Streptococcus suis recipient strain for bacterial conjugation, construction method and application thereof

Abstract

The present invention relates to the field of bioengineering technology, and discloses a Streptococcus suis receptor bacterium for bacterial conjugation, a construction method and an application thereof. Type 1 Streptococcus suis NP4CIP is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on February 27, 2025, at the deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO.33594 and a classification name of Streptococcus suis. Streptococcus suis The present invention adopts the above-mentioned Streptococcus suis receptor bacteria for bacterial conjugation and its construction method and application, and the constructed NP4CIP strain has good stability, can better maintain the integration and expression of drug-resistant genes, overcomes the growth defect of P1 / 7RF, and at the same time, the NP4CIP conjugate can be used for re-conjugation experiments, laying a foundation for the study of the horizontal transfer mechanism of drug-resistant genes, and providing a basis and inspiration for controlling the spread of bacterial resistance.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a Streptococcus suis receptor bacterium for bacterial conjugation, its construction method and application. Background Art

[0002] Streptococcus suis ( Streptococcus suis ) is an important zoonotic pathogen, which not only causes serious infections such as swine meningitis, septicemia and endocarditis, causing significant economic losses to the pig industry, but also can infect humans by contacting diseased pigs or ingesting contaminated pork products, resulting in a toxic shock syndrome with a dangerous course and a very high fatality rate, threatening human health. In recent years, with the widespread use and irrational use of antibacterial drugs, the sensitivity of Streptococcus suis to commonly used antibacterial drugs has decreased.

[0003] Studies have shown that Streptococcus suis mainly generates drug resistance through gene mutations and exogenous drug resistance genes obtained by horizontal gene transfer by mobile genetic elements. In particular, the latter plays an important role in the generation and transmission of drug resistance. Drug resistance genes are spread among different hosts through the HGT (horizontal gene transfer) pathway, which is confirmed by the high homology in drug resistance gene clusters from different sources. The horizontal spread of bacterial drug resistance genes is a key factor in the development and spread of drug resistance.

[0004] Therefore, studying and understanding this process is crucial for formulating effective prevention and control measures. There are many serotypes of Streptococcus suis. Serotypes 1, 2, 7, and 9 are pathogenic bacteria of pigs. In previous studies, Streptococcus suis type 2 P1 / 7RF was mainly selected as the receptor bacterium for bacterial conjugation experiments, and it has the following problems:

[0005] ① The serotype is single. The biological characteristics such as the growth rate of P1 / 7RF and its conjugants are significantly limited compared with the wild strain P1 / 7. The growth-limited P1 / 7RF conjugants cannot accurately reflect the drug resistance characteristics of the wild strain in the natural environment. Due to the differences in its growth rate and metabolic activities from the wild strain, it may lead to inconsistent expression levels or functions of drug resistance genes with the wild strain. This difference will affect the study of the direct relationship between drug resistance genes and drug resistance phenotypes, and further limit the in-depth understanding and research of drug resistance mechanisms.

[0006] ② The growth-limited P1 / 7RF conjugants may lack some key metabolic pathways or enzymes, which are crucial for the expression and transfer of drug resistance genes. Due to its growth limitation and insufficient metabolic activities, it cannot provide enough energy and resources for the expression and transfer of drug resistance genes. This metabolic defect causes the P1 / 7RF conjugants unable to be used as donor bacteria for the re-transfer of drug resistance genes, thus limiting the integrity of the study of drug resistance mechanisms. Summary of the Invention

[0007] The purpose of the present invention is to provide a Streptococcus suis receptor bacterium for bacterial conjugation and a construction method and application thereof. The constructed NP4CIP strain has good stability, can better maintain the integration and expression of drug-resistant genes, overcomes the growth defect of P1 / 7RF, and at the same time, the NP4CIP conjugate can be used for re-conjugation experiments, lays a foundation for the study of the horizontal transfer mechanism of drug-resistant genes, and provides a basis and inspiration for controlling the spread of bacterial resistance.

[0008] To achieve the above purpose, the present invention provides a strain of Streptococcus suis type 1 NP4CIP, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee on February 27, 2025, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO.33594, and is classified and named Streptococcus suis Streptococcussuis , and its 16S rDNA sequence is shown in SEQ ID NO.1.

[0009] The present invention also provides the use of the type 1 Streptococcus suis NP4CIP in bacterial conjugation.

[0010] The present invention also provides a method for constructing a Streptococcus suis receptor bacterium for bacterial conjugation, wherein the Streptococcus suis receptor bacterium is type 1 Streptococcus suis NP4CIP, and the construction method is as follows:

[0011] The strain NP4 was subcultured in THB medium containing 1 / 2×MIC drugs, and the plates were transferred every 3 days to determine its MIC. The drug concentration on the plate was doubled gradually until the MIC of the cultured strain rose to the drug-resistant range, and then the strain was transferred to a drug-free plate containing serum and cultured, and the plates were transferred once a day for continuous subculture for 30 days. The MIC of the drug against the strain was determined every 5 days to determine the stability of the resistant bacteria. The ciprofloxacin-resistant strain NP4CIP was successfully obtained and stored at low temperature for future use.

[0012] Furthermore, the ciprofloxacin resistance of strain NP4CIP was greater than 256 μg·mL -1 .

[0013] The present invention also provides a ciprofloxacin-resistant strain NP4CIP, which is constructed according to the above construction method.

[0014] The present invention also provides the use of the ciprofloxacin-resistant strain NP4CIP in bacterial conjugation.

[0015] Furthermore, bacterial conjugation was performed using strain NP4CIP as a recipient bacterium and AKJ47 as a donor bacterium.

[0016] The present invention also provides the use of the conjugant of the ciprofloxacin-resistant strain NP4CIP in bacterial conjugation.

[0017] Furthermore, using the conjugant of strains NP4CIP and AKJ47 as the donor bacterium and P1 / 7RF as the recipient bacterium, bacterial conjugation was performed.

[0018] The advantages and positive effects of the Streptococcus suis recipient bacterium for bacterial conjugation, its construction method and application according to the present invention are as follows:

[0019] 1. The present invention provides a strain of Streptococcus suis type 1, NP4, and through domestication, a Streptococcus suis recipient bacterium NP4CIP for bacterial conjugation was obtained. Currently, the recipient bacterium P1 / 7RF commonly used in the study of drug resistance gene transfer is Streptococcus suis type 2. There are certain limitations in the study of the transfer of drug resistance genes and virulence genes. The Streptococcus suis type 1 recipient bacterium NP4CIP fills this gap and provides a research basis for the transfer of drug resistance genes and virulence genes between different serotypes.

[0020] 2. In the present invention, NP4CIP does not show an obvious slowdown in the growth rate compared to NP4 and still grows well. Due to drug resistance induction, P1 / 7RF has many mutation sites that limit its own growth, and the growth rate significantly decreases. The good growth of NP4CIP indicates normal metabolic activities, which can provide sufficient energy and resources for the expression and transfer of drug resistance genes, overcoming the problem of limited expression of drug resistance genes caused by insufficient metabolism in the P1 / 7RF conjugant; at the same time, the genomic structure of NP4CIP is relatively stable, which can effectively maintain the integration and expression of drug resistance genes and provides a reliable basis for studying the transmission mechanism of drug resistance genes between different hosts.

[0021] 3. In the present invention, using the conjugants ICE Ssu AKJ47_ Ssu1797 and ICE Ssu AKJ47_ rplL as the donor bacteria and P1 / 7RF as the recipient bacterium for a re-conjugation experiment, the drug resistance genes optrA and erm (B) can still be transferred, and the conjugation frequencies are (4.94 ± 1.14) × 10 -4 and (2.22 ± 1.21) × 10 -4Although P1 / 7RF can complete the initial conjugation, its conjugants cannot be used as donor bacteria for re-conjugation experiments. This limitation stems from its restricted growth rate and metabolic activities, resulting in insufficient energy and resources for the transfer of drug-resistant genes. In contrast, NP4CIP not only has a growth rate and metabolic activities highly consistent with the wild strain NP4, but its conjugants also possess the ability to serve as donor bacteria for the re-transfer of drug-resistant genes. The genomic stability of NP4CIP enables it to better maintain the integration and expression of drug-resistant genes, thereby overcoming the defects of P1 / 7RF. Therefore, NP4CIP has important application value in the study of horizontal transfer of drug-resistant genes, can more comprehensively simulate the transmission mechanism of drug-resistant genes in bacterial populations, and provides a more reliable model for in-depth study of the spread of drug-resistant genes. Studying bacterial re-conjugation can reveal the transmission mechanism of drug-resistant genes, evaluate their transmission potential, understand bacterial adaptation and evolution, and provide a scientific basis for developing new antibacterial strategies and optimizing drug-resistant monitoring and prevention and control measures, thus effectively addressing the problem of antibiotic resistance.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Growth curves of two recipient bacteria before and after induction and conjugants in the embodiments of the present invention, where A is P1 / 7RF and B is NP4CIP;

[0024] Figure 2 Verification results of conjugants in the embodiments of the present invention.

[0025] Biological material preservation information

[0026] A strain of Streptococcus suis type 1 NP4CIP, preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on February 27, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number CGMCC NO. 33594 and the taxonomic name Streptococcus suis Streptococcussuis . DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and examples.

[0028] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs.

[0029] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention. For the experimental methods without specific conditions indicated in the following embodiments, they are generally determined in accordance with national standards. The experimental instruments, equipment and reagents without sources indicated in the following embodiments are all commercially available raw materials.

[0030] Unless otherwise defined or stated, all the professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the methods of the present invention.

[0031] A Streptococcus suis type 1 strain NP4CIP was deposited in the China General Microbiological Culture Collection Center on February 27, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 33594. Its taxonomic name is Streptococcus suis Streptococcussuis , and its 16S rDNA sequence is as shown in SEQ ID NO. 1 as follows:

[0032]

[0033] Example

[0034] 1) Induction of ciprofloxacin resistance in NP4:

[0035] One strain of Streptococcus suis type 1 NP4 isolated from diseased pigs in Shanghai and sensitive to ciprofloxacin (0.125 μg·mL -1 ) was obtained through screening. The method of gradually increasing the drug concentration in vitro was used to induce drug resistance in the sensitive strain. NP4 was subcultured in THB medium containing 1 / 2×MIC drug, transferred every 3 days, and its MIC was determined. The drug concentration on the plate was gradually increased until the MIC of the cultured strain rose to the resistant range, and then it was transferred to a drug-free plate containing serum for culture, transferred once a day, continuously subcultured for 30 days, and the MIC of the drug against the strain was determined every 5 days to judge the stability of the drug-resistant bacteria. The ciprofloxacin-resistant strain NP4CIP (>256 μg·mL -1 ) was successfully obtained and stored at low temperature for later use.

[0036] 2) Growth curves of recipient bacteria and transconjugants:

[0037] Single colonies of NP4, NP4CIP, ICE Ssu AKJ47_ Ssu1797, ICE Ssu AKJ47_ rplL , P1 / 7, P1 / 7RF, and P1 / 7RF transconjugants were inoculated into THB liquid medium and cultured at 37°C until the logarithmic growth phase. The initial concentration of the bacterial solution was adjusted to make its OD 600 = 0.1, and 1% of the bacterial solution was transferred to fresh THB medium, cultured at 37°C with shaking at 180 rpm, vortexed and mixed evenly, added to a flat-bottom 96-well culture plate, and cultured in an enzyme-labeling instrument at 37°C. The OD 600 value was measured every 1 h, shaken for 10 s before measurement, and continuously measured for 24 h. With time as the abscissa and the OD 600 value as the ordinate, the growth curves of each strain were plotted, and the results are as shown in Figure 1 .

[0038] Figure 1Among them, P1 / 7RF and its conjugants showed significant differences in growth characteristics compared with the wild strain P1 / 7, mainly manifested as limited growth rate, significantly later time to reach the plateau phase of the growth curve than the wild strain P1 / 7, and the bacterial density at the plateau phase was only about half of that of the wild strain. Compared with the wild strain NP4, NP4CIP and its conjugants did not show obvious changes in growth rate, had approximately the same time to reach the plateau phase, and the bacterial density at the plateau phase was also comparable. This result indicates that NP4CIP can better simulate the growth state of the wild strain, overcome the problem of growth limitation of P1 / 7RF and its conjugants, and provide a more ideal model for the study of horizontal transfer of drug resistance genes.

[0039] 3) NP4CIP was used for bacterial conjugation experiments:

[0040] Streptococcus suis NP4CIP (resistant to ciprofloxacin, sensitive to florfenicol and erythromycin) was used as the recipient bacterium, and AKJ47 (sensitive to ciprofloxacin, resistant to florfenicol and erythromycin) was used as the donor bacterium.

[0041] The conjugation method was as follows: Single colonies of the donor bacterium and the recipient bacterium were separately picked and cultured at 37 °C until the logarithmic phase. The OD of the donor bacterium and the recipient bacterium was adjusted 600 to be equal to 0.2, and they were mixed at a ratio of 1:10. The mixture was centrifuged at 4,000 rpm for 5 min at room temperature, and then the supernatant was discarded. The bacterial suspension was resuspended with 100 μL of THB without resistance, and the smooth side of the nitrocellulose membrane was attached to the THA plate without resistance and preheated at 37 °C for 30 min. The mixed bacterial suspension was evenly spread on the rough surface of the nitrocellulose membrane to allow the bacteria to fully adhere to the membrane, and then placed in an incubator at 37 °C and incubated statically for 4 h. The donor bacterium was diluted to an appropriate multiple with sterile PBS and plated for counting. The bacteria were washed off the filter membrane with 4 mL of THB, centrifuged and the supernatant was discarded. The bacterial suspension was resuspended with 100 μL of THB without resistance and transferred to a plate containing the corresponding resistance (50 μg·mL -1 ciprofloxacin and 8 μg·mL -1 florfenicol / 50 μg·mL -1 ciprofloxacin and 50 μg·mL -1 erythromycin) and cultured until single colonies grew. All the colonies grown on the plate were counted, and the colonies on the plate were picked to identify whether they were conjugants.

[0042] 4) Verification of conjugants and statistical analysis of conjugation frequency:

[0043] Conventional PCR technology was used to detect serotype genes (CPS1I and CPS3L) and drug resistance genes ( erm (B) and optrA ) to identify the donor bacterium, the recipient bacterium and the conjugants. Information such as the sequences of the primers, annealing temperatures and product lengths is shown in Table 1.

[0044] Table 1 Primer Sequences and Annealing Temperatures

[0045]

[0046] The PCR results were as Figure 2 shown, indicating that NP4CIP could serve as the recipient bacterium in the bacterial conjugation experiment, and the drug-resistant gene was transferred from Streptococcus suis serotype 3 AKJ47 to this recipient bacterium (serotype 1) by conjugation.

[0047] Determination of Antimicrobial Susceptibility (MIC):

[0048] According to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2024), the microbroth dilution method was used to detect the drug susceptibilities of erythromycin and ciprofloxacin for the donor bacterium, recipient bacterium, and transconjugants in the conjugation experiment using MH broth containing 5% newborn bovine serum. The transconjugants successfully acquired the drug-resistant gene phenotype. The detailed information is shown in Table 2.

[0049] Table 2 Relevant Characteristics of Strains in the Conjugation Experiment

[0050]

[0051]

[0052] 5) Re-conjugation frequency:

[0053] Using transconjugant ICE Ssu AKJ47_ Ssu1797 and ICE Ssu AKJ47_ rplL as the donor bacteria and P1 / 7RF as the recipient bacterium for the re-conjugation experiment, the conjugation frequencies were (4.94 ± 1.14)×10 -4 and (2.22 ± 1.21)×10 -4 . The verification method for the transconjugants was the same as above.

[0054] In summary, in the present invention, NP4CIP did not show an obvious slowdown in the growth rate compared to NP4 and still grew well. Due to drug resistance induction, P1 / 7RF had many mutation sites that restricted its own growth, and the growth rate decreased significantly ( Figure 1 shown). The good growth of NP4CIP indicates normal metabolic activities, which can provide sufficient energy and resources for the expression and transfer of drug-resistant genes, overcoming the problem of limited drug-resistant gene expression caused by insufficient metabolism in P1 / 7RF transconjugants; at the same time, the genomic structure of NP4CIP is relatively stable, which can effectively maintain the integration and expression of drug-resistant genes, providing a reliable basis for studying the transmission mechanism of drug-resistant genes between different hosts.

[0055] To analyze the potential for the continuous spread of mobile genetic elements (MGEs) in bacterial populations, the conjugative integrative and conjugative element (ICE) Ssu AKJ47_ Ssu1797 and ICE Ssu AKJ47_ rplL were used as donor bacteria, and P1 / 7RF was used as the recipient bacterium for a re-conjugation experiment. The resistance genes optrA and erm (B) could still be transferred, with conjugation frequencies of (4.94 ± 1.14) × 10 -4 and (2.22 ± 1.21) × 10 -4 respectively. Although P1 / 7RF could complete the initial conjugation, its conjugants could not be used as donor bacteria for re-conjugation experiments. This limitation was due to restricted growth rate and metabolic activities, resulting in insufficient energy and resources for the transfer of resistance genes. In contrast, NP4CIP not only had a growth rate and metabolic activities highly consistent with the wild strain NP4, but its conjugants also had the ability to serve as donor bacteria for the re-transfer of resistance genes. The genomic stability of NP4CIP enabled it to better maintain the integration and expression of resistance genes, thus overcoming the defects of P1 / 7RF.

[0056] In summary, NP4CIP has important application value in the study of horizontal transfer of resistance genes. It can more comprehensively simulate the transmission mechanism of resistance genes in bacterial populations, providing a more reliable model for in-depth study of the spread of resistance genes. Studying bacterial re-conjugation can reveal the transmission mechanism of resistance genes, evaluate their transmission potential, understand bacterial adaptation and evolution, and provide a scientific basis for developing new antibacterial strategies and optimizing resistance monitoring and prevention and control measures, thus effectively addressing the problem of antibiotic resistance.

[0057] Therefore, the present invention uses the above-mentioned Streptococcus suis recipient bacterium for bacterial conjugation, its construction method and application. The constructed NP4CIP strain has good stability, can better maintain the integration and expression of resistance genes, overcomes the growth defects of P1 / 7RF, and at the same time, NP4CIP conjugants can be used for re-conjugation experiments, laying a foundation for the study of the horizontal transfer mechanism of resistance genes and providing a basis and inspiration for controlling the spread of bacterial resistance.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A strain of Streptococcus suis type 1 NP4CIP, characterized in that: Streptococcus suis type 1 NP4CIP was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on February 27, 2025. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO.33594 and the classification name is Streptococcus suis Streptococcussuis , whose 16S rDNA sequence is shown in SEQ ID NO.1; the ciprofloxacin resistance of Streptococcus suis type 1 NP4CIP is greater than 256 μg·mL -1 .

2. Use of the type 1 Streptococcus suis NP4CIP in bacterial conjugation as claimed in claim 1, characterized in that: The application is to use type 1 Streptococcus suis NP4CIP as a recipient bacterium and type 3 Streptococcus suis AKJ47 as a donor bacterium to perform bacterial conjugation. The use of the conjugator of Streptococcus suis NP4CIP type 3.1 in bacterial conjugation is characterized by: The conjugate of Streptococcus suis type 1 NP4CIP is a conjugate obtained by using Streptococcus suis type 1 NP4CIP as a recipient bacterium and Streptococcus suis type 3 AKJ47 as a donor bacterium for bacterial conjugation. The application is to use the conjugate as a donor bacterium and Streptococcus suis type 2 P1 / 7RF as a recipient bacterium for bacterial conjugation. Streptococcus suis type 1 NP4CIP is deposited in the General Microbiology Center of the China National Committee for the Preservation of Microorganisms, the preservation time is February 27, 2025, the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC NO.33594, and the classification name is Streptococcus suis Streptococcussuis , whose 16S rDNA sequence is shown in SEQ ID NO.1; the ciprofloxacin resistance of Streptococcus suis type 1 NP4CIP is greater than 256 μg·mL -1 .

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