Mixed bacterial agent for reducing antibiotic resistance genes and application of mixed bacterial agent

Abstract

The invention belongs to the field of microorganism application, and discloses a mixed bacterial agent for reducing antibiotic resistance genes and application of the mixed bacterial agent, the mixed bacterial agent comprises Neobacillus sementimangrovii J35 and Brevundimonas terrae KanS2, and the preservation numbers of the Neobacillus sementimangrovii J35 and Brevundimonas terrae KanS2 are respectively CCTCC NO: M 20241436 and CCTCC NO: M 20241435. The two bacteria in the mixed inoculant are symbiotic and secreted to generate H2O2, the ARGs in the compost are efficiently degraded by utilizing the strong oxidizing property of the secreted H2O2, the reduction efficiency of the ARGs can reach 94.5% at most, the propagation of the ARGs in the compost product can be effectively controlled, the safety and environmental protection property of compost treatment are improved, and the mixed inoculant is simple, feasible, efficient and reliable and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically, to a mixed bacterial agent for reducing antibiotic resistance genes and application thereof. Background Art

[0002] The widespread spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) has become one of the major threats to human health in the 21st century. Due to the abuse of antibiotics, compost has become an important reservoir of ARGs, with an absolute abundance of up to 10 11 -10 13 The traditional composting method is often difficult to effectively remove ARGs. If ARGs-rich compost products are introduced into farmland soil as soil conditioners, ARGs may spread rapidly through the propagation of ARB, posing a potential threat to the benign homeostasis of soil environmental microorganisms and human health.

[0003] At present, the methods for removing ARGs in composting process at home and abroad include physical adsorption method such as adding activated carbon, but the removal effect varies greatly for different composts and the cost is high; external heating method such as adding external heating device in composting process, this method has the disadvantages of high energy consumption and difficult maintenance while removing ARGs; chemical agent method such as adding strong oxidant, but it is very easy to cause secondary pollution; inoculation of single strain method is low cost, but the removal effect is unstable, and the inoculated strain is difficult to colonize in the complex community of compost. Therefore, there is an urgent need for a safe, environmentally friendly and efficient mixed bacterial agent to reduce ARGs in composting process. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. The present invention provides a mixed bacterial agent for reducing antibiotic resistance genes, which can significantly reduce the abundance of resistance genes in compost.

[0005] The first aspect of the present invention provides a mixed bacterial agent for reducing antibiotic resistance genes, wherein the mixed bacterial agent comprises Neobacillus sedimentimangrovi J35 and Brevundimonas terrae KanS2, and the deposit numbers are CCTCC NO: M 20241436 and CCTCC NO: M20241435, respectively.

[0006] The second aspect of the present invention provides a method for preparing a mixed bacterial agent for reducing antibiotic resistance genes, comprising the following steps:

[0007] S1. Cultivating Neobacillus sedimentimangrovi J35 and Brevundimonas terrae KanS2 in corresponding nutrient fermentation broths, respectively. When the colony forming units of the two bacteria reach a certain standard, centrifuging and concentrating the two bacterial broths, respectively, freeze-drying to obtain original bacterial powder of Neobacillus sedimentimangrovi J35 and original bacterial powder of Brevundimonas terrae KanS2, and freezing and storing them at a certain temperature;

[0008] S2. The original bacterial powder of Neobacillus sedimentimangrovi J35 and the original bacterial powder of Brevundimonasterrae KanS2 were mixed in a certain mass ratio to obtain a bacterial powder mixture, and a carrier was added and mixed evenly to prepare a mixed bacterial agent.

[0009] In some embodiments, the colony forming units of the two bacteria in step S1 reach 10 8 ~10 9 CFU / mL, the two bacterial solutions were centrifuged and concentrated respectively, and the temperature for freezing and storage in step S1 was -20°C.

[0010] In some embodiments, the mass ratio of the bacterial powder mixture to the carrier in step S2 is 1:1, and the mass ratio of the original bacterial powder of Neobacillus sedimentimangrovi J35 to the original bacterial powder of Brevundimonas terrae KanS2 in the bacterial powder mixture is 2:3.

[0011] In some embodiments, the carrier is a mixture of kaolin and glycerol, and the mass ratio of kaolin to glycerol in the carrier is 8:1.

[0012] The third aspect of the present invention provides a use of a mixed bacterial agent for reducing antibiotic resistance genes in reducing resistance genes.

[0013] The fourth aspect of the present invention provides a use of a mixed bacterial agent for reducing antibiotic resistance genes in reducing resistance genes in composting.

[0014] In some embodiments, the resistance genes are tetA, tetG, strA, strB, aacA4, aadE, ermT, mefA, and ereA genes.

[0015] In some embodiments, when the temperature of the compost raw material is lower than 40° C., a mixed bacterial agent for reducing antibiotic resistance genes is uniformly inoculated into the compost at a mass ratio of 1%.

[0016] A fifth aspect of the present invention provides a method for reducing resistance genes in compost, comprising the following:

[0017] Select organic waste as compost raw material, add a mixed bacterial agent for reducing antibiotic resistance genes into the compost raw material at a mass ratio of 1% when the temperature of the compost raw material is lower than 40°C, ensure that the mixed bacterial agent is evenly distributed in the compost raw material, adjust the moisture content of the compost raw material to 60%, and carry out composting fermentation. The organic waste is livestock and poultry manure or crop straw.

[0018] Beneficial effects of the present invention: the mixed bacterial agent of the present invention comprises Neobacillus sedimentimangroviJ35 and Brevundimonas terrae KanS2, the two bacterial strains coexist in a symbiotic relationship, secrete and produce H2O2, and utilize the strong oxidizing property of the secreted H2O2 to efficiently degrade ARGs in compost, with the highest ARGs reduction efficiency reaching 94.5%. By adding the mixed bacterial agent, the present invention can effectively control the spread of ARGs in compost products, improve the safety and environmental protection of composting treatment, is simple, easy to operate, efficient and reliable, and has broad application prospects. DETAILED DESCRIPTION

[0019] The present invention is further described in detail through specific implementation cases, and it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, the modifications of various equivalent forms of the present invention by those skilled in the art are all limited by the claims attached to this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0020] Test materials

[0021] Neobacillus sedimentimangrovi J35 has been deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20241436 and the deposit date July 1, 2024; Brevundimonas terraeKans2 has been deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20241435 and the deposit date July 1, 2024.

[0022] Example 1

[0023] A method for preparing a mixed bacterial agent for reducing antibiotic resistance genes comprises the following steps:

[0024] S1. Cultivate Neobacillus sedimentimangrovi J35 and Brevundimonas terrae KanS2 in the corresponding LB medium. When the colony forming units of the above two bacteria reach 10 8 ~10 9 CFU / mL, the above two bacterial solutions were centrifuged and concentrated, freeze-dried to obtain the original bacterial powder of Neobacillus sedimentimangrovi J35 and the original bacterial powder of Brevundimonas terrae KanS2, and stored at -20°C;

[0025] S2. The original bacterial powder of Neobacillus sedimentimangrovi J35 and the original bacterial powder of Brevundimonasterrae KanS2 were mixed in a mass ratio of 2:3 to obtain a bacterial powder mixture, kaolin and glycerol were mixed in a mass ratio of 8:1 to obtain a carrier, and the carrier and the bacterial powder mixture were uniformly mixed in a mass ratio of 1:1 to prepare a mixed bacterial agent.

[0026] Example 2

[0027] A mixed bacterial agent for reducing antibiotic resistance genes comprises mixed bacterial powder composed of Neobacillus sedimentimangrovi J35 and Brevundimonas terrae KanS2 and a carrier, wherein the mass ratio of the mixed bacterial powder to the carrier is 1:1, the mass ratio of Neobacillus sedimentimangrovi J35 and Brevundimonas terrae KanS2 in the mixed bacterial powder is 2:3, and the mass ratio of kaolin to glycerol in the carrier is 8:1.

[0028] Example 3 Comparative test of the mixed bacterial agent's ability to remove resistance genes

[0029] Experimental Design:

[0030] Treatment Group 1

[0031] Livestock and poultry manure was selected as the compost raw material, and a mixed bacterial agent for reducing antibiotic resistance genes prepared in Example 1 was added to the compost raw material at a mass ratio of 1%, and the mixed bacterial agent was ensured to be evenly distributed in the compost raw material, and the moisture content of the compost raw material was adjusted to 60%, and composting fermentation was performed. During the composting fermentation process, the increase in temperature can promote the reproduction of Neobacillus sedimentimangrovi J35 and Brevundimonas terrae KanS2 in the mixed bacterial agent. When the temperature is raised too high, the above two bacteria in the mixed bacterial agent will be in a dormant state, and after the temperature drops, the above two bacteria will resume activity.

[0032] Control group 1

[0033] Referring to the method of treatment group 1, the added mixed bacterial agent was replaced with an equal amount of carrier, that is, a mixture of kaolin and glycerol in a mass ratio of 8:1.

[0034] Treatment Group 2

[0035] Livestock and poultry manure was selected as the compost raw material, and composting fermentation was carried out under the condition that the moisture content of the compost raw material was 60%. When the composting temperature dropped to 40°C, a mixed bacterial agent for reducing antibiotic resistance genes prepared in Example 1 was added to the compost raw material at a mass ratio of 1%, and the mixed bacterial agent was ensured to be evenly distributed in the compost raw material for composting fermentation.

[0036] Control group 2

[0037] Referring to the method of treatment group 2, after the composting temperature dropped to 40°C, the added mixed bacterial agent was replaced with an equal amount of carrier, that is, a mixture of kaolin and glycerol in a mass ratio of 8:1.

[0038] Control group 3

[0039] Referring to the method of treatment group 2, after the composting temperature dropped to 40°C, the added mixed bacterial agent was replaced with an equal amount of original bacterial powder of Neobacillus sedimentimangrovi J35.

[0040] Control group 4

[0041] Referring to the method of treatment group 2, after the composting temperature dropped to 40°C, the added mixed bacterial agent was replaced with an equal amount of original bacterial powder of Brevundimonas terrae KanS2.

[0042] For the above-mentioned treatment groups 1, 2 and control groups 1-4, the H2O2 content in the compost was regularly monitored on the 0th, 6th, 24th and 35th days of compost fermentation after adding the mixed bacterial agent or carrier or single bacterial powder. The specific results are shown in Table 1.

[0043] Table 1 Determination of H2O2 content in compost

[0044]

[0045] As shown in Table 1, the H2O2 content produced in treatment groups 1 and 2 is much higher than that in control groups 1 to 4. The comparison of the results of treatment groups 1 and 2 shows that after the composting temperature drops to 40°C, the H2O2 content produced by adding the mixed bacterial agent prepared in Example 1 is significantly higher than that by adding the mixed bacterial agent to the unfermented compost raw materials. The comparison of treatment group 2 with control groups 2-4 shows that only adding a carrier or a single addition of the original bacterial powder of Neobacillus sedimentimangrovi J35 or the original bacterial powder of Brevundimonas terraeKanS2 cannot produce H2O2 during the composting fermentation process, indicating that only adding a carrier or a single strain cannot achieve the purpose of synergistically promoting the production of H2O2. Therefore, it is proved that a mixed bacterial agent for reducing antibiotic resistance genes prepared in Example 1 of the present invention has the beneficial effect of promoting the synergistic symbiosis of two bacterial strains of Neobacillus sedimentimangrovi J35 and Brevundimonas terraeKanS2 and secreting H2O2.

[0046] The content of resistance genes (such as tetA, tetG, strA, strB, aacA4, aadE, ermT, mefA, ereA) and the removal rate of resistance genes were detected by real-time fluorescence quantitative PCR reaction on the 0th and 35th days of composting, and the results are shown in Tables 5 and 6. The real-time fluorescence quantitative PCR reaction system is shown in Table 2, the reaction conditions are shown in Table 3, and the primer sequences, annealing temperatures and fragment lengths are shown in Table 4.

[0047] Table 2 qPCR reaction system

[0048]

[0049] Table 3 qPCR reaction conditions

[0050]

[0051] Table 4 Primer sequences, annealing temperature and fragment lengths of qPCR reactions

[0052]

[0053] Table 5 Comparison of resistance gene removal rates in different composting methods

[0054]

[0055] The results of treatment group 1 and control group 1 in Table 5 are compared, indicating that the addition of the mixed bacterial agent prepared in Example 1 has a good removal effect on the resistance genes in the compost. The results of treatment group 2 and control group 2 are compared, indicating that the addition of the mixed bacterial agent prepared in Example 1 has a good removal effect on the resistance genes in the compost. The results of treatment 1 and treatment 2 are compared, indicating that when the temperature is lower than 40°C after the compost is fermented, the effect of adding the mixed bacterial agent prepared in Example 1 on reducing the resistance genes is significantly better than the treatment method of adding the mixed bacterial agent to the compost raw material and then fermenting it.

[0056] Table 6 Comparison of the removal of resistance genes in compost by adding different bacterial agents

[0057]

[0058] As shown in Table 6, the effect of adding the mixed bacterial agent prepared in Example 1 on removing resistance genes in compost is significantly better than that of adding the original bacterial powder of Neobacillus sedimentimangrovi J35 or the original bacterial powder of Brevundimonas terrae KanS2 alone. This shows that the mixed bacterial agent prepared in Example 1 has the effect of synergistic symbiosis and promoting the secretion of H2O2 for the two strains of Neobacillus sedimentimangroviJ35 and Brevundimonas terrae KanS2. The secreted H2O2 has strong oxidizing properties and can efficiently degrade resistance genes in compost. The reduction rate of resistance genes is as high as 94.5%. However, adding a single strain cannot achieve this technical effect.

[0059] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A mixed bacterial agent for reducing antibiotic resistance genes, characterized in that: The mixed bacterial agent comprises Neobacillus sedimentimangrovi J35 and Brevundimonas terrae KanS2, and the preservation numbers are CCTCC NO: M 20241436 and CCTCC NO: M 20241435 respectively.

2. The method for preparing a mixed bacterial agent for reducing antibiotic resistance genes according to claim 1, characterized in that: The following steps are involved: S1. Cultivating Neobacillus sedimentimangrovi J35 and Brevundimonas terrae KanS2 in corresponding nutrient fermentation broths, respectively. When the colony forming units of the two bacteria reach a certain standard, centrifuging and concentrating the two bacterial broths, respectively, freeze-drying to obtain original bacterial powder of Neobacillus sedimentimangrovi J35 and original bacterial powder of Brevundimonas terrae KanS2, and freezing and storing them at a certain temperature; S2. The original bacterial powder of Neobacillus sedimentimangrovi J35 and the original bacterial powder of Brevundimonas terraeKanS2 were mixed in a certain mass ratio to obtain a bacterial powder mixture, and a carrier was added and mixed evenly to prepare a mixed bacterial agent.

3. The preparation method according to claim 2, characterized in that: In step S1, the colony forming units of the two bacteria reached 10 8 ~10 9 CFU / mL, the two bacterial solutions were centrifuged and concentrated respectively, and the temperature of the frozen storage in step S1 was -20°C.

4. The preparation method according to claim 2, characterized in that: The mass ratio of the bacterial powder mixture to the carrier in step S2 is 1:1, and the mass ratio of the original bacterial powder of Neobacillus sedimentimangrovi J35 to the original bacterial powder of Brevundimonas terrae KanS2 in the bacterial powder mixture is 2:

3.

5. The preparation method according to claim 4, characterized in that: The carrier is a mixture of kaolin and glycerol, and the mass ratio of kaolin to glycerol in the carrier is 8:

1.

6. Use of the mixed bacterial agent for reducing antibiotic resistance genes as claimed in claim 1 in reducing resistance genes.

7. Use of the mixed bacterial agent for reducing antibiotic resistance genes as claimed in claim 1 in reducing resistance genes in composting.

8. The use according to claim 6 or 7, characterized in that: The resistance genes are tetA, tetG, strA, strB, aacA4, aadE, ermT, mefA and ereA genes.

9. The use according to claim 7, characterized in that: When the temperature of the compost raw material is lower than 40° C., the mixed bacterial agent for reducing antibiotic resistance genes as claimed in claim 1 is uniformly inoculated into the compost at a mass ratio of 1%.

10. A method for reducing resistance genes in compost, characterized in that: Includes the following: Select organic waste as compost raw material, add a mixed bacterial agent for reducing antibiotic resistance genes as claimed in claim 1 to the compost raw material at a mass ratio of 1% when the temperature of the compost raw material is lower than 40°C, ensure that the mixed bacterial agent is evenly distributed in the compost raw material, adjust the moisture content of the compost raw material to 60%, and perform composting fermentation, wherein the organic waste is livestock and poultry manure or crop straw.

Images