Klebsiella pneumoniae H2208 strain and application thereof

Abstract

The invention relates to the field of bioengineering, and discloses a klebsiella pneumoniae H2208 strain and application thereof. The preservation number of the strain is GDMCC No: 65187; the kit comprises a tool plasmid pMar, has two T4SS gene clusters, and has efficient autonomous transfer efficiency. The strain or plasmid has the following characteristics: reverse mobilization of large non-zygotic plasmids enables host bacteria to directly obtain the large non-zygotic plasmids; and directly mobilizing the large non-zygotic plasmids to join and transfer the large non-zygotic plasmids into the recipient bacteria. According to the application, the pMar plasmid is used as the helper plasmid, large virulence plasmids in H2208 and large non-zygotic plasmids with homologous oriT sequences in other enterobacteriaceae bacteria can be efficiently transferred to recipient bacteria, and an effective tool is provided for researching the evolution and evolution laws and virulence and drug resistance formation mechanisms of various pathogenic bacteria.

Description

Technical Field

[0001] The present application relates to the field of bioengineering, and more specifically, to a Klebsiella pneumoniae H2208 strain and its application. Background Art

[0002] Large, non-conjugative plasmids are widely distributed among Enterobacteriaceae. Under natural conditions, their transfer can enable pathogens to acquire multiple drug resistance and virulence genes. Furthermore, large, non-conjugative plasmids are typically less likely to be targeted by CRISPR-Cas9 upon entry into a new host, allowing them to remain stable in the new host. Consequently, the spread of these plasmids has contributed to the emergence and subsequent clonal spread of highly virulent and resistant strains that are clinically hazardous. However, studying the transfer of these plasmids and their subsequent adaptive evolution under laboratory conditions is currently extremely difficult. Due to the large size of these non-conjugative plasmids, intact transfer into recipient cells via electroporation is difficult. Genetic material transfer through transformation and conjugation is not only a crucial step in microbial evolution but also a crucial tool for studying pathogenicity and drug resistance mechanisms in pathogens. However, the difficulty of transferring large, non-conjugative plasmids under laboratory conditions has severely hampered research into the evolutionary patterns and virulence and drug resistance mechanisms of various important pathogens.

[0003] Taking Klebsiella pneumoniae as an example, it can be divided into classical Klebsiella pneumoniae (cKP) and hypervirulent Klebsiella pneumoniae (hvKP) based on their different genetic backgrounds and evolutionary paths. cKP, which includes multidrug-resistant Klebsiella pneumoniae (MDR-KP) and carbapenem-resistant Klebsiella pneumoniae (CRKP), often causes hospital-acquired infections in immunocompromised patients. hvKP, on the other hand, is characterized by high virulence levels. Its infection characteristics include the ability to infect healthy individuals of any age and an extremely high risk of developing invasive infections.

[0004] The high pathogenicity of hvKP is primarily mediated by a conserved, large, non-conjugative virulence plasmid, pLVKP-like, which encodes multiple virulence factors, including siderophores and regulators of the hyperadhesive phenotype. The classic pLVKP-like virulence plasmid is commonly found in hvKP serotypes K1 / K2. Because this plasmid lacks a complete type IV secretion system (T4SS) required for conjugative transfer, it was considered non-conjugative. Prior to 2015, there were no reports of horizontal transfer of virulence genes between K. pneumoniae strains. However, recent studies have demonstrated that pLVKP-like virulence plasmids can be transferred with the assistance of a conjugative helper plasmid. Transfer of this virulence plasmid has led to the acquisition of pLVKP-like virulence plasmids by cKP strains, including CRKP, resulting in the emergence of highly virulent carbapenem-resistant K. pneumoniae (hv-CRKP).

[0005] Because ST11 CRKP is the predominant Klebsiella pneumoniae clone in China, the interbacterial transfer of the pLVKP-like virulence plasmid has led to the emergence of ST11 hv-CRKP clones carrying this plasmid. Currently, hv-CRKP is prevalent in clinical settings in China. The emergence of this superbug has further increased the public health burden. With the rampant spread of hv-CRKP in clinical settings, a growing number of researchers are investigating the mechanisms of hv-CRKP formation. Although studies have shown that the transfer of pLVKP-like virulence plasmids relies on the assistance of conjugative transfer plasmids, the transfer efficiency of "helper plasmids" found in existing studies is very low, ranging from 10-6 to 10-9. Evidence suggests that the fitness cost of CRKP acquiring the pLVKP-like virulence plasmid is low, and that the CRKP chromosome undergoes a series of adaptive evolutionary changes after acquiring the pLVKP-like virulence plasmid. However, no research has yet examined the adaptive evolution of CRKP strains after acquiring the pLVKP-like virulence plasmid. This is because the pLVKP-like virulence plasmid is approximately 200 kb in size, making it difficult to transfer into recipient bacteria via electroporation. Therefore, to conduct relevant research, it is necessary to use a suitable helper plasmid to efficiently introduce the pLVKP-like virulence plasmid into the recipient bacteria via conjugation. Summary of the Invention

[0006] The purpose of the present invention is to provide a Klebsiella pneumoniae H2208 strain, which carries a tool plasmid for efficiently introducing a non-conjugative pLVKP-like virulence plasmid and other large non-conjugative plasmids with a homologous transfer initiation site (oriT) sequence in Enterobacteriaceae into a recipient bacterium.

[0007] The invention objectives of this application are achieved through the following technical solutions:

[0008] In the first aspect, the present application provides a Klebsiella pneumoniae H2208 strain, which is classified and named: Klebsiella pneumoniae; deposited in Guangdong Provincial Microbiological Culture Collection Center; storage address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; storage date: September 23, 2024; storage number: GDMCC No: 65187.

[0009] Furthermore, the Klebsiella pneumoniae H2208 strain includes a tool plasmid of 270,455 bp in size, named pMar, which has two T4SS gene clusters and has a high autonomous transfer efficiency of (4.73±2.62)×10 -3 .

[0010] In a second aspect, the present application provides a tool plasmid, which is obtained from the Klebsiella pneumoniae H2208 strain described in the first aspect. The tool plasmid has two T4SS gene clusters and has a high autonomous transfer efficiency of (4.73±2.62)×10 -3 .

[0011] In a third aspect, the present application provides the use of the Klebsiella pneumoniae H2208 strain described in the first aspect or the tool plasmid described in the second aspect in assisting the rapid transfer of large non-conjugative plasmids in Enterobacteriaceae.

[0012] In a fourth aspect, the present application provides a method for rapidly transferring large non-conjugative plasmids in Enterobacteriaceae bacteria using the Klebsiella pneumoniae H2208 strain described in the first aspect or the tool plasmid described in the second aspect, wherein the Klebsiella pneumoniae H2208 strain or the tool plasmid reversely mobilizes large non-conjugative plasmids so that the host bacteria directly acquire the large non-conjugative plasmids.

[0013] Furthermore, through conjugation transfer experiments, the Klebsiella pneumoniae H2208 strain was used as the recipient strain and hvKPRJF293CHm R As the donor bacteria, RJF293CHm R Co-cultured with H2208 for conjugative transfer, H2208 was transferred from hvKPRJF293CHm R The pRJF293C virulence plasmid was directly obtained by reverse mobilization.

[0014] In the fifth aspect, the present application provides a method for rapidly transferring large non-conjugative plasmids in Enterobacteriaceae bacteria assisted by the Klebsiella pneumoniae H2208 strain described in the first aspect or the tool plasmid described in the second aspect, wherein the Klebsiella pneumoniae H2208 strain or the tool plasmid directly mobilizes the large non-conjugative plasmid to transfer the large non-conjugative plasmid into the recipient bacteria by conjugation.

[0015] Furthermore, the tool plasmid was transferred from the Klebsiella pneumoniae H2208 strain into hvKP RJF293CHm through conjugation transfer experiments. R , producing the conjugate RJF293CHm R -pMar, and then directly mobilize the tool plasmid and pRJF293C virulence plasmid from RJF293CHmR -pMar donor bacteria were transferred to ST11 CRKPDD02424 recipient bacteria by conjugation.

[0016] In the present invention, hvKP RJF293CHm R The donor bacteria were provided by Ou Hongyu from Shanghai Jiao Tong University. Contact information and strain characteristics can be found in Genome Medicine. 2021, 13(1): 119. Hygromycin resistance was artificially constructed on the pLVPK-like non-conjugative virulence plasmid pRJF293C of the hvKP strain, which can be used to screen for conjugants that have obtained pRJF293C. Since the hvKP chromosome does not contain a drug-resistant gene that can be used for screening, a non-transferable plasmid pACYC184-Apr (which carries apramycin resistance) was transferred into the strain by electroporation to simulate a chromosomal selection marker.

[0017] ST11 CRKP DD02424 was provided by the Antibiotic Research Institute of Huashan Hospital.

[0018] The public can obtain the above-mentioned strains through the above channels.

[0019] In summary, this application has the following beneficial effects:

[0020] This application utilizes the pMar plasmid as a support plasmid for large non-conjugative plasmid transfer, enabling efficient transfer of large virulence plasmids, such as pLVPK-like from Klebsiella pneumoniae, as well as large non-conjugative plasmids with oriT sequences homologous to the pMar plasmid, from other Enterobacteriaceae into recipient bacteria. This provides an effective tool for studying the evolutionary patterns and virulence and drug resistance mechanisms of various important pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :pMar helps plasmid to be mobilized in reverse ( Figure 1 -a) and direct mobilization ( Figure 1 -b) method to assist the transfer of pLVPK-like virulence plasmid (pV) into CRKP or cKP; when the pMar plasmid and the pV plasmid are conjugated and transferred into CRKP together, the pMar plasmid is unstable, while the pV plasmid can exist stably ( Figure 1 -c). DETAILED DESCRIPTION

[0022] The technical solutions and effects of the present application are further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0023] In the following examples, the strains were derived from the following sources:

[0024] In the present invention, hvKP RJF293CHm R The donor bacteria were provided by Ou Hongyu from Shanghai Jiao Tong University. Contact information and strain characteristics can be found in Genome Medicine.2021,13(1):119.

[0025] ST11 CRKP DD02424 was provided by the Antibiotic Research Institute of Huashan Hospital.

[0026] The public can obtain the above-mentioned strains through the above channels.

[0027] Example 1: Utilizing the ability of the pMar plasmid to reversely mobilize the pV virulence plasmid to enable the host bacteria to directly acquire the pV plasmid

[0028] Step 1: Screening clinical strains carrying pMar plasmids from clinical strains;

[0029] Primers were designed for IncFIB (Mar) plasmid replicon using the Primer3 online primer design platform, and the expected product size was 475 bp;

[0030] The primer sequences are as follows:

[0031] pMar-AF 5'-TAACAATGGTACCGTTCAACCT-3' (SEQ ID NO: 1);

[0032] pMar-AR 5'-GGGATCTCCCTCTATCTCAATCT-3' (SEQ ID NO: 2);

[0033] The PCR amplification system and conditions are:

[0034] Components Volume (μL) template 1 Primer-F (10 μM) 1 Primer-R (10 μM) 1 2×HieffTM PCR Master Mix 12.5 Ultrapure water 9.5 Total volume 25

[0035] Among them, the template was obtained by the bacterial boiling lysis method, that is, a bacterial single clone was selected by inoculation loop and put into 50 μL ultrapure water for boiling;

[0036] condition Temperature (℃) time Pre-denaturation 94 1min transsexual 98 10sec annealing 58 5sec extend 72 40sec Final Extension 72 1min 4 Keep

[0037] Step 2: The pV virulence plasmid was transferred into the cKP clinical strain H2208 containing pMar by reverse mobilization;

[0038] The clinical isolate H2208 carrying the pMar plasmid screened in step 1 was used as the recipient strain, and the hvKP strain RJF293CHm R Perform conjugative transfer experiments on donor bacteria.

[0039] The specific steps of the conjugative transfer experiment are as follows:

[0040] (1) Preparation: Before the experiment began, we used the microbroth dilution method to determine the minimum inhibitory concentration (MIC) of rifampicin for the donor and recipient bacteria. The donor and recipient bacteria were inoculated on screening plates containing different concentrations of rifampicin and cultured overnight at 35°C to observe their growth. R , showed no growth on monoclonal antibody plates containing 8 μg / mL levofloxacin, but was able to grow on monoclonal antibody plates containing 200 μg / mL hygromycin. On the other hand, the recipient strain H2208 was able to grow on monoclonal antibody plates containing 8 μg / mL levofloxacin, but was unable to grow on monoclonal antibody plates containing 200 μg / mL hygromycin. Therefore, for the screening of conjugates, the primary screening dual-antibody plates contained 8 μg / mL levofloxacin and 200 μg / mL hygromycin.

[0041] (2) Strain recovery: On the first day, the glycerol bacteria (H2208 and RJF293CHm) stored in a -80℃ refrigerator were R ) were taken out, inoculated onto MHA plates, and cultured overnight in a 35°C incubator. The next day, single clones were selected from the MHA plates and inoculated into LB medium, and cultured overnight at 37°C and 180 rpm in a shaking incubator.

[0042] (3) Co-culture: On the third day, take 500 μL of overnight culture solution and add it to 4500 μL of fresh LB medium. Place the recipient bacteria in a shaker at 37°C and 180 rpm for 4 hours, and the donor bacteria in a constant temperature box at 35°C for 4 hours. Then mix the donor and recipient bacteria in a ratio of 1:3, that is, take 250 μL of donor bacteria and 750 μL of recipient bacteria and mix them in a 1.5 mL autoclaved EP tube and shake evenly. Then centrifuge at 8000g for 3 minutes, remove the supernatant, retain 100 μL of liquid at the bottom of the tube, and mix thoroughly. Use sterile tweezers to pick up the filter membrane, cover it on the MHA plate, drop 100 μL of mixed bacterial solution on the filter membrane, wait for absorption, and place the plate in a constant temperature box at 35°C for overnight culture.

[0043] (4) Initial screening of resistance plates: On the fourth day, observe the growth of bacterial moss under the filter membrane. Use sterile tweezers to transfer the filter membrane to the sterile culture dish cover, then use a sterile cotton swab to scrape the bacterial moss on the filter membrane and the plate, transfer it to a 1.5mL sterile EP tube containing 1mL sterile saline, and shake to mix. Take 100μL of bacterial solution and add it to another 1.5mL sterile EP tube containing 900μL sterile saline, shake to mix, and prepare a 10-fold dilution. Prepare 100-fold and 1000-fold dilutions in the same way. Take 100μL from each of the three dilutions, drop it onto the initial screening double-resistance plate, spread it evenly with an L-shaped coating rod, and then place it in a constant temperature box at 35℃ for overnight culture. On the fifth day, select monoclonal strains from the initial screening double-resistance plate and continue to subculture on the double-resistance plate.

[0044] (5) PCR confirmation of conjugants: Select colonies and extract the bacterial genome template by boiling. Use specific primers for strain H2208 and plasmid pRJF293C to screen for conjugants. The primer sequences are as follows:

[0045] H2208-AF 5'-CTTGTAGCCTTGGTAATAAATCCACC-3' (SEQ ID NO: 3);

[0046] H2208-AR 5'-GGCAAATGCTGGTATCCAGTATCTTAT-3' (SEQ ID NO: 4);

[0047] pRJF293C-AF 5'-AAAATAACAGCTTACCTCTCAATCTTCAG-3' (SEQ ID NO: 5);

[0048] pRJF293C-AR 5'-TCAAATCCACACATCTTAACGAATTCAAC-3' (SEQ ID NO: 6).

[0049] Example 2: Utilizing the ability of the pMar plasmid to directly mobilize the pV virulence plasmid to transfer the pV plasmid into the CRKP recipient bacteria

[0050] Step 1: Construction of hvKP carrying pMar plasmid;

[0051] The H2208 strain carrying the pMar plasmid obtained in Example 1 was used as a donor bacterium, and the hvKP strain RJF293CHm was used as a R The pMar conjugative plasmid was transferred to the hvKP recipient bacteria RJF293CHm through the above conjugative transfer experiment. RThe conjugate RJF293CHm was screened using a double-antibody broth agar plate containing 50 μg / ml apramycin and 50 μg / ml rifampicin. R -pMar( Figure 1 b), the conjugant was obtained by strain RJF293CHm R Specific primers and pMar plasmid specific primers confirmed. RJF293CHm R The specific primer sequences are as follows:

[0052] RJF293CHm R -AF 5'-ACATTATGGTGATTGGTTCTTTTCAAAATG-3' (SEQ ID NO:7);

[0053] RJF293CHm R -AR 5'-TTGAAAATACAATGGTTTCAGAATCATTGG-3' (SEQ ID NO: 8).

[0054] Step 2: pMar plasmid and pV virulence plasmid are co-transferred into CRKP recipient bacteria;

[0055] The conjugate RJF293CHm obtained in step 1 R -pMar was used as the donor strain, and the CRKP clinical isolate DD02424 was used as the recipient strain. The pMar plasmid and pV plasmid were conjugated to the CRKP recipient strain DD02424 by conjugation transfer experiment. The conjugates DD02424-pMar-pV ( Figure 1 b) Confirm the zygote using primers specific for strain DD02424 and pRJF293C. The sequences of the primers specific for strain DD02424 are as follows:

[0056] DD02424-AF 5'-ATGCCAGCGTGAATTATTCTTG-3' (SEQ ID NO:9);

[0057] DD02424-AR 5'-TCCAGGCCTTAATTTTTCAACACC-3' (SEQ ID NO: 10).

[0058] Step 3: Eliminate the pMar plasmid in the conjugate DD02424-pMar-pV by serial passage;

[0059] The conjugate DD02424-pMar-pV was serially passaged. First, a single DD02424-pMar-pV clone was selected on a double-antibiotic broth agar plate containing 2 μg / ml meropenem and 200 μg / ml hygromycin and cultured overnight in 4 ml of broth liquid medium. The next day, 1 μl of the overnight broth was transferred to 4 ml of fresh broth for passage. After repeating the above steps for 5 days, the pMar plasmid began to disappear, while the pV plasmid remained stable ( Figure 1 c) This method constructs an hv-CRKP strain with a plasmid profile consistent with clinical hv-CRKP, which can be used to study the adaptive evolution of pV plasmids in CRKP strains.

[0060] The present invention has the beneficial effect of using the pMar plasmid as a support plasmid for large non-conjugative plasmid transfer. It can efficiently transfer the large pLVPK-like virulence plasmid from Klebsiella pneumoniae, as well as large non-conjugative plasmids from other Enterobacteriaceae that have oriT sequences homologous to the pMar plasmid, into recipient bacteria. This provides an effective tool for studying the evolutionary patterns and virulence and drug resistance mechanisms of various important pathogens.

[0061] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A Klebsiella pneumoniae H2208 strain, characterized in that The strain is classified and named: Klebsiella pneumoniae; deposited in Guangdong Provincial Microbiological Culture Collection Center; preservation date: September 23, 2024; preservation number: GDMCC No: 65187.

2. The Klebsiella pneumoniae H2208 strain according to claim 1, characterized in that The Klebsiella pneumoniae H2208 strain includes a tool plasmid of 270,455 bp in size, which has two T4SS gene clusters and has a high autonomous transfer efficiency of (4.73±2.62)×10 -3 .

3. A tool plasmid, characterized in that The tool plasmid is obtained from the Klebsiella pneumoniae H2208 strain according to claim 1, and the tool plasmid has two T4SS gene clusters and has a high autonomous transfer efficiency of (4.73±2.62)×10 -3 .

4. Use of the Klebsiella pneumoniae H2208 strain according to claim 1 or the tool plasmid according to claim 3 in assisting the rapid transfer of large non-conjugative plasmids in Enterobacteriaceae.

5. The method for rapidly transferring large non-conjugative plasmids in Enterobacteriaceae using the Klebsiella pneumoniae H2208 strain of claim 1 or the tool plasmid of claim 3, characterized in that: The Klebsiella pneumoniae H2208 strain or the tool plasmid reversely mobilizes the large non-conjugative plasmid so that the host bacteria directly obtains the large non-conjugative plasmid.

6. The method according to claim 5, characterized in that Through conjugation transfer experiments, the Klebsiella pneumoniae H2208 strain was used as the recipient strain and hvKP RJF293CHm R As the donor bacteria, RJF293CHm R Co-cultured with H2208 for conjugative transfer, H2208 was transferred from hvKP RJF293CHm R The pRJF293C virulence plasmid was directly obtained by reverse mobilization.

7. The method for rapidly transferring large non-conjugative plasmids in Enterobacteriaceae using the Klebsiella pneumoniae H2208 strain of claim 1 or the tool plasmid of claim 3, characterized in that: The Klebsiella pneumoniae H2208 strain or the tool plasmid directly mobilizes the large non-conjugative plasmid to transfer the large non-conjugative plasmid into the recipient bacteria.

8. The method according to claim 7, characterized in that By conjugation transfer experiment, the tool plasmid was transferred from the Klebsiella pneumoniae H2208 strain into hvKP RJF293CHm R , producing the conjugate RJF293CHm R -pMar, and then directly mobilize the tool plasmid and pRJF293C virulence plasmid from RJF293CHm R -pMar donor bacteria were transferred to ST11 CRKP DD02424 recipient bacteria by conjugation.

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