CN113913318B - Salmonella typhimurium carrying four quinolone drug-resistant mutation sites simultaneously and application thereof - Google Patents

Salmonella typhimurium carrying four quinolone drug-resistant mutation sites simultaneously and application thereof Download PDF

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CN113913318B
CN113913318B CN202011495870.2A CN202011495870A CN113913318B CN 113913318 B CN113913318 B CN 113913318B CN 202011495870 A CN202011495870 A CN 202011495870A CN 113913318 B CN113913318 B CN 113913318B
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salmonella typhimurium
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杨小鹃
黄嘉慧
吴清平
张菊梅
吴诗
柏建玲
王涓
薛亮
庞锐
韦献虎
张友雄
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Guangdong Detection Center of Microbiology of Guangdong Institute of Microbiology
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Abstract

The invention relates to the technical field of drug-resistant strains, in particular to salmonella typhimurium carrying four quinolone drug-resistant mutation sites and application thereof. The salmonella typhimurium carries four quinolone drug resistance mutation sites, and the four quinolone drug resistance mutation sites are respectively: serine at 83 of the gyrA gene coding product is mutated into phenylalanine; aspartic acid at position 87 of the coded product of gyrA gene is mutated into glycine; serine at position 80 of the parC gene coding product is mutated into arginine; serine 458 of the parE gene coding product is mutated to proline. These mutation sites result in the salmonella strain exhibiting high levels of resistance to quinolones. In view of the above, sal17241 of Salmonella can be used as a model material for screening novel antibacterial drugs, and has good application prospect.

Description

Salmonella typhimurium carrying four quinolone drug-resistant mutation sites simultaneously and application thereof
Technical Field
The invention relates to the technical field of drug-resistant strains, in particular to salmonella typhimurium carrying four quinolone drug-resistant mutation sites and application thereof.
Background
Salmonella spp is a zoonotic pathogen with important significance in public health, and can cause a plurality of serious diseases such as typhoid, paratyphoid, gastroenteritis, septicemia and the like of people and animals, so that huge economic losses are suffered worldwide. Currently, about 2610 serotypes have been found worldwide. Among them, salmonella typhimurium (Salmonella enterica subsp. Enterica serovar Typhimurium) is a dominant serotype existing in livestock and poultry and foods, and is also a main serotype causing human infection.
Quinolones, particularly fluoroquinolones, are a last-selectable class of important drugs for the treatment of potentially life-threatening infections caused by multidrug-resistant salmonella. With the wide application of the medicines in clinic and production, the problem of drug resistance of bacteria to the medicines is increasingly serious. Research shows that the drug resistance of salmonella to quinolones rises year by year, while the drug resistance rate of salmonella typhimurium is generally higher than that of other salmonella.
The most important mechanism of the salmonella for generating drug resistance to quinolone drugs is that DNA helicase (coding genes: gyrA and gyrB) and topoisomerase IV (coding genes: parC and parE) at target sites of action of the quinolone drugs are subjected to gene mutation, so that the structure of the enzyme is changed, and the combination with the quinolone drugs is affected. gyrA, gyrB, parC and parE are also known as quinolone resistance determining regions (quinolone resistance-determining regions, QRDR). In addition, plasmid-mediated quinolone drug resistance mechanisms (plasmid-mediated quinolone resistance, PMQR), including the QNR protein (coding gene: qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC), efflux pump transporters (coding genes: qepA and oqxAB), and acetyltransferases (coding genes: aac (6') -Ib-cr) are also responsible for the reduced susceptibility of bacteria to quinolones.
The extent of resistance of salmonella to quinolones is generally related to the location and number of mutation sites. Single site mutations can cause low levels of resistance, and if 2 or more mutations occur simultaneously, high levels of resistant strains can result. Currently, there have been reports on salmonella QRDR mutant strains, in which single site mutation is the dominant, and multiple mutations occur simultaneously, resulting in a relatively rare high-level drug-resistant strain.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides salmonella typhimurium carrying four quinolone drug-resistant mutation sites and application thereof, and the salmonella typhimurium can be used as a model material for screening functional microorganisms/novel antibacterial drugs, and has good application prospect.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the salmonella typhimurium is provided, and carries four quinolone drug resistance mutation sites at the same time, wherein the four quinolone drug resistance mutation sites are respectively:
serine at 83 of the gyrA gene coding product is mutated into phenylalanine;
aspartic acid at position 87 of the coded product of gyrA gene is mutated into glycine;
serine at position 80 of the parC gene coding product is mutated into arginine;
serine 458 of the parE gene coding product is mutated to proline.
As a preferred embodiment of the Salmonella typhimurium of the present invention, the gyrA gene has NCBI accession number AE006468 REGION 2373710..2376422, the parC gene has NCBI accession number AE006468 REGION 3336954..3339227, and the parE gene has NCBI accession number AE006468 REGION 3343969..3345861.
As a preferred embodiment of the Salmonella typhimurium of the present invention, salmonella typhimurium (Salmonella enterica subsp. Enterica serovar Typhimurium) Sal17241, which was classified as Salmonella typhimurium (Salmonella enterica subsp. Enterica serovar Typhimurium), was deposited in the microorganism strain deposit center of Guangdong province at 9 months 30 in 2020, the deposit address: guangzhou city first middle road 100 # college 59 # building 5, deposit number: GDMCC No:61226.
the invention discovers that a strain carries four quinolone drug-resistant mutation sites gyrA simultaneously S83F 、gyrA D87G 、parC S80R And parE S458P The salmonella typhimurium, the gyrA gene coding product of the strain has double mutation: ser83→phe (serine mutation to phenylalanine, abbreviated as S83F), asp87→gly (aspartic acid mutation to glycine, abbreviated as D87G), single site mutation of the parC gene coding product (Ser 80→arg) (serine mutation to arginine, abbreviated as S80R), and single site mutation of the parE gene coding product (Ser 458→pro) (serine mutation to proline, abbreviated as S458P). This is the first discovery of four quinolone drug-resistant mutation sites gyrA S83F 、gyrA D87G 、parC S80R And parE S458P Meanwhile, 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 mutation sites cause the strain to show high-level drug resistance to quinolones, and the minimum antibacterial concentration of nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin to salmonella Sal17241 strain is 8192 mug/mL, 32 mug/mL, 16 mug/mL and 8 mug/mL respectively.
The Salmonella Sal17241 of the invention is gram negative and Brevibacterium, and the API 20E is identified as Salmonella, and the coincidence rate is 89.4%. 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, melibiose, arabinose, urease, phenylalanine deaminase, oxidase, ONPG test, indole test and VP test are negative, gelatin cannot be liquefied, inositol, sucrose and amygdalin cannot be fermented, and the biological and biochemical characteristics of salmonella are typical. The serum antigenicity was identified as 1,4,5,12:i:1,2, which is a typical Salmonella typhimurium serotype.
Salmonella Sal17241 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 mode strains of novel antibacterial drugs.
The invention has the beneficial effects that:
the invention provides a novel quinolone medicine which simultaneously carries four drug-resistant mutation sites gyrA S83F 、gyrA D87G 、parC S80R And parE S458P Salmonella enterica Sal17241. The simultaneous existence of a plurality of mutation sites causes the strain to show high-level drug resistance to quinolone medicines, and the minimum antibacterial concentration of nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin to salmonella Sal17241 strains is 8192 mug/mL, 32 mug/mL, 16 mug/mL and 8 mug/mL respectively. In view of the above, sal17241 of Sal can be used as a model material for screening functional microorganisms/novel antibacterial drugs, and has good application prospect.
Drawings
Fig. 1: colony morphology of Salmonella strain Salmonella 17241 of the present invention.
Fig. 2: morphological images were observed by microscopic examination of Sal17241 strain of Salmonella of the present invention.
Fig. 3: the biochemical identification schematic of the API 20E of the Salmonella Sal17241 strain of the invention.
Fig. 4: the invention relates to a specific locus for the mutation of gyrA gene in Sal17241 of salmonella; gyrA (324 C.fwdarw.T, ser 83.fwdarw.Phe; 336 A.fwdarw.G, asp87.fwdarw.Gly).
Fig. 5: the specific site of the parC gene mutation in Sal17241 of the salmonella; parC (253A. Fwdarw.C, ser 80. Fwdarw.Arg).
Fig. 6: the specific site of the parE gene mutation in Sal17241 of the salmonella; parE (1372 T→C, ser458→Pro).
Detailed Description
In order to more clearly describe the technical solution of the present invention, the following description is further given by way of specific examples, but not by way of limitation, only some examples of the present invention.
EXAMPLE 1 isolation of strains
Sal17241 is separated from fresh pork food in a supermarket in Nanning China, and the collected sample is detected, wherein the detection method refers to salmonella for food safety national standard food microbiology inspection GB 4789.4-2010. 25g (mL) of the sample was taken, homogenized in a sterile homogenizing bag containing 225mL of Buffered Peptone Water (BPW), and incubated at 37℃for 8-18 h. The cultured sample mixture is gently shaken, 1mL of the sample mixture is transferred into 10mL of sodium sulfotetrasulfonate brilliant green (TTB) enrichment medium, the sample mixture is cultured for 18 to 24 hours at the temperature of 42 ℃, and simultaneously, 1mL of the sample mixture is transferred into 10mL of Selenite Cystine (SC) enrichment medium, and the sample mixture is cultured for 18 to 24 hours at the temperature of 37 ℃. Taking 1 loop of enrichment liquid by using an inoculating loop, streaking and inoculating the enrichment liquid to a salmonella chromogenic medium plate, culturing the enrichment liquid for 18 to 24 hours at 37 ℃ respectively, and observing colonies growing on the plate. The typical salmonella colonies were purple, round, moist, and edge-flattened on the chromogenic plate (fig. 1). Colonies of interest were transferred from Nutrient Agar (NA) plates to brain heart infusion nutrient Broth (BHI) and incubated overnight at 37 ℃. Under aseptic condition, the bacterial liquid is added into an glycerol pipe with the final concentration of 50 percent, stored in a refrigerator with the temperature of minus 40 ℃ and freeze-dried pipe storage is carried out, thus obtaining the bacterial strain Sal17241.
EXAMPLE 2 identification and cultivation of strains
The purified strain Sal17241 is identified in aspects of morphological characteristics, physiological biochemistry, serotypes and the like.
(1) Color dyeing and checking: colonies were smeared and gram stained and observed for morphology by microscopic examination. Salmonella was gram negative, short bar-shaped (fig. 2).
(2) API 20E authentication: individual colonies were scraped from NA plates, prepared into a cell suspension of appropriate turbidity with physiological saline and identified using API 20E biochemical identification kit (fig. 3).
(3) Serotype identification: the salmonella isolates were subjected to serotyping using slide agglutination. First, the bacterial antigen (O antigen) of Salmonella is checked, then the phase I and phase II flagella antigens (H antigen) of the strain are sequentially determined, and finally, the serotype diagnosis is made by referring to the Salmonella diagnosis antigen table.
The strain Sal17241 is gram negative and Brevibacterium, the API 20E is identified as Salmonella, and the coincidence rate is 89.4%. 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, melibiose, arabinose, urease, phenylalanine deaminase, oxidase, ONPG test, indole test and VP test are negative, gelatin cannot be liquefied, inositol, sucrose and amygdalin cannot be fermented, and the biological and biochemical characteristics of salmonella are typical. The serum antigenicity was identified as 1,4,5,12:i:1,2, which is a typical Salmonella typhimurium serotype.
The appearance, morphology, gram staining, biochemical reaction and serological reaction of the strain Sal17241 are comprehensively judged, and the strain Sal17241 can be identified as Salmonella enterica subspecies typhimurium serotype (Salmonella enterica subsp.enterica serovar Typhimurium) of Salmonella, and is named Salmonella Sal17241.
Salmonella Sal17241 was deposited at 30/9/2020 with the Guangdong province microbiological bacterial collection center (GDMCC) at the deposit address: guangzhou city first middle road 100 # college 59 # building 5, deposit number: GDMCC No:61226.
EXAMPLE 3 Salmonella Sal17241 drug sensitive profiling
Sand was examined using broth dilution according to the method and results judgment standard of the american clinical laboratory standardization committee (Clinical and Laboratory Standards institute, CLSI) version 2018The Sal17241 strain of the portal fungus has high and low resistance to quinolone drugs, and the drugs tested comprise nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin. The salmonella Sal17241 strain was inoculated into a tube containing 4mL MH broth, incubated to logarithmic phase, and turbidimetric with a 0.5 McMeter tube to give a bacterial suspension concentration of 1X 10 7 cfu/mL. Taking the bacterial liquid, and using fresh MH broth according to the proportion of 1:200, diluting and uniformly mixing. Sterile 96-well flat bottom microplates were selected, 100 μl of MH broth medium was added to each row of wells 1, then 100 μl of the drug to be tested was added to each row of wells 1, mixed well, and 100 μl was removed and transferred to wells 2, and similarly 100 μl was aspirated and discarded after mixing well up to wells 12. Finally, the Salmonella of the present invention Salmonella 17241 (1×10) after being mixed uniformly is added into each well 5 cfu/mL) of 100. Mu.L of the bacterial suspension. Selecting a proper hole site, adding 200 mu L of MH broth culture medium as a negative control; as positive controls, 100. Mu.L of Sal17241 broth and 100. Mu.L of MH broth were added. Each group was run 3 times in parallel. The plates were incubated in an incubator at 37℃for 18-20h for observations and MIC values were determined. And taking out the culture plate from the incubator, and reading an OD value by using an enzyme-labeled instrument to obtain a drug sensitivity result and an analysis report. Or judging the negative and positive results of each hole by naked eyes, wherein the turbidity is positive and the clarity is negative.
The results showed that the minimum inhibitory concentrations (Minimal inhibitory concentration, MIC) of nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin against Sal17241 strain of Salmonella were 8192 μg/mL,32 μg/mL,16 μg/mL,8 μg/mL, respectively. The specific drug sensitivity results of Sal17241 strain of Salmonella are shown in Table 1.
TABLE 1 drug resistance of Sal17241 Strain of Sal to quinolones
Example 4 detection of quinolone drug resistance Gene of Sal17241 Strain of Sal
(1) Whole genome second generation sequencing and Blast sequence analysis target drug resistance gene
The salmonella Sal17241 strain was subjected to genome-wide second-generation sequencing. Genomic DNA was broken into 400bp fragments using a Covaris M200 sonicator and library construction work was performed using Ion Plus Fragment Library Kit. Whole genome sequencing was performed using Ion Torrent S5 sequencer. Genome de novo assembly was performed by SPAdes v 3.6.2. Meanwhile, prokka is used for predicting the genome components such as coding genes, tRNA, rRNA and the like of the spliced genome sequence, and performing functional annotation of the coding genes.
And detecting the drug resistance gene of the quinolone drugs by adopting a local Blast sequence analysis technology. Local Blast analysis: the method comprises the steps of constructing a local database db.nt by using an operation command ' makeblastdb-in 17241.Ffn-dbtype nucleic-service_seqids-out 17241db.nt ' by using a whole genome sequencing sequence information document of a Sal17241 strain of Sal (17241. Ffn is a whole genome sequencing sequence information document name of the strain, 17241db. Nt is a constructed database name), and then outputting a result of the local database 41. Nt by using a sequence information document of a target site (QRDR gene: gyrA, gyrB, parC, parE; PMQR genes: qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC, aac (6 ') -Ib-cr, qepA, oxqAB) as a target gene (using an analysis gyrA gene as an example, gyrA. Txt is a sequence information document of a target gene), and using an operation command ' blastn-db 17241db. Nt-eval fmt 0-5-dim_descum 10-num_pages 64-quegyra-group's data as a result of the local database 41. Mu. Ttry-gyrA. Ttuqunt. And finally, checking the output file and judging the result.
(2) PCR amplification target drug resistance gene and Sanger method first-generation sequencing analysis
And (3) further verifying the detection result of the target drug resistance gene by performing genome-wide second-generation sequencing and Blast sequence analysis on the strain by adopting a method of PCR amplification and Sanger method first-generation sequencing analysis. PCR amplification primers of the target drug-resistant genes are designed according to published gene sequences, and the primer sequences are shown in Table 1. The PCR amplification reaction system (25. Mu.L) was used by the method of single PCR: 12.5. Mu.L of 2 XPremix Taq, 120nmol/L of each of the upstream and downstream primers, 2. Mu.L of template, and ultra pure water; PCR amplification reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 45s, annealing at 55-60℃for 45s, extension at 72℃for 45s, and a total of 30 cycles; extending at 72℃for 10min. And (3) performing electrophoresis detection (120V, 30 min), purification and first-generation sequencing analysis on the amplified fragments to obtain the sequence information of the amplified target genes. Blast analysis and result judgment are carried out on the sequencing result. The PCR amplification primers for each target gene designed in the study are as follows:
TABLE 2 Salmonella gyrA, parC, parE primers and amplified fragment sizes
(3) Detection result of quinolone drug resistance gene of Sal17241 strain of salmonella
Comparing with the standard strain LT2 gene sequence of Salmonella typhimurium, detecting gyrA, parC, parE mutation in the QRDR gene of Salmonella Sal17241 strain; the gyrA gene coding product is subjected to double mutation: ser83→phe (serine mutation to phenylalanine, abbreviated as S83F), asp87→gly (aspartic acid mutation to glycine, abbreviated as D87G), single site mutation of the parC gene coding product (Ser 80→arg) (serine mutation to arginine, abbreviated as S80R), and single site mutation of the parE gene coding product (Ser 458→pro) (serine mutation to proline, abbreviated as S458P). The PMQR gene (qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC, aac (6') -Ib-cr, qepA, oxqAB) is not detected at all. Further adopting PCR amplification and Sanger method first generation sequencing to make verification, and making detection results of two methods be identical.
The meaning of the amino acid symbols is: serine (abbreviated Ser or S), phenylalanine (abbreviated Phe or F), aspartic acid (abbreviated Asp or D), glycine (abbreviated Gly or G), arginine (abbreviated Arginine, abbreviated Arg or R), proline (abbreviated Pro or P).
Details of the gyrA, parC, parE mutation detected in the QRDR gene of Sal17241 strain of Salmonella are shown in FIGS. 4-6, respectively.
EXAMPLE 5 application of quinolone-resistant Salmonella Sal17241 Strain
The specific application method of salmonella Sal17241 is mainly characterized in two aspects:
(1) Sal17241 of Sal and four quinolone drug-resistant mutation sites gyrA are carried simultaneously S83F 、gyrA D87G 、parC S80R And parE S458P High levels of resistance to four quinolone antibacterial drugs (nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin), particularly ciprofloxacin, are considered to be good drugs for treating infections caused by salmonella, and have been remarkable in recent years. The occurrence of multiple mutations of quinolone drug resistance determining region genes in the same strain has important guiding significance for discussing the drug resistance generating mechanism of quinolone drugs and researching a new strategy for treating salmonella infection, and can be used as an important material for seeking a bacterial drug resistance mechanism.
(2) Sal17241 of Sal and four quinolone drug-resistant mutation sites gyrA are carried simultaneously S83F 、gyrA D87G 、parC S80R And parE S458P The novel antibacterial agent can produce high-level drug resistance to four quinolone antibacterial drugs (nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin), and can also be used as an important mode strain for screening novel antibacterial drugs.
The screening method of the antibacterial drugs comprises the following steps:
precisely weighing a certain amount of medicine to be measured, dissolving the medicine with a proper solvent, and preparing a 30mg/mL solution for later use. Then preparing a solution with the concentration of 2000 mug/mL by using a proper solvent, and measuring the minimum inhibitory concentration (minimum inhibitory concentration, MIC) and the minimum bactericidal concentration (minimum bactericidal concentration, MBC) of the solution, and storing the solution in a refrigerator at the temperature of minus 40 ℃ for standby. The day before the experiment, the salmonella Sal17241 of the invention stored in a refrigerator at the temperature of minus 40 ℃ is taken out, and after the salmonella Sal17241 is cooled to the room temperature, a small amount of bacterial colonies are respectively picked up by an inoculating loop, respectively inoculated on MH agar medium, and cultured for 18 to 24 hours in a constant temperature cabinet at the temperature of 37 ℃. On MH agar medium with newly grown colony, small amount of activated colony is selected by inoculating loop, diluted with sterile physiological saline to obtain 1×10 7 cfu/mL of bacterial suspension (standard turbidimetric tube control) is preparedIs used.
The drug to be tested sensitive to the drug resistant strain was screened and the sensitivity of the strain to the different drugs was determined according to the paper diffusion method recommended by the american Clinical Laboratory Standardization Institute (CLSI). Dipping the prepared standard bacteria concentration with a sterile cotton swab to be 1 multiplied by 10 7 cfu/mL of the bacterial suspension was spread evenly onto the corresponding MH agar medium with a spreading bar. Placing the sterilized oxford cup on the coated MH agar medium, and adding 50 mu L of the prepared liquid medicine (30 mg/mL) into the oxford cup by using a micropipette; culturing in a 37 deg.C incubator for 18-24 hr, and measuring the size of the inhibition zone. An equal volume of MH broth medium was taken and vehicle was added as a blank.
Screening for drug-resistant strains for drug-sensitive assays MIC values of the strains for the different drugs were determined according to the american Clinical Laboratory Standardization Institute (CLSI) recommended broth dilution method, and each group was run 3 times in parallel to obtain concentration averages. When MIC values were measured, 1X 10 portions were prepared with sterile physiological saline 7 The cfu/mL bacterial suspension is diluted again to a concentration of 1X 10 5 cfu/mL. Sterile 96-well flat bottom microplates were selected, 100 μl of MH broth medium was added to each row of wells 1, then 100 μl of the drug to be tested was added to each row of wells 1, mixed well, and 100 μl was removed and transferred to wells 2, and similarly 100 μl was aspirated and discarded after mixing well up to wells 12. Finally, the Salmonella of the present invention Salmonella 17241 (1×10) after being mixed uniformly is added into each well 5 cfu/mL) of 100. Mu.L of the bacterial suspension. Selecting a proper hole site, adding 200 mu L of MH broth culture medium as a negative control; as positive controls, 100. Mu.L of Sal17241 broth and 100. Mu.L of MH broth were added. The plates were incubated in an incubator at 37℃for 18-20h for observations and MIC values were determined. And taking out the culture plate from the incubator, and reading an OD value by using an enzyme-labeled instrument to obtain a drug sensitivity result and an analysis report. Or judging the negative and positive 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 in the 3-5 holes before the MIC value is taken, transferred onto MH agar medium, placed in an incubator at 37 ℃, and after 22 hours of culture, taken out for observation, and the minimum drug concentration with average number less than 5 is determined as MBC of the compound.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> Guangdong province microorganism institute (Guangdong province microorganism analysis and detection center)
<120> Salmonella typhimurium carrying four quinolone drug-resistant mutation sites simultaneously and application thereof
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<170> PatentIn version 3.3
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Claims (3)

1. The salmonella typhimurium is characterized in that the salmonella typhimurium carries four quinolone drug resistance mutation sites simultaneously, and the four quinolone drug resistance mutation sites are respectively:
gyrAserine at position 83 of the gene encoding product is mutated to phenylalanine;
gyrAaspartic acid at position 87 of the gene encoding product is mutated to glycine;
parCthe 80 th serine of the gene coding product is mutated into arginine;
parEserine 458 of the gene coding product is mutated into proline;
the salmonella typhimurium is salmonella typhimurium @Salmonella enterica subsp. enterica serovar Typhimurium) Sal17241, classified as Salmonella typhimuriumSalmonella enterica subsp. enterica serovar Typhimurium) has been deposited at 30.9 in 2020 with the cantonese collection of microorganism strains, accession number: guangzhou city first middle road 100 # college 59 # building 5, deposit number: GDMCC No:61226.
2. a microbial agent comprising salmonella typhimurium as defined in claim 1.
3. Use of salmonella typhimurium as defined in claim 1 as a model strain for screening novel antibacterial agents.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014170614A1 (en) * 2013-04-19 2014-10-23 Universite De Reims Champagne-Ardenne Method for characterising the chromosomal mutations of genes coding bacterial topoisomerases
WO2016024098A1 (en) * 2014-08-11 2016-02-18 Redx Pharma Plc Antibiotic-resistant bacteria and their uses
CN107034160A (en) * 2017-05-04 2017-08-11 中国农业科学院上海兽医研究所 The dual-gene gene-deleted strains of Salmonella typhimurtum aroA and luxS and its attenuated vaccine of preparation

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI349704B (en) * 2004-08-10 2011-10-01 Food And Drug Administration Dept Of Health A method for rapidly detecting quinolone-resistant salmonella spp. and the probes and primers utilized therein
WO2016020836A1 (en) * 2014-08-06 2016-02-11 Novartis Ag Quinolone derivatives as antibacterials
TWI778025B (en) * 2017-02-24 2022-09-21 碁米庫斯生物科技股份有限公司 Antibiotic testing and screening system

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014170614A1 (en) * 2013-04-19 2014-10-23 Universite De Reims Champagne-Ardenne Method for characterising the chromosomal mutations of genes coding bacterial topoisomerases
WO2016024098A1 (en) * 2014-08-11 2016-02-18 Redx Pharma Plc Antibiotic-resistant bacteria and their uses
CN107034160A (en) * 2017-05-04 2017-08-11 中国农业科学院上海兽医研究所 The dual-gene gene-deleted strains of Salmonella typhimurtum aroA and luxS and its attenuated vaccine of preparation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CM Bebear 等.Mutations in the gyrA,parC and parE genes addociated with fluoroquinolone resistance in clinical isolates of Mycoplasma hominis.Antimicrob agents chemother.1999,第954-956页. *
Molecular mechanisms of fluoroquinolone resistance in Enterobacteriaceae clinical isolates in Azerbaijan;Azagun R等;Gene reports;第21卷;第100924页 *
食源性沙门氏菌耐药性及质粒介导喹诺酮耐药基因检测;刘贵深 等;生物技术通报(第8期);第202-207页 *

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