Achromobacter yks2 and application thereof

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a bacterium YKS2 and application thereof, the bacterium YKS2 is preserved in the China General Microbiological Culture Collection Center on April 14, 2022, and the preservation number is CGMCC No. 24678, has good acid resistance, bile salt resistance, artificial gastric juice resistance and artificial intestinal juice resistance, can improve immunity, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a kind of achromobacter YKS2 and application thereof. BACKGROUND

[0002] The stomach of ruminants includes four chambers of rumen, reticulum, omasum and abomasum, wherein the rumen as its unique digestive organ is the main place for forage digestion. The reason why yak can better digest cellulose is closely related to the physiological environment of its rumen. In addition, the anaerobic environment of yak rumen also makes it like an efficient living fermentation tank, where various microorganisms coordinate or restrict each other, maintain the balance of rumen microecology, and make the forage digestion process proceed smoothly.

[0003] Achromobacter is a microorganism of Achromobacter genus, which is originally from China and is applied in many fields. For example, the invention patent CN110699279B discloses a kind of achromobacter, which has strong iron carrier production capacity, can convert insoluble iron in soil into effective iron, can increase soil nutrients, promote the absorption of soil iron by plants, especially can successfully colonize in peanut rhizosphere soil, promote the growth of peanuts and increase yield, and has great application potential. The invention patent CN114717144A discloses a strain of achromobacter, which has good degradation effect on metamitron in soil. The achromobacter is used in cooperation with other commonly used soil improvement microbial strains, which expands the performance of existing soil improvement microbial agents. The invention patent CN114164132A discloses a kind of achromobacter. The achromobacter H6 can metabolize to produce compounds phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) with anti-plant pathogenic fungal activity, can be cultured on a large scale in a short period of time, has low fermentation culture cost, and is not limited by time domain and season. The production of PCA and PCN is expanded, and the production cost of PCA and PCN is reduced. The PCA and PCN separated from the fermentation culture of achromobacter H6 have good application prospect for preparing agricultural antibiotics, and can effectively prevent and control fungal diseases of crops. However, no achromobacter with good acid tolerance, bile salt tolerance, artificial gastric juice tolerance and artificial intestinal juice tolerance has been found.

[0004] The present application relates to the field of biotechnology, in particular to a kind of achromobacter YKS2 and application thereof. SUMMARY

[0005] In view of the above technical problems, the primary object of the present application is to provide a kind of achromobacter (Achromobacter YKS2) and application thereof. achromobacter marplatensisYKS2, deposited with the China General Microbiological Culture Collection Center on April 14, 2022, and having the accession number CGMCC No. 24678.

[0006] A second object of the present application is to provide a bacterial agent containing the Achromobacter YKS2.

[0007] Preferably, the bacterial agent contains live bodies of the Achromobacter YKS2.

[0008] Preferably, the bacterial agent is a liquid bacterial agent or a solid bacterial agent.

[0009] Preferably, the bacterial agent is a liquid bacterial agent.

[0010] A third object of the present application is to provide the use of the Achromobacter YKS2 in improving immunity.

[0011] A fifth object of the present application is to provide the use of the Achromobacter YKS2 in preparing an anti-diarrhea drug.

[0012] A sixth object of the present application is to provide the use of the Achromobacter YKS2 in preparing a feed yeast.

[0013] The present application has the following beneficial effects: The Achromobacter YKS2 is unexpectedly screened, and the Achromobacter YKS2 has good acid resistance, bile salt resistance, artificial gastric juice resistance and artificial intestinal juice resistance, and can improve immunity. BRIEF DESCRIPTION OF DRAWINGS

[0014] figure 1 Colony morphology and gram staining of the strain

[0015] Note: A: Colony morphology; B: Gram staining chart (×100 times)

[0016] figure 2 16S rDNA gene amplification results of the strain

[0017] Note: M. DL-2000 Marker; 1. Bacterial sample; 2. Bacterial sample repeat; 3. Negative control

[0018] figure 3 Genetic evolution tree of 16S rDNA gene of the strain

[0019] figure 4 Growth curve and acid production curve of the Achromobacter YKS2

[0020] Note: Left graph: Growth curve; Right graph: Acid production curve

[0021] figure 5 Self-aggregation ability of the Achromobacter YKS2

[0022] figure 6 Hydrophobicity of Achromobacter YKS2

[0023] figure 7 The state of Achromobacter YKS2 and β-hemolytic streptococcus in blood plate culture

[0024] Note: Left: Achromobacter YKS2; Right: β-hemolytic streptococcus

[0025] figure 8 Effect of Achromobacter YKS2 administration on intestinal mucosa of mice.

[0026] Note: (A) HE staining, scale = 100 μm, magnification 100x, the length of intestinal villi (mm) is represented by line segment a, and the depth of crypt (mm) is represented by line segment b. (B) The values of intestinal villus length and crypt depth and the value of V / C, data are expressed as mean ± standard deviation (n = 6). There is no significant difference between groups without superscript letters.

[0027] figure 9 Effect of Achromobacter YKS2 administration on intestinal immune cells of mice.

[0028] (A) HE staining, scale = 100 μm, magnification 100x, red arrows show goblet cells (gc), and yellow arrows show lymphocytes (IEL). (B-Left) The number of duodenal lymphocytes, (B-Right) The number of duodenal goblet cells. Data are expressed as mean ± standard deviation (n = 5). There is a significant difference (p < 0.05) in the bars in the figure without the same superscript letters (a-b). DETAILED DESCRIPTION

[0029] The scope of protection of the present application is described in detail below in combination with specific embodiments, but it should be noted that the scope of protection of the present application is not limited by the following examples.

[0030] In the following examples, all data were analyzed by IBM SPSS 22.0 software, One-Way ANOVA and LSD multiple comparisons, the results were expressed as mean ± standard deviation, and the graphs were drawn by Graphpad prism software. P < 0.05 is significantly different; p < 0.01 is extremely significantly different.

[0031] Example 1, strain screening and identification

[0032] 1. Yak rumen fluid sample collection

[0033] (1) Collection site: yak rumen contents of a pastoral household in Datong County, Xining, Qinghai.

[0034] (2) Collection object: The collection object of the yak rumen fluid sample is a yak with an age of 3-5 years old selected at random, and these yaks are mainly raised by grazing, freely eat and drink water, and are not supplemented with any feed.

[0035] (3) Collection method: The yak rumen fluid sample is extracted by repeatedly inserting a hard PVC plastic tube into different sites of the yak rumen abdominal sac several times, and immediately after the collection is completed, the sample is placed into a carbon dioxide filled thermos bottle, the thermos bottle opening is tightly covered, and the sample is taken back to the laboratory for standby.

[0036] 2. Enrichment culture of rumen bacteria

[0037] (1) Enrichment culture: The collected yak rumen fluid sample is taken out, 1 mL of the yak rumen fluid sample is sucked by a pipette into 9 mL of a liquid enrichment culture medium, and the sample is cultured at a rotation speed of 180 r / min and 39°C on a shaking table for 4 days.

[0038] (2) Dilution culture: The culture liquid is diluted with 0.9% physiological saline at 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , and 10 -7 seven gradients, respectively, and the dilution liquid of the seven gradients is added to the liquid enrichment culture medium at a ratio of 1:10 for culture, and the culture condition is 180 r / min at 39°C for 4 days.

[0039] 3. Strain screening

[0040] (1) Strain primary screening: 100 μL of 10 4 , 10 5 , and 10 6 fold dilution liquid of the bacterial liquid is spread on CMC-Na, LB agar, and blood plate solid culture media, and 3 parallel tests are performed for each dilution gradient. The spread solid plates are respectively placed into a 37°C constant temperature incubator and a 37°C anaerobic incubator, and are inverted cultured for 24 h and 72 h in aerobic and anaerobic environments, respectively, the growth state of the bacteria is observed, and the strains with a good growth state are further rescreened.

[0041] (2) Strain rescreening: The pure culture colonies selected are streaked on a CMC-Na medium, and are cultured at 37°C for 24 h, then a single colony is picked and inoculated on a CMC-Na Congo red solid plate, 4 strains of bacteria are inoculated on each plate, and 3 parallel tests are performed. The strains with obvious transparent hydrolysis rings around the colonies are selected for purification.

[0042] (3) Purification: pick the separated single colony, inoculate on the initial screening medium, and conduct continuous streak culture at 37 ℃ for 3-5 times, pick the single colony with good growth state, streak culture on the CMC-Na medium, after Congo red staining, conduct gram staining and microscopic examination on the strain with obvious transparent circle, observe the uniformity of bacterial staining characteristics and morphological characteristics, and number.

[0043] 4. Strain identification

[0044] (1) Morphological identification

[0045] Colony morphology observation: dilute the bacterial solution and spread on the LB agar plate, and observe the colony morphology after 24 h. Gram staining: refer to the steps in the gram staining kit instruction manual, and observe under a microscope.

[0046] The YKS2 strain from the yak rumen has a colony diameter of about 1-2 mm, is semitransparent, slightly raised, has a light yellow color, and has a smooth surface (see figure 1 A). The gram staining microscopic examination result (see figure 1 B) shows that the bacteria are gram-negative and have a short rod shape.

[0047] (2) Biochemical identification

[0048] Biochemical identification: use the VITEK2 microbial biochemical automatic identification instrument for identification, and refer to the “Bergeier's Systematic Bacteriology Manual” for the identification result. According to the instruction manual, the result is >99.9%, the identification confidence is “excellent”; 99.0%≤result<99.9%, the identification confidence is “very good”; 90.9%≤result<99.0%, the confidence is “good”; 80.0%≤result<90.9%, the confidence is “acceptable”.

[0049] Use the VITEK2 full-automatic microbial biochemical identification instrument to identify the strain, and the identification result is shown in Table 1. According to the “Bergeier's Systematic Bacteriology Manual”, the result of the strain is that the probability of the Enterobacteriaceae Achromobacter genus is 99%, which has a high confidence.

[0050] Table 1 Biochemical identification result of the yak rumen-derived strain

[0051]

[0052] Note: “+” indicates a positive test result, and “-” indicates a negative test result.

[0053] (3) Molecular identification

[0054] Take 2 mL of the third-generation fresh bacterial culture from the incubator, centrifuge at 8000 r / min for 2 min, and collect the bacterial cells. Then, follow the instructions of the bacterial gene extraction kit to extract bacterial DNA.

[0055] PCR amplification, sequencing, and phylogenetic analysis: 16S rDNA gene PCR amplification system: 10 μL of 5×PS Buffer, 5 μL each of 27F and 10 μmol / L 1492R, 4 μL of 2.5 μmol / L dNTPs, 4.5 μL of DNA template, 1.5 μL of PrimeSTAR, and 20 μL of H2O. PCR amplification program: 98 ℃ pre-denaturation for 5 min; followed by denaturation at the same temperature for 10 s, annealing at 55 ℃ for 5 s, extension at 72 ℃ for 90 s, for a total of 30 cycles; final extension at 72 ℃ for 6 min. PCR amplification products were recovered, and gels were prepared using 1% agarose gel electrophoresis. Samples were loaded, processed, analyzed, and the electrophoretic bands were saved using a gel image processing system. PCR products were also sent for assaying. Bacterial 16S rDNA sequences were compared using BLAST in NCBI to analyze bacterial genetic evolution. Select partial 16S rDNA sequences of more than 10 different similar strains, open them in Mega7 software, and create a phylogenetic tree analysis of the target strains.

[0056] Depend on figure 2 As can be seen, 1% agarose gel electrophoresis yielded two single, clear bands (two replicates), bright and free of impurities. PCR amplification showed the target fragment to be 1500 bp in length, consistent with the expected result. The obtained sequences were compared and analyzed using the BLAST online analysis software on the NCBI website. The results showed that the isolated bacteria shared over 99% homology with Achromobacteria (MT078618.1, MN055902.1, etc.).

[0057] Depend on figure 3 It can be seen that strain YKS2 and the verified strain MT078618 belong to the same group of bacteria. Therefore, based on the results of morphological observation, biochemical identification, and 16S sequence analysis, this strain is identified as a subspecies of the genus Achromobacterium in the family Enterobacteriaceae, with the Latin name [missing information]. achromobacter marplatensis It was named Achromobacterium ( achromobacter marplatensis YKS2 was deposited on April 14, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24678. The contact number is 010-64807355, and the address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0058] In the following examples, Achromobacterium ( achromobacter marplatensisYKS2 is abbreviated as Achromobacterium YKS2, and has the same legal effect.

[0059] Example 2: Analysis of the biochemical characteristics of the strain

[0060] 1. Bacterial growth curve and acid production performance

[0061] The fresh third-generation bacterial culture was inoculated into LB broth medium at a rate of 2%. Samples were taken every 2 hours, and the absorbance of the bacterial culture at OD600nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The bacterial growth curve was plotted. The pH value of the bacterial culture was measured every 6 hours, and the acid production status of the bacteria was recorded.

[0062] like figure 4 As shown in the left figure, Achromobacterium YKS2 entered the growth phase after being cultured in LB broth for 2 hours, after which OD... 600nm The OD value continued to rise, reaching a plateau after 20 hours of cultivation, at which point the OD value was measured. 600nm It is 1.03. For example... figure 4 As shown in the right figure, the initial pH of Achromobacterium YKS2 in LB broth medium was 7.07. As the culture time increased, the pH of the bacterial solution showed an upward trend.

[0063] 2. Self-agglomeration

[0064] Collect the fresh third-generation bacterial suspension, centrifuge at 5000 r / min for 10 min, discard the supernatant, wash twice with sterile physiological saline, centrifuge again at the same speed for 10 min to collect the bacterial cells, resuspend in physiological saline and adjust the concentration to OD600nm = 1.0. Take 4 mL of the prepared bacterial suspension and place it in a 10 mL centrifuge tube to allow it to separate into layers. Collect the supernatant at 1 h, 3 h, and 5 h, and measure the OD600nm using an ELISA reader. Calculate the autoagglutination rate of the test strain according to the following formula.

[0065] Χ=(1-At) / A0×100%

[0066] In the formula: A0 and At refer to the values ​​of the supernatant before and after agglutination measured at OD600nm, respectively.

[0067] Studies have shown that the self-aggregation ability of bacterial strains has a significant impact on biofilm formation and is closely related to intestinal colonization. The self-aggregation test results of *Achromobacterium* YKS2 are as follows: figure 5 As shown in the two figures, Achromobacterium YKS2 at OD 600nm The absorbance at the point decreased over time, while the coagulation rate increased continuously, eventually reaching 28.7% at 5 hours.

[0068] 3. Hydrophobic properties

[0069] The 3rd generation fresh bacteria suspension was collected by centrifugation at 5000 r / min for 10 min, the supernatant was discarded, and the bacteria were washed twice with sterile normal saline, and then centrifuged again at the same speed for 10 min. The bacteria were resuspended with normal saline and the concentration was adjusted to OD600nm=1.0. The test bacteria solution was mixed with an equal volume of dimethylbenzene (non-polar solvent), chloroform (polar acidic solvent) and ethyl acetate (polar basic solvent) respectively, and vortexed at high speed for 2 min. After standing for 2 h, the water phase was taken and the OD600nm was measured by an enzyme marker. The hydrophobicity of the test strain was calculated according to the following formula.

[0070] X=(A0-At) / A0x100%

[0071] In the formula, A0 refers to the OD value of the initial bacteria solution at 600 nm wavelength; At refers to the OD value of the water phase at 600 nm wavelength after mixing and standing for t h in the solvent.

[0072] The surface hydrophobicity of bacteria refers to the unstable condition of bacteria in a polar solution, which causes the rearrangement of bacterial cells. Like the self-aggregation ability, the surface hydrophobicity of bacteria is related to the colonization and adhesion ability of bacteria.

[0073] As shown in figure 6 , the hydrophobicity of Achromobacter YKS2 in the three solvents was more than 60% after standing for 2 h, and the hydrophobicity of Achromobacter YKS2 in chloroform was significantly higher than that in the other two solvents (P<0.05), reaching 77.1%, which had good hydrophobicity. P < 0.05), reaching 77.1%, which had good hydrophobicity.

[0074] 4. Hemolytic activity

[0075] Fresh bacteria solution and fresh β-hemolytic streptococcus solution were taken respectively, a small amount of bacteria solution was taken with a sterile inoculation loop and streaked on a blood agar plate, and the plate was incubated in a 37 ℃ incubator for 24 h to observe whether there was a hemolytic ring.

[0076] As shown in figure 7 , by comparing the state of Achromobacter YKS2 and the control strain β-streptococcus cultured at 37 ℃ for 48 h on blood plates, it was found that there was no hemolytic ring on the blood plate of Achromobacter YKS2, and there was obvious hemolytic ring on the blood plate of β-hemolytic streptococcus.

[0077] 5. Drug sensitivity test

[0078] To test the sensitivity of bacteria to antibiotics, the paper disc diffusion method (K-B method) was used. Fresh bacterial suspension of the 3rd generation was collected and adjusted to 0.5 McFarland (about 1.5 × 108 CFU / mL) by a turbidimeter, and then 150 μL of the adjusted bacterial solution was uniformly coated on an MH agar plate, which was allowed to stand for 20 min, and three kinds of drug sensitive sheets were uniformly pasted on each plate, and then placed in a 37 ℃ incubator for 24 h. The diameter (mm) of the inhibition zone was measured. Each group had 3 replicates. The test results were determined according to the NCCLS drug sensitivity test standard, which was divided into resistant (R), susceptible (S) and intermediate (I).

[0079] Antibiotic safety is an important indicator for strain evaluation. A total of 14 kinds of antibiotics such as cefalexin, cefalotin and cefalotin were selected. As shown in Table 2, the achromobacter YKS2 was sensitive to most of the 14 kinds of antibiotics, and only resistant to cefalexin and amikacin, with low safety risk.

[0080] Table 2 Test results of achromobacter YKS2 drug sensitivity test

[0081]

[0082] Note: Refer to the NCCLS drug sensitivity test standard

[0083] 6. Acid and bile salt resistance test

[0084] Acid resistance test: Adjust the pH value of LB broth medium to 2.0, 4.0 respectively, and sterilize for standby. Take 2% of the 3rd generation fresh bacterial suspension and inoculate into LB broth medium, with no hydrochloric acid medium as control. After 3 h of culture at 37 ℃, the colony count was performed.

[0085] Bile salt resistance test: Take No. 3 bile salt and prepare LB broth medium containing 0.1% and 0.3% bile salt respectively, and sterilize for standby. Take the 3rd generation fresh bacterial suspension and inoculate into LB broth medium at a ratio of 2%, and use bile salt-free LB broth medium as control. After 3 h of culture at 37 ℃, the colony count was performed.

[0086] The results of acid resistance test are shown in Table 3. After 3 h of culture in LB broth medium with pH value of 2.0, no bacteria grew after 24 h of plate culture, and the survival rate of bacteria was 0; in LB broth medium with pH value of 4.0, the survival rate of the bacteria of the strain was 61.4%.

[0087] Table 3 Effect of different pH values on the survival rate of achromobacter YKS2

[0088]

[0089] The results of bile salt tolerance are shown in Table 4. After culturing in LB broth medium with 0.1% and 0.3% bile salt for 3 h, the survival rate of Achromobacter YKS2 was 104.69% and 62.94%, respectively. Compared with the culture without bile salt, the survival rate increased to 104.69% at 0.1% bile salt and decreased to 62.94% at 0.3% bile salt, indicating that the bacteria could completely tolerate 0.1% bile salt and had certain adaptability to 0.3% bile salt.

[0090] Table 4 Effect of different bile salt concentrations on the survival rate of Achromobacter YKS2

[0091]

[0092] 7. Tolerance to artificial gastric and intestinal juice

[0093] Artificial gastric and intestinal juice tolerance test: Fresh bacterial suspension of the 3rd generation was washed 3 times with sterile normal saline, and the bacterial concentration was adjusted to 10 5 CFU / mL with sterile normal saline. 500 μL of bacterial suspension was added to 4500 μL of artificial gastric juice and PBS solution, respectively, and mixed well. After incubation in a 37 ℃ incubator for 3 h, the bacterial suspension was diluted by 2-fold, and the colony count was performed to calculate the survival rate of the bacteria. According to the same procedure, the bacterial solution was added to the artificial intestinal juice, and after incubation in a 37 ℃ incubator for 0 h, 4 h, and 8 h, the bacterial suspension was diluted by 2-fold, and the colony count was performed to calculate the survival rate of the bacteria.

[0094] After treatment with artificial gastric juice for 3 h, the viable bacterial concentration of Achromobacter YKS2 strain decreased compared with that at 0 h, and the survival rate was 78.18%, while the survival rate of the control group was 92.30% (Table 5). Compared with the control group, the attenuation rate of YKS2 in artificial gastric juice was faster.

[0095] Table 5 Tolerance of Achromobacter YKS2 to artificial gastric juice

[0096]

[0097] As shown in Table 6, there was no significant difference in the initial viable bacterial concentration of Achromobacter YKS2 in each test group (P<0.05). Compared with the control group, the viable bacterial concentration of Achromobacter YKS2 significantly increased after treatment with artificial intestinal juice for 4 h and 8 h (P<0.05), and the viable bacterial concentration after treatment with artificial intestinal juice for 4 h was about 2 times that of the control group.

[0098] Table 6 Tolerance of Achromobacter YKS2 to artificial intestinal juice

[0099]

[0100] 8. Effects of different doses of Achromobacter YKS2 on intestinal villi and crypts

[0101] Intestinal villi is a structure unique to the small intestine. The length of the villi refers to the length from the top of the villi to the base of the intestinal crypt. The depth of the crypt refers to the distance from the base of the intestinal crypt to the base of the intestinal crypt. The ratio of villus length / crypt depth (V / C value) is often used as an important indicator to measure the structure and morphology of the intestinal tract and the ability of digestion and absorption. In order to study whether different doses of Achromobacter YKS2 can affect the intestinal morphology, jejunal tissue was collected for histopathological analysis.

[0102] The experimental results show that different doses of strains do not cause damage to the morphology of the intestinal mucosa. Each group shows normal physiological morphology. The intestinal villi are arranged in order and are dense, without swelling and shortening. The depth of the crypt is equivalent and there is no deepening. There is no inflammation or epithelial cell damage. figure 8 A). In addition, as shown in Figure ( figure 8 B), although the length of the intestinal villi and the depth of the crypt increased, there was no statistical difference between the groups (P>0.05). After calculating the statistics, the V / C value of the test group increased compared with the control group, and there was no significant difference (P>0.05). The overall trend of the V / C value was similar to that of the intestinal villus length.

[0103] 9. Effects of Achromobacter YKS2 on small intestinal immune cells

[0104] Goblet cells are mainly located between columnar cells and are white vacuole cells in terms of staining morphology. The mucin secreted by goblet cells can repair mucosal damage and prevent the attachment of external conditional pathogenic bacteria, making the intestinal mucosa smooth and maintaining the stability of the intestinal chemical barrier. Intestinal epithelial lymphocytes first contact antigens in the intestinal immune response and are the main immune cells in the intestinal tract, reflecting whether the intestinal mucosal barrier is good.

[0105] As can be seen from figure 9 , the IEL of the Achromobacter YKS2-High group was significantly higher than that of the Cintrol group (P<0.05), and there was no statistical difference between the other groups. In the statistics of goblet cells ( figure 9 ), the Achromobacter YKS2-Middle group was significantly higher than the data of the Achromobacter YKS2-High group, with a statistical difference (P<0.05), and there was no significant difference between the Achromobacter YKS2 test groups and the control group (P>0.05). These results show that Achromobacter YKS2 does not cause damage to the small intestinal barrier. High-dose Achromobacter YKS2 can even increase the number of IELs. Appropriate amounts of Achromobacter YKS2 stimulation can increase the number of intestinal goblet cells, thereby improving the intestinal mucosal immune defense ability of mice.

[0106] In conclusion, the present application unexpectedly screens a strain of achromobacter YKS2, which has good acid tolerance, bile salt tolerance, artificial gastric juice tolerance and artificial intestinal juice tolerance, and can improve immunity.

Claims

1. A type of *Achromobacterium martensii* ( achromobacter marplatensis YKS2, characterized in that, It was deposited on April 14, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 24678.

2. A microbial agent, characterized in that, The bacterial agent contains *Achromobacterium martensii* YKS2 as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The bacterial agent contains live cells of *Achromobacterium martensii* YKS2.

4. The microbial agent as described in claim 2, characterized in that, The bacterial agent can be a liquid bacterial agent or a solid bacterial agent.

5. The use of Achromobacterium martensii YKS2 as described in claim 1 in the preparation of drugs to enhance immunity.

Images