阅读说明：本技术 舍曲林和苯扎氯铵在制备抑制和/或杀灭致病菌的制剂中的应用 (Application of sertraline and benzalkonium chloride in preparation of preparation for inhibiting and/or killing pathogenic bacteria ) 是由 孟赫诚 王一迪 李丽丽 冼洁蓓 谢胜海 于 2021-09-30 设计创作，主要内容包括：本发明提供了舍曲林和苯扎氯铵在制备抑制和/或杀灭致病菌的制剂中的应用,属于公共卫生及养殖环境应用技术领域。舍曲林能够能够抑制金黄色葡萄球菌和单核细胞增生李斯特菌毒力基因的表达,舍曲林与苯扎氯铵联用能够减少苯扎氯铵的用量,降低金黄色葡萄球菌和单核细胞增生李斯特菌对苯扎氯铵的耐药性,有效防止环境金黄色葡萄球菌和单核细胞增生李斯特菌的污染。经过验证,舍曲林添加量为4～32μg/ml,苯扎氯铵的MIC值降低了约1/2。(The invention provides application of sertraline and benzalkonium chloride in preparation of a preparation for inhibiting and/or killing pathogenic bacteria, belonging to the technical field of public health and breeding environment application. The sertraline can inhibit the expression of virulence genes of staphylococcus aureus and listeria monocytogenes, and the combination of sertraline and benzalkonium chloride can reduce the dosage of benzalkonium chloride, reduce the drug resistance of staphylococcus aureus and listeria monocytogenes to benzalkonium chloride, and effectively prevent the pollution of environment staphylococcus aureus and listeria monocytogenes. Proved by verification, the addition amount of sertraline is 4-32 mu g/ml, and the MIC value of benzalkonium chloride is reduced by about 1/2.)

1. The use of sertraline and benzalkonium chloride in the preparation of a formulation for the inhibition and/or killing of pathogenic bacteria.

2. The use of sertraline and benzalkonium chloride in the preparation of a formulation for reducing the expression of virulence genes of pathogenic bacteria.

3. Use according to claim 1 or 2, wherein the pathogenic bacteria comprise gram-positive bacteria.

4. Use according to claim 3, wherein the gram-positive bacteria comprise food-borne gram-positive bacteria.

5. The use according to claim 4, wherein the food-borne gram-positive bacteria comprise food-borne Staphylococcus aureus and/or food-borne Listeria monocytogenes.

6. Use according to claim 2, wherein, when the pathogenic bacterium is food-borne Staphylococcus aureus, the virulence genes include one or more of the hal, pyk and RNAIII genes; when the pathogenic bacteria are food-borne listeria monocytogenes, the virulence genes preferably comprise one or more of hag, argA, degU, luxS, prfA, ztCA, flaA, sigB, itrC, sufS and sufU.

7. The use according to any one of claims 1 to 6, wherein the concentration of sertraline in said formulation is 4 to 32 μ g/mL; the concentration of benzalkonium chloride in the preparation is 300-500 mug/mL.

8. The use according to claim 7, wherein the concentration of sertraline in said formulation is 8-16 μ g/mL; the concentration of benzalkonium chloride in the formulation was 400 μ g/mL.

9. Use according to any one of claims 1 to 6, wherein the formulation comprises a disinfectant, a medicament or a feed additive.

10. Use according to claim 9, wherein the disinfectant comprises an environmental disinfectant.

Technical Field

The invention relates to the technical field of public health and breeding application, in particular to application of sertraline and benzalkonium chloride in preparation of a preparation for inhibiting and/or killing pathogenic bacteria.

Background

Benzalkonium chloride (Benzalkonium chloride) is a quaternary ammonium cationic surfactant, belongs to a non-oxidative bactericide, has broad-spectrum and high-efficiency bactericidal capability, can change the permeability of bacterial cytoplasmic membranes, enables bacterial cytoplasmic substances to permeate outwards, blocks the metabolism of the bacterial cytoplasmic substances to play a role in killing gram-positive bacteria, and has strong effect on gram-positive bacteria. However, antibacterial products containing benzalkonium chloride are often used, which tends to cause resistance to gram-positive bacteria.

Disclosure of Invention

The invention aims to provide application of sertraline and benzalkonium chloride in preparation of a preparation for inhibiting and/or killing pathogenic bacteria.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of sertraline and benzalkonium chloride in preparation of a preparation for inhibiting and/or killing pathogenic bacteria.

The invention also provides application of sertraline and benzalkonium chloride in preparation of a preparation for reducing the expression of virulence genes of pathogenic bacteria.

Preferably, the pathogenic bacteria comprise gram positive bacteria.

Preferably, the gram-positive bacteria comprise food-borne gram-positive bacteria.

Preferably, the food-borne gram-positive bacteria comprise food-borne staphylococcus aureus and/or food-borne listeria monocytogenes.

Preferably, when the pathogenic bacteria is food-borne staphylococcus aureus, the virulence genes comprise one or more of hal, pyk and RNAIII genes; when the pathogenic bacteria are food-borne listeria monocytogenes, the virulence genes preferably comprise one or more of hag, argA, degU, luxS, prfA, ztCA, flaA, sigB, itrC, sufS and sufU.

Preferably, the concentration of sertraline in the preparation is 4-32 mu g/mL; the concentration of benzalkonium chloride in the preparation is 300-500 mug/mL.

Preferably, the concentration of sertraline in the preparation is 8-16 mu g/mL; the concentration of benzalkonium chloride in the formulation was 400 μ g/mL.

Preferably, the formulation comprises a disinfectant, a drug or a feed additive.

Preferably, the disinfectant comprises an environmental disinfectant.

The invention provides application of sertraline and benzalkonium chloride in preparation of a preparation for inhibiting and/or killing pathogenic bacteria. The sertraline can inhibit the expression of virulence genes of staphylococcus aureus and listeria monocytogenes, and the combination of sertraline and benzalkonium chloride can reduce the dosage of benzalkonium chloride, reduce the drug resistance of staphylococcus aureus and listeria monocytogenes to benzalkonium chloride, and effectively prevent the pollution of the environment staphylococcus aureus and listeria monocytogenes. Proved by verification, the addition amount of sertraline is 4-32 mu g/ml, and the MIC value of benzalkonium chloride is reduced by about 1/2.

Drawings

FIG. 1 is a graph of the bactericidal profile of sertraline against USA 300;

FIG. 2 is a graph of sertraline versus Newman sterilization;

FIG. 3 is a graph of the effect of sertraline on virulence gene expression of Staphylococcus aureus;

FIG. 4 shows the combined bacteriostatic effect of benzalkonium chloride and sertraline;

FIG. 5 growth curves for combinations of benzalkonium chloride (400. mu.g/mL) LM001 at different sertraline concentrations;

FIG. 6 growth curves for benzalkonium chloride (400. mu.g/mL) LM11915 in combination at different sertraline concentrations;

FIG. 7 Effect of benzalkonium chloride (400. mu.g/mL) on Listeria monocytogenes virulence gene expression in combination with varying sertraline concentrations.

Detailed Description

The invention provides application of sertraline and benzalkonium chloride in preparation of a preparation for inhibiting and/or killing pathogenic bacteria.

In the present invention, the sertraline is conventionally commercially available and, in the practice of the present invention, is purchased from sertraline, which is available from Shanghai Maxim company. In the present invention, the benzalkonium chloride is commercially available from conventional sources, and in the practice of the present invention, the benzalkonium chloride is available from Sigma-Aldrich.

In the present invention, the pathogenic bacteria preferably include gram-positive bacteria; the gram-positive bacteria preferably include food-borne gram-positive bacteria; the food-borne gram-positive bacteria preferably comprise food-borne staphylococcus aureus and/or food-borne listeria monocytogenes.

The invention also provides application of sertraline and benzalkonium chloride in preparation of a preparation for reducing the expression of virulence genes of pathogenic bacteria.

When the pathogenic bacteria are food-borne staphylococcus aureus, the virulence genes comprise one or more of hal, pyk and RNAIII genes. When the pathogenic bacteria are food-borne listeria monocytogenes, the virulence genes preferably comprise one or more of hag, argA, degU, luxS, prfA, ztCA, flaA, sigB, itrC, sufS and sufU.

In the invention, the concentration of sertraline in the preparation is preferably 4-32 mu g/mL, and more preferably 8-16 mu g/mL; the concentration of benzalkonium chloride in the preparation is preferably 300-500 mug/mL, and more preferably 400 mug/mL.

In the present invention, the formulation preferably comprises a disinfectant, a drug or a feed additive; the disinfectant preferably comprises an environmental disinfectant.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

1. MIC protocol

The method of dilution of the micro medium in a 96-well titer plate was used. Suspending Staphylococcus aureus (ATCC25923) bacteriaThe liquid concentration is adjusted to 10⁶CFU/ml, dilution of SR with double concentration gradient, final yield: 128. 64, 32, 16, 8, 4, 2, 1 mug/ml. SR 100. mu.l and bacterial suspension 100. mu.l were added to each well of the 96-well plate at different concentrations. Blank control and positive control are MH bacterial liquid (hydrolyzed casein culture medium) and bacterial suspension respectively. 96 well plates were incubated overnight at 37 ℃. The MIC is the minimum SR concentration required to inhibit visible growth of the microorganism. The results of the test are shown in Table 1 in three replicates.

TABLE 1 MIC assay of sertraline against Staphylococcus aureus

As can be seen from Table 1, the MIC values of sertraline against Staphylococcus aureus are consistent, and there is no resistance to Staphylococcus aureus.

2. Growth curve experimental protocol

Staphylococcus aureus was grown overnight to an OD600nm of 0.2. 100. mu.l of sertraline Solution (SR) and 100. mu.l of bacterial suspension were added to each well of a 96-well microtiter plate to give a final concentration of SR in each well of 1/2MIC, 2MIC and 4MIC in this order. MH broth and bacterial broth were used as blank and positive controls, respectively. The culture was carried out at 37 ℃ in an incubator, OD600nm values were read every 1 hour by a microplate analyzer, and the effect of different sertraline concentrations on the growth of Staphylococcus aureus was monitored, and the results of the measurement are shown in FIGS. 1 and 2, in which FIG. 1 is a graph showing the bactericidal curve of sertraline against USA300 and FIG. 2 is a graph showing the bactericidal curve of sertraline against Newman.

As can be seen from FIGS. 1 and 2, the activity of both USA300 and Newman gradually decreased with increasing sertraline dosage, and growth stopped at the MIC value.

3. The effect of sertraline on the virulence gene expression of staphylococcus aureus was studied by real-time fluorescent PCR, and the assay results are shown in fig. 3. As can be seen from FIG. 3, the expression of the had, pyk and RNAIII genes of Staphylococcus aureus was greatly inhibited at the level of 1/2MIC of sertraline.

Example 2

5 S.aureus strains (gold 1-gold 5 strains) isolated from a farm (see Schering et al for the study of multiple drug resistance reversal and virulence suppression of Staphylococcus aureus [ D ]]University of southern china, 2019. Note: the names in the literature are: J01-J05 ] for adjusting the concentration of staphylococcus aureus suspension to 10⁶CFU/ml, 100. mu.l sertraline was added to 96-well plate column 2, 50. mu.l MH broth was added to columns 3-12, and double concentration gradient dilutions were performed in columns 2-12. Another 96-well plate was loaded with 100. mu.l of antibiotic solution at row A, 50. mu.l of broth at each of the remaining wells, and diluted with a double concentration gradient at rows A-G. The contents of the two 96-well plates were mixed. In addition to (1, H), 100. mu.l of each bacterial suspension was added to each well of the 96-well plate at different concentrations. Column 1 is SR single drug control, row 12 is antibiotic single drug control. 96 well plates were incubated overnight at 37 ℃. The experiment was repeated three times. See table 2 and fig. 4 for test results. The result shows that when the addition amount of sertraline is 8 mu g/ml, the MIC value of benzalkonium chloride is reduced by about 1/2, the combined use can effectively reduce the dosage of benzalkonium chloride, reduce the drug resistance of environmental strains to benzalkonium chloride and effectively prevent the pollution of environmental microorganisms.

TABLE 2 Combined bacteriostatic test results of benzalkonium chloride and sertraline

Therefore, sertraline not only has a bacteriostatic effect and can inhibit the expression of virulence, but also is an ideal combined bacteriostatic drug, and can reduce the dosage of a disinfectant, reduce the drug resistance of staphylococcus aureus to the disinfectant and reduce the pollution of microorganisms to the environment.

Example 3 chessboard combination of sertraline/benzalkonium chloride to Listeria monocytogenes

1. Study of multiple pairs of different concentrations of sertralineDrug-resistant (benzalkonium chloride-containing) Listeria monocytogenes standard strain and MIC and MBC of LM78 (isolated from commercial raw pork samples, presented to the north river center for disease control as a scientific research use depositor, see [ Lili, Olsen, Heidemann R, et al. The method comprises the following specific steps: adjusting the concentration of bacteria to 10⁶CFU/mL, add 128ul/mL sertraline solution to 96-well plate column 1, and double dilute to 2-10 columns in order. Equal amounts of bacterial suspension were added to each well. 11 is listed as a blank control of MH broth and 12 is listed as a positive control. The culture was incubated overnight at 37 ℃ and the OD600 value was measured.

The results are shown in Table 3, where the MIC and MBC of sertraline were constant for 2 LM strains. The method shows that sertraline has the effect of weakening the drug resistance activity of multiple drug-resistant listeria monocytogenes, and is irrelevant to the drug resistance degree and the genotype of the strain.

TABLE 3 MIC and MBC of sertraline

2. Time-sterilization curve result analysis

The bactericidal effect of sertraline at different concentrations on multiple drug-resistant listeria 001 was determined by time-bactericidal curve. As shown in FIG. 5, the bactericidal curve shows about a 6 log reduction in CFU for the 32 μ g/mL sertraline group compared to the blank control, 4 μ g/mL and 32 μ g/mL sertraline groups. Therefore, 32. mu.g/mL sertraline had a drug-resistant elimination effect on LM 001.

The bactericidal effect of different concentrations of sertraline on multiple-drug resistant listeria 11915 was determined by time-bactericidal curves. As shown in FIG. 6, the bactericidal curves show about 2 and 6 log reductions in CFU compared to the blank control, 4 μ g/mL, 8 μ g/mL sertraline, and 16 μ g/mL sertraline groups, respectively. Thus, 16. mu.g/mL and 32. mu.g/mL sertraline had a drug resistance abrogating effect on LM 11915.

In addition, after the listeria monocytogenes is treated by the sertraline and the benzalkonium chloride with different concentrations together, RNA extraction, CDNA preparation and real-time fluorescence quantitative PCR are sequentially carried out on the bacterial suspension, and the result is shown in figure 7.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.