Bdellovibrio and its application in lysing salmonella enteritidis
By screening and identifying Bdellovibrio HCB006, the problems of antibiotic resistance and side effects in the treatment of Salmonella enteritidis were solved, achieving a highly efficient and safe biological control effect, which is suitable for the control of Salmonella enteritidis in various environments.
Patent Information
- Application Number
- CN202610597985.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-30
- Publication Date
- 2026-06-26
AI Technical Summary
Existing antibiotic treatments for Salmonella enteritidis have led to the emergence of multidrug-resistant strains and have side effects such as intestinal flora imbalance, making them difficult to meet practical application needs. Therefore, there is a need to develop efficient and safe biological control strategies.
A strain of Bdellovibrio HCB006 was screened and identified. It was identified as a novel Bdellovibrio through 16S rRNA gene sequence analysis and genome similarity. It has the ability to efficiently prey on Salmonella enteritidis and a biocontrol agent was developed. It is suitable for environments with pH 6-8 and temperature 25-30℃ and has natural resistance to certain antibiotics.
Bdellovibrio HCB006 can lyse more than 58% of Salmonella enteritidis within 48 hours. It is adaptable to a variety of environments, has good biocontrol stability and industrialization prospects, and is suitable for food processing, aquaculture water bodies and indoor environments.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbiology and relates to a new strain of Bdellovibrio and its application in lysing Salmonella enteritidis. Background Technology
[0002] Salmonella enteritidis ( Salmonella enteritidis Salmonella enteritidis is an important foodborne zoonotic pathogen that can infect poultry, causing significant economic losses in the livestock industry, and can also infect humans through contamination of eggs, meat, and food processing surfaces, leading to diseases such as acute gastroenteritis and seriously threatening public health. Currently, the control of Salmonella enteritidis mainly relies on antibiotic treatment, with commonly used drugs including ciprofloxacin, azithromycin, and cephalosporins.
[0003] However, the widespread and repeated use of antibiotics has led to the emergence of multidrug-resistant strains, significantly reducing the effectiveness of traditional antibiotic treatments. Furthermore, antibiotic abuse can cause side effects such as gut microbiota dysbiosis and superinfections, increasing the difficulty of clinical treatment. Therefore, developing safe, efficient, and less likely to induce drug resistance novel biocontrol strategies has become a current research hotspot.
[0004] Bdellovibrio ( Bdellovibrio sp. Bdellovibrio is a type of parasitic bacterium that obligately preys on Gram-negative bacteria. It proliferates by rapidly impacting and invading the periplasmic space of its prey bacteria, ultimately lysing the host bacteria. Its unique predation mechanism makes it a promising candidate for pathogen control, particularly as an alternative to or supplement to antibiotic therapy. However, the currently available Bdellovibrio strains are limited, exhibiting significant differences in adaptability to various environmental conditions (such as temperature and pH), and displaying inconsistent lysis efficiency against Salmonella enteritidis, making it difficult to meet practical application needs. Therefore, screening for novel Bdellovibrio strains with highly efficient lysis of Salmonella enteritidis has significant scientific research value and application prospects.
[0005] Purpose of the invention The purpose of this invention is to provide a novel Bdellovibrio strain that can efficiently prey on Salmonella enteritidis and can be used as an alternative to antibiotics for the biocontrol of Salmonella enteritidis.
[0006] To achieve the above objectives, the applicant isolated a strain of Salmonella enteritidis from the soil that is capable of efficiently preying on enteritis-like Salmonella. Based on classical microbial morphological classification and ribosomal 16S rRNA gene sequence analysis, it was identified as Bdellovibrio (…). Bdellovibrio sp.) HCB006. This strain was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China on November 23, 2023, with accession number: CCTCC NO: M 20232311.
[0007] The present invention further provides a biological control agent containing the above-mentioned Bdellovibrio HCB006.
[0008] The present invention further provides the application of the above-mentioned Bdellovibrio HCB006 or its biocontrol agent in the lysis of Salmonella enteritidis.
[0009] The present invention also provides a method for lysing Salmonella enteritidis, comprising the steps of inoculating a sample infected with Salmonella enteritidis with Bdellovibrio HCB006 and culturing it.
[0010] Preferably, the pH of the culture is 6 to 8.
[0011] Preferably, the culture temperature is 25–30°C.
[0012] The beneficial effects of this invention are: Bdellovibrio HCB006 has been identified as a novel species within the genus Bdellovibrio through multi-dimensional analysis including 16S rRNA gene sequencing, average nucleotide similarity (ANI), digital DNA-DNA hybridization (dDDH), and predation diet. It possesses unique genetic background, phenotypic characteristics, and predation diet. Experiments show that HCB006 can lyse over 58% of Salmonella enteritidis within 48 hours, adapting to various practical scenarios such as food processing, aquaculture water, and indoor environments. Furthermore, it exhibits natural resistance to carbenicillin, chloramphenicol, and polymyxin B, facilitating its continued effectiveness in environments with antibiotic residues and enhancing the stability of biocontrol. This invention provides a novel solution for the control of Salmonella enteritidis, possessing promising prospects for industrialization and widespread application. Attached Figure Description
[0013] Figure 1 This is a double-layer plate image of Bdellovibrio HCB006 lysed Salmonella enteritidis.
[0014] Figure 2 This is a phylogenetic tree diagram of Bdellovibrio HCB006.
[0015] Figure 3 Line graph showing the temperature sensitivity of the predation ability of Bdellovibrio HCB006.
[0016] Figure 4 Line graph showing the pH sensitivity of the predation ability of Bdellovibrio HCB006.
[0017] Figure 5 This is a graph showing the residual proportion of Bdellovibrio HCB006 lysed Salmonella enteritidis. Detailed Implementation
[0018] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments and accompanying drawings. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.
[0019] The culture medium and buffer solution used in this invention: LB liquid medium: 10g NaCl; 5g yeast extract; 10g tryptone dissolved in 1L ultrapure water. NB upper solid medium: 0.8g nutrient broth; 6g agar; 6mL 0.5M CaCl2 and 3.33mL 0.6M MgCl2 dissolved in 1L ultrapure water. DNB lower solid medium: 0.8g nutrient broth; 15g agar; 6mL 0.5M CaCl2 and 3.33mL 0.6M MgCl2 dissolved in 1L ultrapure water.
[0020] Example 1: Isolation, purification, and preservation of strain HCB006 Weigh 10 grams of soil sample and add it to a 50 ml centrifuge tube containing 20 g of sterile water (pre-sterilized at high temperature). Shake at 28°C and 180 rpm for several hours. Remove the centrifuge tube and centrifuge at 3000 rpm for 30 minutes. Filter the centrifuged soil sample by placing the filter head on a sterilized PA bottle and filtering the soil solution into the PA bottle. Add Salmonella enterica as pre-cultured bacteria and incubate at 28°C and 180 rpm in a shaker. Pre-culture the pre-cultured Salmonella enterica solution to prepare an OD value. 600 =1. Pour the bottom culture medium onto a glass petri dish and let it cool. Then, mix 500 μL of prey culture medium, 100 μL of soil culture medium, and 6 ml of top culture medium in a PA bottle, and pour the mixture into a petri dish to prepare a double-layer plate. After plaques appear on the double-layer plate, pick a single plaque and dilute it into a series of gradient Bdellovibrio suspensions. Perform a double-layer plate test. Pick a single plaque again from the appropriate dilution gradient plates, and repeat this process several times until the plaques become stable plaques with consistent shape, size, and transparency after multiple passages. This yields the pure Bdellovibrio strain of the present invention, which is named... Bdellovibrio sp.HCB006 was deposited on November 23, 2023, at the China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number: CCTCC NO: M 20232311.
[0021] This strain (HCB006) is one of 136 Bdellovibrio strains isolated from 350 soil samples across China. This study systematically isolated, identified, and sequenced the genomes of obligate predatory bacteria from 350 soil samples (18.47°–49.86° N, 76.74°–130.1° E) over a three-year period, resulting in the isolation of 136 Bdellovibrio strains and similar bacteria (including…). Bdellovibrio sp.、 Pseudobdellovibrio sp.、 Peredibacter sp. and Micavibrio Using an average nucleotide identity (ANI) threshold of <95%, these 136 obligate predatory bacteria strains can be divided into 39 distinct species at the genomic level. Among these, 33 species branches at the genomic level differ from previously published Bdellovibrio species with complete genomes. In other words, based solely on the genomic ANI dimension, this study identified 33 potentially unique new species (all strains mentioned in this paper with names beginning with HCB are Bdellovibrio strains isolated and identified by us).
[0022] Example 2 Bdellovibrio ( Bdellovibrio sp.) Identification of a new species HCB006 1. Molecular identification of 16S rRNA gene and phylogenetic relationship A complete Bdellovibrio bacteria was assembled using second-generation Illumina and third-generation Nanopore sequencing data. Bdellovibrio sp. The HCB006 genome was analyzed, and its complete 16S rRNA gene sequence was extracted using Barrnap software. The HCB006 16S rRNA gene sequence was submitted to the NCBI nucleic acid database for BLAST alignment. Simultaneously, a phylogenetic tree of the 16S rRNA genes of HCB006 and representative strains of Bdellovibrio was constructed based on the Neighbor-Joining (NJ) method. Figure 2 16S identification results showed that the strains most closely related to HCB006 were HCB274 and HCB185ZH isolated in this study. HCB006 and Bdellovibrio sp. HCB274 showed the highest consistency, at 99.60%; and Bdellovibrio sp. HCB185ZH had the second highest consistency rate at 99.21%; the third highest was... Bdellovibrio sp. KM01 had a success rate of 99.14%. Following closely behind were HCB290 (98.94%), ZAP7 (98.87%), SKB1291214 (98.87%), HCB209 (98.81%), and uncultured strains. Bdellovibriosp. (Accession number KP183074.1; 98.58%).
[0023] In the latest bacterial species identification standards, when the 16S rRNA gene similarity of an isolated bacterial species to a known bacterial species is less than 98.65%, the isolated species can be considered a potential new species; while when the similarity is higher than 98.65%, the two may belong to the same species or different species. However, in taxonomic studies of diverse bacteria such as Bacillus and Bdellovibrio, the 98.65% 16S similarity threshold cannot accurately identify species. Even if two strains have 100% 16S similarity, they may still be two completely different species. Therefore, it is necessary to further investigate whether Bdellovibrio HCB006 is significantly different from these highly similar Bdellovibrio strains (HCB274, HCB185ZH, KM01, HCB290, ZAP7, SKB1291214, and HCB209) by combining genomic and phenotypic analysis.
[0024] 2. Genomic Average Nucleotide Identity (ANI) and Digital DNA-DNA Hybridization Molecular Identification To further determine the molecular taxonomic position of strain HCB006 and accurately identify whether it is HCB274 、 HCB185ZH, KM01, HCB290, ZAP7, SKB1291214, or HCB209 are novel Bdellovibrio species distinct from these strains. The average nucleotide identity (ANI) between strain HCB006 and these seven bacterial genomes was analyzed, and digital DNA analysis was performed. DNA hybridization (digital DNA) DNA hybridization (dDDH) calculation. dDDH calculation is an important method for definitively identifying new bacterial species; a dDDH ratio >70% is considered to indicate that the two genomes belong to the same species. ANI calculation is another method commonly used to determine the evolutionary relationship of bacterial genomes; generally, an ANI >95% indicates that the two genomes belong to the same species. The calculation results show that strain HCB006 and strain HCB274... 、 The ANI values of HCB185ZH, KM01, HCB290, ZAP7, SKB1291214, and HCB209 were all far below the 95% threshold, at 86.07%, 81.76%, 81.52%, 88.35%, 81.51%, 81.23%, and 81.62%, respectively. Strains HCB006 and HCB274... 、The dDDH values of HCB185ZH, KM01, HCB290, ZAP7, SKB1291214, and HCB209 were all far below the 70% threshold, at 27.60%, 21.60%, 21.40%, 33.30%, 21.50%, 21.00%, and 21.20%, respectively. These genomic results strongly suggest that strain HCB006 is distinct from the known species HCB274. 、 New species of HCB185ZH, KM01, HCB290, ZAP7, SKB1291214 or HCB209.
[0025] 3. Phenotypic identification based on Bdellovibrio's trapping diet and antibiotic resistance. Regarding our own isolated strain HCB274 、 In addition to the genomic comparisons mentioned above, we also conducted a comparative study on the predation range of HCB185ZH, HCB290, and HCB209 based on their most important obligate predation characteristics. The results showed that among the more than 80 tested prey bacteria in this study, strain HCB006 targeted 9 of them (…). Pseudomonas alloputida C96-2 Pseudomonas alloputida KT, Pseudomonas atacamensis M47 Pseudomonas iranensis M49 Pseudomonas chengduensis M61 Ralstonia nicotianae rs1、 Aeromonas hydrophila subsp. hydrophila t25、 Aeromonas veronii t26 and Raoultella ornithinolytica The predation ability of HCB006 (X13) differed significantly from the other four Bdellovibrio strains. HCB006 could only prey on C96-2 and X13, exhibiting a relatively narrow predation diet. In contrast, strain HCB185ZH could prey on C96-2, X13, and t26; strain HCB209 had a wider predation range, preying on C96-2, M47, M49, M61, rs1, t25, and t26, but not X13; strain HCB274 could only prey on t25 and t26; and strain HCB290 could prey on C96-2, KT, M47, and X13. Therefore, HCB006's predation diet differed significantly from that of HCB185ZH, HCB209, HCB274, and HCB290, exhibiting unique predation phenotypes. This difference further confirms at the phenotypic level that strain HCB006 is a new species of Bdellovibrio, distinct from HCB274, HCB185ZH, HCB290, and HCB209.
[0026] For strains already publicly available in the database, because Bdellovibrio sp. KM01 and BdellovibrioOnly the genome sequences of strains sp. ZAP7 are publicly available; phenotypic data are lacking. Therefore, a phenotypic comparison with strain HCB006 cannot be made here. The optimal growth pH, temperature, salt concentration, and antibiotic resistance of strain SKB1291214 are also unreported. We compared its reported predatory diet data. Among the nine comparable indicators, both strains HCB006 and SKB1291214 were found to prey on... Enterobacter sp. Klebsiella sp. and Salmonella sp. has 3 prey indicators, all of which indicate that the prey cannot be hunted. Agrobacterium tumefaciens , Pseudomonas aeruginosa , Pseudomonas putida and Vibrio sp. has a total of 4 prey indicators, two of which are related to Serratia marcescens and Stenotrophomonas maltophilia The predatory abilities of these strains differ significantly. Strain HCB006 is able to prey on... Serratia marcescens and Stenotrophomonas maltophilia The HCB006 strain forms plaques, while strain SKB1291214 does not; the two strains differ in two out of only nine prey indicators. This result further suggests that strain HCB006 is a novel species distinct from the known species SKB1291214.
[0027] In summary, combining physiological and biochemical characteristics, predatory diet characteristics, and molecular biological classification techniques, *Bdellovibrio* (… Bdellovibrio sp. HCB006 can be identified as Bdellovibrio A new species of the genus.
[0028] Example 3: Application Test of Strain HCB006 (1) Effect of temperature on the elimination of Salmonella enteritidis by strain HCB006 First, the Salmonella enteritidis precipitate was centrifuged and the supernatant was removed using HEPES buffer, and then resuspended in OD200. 600 = 1. 500 μL of *Salmonella enteritidis* suspension was mixed thoroughly with 6 mL of melted DNB upper layer medium (approximately 60°C) and poured into a petri dish containing cooled bottom medium to prepare a double-layer plate. Then, 2 μL of *Bdellovibrio* HCB006 suspension was spotted three times onto the medium, and the same petri dish was placed in incubators at 28°C and 37°C for incubation. Plaque diameter was observed and recorded every 24 hours. Results are as follows: Figure 3 As shown, Bdellovibrio HCB006 has a stronger predatory ability at a temperature of 28℃.
[0029] (2) Effect of pH on the elimination of Salmonella enteritidis by strain HCB006 First, adjust the pH to 3-11 using HEPES buffer, disodium hydrogen phosphate, citric acid, sodium bicarbonate, sodium carbonate, hydrochloric acid, or sodium hydroxide solution. Centrifuge the Salmonella enteritidis solution, remove the supernatant, and add the precipitate and supernatant of the Bdellovibrio HCB006 bacterial solution sequentially to the above-mentioned buffer solutions of different pH values to prepare OD. 600 A mixed culture medium with a concentration of approximately 1 was incubated at 28°C. Samples were taken every 6 hours to measure the OD value. 600 In the buffer system, Bdellovibrio is almost undetectable by OD600 due to its small size, while prey bacteria (Salmonella enteritidis) are detectable by OD600 due to their larger size. Therefore, the OD600 value of the buffer system can reflect the degree to which prey bacteria (Ralstonia solanacearum) are preyed upon by Bdellovibrio. In the control group without Bdellovibrio or under conditions where Bdellovibrio cannot prey on its prey, the OD600 value of the buffer system remains almost stable. However, under conditions where Bdellovibrio can prey on its prey, the OD600 value of the buffer system decreases over time.
[0030] The results are as follows Figure 4 As shown, Bdellovibrio HCB006 can grow in near-neutral environments of pH 6, 7, and 8 and has a good ability to prey on Salmonella enteritidis.
[0031] (3) Effects of antibiotics on the elimination of Salmonella enteritidis by strain HCB006 First, the Salmonella enteritidis precipitate and the supernatant supernatant from the Bdellovibrio HCB006 bacterial suspension were centrifuged using HEPES buffer and added sequentially to a PA bottle in a certain ratio to prepare OD. 600 A mixed culture medium with a concentration of approximately 1 was prepared, and then eight different types of antibiotics were added to the Bdellovibrio bacterial culture and mixed thoroughly. The culture was incubated at 28°C. Samples were taken every 6 hours to measure the OD600. Bdellovibrio HCB006 showed resistance to carbenicillin, chloramphenicol, and polymyxin B; however, it showed sensitivity to kanamycin, gentamicin, tigecycline, spectinomycin, and tetracyclines.
[0032] (4) Application test of strain HCB006 in eliminating Salmonella enteritidis Activate pathogenic Salmonella enteritidis and incubate overnight at 37°C. The next day, remove the prey and resuspend it in HEPES buffer to OD200. 600=1. Prepare a Bdellovibrio bacterium suspension (HCB006) and add it to the pathogenic Salmonella enteritidis suspension in the experimental group. The control group does not receive a Bdellovibrio bacterium suspension. Each group has three replicates. The mixture is incubated with shaking at 28℃ and 180 rpm. Samples are taken every 6 hours to measure OD. 600 Continuous measurements were taken for 48 hours, data were processed, and a line graph showing the residual proportion of pathogenic Salmonella enteritidis was plotted. Figure 5 As shown, the prey Salmonella enteritidis began to lyse at 18 hours, the lysis rate accelerated at 42 hours, and only 42% remained at 48 hours, significantly lower than the control group. This indicates that the HCB006 strain has a very high lysis effect on Salmonella enteritidis.
Claims
1. A strain of Bdellovibrio ( Bdellovibrio sp.)HCB006, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20232311.
2. A biological control agent containing the Bdellovibrio HCB006 as described in claim 1.
3. The Bdellovibrio HCB006 of claim 1 or the biocontrol agent of claim 2 in the lysis of Salmonella enteritidis ( Salmonella enteritidis Applications in ).
4. A method for lysing Salmonella enteritidis, comprising the steps of inoculating a sample infected with the pathogen with Bdellovibrio HCB006 as described in claim 1 and culturing it.
5. The method according to claim 4, characterized in that: The culture was carried out under conditions of pH 6–8.
6. The method according to claim 4, characterized in that: The culture temperature is 25-30℃.