Phage and use thereof in soil remediation

Abstract

A phage and use thereof in soil remediation are disclosed. The phage φYSZPK has been deposited at the China Center for Type Culture Collection on Aug. 1, 2018 under Accession No. CCTCC M 2018516, and its taxonomic designation is Pseudomonas aeruginosa and Klebsiella phage φYSZPK. Biochar and the screened phage are combined and returned into contaminated soil to synergistically control and deeply track and inactivate transmission and spread of antibiotic resistance pathogenic bacteria and resistance genes in a soil-vegetable system. The combination of the biochar and the phage φYSZPK not only clearly improves the functional stability of microbial community in the soil-vegetable system, but also significantly alleviates the dissemination of the antibiotic resistance pathogenic bacteria in the soil-vegetable system to prevent secondary pollution, thereby providing a new solution for biological remediation and control of farmland soil contaminated by antibiotic resistance pathogenic bacteria in China.

Claims

1. A method of using a polyvalent phage φYSZPK for controlling and inactivating antibiotic resistant pathogenic bacteria in a soil-vegetable system, wherein the polyvalent phage is active against both Pseudomonas aeruginosa and Klebsiella, and wherein the polyvalent phage φYSZPK is mixed with a biochar.

2. The method of claim 1, wherein the biochar is produced by burning wheat straw as a raw material at a high temperature of 450° C., and with basic physical and chemical properties being total carbon: 548.4 g/kg, total nitrogen: 13.4 g/kg, C/N: 33.6, ash content: 175.5 g/kg, total phosphorus: 2.1 g/kg, total potassium: 10.3 g/kg, and pH: 8.5.

3. The method of claim 1, wherein the polyvalent phage φYSZPK and the biochar are mixed in a mass ratio of 1:1000, and wherein the mixture of the polyvalent phage φYSZPK and the biochar is applied to a contaminated soil.

4. The method of claim 1, wherein the polyvalent phage φYSZPK is used for preparing a product for control and inactivation of antibiotic resistant pathogenic bacteria in a soil-vegetable system.

5. The method of claim 4, wherein the product for control and inactivation of antibiotic resistant pathogenic bacteria in a soil-vegetable system, comprises the polyvalent phage φYSZPK and a biochar.

6. The method of claim 5, wherein the product comprises a mixture of the polyvalent phage φYSZPK and the biochar in a mass ratio of 1:1000, and wherein the product is applied to a contaminated soil.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a scanning electron micrograph of biochar;

(2) FIG. 2 is a transmission electron micrograph of a phage φYSZPK;

(3) FIG. 3 is a flat graph of forms of phage plaques of the phage φYSZPK;

(4) FIG. 4 is a verification diagram of control and inactivation effects on Klebsiella pneumoniae and Pseudomonas aeruginosa in a contaminated soil-vegetable system of a manure accumulation place of the Hengliang dairy farm in Nanjing, Jiangsu Province, when carrots are planted on contaminated soil by using the technical solution of the present invention;

(5) FIG. 5 is a verification diagram of control and inactivation effects on Klebsiella pneumoniae and Pseudomonas aeruginosa in a contaminated soil-vegetable system of the manure accumulation place of the Hengliang dairy farm in Nanjing, Jiangsu Province, when pod peppers are planted on the contaminated soil by using the technical solution of the present invention;

(6) FIG. 6 is a verification diagram of control and inactivation effects on Klebsiella pneumoniae and Pseudomonas aeruginosa in a contaminated soil-vegetable system of the manure accumulation place of the Hengliang dairy farm in Nanjing, Jiangsu Province, when lettuces are planted on the contaminated soil by using the technical solution of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

(7) The following specific implementation manners do not limit the technical solution of the present invention in any form. Any technical solution obtained by means of equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

(8) Biochar B is produced by burning wheat straw as a raw material at the high temperature of 450° C., and the pH of the biochar B is alkaline.

(9) The Accession No. of the phage φYSZPK is CCTCC M 2018516. An electron micrograph shows that the phage φYSZPK has an elliptical head and a shrinkable tail sheath, and has a head long diameter of about 110 nm, a transverse diameter of about 80 nm, and a tail length of about 120 nm, and phage plaques on a culture dish are transparent in the middle, have no halo around, and have a diameter of about 2-3 mm.

(10) The resistance gene ampC refers to a resistance gene carrying chloramphenicol on plasmids in Pseudomonas aeruginosa PAO1 cells.

(11) The resistance gene tetW refers to a resistance gene carrying tetracycline antibiotics on plasmids in Klebsiella pneumoniae cells.

(12) The potting soil is obtained by respectively adding the same abundance of pathogenic bacteria (Pseudomonas aeruginosa PAO1 and Klebsiella pneumoniae) to collected raw soil.

Example 1

(13) Test potting soil was collected from contaminated soil around a manure accumulation pool of the Hengliang dairy farm in Nanjing, Jiangsu Province. Basic physical and chemical properties of the soil were as follows: sand grain: 23.8%, soil grain: 45.4%, clay grain: 31.8%, pH: 7.7, total nitrogen: 1.7 g.Math.kg.sup.−1, water-soluble nitrogen: 1.7 g.Math.kg.sup.−1, total phosphorus: 1.3 g.Math.kg.sup.−1, total potassium: 17.5 g.Math.kg.sup.−1, and CEC: 19.4 cmol.Math.kg.sup.−1.

(14) 5 g of fresh soil samples were taken and added to 50 mL of sterile water, shake culture was performed for 5 h at 28° C. and 150 rpm, centrifugation was performed for 5 min at 10000 rpm, the supernatant liquid was sterilized by a 0.22 μm filter membrane, 9 mL of filtrate and 1 mL of a suspension of Pseudomonas aeruginosa PAO1 growing to a logarithmic phase were taken and added to 40 mL of LB liquid culture medium, calcium chloride solids were added until the final concentration of the solution was 1 mmol.Math.L.sup.−1, shake culture was performed for 12 h at 30° C. and 150 rpm, the obtained culture solution was centrifuged for 5 min at 10000 rpm, and then, the centrifuged culture solution was sterilized by a 0.22 μm filter membrane to obtain a phage stock solution; phages were screened and purified by using a double-layer flat plate method, 100 μL of filtrate and 100 μL of Klebsiella pneumoniae suspension were taken and mixed uniformly, the mixture was allowed to stand for 15 min at room temperature, the mixture was added to 3 mL of 0.7% LB agar culture medium and horizontally poured on an LB solid flat plate after uniform mixing, culture was performed for 10-12 h at 30° C., phage plaques were observed, after the phage plaques occurred, a single phage plaque with clear and transparent edges was taken in LB liquid containing host bacteria so as to be purified, and the purified product was refrigerated at 4° C.; and 600 pt of preserved phage stock solution was taken and added to 99 mL of LB liquid culture medium together with 200 mL of PAO1 and 200 pt of Klebsiella pneumoniae mixed suspension respectively, then, calcium chloride solids were added until the final concentration was 1 mmol.Math.L.sup.−1, shake culture was performed for 96 h at 37° C. and 150 rpm, samples were taken every 8 h, the phage obtained by centrifugal filtration and PAO1 were poured into a double-layer flat plate to be verified, phage plaques were observed, if phage plaques occurred, it was proved that the directed evolution was successful, a polyvalent phage φYSZPK was obtained, a single clear and transparent phage plaque was selected and enriched and mixed with 50% glycerol in a volume ratio of 1:1, and the mixture was preserved at low temperature of −80° C. for later use.

(15) Wheat straw was used as a raw material for preparation of the biochar. 10 kg of wheat straw was weighed and put into a pulverizer and then preliminarily pulverized, the pulverized straw was sieved, 2 kg of sieved wheat straw with 100 meshes was weighed and put into a ceramic crucible, the wheat straw was carbonized for 8 h at the high temperature of 450° C. in a muffle furnace, and then, the carbonized wheat straw was taken out, cooled and stored in a dry place for later use. The measured basic physical and chemical properties were as follows: total carbon: 548.4 g/kg, total nitrogen: 13.4 g/kg, C/N: 33.6, ash content: 175.5 g/kg, total phosphorus: 2.1 g/kg, total potassium: 10.3 g/kg, and pH: 8.5.

Example 2

(16) Test potting soil was collected from the contaminated soil around the manure accumulation pool of the Hengliang dairy farm in Nanjing, Jiangsu Province. Planting vegetables were carrots Seoul six-inch (Daucus L.), and were derived from Beijing Zhongnong Tianteng Vegetable Seed Company. Basic physical and chemical properties of the soil were as follows: sand grain: 23.8%, soil grain: 45.4%, clay grain: 31.8%, pH: 7.7, total nitrogen: 1.7 g.Math.kg.sup.−1, water-soluble nitrogen: 1.7 g.Math.kg.sup.−1, total phosphorus: 1.3 g.Math.kg.sup.−1, total potassium: 17.5 g.Math.kg.sup.−1, and CEC: 19.4 cmol.Math.kg.sup.−1.

(17) Four groups of treatment were set in experiments: (1) control group (CK): 3 carrots were planted per pot (0.5-1 cm of soil was covered on seeds, and the room temperature was 20±2° C.); (2) biochar treatment (B): the biochar (1 g/kg) was applied on the basis of the control group; (3) phage φYSZPK treatment (P): 100 mL of phage φYSZPK with a concentration of 10.sup.6 pfu.Math.mL.sup.−1 was inoculated on the basis of the control group; (4) biochar and phage φYSZPK combined treatment (BP): the biochar (1 g/kg) was applied and 100 mL of the phage φYSZPK with a concentration of 10.sup.6 pfu.Math.mL.sup.−1 was inoculated on the basis of the control group. The soil and carrots were sampled on the site after the 70th day of carrot growth, the measured quantity of Pseudomonas aeruginosa PAO1 in the contaminated soil under the four groups of treatment (CK, B, P, and BP) was respectively 5.1×10.sup.7 cfu.Math.g.sup.−1, 1.5×10.sup.5 cfu.Math.g.sup.−1, 8.3×10.sup.5 cfu.Math.g.sup.−1, and 1.2×10.sup.3 cfu.Math.g.sup.−1, and the abundance of the chloramphenicol resistance gene ampC was respectively 1.4×10.sup.9 copies.Math.g.sup.−1, 2.5×10.sup.6 copies.Math.g.sup.1, 1.3×10.sup.7 copies.Math.g.sup.−1, and 7.8×10.sup.5 copies.Math.g.sup.−1. Under the three groups of treatment (B, P, and BP), compared with the control group, the quantity of Pseudomonas aeruginosa PAO1 in the contaminated soil was respectively reduced by 2.3, 1.9, and 4.3 orders of magnitude, and the abundance of the resistance gene ampC was respectively reduced by 2.8, 2.1 and 3.8 orders of magnitude. The measured related quantity of Klebsiella pneumoniae in carrot root tubers under the four groups of treatment (CK, B, P, and BP) was respectively 6.2×10.sup.4 cfu.Math.g.sup.1, 9.2×10.sup.2 cfu.Math.g.sup.1, 3.3×10.sup.2 cfu.Math.g.sup.1, and 4.1×10.sup.1 cfu.Math.g.sup.−1, and the abundance of the resistance gene ampC was respectively reduced to 1.6×10.sup.6 copies.Math.g.sup.1, 1.4×10.sup.4 copies.Math.g.sup.1, 3.2×10.sup.3 copies.Math.g.sup.1, and 8.4×10.sup.2 copies.Math.g.sup.1. In carrot leaves, under the three groups of treatment (B, P, and BP), compared with the control group, the abundance of K12 was respectively reduced by 1.7, 2.3 and 3.2 orders of magnitude, and the abundance of the resistance gene ampC was respectively reduced by 2.1, 2.9 and 3.4 orders of magnitude. The control and inactivation effects of combination of the biochar and the phage on resistance pathogenic bacteria and resistance genes were significantly better than those of single addition of the biochar or inoculation of the phage φYSZPK (p<0.05).

(18) The analysis finds that the ecological diversity indexes (AWCD indexes) of microorganisms in the soil environment under the four groups of treatment (CK, B, P, and BP) were respectively 0.61±0.1, 0.64±0.2, 0.58±0.2, and 0.68±0.2, by application of the biochar, compared with the control group, the diversity of microorganisms in soil was increased to a certain degree, by inoculation of the phage φYSZPK, compared with the control group, the diversity of microorganisms in soil was reduced to a certain degree, and the combination of the biochar and the phage φYSZPK for remediation has the most significant promotion effect on the functional diversity and stability of microorganisms in soil (p<0.05), indicating that the remediation technology has a significant effect on remediation of the spread of resistance bacteria, and is also favorable for maintaining and improving the ecological functional diversity and stability of microorganisms in soil after remediation.

Example 3

(19) Test potting soil was collected from the contaminated soil around the manure accumulation pool of the Hengliang dairy farm in Nanjing, Jiangsu Province. Planting vegetables were Hongpin No. 1 pod peppers (Capsicum frutescens var), and were derived from Qianshu Baihua Seed Industry Company. Basic physical and chemical properties of the soil were as follows: sand grain: 23.8%, soil grain: 45.4%, clay grain: 31.8%, pH: 7.7, total nitrogen: 1.7 g.Math.kg.sup.−1, water-soluble nitrogen: 1.7 g.Math.kg.sup.−1, total phosphorus: 1.3 g.Math.kg.sup.−1, total potassium: 17.5 g.Math.kg.sup.−1, and CEC: 19.4 cmol.Math.kg.sup.−1.

(20) Four groups of treatment were set in experiments: (1) control group (CK): 3 pod peppers were planted per pot (0.5-1 cm of soil was covered on seeds, and the room temperature was 25±2° C.); (2) biochar treatment (B): the biochar (1 g/kg) was applied on the basis of the control group; (3) phage φYSZPK treatment (P): 100 mL of phage φYSZPK with a concentration of 10.sup.6 pfu.Math.mL.sup.−1 was inoculated on the basis of the control group; (4) biochar and phage φYSZPK combined treatment (BP): the biochar (1 g/kg) was applied and 100 mL of the phage φYSZPK with a concentration of 10.sup.6 pfu.Math.mL.sup.−1 was inoculated on the basis of the control group. The soil and pod peppers were sampled on the site after the 70th day of pod pepper growth, the measured contamination concentration of Pseudomonas aeruginosa PAO1 in the contaminated soil under the four groups of treatment (CK, B, P, and BP) was respectively 5.2×0.sup.7 cfu.Math.g.sup.−1, 3.7×10.sup.4 cfu.Math.g.sup.−1, 1.8×10.sup.5 cfu.Math.g.sup.−1, and 2.3×10.sup.3 cfu.Math.g.sup.−1, and the abundance of the chloramphenicol resistance gene ampC was respectively 8.3×10.sup.8 copies.Math.g.sup.−1, 4.8×10.sup.5 copies.Math.g.sup.−1, 3.5×10.sup.6 copies.Math.g.sup.−1, and 4.5×10.sup.4 copies.Math.g.sup.−1. Under the three groups of treatment (B, P, and BP), compared with the control group, the quantity of Klebsiella pneumoniae in the contaminated soil was respectively reduced by 3.1, 2.2 and 4.3 orders of magnitude, and the abundance of the resistance gene tetW was respectively reduced by 3.3, 2.2 and 4.5 orders of magnitude. The measured quantity of PAO1 in pod pepper fruits under the four groups of treatment (CK, B, P, and BP) was respectively reduced to 6.3×10.sup.4 cfu.Math.g.sup.−1, 4.8×10.sup.3 cfu.Math.g.sup.−1, 2.2×10.sup.2 cfu.Math.g.sup.−1, and 4.2×10.sup.1 cfu.Math.g.sup.−1, and the abundance of the resistance gene ampC was respectively reduced to 1.8×10.sup.6 copies.Math.g.sup.−1, 8.3×10.sup.4 copies.Math.g.sup.−1, 4.1×10.sup.3 copies.Math.g.sup.−1, and 8.2×10.sup.2 copies.Math.g.sup.−1. Under the three groups of treatment (B, P, and BP), compared with the control group, the quantity of Klebsiella pneumoniae in fruits was respectively reduced by 1.1, 2.4 and 3.1 orders of magnitude, and the abundance of the resistance gene tetW was respectively reduced by 1.4, 2.8 and 3.6 orders of magnitude. The control and inactivation effects of combination of the biochar and the phage φYSZPK on resistance pathogenic bacteria and resistance genes were significantly better than those of single addition of the biochar or inoculation of the phage φYSZPK.

(21) The analysis finds that the ecological diversity indexes (AWCD indexes) of microorganisms in the soil environment under the four groups of treatment (CK, B, P, and BP) were respectively 0.51±0.1, 0.55±0.2, 0.47±0.2, and 0.57±0.1, by application of the biochar, compared with the control group, the diversity of microorganisms in soil was increased to a certain degree, by inoculation of the phage φYSZPK, compared with the control group, the diversity of microorganisms in soil was reduced to a certain degree, and the combination of the biochar and the phage φYSZPK for remediation has the most significant promotion effect on the functional diversity and stability of microorganisms in soil (p<0.05), indicating that the remediation technology has a significant effect on remediation of the spread of resistance bacteria, and is also favorable for maintaining and improving the ecological functional diversity and stability of microorganisms in soil after remediation.

Example 4

(22) Test potting soil was collected from the contaminated soil around the manure accumulation pool of the Hengliang dairy farm in Nanjing, Jiangsu Province. Planting vegetables were Italian year-round bolting-resistant lettuces (Lactuca sativa L), and were derived from Hebei Jinfa Seed Industry Co., Ltd. Basic physical and chemical properties of the soil were as follows: sand grain: 23.8%, soil grain: 45.4%, clay grain: 31.8%, pH: 7.7, total nitrogen: 1.7 g.Math.kg.sup.−1, water-soluble nitrogen: 1.7 g.Math.kg.sup.−1, total phosphorus: 1.3 g.Math.kg.sup.−1, total potassium: 17.5 g.Math.kg.sup.−1, and CEC: 19.4 cmol.Math.kg.sup.−1.

(23) Four groups of treatment were set in experiments: (1) control group (CK): 3 lettuces were planted per pot (0.5-1 cm of soil was covered on seeds, and the room temperature was 18±2° C.); (2) biochar treatment (B): the biochar (1 g/kg) was applied on the basis of the control group; (3) phage φYSZPK treatment (P): 100 mL of phage φYSZPK with a concentration of 10.sup.6 pfu.Math.mL.sup.−1 was inoculated on the basis of the control group; (4) biochar and phage φYSZPK combined treatment (BP): the biochar (1 g/kg) was applied and 100 mL of the phage φYSZPK with a concentration of 10.sup.6 pfumL.sup.−1 was inoculated on the basis of the control group. The soil and lettuces were sampled on the site after the 60th day of lettuce growth, the measured contamination concentration of Pseudomonas aeruginosa PAO1 in the contaminated soil under the four groups of treatment (CK, B, P, and BP) was respectively 2.8×10.sup.7 cfu.Math.g.sup.−1, 1.3×10.sup.5 cfu.Math.g.sup.−1, 5.6×10.sup.5 cfu.Math.g.sup.−1, and 2.4×10.sup.4 cfu.Math.g.sup.1, and the abundance of the chloramphenicol resistance gene ampC was respectively 1.4×10.sup.8 copies.Math.g.sup.−1, 2.5×10.sup.5 copies.Math.g.sup.−1, 5.3×10.sup.5 copies.Math.g.sup.−1, and 2.8×10.sup.4 copies.Math.g.sup.−1. Under the three groups of treatment (B, P, and BP), compared with the control group, the total quantity of Klebsiella pneumoniae in the contaminated soil was respectively reduced by 2.2, 1.6 and 3.1 orders of magnitude, and the abundance of the resistance gene tetW was respectively reduced by 2.8, 2.6 and 3.8 orders of magnitude. The measured quantity of PAO1 in lettuce leaves under the four groups of treatment (CK, B, P, and BP) was respectively: 8.2×10.sup.3 cfu.Math.g.sup.1, 3.8×10.sup.2 cfu.Math.g.sup.1, 2.3×10.sup.2 cfu.Math.g.sup.1, and 3.2×10.sup.1 cfu.Math.g.sup.1, and the abundance of the resistance gene ampC was respectively 1.6×10.sup.4 copies.Math.g.sup.−1, 8.9×10.sup.2 copies.Math.g.sup.−1, 1.2×10.sup.2 copies.Math.g.sup.−1, and 1.4×10.sup.1 copies.Math.g.sup.1. Under the three groups of treatment (B, P, and BP), compared with the control group, the quantity of Klebsiella pneumoniae in lettuce leaves was respectively reduced by 1.4, 1.9 and 2.5 orders of magnitude, and the abundance of the resistance gene tetW was respectively reduced by 1.3, 2.1 and 3.1 orders of magnitude. The control and inactivation effects of combination of the biochar and the phage φYSZPK on resistance pathogenic bacteria and resistance genes were significantly better than those of single addition of the biochar or inoculation of the phage φYSZPK.

(24) The analysis finds that the ecological diversity indexes (AWCD indexes) of microorganisms in the soil environment under the four groups of treatment (CK, B, P, and BP) were respectively 0.64±0.1, 0.70±0.2, 0.61±0.1, and 0.75±0.2, by application of the biochar, compared with the control group, the diversity of microorganisms in soil was increased to a certain degree, by inoculation of the phage φYSZPK, compared with the control group, the diversity of microorganisms in soil was reduced to a certain degree, and the combination of the biochar and the phage φYSZPK for remediation has the most significant promotion effect on the functional diversity and stability of microorganisms in soil (p<0.05), indicating that the remediation technology has a significant effect on remediation of the spread of resistance bacteria, and is also favorable for maintaining and improving the ecological functional diversity and stability of microorganisms in soil after remediation.

(25) The technology for simultaneous control and inactivation of multiple resistance pathogenic bacteria and resistance genes in soil-vegetable systems by combination of the biochar and the phage therapy has the advantages of high broad spectrum, low ecological risk and environmental friendliness, and is a compound pathogenic bacterium contaminated soil remediation technology with good application prospects.