CN116036101B - Application of trehalose in improving drug-resistant bacteria sensitivity and assisting chicken disease-resistant breeding - Google Patents
Application of trehalose in improving drug-resistant bacteria sensitivity and assisting chicken disease-resistant breeding Download PDFInfo
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Abstract
The application belongs to the technical field of drug-resistant medicines, and particularly relates to application of trehalose in improving drug-resistant bacteria sensitivity and assisting chicken disease-resistant breeding. The drug-resistant bacteria comprise gentamicin enteritis salmonella drug-resistant bacteria (SE-R), escherichia coli K12, edwardsiella tarda EIB202, vibrio alginolyticus VA, pseudomonas aeruginosa PE and klebsiella pneumoniae KPN; the trehalose can improve the content of antibiotics in cells of the drug-resistant bacteria, thereby improving the sensitivity of the bacteria to the antibiotics; the trehalose can cooperate with gentamicin to reduce the content of bacteria in chicken spleen, liver and kidney and improve the survival rate of the Huiyang beard chicken after infection; the trehalose can cooperate with the gentamicin to enhance the bacterial removal capability of chickens and improve the resistance of Huiyang beard chickens to gentamicin enteritis Salmonella resistant bacteria, and the trehalose is combined with the gentamicin to prepare the drugs for improving the drug resistant bacteria sensitivity and assisting the disease resistance breeding of chickens.
Description
Technical Field
The application belongs to the technical field of disease resistance of livestock and poultry, and particularly relates to application of trehalose in improving drug-resistant bacteria sensitivity and assisting chicken disease resistance breeding.
Background
The discovery and widespread use of antibiotics has saved millions of people's lives during the last decades. In fact, antibiotics are not only special drugs for treating infectious diseases, but also important basic stones for the vigorous development of surgical medicine. The fortuitous discovery of fleming in 1929 provides an unprecedented treatment scheme for infections caused by gram-positive bacteria such as staphylococcus aureus, and the discovery of streptomycin in 1943 effectively controls tuberculosis of mycobacterium tuberculosis. Thus, these previous efforts have paved the way for the advent of the golden age of antibiotics. However, overuse and abuse of antibiotics accelerates the development and spread of bacterial resistance. According to World Health Organization (WHO) statistics, about 50% of the global antibacterial drugs are used in the breeding industry, and China is one of the countries with the largest production and use of animal antibacterial drugs worldwide. But solving the problem of bacterial drug resistance, especially in livestock and poultry farming, has become a worldwide problem. Therefore, there is a need to strengthen the research on the prevention and treatment of drug-resistant bacteria, explore novel prevention and control strategies for drug resistance, and promote the quality improvement and synergy of the aquaculture industry.
Trehalose (Trehalose) is a non-reducing sugar, commonly found in bacteria, fungi, yeasts, insects and plants. Can form a special protective film on the surface of a cell under severe conditions such as high temperature, high cold, drying, water loss and the like, and effectively protects the biological molecular structure from being damaged, thereby maintaining the life process and biological characteristics of a living body. Medical uses of trehalose have been made in medicine, including the treatment of huntington's disease and alzheimer's disease, because trehalose can induce apoptosis through atypical mechanisms. Recent studies have shown that trehalose can also prevent damage to mammalian eyes from desiccation and oxidative damage. However, the sterilization effect of the trehalose on bacteria and even drug-resistant bacteria can be improved, and no report is made on the aspect of bacterial infection resistance of chickens.
Disclosure of Invention
In view of the above problems, the application aims to provide the application of trehalose in improving the sensitivity of drug-resistant bacteria and assisting chicken disease-resistant breeding.
The technical content of the application is as follows:
the application provides application of trehalose in preparing a medicament for improving sensitivity of drug-resistant bacteria to gentamicin;
the drug-resistant bacteria comprise gentamicin enteritis salmonella resistant bacteria SE-R, escherichia coli K12, edwardsiella tarda EIB202, vibrio alginolyticus VA, pseudomonas aeruginosa PE and klebsiella pneumoniae KPN;
the use of the trehalose can improve the content of gentamicin in the cells of the drug-resistant bacteria;
the use of the trehalose can improve the proton motive force of drug-resistant bacteria, promote the entry of gentamicin and reverse the sensitivity of gentamicin.
The application also provides application of the trehalose in preparing medicines for improving the anti-infective power of the gentamicin resistant bacteria to the livestock (chickens).
The application also provides application of the trehalose in preparing a chicken disease-resistant breeding medicament.
The application also provides trehalose as a molecular marker for chicken disease-resistant breeding.
The application also provides application of the trehalose combined gentamicin in preparing a medicament for improving the sensitivity of drug-resistant bacteria.
The application also provides an application of the trehalose combined gentamicin in preparing a chicken disease-resistant breeding medicament.
The beneficial effects of the application are as follows:
the application of the small molecular metabolite trehalose in improving the sensitivity of drug-resistant bacteria and assisting chicken disease-resistant breeding can improve the sterilization effect of gentamicin on gentamicin enteritis Salmonella resistant bacteria (SE-R), escherichia coli K12, edwardsiella tarda EIB202, vibrio alginolyticus VA, pseudomonas aeruginosa PE and klebsiella pneumoniae KPN. Meanwhile, the trehalose can improve the content of antibiotics in cells of drug-resistant bacteria, so that the sensitivity of bacteria to antibiotics is improved; gentamicin Salmonella enteritidis resistance is caused by a decrease in intracellular antibiotics due to a decrease in membrane proton motive force. The exogenesis adding of trehalose can improve the proton motive force of drug-resistant bacteria, promote the entry of gentamicin and reverse the sensitivity of gentamicin; experiments in vivo of the Huiyang beard chickens also prove that after the Huiyang beard chickens are infected with gentamicin enteritis Salmonella resistant bacteria, the trehalose can cooperate with the gentamicin to reduce the content of bacteria in the spleen, liver and kidney of the chickens, so that the survival rate of the Huiyang beard chickens after being infected is improved; the trehalose can cooperate with gentamicin to enhance the bacterial removal capability of chickens and improve the resistance of Huiyang beard chickens to gentamicin enteritis Salmonella resistant bacteria. Therefore, according to the trehalose content of organisms or trehalose metabolism related genes as candidate targets for chicken disease resistance breeding, the disease resistance breeding of chickens can be realized from the aspect of antibiotics; therefore, the trehalose can be used as a candidate molecular marker for chicken disease-resistant breeding. Compared with the existing application of only antibiotics as antibacterial drug resistance drugs, the small molecules provided by the application have better effect and higher safety and operability.
Drawings
FIG. 1 is a graph showing the Minimum Inhibitory Concentration (MIC) of gentamicin against gentamicin Salmonella enteritidis (SE-S) and drug resistant bacteria (SE-R);
FIG. 2 shows the survival rate of gentamicin Salmonella enteritidis after adding 10mmol of trehalose to gentamicin of different concentrations;
FIG. 3 shows the survival rate of gentamicin enteritis Salmonella resistant bacteria after adding 100 μg/mL gentamicin to different concentrations of trehalose;
FIG. 4 shows the survival rate of gentamicin enteritis Salmonella resistant bacteria at various times after adding 10mmol of trehalose to 100 μg/mL of gentamicin;
FIG. 5 shows the survival rate of gentamicin enteritis Salmonella resistant bacteria after adding 20mmol of glucose to 100. Mu.g/mL gentamicin;
FIG. 6 shows the Minimum Inhibitory Concentration (MIC) of gentamicin against E.coli K12, edwardsiella tarda EIB202, vibrio alginolyticus VA, pseudomonas aeruginosa PE, klebsiella pneumoniae KPN;
FIG. 7 shows the bactericidal effect of trehalose in combination with gentamicin on E.coli K12, edwardsiella tarda EIB202, vibrio alginolyticus VA, pseudomonas aeruginosa PE, klebsiella pneumoniae KPN;
FIG. 8 shows the proton kinetic energy and intracellular antibiotic content of gentamicin enteritis Salmonella and drug-resistant bacteria (A is proton kinetic energy and B is intracellular antibiotic content);
FIG. 9 is the effect of exogenous trehalose addition on proton kinetic energy and intracellular antibiotic content of Salmonella enteritidis gentamicin resistant bacteria (A is proton kinetic energy and B is intracellular antibiotic content);
FIG. 10 shows the effect of trehalose on the ability of the viscera to clear bacteria after infection of a Huiyang beard chicken with Salmonella enteritidis gentamicin resistant bacteria (A for spleen, B for liver, C for kidney);
FIG. 11 shows the effect of trehalose on survival rate of Huiyang beard chickens infected with Salmonella enteritidis gentamicin resistant bacteria.
Detailed Description
The application is described in further detail below with reference to specific embodiments and the accompanying drawings, it being understood that these embodiments are only for the purpose of illustrating the application and not for the purpose of limiting the same, and that various modifications of the application, which are equivalent to those skilled in the art, will fall within the scope of the appended claims after reading the present application.
All materials and reagents of the application are materials and reagents of the conventional market unless specified otherwise.
Example 1
Application of trehalose in improving sensitivity of gentamicin enteritis Salmonella resistant bacteria to gentamicin
1) Determination of Minimum Inhibitory Concentration (MIC)
After picking up a monoclonal colony from a 2% solid LB plate, inoculating the colony into an LB liquid medium containing 5mL, and culturing the colony at 37 ℃ and 200rpm overnight for 16 hours, transferring the cultured bacterium into another test tube according to a volume ratio of 1:100 until the OD600 is 0.5, and using an LB solution again according to 1:100, diluting; diluting the antibiotic mother solution according to the set gradient multiple ratio, and then adding 100 mu L of the diluted antibiotic mother solution into a 96-well plate; 10 mu mL of diluted bacterial liquid is taken and placed into a 96-well plate with concentration gradient antibiotics, and the 96-well plate is placed into a constant temperature incubator at 37 ℃ for 16 hours, and the concentration of the antibiotics which just has no bacterial growth is the Minimum Inhibitory Concentration (MIC).
2) Obtaining of artificial passage drug-resistant bacteria-gentamicin enteritis Salmonella drug-resistant bacteria
Salmonella enteritidis sensitive strain CMCC 50041 (SE-S) was used as the starting strain, serially passaged in gentamicin liquid bacterial medium containing a Minimum Inhibitory Concentration (MIC) of 1/2, streaked on a 2% agarose plate every five generations to obtain a monoclonal and determine the MIC thereof on gentamicin until Salmonella enteritidis with a MIC of 200 μg/mL was obtained, and designated gentamicin Salmonella enteritidis resistant strain (SE-R) as shown in FIG. 1.
3) Preparation of test samples
Monoclonal colonies of gentamicin Salmonella enterica drug-resistant bacteria were picked from 2% solid LB plates, inoculated into 5mL of LB liquid medium, cultured overnight at 37℃and 200rpm for 16 hours, the bacteria were collected, centrifuged at 7000rpm for 3 minutes, the supernatant was removed, the bacterial cells were washed 3 times with 0.85% physiological saline, suspended in M9 minimal medium, and the bacterial liquid OD600 = 0.2 was adjusted, and finally the adjusted bacterial liquid was packed into test tubes (5 mL/tube) for bacterial survival rate study.
4) The trehalose improves the sensitivity of gentamicin enteritis Salmonella resistance bacteria to gentamicin and has the effect of antibiotic concentration gradient
To understand the synergistic effect of trehalose when gentamicin was added at different concentrations, 10mmol of trehalose and 4 concentrations of gentamicin (25,50,200 and 400. Mu.g/mL) were set to treat bacteria, the number of viable bacteria was counted in a spot plate after 6 hours, and the survival rates of Salmonella-resistant bacteria after trehalose addition and no trehalose addition were compared at the respective concentrations.
The results as shown in fig. 2 indicate that: as the concentration of gentamicin increases, the synergistic bactericidal effect of trehalose is higher, and the specific results are as follows:
after adding 10mmol of trehalose, when the concentration of gentamicin is 25 mug/mL, the sterilization efficiency is improved by 15.7 times (the survival rate is reduced from 100% without adding trehalose to 6.42% after adding trehalose); when the gentamicin is 50 mug/mL, the sterilization efficiency is improved to 52.7 times (the survival rate is reduced from 89.09 percent without adding trehalose to 1.67 percent after adding trehalose); when the concentration of gentamicin is 100 mug/mL, the sterilization efficiency is improved by 69.4 times (the survival rate is reduced from 65.45 percent without adding trehalose to 0.95 percent after adding trehalose); when the ampicillin concentration is 200 mug/mL, the sterilization efficiency is improved by 195.8 times (the survival rate is reduced from 41.81% without adding trehalose to 0.21% after adding trehalose); when the ampicillin concentration was 400. Mu.g/mL, the sterilization efficiency was increased 571.4 times (the survival rate was decreased from 12.85% without trehalose addition to 0.023% after trehalose addition).
5) The trehalose improves the sensitivity of gentamicin enteritis Salmonella resistant bacteria to gentamicin and has the effect of concentration gradient of the trehalose
To study whether the trehalose concentration has a gradient effect between sterilization efficiencies and obtain the optimal trehalose synergistic concentration, 5 trehalose concentrations (0.156, 0.625, 2.5, 10 and 40mmol respectively) are added to treat bacteria on the premise of adding 100 mug/mL gentamicin, viable bacteria number spot plate counting is carried out after 6h of incubation, survival rate is calculated, and the difference between survival rate under the cooperation of succinic acid with different concentrations and antibiotics and survival rate of antibiotics added independently is compared.
The results are shown in FIG. 3, and show that the survival rate of the gentamicin enteritis Salmonella resistant bacteria is 70.86% when 100 mug/mL gentamicin is added singly; when 0.156mmol of trehalose is added while gentamicin is added, the survival rate is 74.83 percent, which is not different from that of a single-addition antibiotic group; when 0.625mmol of trehalose is added, the survival rate is 16.16%, and the sterilization efficiency is improved by 4.39 times; when 2.5mmol of trehalose is added, the survival rate is 6.16%, and the sterilization efficiency is improved by 12.17 times; when 10mmol of trehalose is added, the survival rate is 0.99%, and the sterilization efficiency is improved by 71.51 times; when 40mmol of trehalose is added, the survival rate is 0.14%, and the sterilization efficiency is improved by 515.03 times.
6) Trehalose improves the time effect of gentamicin enteritis Salmonella resistance bacteria on sensitivity of gentamicin
To further investigate whether trehalose and gentamicin have a temporal effect on enhancing the effects of gentamicin enteritis Salmonella-resistant bacteria on antibiotic sensitivity, bacterial spot counts were performed for 0,2, 4, 6, 8 and 10 hours after addition of 10mmol trehalose and 100 μg/mL gentamicin, respectively, and survival was counted.
The results are shown in fig. 4, which demonstrate: along with the extension of the treatment time, the stronger the synergistic effect of the trehalose and the gentamicin is, the more specific is as follows: 2 hours, 7.4 times higher (survival rate reduced from 89.31% of gentamicin to 12.07%); 4 hours, 61.76 times higher (survival rate reduced from 76.10% of gentamicin to 1.23%); for 6 hours, 135.82-fold improvement (survival rate reduced from 65.85% of gentamicin to 0.49%); 8 hours, 270.30 times higher (survival rate reduced from 58.44% of gentamicin to 0.21%); for 10 hours, the survival rate is increased by 256.02 times (the survival rate is reduced from 52.59% of the gentamicin to 0.22%).
7) Influence of glucose and gentamicin on sterilization efficiency of gentamicin enteritis Salmonella resistant bacteria
In view of the fact that glucose has been reported to increase the effect efficiency of gentamicin in other strains, and trehalose is disaccharide, in order to further examine whether trehalose acts through itself or through glucose, in the case of the same antibiotics, a survival rate experiment was performed by adding glucose (20 mmol) at a concentration of 2 times, and it was found that the sterilization efficiency of gentamicin against gentamicin resistant to Salmonella enterica was not enhanced after adding glucose relative to the antibiotic group, and the above results indicate that trehalose did not act through glucose in increasing the sterilization efficiency of gentamicin against gentamicin resistant to Salmonella enterica.
Example 2
Application of trehalose in improving sensitivity of other various bacteria to gentamicin
1) Preparation of test samples
The bacterial viability was studied by picking up E.coli K12, edwardsiella tarda EIB202, vibrio alginolyticus VA, pseudomonas aeruginosa PE, klebsiella pneumoniae KPN monoclonal colonies from 2% solid LB plates, inoculating to 5mL of LB liquid medium, culturing overnight at 37℃or 30℃E.coli K12, pseudomonas aeruginosa PE and Klebsiella pneumoniae KPN (Edwardsiella tarda EIB202 and Vibrio alginolyticus VA) at 200rpm, collecting bacteria, centrifuging at 7000rpm for 3 minutes, removing supernatant, washing the bacterial cells 3 times with 0.85% physiological saline, suspending the bacterial cells with M9 minimal medium, and adjusting the bacterial cell liquid OD600 = 0.2, and finally packaging the adjusted bacterial liquid into test tubes (5 mL/each tube).
2) Minimum inhibitory concentration of gentamicin against other bacteria
The Minimum Inhibitory Concentration (MIC) of gentamicin on E.coli K12, edwardsiella tarda EIB202, vibrio alginolyticus VA, pseudomonas aeruginosa PE, klebsiella pneumoniae KPN was determined by the method of determining the Minimum Inhibitory Concentration (MIC) in example 1, as shown in FIG. 6, and the result shows that K12 is 6.25 μg/mL; EIB202 is 3.125 μg/mL; VA is 3.125 μg/mL; PE is 25 μg/mL; KPN was 50. Mu.g/mL.
3) Trehalose synergistically improves sensitivity of other bacteria to gentamicin
To further verify whether the synergistic effect of trehalose and gentamicin was applicable to other bacteria, the following experiments were performed, adding 10mmol of trehalose and 3. Mu.g/mL, 1.5. Mu.g/mL, 10. Mu.g/mL and 25. Mu.g/mL of gentamicin incubated K12, EIB202, VA, PE and KPN respectively, after 6 hours, counting the plates, counting the survival rate, and comparing the differences between trehalose and gentamicin synergistic group and gentamicin mono-group.
As a result, as shown in FIG. 7, it was found that the survival rate was 146.72-fold higher (the survival rate was reduced from 88.09% of gentamicin alone to 0.62%) with respect to Escherichia coli K12; the Edwardsiella tarda EIB202 can be increased by 523.49 times (the survival rate is reduced to 0.09 percent from 50 percent of the gentamicin monogamy); the VA of the vibrio alginolyticus can be increased by 606.93 times (the survival rate is reduced to 0.14% from 88.05% of the gentamicin monogamca); for pseudomonas aeruginosa, the survival rate can be increased by 72.85 times (the survival rate is reduced to 0.71% from 51.83% of the gentamicin monogamy); for klebsiella pneumoniae, 121.21-fold improvement can be achieved (survival rate is reduced from 74.10% of gentamicin monogamca to 0.61%).
Example 3
Application of trehalose in improving content of salmonella enteritidis intracellular gentamicin
Gentamicin belongs to an aminoglycoside antibiotic, and the sterilization mechanism is that the gentamicin can be combined with 30s subunits of bacterial cell nucleoses so as to block the synthesis of bacterial proteins. Thus, the foundation for gentamicin to perform sterilization is explained by how much it enters intracellular antibiotics. In order to study whether the trehalose plays a role in promoting sterilization by promoting the entry of gentamicin, adding gentamicin into a gentamicin enteritis salmonella sensitive bacterium group and a drug resistant bacterium group, and incubating the gentamicin in a shaking table for 6 hours at 37 ℃ and 200 rpm; and adding trehalose into the drug-resistant bacteria, adding trehalose and antibiotics, incubating in a shaking table in M9 at 37 ℃ and 200rpm for 6 hours, collecting bacteria, adjusting OD600 = 1.0, taking 1 milliliter of the adjusted bacteria, carrying out ultrasonic crushing, centrifuging, and taking the supernatant for measuring the content of the antibiotics. The determination method adopts REAGEN gentamicin enzyme-linked immune reaction test box (purchased from Shenzhen Hospital gold technology Co., ltd., product number: RND 99004) to detect, and comprises the following specific steps: 50 mu mL of supernatant and 100 mu mL of primary antibody are added into a 96-well plate, after being evenly mixed, the mixture is placed at room temperature for incubation for 30 minutes, the mixture is washed 3 times by plate washing liquid, 150 mu mL of secondary antibody is continuously added for incubation for 30 minutes at room temperature, the mixture is washed 3 times by plate washing liquid, 100 mu mL of TMB substrate is added, the mixture is placed at room temperature for 15 minutes in a dark place, 100 mu mL of stop solution is added, and finally OD value is read at a wave band of 450 nanometers.
As shown in FIG. 8, the result shows that the content of gentamicin in the cells of the sensitive bacteria is 1.75ng/mL, and the content of gentamicin in the resistant bacteria is 0.58ng/mL, which is obviously lower than that of the sensitive bacteria (FIG. 8A), and the salmonella enteritidis resistant bacteria realize the resistance to antibiotics by reducing the content of intracellular antibiotics. Further, the intracellular antibiotic content of the drug-resistant bacteria after the addition of trehalose for incubation was detected, and it was found that the intracellular gentamicin was significantly increased from the initial 0.57ng/mL to 1.99ng/mL, and increased by 3.49 times (FIG. 8B). These results indicate that trehalose can increase the intracellular antibiotic content of resistant bacteria, thereby increasing the sensitivity of the bacteria to antibiotics.
Example 4
Application of trehalose in improving proton motive force of salmonella enteritidis
In view of the research, the aminoglycoside antibiotics are found to enter bacterial cells depending on proton motive force, so that the proton motive force of sensitive bacteria, drug-resistant bacteria and drug-resistant bacteria before and after trehalose is added is detected. The test method is as follows: for the gentamicin enteritis salmonella and drug resistant bacteria group, at 37 in M9Incubating for 6h by a shaking table at the temperature of 200 rpm; adding trehalose into the drug-resistant bacteria, incubating the added trehalose in M9 at 37deg.C under 200rpm for 6 hr, collecting bacteria, diluting to make bacterial count about one million CFU/mL, and adding 10 μmL 3mmol DiOC 2 Added to a centrifuge tube containing 1 ml of diluted bacterial liquid, and incubated at 37℃for 30 minutes with shaking at 200 rpm. Detection was then performed using a flow cytometer. The parameters were set as follows: the excitation wavelength of the green fluorescence and the red fluorescence is 488nm, and the emission wavelength is 530nm and 610nm respectively. The PMF value was calculated as LOG (10 3/2 * Y mean/X mean), Y mean and X mean denote red light intensity and green light intensity, respectively. Change in proton motive force= (experimental group-control group)/control group × 100%.
The results are shown in FIG. 9, which shows that the proton potential of the drug-resistant bacteria was reduced by 12.69% with respect to the sensitive bacteria (FIG. 9A), and further corroborates the conclusion that the amount of gentamicin in cells of the drug-resistant bacteria found in example 3 was lower than that of the sensitive bacteria. When trehalose was added, the proton potential of the drug-resistant bacteria was found to be increased by 21.12% (FIG. 9B). These results demonstrate that salmonella enteritidis resistance to gentamicin is caused by a decrease in membrane proton motive force, resulting in a decrease in intracellular antibiotics. The exogenesis adding of trehalose can improve proton motive force of drug-resistant bacteria, promote the entry of gentamicin and reverse sensitivity of gentamicin.
Example 5
Application of trehalose in improving anti-infective power of Huiyang beard chicken to gentamicin enteritis Salmonella resistance bacteria
1) Preparation of challenge bacteria and test chickens
The stored bacterial glycerol strain was removed from the-80℃refrigerator and all added to 100 ml of LB medium (250 ml flask), and cultured at 37℃and 200 rpm. After the bacteria grew to an OD600 of about 1.0, the bacteria were collected by centrifugation and the supernatant was discarded. Washing with physiological saline for 3 times.
The Huiyang beard female chickens (7 days old) were randomly grouped and, for the detection of the bacterial content of each organ, SE-R (1X 10) 9 CFU/each chicken) was given intraperitoneal injection to the chicken, followed by intraperitoneal injection of gentamicin (10 mg/kg) or gentamicin (10 mg) 1 hour after infectionTrehalose (5 mg/kg) or an equal volume of sterile saline was added per kg. The liver, spleen and kidney samples were collected immediately for bacterial load analysis in a sterile environment 6 hours after the last injection. Specifically: the spleen, left kidney and 25mg liver at the same position were added with 1 mL of physiological saline, then homogenized by a mill, 100. Mu.ml of the homogenate was subjected to gradient dilution, and finally 5. Mu.ml of the homogenate was spotted on LB agar plates, and after stationary culture at 37℃for 8 hours, colony counts were performed. To examine survival, chickens were subjected to intraperitoneal injection of SE-R (5X 10) 9 CFU/chicken). After 1 hour, gentamicin (40 mg/kg) or gentamicin (40 mg/kg) plus trehalose (20 mg/kg) was injected intraperitoneally, and the survival rate was counted after 2 days of observation.
2) Trehalose reduces the bacterial load in organs after chicken is infected with bacteria
As shown in FIG. 10, the results of the plate count of the organ homogenates revealed that the bacteria content in the control group (offending bacteria + physiological saline) in the spleen was 5.47X 10 6 CFU, bacterial content in antibiotic group (challenge bacteria+gentamicin) 1.97X10 6 CFU, whereas the bacterial content in the trehalose group (challenge bacteria + gentamicin + trehalose) was 1.17X10 4 CFU, increased the host bacterial removal capacity by 469 fold relative to control, and 169 fold relative to antibiotic; in the liver, the bacteria content in the control group (challenge bacteria + physiological saline) was 2.5X10 6 CFU, bacterial content in antibiotic group (challenge bacteria+gentamicin) 1.57×10 6 CFU, whereas the bacterial content in the trehalose group (challenge bacteria + gentamicin + trehalose) was 9.43×10 3 CFU, 265-fold higher in host bacterial clearance relative to control, 166-fold higher relative to antibiotic; in the kidney, the bacteria content in the control group (offending bacteria+physiological saline) was 1.89×10 5 CFU, bacterial content in antibiotic group (challenge bacteria+gentamicin) 1.52X10 5 CFU, whereas the bacterial content in the trehalose group (challenge bacteria + gentamicin + trehalose) was 7.42×10 2 CFU increased 254-fold over control, and 204-fold over antibiotic.
3) Trehalose improves survival rate of chicken infected with bacteria
As shown in the survival rate test results shown in FIG. 11, it was found that the survival rate of the control group (challenge bacteria + physiological saline) was 0% after infection of SE-R, the survival rate of the chicken was increased to 10% when gentamicin was injected, and the survival rate was increased to 45% when gentamicin and trehalose were injected simultaneously, which was increased by 35% relative to the antibiotic group.
The results show that the trehalose can cooperate with the gentamicin to enhance the bacterial removal capability of chickens and improve the resistance of Huiyang beard chickens to gentamicin enteritis Salmonella resistant bacteria. Therefore, according to the trehalose content of organisms or trehalose metabolism related genes as candidate targets for chicken disease resistance breeding, the disease resistance breeding of chickens can be realized from the aspect of antibiotics.
Claims (5)
1. The application of trehalose in preparing a medicament for improving the sensitivity of drug-resistant bacteria to gentamicin is characterized in that the drug-resistant bacteria comprise gentamicin salmonella enteritidis drug-resistant bacteria SE-R, escherichia coli K12, edwardsiella tarda EIB202, vibrio alginolyticus VA, pseudomonas aeruginosa PE and klebsiella pneumoniae KPN.
2. The application of trehalose in preparing medicines for improving the anti-infective power of gentamicin resistant bacteria of livestock is characterized in that the resistant bacteria comprise gentamicin enteritis salmonella resistant bacteria SE-R, escherichia coli K12, edwardsiella tarda EIB202, vibrio alginolyticus VA, pseudomonas aeruginosa PE and klebsiella pneumoniae KPN.
3. Application of trehalose in preparing medicines for resisting gentamicin enteritis Salmonella resistant bacteria infection of chicken is provided.
4. The application of the trehalose combined gentamicin in preparing the medicine for improving the sensitivity of the drug-resistant bacteria is characterized in that the drug-resistant bacteria comprise gentamicin salmonella enteritidis drug-resistant bacteria SE-R, escherichia coli K12, edwardsiella tarda EIB202, vibrio alginolyticus VA, pseudomonas aeruginosa PE and klebsiella pneumoniae KPN.
5. The application of trehalose and gentamicin in preparing medicines for resisting gentamicin enteritis salmonella infection of chickens.
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