Salmonella phage with high efficiency and broad spectrum and application thereof in prevention and control of biofilm

Abstract

A high-efficiency and broad-spectrum Salmonella bacteriophage and its application in the prevention and control of biofilm belong to the field of biotechnology / bioengineering. The bacteriophage is isolated from the processing water of broiler slaughtering, scalding and dehairing process, belongs to the long tail bacteriophage family, and has the dual characteristics of high efficiency and broad spectrum. It can efficiently lyse multiple Salmonella, with a latent period of 15 minutes, a lysis period of 45 minutes, and an average lysis amount of 253 after lysis is completed. The whole genome of the bacteriophage is 41763 bp, and the genome has no drug resistance and virulence invasion genes, with high food safety. The bacteriophage can significantly inhibit and remove the formation process and the formed biofilm, with an inhibition rate and removal rate of more than 70%, and has a good effect on the prevention and control of biofilm.

Description

Technical Field

[0001] This invention relates to the field of biotechnology / bioengineering, specifically to a Salmonella phage isolated from broiler chickens, and its application as a main component in Salmonella control. Background Technology

[0002] Salmonella is a common foodborne pathogen that causes frequent food poisoning and food recalls each year. The U.S. Centers for Disease Control and Prevention (CDC) and the European Food Safety Authority (EFSA) estimate that Salmonella causes approximately one million infections annually. In China, Salmonella accounts for about 22.2% of food poisoning cases. This demonstrates that Salmonella contamination is global, with meat products being a key source of Salmonella food poisoning. Food can be contaminated with Salmonella through various routes, with cross-contamination from food processing equipment being a primary cause. The root cause of cross-contamination is the formation of biofilms by bacteria on solid contact surfaces. Salmonella biofilms have been detected on various food processing surfaces.

[0003] Biofilms make adherent bacteria approximately 1000 times more resistant to antibiotics than planktonic bacteria, exacerbating food safety instability. Furthermore, biofilms are widely distributed, detectable on food surfaces, processing equipment surfaces, and in food factory environments, creating conditions for food spoilage and disease transmission. Currently, the prevention and control of Salmonella biofilms in food processing mainly relies on chemical disinfectants. However, frequent use has led to significantly increased Salmonella tolerance, making it resistant to various disinfectants. Therefore, new biofilm control methods are urgently needed.

[0004] Bacteriophages are a hot topic in biofilm control research due to their efficient targeted lysis of bacteria and potential degradation of extracellular secretions from biofilms. However, currently commercially available Salmonella phages can only be used for planktonic bacteria in meat, vegetables, and aquatic products. Phages for controlling biofilms on food processing contact surfaces are still relatively scarce. Furthermore, most phages have narrow host profiles, capable of lysing only one serotype, which cannot meet application requirements. Therefore, Salmonella phages with broad host profiles and high lytic activity suitable for biofilm control are currently a focus of research. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned problems by providing a bacteriophage that can be used to remove Salmonella biofilms from food processing contact surfaces. Through long-term technical and practical exploration, the inventors isolated a Salmonella bacteriophage with a broad lysis spectrum from the scalding and defecation water used in broiler slaughter. The optimal multiplicity of infection for this bacteriophage is 0.1. The latency period for infecting the Salmonella host bacterium is approximately 15 minutes, the lysis period is 45 minutes, and the average lysis yield after lysis is 253. The entire genome contains no drug resistance or virulence attack genes. This bacteriophage significantly inhibits the formation of biofilms and also has a strong removal effect on already formed, mature biofilms.

[0006] This invention relates to the following specific technical solutions:

[0007] This invention provides a Salmonella phage strain with highly efficient and broad-spectrum activity against Salmonella and its biofilm. This phage strain was deposited on December 9, 2021, at the China General Microbiological Culture Collection Center (CGMCC) under the number CGMCC NO.22923. The phage was isolated from the scalding and defecation water of broiler chickens and has the following characteristics:

[0008] The bacteriophage head is three-dimensionally symmetrical and belongs to the order Caudata and family Longtailophages. It can form large plaques on solid substrates with clear, regular edges and no halo.

[0009] This bacteriophage is highly lethal to Salmonella typhimurium, Salmonella enteritidis, Salmonella Stanley, Salmonella Argonnae, Salmonella Delphi, Salmonella Indiana, Salmonella Buri, Salmonella Rostock, Salmonella Chester, and Salmonella Brendenloop.

[0010] The optimal pH range for this bacteriophage is 6-9. It is intolerant to extremely acidic or alkaline environments. The optimal temperature range is 30-50℃. It is sensitive to high temperatures of 60℃ and above. The optimal multiplicity of infection for the bacteriophage is 0.1. The latency period for infecting the host bacteria is about 15 minutes. The lysis period is 45 minutes. The average lysis yield after lysis is 253.

[0011] The phage's full genome is 41,763 bp in length and contains no drug resistance or virulence genes, thus possessing high food safety.

[0012] This bacteriophage significantly inhibited the formation of biofilms, substantially reducing the number of Salmonella biofilms after 24 and 48 hours of culture, with inhibition rates as high as 55% and 72%, respectively. This is significantly higher than the inhibition rate of less than 30% of ordinary bacteriophages.

[0013] This bacteriophage also has a significant removal effect on mature biofilms, with a removal rate as high as 71%, which is significantly higher than the removal effect of less than 50% of ordinary bacteriophages or sodium hypochlorite control technology.

[0014] The phage screening process was as follows: 100 μL of Salmonella host bacteria was spread on an LB agar plate, and 10 μL of preserved phage stock solution was added. The plate was incubated at 37°C for 12 h. Clumps showing obvious lysis plaques were selected, and the agar blocks at the edge of the plaques were placed in sterile SM buffer and incubated overnight at 4°C to prepare crude phage solution. Subsequently, the crude phage solution was serially diluted 10-fold using SM buffer. 100 μL of the appropriately diluted phage solution and 100 μL of freshly cultured and activated Salmonella were placed in a centrifuge tube and gently poured onto a semi-solid medium (0.6% agar powder) LB agar plate. After solidification, the plate was incubated at 37°C for 12 h. Single phage plaques with regular shapes were picked and placed in SM buffer, incubated overnight at 4°C. This process was repeated 3-5 times until the plaques were uniform in size and dense. The phages were collected using SM buffer and filtered through a sterile filter to remove bacteria, thus preparing the phage solution. The bacteriophage head is three-dimensionally symmetrical and belongs to the order Caudata and family Longtailophages. It can form large plaques on solid substrates with clear and regular edges and no halo.

[0015] Phage amplification and culture method: Take an appropriate amount of purified phage and incubate it in an activated bacterial suspension at 37℃ for 3-4 hours. Centrifuge the culture (4℃, 8000×g, 20 min), then filter through a 0.22 μm filter to remove bacteria and obtain the supernatant. Take an appropriate amount of filtrate (e.g., 50 ml) into an Erlenmeyer flask, and add 10 mL of Salmonella culture in the logarithmic growth phase and 15 mL of LB broth, respectively. Mix well and let stand at room temperature for 30 min (to facilitate phage binding to the host bacteria). Then, incubate at 37℃ with shaking for 12 hours to enrich the phage. Repeat the above process until the bacterial suspension is clear. After centrifugation and filtration, collect the filtrate. Add an appropriate amount of LB liquid medium and bacterial suspension according to the filtrate volume for further amplification culture to the desired phage culture medium.

[0016] This bacteriophage is highly lethal to Salmonella enteritidis, Salmonella typhimurium, Salmonella Stanley, Salmonella Argonnae, Salmonella Delphi, Salmonella Indiana, Salmonella Buri, Salmonella Rostock, Salmonella Chester, and Salmonella Brendenloop.

[0017] The optimal pH range for this bacteriophage is 6-9, and the optimal temperature range is 30-50℃. It is sensitive to high temperatures of 60℃ and above. The optimal multiplicity of infection for the bacteriophage is 0.1. The latency period for infecting the host bacteria is about 15 minutes, the lysis period is 45 minutes, and the average lysis volume after lysis is 253. Attached Figure Description

[0018] Figure 1 pH stability of bacteriophages.

[0019] Figure 2 Temperature stability of bacteriophages.

[0020] Figure 3 The optimal infection multiple of bacteriophages.

[0021] Figure 4 One-step growth curve of bacteriophage.

[0022] Figure 5 A phage whole genome circle diagram. The meaning of each circle (from the outside to the inside): (1) Genome size in bp; (2) ORF represented by matrices of different sizes, with the matrix arrows representing the direction of gene transcription; (3) The difference between G+C and the mean value, with values ​​higher than the mean value being red (outer peak) and values ​​lower than the mean value being dark blue (inner peak); (4) G+C offset value (GC / G+C), with offset values ​​greater than zero being crystal blue and values ​​less than zero being brownish-yellow.

[0023] Figure 6 The inhibitory effect of bacteriophages on the biofilm formation process.

[0024] Figure 7 The effect of bacteriophages on the removal of mature biofilms.

[0025] Information on the preservation of biological materials

[0026] Salmonella phage, this strain of phage was deposited on December 9, 2021 at the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC NO.22923, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto: Embodiment 1: The screening process of bacteriophages and the biological characteristics of bacteriophages

[0028] Preparation of phage solution: 100 μL of Salmonella host bacteria was spread on an LB agar plate, and 10 μL of preserved phage stock solution was added. The plate was incubated at 37°C for 12 h. Clumps showing obvious lysis plaques were selected, and the agar blocks at the edge of the plaques were placed in sterile SM buffer and incubated overnight at 4°C to prepare crude phage solution. Subsequently, the crude phage solution was serially diluted 10-fold using SM buffer. 100 μL of the appropriately diluted phage solution and 100 μL of freshly cultured and activated Salmonella were placed in a centrifuge tube and gently poured onto a semi-solid medium (0.6% agar powder) LB agar plate. After solidification, the plate was incubated at 37°C for 12 h. Regularly shaped single phage plaques were picked and placed in SM buffer, incubated overnight at 4°C. This process was repeated 3-5 times until the plaques were uniform in size and dense. The phages were collected using SM buffer and filtered through a sterile filter to remove bacteria, thus preparing the phage solution. The bacteriophage head is three-dimensionally symmetrical and belongs to the order Caudata and family Longtailophages. It can form large plaques on solid substrates with clear and regular edges and no halo.

[0029] 900 μL of Salmonella LB broth with a pH range of 2-13 was incubated at 37℃ for 30 min. After the temperature stabilized, 100 μL of bacteriophage fluid was added and the culture was continued for 1 h. The culture was then centrifuged (8000×g, 1 min, 4℃) to remove bacterial precipitate. The titer of the bacteriophages in the supernatant was determined using the double-layer plate method. The results showed that the bacteriophage survival rate was 0% at pH 2, 3, 12, and 13, indicating that it cannot survive in extremely acidic or alkaline environments. However, when the pH was in the range of 6-9, the bacteriophage survival rate was above 85% compared to the control group, indicating that pH 6-9 is the optimal growth pH for bacteriophage CW1. Figure 1 ).

[0030] 900 μL of Salmonella LB broth was placed in an environment with a temperature range of 30℃-80℃ for 1 hour. After the temperature stabilized, 100 μL of phage fluid was added and the culture was continued for another hour. The bacterial precipitate was removed by centrifugation, and the phage titer in the supernatant was determined using the double-layer plate method. The results showed that when the phage was exposed to an environment with a temperature exceeding 60℃, the phage titer was 0, indicating its sensitivity to high temperatures, which is consistent with the survival conditions of phages in nature. When the temperature was in the range of 30-50℃, the phage survival rate was 100%, and the titer remained stable at 10. 11 PFU / mL, which falls within its optimal temperature range for growth. Figure 2 ).

[0031] Phage to Salmonella ratios of 10, 1, 0.1, 0.01, 0.001, and 0.0001 were prepared. Each solution was added to sterile LB medium and incubated at 37°C with shaking at 150 rpm for 6 h. Bacterial precipitate was removed by centrifugation (8000×g, 10 min, 4°C). The titer of phage in the supernatant was determined using the double-layer plate method. The optimal multiplicity of infection (MLI) was the value at which the highest titer was achieved. Results showed that the overall trend of phage titer was an initial increase followed by a gradual decrease. When the MII was 0.1, the number of progeny phages released was the highest, with a titer as high as 1.413 × 10⁻⁶. 11 PFU / mL. Therefore, the optimal multiplicity of infection (MOI) for phages is 0.1. It is worth noting that when the MOI is 10, the phage titer is significantly lower than at other MOI ratios, indicating that multiple phages may be infecting the same bacterium. Figure 3 ).

[0032] Phage and Salmonella were added at a ratio of 0.1 for the multiplicity of infection (MOI). After thorough mixing, the mixture was incubated at 37°C for 15 min. The supernatant was removed by centrifugation at 8000×g. The precipitate was washed twice with sterile LB filtrate, and then an appropriate amount of LB filtrate was added. After mixing, the mixture was rapidly incubated on a shaker at 90 rpm for 10 min. Supernatants were collected at 0, 5, 10, 15, 20, 30, 40, 50, 60, 90, and 120 minutes, and centrifuged (8000×g, 1 min, 4°C) to remove bacterial precipitate. The phage titer of the supernatant was determined using the double-layer plate method. The results showed that the phage titer did not differ significantly in the first 15 minutes, remaining relatively stable at 6.5×10⁻⁶. 8 PFU / ml; subsequently, the phage count began to increase significantly, gradually stabilizing after 60 minutes; however, it still showed an upward trend at 120 minutes. The phage latency period was 15 minutes, the lysis period was 45 minutes, the adsorption capacity in the first 15 minutes was 62.33%, and the average lysis yield after lysis was 253 (PFU / ml). Figure 4 Bacteriophages have a short latency period and a relatively short lysis period, but they exhibit high lysis rates and high titers.

[0033] Using the Illumina sequencing platform, a 2×150bp bidirectional sequencing strategy was employed. The complete genome of the bacteriophage was obtained through database annotation, and the genome circle map was drawn using the Rcirclize package. Results ( Figure 5 The results showed that the bacteriophage was a double-stranded DNA with a total genome length of 41763 bp and an average G+C content of 50.20%. Figure 5The peak direction and height in the third ring from the outside in show the difference and degree of G+C content in each gene segment compared to the average value, which can distinguish the start and end points of the genome sequence. A total of 59 genes were predicted for this phage, accounting for 91.6% of the phage genome sequence; the gene density was 1.412, reflecting the compactness of its genome organization. This phage does not contain tRNA, indicating that it relies entirely on the tRNA of the host bacteria to complete its translation and replication.

[0034] Example 2: Phage lysis spectrum test results

[0035] Each test strain (Table 1) frozen at -80℃ was streaked on a TSA plate and incubated at 37℃ for 24 h. Single colonies were picked and incubated overnight in TSB medium. After the bacterial culture became turbid, the bacterial culture was transferred to fresh TSB at a ratio of 1:100 and incubated at 37℃ with shaking for 3 h until the bacteria reached the early logarithmic growth stage. The host spectrum of the bacteriophages was determined using the double-layer plate drop method, and the bacteriophage titer was adjusted to 10 using SM buffer. 8 PFU / ml, 100 μL of host bacteria was aspirated into a centrifuge tube, 5 ml of 42℃ semi-solid culture medium was added, and the mixture was gently shaken and poured onto an LB plate. After solidification, 10 μL of phage fluid was added, and the plate was incubated at 37℃ for 12 h. LB culture medium was set as a control, and the lysis was observed. The results showed that the phage could efficiently kill Salmonella enteritis, typhus, Stanley, Argonne, Delphi, Indiana, Bury, Rostock, Chester, and Brendenlupp serotypes (Table 1).

[0036] Table 1

[0037]

[0038] Note: + indicates that it has pyrolysis ability, and - indicates that it does not have pyrolysis ability.

[0039] Example 3: Inhibitory effect of bacteriophage on biofilm formation

[0040] 100 μL of activated fresh Salmonella suspension and 100 μL of prepared bacteriophage solution were mixed and transferred to a 96-well sterile microplate. The mixture was incubated at 37℃ for 12 h, 24 h, and 48 h, respectively. After incubation, the microplates were discarded, and the wells were rinsed three times with sterile PBS buffer. The plates were then air-dried, stained with 0.25% crystal violet for 30 min in the dark, and 220 μL of 95% ethanol was added to each well. The absorbance was measured at 570 nm after 30 min. TSB medium without bacteriophage served as the control group. The results showed that after 12 h, 24 h, and 48 h of co-culture with the host bacteria, the OD570 values ​​of the treatment group were significantly different from those of the control group at all observation points, indicating that the bacteriophage inhibited biofilm formation. The inhibition rate of the bacteriophage on biofilm formation gradually increased with prolonged co-culture time. The phage exhibited inhibition rates of 47.69%, 55.76%, and 72.95% against the biofilm at the observed sites, respectively. Figure 6 ).

[0041] Example 4: The effect of bacteriophages on the removal of mature biofilms

[0042] 200 μL of fresh Salmonella suspension was added to a 96-well microplate and incubated at 37°C for 48 h. The plate was then carefully removed and the suspension was discarded. Airborne bacteria were washed away with sterile PBS buffer. At this point, a mature biofilm adhered to the microplate walls. 220 μL of prepared phage solution was then added to each well, and the plate was incubated for another 4 h. The suspension was then discarded, the plate was rinsed with PBS, and the plate was inverted and allowed to air dry. Crystal violet staining was performed as in Example 2. The results showed a significant difference in OD570 values ​​between the treatment group and the control group, indicating that the phage had a degradation effect on the mature biofilm. Figure 7 ).

[0043] Example 5

[0044] Taking the control of mature Salmonella biofilm on the conveyor chain of a poultry slaughtering production line as an example. Currently, the conveyor belts for packaging cut products or whole carcasses after poultry slaughter are made of polypropylene or stainless steel. Salmonella is one of the most prevalent pathogens in poultry meat. Salmonella adhering to the surface of such materials will gradually develop into a biofilm by using meat juices as nutrients. The bacteriophages in this invention can significantly inhibit the number and development of the biofilm. By implementing the specific spraying operation below 2-3 times a week, the biofilm on the conveyor chain can be well controlled. Salmonella bacteriophages were prepared on a large scale according to the method in Example 1, and their concentration was increased to 10. 7For use with a concentration of PFU / L or higher, install a spray device at the conveyor chain's rotation point or an appropriate location, ensuring the spray area completely covers the width of the conveyor chain and that the spray device has sufficient pressure (2-2.5 bar is recommended). After slaughtering, after treating the conveyor chain with cleaning and chemical disinfectants such as sodium hypochlorite, use the spray device to spray bacteriophage fluid onto the surface of the conveyor chain for 10 minutes each time, ensuring that the meat contact surface is within the sprayed area. Repeat this spraying process for 5 cycles. After stopping spraying for 8 hours, thoroughly rinse the conveyor chain with tap water. This operation can achieve a 71% removal rate of mature biofilm growing on the surface of the conveyor chain.

[0045] Example 6

[0046] Taking the control of biofilm on the surface of small meat cutting equipment in a central kitchen as an example. In a central kitchen processing scenario, Salmonella introduced from raw materials and auxiliary materials will form a biofilm on the surface of small cutting equipment. Using this bacteriophage can effectively control its contamination. Salmonella bacteriophage was prepared on a large scale according to the method of Example 1, achieving a concentration of 10. 7 Prepare PFU / L or higher solution. Dilute it in 37°C warm water and place it in a cleaning tank of appropriate volume. Immerse cutting boards, cutting utensils, and other equipment that have been treated with sodium hypochlorite lamp chemical disinfectant in the bacteriophage solution (chemical disinfectants can only kill bacteria on the surface and shallow layer of the biofilm, and have no effect on the bacteria inside the biofilm or the structure of the biofilm. Bacteriophages can effectively destroy the structure of the biofilm). Allow it to act for 4 hours. Repeat this process 3 times a week to completely prevent Salmonella from forming a biofilm on the surface of such utensils and achieve a good control effect.

Claims

1. A highly efficient and broad-spectrum Salmonella phage, with accession number CGMCC 22923, has a genome size of 41763 bp. Whole-genome sequencing showed that the genome does not contain drug resistance genes or virulence genes, and its titer is as high as 1.413 × 10⁻⁶ at an optimal multiplicity of infection of 0.

1. 11 PFU / mL.

2. A Salmonella phage preparation, characterized in that, The phage preparation uses the Salmonella phage as described in claim 1 as the main active ingredient.

3. The use of the Salmonella phage of claim 1 in the preparation of reagents for the prevention and control of one or more of Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Stanley, Salmonella Argonnae, Salmonella Delphi, Salmonella Indiana, Salmonella Buri, Salmonella Rostock, Salmonella Chester, and Salmonella Brendenlupp.

4. The application of the Salmonella phage according to claim 1 in the preparation of products containing biofilms for disease control, wherein the bacteria producing the biofilm are one or more of Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Stanley, Salmonella Argonnae, Salmonella Delphi, Salmonella Indiana, Salmonella Buri, Salmonella Rostock, Salmonella Chester, and Salmonella Brendenlupp.

5. The use of the Salmonella phage of claim 1 in the preparation of a biofilm product for removing bacteria from the surface of food, wherein the bacteria are one or more of Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Stanley, Salmonella Argonnae, Salmonella Delphi, Salmonella Indiana, Salmonella Buri, Salmonella Rostock, Salmonella Chester, and Salmonella Brendenloop.

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