CN113881589B - Klebsiella pneumoniae KP21 strain and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a Klebsiella pneumoniae KP21 strain and application thereof. The invention separates a strain of klebsiella pneumoniae from the donkey with pneumonia, and then obtains a colony with medium size, neat edge, smoothness, wetness and circular protrusion after culture; carrying out a mouse infection experiment, wherein the experiment is consistent with the symptoms of the klebsiella pneumoniae on the donkey; meanwhile, the inactivated vaccine prepared from the strain has important value for effectively preventing donkey pneumonia caused by local epidemic strains, strengthening research and possible application such as pathogen biochemistry and molecular identification.

Description

Klebsiella pneumoniae KP21 strain and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a Klebsiella pneumoniae KP21 strain and application thereof.

Background

Klebsiella pneumoniae is a very important gram-negative bacillus in the enterobacteriaceae genus, is also a pathogenic microorganism which is commonly suffered by people and livestock, is widely distributed in nature, is mainly distributed in respiratory tract, intestinal tract and genitourinary tract in human and animal bodies, is a component part of a normal organism microbiota, and can become pathogenic bacteria to cause opportunistic infection under the conditions of low immune function and the like.

The klebsiella pneumoniae comprises 3 subspecies of klebsiella pneumoniae subspecies, stink subspecies and nasal scleroderma subspecies, wherein the klebsiella pneumoniae subspecies are most widely distributed and are one of pathogenic bacteria for causing serious diseases such as pneumonia, diarrhea, urinary tract infection, biliary tract infection, septicemia, suppurative meningitis and the like of various animals and people. Klebsiella pneumoniae may have a potential threat to donkey industry, and should be considered important. There are few reports of donkey-derived klebsiella pneumoniae in China.

Donkey itself has stronger tolerance to common diseases, especially under traditional free-range conditions, the donkey has low probability of infectious diseases due to the small and scattered quantity of cultivation. However, in a large-scale donkey farm, due to the increase of the population culture density, the incidence of donkey infectious diseases is increased, public health safety crisis is caused to donkey industry once donkey epidemic diseases are exploded and popular, and serious food safety threat and economic loss are brought to industries such as donkey, donkey meat, donkey milk and the like, so that epidemic disease prevention and control are necessary under the background of intensive culture, frequent flow and international animal product transaction. Although klebsiella pneumoniae bacteria have no influence on donkey raising, in order to promote the rapid development of donkey raising, purify the disease as soon as possible and control the occurrence and prevalence of the bacteria, prevent and block infection of the klebsiella pneumoniae on the donkey, separate local klebsiella pneumoniae epidemic strains, strengthen researches and possible applications such as pathogen biochemistry, molecular identification and the like, and realize effective prevention and treatment of the donkey pneumonia caused locally.

Disclosure of Invention

In order to prevent and block infection of the donkey by the klebsiella pneumoniae and promote rapid development of donkey breeding, the invention separates a strain of klebsiella pneumoniae from the donkey with pneumonia, and then obtains colony with medium size, neat edge, smoothness, wetness and circular bulge after culturing; carrying out a mouse infection experiment, wherein the experiment is consistent with the symptoms of the klebsiella pneumoniae on the donkey; meanwhile, the inactivated vaccine prepared from the strain has important value for effectively preventing donkey pneumonia caused by local epidemic strains, strengthening research and possible application such as pathogen biochemistry and molecular identification.

The technical scheme of the invention is as follows:

klebsiella pneumoniaePlant strainKlebsiella pneumoniaeKP21, the Klebsiella pneumoniae strainKlebsiella pneumoniaeThe microorganism preservation number of KP21 is CGMCC22865, and the preservation date is as follows: 2021, 07, 09, deposit unit: china general microbiological culture Collection center (China Committee for culture Collection).

The Klebsiella pneumoniae strainKlebsiella pneumoniaeKP21 is derived from donkey sources; culturing in nutrient agar medium to obtain colony with medium size, neat edge, smoothness, wetness and circular protrusion; inoculating into nutrient broth, and observing with gram stain to obtain gram negative and coarser bacillus.

The Klebsiella pneumoniae strainKlebsiella pneumoniaeThe 16S rDNA sequence of KP21 is shown as SEQ ID No. 1.

The general primers for PCR amplification of the 16S rDNA of the bacteria are as follows:

the F27 sequence is shown in SEQ ID No. 2: 5'-AGAGTTGATCCTGGCTCAG-3';

the sequence of R1492 is shown as SEQ ID No. 3: 5'-AAGGAGGTGATCCAACCGCA-3'.

Klebsiella pneumoniae strains described aboveKlebsiella pneumoniaeApplication of KP21 in preparing inactivated vaccine.

Advantageous effects

The invention provides a dominant epidemic strain of klebsiella pneumoniae which is separated from a donkey-source klebsiella pneumoniae in Shandong region and causes donkey pneumonia. Only the klebsiella pneumoniae pathogenic bacteria are separated from the disease materials of the donkey pneumonia, so that an accurate basis is provided for the pathogenesis of the donkey pneumonia. In a mouse infection experiment, the bacterium has stronger pathogenicity to mice, and the symptoms are consistent with the symptoms of the klebsiella pneumoniae on the donkey; the inactivated vaccine prepared by the method has good immunity, and has important value for effectively preventing donkey pneumonia and other animal pneumonia caused by local epidemic strains.

Drawings

FIG. 1 is a gram stain of Klebsiella pneumoniae strain KP21 of donkey origin;

FIG. 2 shows PCR amplification product M: a Marker;1: strain KP21 was isolated.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the present invention. The test methods in the examples below are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Example 1

1 materials and methods

1.1 disease materials

In 2021, small-scale donkey pneumonia occurs in some donkey field in eastern Donga in 4 months, and the small-scale donkey field is 250 in total, and the donkey is 69 in pneumonia, and the sick donkey has shortness of breath and dyspnea, or has sleepiness, anorexia, and the like, and dies successively. Necropsy results on 30 dead donkeys showed all occurrence of pulmonary congestion and edema, further pulmonary metaplasia such as liver, apparent swelling, off-white color, and massive hemorrhage. 1 sample of mediastinal lymph node, heart, lung, kidney, liver and spleen tissue of foal is collected for bacteriological detection and virus screening.

1.2 major reagents

Common agar and common nutrient broth medium were purchased from Qingdao sea Bo organisms Co., ltd; the biochemical identification tube was purchased from Qingdao sea Bo biology Co., ltd; the drug sensitive tablet was purchased from Oxoid company.

1.3 Isolation culture of strains

The disease tissue collected in 1.1 is placed in an ultra-clean workbench, the inside of the disease tissue is inoculated into a common agar culture medium by an inoculating loop, and the culture is placed in a incubator at 37 ℃ for culturing for 24 hours, and the colony morphology of the disease tissue is observed. Selecting single colony in the chromogenic medium, inoculating to nutrient broth medium, culturing in a 37 deg.C incubator for 24 hr, gram staining, and observing the morphological characteristics of the strain and purifying.

1.4 Biochemical identification

The separated and purified strain is inoculated in a novel microorganism trace biochemical identification tube, placed in a 37 ℃ incubator for culturing for 24 hours, the color change of the bottom and the inclined plane is observed, and the judgment result is according to the standard of the Berger's bacteria identification handbook.

1.5 PCR sequencing of isolate 16S rDNA

1.5.1 Preparation of PCR template the isolated strain is picked up to culture single colony of pathogenic bacteria, and after enrichment in common nutrient broth, the bacterial liquid DNA is extracted by EasyPure Bacteria Genomic DNA Kit.

1.5.2 PCR amplification of 16S rDNA bacterial 16S rDNA general primer sequence is

F27：5′－AGAGTTGATCCTGGCTCAG－3′，

R1492：5′－AAGGAGGTGATCCAACCGCA－3′；

The size of the fragment to be amplified is about 1500bp, and the primers are synthesized by Beijing qing biological science and technology Co., ltd; reaction system (25 μl): 2x Taq PCR Master Mix 12.5 mu L, ddH ₂ O9. Mu.L, 1. Mu.L of each of the upstream and downstream primers (10 mmol/L), 1.5. Mu.L of the template; the reaction procedure was 95℃for 5min,95℃denaturation for 40s, 56℃annealing for 40s, 72℃extension for 90s,35 cycles, 72℃extension for 8min. The PCR products were visualized by 1% agarose gel electrophoresis.

1.5.3 sequencing alignment Single-entry band PCR products were sent to Beijing qing Biotechnology Co., ltd, SEQ ID No. 1. Sequencing results were aligned in Gen-Bank by Blast (https:// Blast. Ncbi. Nlm. Nih. Gov /).

1.6 Drug sensitivity test

Performing drug sensitivity test by adopting a paper sheet diffusion K-B method, selecting single bacterial colony, inoculating the single bacterial colony into a nutrient broth culture medium, culturing the single bacterial colony at 37 ℃ for 18 h, and diluting bacterial liquid by using a sterile PBS buffer solution to ensure that the absorbance of the bacterial liquid at 600 nm wavelength is 0.1; uniformly coating the diluted bacterial liquid on the surface of an HM solid culture medium, pasting 4 paper sheets on each culture medium, culturing at 37 ℃ for 18 h, measuring the diameter of a bacteriostasis ring, repeating for 3 times, and calculating an average value. The outcome determination was made with reference to the U.S. clinical and laboratory standardization committee (Clinical and laboratory standards institute, CLSI) drug susceptibility outcome determination criteria (version 2013).

Example 2 mouse pathogenicity test

2.1 laboratory animals SPF Kunming mice, 42, approximately 20g in weight, were purchased from the university of Shandong laboratory animal center in male and female halves.

2.2 determination of LD50 the mice were randomly divided into 7 groups of 6 trials, 1 control group, 6 each, male and female halves, bacterial solutions according to 1.8X10 ⁹ -1.8×10 ⁴ CFU/mL. Mice were inoculated with equal volume of bacterial solution for 8h at unequal concentrations by intraperitoneal injection, and the control group was injected with equal amount of physiological saline at 0.2 mL/mouse. After the toxin is attacked, the observation is continued for 10 days.

2.3 dissecting mice, recovering bacteria, and identifying dead mice after challenge by PCR, and dissecting and observing lesions of various tissues and organs. Taking lung, liver and spleen of a mouse to inoculate nutrient agar medium, recovering bacteria, and extracting bacterial DNA by the kit. PCR was performed to determine the presence or absence of Klebsiella pneumoniae in organs and aborted mice.

Example 3 mouse immunoassay

3.1, adding 0.4% formaldehyde into the bacteria to inactivate 36-48 h, centrifuging and discarding the clear. The pellet was diluted to 1X 10 with sterile PBS (10 mmol pH7) ⁶ CFU/mL. The qualified inactivated bacteria liquid is emulsified with equal amount of complete Freund's adjuvant and incomplete Freund's adjuvant to prepare the immunogen after sterile test and safety test.

3.2 mice of 15g body weight were divided into 2 groups of 6 with 0.3 mL/subcutaneous injection of the prepared inactivated antigen and 6 with 0.3 mL/subcutaneous injection of 0.9% physiological saline as control, each group being subjected to 2-way after 20 days after the initial administration, and 15d after 2-way to perform immunoprotection experiments according to 1.7.

Results and analysis of examples of effects

1. Results of Strain culture

The strain is named as KP21 and has a preservation number (CGMCC 22865). The bacterial colony is cultured in a nutrient agar culture medium and is provided with medium-sized, smooth and moist round bulges. After 24h incubation at 37℃of the bacterial liquid inoculated to the nutrient broth, the bacteria were purified, gram negative, as shorter, bacillus, single, double or short chain arrangement, spore-free, flagellum free, by gram staining. (FIG. 1).

2. Results of biochemical tests

Determination result: KP21 is consistent with the biochemical characteristics of Klebsiella pneumoniae in the "Burjie bacteria identification Manual". The biochemical identification results are shown in Table 1.

TABLE 1 KP21 Biochemical identification

3. PCR amplification analysis of Strain 16S rDNA

The KP21 strain 16S rDNA PCR amplified product showed a distinct target band at about 1500bp (FIG. 2). The isolated strain is subjected to 16S rDNA PCR sequencing, and the sequences are compared by Blast, so that the result shows that the 16S rDNA sequence of the isolated strain is Klebsiella pneumoniae.

4. Drug sensitivity analysis

The test results of the paper sheet drug sensitivity show that the bacteria of the strain generate stronger drug resistance (Table 2).

TABLE 2 results of drug sensitivity test of bacteria

5. Results of bacterial Strain pathogenicity test

The strain of Klebsiella pneumoniae can infect mice, and after 36h inoculation, the inoculation concentration is 1.8X10% ⁹ The mice in the CUF/mL test group were first shown to develop symptoms, and the affected mice were rough, listless, inappetence, and had infections as shown in Table 3. According to the Reed-Muench method, the LD50 value of KP21 to mice was calculated to be 1.8X10 ⁷ CFU/mL。

TABLE 3 infection lethal conditions of Salmonella on mice at different doses

6. Analysis of mice and recovery of bacterial identification results

The infected mice are subjected to the dissection examination, and the infected mice are found to have the symptoms of bleeding and obvious swelling of the lungs, grey white and splenomegaly; the nutrient agar medium was re-inoculated from the lungs, liver, spleen and aborted mouse embryos of the mice and incubated at 37℃for 24h, consistent with the strain morphology of the previously injected mice by gram staining microscopy. No other pathogenic microorganisms were detected from the disease mass, and the Klebsiella pneumoniae KP21 was considered to be a pathogenic bacterium causing donkey and mouse pneumonia.

7. Immune effect of inactivated vaccine

KP21 is infected through alimentary canal to cause mental depression, dyspnea and death of mice, and the infection causes the mice to have lung lobus such as liver, obvious swelling, off-white color, large-area bleeding and spleen swelling and necrosis. After the strain is used for culturing and enrichment in common nutrient broth (culturing for not less than 6 hours at 36+/-1 ℃), the prepared inactivated vaccine (0.4% formaldehyde) has serum antibody titer of 1:5.2X10 after two immunization ⁴ The immune group has no symptoms of pneumonia, and the immune protective power of the immune group can reach 100 percent.

Sequence listing

<110> Shandong agricultural sciences laboratory animal husbandry and veterinary institute

<120> Klebsiella pneumoniae KP21 strain and application thereof

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ggcgggaggg ctacctgcaa gtcgagcggt agcacagaga gcttgctctc gggtgacgag 60

cggcggacgg gtgagtaatg tctgggaaac tgcctgatgg agggggataa ctactggaaa 120

cggtagctaa taccgcataa tgtcgcaaga ccaaagtggg ggaccttcgg gcctcatgcc 180

atcagatgtg cccagatggg attagctagt aggtggggta acggctcacc taggcgacga 240

tccctagctg gtctgagagg atgaccagcc acactggaac tgagacacgg tccagactcc 300

tacgggaggc agcagtgggg aatattgcac aatgggcgca agcctgatgc agccatgccg 360

cgtgtgtgaa gaaggccttc gggttgtaaa gcactttcag cggggaggaa ggcgttaagg 420

ttaataacct tggcgattga cgttacccgc agaagaagca ccggctaact ccgtgccagc 480

agccgcggta atacggaggg tgcaagcgtt aatcggaatt actgggcgta aagcgcacgc 540

aggcggtctg tcaagtcgga tgtgaaatcc ccgggctcaa cctgggaact gcattcgaaa 600

ctggcaggct agagtcttgt agaggggggt agaattccag gtgtagcggt gaaatgcgta 660

gagatctgga ggaataccgg tggcgaaggc ggccccctgg acaaagactg acgctcaggt 720

gcgaaagcgt ggggagcaaa caggattaga taccctggta gtccacgccg taaacgatgt 780

cgatttggag gttgtgccct tgaggcgtgg cttccggagc taacgcgtta aatcgaccgc 840

ctggggagta cggccgcaag gttaaaactc aaatgaattg acgggggccc gcacaagcgg 900

tggagcatgt ggtttaattc gatgcaacgc gaagaacctt acctggtctt gacatccaca 960

gaactttcca gagatggatt ggtgccttcg ggaactgtga gacaggtgct gcatggctgt 1020

cgtcagctcg tgttgtgaaa tgttgggtta agtcccgcaa cgagcgcaac ccttatcctt 1080

tgttgccagc ggttaggccg ggaactcaaa ggagactgcc agtgataaac tggaggaagg 1140

tggggatgac gtcaagtcat catggccctt acgaccaggg ctacacacgt gctacaatgg 1200

catatacaaa gagaagcgac ctcgcgagag caagcggacc tcataaagta tgtcgtagtc 1260

cggattggag tctgcaactc gactccatga agtcggaatc gctagtaatc gtagatcaga 1320

atgctacggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgggagtgg 1380

gttgcaaaag aagtaggtag cttaaccttc gggagggcgc ttaccacttt gtgattcatg 1440

actgggggaa gtcgatcaga tgcttc 1466

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agagttgatc ctggctcag 19

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aaggaggtga tccaaccgca 20

Claims (2)

1. A Klebsiella pneumoniae strain is characterized in that the Klebsiella pneumoniae strain is Klebsiella pneumoniae strain @Klebsiella pneumoniae) KP21, the microorganism preservation number is CGMCC22865, and the preservation date is as follows: 2021, 07, 09, deposit unit: china general microbiological culture Collection center (China Committee for culture Collection).

2. Use of a klebsiella pneumoniae strain according to claim 1 for the preparation of an inactivated vaccine.

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