| Aditoprim(ADP)is a new type of dihydrofolate reductase inhibitor.Because of its good bacteriostatic effect,the long half-life,large apparent distribution volume,low toxicity and safety,and no mutagenicity,it has excellent potential and research value in clinical veterinary medicine.At present,there are many studies on ADP at home and abroad,but there is a lack of experimental research on the evaluation of its microbiological safety.In order to evaluate it more systematically and comprehensively,it is necessary to study the acceptable daily intake(ADI)of ADP in microbiology and work out a reasonable microbiological ADI.In this study,a continuous culture chemostat model in vitro was used to evaluate the effect of ADP on human intestinal microflora.ADP in different concentrations was added to the model.The colonization resistance and resistance changes under the influence of various drugs’concentrations were measured.The colonization resistance consists of the bacterial population(Escherichia coli,Enterococcus,Bacteroides fragilis,Clostridium perfringens and Bifidobacterium),fatty acids(acetic acid,propionic acid,n-butyric acid,i-butyric acid,n-valeric acid,i-valeric acid,caproic acid,heptanoic acid)concentration,colonization of Salmonella Typhimurium.Finally,no observable adverse effect Concentration(NOAEC)and the microbiology ADI of ADP were determined by colonization resistance and resistance.1 Impact of ADP on colonization resistanceThrough pre-experiment,it was determined that the final four concentrations of ADP in the chemostat model were 0,1,16,128μg/m L.In the low concentration group(1μg/m L),Populations of five dominant bacteria and fatty acids’concentration were all between 95%confidence.The final concentration of Salmonella typhimurium decreased below 10~2CFU/m L.ADP had no side effect on the colonization resistance.In the medium-concentration group(16μg/m L),populations of Escherichia coli decreased significantly from the 18th day,86.61%less than 95%lower confidence limit on the 22nd day.The populations of Enterococci and Clostridium perfringens fluctuated around 95%lower confidence limit;the minimum values were 4.925×10~4CFU/m L and 9.016×10~5CFU/m L,which were only 17/37 and 30/67 of 95%lower confidence limit.Other flora changes were within the normal range.Acetic acid,propionic acid,n-butyric acid,and i-valeric acid had particular time points that were slightly below than 95%lower confidence limit.In comparison,other time points were between 95%confidence.N-valeric acid was below 95%lower confidence limit from the 20th day.Its minimum concentration is 3.970 mmol/m L,which was 19.73%less than 95%lower confidence limit.The concentration of i-butyric acid was lower than 95%lower confidence limit for four days,and the weakest on the 21st day,which was 21.82%less than 95%lower confidence limit.The lowest concentration of caproic acid decreased by about 15%compared with 95%lower confidence limit.The fluctuation range of heptanoic acid increased significantly,and the highest daily range of concentration was 0.405 mmol/m L,which was 1.49 times as much as 95%confidence interval.The final concentration of Salmonella typhimurium was 3.09×10~3CFU/m L,which did not fall below 10~2CFU/m L,indicating that colonization resistance of intestinal flora in the medium-concentration group was damaged to a certain extent.In the high concentration group(128μg/m L),The lowest values of Escherichia coli,Bifidobacterium,Bacteroides fragilis,and Clostridium perfringens were reduced by 94.56%,89.58%,82.11%,and 91.75%respectively from 95%lower confidence limits.The number of Enterococci increased instead of decreasing,and the number was 3.96 times of 95%upper confidence limit on the 22nd day.The concentrations of acetic acid,propionic acid,butyric acid,n-valeric acid,i-valeric acid and i-butyric acid were lower than 95%lower confidence limit by 21.68%,22.55%,11.06%,27.85%,23.45%,17.91%.Compared with 95%confidence,the maximum daily concentration difference of caproic acid and heptanoic acid was 1.62times and 1.61 times,respectively.The number of Salmonella typhimurium was stable above 10~5CFU/m L.These changes indicate that the colonization resistance was destroyed.2 Impact of ADP on the resistance of intestinal floraThe resistance rate of the five dominant bacteria in the low concentration group(1μg/m L)were all between 95%confidence,and there was no significant difference in resistance compared with the control group.In the medium-concentration group(16μg/m L),the resistance rate of Escherichia coli and Enterococcus exceeded 95%confidence from the 18th day and the 17th day,and the highest resistance rate was13.4%and 48.94%.The resistance rate of Bifidobacterium fluctuated greatly,and the difference between the minimum and maximum was 13.31%.The highest resistance rate of Clostridium perfringens was 15.71%,which was 1.58 times of 95%confidence.The resistance rate of Bacteroides fragilis was between 95%confidence except for a few individual values that exceeded the confidence range.The high-concentration group(128μg/m L)ADP had a significant impact on the resistance change of five dominant bacteria.The resistance rate of Escherichia coli,Enterococcus,Bifidobacterium,Bacteroides fragilis,and Clostridium perfringens were 2.65 times,1.68 times,1.5 times,1.78 times and 1.63 times as much as 95%upper confidence,respectively.At 1μg/m L,the colonization resistance and resistance did not change significantly,so the NOAEC of ADP on human intestinal flora was 1μg/m L.According to the ADI value calculation formula of VICH,the microbiological ADI was 91.67μg/kg b.w./day,which was derived from the impact of ADP on human intestinal flora. |
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