Application of glutamine in the preparation of drugs that inhibit gene mutations in Escherichia coli

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of glutamine in the preparation of drugs that inhibit gene mutations in *E. coli*. This application combines glutamine and ampicillin, and after multiple passages, it can inhibit drug-resistance gene mutations in *E. coli*, significantly reducing the mutation probability of drug-resistance genes, decreasing the fold increase in the minimum inhibitory concentration (MIC) of ampicillin after passage, and lowering the survival rate. Animal experiments have also demonstrated that passaged bacteria with added glutamine and ampicillin are more easily cleared by the body, achieving the effect of slowing down the formation of drug-resistance gene mutations in *E. coli* to ampicillin.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology. More specifically, it relates to the application of glutamine in the preparation of drugs that inhibit gene mutations in Escherichia coli. Background Technology

[0002] Gene mutation is a natural phenomenon, a result of thousands of years of biological evolution, with microbial gene mutations being particularly common. Some pathogenic bacteria, such as *E. coli*, undergo gene mutations during their growth and reproduction. Under the selective pressure of antibiotics, bacteria that continue to grow after mutation are selected and proliferate dominantly. With each generation, the inhibitory effect of antibiotics on bacteria gradually weakens, and even superbugs emerge, making the treatment of bacterial infections more difficult. Furthermore, in the culture of experimental bacteria, bacteria contaminated with antibiotics that undergo gene mutations show significant differences in genotype compared to the primary bacteria as the culture progresses, which can have a substantial impact on experimental results.

[0003] Currently, methods for inhibiting the growth and reproduction of bacteria with gene mutations mainly involve increasing the sensitivity of the mutated bacteria to antibiotics. For example, Chinese patent application CN102973542A discloses a small molecule substance that enhances bacterial sensitivity to antibiotics. This small molecule substance, glutamine, can increase the sensitivity of mutant bacteria to antibiotics and exhibits a significant synergistic effect when used in conjunction with glucose, ultimately achieving a bactericidal effect. However, this technical solution primarily improves the bactericidal efficiency of existing antibiotics. There are currently no research reports on how to inhibit bacterial gene mutations and slow down the development of bacterial resistance. Therefore, fundamentally inhibiting bacterial gene mutations is of greater significance than simply improving the bactericidal effect of antibiotics. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiency and inadequacy of the existing technology in the lack of research on inhibiting gene mutations in Escherichia coli, and to provide a novel application of glutamine in the preparation of drugs that inhibit gene mutations in Escherichia coli.

[0005] The purpose of this invention is to provide an application of glutamine in the preparation of drugs that inhibit gene mutations in Escherichia coli.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] Application of glutamine in the preparation of drugs that inhibit gene mutations in Escherichia coli.

[0008] Furthermore, the *Escherichia coli* is a susceptible *Escherichia coli* bacterium.

[0009] Furthermore, the Escherichia coli is a drug-resistant Escherichia coli.

[0010] Furthermore, the gene mutation was generated during passage in the presence of antibiotics.

[0011] Furthermore, the antibiotics include ampicillin, amoxicillin, penicillin G, and carbenicillin.

[0012] Preferably, the antibiotic is ampicillin.

[0013] Furthermore, the dosage ratio of glutamine to antibiotic is 1:(0.001 to 0.3) by weight.

[0014] Preferably, the dosage ratio of glutamine to antibiotic is 1:(0.01 to 0.3) by weight.

[0015] Glutamine, scientifically known as 2-amino-4-carbamoylbutyric acid, is an important amino acid in living organisms. It has many functions, such as providing nitrogen source to promote protein synthesis, enhancing strength and endurance, strengthening the immune system, and synthesizing the antioxidant glutathione in traditional Chinese medicine. Through extensive creative work, the inventors discovered that adding glutamine during the passage of clinically susceptible or drug-resistant Escherichia coli in antibiotic-containing culture media can inhibit the development of drug resistance gene mutations in E. coli, significantly reduce the mutation probability of drug resistance genes, decrease the fold increase in the minimum inhibitory concentration of ampicillin after passage, reduce the survival rate, and slow down the development of resistance to ampicillin in E. coli. Furthermore, when mice were infected with bacteria that had been passaged for 30 generations with or without glutamine, and treated with glutamine and ampicillin, the bacterial counts in the blood, liver, kidneys, and spleen of mice with glutamine-added passaged bacteria were significantly lower than those without glutamine-added passaged bacteria. This indicates that passaged bacteria with added glutamine and ampicillin are more easily cleared by the body, and also demonstrates that passaged bacteria with added glutamine are less likely to develop drug resistance gene mutations when treated with the combined use of ampicillin and glutamine.

[0016] Additionally, this application provides a method for slowing down the development of drug resistance in Escherichia coli by combining glutamine with antibiotics.

[0017] The present invention has the following beneficial effects:

[0018] This invention provides a novel application of glutamine in the preparation of drugs that inhibit gene mutations in Escherichia coli. The combined use of glutamine and ampicillin, after multiple passages, can inhibit drug resistance gene mutations in Escherichia coli, significantly reduce the mutation probability of drug resistance genes, decrease the fold increase of the minimum inhibitory concentration of ampicillin to bacteria after passage, and reduce the survival rate. Animal experiments have also shown that passaged bacteria with added glutamine and ampicillin are more easily cleared by the body, thus achieving the effect of slowing down the formation of drug resistance gene mutations in Escherichia coli to ampicillin. Attached Figure Description

[0019] Figure 1 This is the result of gene sequence determination of glutamine metabolism pathway and purine metabolism pathway related genes in clinically susceptible and clinically drug-resistant Escherichia coli after 30 passages in vitro, as shown in Example 2.

[0020] in, Figure 1 A is clinically susceptible Escherichia coli K12; Figure 1 B is clinically susceptible Escherichia coli S13; Figure 1 C represents clinically drug-resistant Escherichia coli Y9; Figure 1 D represents clinically drug-resistant Escherichia coli Y17.

[0021] Figure 2 The results of the determination of the minimum inhibitory concentration (MIC) of clinically susceptible and clinically resistant Escherichia coli in vitro passages are presented in Example 3.

[0022] in, Figure 2 A is clinically susceptible Escherichia coli K12; Figure 2 B is clinically susceptible Escherichia coli S13; Figure 2 C represents clinically susceptible Escherichia coli S14; Figure 2 D represents clinically drug-resistant Escherichia coli Y9; Figure 2 E represents clinically drug-resistant Escherichia coli Y17; Figure 2 F represents clinically drug-resistant Escherichia coli Y23.

[0023] Figure 3 The results of in vitro passaged Escherichia coli survival rate determination for clinically susceptible and clinically drug-resistant strains in Example 3;

[0024] in, Figure 3 A is clinically susceptible Escherichia coli K12; Figure 2 B is clinically susceptible Escherichia coli S13; Figure 3 C represents clinically susceptible Escherichia coli S14; Figure 3 D represents clinically drug-resistant Escherichia coli Y9; Figure 3 E represents clinically drug-resistant Escherichia coli Y17; Figure 3 F represents clinically drug-resistant Escherichia coli Y23.

[0025] Figure 4 This is the experimental result of the number of bacteria surviving in mouse organs after 30 passages of clinically sensitive and clinically drug-resistant Escherichia coli in vitro, as described in Example 4.

[0026] in, Figure 4 A is clinically susceptible Escherichia coli K12; Figure 4 B is clinically resistant Escherichia coli Y9.

[0027] Figure 5 The results show the determination of the minimum inhibitory concentration in the in vitro passaged strain of threonine in Comparative Example 1, which was used to study the drug resistance of the strain. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] The specific information on the bacterial samples used is detailed in Table 1.

[0030] Table 1 Information on bacterial samples

[0031]

[0032]

[0033] All the bacteria mentioned above were provided by the Affiliated Zhongshan Hospital of Xiamen University and are deposited in the Bacterial Resistance Research Laboratory of the School of Life Sciences, Sun Yat-sen University. Specifically, *Escherichia coli* Y9, Y17, and Y23 are from the doctoral dissertation "Study on Metabolite Reversal of Multidrug-Resistant *Escherichia coli* Resistance to Ampicillin," published by Zhao Xianliang in 2014; *Escherichia coli* K12, Y17, and Y23 are from Chinese patent application CN107929741B.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] Example 1: In vitro passage experiments of clinically susceptible and clinically resistant bacteria

[0036] 1.1 Sample Preparation

[0037] Clinically isolated Escherichia coli susceptible strains K12, S13, and S14, and drug-resistant strains Y9, Y17, and Y23 (initial strain WT) were selected and inoculated into LB broth. The cultures were incubated at 37°C and 200 rpm for 16 hours, centrifuged at 8000 g for 5 minutes, and the bacteria were collected. The cells were then resuspended and washed with sterile physiological saline, and this process was repeated three times. The prepared bacteria were resuspended in physiological saline to OD0.05. 600 The concentration is 0.2, and 5 mL is dispensed into test tubes for later use.

[0038] 1.2 Passage of clinically susceptible and clinically resistant bacteria

[0039] Each strain was divided into two groups: one group was the ampicillin-only group (AMP, abbreviated as A), with antibiotic concentrations of K12: 0.003125 mg / mL, S13: 0.003125 mg / mL, S14: 0.0125 mg / mL, Y9: 0.8 mg / mL, Y17: 0.8 mg / mL, and Y23: 0.8 mg / mL; the other group was the combination group of the same concentration of ampicillin and 20 mM glutamine (AMP+glutamine, abbreviated as AG). The above groups were cultured at 37℃ and 200rpm for 6 hours. The surviving bacteria in the test tubes were designated as the first generation of bacteria passaged with antibiotics alone (A1) and the first generation of bacteria passaged with antibiotics and glutamine in combination (AG1). The first generation bacteria were then inoculated into LB medium containing only antibiotics or both antibiotics and glutamine and cultured overnight. They were then aliquoted and cultured in the same manner to obtain A2 and AG2, respectively. This process was repeated until A30 and AG30 were obtained. As the number of passages increased, the concentration of antibiotics needed to be increased accordingly to match the minimum inhibitory concentration (MIC), while the concentration of glutamine remained unchanged.

[0040] Example 2: Analysis of major gene sequences in the glutamine and purine metabolic pathways of subcultured bacteria

[0041] Sequences were performed on the primary (WT) and 30th generation (ampicillin-carrying and glutamine-co-ampicillin-carrying) genes of clinically susceptible strains K12 and S13 and clinically resistant strains Y9 and Y17, focusing on glutamine metabolism (carA, carB, gdhA, glnA), purine metabolism (purF, purB, purD, purE, purK, purH, purM, purN, purT, yfbR, rihB), dual regulatory systems (cpxA, cpxR), and major resistance genes in the outer membrane antibiotic entry channel (ompF) pathway. The sequencing primers used are listed in Table 2.

[0042] Table 2 Sequencing primers

[0043]

[0044]

[0045] BLAST analysis of the sequenced data, compared with the gene sequences of the corresponding initial bacterial strains, revealed that for strain K12, 1–3 missense mutation sites were found in genes such as carA, purB, purK, purH, purM, and purT in K12-30A, while only purK and purH in K12-30AG showed missense mutations. Figure 1A); For strain Y9, 1-3 missense mutation sites were found in the genes carA, carB, purK, purT, purM, and ompF in Y9-30A, while only carB showed a missense mutation in K12-30AG; For strains S13 and Y17, only the carA, purD, and purT genes of S13-30A and the cpxA gene of Y17-30A showed 1-3 missense mutation sites, while no mutations were found in S13-30AG and Y9-30AG. Figure 1 B). These results indicate that passage of glutamine in conjunction with ampicillin can inhibit the production of drug resistance gene mutations in Escherichia coli.

[0046] Example 3: Drug Resistance Study of In Vitro Passaged Strains

[0047] 3.1. Determination of minimum inhibitory concentration

[0048] Six bacterial species (K12, S13, S14, Y9, Y17, and Y23) were used in the 0th, 5th, 10th, 15th, 20th, 25th, and 30th passages obtained according to the two passage methods in Example 1 (antibiotic alone or antibiotic in combination with glutamine). The minimum inhibitory concentration (MIC) of ampicillin was determined using the micro-broth dilution method, as follows:

[0049] Each generation of bacteria was inoculated into LB liquid medium and cultured at 37°C and 200 rpm for 16 h; then transferred to 5 mL LB medium and cultured until OD... 600 The concentration was 0.5, diluted 10-fold; ampicillin was serially diluted and added to columns 1 to 11 of a sterile 96-well plate, 100 μL per well, with column 12 as a control without antibiotics; 10 μL of bacterial culture (approximately 10 μL per well) was added to each well. 5 CFU (colony-forming units); after sealing, incubate at 37°C for 16–20 h, and use the antibiotic concentration at which no growth occurs as the minimum inhibitory concentration (MIC). Biologically, repeat 3 times.

[0050] See results Figure 2 As can be seen, with the increase of the number of passages, the minimum inhibitory concentration of bacteria against antibiotics gradually increases in both passage methods. However, the minimum inhibitory concentration of the glutamine-antibiotic passage group (AG group) is significantly lower than that of the antibiotic-only passage group (A group), indicating that glutamine inhibits the generation of antibiotic resistance gene mutations in bacteria.

[0051] The specific results are as follows: The MIC of K12 increased from the initial 0.00625 mg / mL to 0.2 mg / mL (Group A, antibiotic-added group) and 0.05 mg / mL (Group AG, antibiotic-added and glutamine-added group). The antibiotic-only group showed a 32-fold increase in resistance, while the antibiotic-and-glutamine-added group showed only an 8-fold increase in resistance, a 4-fold decrease. Figure 2 A); The MIC of S13 increased from the initial 0.00625 mg / mL to 0.8 mg / mL (with antibiotic only) and 0.05 mg / mL (with antibiotic and glutamine). The antibiotic-only group showed a 128-fold increase in resistance, while the antibiotic and glutamine-only group showed only an 8-fold increase in resistance, a 16-fold decrease in resistance. Figure 2 B); The MIC of S14 increased from the initial 0.0125 mg / mL to 1.6 mg / mL (with antibiotic only) and 0.1 mg / mL (with antibiotic and glutamine). The antibiotic-only group showed a 128-fold increase in resistance, while the antibiotic and glutamine-only group showed only a 5-fold increase in resistance, representing a 25.6-fold decrease in resistance. Figure 2 C); The MIC of Y9 increased from the initial 0.4 mg / mL to 3.2 mg / mL (with antibiotics only) and 0.8 mg / mL (with antibiotics and glutamine). The antibiotic-only group showed an 8-fold increase in resistance, while the antibiotic and glutamine-only group showed only a 2-fold increase in resistance and a 4-fold decrease in resistance. Figure 2 D); The MIC of Y17 increased from the initial 1.6 mg / mL to 6.4 mg / mL (with antibiotics only) and 3.2 mg / mL (with antibiotics and glutamine). The antibiotic-only group showed a 4-fold increase in resistance, while the antibiotic and glutamine-only group showed only a 2-fold increase in resistance, with a 2-fold decrease in resistance. Figure 2 E); The MIC of Y23 changed from the initial 1.6 mg / mL to 3.2 mg / mL (with antibiotic only) and 1.6 mg / mL (with antibiotic and glutamine). The antibiotic-only group showed a 2-fold increase in resistance, while the antibiotic and glutamine-only group showed only a 1-fold increase in resistance, with a 2-fold decrease in resistance. Figure 2 F).

[0052] 3.2 Determination of sterilization rate

[0053] The resistance of six bacterial species to ampicillin at generations 1, 5, 10, 20, and 30, generated from two passage methods, was determined using a bactericidal test. The specific methods are as follows:

[0054] These passages of bacteria were inoculated into LB liquid medium and cultured at 37°C and 200 rpm for 16 h; the cells were collected by centrifugation at 8000 g for 5 min, and then resuspended in sterile M9 medium. This process was repeated three times. The cells were then resuspended in M9 medium to a final volume of 5 × 10⁻⁶. 6 CFU / mL, dispensed into 4 mL test tubes; each passaged bacterium was divided into 3 groups: Group 1 was a blank control, with 1 mL of physiological saline added; Group 2 was treated with antibiotics dissolved in the same volume of physiological saline only; Group 3 was treated with antibiotics dissolved in the same volume of physiological saline and glutamine; the biological assay was repeated 3 times; the antibiotic concentrations added were as follows: 0.2 mg / mL for the two K12 passaged bacteria, 0.8 mg / mL for the two S13 passaged bacteria, 1.6 mg / mL for the two S14 passaged bacteria, 3.2 mg / mL for the two Y9 passaged bacteria, 6.4 mg / mL for the two Y17 passaged bacteria, and 3.2 mg / mL for the two Y23 passaged bacteria, with or without 20 mM glutamine added. All test tubes were incubated at 37°C and 200 rpm for 6 hours. After that, 100 μL of each sample was taken and serially diluted. Finally, 10 μL of each serially diluted bacterial solution was spotted onto LB solid culture plates and incubated at 37°C for 16 hours. Colony-forming units (CFU) were counted. The blank control sample was used as the starting bacterial count. Data with 20–200 colonies per plate were used for statistical analysis. Bacterial survival rate was the percentage of CFU of the treated bacteria to that of the blank control bacteria.

[0055] See results Figure 3 It is evident that with increasing passage numbers, the number of surviving bacteria in antibiotics increases in both passage methods, and the survival rate also rises, indicating that passage gradually increases the mutation of antibiotic resistance genes in bacteria. Of particular note is that, for the same generation of passaged bacteria, such as K12-30A (30th generation) passaged with antibiotics only and K12-30AG (30th generation) passaged with glutamine and antibiotics alone, regardless of whether only antibiotics are added or both glutamine and antibiotics are added, the survival rate of K12-30AG is lower than that of K12-30A. This suggests that the resistance of bacteria passaged with glutamine and antibiotics is lower than that of bacteria passaged with antibiotics alone.

[0056] The survival rate of the passage group with added antibiotics and glutamine was significantly lower than that of the passage group with only added antibiotics, indicating that the addition of glutamine slows down the development of antibiotic resistance in the presence of antibiotics, suggesting that glutamine can inhibit the generation of antibiotic resistance mutations in bacteria.

[0057] The specific results are as follows: For strain K12, the survival rates of the 1st, 5th, 10th, 20th, and 30th generations of ampicillin-treated bacteria (K12-A) were 4.32%, 12.34%, 34.63%, 85.67%, and 96.9%, respectively, when ampicillin alone was used for sterilization. The survival rates were 0.01%, 0.05%, 0.26%, 2.52%, and 12.48%, respectively, when glutamine was used in conjunction with ampicillin for sterilization. The survival rates of the 1st, 5th, 10th, 20th, and 30th generations of K12-AG bacteria (with antibiotics and glutamine) were 4.32%, 8.25%, 11.25%, 26.17%, and 56.74% when only ampicillin was added, respectively. The survival rates when glutamine was combined with ampicillin were 0.01%, 0.03%, 0.06%, 0.3%, and 1.18%, respectively. This data indicates that: 1) Bacterial resistance increases after antibiotic passage, but if glutamine is added during passage, it inhibits antibiotic resistance gene mutations in bacteria. The degree of reduction can be calculated using the following formula: Multiple of reduction in antibiotic resistance in the same passage of bacteria = Survival rate of ampicillin-treated passages / Survival rate of passages with both antibiotics and glutamine. Therefore, when ampicillin was used for sterilization, the susceptibility of the first, fifth, tenth, twentieth, and thirtieth generation bacteria in the combination of glutamine and antibiotics was increased by 1, 1.5, 3, 2.7, and 1.7 times, respectively, compared to the ampicillin-only group. 2) Glutamine increased the degree of resistance reduction. When glutamine was used in combination with antibiotics for sterilization, the susceptibility of the first, fifth, tenth, twentieth, and thirtieth generation bacteria in the combination of antibiotics and glutamine was increased by 1, 1.83, 4.54, 8.32, and 10.55 times, respectively, compared to the ampicillin-only group. Figure 3 A).

[0058] Other strains also showed similar results, summarized below:

[0059] For strain S13, 1) when ampicillin was used for sterilization, the susceptibility of the 1st, 5th, 10th, 20th, and 30th generation strains treated with a combination of glutamine and antibiotics was increased by 1, 2.03, 3.47, 3.65, and 4.34 times, respectively, compared to the ampicillin-only group. 2) Glutamine increased the degree of resistance reduction. When glutamine was used in combination with antibiotics for sterilization, the susceptibility of the 1st, 5th, 10th, 20th, and 30th generation strains treated with a combination of antibiotics and glutamine was increased by 1, 2.45, 5.55, 7.75, and 13.24 times, respectively, compared to the ampicillin-only group. Figure 3 B).

[0060] For strain S14, 1) when ampicillin was used for sterilization, the susceptibility of the 1st, 5th, 10th, 20th, and 30th generation strains treated with a combination of glutamine and antibiotics was increased by 1, 1.5, 2.11, 4.62, and 4.73 times, respectively, compared to the ampicillin-only group. 2) Glutamine increased the degree of resistance reduction. When glutamine was used in combination with antibiotics for sterilization, the susceptibility of the 1st, 5th, 10th, 20th, and 30th generation strains treated with a combination of antibiotics and glutamine was increased by 1, 2.23, 3.8, 12.07, and 32.45 times, respectively, compared to the ampicillin-only group. Figure 3 C).

[0061] For strain Y9, 1) when ampicillin was used for sterilization, the susceptibility of the 1st, 5th, 10th, 20th, and 30th generation strains treated with a combination of glutamine and antibiotics was increased by 1, 1.68, 1.87, 2.02, and 1.97 times, respectively, compared to the ampicillin-only group. 2) Glutamine increased the degree of resistance reduction. When glutamine was used in combination with antibiotics for sterilization, the susceptibility of the 1st, 5th, 10th, 20th, and 30th generation strains treated with a combination of antibiotics and glutamine was increased by 1, 1.73, 2.74, 5.38, and 4.73 times, respectively, compared to the ampicillin-only group. Figure 3 D).

[0062] For strain Y17, 1) when ampicillin was used for sterilization, the susceptibility of the 1st, 5th, 10th, 20th, and 30th generation strains treated with a combination of glutamine and antibiotics was increased by 1, 1.5, 1.54, 1.84, and 1.79 times, respectively, compared to the ampicillin-only group. 2) Glutamine increased the degree of resistance reduction. When glutamine was used in combination with antibiotics for sterilization, the susceptibility of the 1st, 5th, 10th, 20th, and 30th generation strains treated with a combination of antibiotics and glutamine was increased by 1, 1.5, 2.57, 5.49, and 5.44 times, respectively, compared to the ampicillin-only group. Figure 3 E).

[0063] For strain Y23, 1) when ampicillin was used for sterilization, the susceptibility of the 1st, 5th, 10th, 20th, and 30th generation strains treated with a combination of glutamine and antibiotics was increased by 1, 1.29, 1.51, 1.6, and 1.49 times, respectively, compared to the ampicillin-only group. 2) Glutamine increased the degree of resistance reduction. When glutamine was used in combination with antibiotics for sterilization, the susceptibility of the 1st, 5th, 10th, 20th, and 30th generation strains treated with a combination of antibiotics and glutamine was increased by 1, 1.73, 2.72, 5.92, and 6.24 times, respectively, compared to the ampicillin-only group. Figure 3 F).

[0064] Example 4: In vitro passaged bacteria in vivo experiment in mice

[0065] Six bacterial strains were used to infect mice: the initial (WT) strains of clinically susceptible K12 and clinically resistant Y9, the 30th generation passages of both strains after ampicillin treatment (30A), and the 30th generation passages after antibiotic supplementation and glutamine treatment (30AG). The infected mice were then treated using four different methods, and the amount of bacteria remaining in different organs of the mice was investigated. The specific methods are as follows:

[0066] BALB / c mice (six weeks old, weighing approximately 20g) were provided by the Experimental Animal Center of Sun Yat-sen University. Each strain of bacteria required 24 mice, half male and half female. The mice were randomly divided into 4 groups (n=6 per group): saline control group, 100mg / kg glutamine treatment group, 320mg / kg ampicillin treatment group, and 100mg / kg glutamine combined with 320mg / kg ampicillin treatment group.

[0067] Each type of bacteria was inoculated into LB liquid medium and incubated at 37°C and 200 rpm for 16 h. The cells were then collected by centrifugation at 8000 g for 5 min, and the cells were resuspended in sterile physiological saline. This process was repeated three times. The cells were resuspended in physiological saline to a final volume of 2 × 10⁻⁶. 7 CFU / mL, 200 μL of bacterial solution was injected intraperitoneally into each mouse; 1 h and 12 h after injection, the mice were treated according to the above groupings; 24 h after the last treatment, 100 μL of mouse serum, 100 mg of liver, kidney and spleen were added to 0.85% physiological saline to prepare a 5% homogenate, which was thoroughly ground and serially diluted. 10 μL was taken for plate counting, and then converted to CFU / mL blood or liver, kidney and spleen / g, and then logarithmic plotting was performed.

[0068] See results Figure 4 Comparison of bacterial counts in mouse tissues from wild-type K12 and Y9 (WT) and their 30th generation (30A and 30AG) bacteria under different treatments revealed no significant differences in bacterial counts among the saline control group, glutamine-treated group, and ampicillin-treated group for WT, 30A, and 30AG. However, after treatment with glutamine combined with ampicillin, bacterial counts in all three groups significantly decreased. Specifically, in the K122 infection treatment trial, compared with the ampicillin-only group, treatment with glutamine combined with ampicillin resulted in a 3.3, 2.3, and 3.2-fold decrease in serum bacterial counts; an 11, 3.2, and 5.8-fold decrease in liver bacterial counts; a 7.4, 2.2, and 3.6-fold decrease in spleen bacterial counts; and a 6.9, 2.8, and 4.2-fold decrease in kidney bacterial counts. Figure 4A). In a treatment trial following Y9 infection, compared to the ampicillin-only group, glutamine combined with ampicillin resulted in a 37.2, 1.7, and 24.7-fold decrease in serum bacterial counts; a 9.1, 3.1, and 6.4-fold decrease in liver bacterial counts; an 8.9, 2.1, and 4.4-fold decrease in spleen bacterial counts; and a 10.4, 2.4, and 5.4-fold decrease in kidney bacterial counts. Figure 4 B). It was also found that in the glutamine-ampicillin antibiotic treatment group, the bacterial content in serum, liver, kidney and spleen was significantly increased in K12-30A compared with K12-WT, and in Y9-30A compared with Y9-WT (P<0.01 or 0.05), while there was no significant change in K12-30AG compared with K12-WT, and in Y9-30AG compared with Y9-WT.

[0069] These results indicate that: 1) the combined use of glutamine and ampicillin in the passaged bacteria is less likely to induce antibiotic resistance; 2) glutamine, in synergy with antibiotics, more effectively eliminates bacteria in mice, especially the passaged bacteria treated with the combined use of glutamine and ampicillin. This suggests that the addition of glutamine to the passaged bacteria can inhibit the development of drug resistance gene mutations in Escherichia coli under the combined use of antibiotics and glutamine.

[0070] Comparative Example 1: Antimicrobial Resistance Study of Threonine-Rich Strains in In Vitro

[0071] According to the literature (Ye JZ, Lin XM, Cheng ZX, et al. Identification and efficacy of glycine, serine and threonine metabolism in potentiating kanamycin-mediated killing of Edwardsiella piscicida[J]. Journal of Proteomics, 2018:S1874391918302082. doi:10.1016 / j.jprot.2018.05.006.), threonine has the effect of enhancing the bactericidal effect of kanamycin. Referring to the experimental methods of Examples 1 and 3 of this invention, the minimum inhibitory concentration of threonine in in vitro passaged strains was determined. The strains used were susceptible Escherichia coli K12 and resistant Escherichia coli Y9. See the results below. Figure 5 .

[0072] Depend on Figure 5It is evident that with increasing passage numbers, the minimum inhibitory concentrations (MICs) of bacteria cultured using either ampicillin alone or ampicillin + threonine gradually increase. Specifically, against the susceptible *E. coli* K12, the MICs of bacteria cultured using threonine + ampicillin were the same as those cultured using ampicillin alone. Furthermore, the MICs of *E. coli* Y9 cultured using threonine + ampicillin at passages 10, 20, 25, and 30 were all higher than those cultured using ampicillin alone. These results indicate that threonine does not slow down the development of bacterial resistance. Therefore, small molecules that promote the bactericidal effect of antibiotics do not necessarily slow down the development of bacterial resistance, and their effects are unpredictable.

[0073] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for slowing down the development of drug resistance in *Escherichia coli* during the culture of experimental bacteria, characterized in that... The experiment involved the combined use of glutamine and ampicillin, with the experimental bacteria being *Escherichia coli*, specifically a susceptible strain of *Escherichia coli*. The method of slowing down the development of drug resistance in *E. coli* was to inhibit gene mutations in *E. coli* during passage in the presence of ampicillin. The dosage ratio of glutamine to ampicillin was 1:(0.001~0.3) by weight.

2. The method according to claim 1, characterized in that... The dosage ratio of glutamine to ampicillin is 1:(0.01-0.3) by weight.

Images