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CN112961804B - Salmonella typhimurium and application thereof - Google Patents

  • ️Tue Nov 23 2021

CN112961804B - Salmonella typhimurium and application thereof - Google Patents

Salmonella typhimurium and application thereof Download PDF

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Publication number
CN112961804B
CN112961804B CN202110309093.6A CN202110309093A CN112961804B CN 112961804 B CN112961804 B CN 112961804B CN 202110309093 A CN202110309093 A CN 202110309093A CN 112961804 B CN112961804 B CN 112961804B Authority
CN
China
Prior art keywords
salmonella
gene
strain
salmonella typhimurium
mutated
Prior art date
2020-12-17
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110309093.6A
Other languages
Chinese (zh)
Other versions
CN112961804A (en
Inventor
杨小鹃
吴诗
杨诗沅
吴清平
张菊梅
丁郁
陈谋通
蔡淑珍
曾海燕
雷涛
古其会
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Microbiology of Guangdong Academy of Sciences
Original Assignee
Institute of Microbiology of Guangdong Academy of Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
2020-12-17
Filing date
2021-03-23
Publication date
2021-11-23
2021-03-23 Application filed by Institute of Microbiology of Guangdong Academy of Sciences filed Critical Institute of Microbiology of Guangdong Academy of Sciences
2021-06-15 Publication of CN112961804A publication Critical patent/CN112961804A/en
2021-11-23 Application granted granted Critical
2021-11-23 Publication of CN112961804B publication Critical patent/CN112961804B/en
Status Active legal-status Critical Current
2041-03-23 Anticipated expiration legal-status Critical

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Abstract

本发明涉及耐药菌株技术领域，尤其涉及一种鼠伤寒沙门菌及其应用。本发明的鼠伤寒沙门菌同时携带四个喹诺酮类药物耐药突变位点，四个喹诺酮类药物耐药突变位点分别为：gyrA基因编码产物的第83位的丝氨酸突变为苯丙氨酸；gyrA基因编码产物的第87位的天冬氨酸突变为天冬酰胺；parC基因编码产物的第80位的丝氨酸突变为精氨酸；parE基因编码产物的第416位的亮氨酸突变为苯丙氨酸。这是首次发现四个喹诺酮类药物耐药突变位点同时存在于一株鼠伤寒沙门菌菌株中，该菌株在国内外均未见报道。这些突变位点导致沙门菌菌株对喹诺酮类药物表现为高水平耐药。本发明的沙门菌可作为筛选新型抗菌药物的模型材料，具有良好的应用前景。

Figure 202110309093

The invention relates to the technical field of drug-resistant strains, in particular to a Salmonella typhimurium and its application. The Salmonella typhimurium of the present invention simultaneously carries four quinolone drug resistance mutation sites, and the four quinolone drug resistance mutation sites are respectively: serine at position 83 of the gyrA gene encoding product is mutated to phenylalanine; The aspartic acid at the 87th position of the gyrA gene was mutated to asparagine; the 80th position of the serine of the parC gene was mutated to arginine; the 416th leucine of the parE gene was mutated to benzene Alanine. This is the first time that four quinolone drug resistance mutation sites are found in a Salmonella typhimurium strain, which has not been reported at home and abroad. These mutation sites result in Salmonella strains exhibiting high levels of resistance to quinolones. The Salmonella of the invention can be used as a model material for screening new antibacterial drugs, and has a good application prospect.

Figure 202110309093

Description

Salmonella typhimurium and application thereof

Technical Field

The invention relates to the technical field of drug-resistant strains, in particular to salmonella typhimurium and application thereof.

Background

Salmonella (Salmonella spp) is a zoonosis pathogenic bacterium with important public health, can cause a plurality of serious diseases such as typhoid fever, paratyphoid fever, gastroenteritis, septicemia and the like of human and animals, and causes huge economic loss in the world. Currently, about 2610 serotypes have been found worldwide. Among them, Salmonella Typhimurium (Salmonella enterica subsp. enterica serovar Typhimurium) is the predominant serotype present in livestock and food, and is also the major serotype responsible for human infection.

Quinolone drugs, especially fluoroquinolone drugs, are the last important class of drugs to be selected for the treatment of potentially life-threatening infections caused by multidrug-resistant salmonella. With the wide clinical and production applications of these drugs, the problem of drug resistance of bacteria to these drugs is becoming more serious. Research shows that the drug resistance of the salmonella to quinolone drugs rises year by year, and the drug resistance rate of the salmonella typhimurium is generally higher than that of other salmonella.

The main mechanism of the salmonella for generating drug resistance to the quinolone drugs is that DNA helicases (coding genes: gyrA and gyrB) and topoisomerase IV (coding genes: parC and parE) at target sites of action of the quinolone drugs generate gene mutation, so that the enzymes generate structural change, and the combination of the enzymes and the quinolone drugs is influenced. gyrA, gyrB, parC and parE are also known as quinolone resistance-determining regions (QRDR). In addition, plasmid-mediated quinolone drug resistance mechanisms (PMQR), including QNR proteins (encoding genes: qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC), efflux pump transporters (encoding genes: qepA and oqxAB), and acetyl transferase (encoding genes: aac (6') -Ib-cr) are also responsible for the decrease in the sensitivity of bacteria to quinolone drugs.

The degree of resistance of salmonella to quinolone drugs is generally related to the position and number of mutation sites. Single site mutations can cause low levels of resistance, and if 2 or more mutations occur simultaneously, they can result in the production of high levels of resistant strains. At present, in the reports about salmonella QRDR mutant strains, single-site mutation is mainly used, and a plurality of mutations occur simultaneously, so that high-level drug-resistant strains are rare.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides salmonella typhimurium and application thereof, and the salmonella carrying four quinolone drug resistance mutation sites can be used as a model material for screening functional microorganisms/novel antibacterial drugs, and has a good application prospect.

In order to achieve the purpose, the invention adopts the technical scheme that: providing salmonella typhimurium, wherein the salmonella typhimurium simultaneously carries four quinolone drug resistance mutation sites, and the four quinolone drug resistance mutation sites are respectively as follows:

the 83 th serine of the encoded product of the gyrA gene is mutated into phenylalanine;

the aspartic acid at the 87 th site of the gyrA gene coding product is mutated into asparagine;

serine at position 80 of the encoded product of the parC gene was mutated to arginine;

the leucine amino acid at position 416 of the product encoded by the parE gene was mutated to phenylalanine.

As a preferred embodiment of the salmonella typhimurium of the present invention, the four quinolone drug resistance mutation sites are specifically:

the 324 th base C of the gyrA gene is mutated into T;

the 335 th base G of the gyrA gene is mutated into A;

the 253 rd base A of the parC gene is mutated into C;

the 1246 th base C of the parE gene was mutated to T.

As a preferred embodiment of the Salmonella typhimurium of the present invention, the NCBI accession number of the gyrA gene is AE006468 REGION:2373710..2376422, the NCBI accession number of the parC gene is AE006468 REGION:3336954..3339227, and the NCBI accession number of the parE gene is AE006468 REGION:3343969.. 3345861.

As a preferred embodiment of the Salmonella Typhimurium of the present invention, the Salmonella Typhimurium is Salmonella Typhimurium (Salmonella enterica subsp. enterica serovar Typhimurium) Sal9541, which is classified and named as Salmonella Typhimurium (Salmonella enterica subsp. enterica serovar Typhimurium), and has been deposited at the southern east province collection center at 9/30 of 2020, deposition address: guangzhou city, Jielizhou 100 large yard, building 59, floor 5, the preservation number: GDMCC No: 61225.

the invention discovers that a strain simultaneously carries four drug-resistant mutation sites gyrA of quinolone drugsS83F、gyrAD87NAnd parCS80ROf Salmonella typhimurium, gy of the strainThe rA gene coding product is subjected to double mutation: ser83 → Phe (serine mutated to phenylalanine, abbreviated S83F), Asp87 → Asn (aspartic acid mutated to asparagine, abbreviated D87N), while the parC gene-encoded product undergoes a single-site mutation (Ser80 → Arg) (serine mutated to arginine, abbreviated S80R), and the parE gene-encoded product undergoes a single-site mutation (Leu416 → Phe) (leucine mutated to phenylalanine, abbreviated L416F). Wherein parEL416FIs the first reported new mutation site found in the present invention. The method discovers four drug-resistant mutation sites gyrA of quinolone drugs for the first timeS83F、gyrAD87N、parCS80RAnd parEL416FMeanwhile, the strain exists in a salmonella typhimurium strain, and the strain is not reported at home and abroad. Meanwhile, the strain does not have any plasmid-mediated quinolone drug resistance gene. Therefore, the mutant sites cause the strain to show high-level drug resistance to quinolone drugs, and the minimum inhibitory concentrations of nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin to the salmonella Sal9541 strain are 4096 mu g/mL, 16 mu g/mL, 8 mu g/mL and 8 mu g/mL respectively.

The salmonella Sal9541 of the invention is gram-negative brevibacterium, API 20E is identified as salmonella, and the coincidence rate is 77.8%. The biochemical characteristics are as follows: arginine double hydrolase, lysine decarboxylase and ornithine decarboxylase are positive, citrate is decomposed, hydrogen sulfide is generated, glucose, mannitol, sorbitol, rhamnose, arabinose, urease, phenylalanine deaminase, oxidase, ONPG test, indole test and VP test are negative, gelatin cannot be liquefied, inositol, sucrose, melibiose and amygdalin are not fermented, and the salmonella-type bacillus strain has typical physiological and biochemical characteristics. The serum antigen is identified as 1,4,5,12: i:1,2, a typical serotype of Salmonella typhimurium.

The Salmonella Sal9541 of the present invention can be cultured in LB, BHI and NA media.

The invention also provides a microbial agent, which comprises the salmonella typhimurium.

The invention also provides application of the salmonella typhimurium in screening a model strain of a novel antibacterial drug.

The invention has the beneficial effects that:

the invention provides a gyrA simultaneously carrying four drug-resistant mutation sites of quinolonesS83F、gyrAD87N、parCS80RAnd parEL416FSalmonella Sal 9541. The simultaneous existence of a plurality of mutation sites causes the strain to show high-level drug resistance to quinolone drugs, and the minimum inhibitory concentrations of nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin to the salmonella Sal9541 strain are 4096 mu g/mL, 16 mu g/mL, 8 mu g/mL and 8 mu g/mL respectively. Therefore, the salmonella Sal9541 can be used as a model material for screening functional microorganisms/novel antibacterial drugs, and has a good application prospect.

Drawings

FIG. 1: a colonial morphology of the salmonella Sal9541 strain of the invention.

FIG. 2: the morphology of the salmonella Sal9541 strain of the invention is observed by microscopic examination.

FIG. 3: API 20E biochemical identification scheme of Salmonella Sal9541 strain of the present invention.

FIG. 4: the specific site of the gyrA gene mutation in the

salmonella Sal

9541; gyrA (324C → T, Ser83 → Phe; 335G → A, Asp87 → Asn).

FIG. 5: the specific site of parC gene mutation in the

salmonella Sal

9541; parC (253A → C, Ser80 → Arg).

FIG. 6: the parE gene in the salmonella Sal9541 has a specific site of mutation; parE (1246C → T, Leu416 → Phe).

Detailed Description

To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention.

EXAMPLE 1 isolation of the strains

The salmonella Sal9541 is separated from chicken leg meat food in a supermarket of Lanzhou city, China, and the collected sample is detected according to food safety national standard food microbiology inspection salmonella GB 4789.4-2010. Sampling 25g (mL) and adding 225mL Buffer Peptone Water (BPW) in a sterile homogenizing bag for homogenizing and shaking, and culturing for 8-18 h at 37 ℃. Gently shaking the cultured sample mixture, inoculating 1mL of the cultured sample mixture into 10mL of sodium tetrasulfate brilliant green (TTB) enrichment solution, culturing at 42 ℃ for 18 h-24 h, and inoculating 1mL of the cultured sample mixture into 10mL of Selenite Cystine (SC) enrichment solution, and culturing at 37 ℃ for 18 h-24 h. Taking

enrichment liquid

1 ring by using an inoculating ring respectively, streaking and inoculating the enrichment liquid to a salmonella chromogenic medium plate, culturing the enrichment liquid for 18-24h at 37 ℃, and observing bacterial colonies growing on the plate. Typical salmonella colonies were purple, round, moist, flat-edged on the chromogenic plates (fig. 1). The target colonies were transferred from Nutrient Agar (NA) plates into brain heart infusion Broth (BHI) and incubated overnight at 37 ℃. The bacterial solution was aseptically added to a glycerin tube having a final concentration of 50%, stored in a refrigerator at-40 ℃ and freeze-dried in a tube, to thereby obtain a

strain Sal

9541.

Example 2 identification and cultivation of Strain Sal9541

The purified strain Sal9541 is subjected to morphological feature, physiological and biochemical identification, serotype identification and the like.

And (3) dyeing microscopic examination: the colonies were smeared, gram stained, and the morphology was visualized by microscopic examination. Salmonella was gram-negative, short rod-shaped (fig. 2).

API 20E identifies: individual colonies were scraped from the NA plate, prepared as a cell suspension of appropriate turbidity with physiological saline, and characterized using an API 20E biochemical identification reagent strip (FIG. 3).

Serotype identification: serotyping the separated strain of salmonella by adopting a slide agglutination method. Checking the thallus antigen (O antigen) of salmonella, then determining the I phase and II phase flagellum antigen (H antigen) of the strain in turn, and finally referring to the salmonella diagnosis antigen table to make serotype diagnosis.

The result shows that the strain Sal9541 is gram-negative brevibacterium, and API 20E is identified as salmonella, and the coincidence rate is 77.8%. The biochemical characteristics are as follows: arginine double hydrolase, lysine decarboxylase and ornithine decarboxylase are positive, citrate is decomposed, hydrogen sulfide is generated, glucose, mannitol, sorbitol, rhamnose, arabinose, urease, phenylalanine deaminase, oxidase, ONPG test, indole test and VP test are negative, gelatin cannot be liquefied, inositol, sucrose, melibiose and amygdalin are not fermented, and the salmonella-type bacillus strain has typical physiological and biochemical characteristics. The serum antigen is identified as 1,4,5,12: i:1,2, a typical serotype of Salmonella typhimurium.

The appearance, morphology, gram stain, biochemical reaction and serological reaction of the strain Sal9541 can be comprehensively judged to identify the strain Sal9541 as a Salmonella enterica subsp.

Salmonella sali 9541 was deposited at the guangdong province collection of microorganisms (GDMCC) at 9/30 of 2020, address: guangzhou city, Jielizhou 100 large yard, building 59, building 5, the number of deposit is: GDMCC No: 61225.

example 3 drug sensitivity profiling

The broth dilution method was used to examine the levels of resistance of the Salmonella Sal9541 strain to quinolone drugs, including nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin, according to the methods and results criteria of the American Clinical Laboratory Standards Institute (CLSI) version 2018. Inoculating Salmonella Sal9541 strain in a test tube containing 4 mM MH broth, incubating to logarithmic phase, and turbidifying with 0.5M turbidimetric tube to adjust the concentration of the bacterial liquid to 1 × 107About cfu/mL. Taking the bacterial liquid, and mixing the bacterial liquid with fresh MH broth according to the weight ratio of 1: diluting with 200 deg.C, and mixing. Selecting a sterile 96-hole flat-bottom microplate, adding 100 mu L of MH broth culture medium into the 1 st hole of each row, then adding 100 mu L of the drug to be detected into the 1 st hole of each row, uniformly mixing, taking out 100 mu L, moving to the 2 nd hole, and repeating the steps until the 12 th hole is uniformly mixed, sucking 100 mu L and discarding. Finally, the mixed salmonella Sal9541 (1X 10) of the invention is added into each hole5cfu/mL) of the suspension. Selecting a proper hole site and adding 200 mu L MH broth culture medium as a negative control; as positive controls, 100. mu.L of Salmonella Sal9541 broth and 100. mu.L of MH broth were added. Each group was run in parallel 3 times. Placing the culture plate in a 37 ℃ incubatorCulturing for 18-20h, and determining MIC value. And taking out the culture plate from the incubator, and reading the OD value by using an enzyme-labeling instrument to obtain a drug sensitivity result and an analysis report. Or judging the positive and negative results of each hole by naked eyes, wherein the turbidity is positive and the clarity is negative. Experiments prove that the Minimum Inhibitory Concentrations (MICs) of nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin on salmonella Sal9541 strain are 4096 mu g/mL, 16 mu g/mL, 8 mu g/mL and 8 mu g/mL respectively. The results of the specific drug sensitivity of the Salmonella Sal9541 strain are shown in Table 1.

TABLE 1 resistance of Salmonella Sal9541 strains to quinolone drugs

Figure BDA0002989084610000061

Example 4 detection of quinolone drug resistance Gene of Salmonella Sal9541 Strain

(1) Whole genome second-generation sequencing and Blast sequence analysis target drug resistance gene

Whole genome secondary sequencing was performed on salmonella Sal9541 strain. The genomic DNA was fragmented into 400bp fragments using a Covaris M200 sonicator and Library construction was performed using the Ion Plus Fragment Library Kit. Whole genome sequencing was performed using an Ion Torrent S5 sequencer. Genomic de novo assembly was performed by SPAdes v3.6.2. Meanwhile, the genome sequence after splicing is subjected to genome component prediction such as coding gene, tRNA, rRNA and the like by using prokka, and coding gene function annotation is carried out.

And detecting the drug resistance gene of the quinolone drugs by using a local Blast sequence analysis technology. Local Blast analysis: constructing a local database db.nt (9541, ffn is a name of a strain whole genome sequencing sequence information document and 9541db.nt is a database name constructed) from a whole genome sequencing sequence information document of a salmonella strain Sal9541 strain by using an operation command ' makeblastdb-in 9541, ffn-dbype nucleus-pars _ seqids-out 9541 d.nt ' (9541, ffn is a name of a database constructed), and then targeting sequence information documents of target sites (QRD genes: gyrA, gyrB, parC, parE; PMQR genes: qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC, aac (6') -Ib-cr, qep A, oxqAB) (in the case of analyzing the gyrA gene, gyrA.txt is a sequence information document of the target gene), blast analysis of the gyrA gene (outgyrAresult is the name of the output result) is performed on the local database 9541db.nt with the operation command "blastn-db 9541db.nt-evalue 1e-5-outfmt 0-num _ descriptions s 10-num _ threads 64-query gyra.txt-outgyrAresult". And finally, checking the output file and judging the result.

(2) PCR amplification target drug resistance gene and Sanger method first generation sequencing analysis

The detection results of the target drug resistance gene of the whole genome sequencing and Blast sequence analysis of the strain are further verified by adopting a PCR amplification and first-generation sequencing analysis method. And designing a PCR amplification primer of the target drug-resistant gene according to the published gene sequence, wherein the primer sequence is shown in Table 2. The PCR amplification reaction system (25. mu.L) was performed by single PCR: 12.5 mu L of 2 XPremix Taq, 120nmol/L of each of the upstream primer and the downstream primer, 2 mu L of template and complete ultra-pure water; PCR amplification reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 45s, annealing at 55-60 ℃ for 45s, and extension at 72 ℃ for 45s for 30 cycles; extension at 72 ℃ for 10 min. And carrying out electrophoresis detection (120V, 30min), purification and first-generation sequencing analysis on the amplified fragment to obtain the sequence information of the amplified target gene. Performing Blast analysis and result judgment on the sequencing result. The PCR amplification primers for each target gene designed in the research are as follows:

TABLE 2 Salmonella gyrA, parC, parE primers and amplified fragment sizes

Figure BDA0002989084610000071

Figure BDA0002989084610000081

(3) Detection result of drug resistance gene of quinolone drugs of salmonella Sal9541 strain

Comparing with a salmonella typhimurium standard strain LT2 gene sequence, and detecting mutations in gyrA, parC and parE in a salmonella Sal9541 strain QRDR gene; the gyrA gene codes a product and is subjected to double mutation: ser83 → Phe (serine mutated to phenylalanine, abbreviated S83F), Asp87 → Asn (aspartic acid mutated to asparagine, abbreviated D87N), while the parC gene-encoded product undergoes a single-site mutation (Ser80 → Arg) (serine mutated to arginine, abbreviated S80R), and the parE gene-encoded product undergoes a single-site mutation (Leu416 → Phe) (leucine mutated to phenylalanine, abbreviated L416F). Whereas PMQR genes (qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC, aac (6') -Ib-cr, qepA, oxqAB) were not detected at all. And further verifying by adopting PCR amplification and Sanger method generation sequencing, wherein the detection results of the two methods are consistent.

The amino acid symbols have the meaning: serine (Serine, abbreviated to Ser or S), Phenylalanine (Phenylalanine, abbreviated to Phe or F), Asparagine (aspartic, abbreviated to Asn or N), Arginine (argine, abbreviated to Arg or R), Leucine (Leucine, abbreviated to Leu or L).

Details of the mutations detected by gyrA, parC and parE in QRDR genes of Salmonella Sal9541 strain are shown in FIGS. 4-6.

Example 5 application of quinolone resistant Salmonella Sal9541 Strain

The specific application method of the salmonella Sal9541 is mainly embodied in two aspects:

(1) the salmonella Sal9541 simultaneously carries four quinolone drug resistance mutation sites gyrAS83F、gyrAD87N、parCS80RAnd parEL416FThe four quinolone antibacterial drugs (nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin) have high-level resistance, particularly ciprofloxacin is considered to be a good drug for treating infection caused by salmonella, and the effect is always remarkable in recent years. Wherein parEL416FIs the first reported new mutation site found in the present invention. The gyrA of four drug-resistant mutation sites of the quinolone drugs is also found for the first timeS83F、gyrAD87N、parCS80RAnd parEL416FMeanwhile, the strain exists in a salmonella typhimurium strain, and the strain is not reported at home and abroad. The occurrence of multiple mutations in the quinolone drug resistance determining region gene in the same strain, for the study of the drug resistance mechanism of quinolone drugs, theThe research on a new strategy for treating salmonella infection has important guiding significance and can be used as an important material for seeking a drug resistance mechanism of bacteria.

(2) The salmonella Sal9541 simultaneously carries four quinolone drug resistance mutation sites gyrAS83F、gyrAD87NAnd parCS80RThe strain can generate high-level drug resistance to four quinolone antibacterial drugs (nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin), and can also be used as an important model strain for screening novel antibacterial drugs.

The screening method of the antibacterial drug comprises the following steps:

precisely weighing a certain amount of the drug to be detected, dissolving the drug with a proper solvent, and preparing the drug into a solution of 30mg/mL for later use. Then, a solution with a concentration of 2000. mu.g/mL is prepared by using a suitable solvent, and the solution is used for determining the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) and is stored in a refrigerator at-40 ℃ for later use. One day before the experiment, the salmonella Sal9541 of the invention stored in a refrigerator at-40 ℃ is taken out, and is placed to the room temperature, a small amount of bacterial colonies are respectively picked by inoculating loops, and are respectively inoculated on MH agar culture media, and are cultured in a constant temperature box at 37 ℃ for 18-24 h. On MH agar medium with newly grown colony, selecting small amount of activated colony with inoculating loop, diluting with sterile normal saline to prepare 1 × 107cfu/mL of bacterial suspension (standard turbiditube control) for use.

Screening the drug to be tested which is sensitive to the drug-resistant strain, and determining the sensitivity of the strain to different drugs according to the paper diffusion method recommended by the American Clinical Laboratory Standards Institute (CLSI). Dipping prepared standard bacteria with a concentration of 1 × 10 with a sterile cotton swab7cfu/mL of bacterial suspension, using a coating rod to evenly spread to the corresponding MH agar medium. Placing the sterilized Oxford cup on the coated MH agar culture medium, and adding 50 microliter of prepared liquid medicine (30mg/mL) into the Oxford cup by using a micropipettor; culturing in 37 deg.C incubator for 18-24 hr, and measuring the size of antibacterial zone. An equal volume of MH broth was taken and vehicle was added as a blank control.

Screening for drug sensitive to drug resistant strains according to the clinical laboratory standards Committee (CLSI) protocolThe recommended broth dilution method was used to determine the MIC values of the strains for different drugs, and each group was run in parallel 3 times to obtain the concentration averages. When determining MIC value, 1X 10 of the total amount of the mixture was mixed with sterile physiological saline7The bacterial suspension was further diluted to a concentration of 1X 10 cfu/mL5cfu/mL. Selecting a sterile 96-hole flat-bottom microplate, adding 100 mu L of MH broth culture medium into the 1 st hole of each row, then adding 100 mu L of the drug to be detected into the 1 st hole of each row, uniformly mixing, taking out 100 mu L, moving to the 2 nd hole, and repeating the steps until the 12 th hole is uniformly mixed, sucking 100 mu L and discarding. Finally, the mixed salmonella Sal9541 (1X 10) of the invention is added into each hole5cfu/mL) of the suspension. Selecting a proper hole site and adding 200 mu L MH broth culture medium as a negative control; as positive controls, 100. mu.L of Salmonella Sal9541 broth and 100. mu.L of MH broth were added. The culture plate is placed in an incubator at 37 ℃ for 18-20h to observe the result, and the MIC value of the culture plate is determined. And taking out the culture plate from the incubator, and reading the OD value by using an enzyme-labeling instrument to obtain a drug sensitivity result and an analysis report. Or judging the positive and negative results of each hole by naked eyes, wherein the turbidity is positive and the clarity is negative. When the MIC value is determined, the mixture of bacteria and MH broth culture medium 3-5 holes before the MIC value is taken and inoculated on MH agar medium, the mixture is placed in an incubator at 37 ℃, the mixture is taken out after being cultured for 22h and observed, and the lowest drug concentration with the average value less than 5 is determined as the MBC of the compound.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> institute of microbiology, academy of sciences of Guangdong province (center for microbiological analysis and detection of Guangdong province)

<120> Salmonella typhimurium and application thereof

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 1

gcgaccttgc gagagaaatt 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 2

ggcagccgtt aatcacttcc 20

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<212> DNA

<213> Artificial Synthesis

<400> 3

ggatcccctg ttaatgagcg 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 4

cgggatatct gttgccatgc 20

<210> 5

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<212> DNA

<213> Artificial Synthesis

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<211> 20

<212> DNA

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Claims (3)

1. A Salmonella typhimurium characterized in that it is Salmonella typhimurium (I)Salmonella enterica subsp. enterica serovar Typhimurium) Sal9541, which is classified and named as Salmonella Typhimurium (A)Salmonella enterica subsp. enterica serovar Typhimurium), which has been deposited at the Guangdong province Collection of microorganisms at 30 months 9 and 2020, at the deposition site: guangzhou city, Jielizhou 100 large yard, building 59, floor 5, the preservation number: GDMCC No: 61225.

2. a microbial agent, comprising the salmonella typhimurium of claim 1.

3. Use of the salmonella typhimurium of claim 1 as a model strain for screening novel antibacterial agents.

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CN114214238B (en) * 2021-12-23 2023-12-05 广东省科学院微生物研究所(广东省微生物分析检测中心) Multi-drug-resistant Indiananas and application thereof
CN114250181B (en) * 2021-12-23 2023-12-05 广东省科学院微生物研究所(广东省微生物分析检测中心) Indiananas with five quinolone drug resistance mutation sites and application thereof

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