Isolation and characterization of Salmonella enteritidis bacteriophage Salmp-p7 isolated from slaughterhouse effluent and its application in food

doi:10.21203/rs.3.rs-5289804/v1

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Isolation and characterization of Salmonella enteritidis bacteriophage Salmp-p7 isolated from slaughterhouse effluent and its application in food

https://doi.org/10.21203/rs.3.rs-5289804/v1

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Salmonella enteritidis is one of the most common pathogens that cause foodborne outbreaks and food spoilage, which seriously threatens human health. In this study, Salmonella enteritidis bacteriophage Salmp-p7 was isolated and characterized from slaughterhouse wastewater. Transmission electron microscopy (TEM) analysis showed that Salmp-p7 belonged to the Siphoviridae family and was active against Salmonella enteritidis and Escherichia coli. Whole genome sequence analysis showed that Salmp-p7 was a lytic bacteriophage with a total length of 60066 bp. In vitro, antimicrobial curves and inhibition of biofilm removal experiments showed that Salmp-p7 could effectively inhibit and eliminate Salmonella enteritidis. The application of Salmp-p7 to the whole liquid of infected eggs resulted in a significant reduction of viable bacteria in the egg liquid. In conclusion, the bacteriophage Salmp-p7 has high stability and lytic activity and has the potential to become a new biological control agent for Salmonella enteritidis in eggs.

Salmonella enteritidis

bacteriophages

biological characterization

genome sequencing

biological control

Salmonella is a Gram-negative facultatively anaerobic bacteria, belonging to the Enterobacteriaceae, without spores and capsules, most of them have flagella and is a relatively common zoonotic pathogenic bacteria (Billah & Rahman, 2024; Medvedev et al., 2024). Salmonellosis has become an epidemic that is a serious threat to human health and can cause infection in humans and animals, mainly through contaminated food (meat, eggs, dairy products, and water) and direct contact with infected animals or their feces (Aleksandrowicz et al., 2024; Chand, Jain, & Singh, 2024). In the 1950s, Salmonella was identified as a serious zoonotic pathogen by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO), causing widespread concern (Akinola, Mwanza, & Ateba, 2019). In addition, Salmonella is the third leading cause of death from foodborne illness, after pathogenic Escherichia coli and norovirus, primarily from animal-derived foods (Ferrari et al., 2019; Sima et al., 2024). According to the data released by the World Health Organization (WHO), Salmonella is highly infectious in humans, usually manifested as acute fever, abdominal pain, diarrhea, and sometimes vomiting, and about 33 million people die from Salmonella infection every year (Scheik et al., 2023). Previous studies have reported that the incidence of human salmonellosis in China is more than 30 times higher than in Europe and the United States (Wang, Qazi, Wang, Zhou, & Han, 2020). Most of these salmonellosis cases are caused by eating contaminated food. So far, there are more than 2,600 serotypes of Salmonella found in many species, and recent epidemiological studies have shown that Salmonella enteritidis serotypes have become an important serotype of concern (Woh et al., 2021; X. Yuan et al., 2022). Salmonella enteritidis is a pathogen with a relatively wide host range. It is easy to be carried and spread during processing and transportation, which seriously damages human health and the development of food breeding. Among them, Salmonella serotypes in eggs and egg products are the most worrying (Majowicz et al., 2010). Despite the obvious epidemiological relationship between Salmonella enteritidis and eggs, the prevention and management of Salmonella enteritidis in eggs is still very important, and it is urgent and necessary to find effective measures to deal with Salmonella enteritidis (Abdelhamid & Yousef, 2023).

In recent years, antimicrobial drugs have been the first choice for the treatment of bacterial infections, but antibiotic resistance has become a thorny problem due to the overuse and misuse of antibiotics in medical and animal production. It is predicted that by 2050, the global death toll due to antibiotic resistance will be close to 10 million (Castro-Vargas, Herrera-Sánchez, Rodríguez-Hernández, & Rondón-Barragán, 2020). Although antibiotic resistance is a natural occurrence, the misuse of antibiotics will accelerate this process (Peterson & Kaur, 2018; Vitale et al., 2019). There is a worrying trend that shows the isolation of Salmonella serotypes resistant to one or more antibiotics has been on the rise globally since 2015 (Ayoola et al., 2024). A previous study showed that 88 strains of Salmonella were isolated from 1231 different human and animal samples, of which 38.64% had multiple drug resistance (MDR) (Borah et al., 2022). A study of antimicrobial resistance to Salmonella in poultry isolates showed prevalence in broilers, raw chicken, eggs, and laying hens of 40.5%, 30%, and 40%, respectively (Castro-Vargas et al., 2020). According to the World Health Organization, 33,000 people die each year in Europe from antibiotic-resistant bacteria (Frost, Kapoor, Craig, Liu, & Laxminarayan, 2021). At the same time, the presence of bacterial biofilm is another important obstacle to the antimicrobial effect. Biofilm is a biologically active matrix composed of persistent cells and extracellular substances formed on the surface of the host body and in vitro, and is a life phenomenon that facilitates the survival of bacteria to adapt to the natural environment (Sharma, Misba, & Khan, 2019). It is widely found on a variety of wet surfaces containing water, such as food, food processing equipment, and even the surface of human tissues and organs in pathological conditions, and is a structural bacterial community composed of bacterial cells attached to the surface of inert or active entities and a hydrated matrix wrapped in bacteria. On the one hand, the overuse of antibiotics will cause bacterial resistance, and on the other hand, it will promote the formation of biofilms, failing traditional antibiotics to destroy bacteria (Rabsch et al., 2000). We are currently facing a crisis of antimicrobial resistance (AMR) and bacterial biofilms, so the search for alternative and sustainable antimicrobials is urgently needed (Sisson, Jackson, Fagerlund, Warring, & Fineran, 2024).

Bacteriophages are viruses that infect and replicate within bacterial cells and can directly destroy the bacterial cell wall and subsequently kill the bacteria but do not act on mammalian or human cells (Janczuk-Richter, Marinović, Niedziółka-Jönsson, & Szot-Karpińska, 2019). Due to their very high selectivity and toxicity, they target specifically pathogenic bacteria with no effect on the normal microflora in the host, so bacteriophages can be safely used in animals and humans (Kim, Kim, Rahman, & Kim, 2018). At the same time, due to the high cost and long research cycle of new synthetic antibiotics, the development of phage-based alternative antibiotics is low-cost and relatively short-term, and the resistance rate is very low compared with conventional antibiotics (Hauser, Mecsas, Moir, & Weinstein, 2016). The phage product has been approved by the United States Food and Drug Administration (FDA) as a Generally Recognized as Safe Product (GRAS) and is used to control Listeria monocytogenes contamination in meat products (Lang, 2006). Therefore, various bacteriophages have been isolated and studied to control food-borne pathogen contamination. However, compared with other bacteriophages, lytic bacteriophages against Salmonella enteritidis are not available in sufficient numbers and food application studies are limited. Therefore, the development of bacteriophage-based antimicrobials has been considered the most promising way to address the problems associated with antibiotics and resistant bacteria in Salmonella enteritidis. Therefore, we aimed to isolate a highly lytic bacteriophage and to be able to reduce the contamination of Salmonella enteritidis in the whole egg fluid. In this study, the host bacterium Salmonella enteritidis and a bacteriophage Salmp-p7 capable of infection with Salmonella enteritidis were isolated, and the antimicrobial resistance of the host bacteria was determined, and the phenotype and genotype of the bacteriophage Salmp-p7 were identified. At the same time, to determine whether the phage can stably exert functional activity in different food systems, we evaluated the stability of the phage at different temperatures and pH. In addition, we measured the effect of the phage on Salmonella enteritidis biofilm formation before and after formation. Finally, we applied the phage to the whole solution of eggs contaminated with Salmonella enteritidis to evaluate the application effect of bacteriophage Salmp-p7 in food. Our results indicate that the bacteriophage Salmp-p7 has good development potential and can be used as a new biocontrol agent to reduce the contamination of Salmonella enteritidis.

2.1 Bacterial strains and growth conditions

The strains used in this study are listed in Table 1. The samples were collected from the sewage of livestock and poultry farms, and Salmonella enteritidis slaughterhouse isolate 1 (from slaughterhouse sewage) was used as host bacteria for phage isolation. All strains were cultured in beef paste peptone liquid medium (LB) at 37°C, 180 rpm shaking for 12 h, and subcultured in beef paste peptone solid medium (LB). The plaque experiment was carried out with beef extract peptone upper medium (LB).

Table 1 Host range analysis of Salmp-p7

Bacteria	Source	Spot test
Salmonella enteritidis 1	Sewage isolates	+
Salmonella enteritidis 2	Sewage isolates	+
Salmonella enteritidis 3	Sewage isolates	+
Salmonella enteritidis 4	Sewage isolates	-
Salmonella enteritidis 5	Sewage isolates	+
Salmonella enteritidis 6	Sewage isolates	+
Salmonella enteritidis 7	Sewage isolates	+
Escherichia coli 1	laboratory isolate	-
Escherichia coli 2	laboratory isolate	-
Escherichia coli 3	laboratory isolate	+
Escherichia coli 4	laboratory isolate	-
Escherichia coli 5	laboratory isolate	-
klebsiella 10	laboratory isolate	-
klebsiella 11	laboratory isolate	-
klebsiella 12	laboratory isolate	-
klebsiella 15	laboratory isolate	-
klebsiella 16	laboratory isolate	-
Staphylococcus aureus	laboratory isolate	-
Proteus	laboratory isolate	-

Host range result: (+) clear lysis, (-) without lysis

2.2 Isolation of bacterial strains and determination of drug resistance

Bacterial strain isolation was carried out as described above, with some modifications (Raza et al., 2018). Collection of non-duplicate foodborne Salmonella isolates from livestock and poultry farm effluents. In short, the sewage collected from farmers' markets and livestock and poultry farms (slaughterhouses) was pre-treated, allowed to settle statically, impurities were filtered using gauze, and the filtrate was centrifuged. Add liquid LB medium to the supernatant and incubate overnight at 37 ℃ at 180 rpm. The incubated liquid was inoculated on selective SS agar plates and HE agar plates by zoning and marking method in an ultra-clean bench, and incubated in a 37℃ constant temperature incubator for 24 h. The growth of the colonies on the selective medium and the size, shape, and color of the colonies were observed. A single colony with different shapes was selected and purified by zoning and streak, respectively. After 8–10 times of purification, the isolated colonies were identified by 16S rDNA sequencing (Shanghai Shenggong Biological Engineering Company). Subsequently, the strains identified by sequencing were expanded and cultured in a liquid LB medium to logarithmic growth phase, and the strains were tested for drug resistance according to the disc diffusion method. Antimicrobial susceptibility testing was determined by agar dilution recommended by the Clinical and Laboratory Standards Institute (CLSI), and the antimicrobials tested are listed in Table S1.

2.3 Bacteriophage Isolation, purification, and propagation

Bacteriophage isolation was performed as previously described but with some modifications (Zhou et al., 2021). Briefly, the sewage sample was allowed to settle and subsequently preliminarily filtered with gauze for large impurities and particles, and the supernatant was filtered through a sterile filter. The filtrate and host bacterial fluid in the logarithmic growth phase were added to 100 mL LB liquid medium and cultured at 37℃ and 180 rpm for 24h by shock. Then the culture medium was centrifuged supernatant was taken, and the filtrate was collected through a sterile filter with an aperture of 0.22um. The filtrate was temporarily stored at 4℃ for later use. Subsequently, the bacteriophage was purified by double-plate method. The filtrate of bacteriophage with different dilution ratios and the host bacterial fluid of the logarithmic growth stage were evenly mixed and poured into a semi-solid medium centrifuge tube containing about 3 mL at 42℃ and mixed well. Then the liquid in the centrifuge tube was poured into a plate, and after condensation, it was placed into a 37℃ incubator for inverted culture to obtain phage plaques. Subsequently, a single translucent phage plaque was selected and continued for multiple rounds of purification, and the phage obtained was named Salmp-p7.

Then, the phage was amplified by the liquid amplification method. The purified phage with a multiple number of infection (MOI) of 1 and logarithmic host bacteria were added to the liquid LB and cultured in a shaking table at 37℃ until the liquid was clarified in the centrifuge tube. After centrifugation at 5000 rpm for 10 min, the supernatant was fed through a sterile filter with a pore size of 0.22 um, and the phage proliferation solution after one round of amplification was obtained. After multiple rounds of amplification, the titer of the phage was determined by the double-layer plate method. Plates with three consecutive gradients and a countable number of phages were selected and counted, and the experiment was repeated three times. The phage can be stored at 4℃ for a short time, or it can be uniformly mixed with 50% glycerol 1:2 and stored in an ultra-low temperature refrigerator at -80℃.

2.4 Transmission electron microscopy (TEM)

The morphological observation of bacteriophages was carried out using transmission electron microscopy according to the method (Ding et al., 2020). A drop of phage stock solution (1×10⁹ PFU/mL) was added to the carbon-coated copper mesh, and after phage adsorption, a drop of 1% phosphotungstic acid was used to negatively stain it. After the mesh was dried, the phage morphology was observed under a transmission electron microscope at 120 kV.

2.5 Phage host range analysis

A total of 20 bacteriophages were evaluated in the host range, including 7 Salmonella enteritidis, 5 Escherichia coli, 5 Klebsiella pneumoniae, 1 Staphylococcus aureus, 1 Proteobacteria and 1 Citrobacter. All strains were obtained from lab-preserved strains that had previously been confirmed by 16S strain identification tests. For each strain, phage plaque formation was assessed using phage plaque assays.

2.6 Optimal multiplicity determination of phage infection

Host bacteria were cultured to the logarithmic stage, and the number of bacteria was measured by plate counting method. The titer of phages in the phage proliferation solution to be tested was determined by the double-layer plate method, and the average value was taken in three replicates. According to the MOI of 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 mixed bacteriophage and host culture, respectively, the total volume of each group was the same with the host bacteriophage solution without host bacteria and the bacteriophage proliferation solution without host bacteria as the control group. The phage titer was cultured at 37℃ in a constant temperature shaking table at 180 rpm for 6 h and then tested in a 0.22 um aseptic filter by double-layer plate method. The average value was obtained after three parallel repeats. MOI, which produced the highest phage titer, was the best infection complex number.

2.7 One-step growth curve determination

As previously shown, a one-step growth curve assay was performed to determine the incubation period and burst size of the phage, with some modifications (Tayyarcan & Boyaci, 2023). The bacteriophage and host bacteria were mixed in the optimal infection complex ratio, and after standing for 15 min, centrifuged at 8000 rpm for 10 min, and discard the supernatant. Washed and precipitated with PBS three times, and then precipitated with liquid LB heavy suspension bacteria, placed in a constant temperature shaking table at 37℃, 200 rpm, and shock culture. Starting from 0 min, 200 ul culture solution was removed every 10 min, and the titer of phage was measured after passing through a 0.22 um sterile filter. The one-step growth curve was drawn with the infection time as the horizontal coordinate and the phage titer as the vertical coordinate. The incubation period, cleavage period, and cleavage amount of phage could be determined according to the one-step growth curve.

2.8 Temperature and pH stability tests

2.8.1 Temperature stability test

Prepare 1.5mL EP tubes, add 500 ul bacteriophage suspension (10⁹ PFU/mL) into them, and place them in the preheated water bath for 2 hours at 40℃, 50℃, 60℃, 70℃, and 80℃, respectively. After incubation, mix with host bacteria according to the optimal infection complex ratio. The phage titer was determined by the double plate method after continuous dilution of liquid LB ten times, and the average value was obtained after three parallel repeats.

2.8.2 pH stability test

In advance, the pH of liquid LB was adjusted to 2–13 with HCl or NaOH solution, and 100 ul liquid LB of different pH was added to 100 ul phage suspension (10⁹ PFU/mL), and incubated in a constant temperature water bath at 37℃ for 1 h. After incubation, phage titer was determined after continuous dilution of each sample, and the average value was obtained after three parallel repeats.

2.9 Bacteriophage antimicrobial activity test in vitro

The host bacteria were added to the liquid LB and incubated at 37℃ at 200 rpm to the logarithmic phase. Two 10 mL EP tubes were added to one with 2 mL of phage suspension (10⁹ PFU/mL) and 2 mL of log-phase host solution in the ratio of optimal multiplicity of infection, and 2 mL of sterile PBS buffer and 2 mL of log-phase host solution to the other tube. Incubate at 37℃ with shaking on a shaker, sample 200 ul to measure the optical density value (OD_600nm) every hour, and the experiment was repeated three times. The infection time was taken as the abscissa, the optical density value (OD_600nm) was used as the ordinate, the PBS buffer group was used as the control group, and the in vitro antibacterial curve was plotted (Kwak, Kim, Ryu, & Bai, 2023).

2.10 Whole genome sequence analysis and phylogenetic analysis

As previously described, phage genomic DNA was extracted using phenol-chloroform (Chang et al., 2015). The phage was amplified, and 0.5% volume of chloroform was added into the phage proliferation solution, and the supernatant was obtained by centrifuge at 4℃. The supernatant was treated with DNase I and RNase A and cultured at 37℃ to remove bacterial DNA and RNA contamination. PEG/NaCl was then used to precipitate, and the supernatant was left for 3 h at 7℃, then the supernatant was centrifuged, and the precipitate was rinsed and re-suspended with PBS buffer. Sodium dodecyl sulfate (SDS) solution was added to the suspended bacteriophage solution, which was allowed to stand at 68℃, and then a mixed buffer solution of phenol/chloroform/isoamyl alcohol = 25:24:1 was added to it, which was gently mixed evenly and centrifuged at 4℃ to obtain the supernatant, and DNA was purified by ethanol precipitation method. The sodium acetate in the supernatant was thoroughly mixed, then pre-cooled ethanol was added, and the supernatant was removed by centrifuge at 4℃. Then 70% ethanol was added to the EP tube, centrifuged at 4℃, and the supernatant was carefully discarded. Dry the EP tube at room temperature and add the appropriate amount of sterile water to dissolve the DNA precipitate.

The phage genome extracted by the above method was sequenced by the sequencing platform of Shenggong Bioengineering (Shanghai) Co., Ltd. Open reading frames (ORFs) of phages were compared and annotated using BLAST. Amino acid sequence using NCBI (https://blast.ncbi.nlm.nih.gov/) online website compared sequence, genome circle diagram drawing in (https://proksee.ca/). The phage evolutionary tree was constructed with MEGA 11 software and Virulence Factor Database(VFDB: http://www.mgc.ac.cn/VFs/) and Comprehensive Antibiotic to hold the Database (CARD: http://arpcard.mcmaster.ca) for screening Resistance and virulence genes.

2.11 Bacteriophage inhibition and clearance tests for Salmonella biofilms

2.11.1 Determination of biofilms

For the determination of biofilm, refer to the crystal violet staining method for quantitative determination of biofilm using 96-well plates (Wang et al., 2020). The bacteria were first inoculated into liquid LB medium, cultured at 37℃ and 180 rpm for 12 h, and then inoculated into 96-well plates for static culture. After culture for a certain period, the medium and non-adherent bacteria were discarded, and then the culture holes were cleaned three times with PBS, and the holes were dried after PBS was discarded. Then 99% methanol was added to fix the bacteria attached to the wall, fixed for 15 min, and then the methanol was discarded and the holes were dried. Add 0.1% crystal violet to stain for 10 min, remove crystal violet after staining, and gently clean each hole with sterile water until the water is not purple, and then dry each hole at room temperature. After 200 ul glacial acetic acid was added to dissolve crystal violet, the optical density value (OD) of each pore was measured at 570 nm to determine the amount of biofilm.

2.11.2 Phage inhibition of biofilm formation

A certain amount of bacteriophage (titer 10², 10⁴, 10⁶, 10⁸ PFU/mL) was added to 200 ul of host bacteria cultured to the logarithmic stage and mixed well, and the blank group was only added with bacteriophage solution, which was respectively added to 96-well plates, and incubated at 37℃ for 24 h, 48 h, 72 h. Finally, the amount of biofilm was measured by method 2.11.1.

2.11.3 Phage clearance after biofilm formation

Add 200 ul of host bacteria cultured to logarithmic stage into 96-well plates, stand at 37℃ for 24 h, 48 h, and 72 h, discard non-adherent bacteria and cultures, gently wash three times with PBS, and add a certain concentration of phage (titer 10², 10⁴, 10⁶, 10⁸ PFU/mL). Then the 96-well plates were placed in an incubator at 37℃ for 5 h, and the amount of biofilm was measured by method 2.11.1.

2.12 Food application

2.12.1 Preparation of samples of contaminated egg liquid

The sterile egg was beaten into a sterilized conical bottle, 5 mL of the egg was sucked with a disposable syringe, and 5 mL of sterile water was mixed to make a sterile egg sample, and then the host bacteria was added to make its concentration in the egg reach 10⁶ CFU/mL, and the egg sample contaminated with Salmonella enteritidis was obtained.

2.12.2 Effect of phages on contaminated egg liquid

Bacteriophage drops were added to the contaminated egg samples at the optimum titer corresponding to the complex number of infections. The same volume of PBS buffer was used as the control group and cultured in a 4℃ refrigerator and a constant temperature incubator at 25℃ for 12 h. Samples were taken from 1 mL to 1.5 mL aseptic centrifuge tubes every 2 hours, and centrifuged at 5000 rpm for 1 min. Another 1 mL of PBS buffer was added, then centrifuged again after suspension and repeated twice. The last suspension was diluted in a double ratio, 100 ul was dropped on an agar plate, evenly coated with a coating rod, absorbed, and transferred to an incubator at 37℃ for overnight culture, and the viable bacteria were counted. The experiment was repeated three times.

2.13 Statistical analysis

SPSS 16.0 and GraphPad Prism 8.0.2.263 were used for statistical analysis, and T-test was used to analyze the statistical differences between the groups. P < 0.01 and P < 0.05 indicated that the differences were extremely significant and significant, respectively.

3.1 Strain resistance testing

The tolerance of phage Salmp-p7 of Salmonella enteritidis to different antibiotics was determined, and the results are shown in Table S1. It can be seen that phage Salmp-p7 of Salmonella enteritidis is sensitive to Neomycin, Gentamicin, Amikacin, Cefoperazone, Ceftriaxone, Cefradine, Cefamezin, Cephalexin, Cefuroxime, Meropenem, Imipenem and Ciprofloxacin. It is resistant to Carbenicillin, Minocycline, Dominocycline, Tetracycline, Erythromycin, Piperacillin, Ampicillin, Oxacillin, and Penicillin. Phage Salmp-p7 showed resistance to a variety of antibiotics, indicating that its resistance is strong, and it is very important to find suitable and effective phages.

3.2 Morphological analysis

TEM images showed that Salmp-p7 belonged to the Siphoviridae family (Fig. 1). It has a non-shrinkable tail length of about 180.62 nm and an icosahedral head diameter of about 59.81 nm (Fig. 1). Similarly, Pacne11-13, a Siphoviridae phage against multi-drug resistant Propionibacterium acne, has a polyhedral head with a diameter of 60 ± 4.5 nm. The tail length of the phage Salmp-p7 was 170 ± 6.4 nm, which supported that the phage belonged to the Siphoviridae family (Liao et al., 2023).

3.3 Host range analysis

To determine the host range of Salmp-p7, all strains listed in Table 1 were tested for Salmp-p7. The results confirmed that Salmp-p7 could inhibit the growth of multiple strains of Salmonella enteritidis. At the same time, it can also inhibit the growth of another gram-negative bacteria, Escherichia coli, but cannot infect Gram-positive bacteria, indicating that Salmp-p7 has high host specificity. The wide host range of Salmp-p7 indicates its potential as a biological control agent for Salmonella enteritidis.

3.4 Optimum infection multiplicity and one-step growth curve

When the phage is mixed with the host bacteria in the optimal ratio, the phage will exert a better lysis effect, and the phage titer is the highest at this time. As shown in Table S2, when Salmp-p7 was mixed with host bacteria titer in a ratio of 0.001, phage titer was the highest, 8.0×10⁸ PFU/mL. Therefore, the optimal MOI for phage Salmp-p7 is 0.001. Under the optimal MOI condition, the one-step growth curve of Salmp-p7 was tested. The incubation time of phage was shorter, about 10 min, and the burst duration was longer, about 85 min, which reached stability at about 90 min (Fig. S1). The burst size was about 55 PFU/cell, and the cracking effect was good.

3.5 Stability assessment at different temperatures and pH values

Under the stress of various conditions such as temperature and pH, the phage host lysis activity should maintain a certain stability (Fig. 2). When Salmp-p7 was incubated at 30, 40, and 50℃ for some time, there was no significant change in phage titer (Fig. 2a). However, when incubated at 60℃, the titer of phage decreased by 0.85 log. After incubation at temperatures of 70 and 80℃ for some time, all phages died. These results indicate that phage Salmp-p7 is thermally stable at temperatures below 50℃. The results of pH sensitivity showed that the titer of phage Salmp-p7 was fairly stable after incubation for a while in the pH range of 4.0–12.0 (Fig. 2b). At pH levels of 2, 3, and 13, all phages died. These results indicated that phage Salmp-p7 had good tolerance to high temperature, acid, and alkali.

Compared with other phages of Salmonella enteritidis, Salmp-p7 showed similar stability at different temperatures. At the same time, it shows relatively high acid and alkaline resistance at both lower and higher pH conditions (Fig. 2). It can be seen that phage Salmp-p7 has good stability and can be widely used as a biological control agent in various food processing processes.

3.6 In vitro antibacterial test

In vitro, bacterial challenge was used to evaluate the antibacterial effect of bacteriophage Salmp-p7 on Salmonella enteritidis (Fig. 3). Under the condition of MOI of 0.001, the growth of Salmonella enteritidis could be significantly inhibited after 7 hours of infection by adding bacteriophage, and the host strain was almost completely cleaved by the bacteriophage Salmp-p7, and the absorbance at this time was 0.035. When the time was delayed to 12 h, the growth of the host strain was significantly inhibited compared to the control group. It can be intuitively seen that the growth of the host bacteria is well inhibited, and the antibacterial effect of Salmp-p7 is remarkable.

3.7 Genome sequence analysis and phylogenetic analysis

Whole genome sequences of Salmp-p7 were analyzed and the results were stored in the GenBank database with accession number PQ189589 (Fig. 4). The genome of Salmp-p7 is double-stranded DNA with a total length of 60066 bp. It contains 72 predicted ORFs with a G + C content of 56.21% The genome size of Salmp-p7 is consistent with the size of phages in the Siphoviridae phage because these phages range in genome size from 51 ± 23 kb. This result corresponded to the results of transmission electron microscopy analysis, indicating that Salmp-p7 was a phage of the Siphoviridae phage (Fig. 1). The annotated ORFs are bacteriophage structure (blue), cell lysis (green), replication (orange), unknown protein (purple), and other functions (pink). Importantly, genes associated with predicted lysogen-related functions, such as integrase, transferase, resection enzyme, suppressor, and genome attachment site, were not found in Salmp-p7, further proving that phage Salmp-p7 is a lytic phage (Gordillo Altamirano & Barr, 2019).

In addition to this, the potent antimicrobial activity of the bacteriophage Salmp-p7 may be related to the presence of two of these lyases, and although the activity of this lyase has not been proven, we venture to speculate that this lyase can hydrolyze the peptidoglycan layer of the bacterial cell wall (Fig. 4). Biogenic analysis of the lyase showed that the presence of active sites allowed it to bind to other antibacterial substances and improve its bactericidal activity, which is expected to be a new direction for phage research. At the same time, phage Salmp-p7 was also tested for toxicity and drug resistance-related genes, and no toxicity and drug resistance-related genes were found in the results, indicating that phage Salmp-p7 is non-toxic and harmless, will not cause harm to the environment and other species, and can be safely used in the food system and other systems.

Based on morphological and biological characteristics, phage Salmp-p7 was classified as a phage of the Siphoviridae phage (Fig. 1). Therefore, the phylogenetic relationship of Salmp-p7 with previously reported phages of Salmonella enteritidis and other different strains was analyzed. Phylogenetic relationships of Salmp-p7 with other phages show that Salmp-p7 is most closely related to the phage SB5 of Salmonella enteritidis, a phage belonging to the long-tailed phage family (Fig. 5). Therefore, phylogenetic tree results indicate that Salmp-p7 is a bacteriophage of the Siphoviridae phage capable of infecting Salmonella enteritidis.

3.8 Inhibition and clearance of Salmonella biofilm by Salmp-p7

Bacterial biofilm (or bacterial biofilm, BF) refers to a large number of bacterial aggregate film-like substances formed by bacteria adhering to contact surfaces, secreting polysaccharide matrix, fibrin, lipid-protein, etc., and wrapping themselves around it. Bacterial biofilm is a life phenomenon formed by the accumulation of microorganisms and their secretions to adapt to the natural environment and facilitate their survival (Kwak et al., 2023), but the existence of biofilm will bring great harm to human production and life. Bacteriophages are viruses that are specifically capable of lysing bacteria, and their use for the prevention and removal of biofilms is a viable approach. To explore the effect of bacteriophage Salmp-p7 on the biofilm of Salmonella enteritidis, Salmp-p7 was inhibited and cleared on the biofilm of Salmonella enteritidis.

Scanning electron microscopy (SEM) results showed that the biofilm without bacteriophage Salmp-p7 treatment showed a smooth and dense surface and uniform bacterial distribution (Fig. 6a). In contrast, the bacteriophage Salmp-p7 was used to inhibit Salmonella enteritidis that had not yet formed a biofilm, and compared with the control biofilm, only a small amount of formed biofilm appeared in the field of view of the SEM image, and a single host was present (Fig. 6b). At the same time, after the use of bacteriophages to remove Salmonella enteritidis that had formed a biofilm, the irregularly shaped, clearly formed biofilm was destroyed in the field of view compared to the control constitutive biofilm (Fig. 6c). These results indicated that the bacteriophage Salmp-p7 could inhibit the formation of Salmonella enteritidis biofilm to a certain extent, and at the same time, it could also remove Salmonella enteritidis that had formed biofilm.

To further verify the above results, changes in the amount of Salmonella enteritidis biofilm before and after the addition of phage Salmp-p7 were measured. With the increase in the concentration of bacteriophage Salmp-p7, its inhibitory effect on the biofilm of Salmonella enteritidis became more and more obvious (Fig. 6d). Bacterial colony count showed that the amount of biofilm was reduced to different degrees after treatment with phage Salmp-p7 for 24, 48, and 72 h compared with the control group. When the concentration of phage Salmp-p7 was 1.0×10⁸ PFU/mL, the reduction of biofilm was the largest after 24 h treatment of host bacteria, and the number of biofilm cells was reduced by about 48.47%. At this concentration, the amount of biofilm decreased by 25.15% and 17.00%, respectively, after 48 and 72 hours of treatment (Fig. 6d). Therefore, Salmp-p7 can inhibit the biofilm formation of Salmonella enteritidis. In addition, the effects of different concentrations of the bacteriophage Salmp-p7 on Salmonella that have formed biofilms were studied. As the concentration of bacteriophage Salmp-p7 increases, its clearance effect on Salmonella enteritidis biofilm becomes more pronounced (Fig. 6e). In general, compared with the control group, the amount of biofilm treated with Salmp-p7 decreased. When the concentration of phage Salmp-p7 was 1.0×10⁸ PFU/mL, the reduction of biofilm was the largest after 24 h treatment of host bacteria, and the number of biofilm cells decreased by about 30.30%. At this concentration, the amount of biofilm decreased by 20.17% and 13.46%, respectively, after 48 and 72 hours of treatment (Fig. 6e). These results indicate that phage Salmp-p7 has a good inhibition and clearance effect on the biofilm of Salmonella enteritidis.

3.9 Application of Salmp-p7 in egg liquid

The efficiency of bacteriophage Salmp-p7 in controlling Salmonella enteritidis contamination in liquid whole eggs was determined. Before the experiment, the food samples used in the experiment were confirmed to be free of Salmonella enteritidis. The addition of the bacteriophage Salmp-p7 to eggs contaminated with Salmonella enteritidis showed a reduction in the number of bacteria at both 4℃ and 25℃ (Fig. 7). At 4℃, the number of bacteria in eggs treated with bacteriophage Salmp-p7 gradually decreased, with the largest decrease at 12 h, when the number of bacteria retained was 4.4 log CFU/mL (Fig. 7a). At 25℃, the number of bacteria in eggs treated with bacteriophage Salmp-p7 decreased significantly compared to the control group, with the largest reduction at 12 h, when the number of bacteria retained was 1.3 log CFU/mL (Fig. 7b). At 25℃, the decrease in bacterial concentration in the egg liquid was more significant than at 4℃, presumably because the suitable temperature required for phage growth and amplification was provided at this temperature.

Eggs are an integral part of the human diet due to their high protein content and convenient cooking methods (Tu et al., 2024). Therefore, the quality of eggs has attracted much attention. Because Salmonella can survive and grow in eggs which can lead to human contamination, it has become a focus of control (Ben-Porat et al., 2024). In this study, we isolated a new bacteriophage, Salmp-p7, which can be used to control whole egg liquids contaminated with Salmonella enteritidis. The phage was isolated from the effluent, followed by characterization of its biology and subsequent determination of its efficiency in food contaminated with Salmonella enteritidis.

The antimicrobial resistance of Salmonella enteritidis, the host bacterium of the bacteriophage Salmp-p7, was found to have a wide range of drug resistance spectrum, indicating that it is necessary and urgent to find a safe and reliable bacteriophage that can lyse Salmonella enteritidis (Forsyth et al., 2023). At the same time, Salmp-p7 can not only affect the host bacterium Salmonella enteritidis but also affect other gram-negative bacteria, namely Escherichia coli, which provides more possibilities for future experiments on phage cocktails and broadening the phage lysis spectrum. However, the isolated bacteriophage Salmp-p7 has not been found to affect Gram-positive bacteria, and the lysis spectrum is relatively narrow, which will be limited when infected by multiple microorganisms, which is the shortcoming of this experiment.

The optimal multiplicity of infection and its one-step growth curve of bacteriophage Salmp-p7 were determined, which could reflect the ratio at which Salmp-p7 could have the best incubation period and outbreak period in the reaction time, which well reflected its lysis effect and advantages. However, it is necessary to pay attention to the limitation of use time during use, and different phages have different outbreak periods. Compared with previous studies with an incubation period of 25 min and an outbreak of 94 PFU/cell, the incubation time of this phage was as short as 10 min, and the outbreak was relatively small (Hou, Tang, Huang, & Luo, 2024). At the same time, the stability of Salmp-p7, i.e., temperature and pH tolerance, was determined. Salmp-p7 can maintain a stable state in the range of 30–60℃ and a highly active state at pH 4.0–13.0. In previous studies, the Salmonella enteritidis bacteriophage Rostam remained active at temperatures below 80℃ and pH values of 4.0–11.0 (Azari et al., 2023). The results indicated that the isolated bacteriophage Salmp-p7 had strong alkali resistance and could maintain a good stable state during egg food processing.

The in vitro antibacterial experiment of bacteriophage Salmp-p7 can reflect the antibacterial effect of Salmp-p7 and the growth of Salmonella enteritidis can be significantly inhibited after 7 hours of infection with Salmp-p7. Previous studies have shown that the Salmonella specific bacteriophage SapYZU01 was treated with the host strain at 37℃ for 21 h with an optical density value of 0.05 (Zhou et al., 2021). However, phage activity appears to be limited to specific isolates of the bacterium (Seal, 2013). Therefore, if you want to apply phages in practice, you should consider the dosage and duration of the phage (Onallah et al., 2024).

Transmission electron microscopy (TEM) analysis and genomic analysis of the bacteriophage Salmp-p7 showed that the genome size of Salmp-p7 was 60066 bp in length, with a long and non-shrinkable tail, and all the structural proteins were highly similar to other phages of the Siphoviridae phage, indicating that the phage was a bacteriophage of the Siphoviridae phage (Cardarelli et al., 2010). At the same time, the genome annotation results of the bacteriophage Salmp-p7 showed that there was no integrase-related gene in Salmp-p7, indicating that the phage was lytic. When phages are used in food and other aspects, their virulence and drug-resistance genes should be tested, and the results show that none of them exist, which is safe and reliable. When analyzing the proteins of the phage, it was found that the potent antimicrobial activity of the bacteriophage Salmp-p7 may be related to the presence of two of the lyases. Bacteriophage lyase is a peptidoglycan hydrolase that lyses host cells after phage replication and multiplication. Because of the structural differences between gram-positive bacteria and gram-negative bacteria, the presence of the outer membrane of gram-negative bacteria leads to the phage lyase being mainly effective against positive bacteria (Schmelcher & Loessner, 2014). However, some Salmonella bacteriophage lyases can be combined with different outer membrane penetrants to penetrate the outer membrane and can also achieve the effect of lysing bacteria (Lim, Shin, Kang, & Ryu, 2012). Therefore, while phages are promising antimicrobial agents targeting bacteria, other strategies using bacteriophage-associated proteins can also be devised (Woudstra, Sørensen, & Brøndsted, 2023). In recent years, bacteriophage lyases have been widely used as a novel biological control agent and a natural food preservative (Wei et al., 2019). Therefore, the design of phage lyases and the continued modification of phages are essential to facilitate our control of Salmonella transmission (Cardim Falcao, Edwards, Hurst, Fraser, & Otterstatter, 2024).

The phylogenetic tree of the bacteriophage Salmp-p7 could accurately reflect the evolutionary relationship between species, and the results showed that Salmp-p7 had the strongest homology with Salmonella enteritidis in other Siphoviridae phages. At the same time, not all species will have tagged genes, so the phylogenetic tree for building bacteriophages is not yet complete (Kwak et al., 2023). To meet the growing demand for large data sets, there is a need to develop more efficient and accurate methods, as well as more in-depth research on the integration of artificial intelligence and machine learning techniques in phylogenetic tree construction (Zou et al., 2024).

To investigate the inhibition and elimination effect of phage Salmp-p7 on the biofilm of Salmonella enteritidis. The results of scanning electron microscopy showed that, compared with the biofilm with a smooth surface and tight structure in the control group, only a small amount of formed biofilm appeared in the biofilm inhibited by phage Salmp-p7, and the rest was single host bacteria. The biofilm treated by Salmp-p7 was irregularly shaped and the formed biofilm was destroyed. These results indicated that phage Salmp-p7 could inhibit and clear the biofilm of Salmonella enteritidis. To further confirm this conclusion, changes in the amount of Salmonella enteritidis biofilm were measured. When the concentration of bacteriophage Salmp-p7 was 1.0×10⁸ PFU/mL, the reduction of biofilm was the largest after 24 h of inhibition treatment of the host bacteria, which was about 48.47%. After 24 h of host bacteria clearance, the reduction of biofilm was the largest, about 30.30%. Therefore, the bacteriophage Salmp-p7 has a good inhibitory and scavenging effect on Salmonella enteritidis biofilm. At the same time, over time, the inhibition and removal of biofilm by bacteriophage Salmp-p7 still existed at 48 h and 72 h, but the effect was weaker than that at 24 h. It is speculated that the reason for this phenomenon is that the host bacteria have developed a certain resistance to bacteriophages, and because bacterial biofilm cells have existed in the phage environment for a long time, with the change of environmental factors and various factors, resistance to phages has appeared in the genes of the host bacteria (Y. Yuan et al., 2019). The bacteriophage Salmp-p7 can exert the maximum effect at 24 h and play a more effective role for a longer time in the future, which is an aspect that needs to be studied in the future and needs to be explored more comprehensively and deeply.

In the past few years, food spoilage has been one of the causes of huge economic losses in the food industry. The main cause of food corruption is the existence of bacteria, and it is a problem that needs to be solved to control and avoid this problem (Costa, Pastrana, Teixeira, Sillankorva, & Cerqueira, 2023). Eggs are a common and very important staple food in the world, and eggs and their derivatives are an important part of all aspects of the food industry and are deeply loved by people (Eissa & Shehata, 2024). Salmonella enteritidis is one of the main pathogenic bacteria causing spoilage of eggs, so in this study, the bacteriophage Salmp-p7 was applied to contaminated eggs to observe its effect on pathogenic bacteria. At 25℃, the bactericidal ability of the bacteriophage Salmp-p7 was well exerted, and at this temperature, the phage could stably exert its antibacterial effect, which is a meaningful application in biological control. At 4℃, the bacteriophage Salmp-p7 can reduce the number of Salmonella enteritidis in the host bacterium, but the antibacterial effect is not as good as at 25℃. The rate of synthesis, assembly, and lysis of bacteriophages varies under different environmental conditions (Kwak et al., 2023). Therefore, the effective antimicrobial activity of bacteriophage Salmp-p7 in eggs may be due to the antimicrobial effect of the phage at a suitable temperature. If the bacteriophage Salmp-p7 is to be more widely used in food in the future, it is necessary to study how to maintain the high antibacterial activity of phages at different temperatures, and further study the mechanism of action between phages and host bacteria, which is also lacking in this study. At the same time, there are shortcomings in phage research, such as the limited number of databases and the imperfect regulatory system, which are also problems that need to be solved (Guo et al., 2024).

In this study, a bacteriophage Salmp-p7 was isolated and purified from wastewater from farmers' markets and livestock farms. According to its morphological observation and whole genome sequencing analysis, the phage was a bacteriophage of the Siphoviridae phage. Its genome length was 60066 bp, and it contained 72 predicted ORFs, with a G + C content of 56.21%, and no toxic factors and drug resistance genes. It has a wide host range and is capable of lysing Salmonella enteritidis. The phage has good tolerance and can maintain good activity in high-acid and alkali environments. The phage can also effectively inhibit and clear Salmonella enteritidis biofilm, and it can significantly reduce the content of Salmonella enteritidis in the whole liquid of eggs. Therefore, the bacteriophage Salmp-p7 is a biological control agent that can effectively control Salmonella enteritidis. Although the mechanism of action of this phage has not been deeply explored, it is still believed that there will be a breakthrough in this aspect in the future.

Funding

This work was supported by the Shandong Provincial Natural Science Foundation (No. ZR2020MC184 and No. ZR2020MC074).

Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author contributions

Mengge Chen: Writing–original draft, Conceptualization, Visualization. Tong Yu: Data curation, Formal analysis. Xiangyu Cao: Methodology, Validation. Jiaqi Pu: Investigation. Deshu Wang: Investigation. Hongkuan Deng: Project administration, Supervision, Funding acquisition, Writing–review and editing.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Isolation and characterization of Salmonella enteritidis bacteriophage Salmp-p7 isolated from slaughterhouse effluent and its application in food

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Bacterial strains and growth conditions

2.2 Isolation of bacterial strains and determination of drug resistance

2.3 Bacteriophage Isolation, purification, and propagation

2.4 Transmission electron microscopy (TEM)

2.5 Phage host range analysis

2.6 Optimal multiplicity determination of phage infection

2.7 One-step growth curve determination

2.8 Temperature and pH stability tests

2.8.1 Temperature stability test

2.8.2 pH stability test

2.9 Bacteriophage antimicrobial activity test in vitro

2.10 Whole genome sequence analysis and phylogenetic analysis

2.11 Bacteriophage inhibition and clearance tests for Salmonella biofilms

2.11.1 Determination of biofilms

2.11.2 Phage inhibition of biofilm formation

2.11.3 Phage clearance after biofilm formation

2.12 Food application

2.12.1 Preparation of samples of contaminated egg liquid

2.12.2 Effect of phages on contaminated egg liquid

2.13 Statistical analysis

3. Results

3.1 Strain resistance testing

3.2 Morphological analysis

3.3 Host range analysis

3.4 Optimum infection multiplicity and one-step growth curve

3.5 Stability assessment at different temperatures and pH values

3.6 In vitro antibacterial test

3.7 Genome sequence analysis and phylogenetic analysis

3.8 Inhibition and clearance of Salmonella biofilm by Salmp-p7

3.9 Application of Salmp-p7 in egg liquid

4. Discussion

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1