation confirms the host’s susceptibility to phages. The phage host range of each test strain was assessed using a scoring system [19]. The experiment was repeated three times.

2.4. Determination of Biological Characteristics of Phage X5

Phage X5 was incubated under different conditions to determine its tolerance to various environments. The purified phage samples (1 mL phage, 1010 PFU/mL) were incubated at 28, 37, 50, 60, and 70 °C to determine the thermal stability, and the titers were determined by the DLA method at 20, 40, and 60 min [20]. To assess pH tolerance, 100 μL phage suspension (1010 PFU/mL) was mixed with 900 μL fresh LB broth at different pH values (pH 2–12, adjusted with HCl or NaOH). After oscillating the phage suspensions at 37 °C for 1 h (160 rpm/min), the phage titer was determined using the DLA technique [21,22].
According to the infection ratio of 1000, 100, 10, 1, 0.1, 0.01, and 0.001, the mixture of Salmonella CVCC519 and phage X5 was supplemented with LB broth, so that the final volume of each tube was the same. Each treatment had three replicates. The phage titer was determined by the DLA method in a shaker (150 rpm) at 37 °C for 4 h [23]. Chloroform was mixed with phage X5 at 0, 25, and 50% [24]. Each mixture was incubated at 220 rpm for 1 h at 37 °C. Phage titer was determined by the DLA technique. The experiments were carried out in triplicate.

2.5. Adsorption Rate Determination

In this experiment, 3 mL of the logarithmic growth stage of S. pullorum (CVCC 519) was transferred into a centrifuge tube. Subsequently, phage X5 (3 mL) was added according to the optimal multiplicity of infection (MOI; 0.01). The mixture was placed on a shaking table at 37 °C (150 rpm/min) and incubated for 50 min. We then removed 200 μL of the mixture every 5 min and placed it on ice for 1 min. The mixture was centrifuged at 12,000 rpm/min for 1 min, and the phage titer in the supernatant was determined using the DLA technique. The experiment was repeated three times. The phage adsorption rate was calculated using the following formula:
Phage adsorption rate = [(total number of initial phages − number of unadsorbed phages)/total number of initial phages] × 100% [25].

2.6. One–Step Growth Curve of Phage X5

One–step growth curve tests were performed as previously reported [26]. The phage lysate with a MOI of 0.01 and its host bacteria were added to LB broth medium and incubated at 37 °C for 10 min. The mixed medium was centrifuged at 12,000 rpm for 8 min, the supernatant was discarded, and the precipitate was washed three times with LB broth medium. Subsequently, 10 mL of preheated LB liquid medium at 37 °C was added to the precipitate and thoroughly mixed. The culture was mixed by oscillation at 160 rpm at 37 °C. Samples (100 μL) were collected every 10 min for 120 min and diluted in LB broth to determine the phage titer. The filtrate titer was determined by the DLA technique. The latency period is defined as the shortest time required for the phage to adsorb to the host bacteria and release progeny phages. The phage burst size represents the late phage burst titer divided by the concentration of host bacteria at the initial infection stage [27]. The experiment was repeated three times.

2.7. Transmission Electron Microscopy (TEM) of Phage X5

Freshly prepared phages (1010 PFU/mL) were centrifuged at 45,000× g for 2 h at 4 °C and precipitated with 500 μL of 0.1 M ammonium acetate. The phage drops were placed on a mesh for 10 min and stained with 2% (w/v) phosphotungstic acid for 15 min. The stained phages were photographed by TEM (Hitachi H–7000FA, Tokyo, Japan) at 75 kV [28]. The head diameter and tail length of phage X5 were measured using Digital Micrograph Demo 3.9.1 (Gatan, Inc., Pleasanton, CA, USA).

2.8. Phage X5 DNA Extraction and Genome Sequence Analysis

The extraction of phage X5 genomic DNA was performed as previously reported [29] and sequenced in an Illumina HiSeq platform (Illumina Inc., San Diego, CA, USA) [30]. Assembly of the reads was performed using SPAdes (version 3.12.0) software [31].
GeneMarkS (http://exon.gatech.edu/GeneMark/, accessed on 5 May 2024) and RAST (https://rast.nmpdr.org/rast.cgi, accessed on 5 May 2024) were used to predict the genome sequence of phage X5. The phage X5 genome map was generated by CGviewServer (http://cgview.ca/ accessed on 7 May 2024) [32]. The comprehensive antibiotic resistance database (CARD, http://arpcard.mcmaster.ca, accessed on 10 May 2024) and virulence factor database (VFDB, http://www.mgc.ac.cn/VFs/, accessed on 10 May 2024) were used to screen antibiotic resistance and virulence factor–related genes, respectively. A visual genome map of phage X5 was generated using an online website (https://cgview.ca/, accessed on 15 May 2024). The BLASTn webline website (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 May 2024) was used to compare the nucleotide sequences of phages and search for highly similar phages in GenBank. BLASTP was used to search the NR (Non–Redundant) protein database and identify the functional annotation of CDS (Coding sequence) [33,34]. The functions of open reading frames (ORFs) were annotated using BLASTP and the Rast database. Using the virus classification of the International Committee on Taxonomy of Viruses and BLASTn of NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 May 2024), their terminal enzyme large subunits were selected to show significant similarity. MEGA 10 was used for phylogenetic and evolutionary analyses [35]. Visual genome comparison of phage X5 and GSP044 was performed using Mauve 2.3.1 [36]. The whole genome sequence of phage X5 has been uploaded to NCBI database (GenBank number: PP502935).

2.9. Ability of Phage X5 to Reduce Salmonella Biofilm Formation

To evaluate the impact of phage X5 on biofilm formation, this study referred to previous research and made slight modifications to the methodology [37,38,39]. First, the concentration of fresh CVCC519 was adjusted to OD600 = 0.5 (about 1 × 109 CFU/mL). Second, 1 mL bacterial solution was added to each well of the 24–well cell culture plate, followed by 1 mL LB medium. Third, following three days of incubation at 37 °C, the medium was discarded, the wells were washed with 1 mL sterile phosphate buffered saline (PBS) three times, and phage X5 (105 PFU and 107 PFU) was added. Fourth, after incubation at 37 °C for 6 h, the media were discarded and washed three times with sterile PBS. Fifth, the bacterial load in the biofilm was measured. For the biofilm determination assay, following the addition of 1 mL of 95% methanol per well and incubation for 30 min, the fixed solution was discarded, and each well was washed three times with PBS. Subsequently, 1 mL crystal violet solution (1%) was added, and the plate was incubated for 30 min at room temperature. After washing with PBS three times, 1 mL of anhydrous ethanol was added for decolorization. Absorbance was measured at OD600. The bacteria were incubated, and the phage was replaced with an equal amount of SM buffer (Tris/NaCl/MgSO4), stained with crystal violet, and decolorized with ethanol. Absorbance was measured at OD600. Three parallel samples were set up for each experiment, and the experiments were repeated three times.

2.10. Inhibitory Effect of Phage X5 on Salmonella in Foods

The effects of the phage on Salmonella in different food substrates were evaluated in raw chicken breast and milk from a supermarket in Wuhan City.
Milk was confirmed to be aseptic by plate coating. Milk (9.8 mL) was mixed with 100 μL phage X5 (108 or 109 PFU/mL) and inoculated with 100 μL S. pullorum 519 (106 CFU/mL, PBS suspension). The control groups consisted of 200 μL PBS. The mixtures were incubated at 25 °C and 4 °C. Samples were collected at 0, 2, 4, 6, 8, 18, and 24 h, and the recoverable bacteria in milk were detected by the Salmonella Shigella (SS) Agar plate counting method [40,41].
The raw chicken breasts were divided into small pieces (2 × 2 cm), placed on a clean workbench, and irradiated by UV for 20 min to ensure disinfection and sterilization. then, 100 μL log–growth Salmonella CVCC 519 (106 CFU/mL) was added to each experimental sample and incubated at room temperature for 15 min. Subsequently, 100 μL of the phage (108 PFU/mL, MOI = 100 and 109 PFU/mL, MOI = 1000) was added, while an equal amount of sterile saline was added as a negative control. The treated samples were incubated at 4 °C for 0, 2, 4, 8, 18, and 24 h. Each piece of chicken was cut with sterile scissors and added to 1 mL sterile normal saline and mixed by oscillation for 3 min. The amount of Salmonella on the chicken pieces was determined by the dilution coated plate counting method [16].

2.11. Statistical Analysis

Data were statistically analyzed using GraphPad Prism 8.0.1 (San Diego, CA, USA). Results are presented as mean ± standard deviation (SD), and significance analysis between groups was performed using one–way ANOVA. Statistical significance was set at p < 0.05.

3. Results

3.1. Isolation and Identification of Salmonella Phage

A strain of Salmonella phage was isolated from the sewage samples of a chicken farm. The morphology and size of the plaque were similar (Figure 1A). The plaque, which was transparent and had no halo, was labeled vB_SpuM_X5 (X5). Phage X5 had a typical icosahedral structure with a head diameter of about 130 nm, a tail diameter of about 210 nm, and a retractable tail, consistent with the characteristics of Myoviridae (Figure 1B).

3.2. Host Range of Phage X5

Three serotypes of Salmonella were used to test the host range of phage X5. Host range analysis showed that phage X5 could form a transparent phagocytic circle when co–cultured with 70 strains of Salmonella. The cleavage rate reached 87.50% (70/80). However, phage X5 could not infect other genera of bacteria (Table 1). Additionally, phage X5 could not infect all the Gram–negative bacteria tested (Klebsiella pneumoniae and Escherichia coli) or two of the Gram–positive strains (Lactobacillus acidophilus and Clostridium perfringens).

3.3. Biological Characteristics of Phage X5

Figure 2 shows the biological characteristics of phage X5. The initial titer of phage X5 was 1010 PFU/mL. The titer of phage X5 was stable at 28 °C and 37 °C (Figure 2A). When the temperature was raised from 50 °C to 60 °C, the titer of the phage decreased and subsequently stabilized. The titers dropped sharply from 0 to 20 min, and the rate of the decline decreased from 20 to 60 min, whereas the phages were completely inactivated after 20 min at 70 °C. Figure 2B shows that phage X5 remained active and stable at pH 4–12; however, its stability decreased sharply and its titer could not detected at pH 2. Figure 2C shows the optimum MOI of phage X5. Phage X5 had the highest titer at MOI = 0.01; hence, the optimal MOI of phage X5 was 0.01, indicating that a small quantity of phages has the potential to lyse a significant number of bacteria, resulting in optimal titers.
Figure 2D shows the results of the chloroform sensitivity test. At ≤25% chloroform, the phage X5 titer was stable and high. At 50% chloroform, phage X5 was significantly reduced, but not completely inactivated.

3.4. Adsorption Rate

The adsorption rate of phage X5 increased from 0 to 20 min and reached its peak at 20 min with an adsorption rate of 81.0%. After 20 min, the adsorption rate began to decline (Figure 2E).

3.5. One–Step Growth Curve

Figure 2F shows a one–step growth curve of phage X5 at MOI = 0.01. The latent period was 10 min, the lysis period was 10–70 min, and the number of phages increased rapidly. In addition, the mean burst size of phage X5 was estimated to be 120 PFU/cell.

3.6. Whole–Genome Sequencing and Coding Gene Prediction of Phage X5

The genome sequencing results for phage X5 are shown in Figure 3. The genome consists of 118,240 bp of double–stranded DNA with a GC content of 39.40%. The protein sequences of all genes were compared with those in CARD and VFDB database. No pathogenic factors, resistance genes, genes related to phage lysogenicity, or virulence genes were found, indicating their safety. BLAST–N analysis showed that the X5 genome had the highest homology with Salmonella phage S124 (GenBank: OK108607) of the Myoviridae family, with 98.51% homology.
Figure 4 shows the multi–genome alignment and phylogenetic tree analysis of phage X5 and Salmonella phage S124, ABTNLsp, E22, SSP1, SE8, SE11, GSP044, Th1, and L6jm.
GSP044 and X5 with the highest similarity were selected for genomic collinearity analysis. Some genes were in different regions, indicating that phage X5 may exhibit gene rearrangement (Figure 5). Similar color blocks represent putative homologous blocks. The height of the internal lines in the block represents the conservatism of the average sequence in the region, and the higher the height, the better the conservatism. The white area inside the block represents a region where homologous blocks cannot be found in other genomes. The top and bottom of the block represent the justice chain and the antisense chain. Red usually indicates a mismatch between two genomes, and green indicates a match. Phage GSP044 (GenBank: OP394141) was isolated from Huizhou City, Guangdong Province, China.
Table 2 shows the protein analysis of phage X5. The phage genome consists of several gene cluster modules: DNA replication; repair and modification enzymes such as DNA helicase (ORF124), DNA replication primase (ORF121), and DNA polymerase (ORF122); DNA packaging proteins such as terminase small subunit (ORF95), portal protein (ORF150), and terminase large subunit (ORF152); lysis compounds such as lysozyme (ORF41) and holin (ORF42); and structural proteins such as tail fiber protein (ORF142), baseplate protein (ORF117), capsid protein (ORF147), and scaffold protein (ORF101). ORF100 encodes the phage principal protein. ORF99 encodes HNH endonuclease, an intron protein with sequence tolerance and site specificity. Thymidylate synthase (ORF93) is involved in nucleotide metabolism. The X5 genome encodes some additional proteins, such as serine/threonine phosphatase (ORF36) for amino acid biosynthesis. ATP-dependent helicase (ORF124) is involved in fundamental biological processes such as transcription, proliferation, and repair of DNA. ORF143 encodes the major tail protein of phage, while ORF142 encodes the tail fiber of phage. The phylogenetic tree of X5 was constructed based on the nucleotide sequence of the terminase large subunit (ORF152). The location and homology of the major ORFs in phage X5 and GSP044 are shown in Table 2. It is worth noting that the X5’s ORF99 does not exist in GSP044, which is thought to be a gene insertion.
No phage transposase, toxins, excision enzyme homology, virulence factors, or repressors were predicted in the X5 genome. According to the sequencing results, phage X5 is a novel phage.

3.7. Scavenging Effect of Phage X5 on Bacterial Biofilm

To evaluate whether phage X5 can destroy the biofilm formed by S. pullorum 519, the biofilm treated with phage X5 was stained using the CV method. Phage X5 significantly removed the biofilm, even at low concentrations (105 PFU/mL; Figure 6A,B (p < 0.001). Compared with the control group, the phage–treatment group had a significantly lower bacterial load (Figure 6C) (p < 0.001). This finding suggests that phage X5 can effectively reduce the biofilm formed by S. pullorum 519. Phage X5 effectively reduced the number of bacteria in the biofilm (Figure 6D).

3.8. Inactivation of Salmonella in Different Food Models by Phage X5

The bactericidal power of phage X5 was evaluated in solid foods (raw chicken) and liquid foods (milk).

3.8.1. Bacteriostasis of Phages in Milk

The inhibitory effect of phage X5 on Salmonella in milk is shown in Figure 7A.
Phage X5 at MOI of 100 and 1000 reduced live Salmonella counts (S. pullorum) compared with untreated controls. At 4 °C, the number of bacteria in the control group did not increase with time and remained at about 4 log10 CFU/mL. Following incubation at 4 °C for 24 h, compared to the control group, the number of viable bacteria in the MOI = 100 and MOI = 1000 groups were reduced by 0.72 log10 CFU/mL (p < 0.01) and 0.93 log10 CFU/mL (p < 0.001), respectively (Figure 6A). The maximum antibacterial efficiency was 80.70 and 88.10%, respectively.
At 25 °C, the inhibitory effect of phage X5 on Salmonella in milk was similar to that at 4 °C. The number of Salmonella in the control group increased significantly with prolonged time within 24 h, from 4.21 log10 CFU/mL to 8.23 log10 CFU/mL (Figure 7B). Salmonella counts were reduced by 0.66 log10 CFU/mL (p < 0.01) and 1.06 log10 CFU/mL (p < 0.001) with MOI of 100 and 1000, respectively. The inhibitory effect was greater at MOI = 1000 than at MOI = 100.

3.8.2. Bacteriostasis of Phages in Chicken Breast

The inhibitory effect of phage X5 on host bacteria on chicken surface is shown in Figure 8. At 4 °C, at 2–24 h phage treatment with an MOI of 100, the Salmonella count on chicken breast meat decreased from 4.6 log10 CFU/mL to 3.47 log10 CFU/mL (p < 0.01; Figure 8A). After treatment with a MOI of 1000 for 2–24 h, the Salmonella count on chicken breast decreased from 4.6 log10 CFU/mL to 2.18 log10 CFU/mL (p < 0.001) (Figure 8B). At 25 °C, the total number of Salmonella bacteria in each group gradually increased with time. At 2 h, the number of Salmonella counts was significantly lower in the phage–treated groups (MOI = 100 and 1000) than in the control group. At 24 h, the total amount of Salmonella bacteria in the phage–treated groups decreased by 0.81 log10 CFU/mL (MOI = 100) and 1.25 log10 CFU/mL (MOI = 1000) compared with the control group. The results showed that phage X5 had an adequate bacteriostatic effect on the chicken surface. Additionally, the bacteriostatic effect was greater at MOI = 1000 than at MOI = 100.

4. Discussion

Phages can efficiently lysate pathogenic bacteria. The application of phages reduces the need for antibiotics and promotes food safety. Phages have been widely used in clinical medicine, animal husbandry, aquaculture, and agriculture [3,42].
S. pullorum causes pullorum disease, which persists in adult chickens without obvious clinical symptoms and can spread vertically and horizontally, endangering the development of the poultry industry [43,44]. Salmonella phage CKT1 effectively controls the vertical transmission of S. pullorum in adult broilers [45]. Specifically, phage CKT1 significantly reduces weight loss in chickens infected with S. pullorum by regulating the abnormal intestinal flora and can be used as a potential substitute for antibacterial growth promoters in poultry farms [46]. Controlling S. pullorum in chicken farms is an effective strategy to prevent human infection.
The application of phages for the control of bacterial pathogens in foods is an emerging area. The commercial interest in this area has gradually increased since the FDA approved the limited use of phages in fresh foods to control Listeria monocytogenes [47]. Phages have host specificity, which is an important factor limiting the therapeutic efficacy of phages against bacterial infections [48]. Therefore, phages with broad host spectrum have broad application prospects. A broad–spectrum Salmonella phage capable of lysing five Salmonella serotypes has been isolated [49].
As research advances, the issues of phage safety, stability, and the development of bacterial resistance will be resolved, and the screening of novel phages and mixed preparations will become a hot spot in the antibacterial realm [50]. The latency of phage X5 was shorter than those of other phages reported, which may be related to the high lytic activity of phage X5 [51,52]. Phages with short latency can lyse more bacteria in a short period of time; hence, they are suitable for biological control [53,54].
Phage X5 was stable at pH 4–12 and had high pH tolerance. The stability of phage X5 in acidic and alkaline conditions allows it to be used in food with different pH values. Phage X5 had better bacteriostatic effects in milk than in chicken breast, which may be due to the adsorption of phage to bacteria in a liquid food matrix as result of the fluidity of milk [55]. Phage X5 has a binary lysis system composed of holin and lysozyme, which destroy the cell membrane and cell wall of bacteria, respectively, and has a completely different mechanism of action from antibiotics [56,57]. The tail fiber protein is responsible for the specific initial recognition of host bacteria and can be a potential biological cognitive element for detecting bacteria [58,59]. The application of phages and their derivatives, such as endolysin, as biological control agents has been documented [60].
In the study, the genomic and biological characteristics of the isolated phage X5 were evaluated. Phage X5 has a broad host range, high tolerance to extreme conditions, and strong antibacterial ability. The lysis rate of phage X5 was 87.50% (70/80), which was higher than that of phage GSP044, at 81.25% (39/48) [61]. For phage-insensitive Salmonella strains, it is speculated that the tail protein of phage X5 cannot recognize these bacteria. The specific recognition mechanism or the use of phage cocktails will be investigated in the future [62]. Additionally, phage X5 had an adequate scavenging effect on the mature bacterial biofilm and effectively reduced the bacterial load in the biofilm. Compared with plant essential oils, enzyme preparation, and lactic acid bacteria, although they have antibacterial effects, their antibacterial spectrum is relatively wide, which may affect or destroy the balance of normal flora. Phages have the advantages of strict host specificity, strong exponential proliferation ability as well as high safety and ecological friendliness in the field of anti–pathogenic biofilms, which gives phages broad application prospects in the field of antimicrobial therapy [63]. The bacterial load was significantly lower in the treatment groups (MOI of 100 and 1000) than in the control group at 24 h, indicating that phage X5 could significantly inhibit the host bacteria in the milk matrix, whether at 4 °C or 25 °C. These findings suggest that phage X5 is a promising and effective biological control agent for controlling Salmonella infection in the food industry. At present, we have studied the recognition receptor of phage X5, preliminarily analyzed the receptor site, and studied the phage resistance.
The application of phages in the food industry has considerable potential but also faces some challenges and comes with potential risks, such as consumer acceptance, security assessment, technical limitations, potential drug resistance, and effective use in food production. Therefore, there are no approved phage food bacteriostatic agents in China. However, the phage product of Qingdao Phagepharm Bio–Tech Co., Ltd. (Qingdao, China) has been approved by The U. S. Food and Drug Administration (FDA). Considered Generally Recognized as Safe (GRAS), this phage product is expected to play an important role in the international market (https://www.fda.gov/media/178868/download, accessed on 30 May 2024). Our future work will focus on the prevention of Salmonella infection by X5 lyase and on the identification of receptor–binding proteins. Phage X5 has the potential to be used in phage preparations for the prevention of Salmonella infection in the food, agriculture, medical, aquaculture, livestock, agroforestry, and environmental industries.

5. Conclusions

A virulent phage X5 that targets S. pullorum was isolated in this study. Phage X5 exhibits a broad host range, resistance to heat and pH variations, and a short latency period. The results revealed that phage X5 has potential application value as a biocontrol agent for removing biofilm formation of S. pullorum and as an alternative for the prevention and control of S. pullorum contamination of milk and chicken meat.

Author Contributions

X.J.: writing–original draft, visualization, data curation, software. X.S. and Q.L. (Qin Lu): investigation, resources, writing–review and editing. Z.W.; Z.Z. and X.L.: investigation and methodology. R.L. and Y.X.: resources. J.Y. and L.L.: formal analysis. T.Z. and Q.L. (Qingping Luo): project administration. G.C. and Q.L. (Qingping Luo): conceptualization, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

Rural revitalization science and technology of Jingchuxing program (2662024FW012), Huazhong Agricultural University. State Key Research and development program (2022YFD1800602), the China Agriculture Research System of MOF and MARA (CARS–41), the Key Projects of Hubei Natural Science Foundation (2021CFA019), and the Key Research and Development Project of Hubei Province 2022BBA0055. The authors would like to thank members of their laboratory for their helpful and constructive advice.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Morphology and microscopic morphology of phage X5 plaque. (A) Phage X5 plaque morphology. (B) Microscopic morphology of phage X5 under transmission electron microscopy. The scale is 100 nm.
Figure 1. Morphology and microscopic morphology of phage X5 plaque. (A) Phage X5 plaque morphology. (B) Microscopic morphology of phage X5 under transmission electron microscopy. The scale is 100 nm.
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Microorganisms 12 02400 g002
Figure 2. Biological characteristics of phage X5. Stability of phage X5 at different temperatures (A) and pH values (B). (C) Optimal MOI of phage X5. (D) Determination of chloroform sensitivity of phage X5. (E) Adsorption rate of phage X5. (F) One–step growth curve.
Figure 2. Biological characteristics of phage X5. Stability of phage X5 at different temperatures (A) and pH values (B). (C) Optimal MOI of phage X5. (D) Determination of chloroform sensitivity of phage X5. (E) Adsorption rate of phage X5. (F) One–step growth curve.
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Figure 3. Genome map of phage X5 generated by CGView. Green areas indicate the distribution of the coding sequence (CDS) regions; arrows indicate the direction of transcription. The total GC.
Figure 3. Genome map of phage X5 generated by CGView. Green areas indicate the distribution of the coding sequence (CDS) regions; arrows indicate the direction of transcription. The total GC.
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Figure 4. Phylogenetic evolutionary tree of phage X5. The phylogenetic tree was constructed based on the neighbor–joining method of the terminase large subunit.
Figure 4. Phylogenetic evolutionary tree of phage X5. The phylogenetic tree was constructed based on the neighbor–joining method of the terminase large subunit.
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Figure 5. Collinearity analysis of phage X5 and GSP044 genomes.
Figure 5. Collinearity analysis of phage X5 and GSP044 genomes.
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Figure 6. Effect of phage X5 on bacterial biofilm. The initial titers of phage X5 were 105 and 107 PFU/well. The biofilm biomass was obtained after incubation for 24 h. (A) Crystal violet staining analysis. (B) Optical density values measured at 600 nm. (C) Plate count results. The results are expressed as the mean ± SD (standard deviation) of three independent experiments. The one–way ANOVA method was used to assess significant differences between control and test samples. *** p < 0.001, **** p < 0.0001. (D) Pattern diagram of phage entry into bacteria.
Figure 6. Effect of phage X5 on bacterial biofilm. The initial titers of phage X5 were 105 and 107 PFU/well. The biofilm biomass was obtained after incubation for 24 h. (A) Crystal violet staining analysis. (B) Optical density values measured at 600 nm. (C) Plate count results. The results are expressed as the mean ± SD (standard deviation) of three independent experiments. The one–way ANOVA method was used to assess significant differences between control and test samples. *** p < 0.001, **** p < 0.0001. (D) Pattern diagram of phage entry into bacteria.
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Figure 7. Application of phage X5 in the biological control of S. pullorum 519 in milk. (A) Effects of phage X5 on the growth of S. pullorum 519 in milk at 4 °C. (B) Effects of phage X5 on the growth of S. pullorum 519 in milk at 25 °C. * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 7. Application of phage X5 in the biological control of S. pullorum 519 in milk. (A) Effects of phage X5 on the growth of S. pullorum 519 in milk at 4 °C. (B) Effects of phage X5 on the growth of S. pullorum 519 in milk at 25 °C. * p < 0.05; ** p < 0.01; *** p < 0.001.
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Microorganisms 12 02400 g008
Figure 8. Application of phage X5 in biological control of S. pullorum 519 in chicken breast. (A) Effects of phage X5 on the growth of S. pullorum 519 in chicken breast at 4 °C. (B) Effects of phage X5 on the growth of S. pullorum 519 in chicken breast at 25 °C. * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 8. Application of phage X5 in biological control of S. pullorum 519 in chicken breast. (A) Effects of phage X5 on the growth of S. pullorum 519 in chicken breast at 4 °C. (B) Effects of phage X5 on the growth of S. pullorum 519 in chicken breast at 25 °C. * p < 0.05; ** p < 0.01; *** p < 0.001.
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Table 1. Lytic activity of phage X5 against the tested strains.
Table 1. Lytic activity of phage X5 against the tested strains.
StrainsX5 aStrainsX5 aStrainsX5 a
S. pullorum CVCC529+S. typhimurium ATCC14028+S. typhimurium 3+
S. pullorum CVCC530+S. typhimurium
CMCC50115		+S. typhimurium 129+
S. pullorum CVCC531+S. typhimurium
SL1344		+S. typhimurium 107+
S. pullorum CVCC534+S. typhimurium 1+S. typhimurium 6+
S. pullorum CVCC535+S. typhimurium 10+S. typhimurium 8+
S. pullorum CVCC540+S. typhimurium 11+S. typhimurium ST24−
S. pullorum 1+S. typhimurium 16+S. typhimurium 27+
S. pullorum 4−S. typhimurium 17−S. typhimurium 130+
S. pullorum 5+S. typhimurium 19+S. typhimurium 5+
S. pullorum 6+S. typhimurium 20+S. typhimurium 78−
S. pullorum 12+S. typhimurium 93+S. typhimurium G7+
S. pullorum 16+S. typhimurium 86+S. typhimurium 74+
S. pullorum 59+S. typhimurium 90+S. typhimurium P17+
S. pullorum 64−S. typhimurium 77+S. typhimurium 21+
S. pullorum 78+S. typhimurium P6+S. typhimurium G20−
S. pullorum 84+S. typhimurium 74+S. typhimurium G10+
S. pullorum 85+S. typhimurium 80+S. typhimurium G7+
S. pullorum 92+S. typhimurium 85+S. typhimurium G1+
S. pullorum 104+S. typhimurium 83+S. typhimurium 24+
S. pullorum
ATCC9120		−S. typhimurium ST1+S. typhimurium ST21+
S. pullorum 127+S. typhimurium 87−S. typhimurium P11+
S. pullorum 153+S. typhimurium 7+S. typhimurium 88+
S. enteritidis
CVCC3375		+S. typhimurium 81+S. typhimurium
ST22		+
S. enteritidis
CMCC50746		+S. typhimurium 11+S. typhimurium
ST25		−
S. enteritidis 7−S. typhimurium 82+L. acidophilus
ATCC832		−
S. enteritidis 23+S. typhimurium P8+C. perfringensa
ATCC13124		−
S. enteritidis25+S. typhimurium 79+K. pneumoniae Y1−
S. enteritidis 61−S. typhimurium 180+E. coli ATCC25922
a Symbols: After the bacteria to be tested are infected with phage X5, the (+) region is clear or the (−) region has no plaques. CMCC: National Center for Medical Culture Collections. ATCC: American Type Culture Collection. The other strains tested were clinical isolates.
Table 2. Functional analysis of major proteins of phage X5.
Table 2. Functional analysis of major proteins of phage X5.
X5 GSP044Function
ORFStrandStartStopStrandStartStopHomology
ORF3−13833137−49,96551,72092%A1 protein
ORF5−33933800−51,97652,38399%A2 protein
ORF34−15,68216,272−65,19065,77896%putative serine/threonine protein phosphatase
ORF36−16,56117,424−66,06766,93094%serine/threonine protein phosphatase
ORF41−19,00519,468−68,56168,97498%lysozyme
ORF42−19,46520,121−68,97169,62799%holin
ORF43−20,27820,877−69,78470,38399%ATP-dependent Clp protease proteolytic subunit
ORF44−20,89021,642−70,39671,14898%deoxynucleotide monophosphate kinase
ORF56−26,08926,457−75,59575,96399%putative acetyltransferase-like protein
ORF91−44,33944,815−93,91194,38597%RNaseH
ORF93−45,19046,044−94,76095,61497%thymidylate synthase
ORF94−46,04446,577−95,61496,14798%putative dyhydrofolate reductase
ORF95−46,57447,719−96,14497,28996%aerobic ribonucleoside diphosphate reductase, small subunit
ORF96−47,82650,156−97,39699,72698%aerobic ribonucleoside diphosphate reductase large subunit
ORF98−50,52251,274−100,093110,56399%phosphate starvation-inducible protein
ORF99−51,46051,951 HNH endonuclease
ORF100+52,14554,019+101,200103,07497%anaerobic ribonucleoside-triphosphate reductase
ORF109+57,83360,622+106,613109,40297%DNA primase C
ORF111+60,90861,612+109,688110,39297%D2 protein
ORF117+63,31264,283+1606257798%DNA ligase
ORF118+64,48665,265+2780355998%DNA ligase
ORF121+67,57768,467+6401729197%putative DNA replication primase
ORF122+68,53071,097+774110,30896%DNA polymerase I
ORF124+71,58472,936+10,79512,14797%putative ATP-dependent helicase
ORF127+74,24475,221+13,96714,94495%calcineurin-like phosphoesterase superfamily domain protein
ORF128+75,20277,040+14,92516,76397%recombination related exonuclease
ORF129+77,04477,526+16,76717,24998%D14 protein
ORF130+77,52678,401+17,24918,12498%flap endonuclease
ORF131+78,39878,844+18,12118,56795%putative deoxyUTP pyrophosphatase
ORF133−79,11181,198−18,83720,92496%chaperone of endosialidase domain-containing protein
ORF134−81,24184,591−20,96724,31799%chaperone of endosialidase domain-containing protein
ORF135−84,59185,013−24,31724,739100%putative tail protein
ORF136−85,01887,075−24,74426,80199%tail protein
ORF137−87,07589,924−26,80129,65098%tail protein Pb3
ORF139−90,64594,352−30,37132,56794%pore-forming tail tip protein
ORF142−95,26796,166−34,96635,86598%tail fibers protein
ORF143−96,17197,577−35,87737,27993%major tail protein
ORF147−99,432100,808−39,13440,51092%major head protein precursor
ORF148−100,826101,458−40,52841,16099%putative prohead protease
ORF149−101,462101,953−41,31541,64686%tail protein
ORF150−101,950103,167−42,08242,55899%portal protein
ORF152−103,719105,035−43,25844,57494%terminase large subunit
ORF153−105,035105,517−44,57445,05698%putative SciB protein
ORF154−105,528107,315−45,06746,85497%receptor binding protein
ORF155+107,401107,667+46,94047,206100%receptor-blocking protein
ORF163−110,115111,869−49,96551,72092%A1 protein
ORF165−112,125112,532−51,97652,38399%A2 protein
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MDPI and ACS Style

Jin, X.; Sun, X.; Lu, Q.; Wang, Z.; Zhang, Z.; Ling, X.; Xu, Y.; Liang, R.; Yang, J.; Li, L.; et al. Salmonella Phage vB_SpuM_X5: A Novel Approach to Reducing Salmonella Biofilms with Implications for Food Safety. Microorganisms 2024, 12, 2400. https://doi.org/10.3390/microorganisms12122400

AMA Style

Jin X, Sun X, Lu Q, Wang Z, Zhang Z, Ling X, Xu Y, Liang R, Yang J, Li L, et al. Salmonella Phage vB_SpuM_X5: A Novel Approach to Reducing Salmonella Biofilms with Implications for Food Safety. Microorganisms. 2024; 12(12):2400. https://doi.org/10.3390/microorganisms12122400

Chicago/Turabian Style

Jin, Xinxin, Xiuxiu Sun, Qin Lu, Zui Wang, Zhenggang Zhang, Xiaochun Ling, Yunpeng Xu, Ruiqin Liang, Junjie Yang, Li Li, and et al. 2024. "Salmonella Phage vB_SpuM_X5: A Novel Approach to Reducing Salmonella Biofilms with Implications for Food Safety" Microorganisms 12, no. 12: 2400. https://doi.org/10.3390/microorganisms12122400

APA Style

Jin, X., Sun, X., Lu, Q., Wang, Z., Zhang, Z., Ling, X., Xu, Y., Liang, R., Yang, J., Li, L., Zhang, T., Luo, Q., & Cheng, G. (2024). Salmonella Phage vB_SpuM_X5: A Novel Approach to Reducing Salmonella Biofilms with Implications for Food Safety. Microorganisms, 12(12), 2400. https://doi.org/10.3390/microorganisms12122400

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