Streptococcus suis recipient bacteria for bacterial conjugation as well as construction method and application of streptococcus suis recipient bacteria

Abstract

The invention relates to the technical field of bioengineering, and discloses streptococcus suis recipient bacteria for bacterial conjugation and a construction method and application thereof. The type 1 streptococcus suis NP4CIP is preserved in China General Microbiological Culture Collection Center, the preservation time is February 27, 2025, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC NO.33594, and the classification name is Streptococcus suis. According to the streptococcus suis recipient bacterium for bacterium conjugation and the construction method and application thereof, the constructed NP4CIP strain is good in stability, integration and expression of drug-resistant genes can be better maintained, the growth defect of P1 / 7RF is overcome, meanwhile, an NP4CIP zygote can be subjected to a re-conjugation experiment, a foundation is laid for research of a drug-resistant gene horizontal transfer mechanism, and the application of the streptococcus suis recipient bacterium for bacterium conjugation and the construction method and application of the streptococcus suis recipient bacterium for bacterium conjugation is developed. And a basis and inspiration are provided for controlling spreading of bacterial drug resistance.

Description

Technical Field

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

[0002] Streptococcus suis ( Streptococcus suis ) is an important zoonosis pathogen that not only causes serious infections such as meningitis, sepsis and endocarditis in pigs, causing significant economic losses to the pig industry, but can also infect humans through contact with sick pigs or ingestion of contaminated pork products, leading to toxic shock syndrome with a dangerous course and high mortality rate, posing a threat to human health. In recent years, with the widespread use of antibiotics and irrational use of drugs, the sensitivity of Streptococcus suis to commonly used antibiotics has decreased.

[0003] Studies have shown that Streptococcus suis develops resistance mainly through gene mutation and exogenous resistance genes acquired by horizontal gene transfer from mobile genetic elements, especially the latter plays an important role in the generation and spread of resistance. Resistance genes spread in different hosts through the HGT (horizontal gene transfer) pathway, which is confirmed by the high homology in resistance gene clusters from different sources. The horizontal transmission of bacterial resistance genes is a key factor in the development and spread of resistance.

[0004] Therefore, studying and understanding this process is crucial for developing effective prevention and control measures. There are many serotypes of Streptococcus suis. Types 1, 2, 7, and 9 are pathogenic bacteria of pigs. In previous studies, type 2 Streptococcus suis P1 / 7RF was mainly selected as the receptor bacteria for bacterial conjugation experiments, which has the following problems: ① The serotype is single, and the biological characteristics of P1 / 7RF and its conjugates, such as growth rate, are significantly limited compared to the wild-type P1 / 7. The growth-restricted P1 / 7RF conjugate cannot accurately reflect the drug resistance characteristics of the wild-type in the natural environment. Due to the differences in its growth rate and metabolic activity from the wild-type, the expression level or function of the drug-resistant gene may be inconsistent with that of the wild-type. This difference will affect the study of the direct association between drug-resistant genes and drug-resistant phenotypes, thereby limiting the in-depth understanding and research of the drug-resistant mechanism.

[0005] ② Growth-restricted P1 / 7RF conjugates may lack certain key metabolic pathways or enzymes that are essential for the expression and transfer of drug-resistant genes. Due to their restricted growth and insufficient metabolic activity, they cannot provide sufficient energy and resources for the expression and transfer of drug-resistant genes. This metabolic defect makes it impossible for P1 / 7RF conjugates to serve as donor bacteria for the retransfer of drug-resistant genes, thus limiting the integrity of research on drug-resistant mechanisms. Summary of the invention

[0006] 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.

[0007] 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 Streptococcus suis , and its 16Sr DNA sequence is shown in SEQ ID NO.1.

[0008] The present invention also provides the use of the type 1 Streptococcus suis NP4 in bacterial conjugation.

[0009] 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: 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.

[0010] Furthermore, the ciprofloxacin resistance of strain NP4CIP was greater than 256 μg·mL -1 .

[0011] The present invention also provides a ciprofloxacin-resistant strain NP4CIP, which is constructed according to the above construction method.

[0012] The present invention also provides the use of the ciprofloxacin-resistant strain NP4CIP in bacterial conjugation.

[0013] Furthermore, bacterial conjugation was performed using strain NP4CIP as a recipient bacterium and AKJ47 as a donor bacterium.

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

[0015] Furthermore, bacterial conjugation was performed using the conjugate of strains NP4CIP and AKJ47 as donor bacteria and P1 / 7RF as recipient bacteria.

[0016] The advantages and positive effects of the Streptococcus suis receptor bacteria for bacterial conjugation and the construction method and application thereof of the present invention are: 1. The present invention provides a strain of type 1 Streptococcus suis NP4, and obtains a strain of type 1 Streptococcus suis receptor NP4CIP for bacterial conjugation through training. The receptor bacteria P1 / 7RF commonly used in the study of drug resistance gene transfer is type 2 Streptococcus suis. There are certain limitations in the study of drug resistance gene and virulence gene transfer. Type 1 Streptococcus suis receptor NP4CIP fills this gap and provides a research basis for the transfer of drug resistance genes and virulence genes between different serotypes.

[0017] 2. In the present invention, NP4CIP has no obvious growth rate slowdown relative to NP4, and the growth is still good. Due to drug resistance induction, P1 / 7RF has produced many mutation sites that limit its own growth, and the growth rate is significantly reduced. The good growth of NP4CIP indicates that the metabolic activity is normal, and it can provide sufficient energy and resources for the expression and transfer of drug-resistant genes, overcoming the problem of limited expression of drug-resistant genes caused by insufficient metabolism of P1 / 7RF conjugates; at the same time, the genome structure of NP4CIP is relatively stable, which can effectively maintain the integration and expression of drug-resistant genes, and provides a reliable basis for studying the transmission mechanism of drug-resistant genes between different hosts.

[0018] 3. In the present invention, the conjugate ICE Ssu AKJ47_ Ssu1797 and ICE Ssu AKJ47_ rplL As donor bacteria, P1 / 7RF was used as recipient bacteria for reconjugation experiments, and drug resistance genes optrA and erm (B) can still transfer, and the bonding frequencies are (4.94±1.14)×10 -4 and (2.22±1.21)×10 -4. Although P1 / 7RF can complete the initial conjugation, its conjugants cannot be used as donor bacteria for reconjugation experiments. This limitation is due to its limited growth rate and metabolic activity, which makes it impossible to provide sufficient energy and resources for the transfer of resistance genes. In contrast, NP4CIP is not only highly consistent with the wild strain NP4 in growth rate and metabolic activity, but its conjugants also have the ability to re-transfer resistance genes as donor bacteria. The genomic stability of NP4CIP enables it to better maintain the integration and expression of resistance genes, thus overcoming the defects of P1 / 7RF. Therefore, NP4CIP has important application value in the study of horizontal transfer of resistance genes, and can more comprehensively simulate the transmission mechanism of resistance genes in bacterial populations, providing a more reliable model for in-depth study of the transmission of resistance genes. Studying bacterial reconjugation can reveal the transmission mechanism of resistance genes, evaluate their transmission potential, understand bacterial adaptability and evolution, and provide a scientific basis for the development of new antibacterial strategies, optimize resistance monitoring and prevention and control measures, so as to effectively deal with the problem of antibiotic resistance.

[0019] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The growth curves of the two receptor bacteria before and after induction and the conjugates in the embodiment of the present invention, where A is P1 / 7RF and B is NP4CIP; Figure 2 This is the result of zygote verification in the embodiment of the present invention.

[0021] Biomaterial Deposit Information A strain of 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 Streptococcus I am . DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0023] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0024] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following examples are usually measured in accordance with national standards. The experimental instruments, equipment and reagents without source indication in the following examples are all commercially available raw materials.

[0025] Unless otherwise defined or indicated, all 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 those described herein can be applied to the present invention.

[0026] A strain of 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 Streptococcus suis , and its 16S rDNA sequence is shown in SEQ ID NO.1.

[0027] Example 1) NP4 ciprofloxacin resistance induction: A strain isolated from a pig with the disease in Shanghai was obtained through screening and was sensitive to ciprofloxacin (0.125 μg mL -1 ) type 1 Streptococcus suis NP4 was used to induce drug resistance in sensitive strains by gradually increasing the drug concentration in vitro. NP4 was subcultured in THB medium containing 1 / 2×MIC drugs, and the culture was transferred every 3 days to measure its MIC. The drug concentration of the plate was gradually increased until the MIC of the cultured strain rose to the resistance range, and then it was transferred to a drug-free plate containing serum for culture, and the culture was transferred once a day for 30 consecutive days. The MIC of the drug to the strain was measured every 5 days to determine the stability of the resistant bacteria. The ciprofloxacin-resistant strain NP4CIP (>256 μg·mL -1 ), store at low temperature for later use.

[0028] 2) Growth curve of recipient bacteria and conjugates: Pick NP4, NP4CIP, and ICE respectively Ssu AKJ47_ Ssu1797、 ICE Ssu AKJ47_ rplL Single colonies of P1 / 7, P1 / 7RF and P1 / 7RF conjugate 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 an OD of 600 =0.1, and transfer 1% of the bacterial solution to fresh THB medium, culture at 37°C, 180rpm, vortex to mix, add the sample to a flat-bottom 96-well culture plate, and culture at 37°C in an ELISA reader. Measure OD every 1h 600 The value was measured by shaking for 10 s before measurement and measuring continuously for 24 h. 600 The value is the vertical axis, and the growth curve of each strain is drawn. The results are as follows Figure 1 shown.

[0029] Figure 1 In the study, P1 / 7RF and its conjugates showed significant differences in growth characteristics from the wild strain P1 / 7, mainly manifested in limited growth rate, the time when the growth curve reached the plateau phase was significantly later than that of the wild strain P1 / 7, and the bacterial density in the plateau phase was only about half of that of the wild strain. Compared with the wild strain NP4, the growth rate of NP4CIP and its conjugates did not change significantly, the time to reach the plateau phase was roughly the same, and the bacterial density in the plateau phase was also comparable. This result shows that NP4CIP can better simulate the growth state of the wild strain, overcome the problem of limited growth of P1 / 7RF and its conjugates, and provide a more ideal model for the study of horizontal transfer of drug-resistant genes.

[0030] 3) NP4CIP is used in bacterial conjugation experiments: S. suis NP4CIP (ciprofloxacin-resistant, florfenicol- and erythromycin-susceptible) was used as the recipient strain, and AKJ47 (ciprofloxacin-susceptible, florfenicol- and erythromycin-resistant) was used as the donor strain.

[0031] The conjugation method is as follows: Pick single colonies of donor bacteria and recipient bacteria and culture them at 37°C until the logarithmic phase. Adjust the OD of the donor bacteria and the recipient bacteria respectively. 600 Make it equal to 0.2 and mix at a ratio of 1:10. Centrifuge the mixture at 4,000 rpm for 5 min at room temperature, discard the supernatant, resuspend the bacterial solution with 100 μL of non-resistant THB, and stick the smooth side of the nitrocellulose membrane on a non-resistant THA plate preheated at 37°C for 30 min. Evenly apply the mixed bacterial solution to the rough surface of the nitrocellulose membrane to allow the bacteria to fully adhere to the membrane, and place it in a 37°C incubator for 4 hours. Dilute the donor bacteria with sterile PBS to an appropriate multiple and count them on the plate. Wash the bacteria from the filter membrane with 4 mL of THB, centrifuge and discard the supernatant, resuspend the bacterial solution with 100 μL of non-resistant THB, and transfer 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 Culture on a plate containing erythromycin until a single colony grows. Count all colonies grown on the plate and pick a colony on the plate to identify whether it is a zygote.

[0032] 4) Zygote verification and zygote frequency statistics: Conventional PCR technology was used to detect serotype genes (CPS1I and CPS3L) and drug resistance genes ( erm (B) and optrA ) to identify donor bacteria, recipient bacteria and conjugates. The information of primer sequences, annealing temperatures and product lengths are shown in Table 1.

[0033] Table 1 Primer sequences, annealing temperatures ;

[0034] PCR results are as follows Figure 2 As shown, the results showed that NP4CIP can be used as a recipient bacterium in bacterial conjugation experiments, and the resistance gene is transferred from serotype 3 S. suis AKJ47 to the recipient bacterium (serotype 1) by conjugation.

[0035] Antimicrobial Susceptibility (MIC) Determination: According to the Clinical and Laboratory Standards Institute (CLSI, 2024) guidelines, the microbroth dilution method was used to test the erythromycin and ciprofloxacin drug sensitivity of the donor bacteria, recipient bacteria, and conjugates in the conjugation test using MH broth containing 5% newborn calf serum. The conjugate successfully acquired the drug-resistant gene phenotype. The detailed information is shown in Table 2.

[0036] Table 2 Relevant characteristics of strains in conjugation test ;

[0037] a: Conjugation transfer frequency = (total number of conjugants / total number of donor bacteria) .

[0038] 5) Reengagement frequency: ICE Ssu AKJ47_ Ssu1797 and ICE Ssu AKJ47_ rplL The reconjugation experiments were conducted with P1 / 7RF as the donor bacteria and P1 / 7RF as the recipient bacteria. The conjugation frequencies were (4.94±1.14)×10 -4 and (2.22±1.21)×10 -4 The method for zygosity verification is the same as above.

[0039] In summary, the growth rate of NP4CIP in the present invention is not significantly slowed down compared to NP4, and the growth is still good. P1 / 7RF has many mutation sites that restrict its own growth due to drug resistance induction, and the growth rate is significantly reduced ( Figure 1 The good growth of NP4CIP indicates that its metabolic activity is normal, which can provide sufficient energy and resources for the expression and transfer of drug-resistant genes, overcoming the problem of limited expression of drug-resistant genes due to insufficient metabolism in P1 / 7RF conjugates. At the same time, the genome 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.

[0040] To analyze the potential of mobile genetic elements (MGEs) to persist in bacterial populations, we used the conjugated ICE Ssu AKJ47_ Ssu1797 and ICE Ssu AKJ47_ rplL As donor bacteria, P1 / 7RF was used as recipient bacteria for reconjugation experiments, and drug resistance genes optrA and erm (B) can still transfer, and the bonding frequencies are (4.94±1.14)×10 -4 and (2.22±1.21)×10 -4. Although P1 / 7RF can complete the initial conjugation, its conjugants cannot be used as donor bacteria for reconjugation experiments. This limitation is due to its limited growth rate and metabolic activity, which makes it impossible to provide sufficient energy and resources for the transfer of drug-resistant genes. In contrast, NP4CIP is not only highly consistent with the wild-type NP4 in growth rate and metabolic activity, but its conjugants also have the ability to act as donor bacteria for 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.

[0041] In summary, NP4CIP has important application value in the study of horizontal transfer of drug-resistant genes. It can more comprehensively simulate the propagation mechanism of drug-resistant genes in bacterial populations, and provides a more reliable model for in-depth study of the propagation of drug-resistant genes. Studying bacterial reconjugation can reveal the propagation mechanism of drug-resistant genes, evaluate their propagation potential, understand bacterial adaptability and evolution, and provide a scientific basis for the development of new antibacterial strategies, the optimization of drug resistance monitoring and prevention and control measures, thereby effectively addressing the problem of antibiotic resistance.

[0042] Therefore, 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 the foundation for the study of the mechanism of horizontal transfer of drug-resistant genes, and providing a basis and inspiration for controlling the spread of bacterial resistance.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution 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 , and its 16Sr DNA sequence is shown in SEQ ID NO.

1.

2. Use of type 1 Streptococcus suis NP4CIP as claimed in claim 1 in bacterial conjugation.

3. A Streptococcus suis receptor bacterium for bacterial conjugation, characterized in that: The receptor bacteria of Streptococcus suis is Streptococcus suis type 1 NP4CIP, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee on February 27, 2025. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCCNO.33594, and the classification name is Streptococcus suis Streptococcussuis , and its 16Sr DNA sequence is shown in SEQ ID NO.

1.

4. The Streptococcus suis receptor bacterium according to claim 3, characterized in that: The ciprofloxacin resistance of Streptococcus suis NP4CIP type 1 was greater than 256 μg·mL -1 .

5. Ciprofloxacin-resistant strain NP4CIP, characterized in that: It is Streptococcus suis type 1 NP4CIP. Streptococcus suis type 1 NP4CIP is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee 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 , and its 16Sr DNA sequence is shown in SEQ ID NO.

1.

6. Use of the ciprofloxacin-resistant strain NP4CIP as claimed in claim 5 in bacterial conjugation.

7. The use according to claim 6, characterized in that: The ciprofloxacin-resistant strain NP4CIP was used as the recipient bacterium, and AKJ47 was used as the donor bacterium for bacterial conjugation.

8. Use of the conjugant of the ciprofloxacin-resistant strain NP4CIP as claimed in claim 5 in bacterial conjugation.

9. The use according to claim 8, characterized in that: Bacterial conjugation was performed using the conjugate of strains NP4CIP and AKJ47 as donor bacteria and P1 / 7RF as recipient bacteria.

Images