Application of inulin in improving the efficacy of ampicillin against drug-resistant Salmonella

Abstract

This invention discloses the application of inulin in enhancing the efficacy of ampicillin against drug-resistant Salmonella, belonging to the field of pharmaceutical biotechnology. The research of this invention shows that inulin itself does not have an inhibitory effect on Salmonella, but when used in combination with ampicillin, it can significantly enhance the inhibitory effect of ampicillin on Salmonella. Both in vitro and in vivo experiments of this invention have yielded the same conclusion. This invention can reduce the use of antibiotics and provides a new research direction for the prevention and control of Salmonella.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine biotechnology, more particularly to the application of inulin in improving the efficiency of ampicillin on drug-resistant salmonella. BACKGROUND

[0002] Salmonella enterica is a conditional gram-negative pathogenic bacteria widely existing in nature, and is also a common disease restricting the sustainable development of poultry farming. Poultry at different growth stages will cause different clinical phenotypes after being infected with Salmonella enterica. The younger the age, the more obvious the symptoms, and the mortality is also the highest. In addition, the most difficult thing is that once infected, it will cause group disease outbreak, and it is difficult to eliminate in the group, leading to the decline of group immunity performance, causing huge economic and human resource loss to the farm. At the same time, Salmonella enterica is an important zoonosis, and Salmonella enterica in the process of poultry farming is the source of Salmonella contamination in the animal source food production chain. Through cross contamination in the slaughtering process, it can cause pollution of animal products, and can spread and infect humans through the food chain, seriously threatening human health and public health safety.

[0003] The current main means to prevent and control Salmonella enterica relies on antibiotics, but due to the large-scale use of antibiotics in livestock and poultry production, it has given birth to the rapid spread and large-scale spread of drug-resistant bacteria in the environment. To deal with drug-resistant bacteria, new antibiotics are needed, but the discovery of new antibiotics takes nearly 10 years, and since the late 20th century, the discovery of new antibiotics has become more and more difficult, and the use of many types of antibiotics will eventually lead to multi-drug resistant bacteria, even super drug-resistant bacteria, seriously threatening the sustainability of the livestock and poultry industry and human life safety.

[0004] Therefore, it is of great significance to excavate synergists that can improve the efficiency of existing antibiotics to prevent and control drug-resistant bacteria. SUMMARY

[0005] Therefore, the present application provides the application of inulin in improving the efficiency of ampicillin on drug-resistant salmonella.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] The application of inulin in improving the efficiency of ampicillin on salmonella.

[0008] Preferably, the salmonella is ampicillin-resistant bacteria.

[0009] Another object of the present application is to provide the application of inulin in preparing a drug for preventing and treating salmonella.

[0010] Preferably, the salmonella is ampicillin-resistant bacteria.

[0011] Another object of the present application is to provide a medicine for preventing and treating Salmonella, which comprises inulin and ampicillin.

[0012] Preferably, the Salmonella is ampicillin-resistant bacteria.

[0013] Beneficial effects:

[0014] The present application discloses the application of inulin in improving the efficiency of ampicillin on resistant Salmonella. The present application shows that inulin itself has no inhibitory effect on Salmonella, but when used together with ampicillin, it can significantly improve the inhibitory effect of ampicillin on Salmonella. The same conclusion is drawn from in vitro and in vivo tests. The present application can reduce the use of antibiotics and provide a new research direction for the prevention and treatment of Salmonella. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0016] Figure 1 Figure 1 is the phenotype determination result of ampicillin-resistant Salmonella enterica bacteria in Example 1 of the present application, wherein A is the minimum inhibitory concentration of Amp-S and Amp-R, B is the growth curve of Amp-S and Amp-R, C is the survival rate of Amp-S and Amp-R under different ampicillin concentrations, and D is the survival of Amp-S and Amp-R under 200 μg / mL ampicillin.

[0017] Figure 2 Figure 2 is the killing effect of inulin in combination with ampicillin on Salmonella, wherein A is that inulin alone does not affect the growth of Salmonella (LB medium), B is that inulin alone does not affect the growth of Salmonella (M9 medium), C is the point plate diagram of in vitro sterilization experiment of inulin in combination with ampicillin, and D is the statistical diagram of survival rate in B.

[0018] Figure 3 Figure 3 is the effect of inulin on improving the sterilization efficiency of ampicillin, including the effect of ampicillin concentration, the effect of inulin concentration and the effect of action time, wherein A is the effect of ampicillin concentration, B is the effect of inulin concentration, and C is the effect of action time.

[0019] Figure 4 Figure 4 is the effect of inulin on improving the efficiency of ampicillin on clinical Salmonella-resistant bacteria.

[0020] Figure 5 The in-vivo synergistic effect of inulin and ampicillin, wherein A is the in-vivo synergistic effect of Huixiang mustache chicken, B is the in-vivo synergistic effect of Kunming mouse, and C is the in-vivo synergistic effect of large wax moth. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0022] The embodiments of the present application disclose the application of inulin in improving the effect of ampicillin on drug-resistant salmonella, and the raw materials used in the present application are commercially available, and their sources are not specifically limited, for example, inulin (C 6n H 10n O 5n ) can be purchased from Shanghai Maikelin Biochemical Technology Co., Ltd. The methods involved in the present application are conventional methods, and are not described one by one here.

[0023] Embodiment 1

[0024] Determination of ampicillin-resistant Salmonella enteritidis phenotype

[0025] 1. Determination of antibiotic minimum inhibitory concentration (MIC)

[0026] The Salmonella enteritidis glycerol strain stored at -80℃ is streaked on 2% solid culture medium using an inoculation loop, and is cultured overnight in a 37℃ incubator until single colony cells are grown. The single colony cells are picked into 5mL of liquid LB culture medium, and are cultured at 37℃ and 200rpm for 16 hours. The cultured bacteria are transferred into new test tubes in a ratio of 1:100, and are continuously cultured until OD 600 =0.5. Then, 10ul of the diluted bacterial solution is added into a 96-well plate containing ampicillin with a concentration gradient, and is cultured in a 37℃ incubator for 16 hours. The antibiotic concentration is observed from low to high, and the antibiotic concentration corresponding to the well without bacterial growth is the minimum inhibitory concentration.

[0027] 2. Acquisition of ampicillin-resistant Salmonella enteritidis

[0028] Take the enteritis Salmonella standard strain (CMCC 50041) as the starting strain, and define it as Amp-S. The minimum inhibitory concentration (MIC) of Amp-S to ampicillin was determined. Amp-S was continuously subcultured in LB medium containing 1 / 2 MIC concentration of ampicillin. Every 5 generations, it was streaked on 2% solid medium, and single colony cells were picked for MIC determination until enteritis Salmonella with MIC of 400 μg / mL was obtained, and was located as enteritis Salmonella ampicillin-resistant bacteria (Amp-R).

[0029] 3. Growth curve determination

[0030] After picking Amp-S and Amp-R single colony cells and inoculating them in 5 mL of LB medium for 16 hours, they were transferred to new 5 mL of LB test tubes at 1:100, and their OD 600 values were determined every 2 hours, with 3 test tubes at each time point. The growth curve was plotted according to the time and the corresponding OD 600 values.

[0031] 4. Survival rate determination

[0032] Amp-S and Amp-R single colony cells were cultured in LB medium with different concentrations of ampicillin for 3 hours. Their respective groups without antibiotics were used as controls to determine the OD 600 values. The OD 600 values of the antibiotic groups and the OD 600 values of the control groups were the survival rates.

[0033] Further, the sensitive bacteria and the resistant bacteria cultured for 16 hours were dropped on solid medium with a concentration of 200 μg / mL of ampicillin (titering volume was 5 μg / mL) to detect their growth.

[0034] The results of this example are shown in the accompanying Figure 1 The minimum inhibitory concentration of ampicillin for sensitive bacteria (Amp-S) was 6.25 μg / mL, and that for resistant bacteria (Amp-R) was 400 μg / mL, which was 64 times that of sensitive bacteria. The growth curve determination results showed that the growth rate of sensitive bacteria was higher than that of resistant bacteria. However, the survival rate of resistant bacteria was significantly higher than that of sensitive bacteria. Finally, the sensitive bacteria and the resistant bacteria cultured for 16 hours were dropped on solid medium with a concentration of 200 μg / mL of ampicillin (titering volume was 5 μg / mL) to detect their growth. The results showed that the resistant bacteria could grow in it, while the sensitive bacteria did not grow.

[0035] Example 2

[0036] Inositol improves the efficiency of ampicillin on enteritis Salmonella ampicillin-resistant bacteria

[0037] 1. Antibiotics kill bacteria in vitro

[0038] First, gradient concentrations of inulin (0-20 mM) were added to LB liquid medium to detect the effect of inulin on the growth of drug-resistant Salmonella.

[0039] Then, single colonies of ampicillin-resistant Salmonella enteritidis were picked and inoculated into 5 mL of LB liquid medium. After overnight incubation at 37°C and 200 rpm for 16 h, the bacteria were collected, centrifuged at 8000 rpm for 3 minutes, the supernatant was removed, and the bacterial cells were washed three times with 0.85% physiological saline. The bacterial cells were then resuspended in M9 basal medium, and the OD of the bacterial culture was adjusted. 600 =0.6, and finally, dispense the prepared bacterial solution into test tubes (5 mL / tube) for later use. The experiment was divided into four groups: inulin group (0-20 mM), ampicillin group, ampicillin + inulin group, and control group (same volume of M9 basal medium). After each experimental group was cultured on a shaker at 37℃ and 200 rpm for 6 hours, the bacteria were serially diluted 10-fold. 5 μL of bacteria from each gradient was dropped into 2% solid medium and incubated overnight at 37℃. The single colonies were then counted to detect the bacterial survival rate of each group. The single colony count of 20-200 was considered reliable and used for subsequent survival rate analysis.

[0040] The results are attached. Figure 2 Inulin itself does not affect the growth of Salmonella. In the subsequent in vitro bactericidal experiment of inulin and ampicillin, the survival rate of ampicillin-resistant Salmonella enteritidis (Amp-R) was 84.49% after adding ampicillin at a concentration of 400 μg / mL alone, while the survival rate of Amp-R was 0.37% after adding 400 μg / mL ampicillin + 5 mM inulin. Inulin, in combination with ampicillin, increased the inhibitory efficiency against drug-resistant Salmonella by 226 times.

[0041] Example 3

[0042] Inulin enhances the bactericidal efficacy of ampicillin through a combination of ampicillin concentration effect, inulin concentration effect, and time-of-action effect.

[0043] 1. Ampicillin concentration effect: Single colonies of ampicillin-resistant Salmonella enteritidis were picked and inoculated into 5 mL of LB liquid medium. After overnight incubation at 37°C and 200 rpm for 16 h, the bacteria were collected, centrifuged at 8000 rpm for 3 min, the supernatant was removed, and the bacterial cells were washed three times with 0.85% physiological saline. The bacterial cells were then resuspended in M9 basal medium, and the OD of the bacterial culture was adjusted. 600=0.6, and finally, dispense the prepared bacterial solution into test tubes (5 mL / tube) for later use. The experiment was divided into twelve groups to investigate the effects of different concentrations of ampicillin (0, 100, 200, 400, 800, 1600 μg / mL) + 5 mmol inulin (with ampicillin without inulin as the control group). After each experimental group was cultured on a shaker at 37℃ and 200 rpm for 6 hours, the bacteria were serially diluted 10-fold. 5 μL of bacteria from each gradient was dropped into 2% solid medium and incubated overnight at 37℃. The single colonies were then counted to detect the bacterial survival rate of each group. The single colony count of 20-200 was considered reliable and used for subsequent survival rate analysis.

[0044] The results of the ampicillin concentration experiment showed (see appendix) Figure 3 In Figure A), the addition of 5 mmol of inulin increased the bactericidal efficiency by 47 times when the ampicillin concentration was 100 μg / mL (the survival rate decreased from 96.51% without inulin to 2.0% with inulin); when the ampicillin concentration was 200 μg / mL, the bactericidal efficiency increased by 105 times (the survival rate decreased from 87.79% without inulin to 0.83% with inulin); and when the ampicillin concentration was 400 μg / mL, the bactericidal efficiency increased by 2... The bactericidal efficiency increased 42 times (survival rate decreased from 82.55% without inulin to 0.34% with inulin); when the ampicillin concentration was 800 μg / mL, the bactericidal efficiency increased 1246 times (survival rate decreased from 62.37% without inulin to 0.05% with inulin); when the ampicillin concentration was 1600 μg / mL, the bactericidal efficiency increased 3932 times (survival rate decreased from 11.8% without inulin to 0.003% with inulin).

[0045] 2. Inulin concentration effect: Single colonies of ampicillin-resistant Salmonella enteritidis were picked and inoculated into 5 mL of LB liquid medium. After overnight incubation at 37°C and 200 rpm for 16 h, the bacteria were collected, centrifuged at 8000 rpm for 3 min, the supernatant was removed, and the bacterial cells were washed three times with 0.85% physiological saline. The bacterial cells were then resuspended in M9 basal medium, and the OD of the bacterial culture was adjusted. 600=0.6, and finally, the prepared bacterial solution was dispensed into test tubes (5 mL / tube) for later use. The experiment was divided into seven groups to investigate the effects of different concentrations of inulin (0, 1.25, 2.5, 5, 10, 20 mmol) + 400 μg / mL ampicillin (with a suspension of Salmonella ampicillin-resistant bacteria as a blank control group). After each experimental group was shaken and cultured on a shaker at 37℃ and 200 rpm for 6 hours, the bacteria were serially diluted 10-fold. 5 μL of bacteria from each gradient was dropped into 2% solid culture medium and incubated overnight at 37℃. The single colonies were then counted to detect the bacterial survival rate of each group. The single colony count of 20-200 was considered reliable and used for subsequent survival rate analysis.

[0046] The results of the inulin concentration experiment showed (see appendix) Figure 3 In (B) of the study, the survival rate of bacteria was 80.15% when 400 μg / mL of ampicillin was added alone. When 1.25 mM inulin was added along with ampicillin, the survival rate was 3.94%, which increased the bactericidal efficiency by 19 times. When 2.5 mmol of inulin was added, the survival rate was 0.98%, which increased the bactericidal efficiency by 81 times. When 5 mmol of inulin was added, the survival rate was 0.35%, which increased the bactericidal efficiency by 228 times. When 10 mmol of inulin was added, the survival rate was 0.24%, which increased the bactericidal efficiency by 333 times. When 20 mmol of inulin was added, the survival rate was 0.08%, which increased the bactericidal efficiency by 1000 times.

[0047] 3. Time-of-action effect: Single colonies of ampicillin-resistant Salmonella enteritidis were picked and inoculated into 5 mL of LB liquid medium. After overnight incubation at 37°C and 200 rpm for 16 h, the bacteria were collected, centrifuged at 8000 rpm for 3 minutes, the supernatant was removed, and the bacterial cells were washed three times with 0.85% physiological saline. The bacterial cells were then resuspended in M9 basal medium, and the OD of the bacterial culture was adjusted. 600 =0.6, and finally, the prepared bacterial solution was dispensed into test tubes (5 mL / tube) for later use. The experiment was divided into four groups: inulin group, ampicillin group, ampicillin + inulin group, and control group. Each experimental group was cultured on a shaker at 37℃ and 200 rpm, and samples were taken at 0, 2, 4, 6, 8, and 10 hours. The bacteria were serially diluted 10-fold, and 5 μL of bacteria from each gradient was dropped into 2% solid medium and incubated overnight at 37℃. The single colonies were counted to detect the bacterial survival rate of each group. The single colony count of 20-200 was considered reliable data for subsequent survival rate analysis.

[0048] The results of the time-effect experiment showed (see appendix) Figure 3(C) After 2 hours of treatment, the survival rate increased 6-fold (from 94.15% with ampicillin monotherapy to 14.09%); after 4 hours, it increased 112-fold (from 90.11% with ampicillin monotherapy to 0.80%); after 6 hours, it increased 214-fold (from 85.88% with ampicillin monotherapy to 0.40%); after 8 hours, it increased 540-fold (from 75.76% with ampicillin monotherapy to 0.14%); and after 10 hours, it increased 546-fold (from 76.64% with ampicillin monotherapy to 0.14%).

[0049] Example 4

[0050] Inulin enhances the efficacy of ampicillin against clinically resistant Salmonella bacteria.

[0051] To further verify whether the synergistic effect of inulin and ampicillin is applicable to clinically resistant Salmonella bacteria, in vitro bactericidal experiments were conducted on seven clinically resistant Salmonella enteritidis strains (SE1, SE2, SE3, SE4, SE5, SE6, and SE7) preserved in our laboratory using 500 μg / mL ampicillin and 5 mM inulin. The results showed that for SE1, the survival rate was 71.7% with ampicillin alone, but increased by 651 times to 0.11% with the addition of inulin; for SE2, the survival rate was 61% with antibiotic alone, but increased by 1016 times to 0.06% with the addition of inulin; and for SE3, the survival rate was 8... The survival rate of SE4 was 97% with antibiotics alone, but increased by 123 times with the addition of inulin to 2.67%. For SE5, the survival rate was 85% with antibiotics alone, but increased by 229 times with the addition of inulin to 0.37%. For SE6, the survival rate was 100% with antibiotics alone, but increased by 18 times with the addition of inulin to 5.25%. For SE7, the survival rate was 79.3% with antibiotics alone, but increased by 239 times with the addition of inulin to 0.33%. These results indicate that inulin can significantly improve the efficacy of ampicillin against clinically resistant bacteria.

[0052] Example 5

[0053] Synergistic effect of inulin and ampicillin in vivo

[0054] 1. Preparation of bacterial strains

[0055] The Amp-R glycerol strain from Example 1, stored at -80°C, was removed from the freezer and added to 100 mL of LB medium (250 mL culture flask), and incubated at 37°C and 200 rpm. The bacteria were allowed to grow to the OD value. 600At approximately 1.0 mmol / L, centrifuge to collect bacteria and discard the supernatant. Wash three times with physiological saline for later use.

[0056] To investigate whether the synergistic effect of inulin and ampicillin is applicable in animals, a challenge experiment was conducted using Huiyang bearded chickens, Kunming rats, and large wax moths as experimental animals. The results are as follows:

[0057] For Huiyang bearded chickens, the challenge dose was 3*10^9 CFU / bird, administered intraperitoneally. One hour after challenge, the antibiotic group received an intraperitoneal injection of 20 mg / kg ampicillin, while the synergistic group received 20 mg / kg ampicillin and 30 mg / kg inulin. The control group received the same volume of physiological saline. Survival results showed that all control group members died, the antibiotic group had a survival rate of 30%, and the synergistic group had a survival rate of 80%, representing a 50% improvement in survival rate. For Kunming rats, the challenge dose was 5*10^8 CFU / bird, administered intraperitoneally. One hour after challenge, the antibiotic group received an intraperitoneal injection of 20 mg / kg ampicillin, while the synergistic group received 20 mg / kg ampicillin. In a study comparing the survival rates of ampicillin and inulin (15 mg / kg) in a control group (same volume of physiological saline), the survival rate was 10% in the control group, 50% in the antibiotic group, and 90% in the synergistic group, representing a 40% improvement in survival rate. For the large wax moth, the challenge dose was 8 x 10^7 CFU / animal, administered intraperitoneally. One hour later, the antibiotic group received an intraperitoneal injection of 15 mg / kg ampicillin, while the synergistic group received both 15 mg / kg ampicillin and 20 mg / kg inulin. The control group received the same volume of physiological saline. Survival rates showed that all animals in the control group died, while the antibiotic group had a 40% survival rate and the synergistic group had a 100% survival rate, representing a 60% improvement in survival rate. These results indicate that inulin can enhance the efficacy of ampicillin in animals and improve the survival rate of animals infected with drug-resistant bacteria.

[0058] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0059] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of inulin and ampicillin in combination in the preparation of a drug for preventing and treating ampicillin-resistant Salmonella enteritidis.

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