CN114250181B - Indiananas with five quinolone drug resistance mutation sites and application thereof - Google Patents
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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Abstract
The invention relates to the technical field of drug-resistant strains, in particular to Indiananas carrying five quinolone drug-resistant mutation sites and application thereof. The invention provides a drug-resistant mutant site gyrA which simultaneously carries five quinolones S83F 、gyrA D87G 、parC T57S 、parC S80R And parE L502F Salmonella-Salmonella 51-38. 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 51-38 strains is 4096 mug/mL, 16 mug/mL, 4 mug/mL and 4 mug/mL respectively. In view of the above, salmonella 51-38 can be used as a model material for screening functional microorganisms/novel antibacterial drugs, and has good application prospects.
Description
Technical Field
The invention relates to the technical field of drug-resistant strains, in particular to Indiananas carrying five 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. It has now been found that 2600 more than one salmonella serotype, indiana (Salmonella enterica subsp. The first report in 1984 was that Ind. Ananadis was found in Guangdong, but only a few reports on indiananas have been made for a long period of time thereafter. In recent years, indiananas has been detected in patients, animals, foods and environments, chicken is the main host, and Indiananas has wide drug resistance, and the drug resistance range is in an increasing trend year by year.
Quinolone drugs are broad-spectrum antibacterial drugs, and the antibacterial effect is generated by identifying strain target proteins and affecting the replication of strain DNA. The reduced sensitivity of salmonella to quinolones is associated with altered targeting. The DNA helicase (encoding genes gyrA and gyrB) and topoisomerase IV (encoding genes parC and parE) of the quinolone drug resistance determining region (quinolone resistance-determining regions, QRDR) are subjected to gene mutation, so that the enzyme structure is changed, and the combination of the quinolone drugs is affected. Plasmid-mediated quinolone resistance gene (plasmid-mediated quinolone resistance, PMQR), comprising: qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC, qepA, oqxAB and aac- (6') -Ib-cr are also responsible for the reduced sensitivity of Salmonella 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 there are 2 or more mutation sites at the same time, this can lead to the production of high levels of resistant strains. Ser at position 83 in the GyrA gene is often mutated into Phe, tyr or Ala, asp at position 87 is often mutated into Gly, asn or Tyr; the 57 th Thr of ParC gene is usually mutated to Ser, and the 80 th Ser is usually mutated to Arg or Ile; glu 453 of ParE gene can be mutated to Gly and His 461 of ParE gene can be mutated to Tyr, but mutation of parE gene is rare.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides Indiananas and application thereof, wherein the Indiananas carries five quinolone drug resistance mutation sites.
The invention discovers that a strain carries five quinolone drug-resistant mutation sites gyrA simultaneously S83F 、gyrA D87G 、parC T57S 、parC S80R And parE L502F Is a strain of Indonesia, and the gyrA gene of the strain is subjected to double-site mutation: ser83 → Phe (serine to phenylalanine, abbreviated S83F), asp87 → Gly (aspartic acid to glycine, abbreviated D87G), and the parC gene also had double site mutations: thr57→Ser (threonine mutated to serine, abbreviated as T57S), ser80→Arg (serine mutated to arginine, abbreviated as S80R), and pThe arE gene is subject to a single point mutation (Leu502→Phe) (leucine to phenylalanine, abbreviated L502F). This is the first discovery of five quinolone resistant mutation sites gyrA S83F 、gyrA D87G 、parC T57S 、parC S80R And parE L502F Meanwhile, the strain exists in a strain of Indonesia, and the strain is not reported at home and abroad. Wherein, parE L502F Is the first reported new mutation site found in the present invention. These mutation sites resulted in the Salmonella strain 51-38 exhibiting high levels of drug resistance to quinolones, with minimum inhibitory concentrations of nalidixic acid, ciprofloxacin, levofloxacin, and moxifloxacin for the Salmonella strain 51-38 being 4096 μg/mL, 16 μg/mL, 4 μg/mL, and 4 μg/mL, respectively.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: provided is Indiananas which carries five quinolone drug resistance mutation sites simultaneously, wherein the 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;
threonine at position 57 of the parC gene encoding product is mutated to serine;
serine at position 80 of the parC gene coding product is mutated into arginine;
leucine at position 502 of the parE gene encoding product was mutated to phenylalanine.
As a preferred embodiment of the Indiananas according to the present invention, the Indiananas further carries the aac (6') -Ib-cr gene in the PMQR gene.
As a preferred embodiment of the Indiananas according to the invention, the Indiananas is Indiananas (Salmonella enterica subsp. Enterica serovar Indiana) 51-38, accession number of which is GDMCC No:62102.
the salmonella 51-38 is in a gram-negative short rod shape with rounded ends, and the mass spectrum identification result is salmonella with a score value of 2.17. Which is arranged on the sliding doorThe bacteria chromogenic plate is purple, round, smooth and regular-edged colony form, and the nutrient agar plate is off-white, round and slightly convex colony form, and has typical colony form characteristics of salmonella. Serum antigen type identification as14,12:z,1,7 are typical Indonesia serotypes. The salmonella 51-38 of the present invention can be cultivated in LB, BHI and NA media.
The invention also provides a microbial agent, which comprises the Indiananas sp.
The invention also provides application of the Indiananas in researching a bacterial drug resistance mechanism.
The invention also provides application of the Indiananas in screening novel antibacterial medicines of salmonella.
As a preferred embodiment of the use according to the invention, the use is in the form of a model strain for screening novel antibacterial agents against Salmonella.
As a preferred embodiment of the application of the invention, the screening is specifically: and preparing the medicine to be tested into a solution, and determining the minimum inhibitory concentration and the minimum bactericidal concentration of the medicine to be tested on the Indiananas.
Wherein, salmonella 51-38 is deposited at the microorganism strain collection, guangdong province, 12 months and 9 days of 2021, address: guangzhou city first middle road 100 # college 59 # building 5, deposit number: GDMCC No:62102.
the invention has the beneficial effects that:
the invention provides a drug-resistant mutant site gyrA which simultaneously carries five quinolones S83F 、gyrA D87G 、parC T57S 、parC S80R And parE L502F Salmonella-Salmonella 51-38. 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 51-38 strains is 4096 mug/mL, 16 mug/mL, 4 mug/mL and 4 mug/mL respectively. In view of the above, salmonella 51-38 can be used as a model material for screening functional microorganisms/novel antibacterial agents, and has good propertiesIs a promising application prospect.
Drawings
FIG. 1 is a colony morphology of Salmonella strain 51-38 on a Salmonella chromogenic plate.
FIG. 2 is a colony morphology of Salmonella strain 51-38 on a nutrition plate.
FIG. 3 is a morphological image of microscopic examination of Salmonella strain 51-38.
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.
The experiments and methods described in the examples (e.g., molecular biology and nucleic acid chemistry experimental methods) are performed substantially in accordance with conventional methods well known in the art and described in various references unless otherwise indicated. See, e.g., sambrook et al Molecular Cloning: A Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (1989); and Ausubel et al, current Protocols in Molecular Biology, greene Publishing Associates (1992), which are incorporated herein by reference in their entirety. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, the terms related to molecular genetics, nucleic acid chemistry and molecular biology and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
As used herein, the meaning of amino acid notations is: serine (Serine, abbreviated Ser or S), phenylalanine (Phe or F), aspartic acid (Asparticacid, abbreviated Asp or D), glycine (Glycine, abbreviated Gly or G), threonine (Thr or T), arginine (Arginine, abbreviated Arg or R), leucine (Leucine, abbreviated Leu or L).
As used herein, the term "mutation," when used to describe a gene or DNA, refers to the addition, deletion, and/or substitution of one or more (e.g., several) bases in a gene sequence or DNA sequence; when used in describing a protein, refers to the addition, deletion, and/or substitution of one or more (e.g., several) amino acid residues in the amino acid sequence of the protein.
EXAMPLE 1 isolation and purification of strains
The salmonella 51-38 is separated from chicken samples of a supermarket in Jinan, shandong province of China, and is subjected to sample detection by referring to a detection method of salmonella in food safety national Standard food microbiology inspection salmonella test GB 4789.4-2016. Sterile operation 25g (mL) of the sample was weighed and placed in a sterile container of 225mL of Buffered Peptone Water (BPW) and mixed homogeneously, and incubated at 37℃for 8-18 h. The cultured sample mixture is gently shaken, 1mL of the mixture solution is removed by a sterile suction head, transferred to 10mL of sodium tetrasulfanilate brilliant green (TTB) enrichment medium, cultured at 42 ℃ for 18-24 h, simultaneously 1mL of the mixture solution is transferred to 10mL of Selenite Cystine (SC) enrichment medium, and cultured at 37 ℃ for 18-24 h. Taking 1 loop of enrichment liquid by an inoculating loop, streaking and inoculating the enrichment liquid on a salmonella chromogenic medium plate, culturing the enrichment liquid at 37 ℃ for 18-24 hours, and observing morphological characteristics of colonies on the plate. Typical salmonella colonies are purple, round, smooth, regular-edged colonies on a salmonella chromogenic medium plate. After streaking target colonies onto Nutrient Agar (NA) plates for purification culture, single colonies were picked and transferred to brain heart extract (BHI) nutrient broth for overnight culture at 37 ℃. Sterile operation the bacterial solution was added to 50% glycerol vials, stored at-40 ℃ and freeze-dried, thus obtaining strains 51-38.
Example 2 identification of strains
And (3) carrying out colony morphological characteristics, mass spectrum identification, serotyping and other identification on the purified strain 51-38.
(1) Color dyeing and checking: and (5) picking a single colony thallus smear, performing sheet making by gram staining, and observing the morphology by microscopic examination. Salmonella is gram negative, blunt ended, short rod (figure 3).
(2) Mass spectrometry identification: picking single colony thalli on a fresh NA flat plate, smearing the single colony thalli on a MALDI target plate, naturally drying, adding 1 mu L of 70% formic acid aqueous solution on the single colony coating, naturally drying, adding 1 mu L of matrix solution, naturally drying, and then placing the mixture into a matrix-assisted laser desorption ionization-time-of-flight mass spectrometer for detection (table 1).
Table 1 Mass Spectrometry identification results of Salmonella strain 51-38
Serotype identification: serotyping is carried out on the salmonella isolate by adopting a slide agglutination method, the thallus antigen (O antigen) of the salmonella, the phase I and phase II flagella antigen (H antigen) are sequentially checked, and finally serodiagnosis is carried out by referring to the salmonella diagnosis antigen table.
Strain 51-38 was gram negative, blunt-ended, short rod-like, and mass spectrometry identified salmonella with a score of 2.17. The salmonella chromogenic medium plate is typically purple, round, smooth and regular-edged colony of salmonella, and the nutrient agar plate is off-white, round and slightly raised colony. Serum antigen type identification as14,12:z,1,7, are typical Indiananas serotypes.
The bacterial strain 51-38 was identified as Salmonella enterica subspecies enterica Indiana serotype (Salmonella enterica subsp. Enterica serovar Indiana) by comprehensive judgment of colony morphology, appearance, gram staining, mass spectrometry, and serum agglutination reaction results, and designated as Salmonella 51-38.
EXAMPLE 3 strain drug sensitivity characterization
According to the experimental method and the result judgment standard of the American clinical laboratory standardization committee (Clinical and Laboratory Standards institute, CLSI) 2018 edition, a trace broth dilution method is adopted to examine the drug resistance level of the salmonella 51-38 strain to quinolones such as nalidixic acid, ciprofloxacin, levofloxacin, moxifloxacin and the like.
Transferring strain 51-38 in glycerol pipe onto NA plate for activating culture, selecting single colony thallus into small pipe containing 4mL hydrolyzed casein (MH) broth, incubating at 37deg.C and 200rpm to logarithmic phase, performing turbidimetry with 0.5 McAb tube, and preparing bacterial liquid with concentration of 1×10 8 cfu/mL. The bacterial liquid is removed by aseptic operation, and fresh MH broth is used for preparing the bacterial liquid according to the following formula 1:200, and diluting and mixing uniformly. 100 mu L of MH broth is added into a sterile 96-well flat-bottom micro-culture plate A1, 100 mu L of antibiotic solution to be detected is added into the same well, the mixture is gently blown and evenly mixed by a sterile micropipette, 100 mu L of the antibiotic solution is removed to A2, the mixture is diluted to A12 in sequence, and 100 mu L of antibiotic solution is sucked from the homogenate of the A12. Finally, adding the uniformly mixed salmonella 51-38 (1X 10) of the present invention into each well 5 cfu/mL) and 200 μl of MH broth medium was added as negative control at the appropriate well site, and 100 μl of salmonella 51-38 diluted bacterial suspension and 100 μl of MH broth medium were added as positive control at the other well. Culturing the culture plate in a constant temperature incubator at 37deg.C for 18-20 hr, observing turbidity of bacterial liquid at each hole site (positive turbidity and negative clarification), and measuring OD with enzyme-labeled instrument 600 Value, MIC value is interpreted.
Experiments prove that the minimum inhibitory concentrations (Minimal inhibitory concentration, MIC) of nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin on the salmonella 51-38 strain are 4096 mug/mL, 16 mug/mL, 4 mug/mL and 4 mug/mL respectively. The results of the drug sensitive assay for Salmonella strain 51-38 are shown in Table 2.
Table 2 drug resistance of salmonella 51-38 strain to 4 quinolones
| Antibiotics | MIC(μg/mL) | Drug resistance condition |
| Naphthyridonic acid | 4096 | R |
| Ciprofloxacin | 16 | R |
| Levofloxacin | 4 | R |
| Moxifloxacin | 4 | R |
Example 4 detection of quinolone drug resistance Gene of Salmonella 51-38 Strain
(1) Strain whole genome second generation sequencing and Blast analysis target drug resistance gene
The salmonella 51-38 strain is subjected to whole genome second generation sequencing through strain DNA extraction, quality inspection, library construction, on-machine sequencing, data processing and the like. Extracting strain DNA by adopting a Meiya D3146-02 bacterial DNA kit, measuring the concentration of the DNA, diluting the DNA to a proper concentration, transferring the DNA into a special 96-well plate of an automatic library constructing instrument, and constructing a library. After constructing the library, the library is subjected to quality control, gradually diluted to a proper concentration, denatured and sequenced on a machine. After the program is finished, copying the sequencing data to a local server, and sequentially operating commands such as 'gunzip gz', 'data/database/command/pre 0', 'data/database/command/ill 00' and the like to assemble and splice the sequencing data after splitting the data. Meanwhile, prokka is used for predicting genome components such as coding genes, rRNA, tRNA and the like of the spliced gene sequences, and functional annotation of the coding genes is carried out.
The quinolone drug resistance gene of salmonella 51-38 strain is detected by adopting a local Blast sequence analysis technology. Local Blast analysis process: 1) Constructing a local database by using a whole genome sequencing sequence information file of the salmonella 51-38 strain (51-38 assembly. Fasta is the name of the whole genome sequencing sequence information file of the strain, 51-38db is the name of the constructed local database), and operating a command of 'makeblastdb-in 51-38assembly. Fasta-dbtypenucl-parameter_seqids-out 51-38 db'; 2) With the target gene locus (QRDR gene: gyrA, gyrB, parC, parE; PMQR gene: qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC, qepA, oqxAB, aac- (6') -Ib-cr) (taking the gyrA gene as an example, the gyrA. Txt as the sequence information file of the target gene locus, and 51-38_gyrA_out as the output result file name) as a running command of "blastn-db51-38 db-event 1e-5-outfmt 0-num_descriptions 10-num_threads 64-query gyrA. Txt-out 51-38_gyrA_out", and performing Blast analysis of the gyrA gene on the local database 51-38 db; 3) And checking the output result file, and judging Blast results.
(2) PCR amplification target drug resistance gene and first generation sequencing analysis
Further adopting a PCR amplification and first generation sequencing analysis method to verify the determination result of the whole genome second generation sequencing and Blast sequence analysis target drug resistance genes of the salmonella 51-38 strain. PCR amplification primers of the target drug resistance genes were designed based on the published gene sequences (primer sequences are shown in Table 3). 25. Mu.L of PCR amplification reaction system (12.5. Mu.L of 2 XPromix Taq, 120nmol/L of each of the upstream and downstream primers, 2. Mu.L of template, and ultra pure water) by the method of single PCR, and the conditions of the PCR amplification reaction: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 45s, annealing at 55-60 ℃ for 45s and extension at 72 ℃ for 45s, and carrying out 30 cycles in total; extending at 72℃for 10min. And (3) performing agarose gel electrophoresis detection (120V, 30 min), purification and first generation sequencing analysis on the amplified fragment product to obtain the sequence information of the amplified target gene, and performing Blast analysis on the sequencing result to judge the result.
TABLE 3 primer sequences and fragment sizes of salmonella gyrA, parC, parE genes
(3) Detection results of quinolone resistance genes of Salmonella strain 51-38.
Comparing with the standard strain LT2 gene sequence of Salmonella typhimurium, and detecting mutation in the QRDR gene gyrA, parC, parE of Salmonella strain 51-38; the gyrA gene is subject to double-site mutation: ser83 → Phe (serine to phenylalanine, abbreviated S83F), asp87 → Gly (aspartic acid to glycine, abbreviated D87G), and the parC gene also had double site mutations: thr57→ser (threonine to serine, abbreviated as T57S), ser80→arg (serine to arginine, abbreviated as S80R), and single site mutation of the parE gene (leu502→phe) (leucine to phenylalanine, abbreviated as L502F). In the PMQR gene, only aac (6') -Ib-cr gene is detected. Further verifying by adopting a PCR amplification method and a first generation sequencing analysis method, wherein the detection results of the two methods are consistent.
The QRDR gene and PMQR gene in the salmonella 51-38 strain were detected as follows:
①gyrA(324C→T，Ser83→Phe；336A→G，Asp87→Gly)
②parC(170C→G，Thr57→Ser；240C→A，Ser80→Arg)
③parE(1504C→T，Leu502→Phe)
④aac(6')-Ib-cr
EXAMPLE 5 use of quinolone-resistant Salmonella strain 51-38
The specific application method of the salmonella 51-38 mainly comprises the following two aspects:
(1) Salmonella 51-38 simultaneously carry five quinolone drug-resistant mutation sites gyrA S83F 、gyrA D87G 、parC T57S 、parC S80R And parE L502F And a plasmid-mediated quinolone drug resistance gene aac (6') -Ib-cr, has high-level drug resistance to four quinolone drugs such as nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin, especially ciprofloxacin, and is clinically used for intestinal tract infection and fine drug resistanceBacterial enteritis, bacillary dysentery and the like are clinical common medicines for salmonella treatment. A plurality of quinolone drug resistance mutation sites appear in salmonella 51-38 at the same time, so that the method has important guiding significance for discussing the mechanism of occurrence of quinolone drug resistance of strains and the formation of a new scheme for preventing and treating salmonella infection, and can be used as an important material for exploring the bacterial drug resistance mechanism.
(2) Salmonella 51-38 strain simultaneously carries five quinolone drug-resistant mutation sites gyrA S83F 、gyrA D87G 、parC T57S 、parC S80R And parE L502F And a plasmid-mediated quinolone drug resistance gene aac (6') -Ib-cr, has high-level drug resistance to four quinolone drugs such as nalidixic acid, ciprofloxacin, levofloxacin and moxifloxacin, and can be used as an important mode strain for screening novel antibacterial drugs of salmonella. Screening a drug with resistance to a strain to be tested, wherein the screening method comprises the following steps:
precisely weighing a certain amount of medicine to be tested, dissolving and mixing uniformly with a proper solvent to prepare mother solution with the concentration of 30mg/mL, preparing use solution with the solvent to prepare the use solution with the concentration of 2000 mug/mL, determining the minimum inhibitory concentration (minimum inhibitory concentration, MIC) and the minimum bactericidal concentration (minimum bactericidal concentration, MBC) of salmonella, and storing in a refrigerator at the temperature of minus 40 ℃. The day before the drug sensitivity experiment, the salmonella 51-38 of the invention is taken out in a refrigerator at the temperature of minus 40 ℃, and after the salmonella is cooled to the room temperature, bacterial liquid is streaked and transferred on an MH agar medium by an inoculating loop, and is cultured for 18-24 hours in a constant temperature incubator at the temperature of 37 ℃. The small amount of activated colony on MH agar culture medium is picked up by inoculating loop, and diluted with sterile physiological saline to prepare 1X 10 7 cfu/mL of bacterial suspension (standard turbidimetric tube control) was mixed well for use.
The sensitivity of the strains to different drugs was determined according to the paper diffusion method recommended by the american Clinical Laboratory Standardization Institute (CLSI). Dipping with sterile cotton stick to obtain 1×10 concentration 7 cfu/mL of standard bacterial suspension was spread evenly onto the corresponding MH agar medium. The sterile oxford cup was placed on the applied MH agar medium, and 50. Mu.L of the drug solution (30 mg/mL) was taken in the oxford cup (in an equal volume of MH broth medium) using a micropipetteAdding solvent as blank control), culturing at 37 deg.C for 18-24 hr, and measuring the size of bacteriostasis area.
MIC values of the strains for the different drugs were determined according to the broth dilution method recommended by the american Clinical Laboratory Standardization Institute (CLSI). In the determination of MIC values, 3 replicates were performed for each group, with the mean of the parallel concentrations being the final result. 1X 10 with sterile physiological saline 7 Dilution of cfu/mL of bacterial suspension to 1X 10 5 cfu/mL, selecting a sterile 96-well flat-bottomed microplate, adding 100 mu L of MH broth into each row of 1 st well, adding 100 mu L of antibiotic use solution to be tested into the same row of wells, gently beating and uniformly mixing by using a sterile micropipette, transferring 100 mu L to 2 nd wells, sequentially diluting to 12 th wells, and sucking 100 mu L from the homogenized solution of the 12 th wells. Finally, the salmonella 51-38 homogenate (1 multiplied by 10) of the invention is taken 5 cfu/mL) 100 μl was placed in each well. And 200. Mu.L of MH broth medium was added to the appropriate well site as a negative control, and 100. Mu.L (1X 10) of the salmonella 51-38 homogenate of the present invention was used 5 cfu/mL) and 100 μl MH broth were added to the same wells as positive controls. Culturing the culture plate in a constant temperature incubator at 37deg.C for 18-20 hr, observing turbidity of bacterial liquid at each hole site (positive turbidity and negative clarification), and measuring OD with enzyme-labeled instrument 600 Value, MIC value is interpreted. After determination of the MIC values, bacterial broth mixtures from 3 to 5 wells prior to the MIC values were plated onto MH agar medium and incubated at 37℃for 22h, and the colony growth on the plates was observed, and the average number of less than 5 corresponding minimum drug concentrations was determined as the MBC value for 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> the institute of microbiology of the academy of sciences of Guangdong province (microbiological analysis and detection center of Guangdong province)
<120> A Indiananas carrying five quinolone drug resistance mutation sites simultaneously and application thereof
<130> 2021.12.21
<160> 6
<170> PatentIn version 3.3
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Claims (5)
1. The indiana salmonella is characterized in that the indiana salmonella carries five quinolone drug resistance mutation sites at the same time, and the 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;
threonine at position 57 of the parC gene encoding product is mutated to serine;
serine at position 80 of the parC gene coding product is mutated into arginine;
leucine at position 502 of the parE gene encoding product is mutated to phenylalanine;
the Indiananas carries aac (6') -Ib-cr gene in PMQR gene;
the Indiananas is Indiananas (Salmonella enterica subsp. Enterica serovarIndiana) 51-38 with deposit number GDMCC No:62102.
2. a microbial agent comprising the indiana salmonella of claim 1.
3. Use of indiana salmonella of claim 1 for studying bacterial resistance mechanisms.
4. The use of indiana salmonella as defined in claim 1 for screening novel antimicrobial agents against salmonella.
5. The use according to claim 4, wherein the screening is in particular: and preparing the medicine to be tested into a solution, and determining the minimum inhibitory concentration and the minimum bactericidal concentration of the medicine to be tested on the Indiananas.
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