Streptococcus agalactiae and application of streptococcus agalactiae in preparation of medicine for treating drug-resistant streptococcus agalactiae infection

Abstract

The invention discloses streptococcus agalactiae and application of the streptococcus agalactiae in preparation of drugs for treating drug-resistant streptococcus agalactiae infection, the streptococcus agalactiae is streptococcus agalactiae S.aXJM001, the streptococcus agalactiae S.aXJM001 is preserved in the China General Microbiological Culture Collection Center, the preservation date is January 17, 2025, and the biological preservation number is CGMCC NO.33461. The invention further discloses a preparation method of the streptococcus agalactiae. According to the invention, a homologous recombination technology is adopted to construct a CovS and CovR double-gene knockout mutant strain, a histidine kinase inhibitor is combined to block a signal channel, qRT-PCR (quantitative reverse transcription-polymerase chain reaction) is adopted to measure signal molecules and virulence and abundance change of drug-resistant genes, and the influence of CovS and CovR genes on the virulence and drug resistance of streptococcus agalactiae is explored; the molecular regulation mechanism of the CovS / CovR system mediated streptococcus agalactiae virulence and drug resistance is clarified, and theoretical and technical bases are provided for analysis and research of streptococcus agalactiae drug treatment targets, so that generation of drug resistance is reduced, and the CovS / CovR system mediated streptococcus agalactiae virulence and drug resistance molecular regulation method has important significance for guaranteeing quality safety of milk and dairy products.

Description

Technical Field

[0001] The present invention relates to the technical field of prevention and treatment of foodborne pathogenic bacteria, and particularly to Streptococcus agalactiae and its application in the preparation of a drug for treating drug-resistant Streptococcus agalactiae infection. Background Art

[0002] Streptococcus agalactiae (S. agalactiae) is an important zoonotic pathogen, which seriously affects the milk yield and quality, poses certain potential hazards to the quality and safety of milk and dairy products. At the same time, as a foodborne pathogenic bacterium, it threatens public health and human health. The pathogenicity of S. agalactiae mainly depends on its various virulence factors. In recent years, with the abuse of antibacterial drugs, the problems of drug resistance and multi-drug resistance (MDR) of S. agalactiae have become increasingly prominent. There have been many studies at home and abroad showing that there is a correlation between bacterial virulence and drug resistance, which may be positive or negative, and there are also rare reports that the acquisition of drug resistance by strains is not related to their virulence. Bacterial virulence and drug resistance are regulated by complex signal systems. The two-component signal transduction system (TCSTS) can form a regulatory cycle for the connection between bacterial drug resistance and virulence. CovS / CovR in TCSTS plays a very important role in the virulence of S. agalactiae, while the related research on CovS / CovR and S. agalactiae drug resistance has not been reported.

[0003] The research team of the present invention found through previous research that S. agalactiae drug resistance and MDR in bovine mastitis sources in China are serious, and different drug resistance profiles of S. agalactiae all carry virulence factor invasion-related gene iagA and fibrin-binding protein fbsA. At present, the relationship and regulatory mechanism between the virulence and drug resistance of bovine-derived S. agalactiae have not been clarified. Therefore, further research on the drug resistance and virulence of bovine-derived S. agalactiae is of great significance for screening reasonable antibacterial drugs for bovine mastitis caused by S. agalactiae, thereby reducing the generation of drug resistance, promoting the healthy development of the dairy industry, and ensuring the quality and safety of milk and dairy products.

[0004] There is a close correlation between bacterial virulence and drug resistance, and both are regulated by complex signal systems. Among them, TCSTS is involved in regulating various aspects such as bacterial growth metabolism, antimicrobial drug resistance, and pathogenicity, and is a key regulatory system for regulating gene expression in various bacterial pathogens. The main TCSTS involved in bacterial virulence and antimicrobial drug resistance are: PhoP / PhoQ, PmrA / PmrB, CbrA / CbrB, etc. These TCSTS are mainly involved in the regulation of resistance and virulence to polymyxin, colistin, quinolones, and antimicrobial peptides in Escherichia coli, Salmonella, Pseudomonas aeruginosa, Shigella dysenteriae, and Mycobacterium tuberculosis, etc.; there are also two-component signal systems ComD / ComE, CiaR / CiaH involved in regulating the resistance of Streptococcus pneumoniae to β-lactam antibiotics, but the impact on bacterial virulence is not clear.

[0005] Regarding the research on TCSTS in S.agalactiae, it mainly focuses on the regulation of virulence genes, and there are few reports on the research of TCSTS in the drug resistance of S.agalactiae. At present, only the roles of four signal systems in the regulation of virulence gene expression in S.agalactiae have been reported at home and abroad. Among them, CovS / CovR (Control of virulence) is a comprehensive virulence regulation system in Group A Streptococcus (GAS), inhibiting genes encoding known virulence factors, mainly acting as a negative regulator of gene expression to control virulence. Mutant strains of the sensor kinase CovS and the response regulator CovR can produce more virulence factors. CovS / CovR can regulate the transcription of about 15% of genes in GAS and about 7% of genes in Group B Streptococcus (GBS), and plays a crucial role in its virulence, pathogenesis, and stress response processes.

[0006] At present, the research on the CovS / CovR system in S.agalactiae is still in its infancy. There are no reports on the research related to the drug resistance of S.agalactiae by CovS / CovR. Whether CovS / CovR is involved in regulating the drug resistance of S.agalactiae and the specific mechanism are not clear, and further research is still needed to prove it.

[0007] Since the report of the Streptococcus agalactiae regulatory factors CovS / CovR in 2002, the research on S. agalactiae CovS / CovR has been very slow. In particular, there is still a blank in the research related to the drug resistance of bovine-derived S. agalactiae. So far, there has been no research report on the binary signal system CovS / CovR regulating the virulence genes of S. agalactiae and affecting its drug resistance. Therefore, the research team of this invention for the first time adopted homologous recombination technology to construct CovS and CovR double gene knockout mutants and their corresponding complementary strains in bovine-derived Streptococcus agalactiae, explored the molecular regulatory mechanism of CovS / CovR on the virulence and drug resistance of bovine-derived Streptococcus agalactiae, and proved that CovSR can be used as a target in the preparation of drugs for treating drug-resistant Streptococcus agalactiae infections, which is of great significance for the prevention and treatment of bovine mastitis caused by Streptococcus agalactiae infection. Summary of the Invention

[0008] The purpose of this invention is to provide a Streptococcus agalactiae and its application in the preparation of drugs for treating drug-resistant Streptococcus agalactiae infections. The research of this invention isolated a strain of Streptococcus agalactiae S.aXJM001 from bovine mastitis sources. From the perspective of virulence genes, taking the two-component regulatory system as a breakthrough point, through methods such as gene knockout, homologous recombination technology, and signal pathway blocking, the correlation between the virulence and drug resistance of S. agalactiae was clarified, and the effects and regulatory mechanisms of CovS and CovR genes on the virulence and drug resistance of S. agalactiae were explored; it provided a theoretical basis for further exploring the pathogenic mechanism of S. agalactiae, and thus provided new ideas for the precise control of bovine mastitis.

[0009] To achieve the above purpose, the technical solutions adopted by this invention are specifically as follows:

[0010] The research team of this invention isolated a strain of Streptococcus agalactiae S.aXJM001 from bovine mastitis sources. It was deposited in the China General Microbiological Culture Collection Center on January 17, 2025, and the biological deposit number is CGMCC NO. 33461.

[0011] The serotype of Streptococcus agalactiae S.aXJM001 is type Ⅰa. It is highly sensitive to ampicillin, amoxicillin / clavulanic acid, oxacillin, cefalotin, sulfisoxazole, and co-trimoxazole, and shows drug resistance to tetracycline, erythromycin, and clindamycin. It mainly carries pbp1a and tetO drug resistance genes and igaA and fbsA virulence genes. There is no significant correlation between the virulence genes and drug resistance of S.aXJM001.

[0012] The Streptococcus agalactiae S.aXJM001 can be used to construct CovS and CovR gene knockout mutants, and further can be used to prepare or screen drugs for treating drug-resistant Streptococcus agalactiae infections, for the prevention and treatment of bovine mastitis diseases caused by Streptococcus agalactiae infections.

[0013] (1) Construction of Streptococcus agalactiae S.aXJM001 CovS and CovR gene knockout mutants

[0014] Using the S.aXJM001 genomic DNA as a template, the upstream and downstream fragments of the CovS / CovR coding genes were amplified by PCR; at the same time, using the pSet4s shuttle plasmid DNA as a template, the spectinomycin resistance gene spc was amplified by PCR. Under the action of restriction enzymes and T4 ligase, they were successively cloned into the multiple cloning site of the pUC19 vector to form a CovS / CovR gene knockout vector pUC19-CovSR with homologous sequences to the target gene on both sides of spc. The pUC19-CovSR was electrotransformed into EcoliDH5alphA competent bacteria, and single colonies of S.aXJM001 with Amp / spc double resistance were screened. After expanded culture, genomic DNA was extracted, and the combination PCR verification method was used to confirm that the CovS / CovR coding genes had been replaced by spc, thus obtaining the CovS / CovR gene knockout mutant SAΔCovS / CovR with spectinomycin resistance. Subsequently, the CovS / CovR coding genes and their upstream promoter sequences were amplified by PCR and cloned into the shuttle plasmid pset4s. The recombinant plasmid was electrotransformed into the mutant SAΔCovS / CovR, and the complementary strain CΔCovS / CovR with CovS / CovR function compensation was obtained through resistance screening and PCR identification.

[0015] (2) Effects of CovS / CovR deletion on the biological characteristics of S.aXJM001

[0016] Under the same conditions, the basic biological characteristics of the wild strain S.aXJM001, the mutant SAΔCovS / CovR, and the complementary strain CΔCovS / CovR were observed for obvious differences. The results showed that the deletion of the CovS / CovR genes: 1) caused the chains of S.aXJM001 to change from the original single or paired arrangement to a short-chain arrangement, and S.aXJM001 grew rapidly; 2) reduced the hemolytic ability of S.aXJM001; 3) when the biofilm formation ability was measured by the crystal violet staining method, its OD 600 value decreased by 54% (P<0.01). It shows that the deletion or expression inhibition of the CovS and CovR genes will reduce the growth ability, biofilm formation ability, and hemolytic ability of S.aXJM001.

[0017] (3) Effects of CovS / CovR deletion on the drug resistance and virulence genes of S. agalactiae XJM001

[0018] After CovS / CovR deletion, sensitive strains to ampicillin, amoxicillin / clavulanic acid, oxacillin, clindamycin, and cephalothin became drug-resistant, and tetracycline-resistant strains became sensitive. It did not affect the drug resistance and sensitivity of each strain to doxycycline, erythromycin, sulfisoxazole, and co-trimoxazole. Except for changing the carriage of pbp1a and tetO drug-resistant genes, it did not affect the expression of other drug-resistant genes, and significantly up-regulated the expression of virulence genes igaA and fbsA (P<0.05). Chlorflumethiazide at 50 μmol / L or 100 μmol / L could significantly down-regulate the CovS / CovR mRNA level (P<0.05), while 25 μmol / L of flumethiazide could not. Chlorflumethiazide at different concentration levels reduced the MIC values of β-lactam and tetracycline antibiotics and the expression of drug-resistant genotypes in Streptococcus agalactiae, indicating that the two-component signal transduction pathway CovS / CovR positively regulated the drug resistance of Streptococcus agalactiae.

[0019] Thus, it can be seen that CovS / CovR significantly affects the basic characteristics of Streptococcus agalactiae, such as growth, morphological structure, hemolytic activity, and biofilm formation ability. CovS / CovR positively regulates the drug resistance of Streptococcus agalactiae. This Streptococcus agalactiae can be used to prepare or screen drugs for treating drug-resistant Streptococcus agalactiae infections.

[0020] Compared with the prior art, the outstanding effects of the present invention are as follows:

[0021] (1) In the present invention, a strain of Streptococcus agalactiae S. agalactiae XJM001 was isolated from the raw milk of dairy cow mastitis, and a double gene mutant strain with CovS and CovR knocked out was constructed by homologous recombination technology. The signal pathway was blocked by a histidine kinase inhibitor, and the changes in the abundance of signal molecules, virulence, and drug-resistant genes were measured by qRT-PCR to explore the effects of CovS and CovR genes on the virulence and drug resistance of Streptococcus agalactiae, and to clarify the molecular regulatory mechanism of the CovS / CovR system mediating the virulence and drug resistance of Streptococcus agalactiae.

[0022] (2) The present invention provides a construction method for homologous recombination gene knockout of Streptococcus agalactiae strains from dairy cows. As an important means for studying bacterial gene functions, homologous recombination technology has been widely applied in animal-derived negative strains (such as Cronobacter, Escherichia coli, etc.). By knocking out specific genes, its roles in pathogenicity, metabolic regulation, etc. can be revealed. However, there are few reports on the application of this technology in animal-derived Gram-positive strains, and there is no research report on homologous recombination technology related to bovine Streptococcus agalactiae. In recent years, homologous recombination technology combined with new vector systems (such as suicide plasmids, Red recombination systems, etc.) has significantly improved the efficiency of gene knockout. The CovS and CovR gene knockout system of the present invention realizes high-efficiency and precise knockout through optimizing vector design and screening methods, which is innovative.

[0023] (3) The present invention provides the use of Streptococcus agalactiae CovSR from dairy cows as a target. Research has confirmed that CovS / CovR plays a crucial role in the virulence, pathogenesis, and stress response processes of group A streptococci. Gene knockout systems of CovS / CovR in multiple animal-derived multi-strains have been used to study the synthesis and regulation mechanisms of their virulence factors. However, for Streptococcus agalactiae, which is a group A streptococcus, there are few related studies on the virulence and drug resistance of CovS / CovR. The present invention finds that knocking out CovS / CovR can reduce the basic characteristics of Streptococcus agalactiae's growth ability, biofilm formation ability, and hemolytic ability, change its sensitivity and drug resistance to antibacterial drugs such as ampicillin, amoxicillin / clavulanic acid, oxacillin, clindamycin, cefalotin tetracycline, as well as the expression of key drug resistance genes, and confirms that CovS / CovR may positively regulate the drug resistance of Streptococcus agalactiae through the igaA and fbsA virulence genes. Therefore, CovSR as a target and gene knockout vector can be applied to the preparation of drugs for treating drug-resistant Streptococcus agalactiae infections, which has important practical significance for the prevention and treatment of bovine mastitis diseases caused by Streptococcus agalactiae infections.

[0024] The following further illustrates the Streptococcus agalactiae described in the present invention and its application in the preparation of drugs for treating drug-resistant Streptococcus agalactiae infections in conjunction with the accompanying drawings and specific embodiments.

[0025] Biological deposit information

[0026] Streptococcus agalactiae S.aXJM001, classified and named as Streptococcus agalactiae, is deposited in the China General Microbiological Culture Collection Center. The abbreviation of the deposit institution is: CGMCC. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is January 17, 2025, and the biological deposit number is CGMCC NO. 33461. Description of the drawings

[0027] Figure 1Colony morphology of Streptococcus agalactiae S.aXJM001 on Columbia blood agar plate.

[0028] Figure 2 Gram staining results of Streptococcus agalactiae S.aXJM001 (b, 1000×).

[0029] Figure 3 Results of identification with specific primers of Streptococcus agalactiae S.aXJM001.

[0030] Figure 4 Amplification results of upstream and downstream fragments of CovS and CovR genes. M: DL2000 DNA Marker; 1 - 3: PCR products of upstream fragment CovS; 4 - 6: PCR products of downstream fragment CovR.

[0031] Figure 5 Map of suicide plasmid pSET4s.

[0032] Figure 6 Results of 1% agarose gel electrophoresis of amplification of CovSR homologous arms.

[0033] Figure 7 Amplification results of suicide vector construction fragments and vector framework.

[0034] Figure 8 Amplification detection of fragments for construction of CovSR UD pset4s.

[0035] Figure 9 Electrophoresis detection of amplification of up - spc - down fragment.

[0036] Figure 10 Schematic diagram of construction of gene knockout vector pSET4s - CovS / CovR.

[0037] Figure 11 Genes to be knocked out and their homologous arm sequences. Among them, the red - shaded area is the CovS gene and the gray - shaded area is the CovR gene.

[0038] Figure 12 Sequences after knockout by pSET4s method.

[0039] Figure 13 Sequences after knockout by suicide vector (the middle magenta - marked area is the spectinomycin resistance gene, derived from pSET4s).

[0040] Figure 14 Results of determination of growth curves of wild - type, mutant and complementary strains of Streptococcus agalactiae.

[0041] Figure 15 Results of determination of biofilm formation of wild - type, mutant and complementary strains of Streptococcus agalactiae.

[0042] Figure 16 Results of hemolytic ability determination of wild-type, mutant and complementary strains of Streptococcus agalactiae. Among them, A: S.aXJM001; B: CΔCovS / CovR; C: SAΔCovS / CovR.

[0043] Figure 17 Results of drug resistance gene detection of wild-type, mutant and complementary strains of Streptococcus agalactiae. Among them, 1: S.aXJM001 pbp1a; 2: CΔCovS / CovR pbp1a; 13: S.aXJM001 tetO; 14: CΔCovS / CovR tetO.

[0044] Figure 18 Results of the expression levels of virulence genes of wild-type, mutant and complementary strains of Streptococcus agalactiae.

[0045] Figure 19 Results of drug resistance gene detection of wild-type and mutant strains of Streptococcus agalactiae after adding different concentrations of closantel blocking agent. In Figure (a), 1: S.aXJM001 25 pbp1a; 2: S.aXJM001 50 pbp1a; 3: S.aXJM001 100 pbp1a; In Figure (b), 19: SAΔCovS / CovR 25 tetO; 20: SAΔCovS / CovR 50 tetO; 21: SAΔCovS / CovR 100 tetO.

[0046] Figure 20 Results of real-time fluorescence quantitative RT-PCR of wild-type and mutant strains of Streptococcus agalactiae after adding different concentrations of closantel blocking agent. Specific implementation manners

[0047] The experimental strains, reagents and instruments used in the following examples are as follows.

[0048] (1) Experimental strains

[0049] Standard strain: Streptococcus agalactiae ATCC13813; Experimental strain: S.aXJM001.

[0050] (2) Main reagents and instruments

[0051] Columbia blood agar plates, tryptone soy broth (TSB), MH medium, and Gram staining solution were all purchased from Beijing Land Bridge Technology Co., Ltd.; Kirby-Bauer (K-B) drug susceptibility test strips and E-test drug susceptibility test strips were purchased from Yinuokang (Tianjin) Technology Development Co., Ltd. Bacterial DNA extraction kits, nucleic acid dyes, 5×TBE electrophoresis buffer, and DL2000 DNA Marker were all purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; agarose was purchased from Thermo Fisher Scientific (China) Co., Ltd.; 2×Taq PCR MasterMix was purchased from Beijing Bioteke Corporation; 16S rDNA universal primers were purchased from Dingguo Changsheng Biotechnology Co., Ltd.; drug susceptibility plates were purchased from Tianjin Jinzhang Technology Development Co., Ltd.; 2×Superpfu PCR mix, PCR product purification kit, suicide plasmid pSET4s, Trans2K DNA marker,

[0052] 2×Taq PCR mix, E. coli DH5α competent cells, Trans2k plus DNA marker, Trans2kplus II DNA marker, and 5×infusion cloing mix were all purchased from Hangzhou Bioscience Co., Ltd. PCR instrument (Agilent Technologies Co., Ltd.), electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), molecular imaging system (Shanghai Ouxiang Scientific Instruments Co., Ltd.), bench-top high-speed centrifuge (Hunan Xiangxin Instrumentation Co., Ltd.), electrothermal constant temperature incubator (Shanghai Yiheng Scientific Instruments Co., Ltd.), electrophoresis instrument (Beijing Liuyi Biotechnology Co., Ltd.), biological safety cabinet (Artel High-Tech Co., Ltd., Singapore), vertical autoclave (Shanghai Shen'an Medical Instrument Co., Ltd.).

[0053] Example 1 Isolation and Identification of Streptococcus agalactiae S.aXJM001

[0054] 1. Isolation of Wild Streptococcus agalactiae S.aXJM001

[0055] A wild strain of Streptococcus agalactiae S.aXJM001 was isolated from raw milk from cow mastitis. It showed white, milky white or grayish white, smooth and convex circular colonies on Columbia blood agar plates, and was Gram-positive cocci arranged in purple, short chains, as Figure 1 shown. The results of Gram staining were as Figure 2 shown. The cfb specific gene of Streptococcus agalactiae could be amplified, with a size of 600 bp. The results of identification with specific primers were as Figure 3 shown. It was preserved in the Institute of Agricultural Quality Standards and Testing Technology, Xinjiang Academy of Agricultural Sciences.

[0056] 2. Molecular Serotype, Drug Resistance and CovS / CovR Expression of Streptococcus agalactiae S.aXJM001

[0057] (1) Serotyping Experiment of S.aXJM001

[0058] Referring to the multiplex PCR method of Monica Imperi, the bacterial serotype of S.aXJM001 was accurately identified. 19 primer sequences (SEQ ID NO: 1 - 19) were synthesized by Beijing New Era Zhonghe Technology Co., Ltd. (see Table 1). The PCR reaction procedure was as follows: pre-denaturation at 95°C for 5 min, 95°C for 60 s, 54°C for 60 s, 72°C for 2 min, for a total of 15 cycles, 95°C for 60 s, 56°C for 60 s, 72°C for 2 min, for 25 cycles, 72°C for 10 min. The PCR reaction volume (50 μL) was: 2×RAPA3G Multiplex PCR Mix 25 μL, 1 μL of each of the 19 primers, 5 μL of DNA template, and ddH 2 O 1 μL. After PCR amplification, the size of the PCR bands was detected by 1.5% agarose gel electrophoresis, and the serotype of the strain was identified according to the size of the amplified fragment.

[0059] The serotyping results of S.aXJM001 showed that the serotype of S.aXJM001 was type Ia, which was different from the serotypes reported abroad. The distribution of Streptococcus agalactiae serotypes varies worldwide. According to existing research reports, the main serotypes of Streptococcus agalactiae strains isolated from dairy cows in the Argentine region are mainly types III and II; the main serotypes of Canadian bovine Streptococcus agalactiae are type V (54%) and type IV (34%); the main serotype in Poland is type II. This indicates that the serotypes of Streptococcus agalactiae from dairy cows vary due to different geographical locations.

[0060] Table 1 Primer Sequences for Molecular Serotypes of Streptococcus agalactiae

[0061]

[0062] (2) Drug Sensitivity Test of Streptococcus agalactiae

[0063] The susceptibility of the strains to 16 antimicrobial agents was tested by the broth dilution method recommended by the Clinical and Laboratory Standards Institute of the United States (CLSI, 2018). The bacterial suspension was adjusted to 0.5 McFarland turbidity using a McFarland nephelometer. The bacterial suspension adjusted to 0.5 McFarland turbidity was inoculated into a 96-well plate containing different concentrations of drugs. The following antimicrobial agents were tested, and the dilution concentrations in each well were as follows: penicillin (PEN), ampicillin (AMP), clindamycin (CLI), and ciprofloxacin (CIP) (0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, and 64.0 mg / mL), erythromycin (ERY), cefalotin (CEF), ceftiofur (CET), oxacillin (OXA), tetracycline (TE), doxycycline (DOX), gentamicin (GM), and florfenicol (FFC) (0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, and 128 mg / mL), kanamycin (Ka) (0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, 128, and 256 mg / mL), sulfisoxazole (SMZ) (2, 4, 8, 16, 32, 64, 128, 256, 512, 1024 mg / mL), amoxicillin / clavulanic acid (A / C) (0.25 / 0.12, 0.5 / 0.25, 1 / 0.5, 2 / 1, 4 / 2, 8 / 4, 16 / 8, 32 / 16, 64 / 32, 128 / 64 mg / mL), and co-trimoxazole (SXT) (0.12 / 2.4, 0.25 / 4.8, 0.5 / 9.5, 1 / 19, 2 / 38, 4 / 76, 8 / 152, 16 / 304, 32 / 608, and 64 / 1216 mg / mL).

[0064] Streptococcus agalactiae (ATCC13813) was used as the quality control. Since there are no specific resistance breakpoints for streptococci for some of the tested antimicrobial agents, the resistance breakpoints of any antimicrobial agent in the same antimicrobial agent class recommended by CLSI (2024) were referred to.

[0065] The results showed that Streptococcus agalactiae S.aXJM001 was highly sensitive to ampicillin, amoxicillin / clavulanic acid, oxacillin, cefalotin, sulfisoxazole, and co-trimoxazole; showed intermediate susceptibility to doxycycline; and was resistant to tetracycline, erythromycin, and clindamycin. The specific results are shown in Table 2.

[0066] Table 2 Results of drug susceptibility test

[0067]

[0068] (3) Expression of CovS / CovR genes in S.aXJM001

[0069] The genomic DNA of S. a XJM001 was extracted by the CTAB method, and its concentration was determined by spectrophotometry. According to the target gene sequence, primers were designed using Primer Premier 5.0 software. The primers were synthesized by Beijing New Era Zhonghe Technology Co., Ltd. The reaction procedure was as follows: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 45 s, for 30 cycles, and then extension at 72 °C for 10 min. The reaction system (total volume 30 μL) was: 2×Master Mix 15 μL, upstream and downstream primers 1 μL each, DNA template 2 μL, ddH 2 O 11 μL, and the amplification products were detected by 1.5% agarose gel electrophoresis.

[0070] The results showed that using the genomic DNA of Streptococcus agalactiae S. a XJM001 as a template, the upstream fragment of the CovS gene and the downstream fragment of the CovR gene were successfully amplified. The fragment size of the CovS gene was 547 bp, and the fragment size of the CovR gene was 523 bp( Figure 4 ).

[0071] Example 2 Construction and identification of CovS / CovR gene deletion vector

[0072] 1. Construction of CovS / CovR gene deletion vector

[0073] The structure of the suicide plasmid pSET4s is shown in Figure 5 . Primers for constructing the CovS / CovR gene deletion mutant were designed using Primer 5.0, and the primer sequences are shown in Table 3 (SEQ ID NO: 20 - 33).

[0074] Table 3 Primers for constructing CovS / CovR gene deletion mutant

[0075]

[0076] (1) PCR amplification of upstream and downstream homologous arms

[0077] According to the principle and method of the homologous recombination system, using the genomic DNA of the wild strain of Streptococcus agalactiae S. a XJM001 as a template, the upstream and downstream homologous arms of CovSR were amplified using the primers CovSR-19-F / CovSR-up-R, CovSR-down-F / CovSR-19-R according to the following procedure (Table 4). Amplification conditions: 94 °C for 5 min, 32 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 40 s), and hold at 10 °C. The amplification products were recovered using a PCR product purification kit and reserved for use( Figure 6 ).

[0078] Seamless cloning, ligation, transformation and identification: Mix 2 μL of 5×infusion Cloning mix, 3 μL of the amplified and recovered Up amplification fragment, 3 μL of the amplified and recovered Down amplification fragment, and 2 μL of the linearized vector pUC19 [EcoRI and HindIII restriction enzymes]. Place the mixture on ice for 30 min, transform competent E. coli DH5α cells, spread on an Amp-IPTG-Xgal plate, pick white colonies for sequencing, and the correctly sequenced one is CovSR-UD-pUC19.

[0079] Table 4 Homologous recombination amplification system

[0080]

[0081]

[0082] (2) Construction of the suicide vector with the middle Spc resistance cassette

[0083] Inverse amplification of the vector framework: Using the CovSR-UD-pUC19 plasmid as a template, reverse amplify the vector framework with primers Cov-spc-F / Cov-spc-R. The amplification reaction system is 50 μL: 25 μL of 2×Superpfu PCR mix, 2 μL of Primer P1 (10 μM), 2 μL of Primer P2 (10 μM), 1 μL of CovSR-UD-pUC19, and 20 μL of dd H 2 O. Amplification conditions: 94°C for 5 min, 32 cycles (94°C for 30 sec, 55°C for 30 sec, 68°C for 2 min), and hold at 10°C.

[0084] Amplification of the Spc resistance gene: Using primers Spc-CovSR-F / Spc-CovSR-R, amplify the Spc resistance gene with the pSet4s plasmid stored in the laboratory as a template. The amplification reaction system is 50 μL: 25 μL of 2×Superpfu PCR mix, 2 μL of Primer P1 (10 μM), 2 μL of Primer P2 (10 μM), 1 μL of pset4s, and 20 μL of dd H 2 O. Amplification conditions: 94°C for 5 min, 32 cycles (94°C for 30 sec, 55°C for 30 sec, 68°C for 30 sec), and hold at 10°C.

[0085] Seamless cloning, ligation, transformation and identification: Mix 2 μL of 5×infusion Cloning mix, 6 μL of the amplified and recovered spc amplified fragment, and 2 μL of the CovSR-pUC19 reverse amplified vector framework on ice for 30 min. Transform competent E. coli DH5α cells, spread on Amp / spc plates, pick the clones grown on the double-antibody plates for sequencing. The clones with correct sequencing are the prepared pUC19-CovSR-up-spc-down suicide vector. The amplification results of the suicide vector construction fragments and vector framework are as shown in Figure 7 。

[0086] (3) Construction of the temperature-sensitive knockout vector pSET4s

[0087] Amplification of homologous arms: Using the CovSR-UD-pUC19 plasmid with correct sequencing as a template, amplify the fused homologous arm fragments with primers CovSR-smaI-F / cosvR-smaI-R. The amplification reaction system is 50 μL: 25 μL of 2×Superpfu PCR mix, 2 μL of Primer P1 (10 μM), 2 μL of Primer P2 (10 μM), 1 μL of CovSR-UD-pUC19, and 20 μL of ddH 2 O₂. Amplification conditions: 94°C for 5 min, 32 cycles (94°C for 30 sec, 55°C for 30 sec, 68°C for 30 sec), and hold at 10°C. The extracted pSET4s plasmid is digested with smaI alone under the following conditions (Table 5), digested at 37°C for 1 h, and recovered by gel electrophoresis. The seamless cloning ligation system is shown in Table 6. Place on ice for 30 min, transform competent E. coli DH5α cells, and perform sequencing. The PCR fragment amplification electrophoresis detection results are shown in Figure 8 。

[0088] Table 5 Single enzyme digestion system

[0089]

[0090] Table 6 Seamless cloning ligation system

[0091]

[0092] 2. CovS / CovR knockout screening

[0093] Use primers CovSR-up-F / COVSR-down-F to amplify the knockout fragment using the correctly sequenced CovSR-Up-spc-down-pSet4s plasmid as a template. The amplification reaction system is 50 μL: 2×Superpfu PCR mix 25 μL, Primer P1 (10 μM) 2 μL, Primer P2 (10 μM) 2 μL, CovSR-Up-spc-downD-pUC19 1 μL, ddH 2 O 20 μL. Amplification conditions: 94°C for 5 min, 32 cycles (94°C for 30 sec, 55°C for 30 sec, 68°C for 2 min), and hold at 10°C. The amplified suicide fragment is transformed into S.aXJM001 competent cells, and those with spc resistance are obtained for identification. The up-spc-down fragment amplification is detected by electrophoresis as shown in Figure 9 . Extract the plasmid and transform it into S.aXJM001 to obtain clones with spc resistance, which require single exchange and double exchange screening. The schematic diagram of the gene knockout vector pSET4s-CovS / CovR is as shown in Figure 10 . Among them, Figure 11 is the gene to be knocked out and its homologous arm sequence (shown in SEQ ID NO: 38), with the CovS gene in red shading and the CovR gene in gray shading. Figure 12 is the sequence after knockout by the pSET4s method (shown in SEQ ID NO: 39). Figure 13 is the sequence after knockout by the suicide vector (shown in SEQ ID NO: 40). The middle magenta-marked is the spectinomycin resistance gene, which is derived from pSET4s. Construct the deletion mutant SAΔCovS / CovR, select monoclonal colonies as templates, the wild strain as a positive control, and the mutant strain as a negative control, and use the internal primers of the deleted gene for PCR identification to construct the complementary strain CΔCovS / CovR.

[0094] Example 3 Biological characteristics study of Streptococcus agalactiae wild strain, mutant strain and complementary strain

[0095] 1. Determination of the growth rates of Streptococcus agalactiae wild strain, mutant strain and complementary strain

[0096] The wild strain S.aXJM001, the gene mutant strain SAΔCovS / CovR, and the gene complementary strain CΔCovS / CovR were inoculated into fresh BHI liquid medium without antibiotics. After culturing overnight at 37°C with shaking at 160 r / m, equal amounts of the wild strain and the mutant strain were taken and inoculated into 200 mL of fresh BHI liquid medium without antibiotics, and cultured at 37°C with shaking at 160 r / m. During the culturing process, equal amounts of the bacterial liquid were taken every 1 h, and the absorbance value at OD600 was measured for a total of 24 h, and then measured every 3 h until 24 h was completed, and the measurement was repeated three times. Finally, with the average value of OD 600 as the ordinate and the culturing time as the abscissa, the growth curves of the wild strain, the mutant strain, and the complementary strain were plotted to compare their growth rates.

[0097] (1) Effects of CovS / CovR deletion on the morphological structure of Streptococcus agalactiae

[0098] There was no difference in the growth morphology of the wild strain S.aXJM001 and the complementary strain CΔCovS / CovR on blood agar plates. The colonies formed by the mutant strain SAΔCovS / CovR were grayish-white, smooth on the surface, with opalescence, round, and the diameter increased; Gram staining showed that the wild strain S.aXJM001 and the complementary strain CΔCovS / CovR were single or paired Gram-positive cocci, and the mutant strain SAΔCovS / CovR was arranged in short chains, indicating that the deletion of the CovS and CovR genes changed the morphological structure of the strain.

[0099] (2) Effects of CovS / CovR deletion on the growth curve of Streptococcus agalactiae

[0100] The growth rate of the mutant strain SAΔCovS / CovR was extremely significantly higher than that of the wild strain S.aXJM001 and the complementary strain CΔCovS / CovR( Figure 14 ), indicating that the deletion of the CovS and CovR genes had a greater impact on the growth of Streptococcus agalactiae.

[0101] 2. Biofilm formation ability of the wild strain, mutant strain, and complementary strain of Streptococcus agalactiae

[0102] Each bacterial liquid to be tested was cultured until OD 600 = 0.2. 200 μL of each bacterial liquid was added to a sterile 96-well cell culture plate, with 3 parallels for each strain, and an equal amount of LB liquid medium was used as a control. The 96-well plate was placed in a wet box and cultured at 37°C for 36 h; the bacterial liquid and the culture medium were aspirated, 200 μL of methanol was added to each well to fix for 15 min, the methanol was aspirated, and the wells were washed 3 times with sterile PBS and air-dried naturally; 200 μL of 0.5% crystal violet was added to each well and stained for 30 min; washed with 200 μL of sterile PBS and air-dried naturally, and repeated 3 times; 200 μL of acetic acid was added to each well to dissolve for 30 min, and OD was measured570 . Quantitatively study the biofilm formation ability of wild strains, deletion strains, and complementary strains. The experiment was repeated 3 times, and the average value was taken. The data was subjected to a t-test, and a P value < 0.05 was considered statistically significant.

[0103] The results showed that the biofilm formation ability of the wild strain S.aXJM001 was extremely significantly higher than that of the complementary strain CΔCovS / CovR and the mutant strain SAΔCovS / CovR ( Figure 15 ). It indicated that the deletion of the CovS / CovR gene led to a decrease in the biofilm formation ability of Streptococcus agalactiae, and the biofilm formation ability was closely related to drug resistance, suggesting that the deletion of the CovS / CovR gene might regulate the drug resistance of Streptococcus agalactiae by reducing its biofilm formation ability.

[0104] 3. Detection of the hemolytic ability of wild strains, mutant strains, and complementary strains of Streptococcus agalactiae

[0105] Blood agar plate detection: Streak-inoculate the overnight-cultured wild strains, mutant strains, and complementary strains on blood agar plates, and incubate them in an inverted position at 37 °C for 48 h to form obvious hemolytic zones. Observe the hemolytic rings and colony morphology and take pictures.

[0106] The results showed that the hemolytic ability of the wild strain S.aXJM001 was stronger than that of the complementary strain CΔCovS / CovR; the mutant strain SAΔCovS / CovR had no hemolytic ability ( Figure 16 ). It indicated that the deletion of CovS / CovR reduced the hemolytic ability of Streptococcus agalactiae.

[0107] 4. Detection of drug resistance genes of wild strains, mutant strains, and complementary strains of Streptococcus agalactiae

[0108] Before and after gene knockout, the drug sensitivity test was performed. The minimum broth dilution method was used to determine the sensitivity of S.aXJM001 and SAΔCovS / CovR to four major categories of antibacterial drugs: β-lactams, lincosamides, macrolides, and tetracyclines. The results showed that the wild strain S.aXJM001 and the complementary strain CΔCovS / CovR were sensitive to ampicillin, amoxicillin / clavulanic acid, oxacillin, clindamycin, and cephalothin, while the mutant strain SAΔCovS / CovR was resistant to these drugs; the wild strain S.aXJM001 and the complementary strain CΔCovS / CovR were resistant to tetracycline, and the mutant strain SAΔCovS / CovR was sensitive to these drugs (Table 7). It indicated that the deletion of CovS / CovR positively regulated the phenotypic drug resistance of Streptococcus agalactiae.

[0109] Table 7 Experimental results of the drug resistance of wild strains, mutant strains, and complementary strains of Streptococcus agalactiae

[0110]

[0111]

[0112] PCR was used to detect the relevant drug-resistant genes pbp1a, pbp2b, lnuA, lnuB, ermA, ermB, ermC, mefA, tetM, tetK, tetS, and tetO in each strain, and agarose gel imaging was used to observe the amplification effect.

[0113] The results showed that only S. a XJM001 and CΔCovS / CovR strains carried the pbp1a and tetO drug-resistant genes, and no other drug-resistant genes were detected ( Figure 17 ). It was shown that the deletion of CovS / CovR lost the expression of the pbp1a and tetO drug-resistant genes, which was consistent with the phenotypic drug resistance.

[0114] 5. Detection of virulence genes in wild-type, mutant, and complementary strains of Streptococcus agalactiae

[0115] (1) RNA extraction from strains: For bacterial RNA extraction, first treat with 20 mg / mL lysozyme, and then use the Trizol method for RNA extraction. The steps of the Trizol method are as follows.

[0116] 1) After adding Trizol reagent to the bacteria, let it stand at room temperature for 5 min to allow sufficient lysis.

[0117] 2) Centrifuge at 12000 rmp for 5 min and discard the precipitate.

[0118] 3) Add chloroform at 200 μL chloroform / mL Trizol, mix well by shaking, and let it stand at room temperature for 15 min. Note: Do not use a vortex oscillator to avoid breaking genomic DNA. (Chloroform is an organic solvent that effectively separates the organic and inorganic phases. In the organic phase, phenol mainly binds to proteins, thus separating proteins from RNA, and RNA enters the aqueous phase).

[0119] 4) Centrifuge at 12000 rmp for 15 min in a 4°C low-temperature centrifuge.

[0120] 5) Pipette the upper aqueous phase into another centrifuge tube. Note: Do not pipette the middle interface. If both RNA and protein are extracted, retain the lower phenol phase and store it in a 4°C refrigerator. If only RNA is extracted, discard the lower phenol phase.

[0121] 6) Add isopropanol at 0.5 mL isopropanol / mL Trizol (to precipitate RNA), mix well, and let it stand at room temperature for 5 - 10 min.

[0122] 7) Centrifuge at 12000 rmp for 10 min at 4°C, discard the supernatant, and the RNA precipitates at the bottom of the tube.

[0123] 8) Add 75% ethanol (to precipitate RNA) at a ratio of 1 mL of 75% ethanol per mL of Trizol. Gently vortex the centrifuge tube to suspend the precipitate.

[0124] 9) Centrifuge at 8000 rmp for 5 min at 4°C and discard the supernatant as much as possible.

[0125] 10) Air-dry at room temperature or vacuum-dry for 5 - 10 min. Note: The RNA sample should not be overly dried, otherwise it will be difficult to dissolve.

[0126] 11) The RNA sample can be dissolved in 50 μL of H2O, TE buffer, or 0.5% SDS at 55 - 60°C for 5 - 10 min.

[0127] (2) Design of fluorescence quantitative primers

[0128] The primer sequences are shown in Table 8 (SEQ ID NO: 34 - 37).

[0129] Table 8 Fluorescence quantitative primer sequences of Streptococcus agalactiae virulence genes

[0130]

[0131] (3) Fluorescence quantitative reaction system and reaction program

[0132] The fluorescence quantitative PCR amplification reaction system is a 20 μL reaction system as shown in Table 9. The reaction conditions are shown in Table 10.

[0133] Table 9 Fluorescence quantitative PCR reaction system

[0134]

[0135] Table 10 Fluorescence quantitative PCR reaction program

[0136]

[0137] The results are as Figure 18 , According to the expression levels of the igaA and fbsA genes, the mutant strain and the complementary strain were significantly higher than the wild strain S.aXJM001 (P < 0.05). It indicates that CovS / CovR negatively regulates the expression of the igaA and fbsA virulence genes of Streptococcus agalactiae.

[0138] Example 4 Closantel blocking test

[0139] The mutant strain was inoculated into BHI liquid medium containing ampicillin (2 mg), and the wild strain was inoculated into BHI liquid medium without ampicillin. After culturing at 37 °C for 24 h, they were transferred to fresh BHI medium. Under appropriate conditions, mutant and wild strains were treated with closantel solutions at different final concentration gradients (25, 50, 100 μmol / L). SAΔCovS / CovR and S.a XJM001 without treatment were set as control groups. After culturing at 37 °C for 1 h, the bacterial precipitates were collected by centrifugation at 14 000 r / min for 1 min.

[0140] Detect the changes and correlations of phenotypic drug resistance, drug resistance genes and virulence genes in the three strains before and after the CovS / CovR signal blockade. Real-time fluorescence quantitative PCR was used to detect the expression abundances of the key genes CovS and CovR mRNA in each signal pathway, explore the blocking effect of closantel on the two-component signal system CovS / CovR, and screen the optimal conditions for closantel to cut off the two-component signal transduction pathway CovS / CovR.

[0141] (1) Determination results of drug sensitivity of wild and mutant strains of Streptococcus agalactiae after adding blockers of different concentrations of closantel

[0142] It was found that when 25 μmol / L, 50 μmol / L, and 100 μmol / L of closantel were added, the MIC values of the wild strain S.a XJM001 against tetracycline, doxycycline, and sulfisoxazole decreased significantly. The MIC value of tetracycline decreased by 50% from 32 mg / mL. With the increase of the blocking concentration, the MIC value of doxycycline decreased from the original 16 mg / mL to ≤0.25 mg / mL, and the MIC value of sulfisoxazole decreased from 16 mg / mL to 4 mg / mL. This indicates that after the two-component signal pathway CovS / CovR is blocked, Streptococcus agalactiae becomes more sensitive to these three types of antibacterial drugs. For the mutant strain SAΔCovS / CovR, the MIC values of β-lactam antibacterial drugs decreased, while the MIC values of tetracycline antibacterial drugs increased. This shows that after the two-component signal pathway CovS / CovR is blocked, there is an additive effect with gene knockout, and the drug resistance to some antibacterial drugs has not been completely eliminated. The blocker further inhibits the compensation mechanism. Even if the CovS / CovR gene is knocked out, this pathway can still maintain partial drug resistance through other ways. This means that the two-component signal transduction pathway CovS / CovR is positively correlated with the drug resistance of Streptococcus agalactiae.

[0143] Table 11 Results of drug resistance experiments of wild and mutant strains of Streptococcus agalactiae after adding blockers of different concentrations of closantel (unit: μmol / L)

[0144]

[0145] (2) Detection results of drug resistance genes of Streptococcus agalactiae wild strains and mutant strains after adding different concentrations of closantel blockers

[0146] After PCR detection of pbp1a, pbp2b, lnuA, lnuB, ermA, ermB, ermC, mefA, tetM, tetK, tetS, and tetO drug resistance genes in S.aXJM001 25, S.aXJM001 50, S.aXJM001 100, SAΔCovS / CovR25, SAΔCovS / CovR 50, and SAΔCovS / CovR 100 after closantel blocking, it was found that wild strains with different concentrations of blockers all carried the pbp1a drug resistance gene, mutant strains with different concentrations of blockers all carried the tetO gene, and other strains did not carry each drug resistance gene, which was consistent with some of the results of drug resistance performance ( Figure 19 ).

[0147] (3) Real-time fluorescence quantitative RT-PCR detection results of Streptococcus agalactiae wild strains and mutant strains after adding different concentrations of closantel blockers

[0148] When the concentration of closantel was 25 μmol / L, there was no significant difference in the mRNA levels of CovS and CovR genes in wild strains compared with those without closantel treatment (P>0.05). When the drug concentration increased to 50 μmol / L or 100 μmol / L, the mRNA levels of CovS and CovR genes in wild strains were significantly down-regulated (P<0.05)( Figure 20 ).

[0149] In summary, in the present invention, a strain of Streptococcus agalactiae S.aXJM001 was isolated from the source of cow mastitis. An innovative method for homologous recombination gene knockout of animal-derived positive strains was constructed. It was found that CovS / CovR could significantly improve the growth and hemolytic ability of Streptococcus agalactiae, inhibit the expression of key virulence genes of Streptococcus agalactiae, and increase the drug resistance of Streptococcus agalactiae. This may be because the key virulence of Streptococcus agalactiae is weakened, resulting in the weakening of bacterial pathogenicity. Low-pathogenic strains can colonize or coexist in the immune-compromised body for a longer time, thus increasing the chance of acquiring bacterial drug resistance. CovSR can be used as a target and gene knockout vector in the preparation of drugs for the treatment of drug-resistant Streptococcus agalactiae infections, which is of great significance for the prevention and treatment of cow mastitis diseases caused by Streptococcus agalactiae infections.

[0150] The above-described embodiments are only used to describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A Streptococcus agalactiae, characterized in that: It is Streptococcus agalactiae S.aXJM001 isolated and identified from milk, and deposited in the China General Microbiological Culture Collection Center. The deposit date is January 17, 2025, and the biological deposit number is CGMCC NO.33461.

2. Use of the Streptococcus agalactiae according to claim 1 in the preparation or screening of drugs for treating drug-resistant Streptococcus agalactiae infection.

3. The use of Streptococcus agalactiae according to claim 2 in the preparation or screening of drugs for treating drug-resistant Streptococcus agalactiae infection, characterized in that: It includes constructing CovS and CovR gene knockout mutant strains of Streptococcus agalactiae S.aXJM001.

4. A method for constructing a CovS and CovR gene knockout mutant of Streptococcus agalactiae, characterized in that: Using the genomic DNA of Streptococcus agalactiae as a template, PCR was used to amplify the upstream and downstream fragments of the CovS / CovR coding gene; at the same time, using the pSet4s shuttle plasmid DNA as a template, PCR was used to amplify the spectinomycin resistance gene spc, and under the action of restriction endonucleases and T4 ligase, they were cloned into the multiple cloning site of the pUC19 vector in sequence to form a CovS / CovR gene knockout vector pUC19-CovSR with homologous sequences to the target gene on both sides of spc; pUC19-CovSR was electroporated into Ecoli DH5alphA competent bacteria were used to screen single colonies of Streptococcus agalactiae with dual resistance to Amp / spc. After expansion culture, genomic DNA was extracted, and a combined PCR verification method was used to confirm that the CovS / CovR coding gene had been replaced by spc, thereby obtaining the CovS / CovR gene knockout mutant strain SAΔCovS / CovR with spectinomycin resistance.

5. The CovS and CovR gene knockout mutants of Streptococcus agalactiae obtained by the construction method of claim 4.

6. Use of the CovS and CovR gene knockout mutants of Streptococcus agalactiae according to claim 5 in the preparation or screening of drugs for treating drug-resistant Streptococcus agalactiae infection.

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