Method for evaluating intestinal horizontal transfer degree of transferable drug-resistant genes of bacteria

Abstract

The invention relates to the technical field of animal husbandry. The invention provides a method for evaluating the intestinal horizontal transfer degree of transferable drug-resistant genes of bacteria, which comprises the following steps: (1) sequencing probiotics to determine transferable drug-resistant genes in the probiotics; (2) feeding animals with probiotics, collecting excrement samples of the fed animals, and extracting total DNA data in the excrement samples; (3) carrying out quality control and screening on the total DNA data by utilizing fastp, and carrying out sequence assembly on the data subjected to quality control by utilizing megahit, so as to obtain corresponding contigs; (4) carrying out box separation on the contigs by utilizing a box separation module in the metatrap; and (5) after binning, calculating the transfer rate of the transferable drug-resistant gene in the intestinal tract level. According to the method, the safety of the feed probiotics entering the intestinal tract can be evaluated, and safe and effective feed probiotic strains are developed.

Description

Technical Field

[0001] The present invention relates to the field of animal husbandry technology, and in particular to a method for evaluating the degree of intestinal horizontal transfer of transferable antibiotic resistance genes in bacteria. Background Art

[0002] In order to improve the production performance of animals, the breeding industry usually adds antibiotics to animal feed to inhibit pathogenic microorganisms in animals, maintain the intestinal health of animals, and improve the production performance of animals. However, with the extensive use of antibiotics, antibiotic resistance genes are continuously generated, posing a serious threat to human health.

[0003] In the method of antibiotic-free breeding, probiotics for feeding are widely used in the breeding industry. However, some probiotic strains contain plasmids carrying antibiotic resistance genes, posing a risk of horizontal transfer in the animal intestine. Currently, 36 species of probiotics are allowed to be used, of which up to 30 are bacterial species, and the wild type of most bacteria has natural plasmids carrying antibiotic resistance genes. In 2024, the output of probiotic products related to feeding was approximately 7,288 tons. The huge output requires the safety of feeding strains. At the same time, it is necessary to fully evaluate the degree of horizontal transfer of antibiotic resistance genes caused by probiotics containing transferable antibiotic resistance genes in use and the risks brought, in order to guide the development of safe and effective probiotics for feeding.

[0004] However, at present, the method for evaluating the degree of horizontal transfer of antibiotic resistance genes caused by probiotic strains containing transferable antibiotic resistance genes after entering the intestine is not yet mature. There is mainly ABRicate, which can lock contigs containing both mobile elements and antibiotic resistance genes, that is, transferable antibiotic resistance genes, by using the embedded ResFinder and PlasmidFinder databases. However, the species tracing of transferable antibiotic resistance genes in the intestine is only based on blastn for species identification. However, since some plasmids contain a small number of nucleotides, the number of species returned by blastn alignment is large, making it impossible to accurately trace the microbial distribution of transferable antibiotic resistance genes in the intestine and further evaluate the degree of intestinal horizontal transfer of transferable antibiotic resistance genes.

[0005] Therefore, it is extremely important to develop a method for accurately evaluating the degree of intestinal horizontal transfer of transferable antibiotic resistance genes in bacteria. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for evaluating the degree of intestinal horizontal transfer of transferable antibiotic resistance genes in bacteria, to help enterprises evaluate the safety of probiotics for feeding after entering the intestine and assist enterprises in quickly developing safe and effective probiotic strains.

[0007] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0008] The present invention provides a method for evaluating the intestinal horizontal transfer degree of transferable antibiotic resistance genes in bacteria, comprising the following steps:

[0009] (1) Sequencing probiotics to determine the transferable antibiotic resistance genes in the probiotics;

[0010] (2) Feeding animals with probiotics, collecting fecal samples of the animals after feeding, and extracting total DNA data from the fecal samples;

[0011] (3) Using fastp to perform quality control and screening on the total DNA data, and using megahit to perform sequence assembly on the quality-controlled data to obtain corresponding contigs;

[0012] (4) Using the binning module in metawrap to bin the contigs;

[0013] (5) Calculating the intestinal horizontal transfer rate of the transferable antibiotic resistance genes after binning.

[0014] Preferably, in step (2), the animals are 10-day-old white - feather broilers, the period for fecal collection is when the white - feather broilers are 21 - day-old, and the data volume of a single sample in the samples is ≥5 GB, and Q30≥80%.

[0015] Preferably, the method for extracting the total DNA data from the fecal samples in step (2) comprises the following steps:

[0016] ① Mixing the fecal samples and PBS, centrifuging and collecting the precipitate;

[0017] ② Washing the precipitate and extracting total DNA using a fecal kit;

[0018] ③ Conducting quality identification and concentration detection on the total DNA;

[0019] ④ Establishing a sequencing library for the total DNA.

[0020] Preferably, in step ①, the dosage ratio of the fecal samples to PBS is 0.2 g:0.8 - 1.2 mL, and the centrifugation conditions are 11000 - 13000 r / min for 100 - 140 s;

[0021] In step ②, the number of washing times is 2 - 3 times.

[0022] Preferably, the method for quality identification and concentration detection in step ③ is: detecting the concentration of DNA using qubit4.0, and analyzing the purity and integrity of DNA by agarose gel electrophoresis with 0.8 - 1.2% agarose.

[0023] Preferably, the method for constructing the sequencing library of total DNA in step ④ is as follows: Use a Tn5 transposase kit to construct a library for the total DNA, introduce index sequences by PCR, and then perform high-throughput sequencing on each constructed library using a high-throughput sequencing platform, with the data volume ≥ 5 GB.

[0024] Preferably, the software used for binning in step (6) is one or more of metabat, maxbin, or concoct.

[0025] Preferably, the method of calculation in step (7) is as follows:

[0026] Use checkm or gtdbtk to perform phylogenetic tree analysis on the above-mentioned bins to identify the strains of each bin, and then use abricate to identify whether each bin contains transferable resistance genes, and detect whether plasmid elements and resistance genes appear on the same contig at the same time. By calculating the ratio of the number of bins containing transferable resistance genes to the total number of bins, the intestinal horizontal transfer rate of transferable resistance genes can be obtained to evaluate the degree of intestinal horizontal transfer of transferable resistance genes in feed probiotics.

[0027] Preferably, the method of calculation in step (7) is as follows:

[0028] Use abricate to identify whether each bin contains transferable resistance genes, and detect whether plasmid elements and resistance genes appear on the same contig at the same time. Then use checkm or gtdbtk to perform phylogenetic tree analysis on the above-mentioned bins to identify the strains of each bin. By calculating the ratio of the number of bins containing transferable resistance genes to the total number of bins, the intestinal horizontal transfer rate of transferable resistance genes can be obtained to evaluate the degree of intestinal horizontal transfer of transferable resistance genes in feed probiotics.

[0029] Preferably, the method of calculation in step (7) is as follows:

[0030] Use abricate to identify whether each bin contains transferable resistance genes, and detect whether plasmid elements and resistance genes appear on the same contig at the same time. Then, by calculating the ratio of the number of bins containing transferable resistance genes to the total number of bins, the intestinal horizontal transfer rate of transferable resistance genes can be obtained to evaluate the degree of intestinal horizontal transfer of transferable resistance genes in feed probiotics.

[0031] The present invention provides a method for evaluating the degree of intestinal horizontal transfer of transferable antibiotic resistance genes in bacteria, comprising the following steps: (1) sequencing probiotics to determine the antibiotic resistance genes in the probiotics; (2) feeding animals with the probiotics, collecting fecal samples of the animals after feeding, and extracting the total DNA data in the fecal samples; (3) using fastp to perform quality control and screening on the total DNA data, and using megahit to perform sequence assembly on the quality-controlled data to obtain corresponding contigs; (4) using the binning module in metawrap to bin the contigs; (5) calculating the intestinal horizontal transfer rate of the transferable antibiotic resistance genes after binning.

[0032] The present invention can accurately evaluate the degree of intestinal horizontal transfer of transferable antibiotic resistance genes in feed probiotics, and provide guidance for the development of safe and effective feed probiotics.

[0033] The global animal feed probiotic industry has maintained rapid growth in recent years. It is expected that the output value of the global probiotic industry will exceed 77 billion US dollars in 2025, of which the Chinese market accounts for about 25%, that is, 19.25 billion US dollars (about 130 billion yuan). As an important sub-sector, feed probiotics are expected to account for 15% - 20% of the total output value of probiotics in China, that is, 19.5 - 26 billion yuan. Using the present invention to evaluate the safety of feed probiotics after entering the intestine for enterprises, and helping enterprises quickly develop safe and effective feed probiotic strains. Calculated at 1%, there is at least 200 million yuan of economic value every year.

[0034] In the long run, reducing the horizontal transfer of transferable antibiotic resistance genes in feed probiotics in the animal intestine from the source can greatly reduce the spread of antibiotic resistance genes and reduce the threat of drug-resistant bacteria to human health, with great social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a process flow chart of the present invention, where the green part is the box containing transferable antibiotic resistance genes. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention provides a method for evaluating the degree of intestinal horizontal transfer of transferable antibiotic resistance genes in bacteria, comprising the following steps:

[0037] (1) Sequencing probiotics to determine the transferable antibiotic resistance genes in the probiotics;

[0038] (2) Feeding animals with the probiotics, collecting fecal samples of the animals after feeding, and extracting the total DNA data in the fecal samples;

[0039] (3) Using fastp to perform quality control and screening on the total DNA data, and using megahit to perform sequence assembly on the quality-controlled data to obtain corresponding contigs;

[0040] (4) Use the binning module in metawrap to bin the contigs;

[0041] (5) Calculate the horizontal transfer rate of transferable drug resistance genes at the intestinal level after binning.

[0042] In the present invention, the animal described in step (2) is a 10-day-old white - feather broiler, the period for feces collection is when the white - feather broiler is 21 days old, the data volume of a single sample in the sample ≥ 5 GB, and Q30 ≥ 80%.

[0043] In the present invention, the method for extracting total DNA data from fecal samples in step (2) includes the following steps:

[0044] ① Mix the fecal sample and PBS, and collect the precipitate after centrifugation;

[0045] ② Wash the precipitate and extract total DNA using a feces kit;

[0046] ③ Conduct quality identification and concentration detection on the total DNA;

[0047] ④ Establish a sequencing library for the total DNA.

[0048] In the present invention, in step ①, the dosage ratio of the fecal sample to PBS is 0.2 g: 0.8 - 1.2 mL, and the centrifugation conditions are 11000 - 13000 r / min for 100 - 140 s;

[0049] The number of washing times in step ② is 2 - 3 times.

[0050] In the present invention, the method for quality identification and concentration detection in step ③ is: use qubit4.0 to detect the concentration of DNA, and use 0.8 - 1.2% agarose gel electrophoresis to analyze the purity and integrity of DNA.

[0051] In the present invention, the method for establishing a sequencing library for the total DNA in step ④ is: use the Tn5 transposase kit to construct a library for the total DNA, introduce the index sequence by PCR, and then perform high - throughput sequencing on each constructed library using a high - throughput sequencing platform, with the data volume ≥ 5 GB.

[0052] In the present invention, the software used for binning in step (6) is one or more of metabat, maxbin, or concoct.

[0053] In the present invention, the calculation method in step (7) is:

[0054] The above boxes were analyzed by phylogenetic tree using CheckM or GTDBTk to identify the strains of each box. Then,abricate was used to identify whether each box contained transferable drug-resistant genes, and whether plasmid elements and drug-resistant genes appeared on the same contig. By calculating the ratio of the number of boxes containing transferable drug-resistant genes to the total number of boxes, the intestinal horizontal transfer rate of transferable drug-resistant genes was obtained to evaluate the degree of intestinal horizontal transfer of transferable drug-resistant genes in feed probiotics.

[0055] In the present invention, the calculation method described in step (7) is as follows:

[0056] abricate was used to identify whether each box contained transferable drug-resistant genes, and whether plasmid elements and drug-resistant genes appeared on the same contig. Then, the above boxes were analyzed by phylogenetic tree using CheckM or GTDBTk to identify the strains of each box. By calculating the ratio of the number of boxes containing transferable drug-resistant genes to the total number of boxes, the intestinal horizontal transfer rate of transferable drug-resistant genes was obtained to evaluate the degree of intestinal horizontal transfer of transferable drug-resistant genes in feed probiotics.

[0057] In the present invention, the calculation method described in step (7) is as follows:

[0058] abricate was used to identify whether each box contained transferable drug-resistant genes, and whether plasmid elements and drug-resistant genes appeared on the same contig. Then, by calculating the ratio of the number of boxes containing transferable drug-resistant genes to the total number of boxes, the intestinal horizontal transfer rate of transferable drug-resistant genes was obtained to evaluate the degree of intestinal horizontal transfer of transferable drug-resistant genes in feed probiotics.

[0059] The technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0060] Example 1

[0061] 1. Selection of test materials

[0062] Enterococcus faecium strains without plasmids (purchased from China Center of Industrial Culture Collection, numbered: CICC 23188), Enterococcus faecium strains carrying the cat drug-resistant gene on the pCW7 plasmid (purchased from China Center of Industrial Culture Collection, numbered: CICC 24252), were preserved in the laboratory.

[0063] 2. Preparation of fermentation broth

[0064] Streak CICC 23188 and CICC 24252 on MRS medium plates respectively, and culture at 37°C for 16 hours for strain activation. Use a sterile toothpick to pick single colonies on the plate and inoculate them into test tubes of MRS liquid medium, and culture at 37°C and 250 rpm for 8 hours. Then transfer them to a triangular flask containing 100 mL of MRS liquid medium at an inoculation amount of 1%, and culture at 37°C and 250 rpm overnight to obtain the fermentation broth of CICC 23188 and CICC 24252 respectively.

[0065] 3. Experimental animals and experimental design

[0066] Thirty 1-day-old Arbor Acres Plus (AA+) broiler chickens with similar body weights and healthy and disease-free were selected for the experiment and randomly divided into 3 groups with 10 chickens in each group. The control group was fed a basal diet and given normal drinking water. In experimental group 1 and experimental group 2, 3% of the fermentation broth of CICC 23188 and 3% of the fermentation broth of CICC 24252 were added to the drinking water respectively on the 10th day, and the experimental period was 21 d. On the 21st day, 6 white - feather broiler chickens were randomly selected from each group to collect feces. The experimental broiler chickens were provided by Miyun Branch of Beijing Dafa Zhengda Co., Ltd.

[0067] 4. Total DNA extraction and quality control

[0068] Suspend 0.2 g of chicken feces sample with 1 mL of 1×PBS, centrifuge at 12000 r / min for 2 min, collect the precipitate, and repeat 3 times to wash the sample. Then use the Qiagen feces kit to extract the total DNA of the sample according to the steps in the instruction manual. The quality identification and concentration detection of microbial DNA were carried out by detecting the concentration of DNA with qubit4.0. The purity and integrity of DNA were analyzed by 1% agarose gel electrophoresis.

[0069] 5. Construction of sequencing library and sequencing

[0070] Use the TransGen AP221 - 02 / Tn5 Transposase Kit to construct a library for the total DNA, and introduce the index sequence by PCR. Then use the illumina NovaSeq 6000 to perform high - throughput sequencing on each constructed library in the PE150 mode, and the data volume is ≥5 GB.

[0071] 6. Data quality control, sequence assembly and binning

[0072] Use fastp to perform quality control and screening on the data, and use megahit (or sequence assembly software such as SPAdes) to perform sequence assembly on each sample. Use the binning module in metawrap (binning, the binning software can be selected from metabat, maxbin, concoct, one of them or a random combination) to bin the above - mentioned contigs.

[0073] 7. Calculate the horizontal transfer rate of transferable drug resistance genes in the intestine (any one of the following three methods can be selected).

[0074] A. Use CheckM or GTDBTk to perform phylogenetic tree analysis on the above bins to identify the strains of each bin, and then useabricate to identify whether each bin contains transferable drug resistance genes. Detect whether plasmid elements and drug resistance genes appear on the same contig at the same time. By calculating the ratio of the number of bins containing transferable drug resistance genes to the total number of bins, the horizontal transfer rate of transferable drug resistance genes in the intestine can be obtained to evaluate the horizontal transfer degree of transferable drug resistance genes in feed probiotics.

[0075] B. Useabricate to identify whether each bin contains transferable drug resistance genes. Detect whether plasmid elements and drug resistance genes appear on the same contig at the same time, and then use CheckM or GTDBTk to perform phylogenetic tree analysis on the above bins to identify the strains of each bin. By calculating the ratio of the number of bins containing transferable drug resistance genes to the total number of bins, the horizontal transfer rate of transferable drug resistance genes in the intestine can be obtained to evaluate the horizontal transfer degree of transferable drug resistance genes in feed probiotics.

[0076] C. Useabricate to identify whether each bin contains transferable drug resistance genes. Detect whether plasmid elements and drug resistance genes appear on the same contig at the same time, and then by calculating the ratio of the number of bins containing transferable drug resistance genes to the total number of bins, the horizontal transfer rate of transferable drug resistance genes in the intestine can be obtained to evaluate the horizontal transfer degree of transferable drug resistance genes in feed probiotics.

[0077] As can be seen from Table 1, the plasmids containing chloramphenicol resistance genes are mainly distributed in Enterococcus and Clostridium, and the transfer rate is 15.56%.

[0078] Table 1 Binning results of the CICC 24252 strain group

[0079]

[0080]

[0081]

[0082] As can be seen from the above embodiments, the present invention provides a method for evaluating the intestinal horizontal transfer degree of transferable antibiotic resistance genes in bacteria, comprising the following steps: (1) sequencing probiotics to determine the antibiotic resistance genes in the probiotics; (2) feeding animals with the probiotics, collecting fecal samples of the animals after feeding, and extracting the total DNA data in the fecal samples; (3) using fastp to perform quality control and screening on the total DNA data, and using megahit to perform sequence assembly on the data after quality control to obtain the corresponding contigs; (4) using the binning module in metawrap to bin the contigs; (5) calculating the intestinal horizontal transfer rate of the transferable antibiotic resistance genes after binning. The present invention can evaluate the safety of feed probiotics after entering the intestine and develop safe and effective feed probiotic strains. In the long run, reducing the horizontal transfer of transferable antibiotic resistance genes in feed probiotics at the source in the animal intestine can greatly reduce the spread of antibiotic resistance genes and reduce the threat of antibiotic-resistant bacteria to human health, having great social benefits.

[0083] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for evaluating the degree of intestinal horizontal transfer of mobile antibiotic resistance genes in bacteria, characterized in that It includes the following steps: (1) Sequencing the probiotics to determine the transferable resistance genes in the probiotics; (2) Feeding the animals with the probiotics, collecting the fecal samples of the animals after feeding, and extracting the total DNA data from the fecal samples; (3) Using fastp to perform quality control and screening on the total DNA data, and using megahit to perform sequence assembly on the quality-controlled data to obtain the corresponding contigs; (4) Using the binning module in metawrap to bin the contigs; (5) Calculating the intestinal horizontal transfer rate of the transferable resistance genes after binning.

2. The method according to claim 1, wherein The animal described in step (2) is a 10-day-old white broiler chicken, and the feces are collected when the white broiler chicken is 21 days old. The data volume of a single sample in the sample is ≥5GB, and Q30≥80%.

3. The method according to claim 1, wherein The method for extracting the total DNA data from the fecal samples described in step (2) includes the following steps: ① Mix the fecal sample and PBS, and collect the precipitate after centrifugation; ② Wash the precipitate and extract the total DNA using a feces kit; ③ Perform quality identification and concentration detection on the total DNA; ④ Establish a sequencing library for the total DNA.

4. The method according to claim 1, characterized in that, In step ①, the dosage ratio of the fecal sample to PBS is 0.2g:0.8 - 1.2mL, and the centrifugation conditions are 11000 - 13000r / min for 100 - 140s; In step ②, the number of washing times is 2 - 3 times.

5. The method according to claim 1, characterized in that The method for quality identification and concentration detection in step ③ is: detecting the concentration of DNA using qubit4.0, and analyzing the purity and integrity of DNA using 0.8 - 1.2% agarose gel electrophoresis.

6. The method according to claim 1, characterized in that, The method for establishing a sequencing library for the total DNA in step ④ is: constructing a library for the total DNA using the Tn5 transposase kit, introducing an index sequence using PCR, and then performing high-throughput sequencing on each constructed library using a high-throughput sequencing platform, with the data volume ≥5GB.

7. The method according to claim 6, characterized in that, The software used for binning in step (6) is one or more of metabat, maxbin, or concoct.

8. The method according to any one of claims 1 to 7, characterized in that The calculation method described in step (7) is: Using checkm or gtdbtk to perform phylogenetic tree analysis on the above bins, identifying the strains of each bin, and then using abricate to identify whether each bin contains transferable resistance genes, detecting whether the plasmid element and the resistance gene appear on the same contig at the same time. By calculating the ratio of the number of bins containing transferable resistance genes to the total number of bins, the intestinal horizontal transfer rate of the transferable resistance genes is obtained to evaluate the intestinal horizontal transfer degree of the transferable resistance genes in the probiotics for feeding.

9. The method according to any one of claims 1 to 7, characterized in that The calculation method described in step (7) is: Use abricate to identify whether each bin contains transferable resistance genes, and detect whether plasmid elements and resistance genes appear on the same contig at the same time. Then use checkm or gtdbtk to perform phylogenetic tree analysis on the above-mentioned bins to identify the strains of each bin. By calculating the ratio of the number of bins containing transferable resistance genes to the total number of bins, the intestinal horizontal transfer rate of transferable resistance genes is obtained to evaluate the degree of intestinal horizontal transfer of transferable resistance genes in feed probiotics.

10. The method according to any one of claims 1 to 7, characterized in that The calculation method described in step (7) is as follows: Use abricate to identify whether each bin contains transferable resistance genes, and detect whether plasmid elements and resistance genes appear on the same contig at the same time. Then, by calculating the ratio of the number of bins containing transferable resistance genes to the total number of bins, the intestinal horizontal transfer rate of transferable resistance genes is obtained to evaluate the degree of intestinal horizontal transfer of transferable resistance genes in feed probiotics.

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