CN109486739B - Method for inducing vibrio parahaemolyticus to generate levofloxacin drug resistance - Google Patents
Method for inducing vibrio parahaemolyticus to generate levofloxacin drug resistance Download PDFInfo
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Abstract
本发明公开了一种诱导副溶血性弧菌产生左氧氟沙星耐药性的方法，包括：敏感菌株筛选、亚致死抗生素浓度初步诱导和连续诱导、耐药性菌株保藏步骤，本发明诱导后的副溶血性弧菌经验证获得了左氧氟沙星耐药性，其MIC值均达到了CLSI的耐药标准，即副溶血性弧菌的MIC值≥8mg/L，且这种耐药性能够稳定地遗传；本发明的诱导方法可快速获得具有稳定遗传特性的耐药性菌株，为副溶血性弧菌对左氧氟沙星耐药机制的研究提供了可靠工具，具有良好的参考价值和实际意义。
The invention discloses a method for inducing Vibrio parahaemolyticus to produce levofloxacin resistance, comprising: screening of sensitive strains, preliminary and continuous induction of sub-lethal antibiotic concentration, preservation of drug-resistant strains, Vibrio parahaemolyticus has been verified to have acquired levofloxacin resistance, and its MIC values have reached the CLSI resistance standard, that is, the MIC value of Vibrio parahaemolyticus ≥ 8 mg/L, and this resistance can be stably inherited; The inducing method of the invention can quickly obtain drug-resistant strains with stable genetic characteristics, provides a reliable tool for the study of the resistance mechanism of Vibrio parahaemolyticus to levofloxacin, and has good reference value and practical significance.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for inducing vibrio parahaemolyticus to generate levofloxacin drug resistance.
Background
Vibrio parahaemolyticus (Vp) is a common gram-negative halophilic bacterium, widely distributed in seawater, submarine sediments and marine products near the coast, and eating marine products polluted by the pathogenic bacterium is easy to cause severe acute gastroenteritis and primary septicemia, accompanied by clinical symptoms such as abdominal colic, nausea, vomiting, headache, low fever, chill and the like, and is considered as one of main causes of diarrhea diseases outbreak in the global range. According to the data of the food-borne disease monitoring network in China, the vibrio parahemolyticus pollution is an important factor in the food-borne disease outbreak event caused by microorganisms, is a very important food-borne pathogenic bacterium, and poses great threats to the safety of aquatic products and public health.
The antibiotic is used as a good medicament and can effectively cure common food-borne diseases. However, with the excessive and irregular use of antibiotics, bacterial resistance is increasing. The emergence of bacterial resistance and the spread of bacterial resistance in the treatment of human and animal diseases has brought about great difficulties and has attracted widespread attention worldwide. The vibrio parahaemolyticus is a common zoonosis pathogen, has frequent drug resistance and multiple drug resistance phenomena, and has the characteristics of high drug resistance rate and wide drug resistance spectrum. The quinolone has an obvious antibacterial effect on vibrio parahaemolyticus as a widely used broad-spectrum antibacterial drug. However, with the wide use of the industries such as clinic, agriculture and breeding, the resistance of vibrio parahaemolyticus to quinolone antibacterial drugs is more and more serious. The method for inducing the vibrio parahaemolyticus to generate the drug resistance of the levofloxacin is constructed, the research on the drug resistance mechanism of the vibrio parahaemolyticus to the levofloxacin is facilitated, the scientific research value and the practical significance are good, and related technical means are still lacked at the present stage.
Disclosure of Invention
In order to make up for the technical blank, the invention aims to provide a method for inducing vibrio parahaemolyticus to generate levofloxacin drug resistance.
The above object of the present invention is achieved by the following technical solutions:
a method for inducing vibrio parahaemolyticus to generate levofloxacin drug resistance comprises the following steps:
s1, preparing a seed solution: taking out the vibrio parahaemolyticus strain from a glycerol tube stored at-80 ℃, streaking and inoculating the vibrio parahaemolyticus strain on a TCBS flat plate, and culturing the vibrio parahaemolyticus strain in a constant-temperature incubator at 37 ℃ for 16 h; selecting well-grown single colony, culturing and activating in 9mL of TSB test tube containing NaCl and with mass concentration of 3% for 16h in a shaker at 37 ℃ and rotation speed of 180r/min, and continuously activating for 2 times to serve as seed liquid for later use;
s2, sensitive strain screening: taking the seed liquid in the step S1, and adjusting OD600The inoculation amount is 1.5 multiplied by 108CFU/mL, adjusting the mass concentration of NaCl to 6Log CFU/mL, inoculating the strain into a 96-pore plate containing levofloxacin antibiotic with different concentrations, placing 100ul of seed solution in each pore, culturing for 16h in an incubator at 37 ℃, measuring the OD value of each pore by using an enzyme labeling instrument, determining the Minimum Inhibitory Concentration (MIC) according to a drug resistance standard, and screening a levofloxacin sensitive vibrio parahemolyticus strain which can grow in the MIC value of 1/2;
s3, preliminary induction of sublethal antibiotic concentration: sucking the levofloxacin sensitive vibrio parahaemolyticus bacterial liquid screened in the step S2 into a 10mL centrifugal tube, adjusting the concentration of the bacterial liquid to 6Log CFU/mL by using NaCl with the mass concentration of 0.85%, inoculating the bacterial liquid into levofloxacin antibiotic 96-pore plates with different concentrations, and culturing the bacterial liquid in a 37 ℃ constant temperature incubator for 16 hours;
s4, continuous induction of sublethal antibiotic concentration: continuously growing the levofloxacin-sensitive vibrio parahaemolyticus strain subjected to the primary induction in the step S3 under the condition of sublethal antibiotic concentration until the MIC value of the vibrio parahaemolyticus is more than or equal to 8mg/L to obtain a drug-resistant vibrio parahaemolyticus strain;
s5, preserving the drug-resistant vibrio parahemolyticus strain in the step S4;
s6, verifying that the vibrio parahaemolyticus levofloxacin obtains drug resistance: and (3) reactivating the mutant strain preserved in the step 5, and detecting the drug resistance of the mutant strain by using a K-B paper method and a broth multiple dilution method to verify that the mutant strain obtains the drug resistance capable of being stably inherited.
Further, in step S1, the concentration of the seed solution is 1 × 108～9×109CFU/mL。
Further, in step S2, the preparation process of the 96-well plate containing levofloxacin antibiotics at different concentrations is as follows: taking 1mL of levofloxacin mother liquor to be put into 19mL of MHB liquid culture medium, fully shaking and uniformly mixing to obtain levofloxacin antibiotic with the concentration of 256 mg/L; then 200ul of the levofloxacin antibiotic is taken to be put into each hole of the 1 st row of the 96-well plate, 100ul of MHB culture medium is added into each hole of the 2 nd row to the 11 th row, and 200ul of MHB culture medium is added into each hole of the 12 th row to be used as blank control; and then sucking 100ul of liquid from the 1 st row to the 2 nd row by using a 100ul row gun, sucking 100ul of liquid to the 3 rd row after full sucking and beating, performing gradient dilution to 11 rows in sequence, sucking 100ul of liquid after full blowing and beating of the row to obtain a levofloxacin antibiotic 96 pore plate with different concentrations, and placing the levofloxacin antibiotic 96 pore plate in a refrigerator at the temperature of minus 80 ℃ for refrigeration for later use.
Further, in step S2, the levofloxacin antibiotic 96-well plates with different concentrations have the levofloxacin antibiotic concentrations of 256mg/L, 128mg/L, 64mg/L, 32mg/L, 16mg/L, 8mg/L, 4mg/L, 2mg/L, 1mg/L, 0.5mg/L and 0.25mg/L from row 1 to row 11 in sequence, and each pore volume is 100 ul.
Further, in step S4, the levofloxacin-sensitive Vibrio parahaemolyticus continues to grow for 12-30 days until the MIC value of the Vibrio parahaemolyticus is greater than or equal to 8 mg/L.
Further, in step S5, the deposited drug-resistant Vibrio parahaemolyticus strain is a mutant strain cultured to 9LogCFU/mL after continuous induction in step S4.
Further, in step S5, the preservation method specifically includes: and (3) carrying out streak culture on the vibrio parahaemolyticus mutant strain which obtains the drug resistance after induction, selecting a well-grown single colony to be cultured in 9mL of TSB test tube containing NaCl and having the mass concentration of 3% to 9LogCFU/mL, mixing the bacterial suspension and glycerol according to the volume ratio of 1:1, and then preserving at-80 ℃.
Further, in step S6, the method for verifying drug resistance specifically includes: reactivating the mutant strain preserved in the step S5, streaking the mutant strain on an MHA solid culture medium containing 8mg/L levofloxacin, culturing for 16h, selecting a single colony which grows well in a test tube of 9ml LTSB (3% NaCl), and culturing for 16h in a shaking table at 37 ℃ and the rotating speed of 180 r/min; sucking 10ul of the bacterial suspension into a test tube of 9ml LTSB (3% NaCl), and culturing for 16h in a shaking table at 37 ℃ and the rotating speed of 180 r/min; repeating the operation, continuously subculturing for 5 days, detecting the drug resistance of the mutant strain by using a K-B paper method and a broth multiple dilution method, and verifying that the minimum inhibitory concentration of each generation of vibrio parahaemolyticus strains after induction is more than 8mg/L, so that the vibrio parahaemolyticus strains capable of stably inheriting the drug resistance of levofloxacin can be obtained by the method.
Furthermore, in step S6, the K-B paper method for detecting drug resistance of the mutant strain specifically comprises the steps of: adjusting OD by taking seed liquid600So that the inoculation amount is 1.5X 108CFU/mL, using a cotton swab to absorb bacterial liquid, coating the bacterial liquid on the surface of a Mueller-Hinton agar (MHA) culture medium, sticking an antibiotic paper sheet after the bacterial liquid on the surface of the culture medium is completely dried, placing the culture medium in an incubator at 37 ℃ for overnight culture for 16 hours, and observing and recording a test result; according to the standard of the American society for standardization of Clinical Laboratories (CLSI), the diameter of a bacteriostatic zone is measured, the drug resistance of the vibrio parahaemolyticus is judged to be drug resistance, medium and sensitivity, the minimum bacteriostatic concentration of the induced vibrio parahaemolyticus reaches the drug resistance standard (the diameter is less than or equal to 13mm) specified by CLSI, and the vibrio parahaemolyticus capable of stably inheriting the drug resistance of levofloxacin is obtained through induction.
Furthermore, in step S6, the broth dilution method for detecting drug resistance of the mutant strain comprises the following steps: adjusting OD by taking seed liquid600So that the inoculation amount thereof is 1.5×108CFU/mL, adjusting the concentration to 6LogCFU/mL by using 0.85% NaCl, inoculating the solution into 96-well plates containing levofloxacin antibiotics with different concentrations, setting 100ul bacterial solution in each well, setting the last column as a blank control, placing the solution in an incubator at 37 ℃ for overnight culture for 16h, and observing and recording test results; according to the standard of the American Clinical Laboratory Standardization Institute (CLSI), the Minimum Inhibitory Concentration (MIC) is measured, and the drug resistance of the vibrio parahaemolyticus is judged to be drug resistance, medium and sensitivity; further proves that the minimum inhibitory concentration of the induced vibrio parahaemolyticus reaches the drug resistance standard (MIC is more than or equal to 8mg/L) specified by CLSI, which indicates that the vibrio parahaemolyticus capable of stably inheriting levofloxacin drug resistance can be obtained through induction.
Compared with the prior art, the invention has the positive improvement effects that:
the method can quickly obtain drug-resistant strains with stable genetic characteristics, fills the blank of research on a method for inducing vibrio parahaemolyticus to generate levofloxacin drug resistance, provides a reliable tool for the research on a levofloxacin drug resistance mechanism by the vibrio parahaemolyticus, and has good scientific research value and practical significance.
Drawings
FIG. 1 is a flow chart of the method of the present invention;
FIG. 2 is a schematic diagram showing the drug resistance change of Vibrio parahaemolyticus levofloxacin in example 2 of the present invention;
FIG. 3 is a K-B paper sheet method for verifying the drug resistance of the Vibrio parahaemolyticus levofloxacin in example 3 of the present invention;
FIG. 4 shows that the broth dilution method in example 4 of the present invention verifies the drug resistance of the Vibrio parahaemolyticus levofloxacin.
Detailed Description
The following description will be given with reference to the accompanying drawings to describe the preferred embodiments of the present invention in detail, but the scope of the present invention is not limited to the following embodiments.
1 origin of the Strain
The Vibrio parahaemolyticus is isolated from marine products, and the quality control strain is Escherichia coli ATCC 25922.
2 instruments and reagents
The instrument comprises the following steps: MLS-3750 model autoclave, Sanyo electric Motor biomedical corporation, Japan;
a vertical flow super-clean workbench is provided,
model a2 secondary biosafety cabinet, Esco China; a QL-901 type vortex oscillator, linbel instruments manufacturing ltd, hampson, hamamatsu; GHP-9270 type water-proof constant temperature incubator, Shanghai-Hengshi Co., Ltd; shake culture box (ZQZY-70B), Shanghai ZhiChu instruments Co., Ltd; bio Tek multifunctional microplate reader (Synergy2), Boteng instruments, Inc. USA.
Reagent: thiosulfate-citrate-bile salt sucrose agar (TCBS), tryptone soy broth (TSB, 3% NaCl), 0.85% NaCl, beijing land bridge technology ltd; Mueller-Hinton agar (MHA), Mueller-Hinton broth (MHB), Oxoid, UK; levofloxacin (LEV) powder, sigma aldrich trade ltd; levofloxacin (LEV) drug sensitive paper, Oxoid, uk.
Example 1 Induction method
The method for inducing vibrio parahaemolyticus to generate levofloxacin drug resistance comprises the following specific steps:
step 1, preparation of levofloxacin antibiotic 96 pore plate
Preparing a levofloxacin antibiotic mother liquor (5120mg/L, 25 ml): wherein the mass of antibiotics is calculated as follows: mass (mg) × mother liquor concentration (mg/L) × solvent (L)/purity (%) (5120mg/L × 25 × 10)-3L/98% ═ 131 mg. Dissolving levofloxacin in sterilized 25mL ddH, weighing 131mg with ten-thousandth analytical balance2And O, filtering and sterilizing the mixture by using a filter membrane with the diameter of 0.22um, placing the mixture into a 50mL centrifuge tube, and storing the mixture at the temperature of minus 80 ℃ for later use.
Wherein, the preparation of a levofloxacin antibiotic 96 pore plate comprises the following steps: 1mL of levofloxacin mother liquor is taken to be put into 19mL of MHB liquid culture medium, and the antibiotic with the concentration of 256mg/L is obtained after sufficient shaking and uniform mixing for standby. 200ul of levofloxacin at this concentration was taken in each well of column 1 of a 96-well plate. Column 2 to column 11 were filled with 100ul of MHB medium per well and column 12 with 200ul of MHB medium per well as a blank. Then 100ul of liquid is sucked from the 1 st row to the 2 nd row by a 100ul row gun, 100ul of liquid is sucked to the 3 rd row after full suction and beating, gradient dilution is sequentially carried out to the 11 rows in this way, and 100ul of liquid is sucked out after full blow beating of the rows. The prepared levofloxacin antibiotic plate has the concentration of 256mg/L, 128mg/L, 64mg/L, 32mg/L, 16mg/L, 8mg/L, 4mg/L, 2mg/L, 1mg/L, 0.5mg/L and 0.25mg/L from the 1 st row to the 11 th row in sequence, and the pore volume is 100 ul. And (3) placing the prepared levofloxacin antibiotic plate in a refrigerator at the temperature of-80 ℃ for refrigeration for later use.
Step 2, strain activation and inoculum preparation
Taking out Vibrio parahaemolyticus strain from glycerol tube stored at-80 deg.C, streaking and inoculating on TCBS plate, culturing in constant temperature incubator at 37 deg.C for 16h, selecting well-grown single colony in 9mL TSB (3% NaCl) test tube, culturing in shaker at 37 deg.C and rotation speed of 180r/min for 16h, continuously activating for 2 times, and using as seed solution (about 10 times)9CFU/mL), ready for use.
Adjusting OD by taking seed liquid600So that the inoculation amount is 1.5X 108CFU/mL, then using 0.85% NaCl to adjust it to 6Log CFU/mL, inoculated in containing different concentrations of levofloxacin antibiotic 96-well plate, each hole 100u bacterial liquid, and the last column as blank control, placed in 37 degrees C incubator culture for 16 h. After culturing, measuring the OD value of each well by using a microplate reader, observing and recording the test result, and measuring the Minimum Inhibitory Concentration (MIC) of 17 vibrio parahaemolyticus to levofloxacin by using a broth multiple dilution method, wherein the standards of sensitivity, intermedium and drug resistance of the levofloxacin refer to CLSI standard, and are as follows: MIC (mg/L) is less than or equal to 2, 4 and more than or equal to 8, and the 17 vibrio parahaemolyticus strains are judged to be sensitive strains. As shown in table 1.
Table 117 strain vibrio parahaemolyticus drug sensitivity test results for levofloxacin
Step 4, preliminary induction
An experimental procedure for inducing vibrio parahaemolyticus to obtain levofloxacin resistance is shown in fig. 1, after an initial MIC value of a wild strain is determined according to a broth multiple dilution method, vibrio parahaemolyticus liquid capable of growing in 1/2MIC is sucked into a 10mL centrifuge tube, the concentration of the vibrio parahaemolyticus liquid is adjusted to 6LogCFU/mL by 0.85% NaCl, and the vibrio parahaemolyticus liquid is inoculated into 96-well plates containing levofloxacin with different concentrations again and cultured in a constant temperature incubator at 37 ℃ for 16 hours.
Step 5, continuous induction
Continuously culturing the vibrio parahaemolyticus strain under the condition of sublethal antibiotic concentration for 12-30 days until the minimum inhibitory concentration of the antibiotic for inhibiting the vibrio parahaemolyticus reaches the drug resistance standard specified by CLSI, and obtaining the vibrio parahaemolyticus with different drug resistance degrees.
Step 6, preservation
Re-streak-culturing the drug-resistant vibrio parahaemolyticus mutant strain with the induced MIC value larger than 8, 16, 32, 64, 128 and 256mg/L, picking out a well-grown single colony in a test tube of 9mL LTSB (3% NaCl), culturing to 9LogCFU/mL, mixing the bacterial suspension with glycerol 1:1, and preserving at-80 ℃, wherein the preserved strain information is as follows: VPD8M, VPD14M, VPD31M, VPD33, VPD34M, VPD58M, VPD61M, VPR102M, VPR103M, VPR104M, VPR105M, VPR106M, VPR107M, VPR108M, VPR110M and VPR 111M.
Example 2 verification method
Reactivating the deposited mutant strain, streaking the mutant strain on an MHA solid culture medium containing 8mg/L levofloxacin, culturing for 16h, selecting a well-grown single colony in a test tube of 9ml LTSB (3% NaCl), and culturing for 16h in a shaking table at 37 ℃ and the rotating speed of 180 r/min; sucking 10ul of the bacterial suspension into a test tube of 9ml LTSB (3% NaCl), and culturing for 16h in a shaking table at 37 ℃ and the rotating speed of 180 r/min; this operation was repeated and subcultured continuously for 5 days. Then, the K-B paper method and the broth multiple dilution method are used for detecting the drug resistance of the mutant strains, and the minimum inhibitory concentration of each generation of vibrio parahaemolyticus strains after induction is more than 8mg/L, so that the vibrio parahaemolyticus strains capable of stably inheriting the drug resistance of levofloxacin can be obtained by the induction method.
The K-B paper method for detecting the drug resistance of the mutant strain comprises the following specific steps: adjusting OD by taking seed liquid600So that the inoculation amount is 1.5X 108CFU/mL, using a cotton swab to absorb bacterial liquid, coating the bacterial liquid on the surface of a Mueller-Hinton agar (MHA) culture medium, sticking an antibiotic paper sheet after the bacterial liquid on the surface of the culture medium is completely dried, placing the culture medium in an incubator at 37 ℃ for overnight culture for 16 hours, and observing and recording a test result; according to the standard of the American society for standardization of Clinical Laboratories (CLSI), the diameter of a bacteriostatic zone is measured, the drug resistance of the vibrio parahaemolyticus is judged to be drug resistance, medium and sensitivity, the minimum bacteriostatic concentration of the induced vibrio parahaemolyticus reaches the drug resistance standard (the diameter is less than or equal to 13mm) specified by CLSI, and the vibrio parahaemolyticus capable of stably inheriting the drug resistance of levofloxacin is obtained through induction.
The method comprises the following specific steps of detecting the drug resistance of the mutant strain by a broth multiple dilution method: adjusting OD by taking seed liquid600So that the inoculation amount is 1.5X 108CFU/mL, adjusting the concentration to 6LogCFU/mL by using 0.85% NaCl, inoculating the solution into 96-well plates containing levofloxacin antibiotics with different concentrations, setting 100ul bacterial solution in each well, setting the last column as a blank control, placing the solution in an incubator at 37 ℃ for overnight culture for 16h, and observing and recording test results; according to the standard of the American Clinical Laboratory Standardization Institute (CLSI), the Minimum Inhibitory Concentration (MIC) is measured, and the drug resistance of the vibrio parahaemolyticus is judged to be drug resistance, medium and sensitivity; further proves that the minimum inhibitory concentration of the induced vibrio parahaemolyticus reaches the drug resistance standard (MIC is more than or equal to 8mg/L) specified by CLSI, which indicates that the vibrio parahaemolyticus capable of stably inheriting levofloxacin drug resistance can be obtained through induction.
In conclusion, the sensitive vibrio parahaemolyticus developed levofloxacin resistance on day 12 by continuous induction under the condition of sub-lethal antibiotic concentration, and the MIC value thereof increased with the increase of induction days and reached 256mg/L on day 30. The verification proves that the drug resistance of the vibrio parahaemolyticus inducing the drug resistance of the levofloxacin can be stably inherited, and the MIC value of each generation of the strain to the levofloxacin is more than 8mg/L and reaches the drug resistance standard specified by CLSI. The invention fills the blank of research on a method for inducing vibrio parahemolyticus to generate levofloxacin drug resistance, provides a reliable tool for research on a levofloxacin drug resistance mechanism of the vibrio parahemolyticus, and has good scientific research value and practical significance.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the present invention should not be limited by the disclosure of the preferred embodiments. Therefore, it is intended that all equivalents and modifications which do not depart from the spirit of the invention disclosed herein are deemed to be within the scope of the invention.
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