WO2017067089A1

WO2017067089A1 - Neovison vison klebsiella peneumoniae

Info

Publication number: WO2017067089A1
Application number: PCT/CN2016/000100
Authority: WO
Inventors: 王贵升; 王金宝; 田夫林; 单虎
Original assignee: 王贵升
Priority date: 2015-10-19
Filing date: 2016-02-26
Publication date: 2017-04-27
Also published as: CN105199991A

Abstract

Provided is a neovison vison klebsiella peneumoniae strain which is named as a WMOK strain with the preservation number of CGMCC NO.11222. The strain is K2 type, is Gram-negative (G-), facultatively anaerobic, atrichous and asporous, and has an obvious capsule; most strains have fimbriae and are rod-shaped with finely rounded terminuses. The WMOK strain has relatively high toxicity, and has a half lethal dose of 2.1×10^0.5 CFU to mice and a half lethal dose of 3.8×10^0.7 CFU to neovison vison.

Description

Klebsiella pneumoniae

Technical field

The invention relates to a strain of Klebsiella pneumoniae, belonging to the technical field of microorganisms.

Background technique

Klebsiella pneumoniae (Klebsiella peneumoniae) belongs to the family Enterobacteriaceae, Klebsiella, Gram-negative (G ^-). Facultative anaerobic, no flagella and spores, with obvious capsules, most strains have pili. Klebsiella pneumoniae has K (capsular) antigen and O (bacteria) antigen, the former being lipopolysaccharide and the latter being polysaccharide. K antigen can be used for the identification of bacterial types. According to the capsular polysaccharide (K antigen) typing, Klebsiella pneumoniae can be divided into 82 K serotypes, wherein the K1, K2 and K5 serotypes are closely related to severe infectious diseases in humans and animals. The strain was first isolated from the lung tissue of patients with lobar pneumonia in 1883. It is a common pathogen of hospital-acquired pneumonia (HAP). In recent years, in human infection, the strain is in addition to E. coli. The most important condition of iatrogenic infection is pathogenic bacteria, which can cause infection in multiple parts of the body, but the highest incidence in the urinary tract and respiratory tract, when the body's immunity is reduced or due to long-term use of antibiotics, the body Infection caused by flora imbalance can lead to pneumonia, meningitis, liver abscess, endophthalmitis, urinary tract inflammation, wound infection and systemic sepsis, which brings great difficulties to clinical treatment, and the morbidity and mortality are extremely high. In animal infections, various livestock, poultry, wild animals and aquatic animals can be infected. Broilers and laying hens in poultry are susceptible to infection. Pigs, cattle, sheep and rabbits can be infected in livestock, in addition to wild and aquatic animals. The giant panda, golden monkey, Chinese sturgeon, etc. can also be infected, among which the white mice and the chickens within 15 days old are most susceptible to the bacteria. In recent years, there have been reports of animal infection with klebsiella, which can cause liver necrosis in chickens, pneumonia in rabbits, enteritis, diarrhea in giant pandas, hepatitis, diarrhea in pigs, death of cattle and sheep, etc. Caused a large number of deaths such as fur otters, moles, and bamboo rats. Due to the abuse of antibiotics, the drug resistance of the bacteria is increasing, and the emergence of multi-drug resistant strains brings great difficulties to clinical treatment.

Klebsiella pneumoniae is not only more harmful to livestock and poultry, but also causes the incidence of fur animals (fox, scorpion, leeches) to rise, leading to pneumonia, metritis, mastitis and other diseases in fur animals. Suppurative inflammation. In foreign countries, Morris first reported rickettsial disease in the United States in 1947. It is characterized by posterior numbness and sepsis of abscess cellulitis. In China, Zhao Chunde et al first reported two 貂 in Zhoushan City in 1987. Klebsiella pneumoniae occurred in the field, with acute abscess in the neck as the main feature, and the mortality rate was higher. Since then, there have been reports of the incidence of fur animals caused by Klebsiella pneumoniae at home and abroad. At present, scholars in China have reported the median lethal dose of Klebsiella pneumoniae in mice. The median lethal dose of Klebsiella isolated from bamboo rats to horses is 4.5×10 ⁷ CFU/ Only; Zhang Shizhen et al. determined the median lethal dose of the isolate KPm0912 and the standard strain KP1300, which were 1.1×10 ⁵ CFU/mL and 1.3×10 ⁶ CFU/mL, respectively; and the Klebsiella pneumoniae K1 isolated from fur animals such as Zhong Shixun et al. The median lethal dose to mice was 1.53×10 ⁸ CFU/mL; the half lethal doses of two strains of K. pneumoniae KP-ZC1 and KP-ZC2 isolated from leeches were 4.9×10 ⁶ CFU/mL, respectively. , 3.1 × 10 ⁶ CFU / mL; Li Fuxiang and other isolated KP14013 strain on the mice lethal dose of 3 × 10 ^1.8 CFU.

Summary of the invention

The invention separates Klebsiella from the Klebsiella sinensis disease material, and isolates and purifies and cultures to obtain a highly toxic Klebsiella; polymerase chain reaction (PCR), transmission Electron microscopy and nucleic acid sequence analysis methods were used to identify and confirm the isolates of Klebsiella pneumoniae; it provided a basis for further research and control of Klebsiella pneumoniae.

Technical solutions

A strain of Klebsiella pneumoniae, named WMOK strain, accession number: CGMCC NO.11222.

The WMOK strain of K. pneumoniae of the present invention is K2 type, Gram stain is negative (G-), facultative anaerobic, no flagella and spore, and has obvious capsule, and most strains have pili. Both ends are obtusely rounded.

The drug resistance of the K. pneumoniae WMOK strain of the present invention is as follows:

Resistance: ampicillin, ampicillin, cefazolin, ceftazidime, ceftriaxone, cefepime, antrexam, gentamicin, cotrimoxazole;

Zhongmin: tobramycin, ciprofloxacin;

Sensitive: piperacillin, cefotetan, ertapenem, imipenem, amikacin, levofloxacin, nitrofurantin.

Method for obtaining Klebsiella pneumoniae of the present invention:

The diseased sputum (mainly characterized by lack of energy, weight loss, etc.) was cultured on a urinary tract chromogenic medium at 37 ° C for 12 hours; a single blue-gray colony was picked (Klebsiella pneumoniae) The bacteria were grown as blue-gray colonies on the urinary tract chromogenic medium, and they were streaked on tryptone soy agar medium to purify the colonies.

The purified colonies were subjected to PCR amplification, and the amplified products were sequenced. The results of sequencing the PCR reaction products were Blast, and compared with the published gene sequence of K. pneumoniae in GenBank, homology. It reaches 99%-100%.

The K. pneumoniae WMOK strain of the present invention has an LD ₅₀ of 2.1 × 10 ^0.5 CFU for KM mice. The virulence is significantly stronger than other Klebsiella strains that have been reported so far.

The present invention measures the LD ₅₀ of Klebsiella pneumoniae on leech for the first time, and the WMOK strain of K. pneumoniae of the present invention has an LD ₅₀ of 3.8 × 10 ^0.7 CFU for leech.

Beneficial effect

The K. pneumoniae WMOK strain of the present invention is highly toxic and can be used as an attacking strain for the development of a subsequent product.

Deposit information

Deposit time: August 11, 2015

Name of the depository: General Microbiology Center of China Microbial Culture Collection Management Committee

Deposit number: CGMCC NO.11222

Depository Address: No. 3, No.1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences

Classification: Klebsiella peneumoniae

DRAWINGS

Figure 1. Electropherogram of PCR amplification products of Klebsiella pneumoniae: M. pneumoniae DNA standard DL 2 000; 1-6. PCR product designated as K. pneumoniae WMOK strain colony; 7 positive control (take K. pneumoniae ATCC 700603 purchased from the US Standard Biological Collection Center as a control); 8. Negative control (take E. coli ATCC 25922 purchased from the American Standard Biological Collection Center as a negative control);

Fig. 2 is a photograph of the K. pneumoniae WMOK strain of the present invention observed under an electron microscope.

detailed description

In the following examples, the diseased material came from a large-scale mink farm in Shandong Province. Before the death of the disease, the main manifestations were lack of energy, difficulty in breathing and so on. SPF Kunming mice were purchased from Shandong Experimental Animal Center. The tryptone soy agar medium, the tryptone soy broth medium and the urinary tract group chromogenic medium are all commonly used in the field; the components of the tryptone soy agar medium are tryptone, soybean meal, sodium chloride, The agar; tryptone soy broth medium components are tryptone, soy peptone, sodium chloride, dipotassium hydrogen phosphate, glucose; urinary flora group chromogenic medium components are agar, peptone, yeast powder, pigment.

Example 1

1. Bacterial isolation and purification

Sterile operation with a scalpel, open the chest and abdomen of the sick sputum, expose the main organs. Use the scissors to make the "well" shape to cut the liver of the diseased sputum, fully expose the internal tissues; use a sterile disposable cotton swab to take the inside of the liver and make a "Zi" on the urinary tract chromogenic medium. Line, the bacteria dish marked with bacteria was marked and placed in a biochemical incubator. After incubating at 37 ° C for 12 hours, the growth of the bacteria was observed. A single blue-gray colony (Klebsiella pneumoniae was grown as a blue-gray colony on the urinary tract chromogenic medium) was picked and streaked on tryptone soy agar medium to purify the colonies. If the bacterial colonies are still impure after a pure culture, continue to pick a single colony for bacterial bacteria. Purify until pure colonies grow.

2. PCR identification

Primers: According to the specific gene Khe of K. pneumoniae published by GenBank, two pairs of primers were designed by Primer software and synthesized by Shanghai Shenggong Biotechnology Service Co., Ltd. The primer sequences are:

As shown in SEQ ID NO.

As shown in SEQ ID NO. 2;

As shown in SEQ ID NO. 3;

As shown in SEQ ID NO.

The Klebsiella pneumoniae colonies purified in operation 1 were subjected to PCR amplification using the above primers, and the PCR products obtained by PCR amplification were sequenced to Shandong Academy of Agricultural Sciences, and the results of sequencing the PCR reaction products were subjected to Blast. Compared to the published gene sequences in GenBank, the homology is 99%-100%. The gene sequence of the amplified product is shown in SEQ ID NO. 5, SEQ ID NO.

Primer pair K1 amplified gene sequence:

Primer pair K2 amplified gene sequence:

3. Electron microscopic observation

The obtained K. pneumoniae WMOK strain was washed with tryptone soy broth medium for 14 hours, and the conditions were as follows: centrifugation at 3000 rpm for 10 min, the supernatant was discarded, and the slime was resuspended in physiological saline. After repeated washing 5 times, The morphology of the bacteria was observed by transmission electron microscopy. The bacteria observed under the transmission electron microscope were rod-shaped at both ends, as shown in Fig. 2.

4. Determination of bacterial LD50

1 strain recovery

The strains stored in glycerol were taken out from the refrigerator, naturally thawed at room temperature, and a loop of bacteria was aseptically picked and streaked onto tryptone soy agar medium, and cultured at 37 ° C for 16-18 h to carry out strain recovery. .

2 seed liquid preparation

A single colony of the resuscitation on tryptone soy agar medium was inoculated into a triangular flask containing 100 mL of tryptone soy broth medium, and placed in a constant temperature shaker at 180 r/min and 37 ° C for 12 hours to prepare a seed liquid.

3 expansion training

The prepared seed solution was inoculated into 200 mL of tryptone soy broth medium at a seeding ratio of 1% by volume, and placed in a constant temperature oscillator at 180 r/min and 37 ° C for 14 hours. The Maid turbidity measurement and the viable count were performed.

4 bacteria LD ₅₀ in mice

Sixty SPF KM mice weighing approximately 20 g, half male and half female, were randomly divided into 6 groups, ten in each group. Each group was injected with different dilutions of bacterial liquid (in the case of undiluted original bacterial solution, 2.1×10 ⁹ CFU/mL), 0.2 mL/only, and the control group was injected with the same amount of physiological saline for 7 days. The results are shown in Table 1:

Table 1

稀释度Dilution	10^-5 10 ^-5	10^-6 10 ^-6	10^-7 10 ^-7	10^-8 10 ^-8	10^-9 10 ^-9
死亡率 mortality rate	100％100%	100％100%	100％100%	100％100%	30％30%

The mortality rate was 100% in the 10 ^-5 , 10 ^-6 , 10 ^-7 and 10 ^-8 dilution groups , and 30 % in the 10 ^-9 dilution group . The median lethal dose was 2.1 × 10 ^0.5 CFU measured by the 寇 method . That is, intraperitoneal injection of mice at a dose of 2.1 × 10 ^0.5 CFU / 0.2 mL can cause 50% of mice to die.

5 LD _{50 of} bacteria on leech

Thirty healthy, susceptible leeches of 4 months old were randomly divided into 6 groups of 5 animals each. Each group was injected with different dilutions of bacterial solution (in the case of undiluted original bacterial solution, 3.8×10 ⁹ CFU/mL), 1.0 mL/only, and the control group was injected with the same amount of physiological saline for 14 days. The result is shown in Figure 2:

Table 2

稀释度Dilution	10^-5 10 ^-5	10^-6 10 ^-6	10^-7 10 ^-7	10^-8 10 ^-8	10^-9 10 ^-9
死亡率 mortality rate	100％100%	100％100%	100％100%	80％80%	00

Results The mortality rate was 100% in the 10 ^-5 , 10 ^-6 , 10 ^-7 dilution group , 80 % in the 10 ^-8 dilution group , and 0 in the 10 ^-9 dilution group . The median lethal dose measured by the method of 寇 is 3.8×10 ^0.7 CFU, that is, intraperitoneal injection of leeches at a dose of 3.8×10 ^0.7 CFU/mL can cause 50% of leeches to die.

Claims

A strain of Klebsiella pneumoniae, named WMOK strain, accession number: CGMCC NO.11222.
The Klebsiella pneumoniae according to claim 1, characterized in that it is K2 type, Gram stain is negative, facultative anaerobic, flagellar and spore-free, with obvious capsule, and most strains have bacteria The hair is in the shape of a rod that is obtuse at both ends.
The Klebsiella pneumoniae strain according to claim 1 or 2, wherein the drug resistance is as follows:

Resistance: ampicillin, ampicillin, cefazolin, ceftazidime, ceftriaxone, cefepime, antrexam, gentamicin, cotrimoxazole;

Zhongmin: tobramycin, ciprofloxacin;

Sensitive: piperacillin, cefotetan, ertapenem, imipenem, amikacin, levofloxacin, nitrofurantin.
The Klebsiella pneumoniae strain according to claim 3, wherein the LD 50 for KM mice is 2.1 × 10 0.5 CFU.
The Klebsiella pneumoniae strain according to claim 3, wherein the LD 50 for leech is 3.8 × 10 0.7 CFU.