CN115612653A - Method for inducing salmonella enteritidis to generate resistance by sodium hypochlorite - Google Patents

Method for inducing salmonella enteritidis to generate resistance by sodium hypochlorite Download PDF

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CN115612653A
CN115612653A CN202211144347.4A CN202211144347A CN115612653A CN 115612653 A CN115612653 A CN 115612653A CN 202211144347 A CN202211144347 A CN 202211144347A CN 115612653 A CN115612653 A CN 115612653A
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sodium hypochlorite
mic
strain
salmonella enteritidis
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汪雯
肖兴宁
肖英平
杨华
王文思
吕文涛
马灵燕
马洁乐
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Zhejiang Academy of Agricultural Sciences
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Abstract

The invention discloses a method for inducing salmonella enteritidis to generate resistance by sodium hypochlorite, which relates to the field of biology correlation and comprises the steps of selecting test strains, activating primary strains, preparing bacterial suspension, measuring MIC value of the strains, adding 10 mu L of culture solution into 10ml of MH broth (MHB) for dilution, then adding 100 mu L of disinfectant with different concentration gradients into a 96-hole enzyme label plate, and then adding 100 mu L of diluted bacterial suspension (the bacterial number is about 10) 6 CFU), continuously inducing the strain by adopting a sodium hypochlorite solution with the lowest inhibitory concentration (MIC) of 1/2, inducing the bacteria to generate antibodies by using the sodium hypochlorite, continuously inducing the strain by adopting the sodium hypochlorite solution with the lowest inhibitory concentration (MIC) of 1/2, researching the physiological characteristics of the sodium hypochlorite resistant strain, and generating and preparing a resistance generation mechanismThe targeted intervention strategy has important significance.

Description

Method for inducing salmonella enteritidis to generate resistance by sodium hypochlorite
Technical Field
The invention relates to the field of biology correlation, in particular to a method for inducing resistance of salmonella enteritidis by sodium hypochlorite.
Background
Salmonella-induced food poisoning ranks the leading position in the bacterial food poisoning event in our country, with over 300 million cases per year, of which Salmonella Enteritidis (s.enteritidis) is one of the most prominent serotypes. Sodium hypochlorite (NaClO) is a raw material component of a disinfectant for food, and is widely used for washing and disinfecting food containers, food production and management tools, equipment, vegetables and fruits. However, long-term exposure of bacteria to sub-lethal concentrations of disinfectants induces tolerance, affects the actual disinfection effect, and poses a threat to food quality safety. The research on the physiological characteristics of the sodium hypochlorite resistant strain has important significance on a resistance generation mechanism and the establishment of a targeted intervention strategy. At present, no patent or literature specifically reports how to utilize sodium hypochlorite to induce and detect the resistance of salmonella enteritidis.
Disclosure of Invention
The invention aims to provide a method for inducing resistance of salmonella enteritidis by using sodium hypochlorite so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a method for inducing resistance of Salmonella enteritidis by sodium hypochlorite, comprising the steps of:
the method comprises the following steps: test Strain selection
Selecting salmonella enteritidis S.Enteritidis CVCC1806 from China center for culture collection of microorganisms, wherein the culture is preserved at the temperature of below 80 ℃ below zero for 1 to 5 times;
step two: activation of primary strains
Taking out the frozen strain from-80 ℃, thawing at room temperature, sucking 0.4ml, inoculating into 20ml of TSB culture medium, controlling the temperature at 37 ℃, and incubating for 24h;
step three: preparation of bacterial suspension
In a sterile environment, performing centrifugal treatment on bacterial cells incubated for 24 hours, controlling the temperature at 4 ℃, controlling the rotating speed at 10000rpm, centrifuging for 5min, washing for 3 times by using 0.85% (w/v) sterile physiological saline, and then suspending in 0.85% (w/v) sterile physiological saline to obtain 109CFU/mL bacterial suspension;
step four: determination of MIC value of Strain
mu.L of the culture broth was diluted in 10ml MH broth (MHB), and then 100. Mu.L of a disinfectant with a gradient of different concentrations was added to a 96-well microplate, followed by 100. Mu.L of the diluted bacterial suspension (about 10 bacteria count) 6 CFU)；
Setting positive control (inoculating 100 μ L bacterial suspension and 100 μ L blank MHB) and negative control (directly inoculating 100 μ L blank MHB);
setting each sample for 2 times, sealing with disposable sealing film of enzyme labeling plate, culturing at 37 deg.C for 18 hr, and determining OD with enzyme labeling instrument 600 A value;
step five: continuous induction of strains
Continuously inducing the strains by adopting a sodium hypochlorite solution with the Minimum Inhibitory Concentration (MIC) of 1/2;
the method comprises the following specific steps:
s5.1, taking 10ml of NaClO solution of an original strain MIC, mixing the NaClO solution with a trypticase soy peptone liquid medium (TSB medium) in an equal volume, uniformly mixing by vortex to obtain a TSB medium containing 1/2MIC NaClO solution, inoculating 0.4ml of incubated S.enteritidis CVCC1806, placing at 37 ℃ for incubation for 24 hours, counting as the 1 st generation, and determining the MIC value;
s5.2, taking 10ml of solution of the 1 st generation bacterium MIC NaClO, mixing the solution with a trypticase soy peptone liquid medium (TSB medium) in equal volume, uniformly mixing the solution in a vortex mode to obtain a TSB medium containing the 1/2MIC NaClO solution, inoculating 0.4ml of the first generation bacterium solution, placing the TSB medium at 37 ℃ for incubation for 24 hours to obtain the 2 nd generation, and determining the MIC value of the second generation bacterium solution;
s5.3, repeating the operations, and when the MIC value of the strain is increased in the induction process, correspondingly increasing the concentration (1/2 MIC) of the stress solution;
s5.4 after the induction is finished, placing the induced strain in a TSB culture medium at 37 ℃ for incubation for 24h, carrying out subculture, carrying out continuous passage for 5 times, measuring an MIC value after each passage, and observing the stability of the induced strain;
step six: determination of results
MIC value determination, wherein the MIC is the lowest concentration of the disinfectant in which no visible growth of bacteria (clear wells) is observed in a 96-well plate, and the MIC values are 256 and 512mg/L respectively by taking bacteria A and B as examples;
OD 600 and (3) value determination, namely determining the OD600 value by using a microplate reader, and determining clear holes when the OD is less than or equal to 0.1, otherwise, determining turbid holes.
In conclusion, the beneficial effects of the invention are as follows:
1. the invention induces the bacteria to generate antibodies through sodium hypochlorite, adopts sodium hypochlorite solution with the Minimum Inhibitory Concentration (MIC) of 1/2 to continuously induce the strains, researches the physiological characteristics of the resistant strains of the sodium hypochlorite, and has important significance on a resistance generation mechanism and formulation of a targeted intervention strategy.
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In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the invention, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a schematic diagram illustrating the judgment of the resistance result of the method for inducing the resistance of the salmonella enteritidis by using sodium hypochlorite.
Detailed Description
All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.
Any feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving equivalent or similar purposes, unless expressly stated otherwise. That is, unless expressly stated otherwise, each feature is only an example of a generic series of equivalent or similar features.
The invention will now be described in detail with reference to fig. 1, wherein for the sake of convenience of description the orientations referred to below are now defined as follows: the up, down, left, right, front and rear directions described below correspond to the front, back, left, right, top and bottom directions of the view direction of fig. 1, fig. 1 is a front view of the apparatus of the present invention, and the directions shown in fig. 1 correspond to the front, back, left, right, top and bottom directions of the apparatus of the present invention.
Referring to fig. 1, an embodiment of the present invention: a method for inducing resistance of Salmonella enteritidis by sodium hypochlorite, comprising the steps of:
the method comprises the following steps: test Strain selection
Selecting salmonella enteritidis CVCC1806 from China center for culture Collection of microorganisms, wherein the salmonella enteritidis is preserved at the temperature of below-80 ℃ for 1-5 times;
step two: activation of primary strains
Taking out the frozen strain from-80 ℃, thawing at room temperature, sucking 0.4ml, inoculating into 20ml of TSB culture medium, controlling the temperature at 37 ℃, and incubating for 24h;
step three: preparation of bacterial suspension
In a sterile environment, performing centrifugal treatment on bacterial cells incubated for 24 hours, controlling the temperature at 4 ℃, controlling the rotating speed at 10000rpm, centrifuging for 5min, washing for 3 times by using 0.85% (w/v) sterile physiological saline, and then suspending in 0.85% (w/v) sterile physiological saline to obtain 109CFU/mL bacterial suspension;
step four: determination of MIC value of Strain
mu.L of the culture broth was diluted in 10ml MH broth (MHB), and then 100. Mu.L of a disinfectant with a gradient of different concentrations was added to a 96-well microplate, followed by 100. Mu.L of the diluted bacterial suspension (about 10 bacteria count) 6 CFU)；
Setting positive control (inoculating 100 μ L bacterial suspension and 100 μ L blank MHB) and negative control (directly inoculating 100 μ L blank MHB);
setting each sample for 2 times, sealing with disposable sealing film of enzyme labeling plate, culturing at 37 deg.C for 18 hr, and determining OD with enzyme labeling instrument 600 A value;
step five: continuous induction of strains
Continuously inducing the strains by adopting a sodium hypochlorite solution with the Minimum Inhibitory Concentration (MIC) of 1/2;
the method specifically comprises the following steps:
s5.1, taking 10ml of NaClO solution of an original strain MIC, mixing the NaClO solution with trypticase soy peptone liquid medium (TSB medium) in equal volume, uniformly mixing in a vortex mode to obtain a TSB medium containing 1/2MIC NaClO solution, inoculating 0.4ml of incubated S.enteritidis CVCC1806, placing at 37 ℃ for incubation for 24 hours, counting as the 1 st generation, and determining the MIC value;
s5.2, taking 10ml of a 1 st generation bacterium liquid MIC NaClO solution, mixing the solution with a trypticase soy peptone liquid medium (TSB medium) in an isometric mode, uniformly mixing in a vortex mode to obtain a TSB medium containing a 1/2MIC NaClO solution, inoculating 0.4ml of the first generation bacterium liquid, placing at 37 ℃ for incubation for 24 hours to obtain a 2 nd generation, and determining the MIC value of the first generation bacterium liquid;
s5.3, repeating the operations, and when the MIC value of the strain is increased in the induction process, correspondingly increasing the concentration (1/2 MIC) of the stress solution;
s5.4 after the induction is finished, placing the induced strain in a TSB culture medium at 37 ℃ for incubation for 24h, carrying out subculture, carrying out continuous passage for 5 times, measuring an MIC value after each passage, and observing the stability of the induced strain;
step six: determination of results
MIC value determination, wherein the MIC is the lowest concentration of the disinfectant in which no visible growth of bacteria (clear wells) is observed in a 96-well plate, and the MIC values are 256 and 512mg/L respectively by taking bacteria A and B as examples;
OD 600 determining the value, namely determining the OD600 value by using an enzyme labeling instrument, and determining clear holes when the OD is less than or equal to 0.1, otherwise determining turbid holes;
in addition, in one example, it can be seen from Table I that after 25 induction passages and 5 stable passages, the S.Enteritidis CVCC1806 induced strain reached 512ppm sodium hypochlorite resistance and the MIC value increased 4-fold compared to 128ppm for the standard strain
TABLE I sodium hypochlorite MIC values before and after Salmonella induction
Figure BDA0003854691950000051
The above description is only an embodiment of the invention, but the scope of the invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the invention. Therefore, the protection scope of the invention should be subject to the protection scope defined by the claims.

Claims (7)

1. A method for inducing resistance of salmonella enteritidis by sodium hypochlorite is characterized in that: the method comprises the following steps:
the method comprises the following steps: test Strain selection
Selecting Salmonella enteritidis CVCC1806, and storing at below-80 deg.C for 1-5 times;
step two: activation of primary Strain
Taking out the frozen strain from-80 ℃, thawing at room temperature, sucking 0.4ml, inoculating into 20ml of TSB culture medium, controlling the temperature at 37 ℃, and incubating for 24h;
step three: preparation of bacterial suspension
Washing with 0.85% (w/v) sterile physiological saline for 3 times, and resuspending in 0.85% (w/v) sterile physiological saline to obtain 10 9 CFU/mL bacterial suspension;
step four: determination of MIC value of Strain
mu.L of the culture broth was diluted in 10ml MH broth (MHB), and then 100. Mu.L of a disinfectant with a gradient of different concentrations was added to a 96-well microplate, followed by 100. Mu.L of the diluted bacterial suspension (about 10 bacteria count) 6 CFU)；
Step five: continuous induction of strains
Continuously inducing the strains by adopting a sodium hypochlorite solution with the Minimum Inhibitory Concentration (MIC) of 1/2;
step six: and (6) judging the result.
2. The method of claim 1, wherein the method comprises inducing resistance to Salmonella enteritidis with sodium hypochlorite, the method comprising: in the third step, the environmental temperature is kept at 4 ℃ during centrifugation, the centrifugation speed is 10000rpm, and the centrifugation lasts for 5min.
3. The method of claim 1, wherein the method comprises inducing resistance to Salmonella enteritidis by sodium hypochlorite, the method comprising: in step four, a positive control (inoculated with 100. Mu.L of the bacterial suspension and 100. Mu.L of blank MHB) and a negative control (directly inoculated with 100. Mu.L of blank MHB) were set simultaneously.
4. The method of claim 1, wherein the method comprises inducing resistance to Salmonella enteritidis with sodium hypochlorite, the method comprising: setting each sample for 2 times in the fourth step, sealing with disposable sealing film of enzyme label plate, culturing at 37 deg.C for 18h, and determining OD with enzyme label 600 The value is obtained.
5. The method of claim 1, wherein the method comprises inducing resistance to Salmonella enteritidis with sodium hypochlorite, the method comprising: the fifth step also comprises the following steps:
s5.1, taking 10ml of NaClO solution of an original strain MIC, mixing the NaClO solution with a trypticase soy peptone liquid medium (TSB medium) in an equal volume, uniformly mixing by vortex to obtain a TSB medium containing 1/2MIC NaClO solution, inoculating 0.4ml of incubated S.enteritidis CVCC1806, placing at 37 ℃ for incubation for 24 hours, counting as the 1 st generation, and determining the MIC value;
s5.2, taking 10ml of a 1 st generation bacterium liquid MIC NaClO solution, mixing the solution with a trypticase soy peptone liquid medium (TSB medium) in an isometric mode, uniformly mixing in a vortex mode to obtain a TSB medium containing a 1/2MIC NaClO solution, inoculating 0.4ml of the first generation bacterium liquid, placing at 37 ℃ for incubation for 24 hours to obtain a 2 nd generation, and determining the MIC value of the first generation bacterium liquid;
s5.3, repeating the operations, and when the MIC value of the strain is increased in the induction process, correspondingly increasing the concentration (1/2 MIC) of the stress solution;
and (5) after S5.4 induction is finished, incubating the induced strain in a TSB culture medium at 37 ℃ for 24h, carrying out subculture, carrying out continuous subculture for 5 times, measuring an MIC value after each subculture, and observing the stability of the induced strain.
6. The method of claim 1, wherein the method comprises inducing resistance to Salmonella enteritidis by sodium hypochlorite, the method comprising: and judging the MIC value in the sixth step, wherein the MIC value is 256 and 512mg/L respectively by taking the lowest disinfectant concentration of no visible bacteria growth (clear wells) in a 96-well plate as the MIC and taking bacteria A and B as examples.
7. The method of claim 1, wherein the method comprises inducing resistance to Salmonella enteritidis by sodium hypochlorite, the method comprising: OD in step six 600 And (4) determining the value, namely determining the OD600 value by using a microplate reader, and determining that the well is clear when the OD is less than or equal to 0.1, otherwise, determining that the well is turbid.
CN202211144347.4A 2022-09-20 2022-09-20 Method for inducing salmonella enteritidis to generate resistance by sodium hypochlorite Pending CN115612653A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115232779A (en) * 2022-07-18 2022-10-25 浙江省农业科学院 Method for inducing salmonella enteritidis to enter VBNC state by utilizing sodium hypochlorite

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115232779A (en) * 2022-07-18 2022-10-25 浙江省农业科学院 Method for inducing salmonella enteritidis to enter VBNC state by utilizing sodium hypochlorite

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